Abstract book - ITQB - Universidade Nova de Lisboa

3 rd European Meeting in Oxizymes
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa

OXIZYMES IN OEIRAS
3 rd European Meeting in Oxizymes
Abstract Book
Oxizymes in Oeiras – 3 rd European Meeting in Oxizymes
September, 7-9, 2006
Oeiras, Portugal
Editors: Lígia O. Martins, André T. Fernandes, Paulo Durão
Microbial and Enzyme Technology Lab.
Instituto de Tecnologia Química e Biológica, Oeiras, Portugal
Printing: Reprocromo Sociedade de Fotolito, Lda., Amadora, Portugal
This book of abstracts was carefully produced. Nevertheless we do not warrant the information
contained therein to be free of errors

WELCOME TO OEIRAS
The Organizing Committee of the 3 rd European Meeting in Oxizymes – OXIZymes in Oeiras
welcomes you to Oeiras, Portugal.
OXIZymes in Oeiras comes in the sequence of previous two meetings organized respectively by
Thierry Tron at Cassis, France, in 2002, and by Giovanni Sannia at Naples, Italy, in 2004.
These meetings have, from the beginning, an idea of not only being a “melting pot” of European
Scientists working in the field of oxidative enzymes, but also to be the backbone of an European
Network of Excellence, hoping to constitute an “incubator” for many application proposals to the
European Commission.
Thanks to the contributions of participants we were able to design a programme to OXIZymes in
Oeiras that will cover recent developments on oxidases, oxygenases and peroxidases, from
microbial physiology and genetics, enzymology, protein structure and structure-function studies
to environmental and biotechnological applications. OXIZymes in Oeiras will present three
eventfull days where thirty six lectures will take place and near seventy posters will be
accessible.
On behalf of the Organizing Committee I would like to to express my gratitude to all people that
contribute to the organization of this Meeting, to the members of the Scientific Committee, in
particular Prof. Giovanni Sannia, to Ms. Rosina Gadit from the ITQB secretariat, to all our
sponsors that provide us extra-funds and finally to the institutional support of Instituto de
Tecnologia Química e Biológica, Universidade Nova de Lisboa.
We wish you all a fruitful meeting of effective scientific exchange and an enjoyable stay in
Portugal.
Oeiras, 20 August, 2006
4

OXIZymes in Oeiras SPONSORS
5

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Scientific Committee
Angel T. Martínez CIB, CSIC, Madrid, Spain
Antonio Sanchez-Amat Univ Murcia, Spain
Artur Cavaco-Paulo Univ Minho, Portugal
Cláudio M. Soares ITQB, Univ Nova de Lisboa, Portugal
Dietmar Schlösser UFZ Center Environm. Res., Germany
Georg M. Güebitz Graz Technical Univ, Austria
Giovanni Sannia Univ di Napoli “Federico II”, Italy
Kristiina Kruus VTT Biotechnology, Finland
Lígia O. Martins ITQB, Univ Nova de Lisboa, Portugal
Maria Jesus Martínez CIB, CSIC, Madrid, Spain
Paola Giardina Uiv di Napoli ”Federico II”, Italy
Peter F. Lindley ITQB, Univ Nova de Lisboa, Portugal
Riccardo Basosi Univ di Siena, Italy
Sophie Vannhule Univ Catholique LLN, Belgium
Tajalli Kershavarz Univ of Westminster, United Kingdom
Thierry Tron CNRS, Marseille, France
Willem van Berkel Wageningen Univ, The Netherlands
Organizing Committee (ITQB/UNL)
Lígia O. Martins
André T Fernandes
Paulo Durão
Luciana Pereira
Cláudio M. Soares
Isabel Bento
Manuela M. Pereira
6 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
OXIZymes in Oeiras TIMETABLE
THURSDAY
SEPTEMBER, 7
8.00-9.00
REGISTRATION
9.00-9.30
OPENING
9.30-11.00
S1
MICROBIAL PHYSIOLOGY I
9.00-10.30
FRIDAY
SEPTEMBER, 8
S5
STRUCTURE-FUNCTION
RELATIONSHIPS I
9.00-10.30
SATURDAY
SEPTEMBER, 9
S9
APPLICATIONS III
coffee-break coffee-break coffee-break
11.30-13.00
S2
MICROBIAL PHYSIOLOGY II
11.00-12.30
S6
STRUCTURE-FUNCTION
RELATIONSHIPS II
11.00-12.30
Round Table
“Which Future for Oxizymes in the 7 th FP?”
15.00-16.30
S3
ENZYMOLOGY I
lunch
poster attendance
lunch
poster attendance
14.30-16.00
S7
APPLICATIONS I
coffee-break
17.00 -18.30
S4
ENZYMOLOGY II
coffee-break
16.30 -18.00
S8
APPLICATIONS II
18.30-19.00
Welcome Drink
“Porto de Honra”
20.30
Oxizymes dinner
7 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Thursday, September 7, 2006
8.00-9.00 Registration
9.00-9.30 Opening
9.30-11.00
S1 - MICROBIAL PHYSIOLOGYI
CHAIRPERSON: TAJALLI KESHAVARZ
9.30-9.55
9.55-10.20
10.20-10.40
10.40-11.00
L1 - Antimicrobial and Biochemical Properties of a Novel Type of Lysine Oxidase
Expressed by Marinomonas mediterranea
Antonio Sanchez-Amat, Murcia Univ, Spain
L2 - Lignin-Modifying Peroxidases and Laccases of the White Rot Basidiomycete
Phlebia radiata
Taina Lundell, Helsinki Univ, Finland
L3 - Essential Role of the LPR1 Family of Metallo-Oxidases in the Arabidopsis
thaliana Root Growth Response to Low-Phosphate Media
Thierry Desnos, DEVM, St Paul-les-Durance, France
L4 - Recent Advances in the Physiology of Ligninolytic Enzymes Produced by
White-Rot Basidiomycetes
Vladimir Elisashvili, Inst Biochem and Biotechnology, Tbilisi, Georgia
11.00-11.30 Coffee
11.30-13.00
S2 - MICROBIAL PHYSIOLOGY II
CHAIRPERSON: ANNELE HATAKKA
11.30-11.55
11.55-12.20
12.20-12.40
12.40-13.00
L5 - Biological Functions and Regulation of the Multicopper Oxidase LcsA from
Myxococcus xanthus
Juana Pérez-Torres, Granada Univ, Spain
L6 - Oxizymes in Hardwood Forest Soil: Production of Oxidases and Peroxidases
by Exploratory Mycelium of Saprotrophic Soil Basidiomycetes
Petr Baldrian Inst Microbiol ASCR, Prague, Czech Republic
L7 – Novel Efficient Producers of Blue Laccases
Ludmila Golovleva, GKS Inst Biochem Physiolo Microorg, Moscow,
Russia
L8 – Discovery of an Epoxide Forming Monooxygenase from the Metagenome
Erik van Hellemond, Groningen Univ, The Netherlands
13.00-15.00 Lunch/poster attendance
8 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Thursday, September 7, 2006
15.00-16.30
S3 – ENZYMOLOGY I
CHAIRPERSON: THIERRY TRON
15.00-15.25
15.25-15.50
15.50-16.10
16.10-16.30
L9 – Production and Characterization of a Secreted C-terminally Processed
Tyrosinase from the Filamentous Fungus Trichoderma reesei
Kristiina Kruus, VTT, Finland
L10 - Understanding the Selection of Arene Dioxygenase Enzymes for Optimal
Chemo-, Stereo- and Regio-Selectivity of Biotransformation Processes
Christopher C. R. Allen, Queen’s Univ Belfast, Northern Ireland
L11 – Redesign of AtGALDH, a Flavoprotein Involved in Vitamin C Biosynthesis
Nicole G. H. Leferink, Wageningen Univ, The Netherlands
L12 – Acetate Inhibition of Laccase Activity
Ewald Srebotnik, Vienna Univ Technol, Austria
16.30-17.00 Coffee
17.00-18.30
S4 – ENZYMOLOGY II
CHAIRPERSON: STEFFEN DANIELSEN
17.00-17.25
17.25-17.50
17.50-18.10
18.10-18.30
L13 – Cofactor Incorporation and Cofactor-Induced Stabilization of Oxizymes
Willem J.H. van Berkel, Wageningen Univ, The Netherlands
L14 – A Flavin-Dependent Tryptophan 6-Halogenase and its use in Combinatorial
Biosynthesis
Karl Heinz Van Pée, Dresden Univ, Germany
L15 – A Plant Peroxidase Intrinsically Stable Towards Hydrogen Peroxide
Brenda Valderrama, Aut. Nac Mexico Univ, éxico
L16 – Functional Hybrids of Haloperoxidases and Cytochrome P450
Monooxygenases from Alkaliphilic Mushrooms
Martin Hofrichter, Int Grad School Zittau, Germany
18.30-19.00 Welcome Drink – “Porto de Honra”
9 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Friday, September 8, 2006
9.00-10.30
S5 - STRUCTURE-FUNCTION RELATIONSHIPS I
CHAIRPERSON: CLÁUDIO M. SOARES
9.00-9.25
9.25-9.50
9.50-10.10
10.10-10.30
L17 - Laccase Engineering by Rational and Random Mutagenesis
Giovanni Sannia, “Federico II” Napoli Univ, Italy
L18 - Structure-Function Studies of Pleurotus Versatile Peroxidase, A Model
Ligninolytic Enzyme
Angel T. Martínez, CSIC, CIB, Spain
L19 - Structure-Activity Relationship of the Laccase Mediator System
Rebecca Pogni, Siena Univ, Italy
L20 – 'Titrating' Steric and Redox Features of the Active Site of Laccase
Carlo Galli, “La Sapienza” Roma Univ, Italy
10.30-11.00 Coffee
11.00-12.30
S6 - STRUCTURE-FUNCTION RELATIONSHIPS II
CHAIRPERSON: CLÁUDIO M. SOARES
11.00-11.25
11.25-11.50
11.50-12.10
12.10-12.30
L21 – A Near-Atomic Resolution Crystal Structure of Melanocarpus
albomyces Laccase
Nina Hakulinen, Joensuu Univ, Finland
L22 - Structure-Function Studies in Bacterial Multicopper Oxidases
Lígia O. Martins, ITQB, UNL, Portugal
L23 - Crystal Structures of Three New Fungal Laccases: Implications on the
Catalytic Mechanism and on the Dynamics of the Copper Sites Redox States
Fabrizio Briganti, Firenze Univ, Italy
L24 - Construction and Characterisation of Horseradish Peroxidase Mutants
that Mimic Some of the Properties of Cytochromes P450
Andrew T. Smith, Sussex Univ, UK
12.30-14.30 Lunch/poster attendance
10 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Friday, September 8, 2006
14.30-16.00
S7 – APPLICATIONS I
CHAIRPERSON: LIISA VIIKARI
14.30-14.55
14.55-15.20
15.20-15.40
15.40-16.00
L25 - Immobilisation of Laccases for Biotransformations in Environmental
and Food-Technology
Georg M. Güebitz , Techn Univ Graz, Austria
L26 - Laccase-Catalyzed Polymerization for Coating and Material
Modification
Artur Cavaco-Paulo, Minho Univ, Portugal
L27 – Potential of White-Rot Fungi for Decolourisation and Detoxification of
Dyes
Sophie Vanhulle, Univ Catholique LLN, Belgique
L28 – Biotransformation of Environmental Pollutants by Aquatic Fungi –
The Role of Laccases
Dietmar Schlosser, UFZ, Leipzig, Germany
16.00-16.30 Coffee
16.30-18.00
S8 – APPLICATIONS II
CHAIRPERSON: PAUL ANDER
16.30-16.55
16.55-17.20
17.20-17.40
17.40-18.00
L29 - Transformation of Textile Dyes by Oxidoreductases
Feng Xu, Novozymes, USA
L30 - Free, Supported and Insolubilized Laccases : Novel Biocatalysts for
the Elimination of Micropollutants and Xenoestrogens
Spiros N. Agathos, Univ Catholique LLN, Belgium
L31 - Olive Mill Wastewater Transformation and Detoxification by White-
Rot Fungi: Role of the Laccase in the Process
Maria Jesus Martínez, CISC, CIB, Madrid, Spain
L32 - Combined Application of Glucose Oxidases and Peroxidases in
Bleaching Processes
Klaus Opwis, Deutsches Textilforschungszentrum, Germany
20.30 OxiZymes DINNER
11 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Saturday, September 9, 2006
9.00-10.30
S9 – APPLICATIONS III
CHAIRPERSON: CHRISTIAN-MARIE BOLS
9.00-9.25
9.25-9.50
9.50-10.10
10.10-10.30
L33 - Laccase-Mediator System: the Definitive Solution to Pitch Problems in
the Pulp and Paper Industry?
Ana Gutiérrez, CSIC, Seville, Spain
L34 - Optimization of a Laccase-based Delignification System which uses as
Mediators Fatty Hydroxamic Acids in situ Generated by Lipases
Hans-Peter Call, Bioscreen, Germany
L35 - Studies on the effect of the laccase mediator system on ageing
properties of hand sheets of different origin
Maria Costa-Ferreira, INETI, Lisboa, Portugal
L36 - Laccase in Pulp Activation and Functionalisation
Anna Suurnäkki, VTT, Finland
10.30-11.00 Coffee
11.00-12.30 Round Table
“Which future for the OXIZYMES in the 7 th FP?”
Chairpersons: Liisa Viikari and Giovanni Sannia
Angel T Martínez
Georg M. Gübitz
Christian-Marie Bols
12 September 7-9, 2006
Oeiras, Portugal

ORAL PRESENTATIONS

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Antimicrobial and Biochemical Properties of a Novel type
of Lysine Oxidase Expressed by Marinomonas
mediterranea.
Daniel Gómez a , Patricia Lucas-Elío a , Francisco Solano b , Antonio Sanchez-Amat a
a
Department of Genetics and Microbiology, Faculty of Biology; b Department of Biochemistry
and Molecular Biology, School of Medicine, University of Murcia, Campus de Espinardo,
Murcia 30100, Spain
E-mail: antonio@um.es
Traditionally, the pharmaceutical industry looking for new antibiotics has focused in the study
of small molecules (< 1 kDa). However, the need of new compounds, driven for example by
the increase of antibiotic resistance in many pathogens, is determining an increase in the study
of alternative sources of molecules with biological properties. Proteins are of interest because
they can be expressed in heterologous hosts, and molecular techniques facilitate their
improvement and characterization. Two sources of this kind of proteins are marine see hares
and the venom of snakes. From both sources, L-amino acid oxidases (L-AAOs) have been
isolated. L-AAOs are flavoenzymes that catalyze the oxidative deamination of L-amino acids
to the respective enzymes α-ketoacids with the release of hydrogen peroxide, which
determines their antimicrobial properties.
Marinomonas mediterranea is a melanogenic marine bacterium isolated by our group that
expresses two polyphenol oxidases (PPOs) a laccase and a tyrosinase. We have recently
demonstrated that M. mediterranea also synthesizes an antimicrobial protein, named
marinocine, showing a broad range of antibacterial activity 1 . The gene coding for this enzyme
has been cloned, and it has been demonstrated that the antimicrobial activity is due to the
hydrogen peroxide generated by its lysine oxidase activity 2 . Sequence analysis revealed that
marinocine shows similarity to other bacterial proteins, most of them hypothetical, but not to
the previously characterized L-AAOs. Moreover, marinocine catalyzes a novel reaction: the
deamination of lysine generating semialdehyde 2-aminoapidic acid and releasing H 2 O 2 . The
characteristics of marinocine in comparison with other proteins also able to catalyze the
oxidation or transformation of L-lysine will be discussed.
[1] Lucas-Elío, P., Hernández, P., Sanchez-Amat, A., & Solano, F. 2005. Purification and partial characterization
of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea. Biochim.
Biophys. Acta. 1721: 193-203.
[2] Lucas-Elío, P., Gómez, D., Solano, F. & Sanchez-Amat, A. 2006. The antimicrobial activity of marinocine,
synthesized by Marinomonas mediterranea,is due to the hydrogen peroxide generated by its lysine oxidase
activity. J. Bacteriol. 188: 2493-2501.
L1
16 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L2
Lignin-Modifying Peroxidases and Laccases of the White
Rot Basidiomycete Phlebia radiata
Taina Lundell a , Kristiina S. Hildén a , Miia R. Mäkelä a , Annele Hatakka a
a Department of Applied Chemistry and Microbiology, Division of Microbiology, University
of Helsinki, Finland;
E-mail: taina.lundell@helsinki.fi
The naturally wood-colonising, saprophytic white rot fungus Phlebia radiata (Corticiaceae,
Aphyllophorales, Homobasidiomycetes) is an efficient degrader of hardwood and softwood
lignin, synthetic lignin (DHP) and lignin-like model compounds. Our own isolate P. radiata
79 produces a versatile set of extracellular lignin-modifying enzymes (LMEs) including two,
structurally and genetically divergent manganese peroxidases (MNPs), [1] three lignin
peroxidases (LIPs) [2] and at least one laccase upon growth in liquid media or in cultures
supplemented with milled hardwood.
Molecular evolutionary sequence analysis of the lignin-modifying peroxidases (LMPs)
reveals clustering of the P. radiata lip genes but significant divergence with the two mnp
genes, one short and the other long, thereby supporting at least three main evolutionary fungal
peroxidase gene families within the class II heme peroxidases. [1,3] Phylogeny of LMP
supports more functional than fungal species-based evolution, and peroxidase gene intronexon
organisation indicates a more recent gene duplication or lateral gene transfer, in
particular for the short LIP-MNP-VP-encoding genes irrespective of fungal taxons. Structurefunction
relationship of the LMPs is also discussed based on in vitro reactions and differential
expression upon degradation and growth on wood.
We recently identified a new laccase-encoding gene of P. radiata when the fungus is growing
in the presence of wood. The second predicted laccase Lac2 displays a higher pI value (5.8)
than the previously isolated Lac1 (pI 3.2-3.5). Preliminary protein analysis demonstrates that
Lac2 may be retained by the hyphae or it is secreted only in minor amounts. On spruce wood
chips, the two laccases (genes Pr-lac1 and Pr-lac2) were expressed within three weeks of
growth together with the MNP and LIP-encoding genes. Our results indicate synchronous,
time-dependent regulation of expression for the P. radiata laccases, together with the two
divergent MNPs and the three LIPs. These findings also implicate that the complete assembly
of all the so far characterised P. radiata LMPs and laccases are involved in the processes of
wood colonisation and decomposition of wood lignin, although the individual functions for
each enzyme is not known yet.
[1] Hildén K, Martínez AT, Hatakka A, Lundell T (2005) The two manganese peroxidases Pr-MnP2 and Pr-
MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent.
Fungal Genetics and Biology 42: 403-419
[2] Hildén KS, Mäkelä MR, Hakala TK, Hatakka A, Lundell T (2006) Expression on wood, molecular cloning
and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata.
Current Genetics 49: 97-105
[3] Martínez AT (2002) Molecular biology and structure-function of lignin-degrading peroxidases. Enzyme and
Microbial Technology 30: 425-444
17 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Essential Role of the LPR1 Family of Metallo-Oxidases in
the Arabidopsis thaliana Root Growth Response to Low-
Phosphate Media
Sergio Svistoonoff a,1 , Cécile Sigoillot-Claude a , Matthieu Reymond a,2 , Audrey Creff a , Lilian
Ricaud a , Aline Blanchet a , Laurent Nussaume a and Thierry Desnos a
a Laboratoire de Biologie du Développement des Plantes, DEVM, CEA cadarache, 13108 St
Paul-lez-Durance cedex, France;
1 Present address: Federal Institute of Technology (ETH) Zurich, Institute of Plant Sciences,
Experimental Station Eschikon 33, CH-8315 Lindau, Switzerland.
2 Present address: Department of Plant Breeding and Genetics, Max Planck Institute for Plant
Breeding Research (MPIZ), Carl-von-Linné-Weg 10, D-50829 Cologne, Germany.
E-mail: thierry.desnos@cea.fr
The search for nutrients is an essential activity for all organisms. In plants, the roots are able
to sense nutrient availability and the root architecture optimizes exploration of the soil to
acquire heterogeneously distributed water and minerals. One well-known plant response to
soil phosphate (Pi)-deficiency is a reduction in primary root growth with an increase in the
number and length of lateral roots. We show that loss-of-function mutations in LPR1 (Low
Phosphate Root1) and its close paralogue LPR2 strongly reduce this inhibition. LPR1 was
previously mapped as a major quantitative trait locus (QTL) 1 ; the molecular origin of this
QTL is explained by the differential allelic expression of LPR1 in the root tip. LPR1 and
LPR2 encode metallo-oxidases and pharmacological inhibition of these oxidases activity in
the wild type phenocopies the Lpr - root. The enzymatic characteristics of LPR1 have been
analyzed in vitro. Our results demonstrate the essential role of these oxidases in plant growth
plasticity and provide evidence for their involvement in sensing and/or responding to nutrient
deficiency.
[1] Reymond et al., Plant Cell, & Environment (2006) 29, 115-125.
L3
18 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L4
Recent Advances in the Physiology of Ligninolytic
Enzymes Produced by White-Rot Basidiomycetes
V. Elisashvili, E. Kachlishvili, N. Mikiashvili, N. Tsiklauri, E. Metreveli, G. Kvesitadze
Institute of Biochemistry and Biotechnology, 10 km Agmashenebeli kheivani, 0159 Tbilisi,
Georgia
E-mail: velisashvili@hotmail.com
Ligninolytic enzymes have potential use in a wide range of industrial and environmental
purposes. However, the cost of production and low yields of these enzymes are the major
problems for their bulk industrial application. Numerous reports have been published recently
on the strategies improving the production of ligninolytic enzymes, such as the isolation of
new fungal strains, optimization of growth conditions, use of inducers and stimulators, as well
as use of cheap growth substrates such as agricultural and food industry wastes. In this
communication, these recent advances in the production of extracellular laccases and
peroxidases by white-rot fungi will be critically discussed.
Some recent developments of our laboratory in laccase and manganese peroxidase production
will be considered. A broad diversity among white-rot basidiomycetes from various
taxonomic groups and ecological niches was revealed in evaluation of their ability to produce
laccase and manganese peroxidase under identical laboratory conditions. The crucial effect of
carbon source and especially of lignocellulosic material on the secretion and ratio of
individual enzymes will be underlined. The contribution of extractable with water and organic
solvents compounds from lignocellulosic substrates in secretion of ligninolytic enzymes
production will be discussed. Some strategies of these extracts utilization to enhance enzyme
production and to improve the rheological properties of fermentation medium will be
suggested. A special attention will be paid to the regulation of laccase and manganese
peroxidase by microelements and aromatic compounds/dyes (effects of their concentration,
time addition, and cumulative effect).
19 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Biological Functions and Regulation of the Multicopper
Oxidase LcsA from Myxococcus xanthus
María Celestina Sánchez-Sutil, Aurelio Moraleda-Muñoz, Nuria Gómez-Santos, José Muñoz-
Dorado and, Juana Pérez-Torres
Departamento de Microbiología. Facultad de Ciencias. Universidad de Granada. Avda.
Fuentenueva s/n. E-18071 Granada. Spain.
E-mail: jptorres@ugr.es
Myxococcus xanthus is a soil-dwelling bacterium that undergoes a developmental cycle upon
starvation that culminates with the formation of multicellular macroscopic structures, fruiting
bodies, filled of myxospores. This behaviour is unique among the prokaryotes. M. xanthus
genome has been sequenced by TIGR/Monsanto, and the analysis of the genome has revealed
that it encodes three multicopper oxidases, which have been designated LcsA, LcsB and
LcsC. lcsA is forming an operon (named as curA) with other 8 genes, which encode several
proteins with similarities to other deposited in the databases which have been reported to be
involved in copper resistance and homeostasis. The curA promoter is induced in a stepwise
fashion as the external Cu(II) ions are increased, reaching the maximum levels to
subinhibitory copper concentration. Surprisingly, vegetative cells need almost ten-fold more
copper compared to developing ones to reach similar expression levels. This different copper
sensitivity of curA promoter can not be attributed to intracelular copper accumulation. The
operon also responds to other divalent borderline soft/hard metals that are biologically
required such as nickel, cobalt or zinc, but to a lower induction ratio compared to copper. We
have identified a two-component system (CusSR) responsible for the expression and
induction of the curA operon during both growth and development The phenotype
characterization of an in-frame deletion mutant ∆lcsA evidences that LcsA plays an important
role in detoxification of periplasm and in the normal differentiation of cell to spores during
development. More details will be presented at the conference.
L5
20 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L6
Oxizymes in Hardwood Forest Soil: Production of Oxidases
and Peroxidases by Exploratory Mycelium of Saprotrophic
Soil Basidiomycetes
Jaroslav Šnajdr a , Vendula Valášková a , Tomáš Cajthaml a , Věra Merhautová a , Petr Baldrian a
a Institute of Microbiology ASCR, Vídeňská 1083, 14220 Prague 4, Czech Republic
E-mail: baldrian@biomed.cas.cz
Ligninolytic oxidases and peroxidases of saprotrophic fungi are the enzymes responsible for
the transformation of lignin – the second most abundant biopolymer. In forest soil,
ligninolytic enzymes contribute to the degradation of lignin in decaying leaf litter and to the
transformation of humic substances with a similar chemical structure [1,2]. The aims of this
work were to detect and quantify the activity of ligninolytic enzymes found in oak forest soil
with respect to their spatial distribution and temporal variability and to identify the changes of
oxidative enzymes activities during the colonization of soil by saprotrophic basidiomycetes.
Enzyme activity was measured in environmental samples from oak (Quercus robur) forest
(Xaverov Natural Reserve, Czech Republic) and linked with fungal occurrence and biomass
and the production of other extracellular enzymes. The species producing ligninolytic
enzymes were isolated from the studied soil and tested for their ability to produce oxidative
enzymes. The production of ligninolytic and hydrolytic enzymes of two indigenous
basidiomycete strains – PL13 and PL33 – was studied in nonsterile soil during a 10-week
colonization of soil profile microcosms with L (litter-upper), H (humic-middle) and S (soillower)
layers.
Laccase and Mn-peroxidase (MnP) but not lignin peroxidase were found in the studied soil
with laccase activity being by far higher. Activity of both enzymes decreased with the soil
depth and showed a patchy pattern of horizontal distribution with “hotspots”. In the season of
fruitbody production, laccase activity hotspots were associated with the occurrence of fruit
bodies of saprotrophic basidiomycetes.
Activity of oxidative enzymes in soil profile microcosms was significantly altered during
colonization by the basidiomycetes PL13 and PL33 compared to noninoculated control. The
activity of Mn-peroxidase (MnP) increased was 300-1800 mU/g soil d.w. during fungal
colonization of L layer, while it was only 0-44 mU/g in the control. MnP activity also
increased in H and S layers and coincided with mycelial colonization. Activity of laccase was
significantly increased only in L layer (200-350 mU/g compared to 40-150 mU/g in control).
The colonization of soil profile by saprotrophic basidiomycetes also resulted in the decrease
of microfungi counts in L and S layers, the increase of the counts of soil microfungi and
bacteria in the middle (humic) layer and increase in the activity of hydrolytic enzymes and
phosphatases.
Laccase and MnP play important roles in the turnover of carbon in the soil environment
during the transformation of lignin in the fresh biomass (fallen litter) and nutrients liberation
from the recalcitrant humic material. This study shows that a patchy pattern of oxizymes
activity is present in native soils, the activity sharply decreases with soil depth and that it can
be associated with the mycelium of saprotrophic fungi colonizing soil.
This work was supported by the Czech Science Foundation (526/05/0168) and by the Grant
Agency of ASCR (B600200516).
[1] Hofrichter M. Enzyme Microb. Technol. 30: 454 (2002).
[2] Baldrian P. FEMS Microbiol. Rev. 30: 215 (2006).
21 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L7
Novel Efficient Producers of Blue Laccases
A. Chernykh a , L. Golovleva a , N. Myasoedova a , N. Psurtseva b , N. Belova b , M. Ferraroni c , A.
Scozzafava c , F. Briganti c
a G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, Russia;
b Komarov Botanical Institute RAS, Russia; c Universitá degli Studi di Firenze, Italy
E-mail: golovleva@ibpm.pushchino.ru
Laccase is one of the very important ligninolytic enzyme of basidiomycetes, which are
responsible for many biotechnological processes, such as pulp and paper bleaching, textile
delignification, degradation of great variety of persistent pollutants. That is why the screening
and studying of new efficient producers of this enzyme are very actual.
Screening between 220 cultures of aphyllophoroid and agaricoid species permitted to find two
active strains - Steccherinum ochraceum 1833 and Lentinus strigosus 1566, which produce
the high level of laccase activity. Optimal conditions for laccase production by both strains
were carried out. Different culture conditions and inducers were studied, including 20
aromatic compounds, CuSO 4 , and polycaproamide tissue (PCA) for immobilization of
mycelium. Blue laccase production for S. ochraceum was optimal in such conditions -
glucose-peptone medium with 2,4-dimethylphenol or tannic acid as inducer, 2mM CuSO 4 ,
immobilization on PCA and aeration. In these conditions laccase activity was very high and
equal 33.1 U/ml. The best conditions for laccase production by L. strigosus 1566 were “rich”
medium with high Cu 2+ concentration, 1mM 2,6-dimethylphenol as an optimal inducer,
immobilization on PCA, and aeration. Maximal laccase activity in such conditions was 186,5
U/ml. Dominating laccase from S. ochraceum 1833 was purified to apparent electrophoretic
homogeneity. It has a molecular mass 64 kDa. Concentrated solution has blue colour (“blue
laccase”), and absorption spectrum typical for blue laccases with maximum in 610 nm, that
indicates the presence of Cu 2+ metal centres of T1 type. N-amino acid sequence of S.
ochraceum laccase (VQIGPVTDLH) has a high homology with sequences of other fungal
laccases. The crystallization of blue laccase of S. ochraceum and preliminary structural
analysis were performed.
The work was supported by grant INTAS 03-51-5889.
22 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L8
Discovery of an Epoxide Forming Monooxygenase from the
Metagenome
E.W van Hellemond, D.B. Janssen, M.W.Fraaije
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute,
University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
E-mail: E.W.van.Hellemond@rug.nl
Two-component flavin-dependent monooxygenases form an interesting class of
flavoenzymes. They consist of two separate proteins; a monooxygenase component, which
catalyses an oxygenation reaction in the presence of reduced flavin, and a flavin reducing
component, which reduces flavin (FAD or FMN) using NAD(P)H as an electron donor. A
well-known example of this class of monooxygenases is styrene monooxygenase 1 . Due to the
ability to form enantiopure epoxides, which are relevant building blocks for the
pharmaceutical industry, styrene monooxygenases form a valuable class of enzymes for
biocatalysis.
O
O 2
StyA
O H 2
FADH 2
FAD
StyB
NAD + NADH, H +
Figure 1. Reaction catalyzed by two-component flavin dependent styrene monooxygenase (StyAB) 1
While screening a metagenomic library for oxidative enzymes, an indigo-producing clone was
found. Sequencing the particular clone revealed an inserted fragment of environmental DNA
encoding a two-component monooxygenase (StyAB), consisting of a monooxygenase (StyA)
and a flavin reductase (StyB) component (Figure 1). The monooxygenase component shows
homology with known styrene monooxygenases. While sequence homology among the
styrene monooxygenases is high (>95% seq. id.), SmoA only displays a moderate sequence
homology (~ 50 % seq. id.). The substrate specificity for SmoAB is currently being
investigated.
[1] Otto, K., Hofstetter, K., Rothlisberger, M., Witholt, B. and Schmid, A. J.Bacteriol., 186,16 (2004): 5292-
5302.
23 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Production and Characterization of a Secreted
C-terminally Processed Tyrosinase from the Filamentous
Fungus Trichoderma reesei
Kristiina Kruus a , Emilia Selinheimo a , Markku Saloheimo a , Elina Ahola b , Ann Westerholm-
Parvinen a , Nisse Kalkkinen b and Johanna Buchert a
a VTT Technical Research Centre of Finland, P.O. Box 1500, Espoo FIN-02044 VTT, Finland;
b Protein Chemistry Research Group and Core Facility, Institute of Biotechnology, P.O. Box
65, FIN-00014 University of Helsinki, Finland
E-mail: kristiina.kruus@vtt.fi
Tyrosinases (monophenol, o-diphenol:oxygen oxidoreductase, EC 1.14.18.1) are type 3
copper proteins having a diamagnetic spin-coupled copper pair in the active centre. They
catalyze the o-hydroxylation of monophenols and subsequent oxidation of o-diphenols to
quinones and can thus oxidize both mono- and diphenols. Molecular oxygen is used as an
electron acceptor and it is reduced to water in tyrosinase-catalyzed reactions. Tyrosinases are
ubiquitously distributed enzymes in nature. They are found in prokaryotic as well as in
eukaryotic microbes, and in mammals, invertebrates and plants. In mammals, tyrosinases
catalyze reactions in the multi-step biosynthesis of melanin pigments, being responsible, for
instance, for skin and hair pigmentation. They are also related to browning reactions of fruit
and vegetables
Homology search of the filamentous fungus Trichoderma reesei genome database resulted in
a new T. reesei TYR2 tyrosinase gene with a signal sequence. The gene was over-expressed
in the native host under a strong cbh1 promoter in high yields. The purified TYR2 protein
showed significantly lower molecular weight, 43.2 kDa, than was expected according to the
putative amino acid sequence, 61.151 kDa. The exact cleavage site was determined using
chromatographic and mass spectrometric analysis. The protein properties of the Trichoderma
TYR2 will be discussed.
L9
24 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L10
Understanding the Selection of Arene Dioxygenase
Enzymes for Optimal Chemo-, Stereo- and Regio-Selectivity
of Biotransformation Processes
Christopher CR Allen a , Derek R Boyd b , Leonid L Kulakov a,c , Narain D Sharma b .
a School of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97
Lisburn Road, Belfast BT9 7BL, Northern Ireland. b School of Chemistry & Chemical
Engineering, Queen’s University Belfast, David keir Building, Stranmillis Road, Belfast BT9
5AG, Northern Ireland. c The QUESTOR Centre, Queen’s University Belfast, David keir
Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland.
E-mail: c.allen@qub.ac.uk
Dioxygenase enzymes are versatile biotransformation catalysts, that can be utilised for the
preparation of chiral cis-dihydrodiol, benzylic alcohol and sulfoxide metabolites for use in
many synthetic applications in the pharmaceutical and agrochemical industries 1 . These
enzymes are generally obtained from environmentally-significant soil bacteria – where they
have evolved for the biodegradation of aromatic hydrocarbons such as benzene, naphthalene,
toluene and azaarenes 2 .
When considering the use of cis-dihydrodiol metabolites in a synthetic role, a number of key
factors will limit successful application. These include product yield; metabolite enantio- and
regio-purity; the availability of both enantiomers of target compounds; and other processrelevant
parameters that will have an impact on eventual scale-up – such as enzyme and
genetic stability. Therefore, if the full potential of dioxygenase enzymes in biocatalysis is to
be addressed, it is imperative that research into factors that affect these variables is
considered.
We have conducted extensive studies on the biotransformation of mono- and poly-cyclic
compounds with benzene, toluene, naphthalene and biphenyl dioxygenase-expressing
microorganisms. These experiments have delivered several insights into the relationship
between enzyme choice and the ultimate structure and stereochemistry of biotransformation
products.
In this report, we will summarise our recent observations regarding the impact of dioxygenase
enzyme choice on the ‘key factors’ described above, and also propose modifications to
biotransformation process design that may be considered when coupled with judicious choice
of enzyme biocatalyst.
[1] Boyd DR, Sharma ND, Allen CCR. (2001).Aromatic dioxygenases: molecular biocatalysis and applications.
Current Opinion in Biotechnology. 12 564-573.
[2] Boyd DR and Bugg T (2006). Arene cis-dihydrodiol formation: from biology to application. Org. Biomol.
Chem. 4 181-192.
25 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L11
Redesign of AtGALDH, a Flavoprotein Involved in Vitamin
C Biosynthesis
Nicole G. H. Leferink a , Yu Lu a , Willy A. M. van den Berg a , Willem J. H. van Berkel a
a Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen,
The Netherlands
E-mail: nicole.leferink@wur.nl
Vitamin C (L-ascorbic acid) is an important antioxidant and an essential component of the
human diet. Animals, plants, yeasts and fungi produce vitamin C from different precursors.
The final step in the biosynthesis of vitamin C and its analogs is catalyzed by L-gulono-1,4-
lactone oxidase (GUO), L-galactono-1,4-lactone dehydrogenase (GALDH), D-arabinono-1,4-
lactone oxidase (ALO) and D-gluconolactone oxidase (GLO), in animals, plants, yeasts and
fungi, respectively. These homologous enzymes belong to the VAO family of flavoproteins [1] .
Many members of this family contain a covalently bound FAD, including GUO, ALO and
GLO. Though isolated from various sources, detailed characterization and structural
information is lacking for these enzymes. GUO, GALDH, ALO and GLO catalyze similar
reactions, but have different substrate specificities and reactivities towards molecular oxygen.
There is no general structural rule that enables the prediction of the reactivity of flavoenzymes
towards dioxygen [2] . Our aim is to unravel the molecular determinants for the substrate
specificity and oxygen reactivity of the mitochondrial Arabidopsis thaliana GALDH and
related enzymes.
Mature Arabidopsis GALDH (58 kDa) is expressed as soluble protein in E. coli. The enzyme
contains a non-covalently bound FAD as redox active center and is highly active with L-
galactono-1,4-lactone (K m = 80 µM, k cat = 87 s -1 ) and cytochrome c (K m = 41 µM), but not
with molecular oxygen. The enzyme has been crystallized and detailed biochemical
characterization is ongoing.
Recombinant AtGALDH is rather stable but inactivated by hydrogen peroxide. The
galactonolactone substrate protects the enzyme from hydrogen peroxide inactivation. The
inactivation is due to the modification of a single thiol. Oxidative stress resistant GALDH will
be produced by replacing the reactive cysteine residue.
Future work will be directed towards the design of a galactonolactone oxidase with a relaxed
substrate specificity. Enzyme variants aimed at covalent attachment of the flavin have already
been constructed.
[1] Fraaije MW & Van Berkel WJH et al. (1998). A novel oxidoreductase family sharing a conserved FADbinding
domain. Trends Biochem Sci, 23:203-207
[2] Mattevi A (2006). To be or not to be an oxidase: challenging the oxygen reactivity of flavoenzymes. Trends
Biochem Sci, in press
26 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L12
Acetate Inhibition of Laccase Activity
Thomas Ters, Thomas Kuncinger, Ewald Srebotnik
Competence Centre for Wood Composites and Wood Chemistry, St.-Peter-Strasse 25, 4021
Linz, Austria; and Institute of Chemical Engineering, Vienna University of Technology,
Getreidemarkt 9, 1060 Wien, Austria
E-mail: ewald.srebotnik@tuwien.ac.at
We have observed unsatisfactory linearity and reproducibility in routine assays for fungal
laccase activity. It was found that these problems were due to a slight but significant
inhibition of laccase by acetate, the most commonly used buffering substance in laccase
assays.
Kinetic measurements performed with recombinant laccase from Trametes villosa (44008,
Novo Nordisk) and ABTS as a substrate revealed an s-linear, i-parabolic mixed inhibition
type for acetate at pH 5.0 with calculated Ki and Ki’ values of 38.8 mM and 117.5 mM,
respectively. Thus the affinity of acetate for laccase was very low compared to the classical
inhibitor azide which exhibited Ki and Ki’ values of 0.0176 mM and 0.0106 mM,
respectively. Similar effects were observed at pH 4.0 and also for wild-type laccases from
several other Trametes species such as T. pubescens CBS 696.94. However, due to the
relatively high concentrations used in routine assays, inhibition levels were substantial
ranging from 15% to 50% of initial activity at acetate concentrations from 10 mM to 100 mM,
respectively. The first order inactivation rate constant was rather low (k ~0.1 min -1 ). In
practice this means that upon contact with acetate, 90% of the final (stable) inactivation level
is reached only after ~23 min.
No correlation was found between the size of the carboxyl anion and the extent of inhibition -
formiate, propionate as well as butyrate were stronger inhibitors than acetate. Moreover, the
results indicated a simple linear non-competitive type for formiate in contrast to acetate.
Several α-hydroxycarboxylic, di- and tricarboxylic acids were also tested at pH values from
3.0 to 5.0. While inhibition characteristics of lactate and glycolate were similar to those of
acetate, the inhibition characteristics of citrate and succinate were not: generally the extent of
inhibition by citrate and succinate was much lower (

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L13
Cofactor Incorporation and Cofactor-Induced Stabilization
of Oxizymes
Willem J.H. van Berkel
Laboratory of Biochemistry, Wageningen University,
Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
E-mail: willem.vanberkel@wur.nl
Oxizymes need cofactors for their functioning. In many cases the cofactor is spontaneously
incorporated after folding and assembly of the apoprotein. However, cofactors may also bind
to a folding intermediate or preprotein and induce protein maturation. Even more complicated
are the cases where cofactor insertion is guided by chaperones or the cofactor is made by the
redoxenzyme itself.
Many oxizymes are most stable in their holoenzyme form. For biotechnological
applications this means that we need more insight in how oxizymes deal with (artificial)
cofactor binding and how this binding affects the functioning and stability of the biocatalyst.
Mutant proteins with the desired catalytic properties are often unstable due to cofactor loss.
How can we improve these enzymes without losing their beneficial properties?
In flavoprotein oxidases the flavin prosthetic group is covalently or non-covalently
bound to the protein. In this presentation I will illuminate how flavoprotein biocatalysts such
as aryl-alcohol oxidase (AAO), vanillyl-alcohol oxidase (VAO), D-amino acid oxidase (DAO)
and glucose oxidase (GOX) bind their cofactor and how this binding influences the protein
stability. For introducing new properties, the natural cofactor might be replaced by an articial
cofactor. Strategies towards a reversible cofactor exchange will be discussed.
28 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L14
A Flavin-Dependent Tryptophan 6-Halogenase and Its Use
In Combinatorial Biosynthesis
C. Schmid a , H. Schnerr a J. Rumpf a , A. Kunzendorf a , C. Hatscher a , T. Wage a , A. Ernyei a , C.
Dong b , J. H. Naismith b , K.-H. van Pée a
a Biochemie, Technische Universität Dresden, D-01062 Dresden, Germany, and b Centre for
Biomolecular Sciences, EaStchem, University of St. Andrews, St. Andrews KY16 9ST, UK
E-mail: karl-heinz.vanpee@chemie.tu-dresden.de
Regioselective halogenation of electron rich substrates is catalysed by flavin-dependent
halogenases. These halogenases require reduced flavin which is provided by a flavin
reductase for halogenating activity. Investigations of the tryptophan 7-halogenase from
pyrrolnitrin biosynthesis have shown that reduced flavin is bound by the halogenases and it is
suggested that, like in monooxygenases, flavin hydroperoxide is formed. In contrast to
monooxygenases, where this flavin hydroperoxide reacts with an organic substrate leading to
hydroxylation reactions, the flavin hydroperoxide in halogenases reacts with halide ions. This
leads to the formation of hypohalous acid at the active site. Since the exit of the tunnel which
is formed by the active site amino acids is blocked by the isoalloxazine ring of FAD, the
hypohalous acid cannot leave the active site but is guided to the organic substrate position at
the other end of the 10 Å long tunnels. To achieve regioselective incorporation of the halide
the substrate must be positioned in such a way that the position at which halogenation should
occur is presented to the oncoming hypohalous acid [1].
Thienodolin produced by Streptomyces albogriseolus contains a chlorine atom in the 6-
position of the indole ring system and is believed to be derived from tryptophan. Using the
gene of the tryptophan 7-halogenase (PrnA) from pyrrolnitrin biosynthesis the gene for a
tryptophan 6-halogenase was cloned, sequenced and heterologously overexpressed in
Pseudomonas strains. In vitro activity of the purified enzyme could only be shown in a twocomponent
system consisting of the halogenases, a flavin reductase, NADH, FAD and halide
ions. The enzyme catalysis the regioselective chlorination and bromination of L- and D-
tryptophan. In its native form, the enzyme is probably a homodimer with a relative molecular
mass of the subunits of 64,000. All the amino acids found to be involved in the binding and
positioning of the substrate and to be involved in catalysis in tryptophan 7-halogenase are also
present in tryptophan 6-halognase. This suggests that, while the overall mechanism of the
reaction is identical to that of tryptophan 7-halogenase, the position that is presented to the
hypohalous acid must be different. Transformation of the pyrrolnitrin producer Pseudomonas
chlororaphis ACN with a plasmid containing the tryptophan 6-halogenase gene lead to the
formation of the new aminopyrrolnitrin derivative 3-(2-amino-4-chlorophenyl)pyrrole.
[1] Dong C., S. Flecks, S. Unversucht, C. Haupt, K.-H. van Pée, J. H. Naismith (2005) Tryptophan 7-halogenase
structure suggests a mechanism for regioselective chlorination. Science 309, 2216-2219.
29 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L15
A Plant Peroxidase Intrinsically Stable Towards Hydrogen
Peroxide
Paloma Gil a,b , Cesar Ferreira Batista c , Rafael Vazquez-Duhalt a and Brenda Valderrama a,b
a Departamento de Ingeniería Celular y Biocatálisis , b Departamento de Medicina Molecular
y Bioprocesos, c Unidad de Proteómica. Instituto de Biotecnología, Universidad Nacional
Autónoma de México. AP 510-3 Cuernavaca, Morelos 62250, México
E-mail brenda@ibt.unam.mx
Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and
are thus potentially useful for multiple applications. However, hemeperoxidases are unusually
susceptible to self-inflicted oxidative damage [1]. The search for more stable
hemeperoxidases has been actively pursued by different methods in the past, including redoxbased
protein engineering [2]. Here we report the identification and biochemical
characterization of a novel hydrogen peroxide-resistant hemeperoxidase isolated from roots of
Japanese radish (Raphanus sativus L. cv. daikon) and named Zo peroxidase (ZoP), after the
Greek word meaning permanence. ZoP accounts for only 0.01% of the total peroxidase
activity detected in a crude extract and its identification was possible after the systematic
separation and study of each activity fraction. Pure ZoP was shown to be a monomeric
hemeprotein with a molecular size of 50 kDa and an isolectric point of pH 6.0. Partial protein
sequencing by mass-spectrometry demonstrated that ZoP is more related to isoenzymes A2
from Arabidopsis thaliana and Armoracia rusticana than to any other known peroxidase. The
stability behavior of ZoP was evaluated by four different methods: 1) Catalytic stability
during the continuous incubation with 1 mM hydrogen peroxide in the absence of exogenous
reducing substrate, 2) Significant activity tolerance after 12h incubation against different
molar ratios of hydrogen peroxide in the absence of exogenous reducing substrate, 3) High
yield under operation conditions, and 4) Resistance to heme bleaching in the presence of
1mM hydrogen peroxide, indicating porphyrin integrity. The performance of ZoP was
concurrently compared with that of the horseradish isoenzyme A2 (HRPA2), an isoenzyme
known for its relative oxidative stability. Independently of the method used, ZoP
outperformed HRPA2. The Michaelis-Menten catalytic constants of ZoP were calculated
using guaiacol and hydrogen peroxide. ZoP presented lower affinity for both substrates
compared with HRPA2 but higher turnover, which rendered all catalytic efficiencies within
the same order of magnitude. A catalytic model based on our experimental data will be
proposed as well as potential applications for a stable peroxidase.
The authors acknowledge R. Tinoco, R. Roman and S. Rojas for technical assistance. This
study was supported by grants IFS F/3562-1 and PAPIIT IN202305.
[1] Valderrama, Ayala and Vazquez (2002) Chemistry & Biology 9, 555-565.
[2] Valderrama et al. (2006) FASEB Journal In Press
30 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L16
Functional Hybrids of Haloperoxidases and Cytochrome
P450 Monooxygenases from Alkaliphilic Mushrooms
René Ullrich a , Dau Hung Anh a,b , Martin Kluge a , Matthias Kinne a , Katrin Scheibner c , Martin
Hofrichter a
a Int. Graduate School of Zittau, Environ. Biotech. Unit, Markt 23, 02763 Zittau Germany;
b Vietnamese Acad of Sci. & Techn., Dept of Biotechnology,18-Hoang Quoc Viet Rd., Hanoi,
Vietnam; c Lausitz Univ of Appl. Sci.s, Dept of Biotechnology, 01958 Senftenberg, Germany
E-mail: hofrichter@ihi-zittau.de
The family of heme-thiolate proteins comprises versatile oxidoreducatases (primarily
cytochrome P450 monooxygenases) catalyzing amongst others different oxygen transfer
reactions (hydroxylations, epoxidations, sulfoxidations). Until recently, only one peroxidase
of this type has been known – chloroperoxidase (CPO) from the ascomycete Caldariomyces
fumago. This heme-thiolate peroxidase is able to halogenate organic substrates unspecifically
via free hypohalous acids and it can act as a peroxygenase in P450-like reactions [1].
We have isolated a second haloperoxidase of this type from the agaric basidiomycete
Agrocybe aegerita, that shares even more spectral and catalytic properties with cytochrome
P450s than CPO does [2, 3]. During the growth in complex media, the alkaliphilic fungus
secretes an unusual peroxidase that oxidizes aromatic alcohols into the corresponding
aldehydes and carboxylic acids. The enzyme, termed AaP (Agrocybe aegerita peroxidase),
was purified to homogeneity and characterized [2]. There are multiple forms differing in the
carbohydrate content (15-20%) and isoelectric points (4.9-5.7) but having the same
molecular mass of 46 kDa. The N-terminal amino acid sequence of AaP shows hardly
similarity to any known sequence of a basidiomycete peroxidase. On the other hand, 5 and 3
out of 14 amino acids are identical to amino acids at the N-terminus of CPO and a fungal
P450 nor , respectively. AaP has a strong brominating and a weak chlorinating activity. The
UV-Vis spectrum of native AaP differs noticeably from that of CPO but is almost identical to
a resting-state P450 [3]. The reduced CO complex of AaP has its Soret maximum at 445 nm
which proves its heme-thiolate affiliation and the similarity to P450s (446-453 nm). The
hydroxylating activity of AaP was tested with toluene, ethylbenzene, anisole and naphthalene
as substrates. Benzyl alcohol was found to be the major product of toluene oxidation (along
with traces of o- and p-cresol); ethylbenzene, anisole and naphthalene were selectively
converted into (R)-1-phenylethanol, p-methoxyphenol and 1-naphthol, respectively. Thus,
AaP is in fact capable of catalyzing benzylic and aromatic hydroxylations merely with H 2 O 2
as cosubstrate (peroxygenase); complex electron donors (e.g. NADPH) as well as auxiliary
proteins as in classic P450 reactions are not required.
Since AaP halogenates and hydroxylates aromatic substrates, it can be regarded as a
functional hybrid that is closer to P450 monooxygenases than to classic CPO. Such hybrids
are seemingly widespread and we have recently identified four further strains of the genus
Agrocybe as well as two coprophilic Coprinus species producing similar AaP-like enzymes.
As selective hydroxylations are among the most desired reactions in chemical synthesis,
mushroom peroxygenases could become interesting biocatalytic tools. Respective
biochemical and molecular studies are currently under investigation in our laboratories.
[1] Hofrichter, M. & Ullrich, R. (2006) Appl. Microbiol. Biotechnol.: DOI 10.1007/s00253-006-0417-3
[2] Ullrich, R. et. al. (2004) Appl. Environ. Microbiol. 70: 4575-4581
[3] Ullrich, R. & Hofrichter, M. (2005) FEBS Lett. 579: 6247-6250
31 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L17
Laccase Engineering by Rational and Random Mutagenesis
Giovanna Festa 1 , Alessandra Piscitelli 1 , Vincenza Faraco 1 , Paola Giardina 1 , Flavia Autore 1 ,
Franca Fraternali 2 and Giovanni Sannia 1
1 Department of Organic Chemistry and Biochemistry, Complesso Universitario Monte
S.Angelo, via Cintia 4, 80126 Naples, Italy 2 Randall Division of Cell and Molecular
Biophysics, King's College London, Guy's Campus, SE1 1UL London UK
E-mail: sannia@unina.it
The white-rot fungus Pleurotus ostreatus is able to express multiple laccase genes encoding
isoenzymes with different properties: POXC [1], POXA1w [2], POXA1b [3], and the
heterodimeric proteins POXA3a and POXA3b [4]. However, because of the multiplicity of
applicative potentialities of these enzymes, it would be desirable to have a large range of
enzymes endowed with different characteristics to select proteins for specific applications.
Directed evolution by random mutagenesis and recombination followed by appropriate
screening is a valuable tool for tailoring enzymes. On the other hand, a deep knowledge of the
structure-activity-stability relationships of laccases can be achieved through the rational
design and characterization of point-mutated proteins.
Functional gene expression in a suitable host is a prerequisite for protein engineering through
both rational and random mutagenesis. P. ostreatus laccases POXC and POXA1b were
successfully expressed in two yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae [5].
Moreover recombinant expression in K. lactis of the large POXA3 subunit and of the whole
heterodimeric complex was achieved.
P. ostreatus laccase aminoacidic sequences were aligned with those of other laccases whose
3D structures are known; therefore their structures were modelled by homology. POXA1b
and POXC present a longer C-terminal region. In order to investigate role of this additional
segment, site directed mutagenesis experiments tailored for these regions were performed and
the mutated enzymes characterised. Analysis of the laccases alignment and of the 3D models
has led to the design, expression and characterization of other point mutated enzymes.
S. cerevisiae was chosen as host for construction of random mutated laccase libraries on the
basis of its transformation efficiency, stability of plasmid DNA, and growth rate. Two
mutagenesis methods were explored: error-prone PCR and DNA shuffling. Libraries of low,
medium and high range mutants (from 0 to more than 7 mut/kbase) were generated by errorprone
PCR. Furthermore, a library from poxc and poxa1b shuffling was also produced.
Positive clones were selected on the basis of their ability to express high levels of laccase
activity. Structural and catalytic characterization of these mutants is in progress.
[1] Palmieri G., et al., 1993, Appl. Microbiol. Biotechnol., 39,632-636
[2] Palmieri G. et al., 1997, J. Biol. Chem., 272,31301-31307
[3] Giardina P. et al., 1999, Biochem. J., 34,655-663
[4] Palmieri G., et al., 2003, Enzyme. Microb. Technol., 33,220-230
[5] Piscitelli A., et al., 2005,. Appl. Microbiol. Biotechnol., 69,428-39
32 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L18
Structure-Function Studies of Pleurotus Versatile
Peroxidase, a Model Ligninolytic Enzyme
F.J. Ruiz-Dueñas a , M. Pérez-Boada a , M. Morales a , R. Pogni b , R. Basosi b , T. Choinowski c ,
M.J. Martínez a , K. Piontek c , Á.T. Martínez a
a Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain;
b Department of Chemistry, University of Siena, via Aldo Moro, I-53100 Siena, Italy; c Institute
of Biochemistry, ETH, Schafmattstrasse 18, CH-8093 Zürich, Switzerland
E-mail: ATMartinez@cib.csic.es
Versatile peroxidase (VP) represents a third type of fungal ligninolytic peroxidase together
with lignin peroxidase and manganese peroxidase [1]. Ligninolytic peroxidases differ from
peroxidases from saprophytic basidiomycetes (e.g. Coprinopsis cinerea) and plant
peroxidases involved in monolignol polymerization, by their high redox potential enabling
oxidative degradation of lignin. This property makes them the biocatalyst of choice for
industrial applications requiring enzymatic oxidation of recalcitrant aromatic compounds,
including simple and complex/polymeric substrates. However, the native enzymes are far
from optimally operating under industrial conditions. Therefore, enzyme structure-function
studies are required as a first step for designing new tailor-made biocatalysts. VP has been
described in fungi from the genera Pleurotus and Bjerkandera. The VP from Pleurotus
eryngii, a species of biotechnological interest, is the most extensively investigated [2,3].
Using structure-function studies the catalytic properties of this new enzyme can be explained,
and provide general information on peroxidases. VP versatility is related to different substrate
oxidation sites that have been identified in high-resolution crystal structures, and were
confirmed by site-directed mutagenesis in combination with spectroscopic techniques [4-6].
Mn 2+ oxidation to Mn 3+ , which acts as a diffusible oxidizer of phenolic and non-phenolic
lignin (via lipid peroxidation), implies binding to three acidic residues (two of them acting as
a gate controlling cation binding and release) and direct electron transfer to the internal
propionate of heme. Oxidation of high redox potential aromatic compounds, including
veratryl alcohol and Reactive Black 5 (and possibly also polymeric lignin) is produced by
long-range electron transfer to the heme methyl-3 from a surface tryptophan residue. The
oxidation of the latter to a protein radical has been detected by EPR of H 2 O 2 -activated VP. In
contrast to that found in the equivalent tryptophan of LiP, in VP no indication of a β-
hydroxylation of the tryptophan residue was found. Moreover, by multifrequency EPR and
density-functional-theory calculations it was demonstrated that the tryptophan radical is in the
neutral form. The ability to directly oxidize high redox potential substrates that are not
oxidized by lignin peroxidase in the absence of veratryl alcohol (as a mediator) seems to be
related to the environment of this exposed residue in VP. Low redox potential dyes, such as
ABTS, can be oxidized by VP at the above exposed tryptophan but also at the heme access
channel. Therefore, the VP molecular architecture combines in the same protein the substrate
oxidation sites characteristics of the three other fungal peroxidases mentioned above. The
structural bases for the catalytic versatility of this enzyme are already understood in quite
some detail enabling enzyme tailoring using protein engineering tools.
[1] Martínez, A. T. (2002) Enzyme Microb Technol 30, 425
[2] Camarero, S., Sarkar, S., Ruiz-Dueñas, F. J. et al. (1999) J Biol Chem 274, 10324
[3] Ruiz-Dueñas, F. J., Martínez, M. J., and Martínez, A. T. (1999) Mol Microbiol 31, 23
[4] Pérez-Boada, M., Ruiz-Dueñas, F. J., Pogni, R. et al. (2005) J Mol Biol 345, 385
[5] Banci, L., Camarero, S., Martínez, A. T. et al. (2003) J Biol Inorg Chem 8, 751
[6] Pogni, R., Baratto, M. C., Teutloff, C. et al. (2006) J Biol Chem 281, 9517
33 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Structure-Activity Relationship of the Laccase-Mediator
System
R. Pogni a , B. Brogioni a , A. Sinicropi a , M.C. Baratto a , P. Giardina b , G. Sannia b , R. Basosi a
a Chemistry Department, University of Siena, Via A. Moro, Italy; b Organic Chemistry and
Biochemistry Department, University of Naples, via Cinthia 4, Italy.
E-mail: pogni@unisi.it
L19
Laccase, a multi-copper enzyme, belongs to the lignin degrading system and catalyzes the
one-electron oxidation of different substrates, with the simultaneous four electron reduction of
molecular oxygen to water [1]. The broad substrate specificities of laccases, together with the
fact that they use molecular oxygen as the final electron acceptor, make these enzymes highly
interesting for industrial and environmental applications. However, the low redox potentials
of laccases (0.5 to 0.8 mV) only allow the direct degradation by laccases of low redox
potentials phenolic compounds. The range of chemical structures oxidized by the enzyme can
be even increased by employing different natural and synthetic redox mediators[2].
The basis of the laccase-mediator concept is the use of low-molecular weight compounds
that, once oxidized by the enzyme to stable radicals, act as redox mediators, oxidizing other
compounds that in principle are not substrates of laccase.
An important role in determining the mechanism of substrate oxidation may be played by the
stability of the oxidized form of the radical mediator, as well as by its redox potential.
In this work the reaction between fungal laccases from the wood-rot fungi Pleurotus ostreatus
[3] and Trametes versicolor and the synthetic redox mediator Violuric Acid (VIO) has been
analyzed by EPR spectroscopy. An intense and stable radical species has been generated and
detected during this reaction [4]. Comparative density functional calculations indicate the
presence of a deprotonated neutral radical species. Simulation of a cluster consisting of VIO
in presence of H 2 O has shown the possibility of H-bonds formation.
The bleaching activity of the laccase and laccase-mediator systems has been tested towards
various dyes with different structures and using different redox mediators (VIO, 1-
hydroxybenzotriazole, 2,2’-Azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid ).
[1] Basosi,R., Della Lunga, G., Pogni, R. (2005), 385 - 416 in: Biomedical EPR - Part A: Free Radicals, Metals,
Medicine and Physiology, Kluwer Academic / Plenum Publishers, New York, USA
[2] Camarero, S., Ibarra, D., Martinez, M.J. and Martinez, A. T. (2005) Appl. Env. Microbiol. 1775-1784.
[3] Palmieri, G., Cennamo, G., Sannia G. (2005) Enzyme Microbiol. Technol. 36, 17-24
[4] Kim, H.-C., M. Mickel, N. Hampp. (2003) Chem. Phys. Lett. 371: 410-416.
34 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L20
'Titrating' Steric and Redox Features of the Active Site of
Laccase
Mahelet Aweke Tadesse, Alessandro D'Annibale, Carlo Galli, Patrizia Gentili,
Ana Sofia Nunes Pontes and Federica Sergi
Dipartimento di Chimica, Università 'La Sapienza', Roma, Italy
E-mail: carlo.galli@uniroma1.it
Steric and redox issues of the substrate are investigated for a better insight of the reactivity
features of the phenoloxidase laccase. A few bulky phenols and anilines are not susceptible to
oxidation, in spite of being ‘putative substrates’ for laccase. By exploiting crystallographic
data of the enzyme available from the literature, 1-3 it becomes possible to appraise the
maximum width of the substrate, and to outline its proper alignment in the binding site,
besides other steric requirements, which enable a successful monoelectronic oxidation.
With regard to the redox issue, any substrate could be a candidate for monoelectronic
oxidation by laccase, regardless its phenolic or non-phenolic nature, provided that the
electrochemical potential is suited. For example, being 0.78 V vs NHE the redox potential of
Trametes villosa laccase, a non-phenolic compound such as 1,2,4,5-tetramethoxybenzene (E½
1.05 V vs NHE) can be quantitatively oxidised. 4 In contrast, phenols substituted with electronwithdrawing
groups become progressively resistant to the oxidation as their electrochemical
potential gradually increases. Myceliophthora thermophila laccase, having a redox potential
of only 0.48 V vs NHE, suffers from unfavourable redox features of the substrate more
crucially.
[1] K. Piontek, M. Antorini, T. Choinowski, J. Biol. Chem., 277 (2002) 37663-37669.
[2] T. Bertrand, C. Jolivalt, P. Briozzo, E. Caminade, N. Joly, C. Madzak, C. Mougin, Biochemistry, 41 (2002)
7325-7333.
[3] F.J. Enguita, D. Marçal, L.O. Martins, R. Grenha, A.O. Henriques, P.F. Lindley, M.A. Carrondo, J. Biol.
Chem., 279 (2004) 23472-23476.
[4 ] F.d'Acunzo, C.Galli, P.Gentili, F.Sergi, New J. Chem., 30 (2006) 000.
35 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L21
A Near-Atomic Resolution Crystal Structure of
Melanocarpus albomyces Laccase
Nina Hakulinen a , Martina Andberg b , Anu Koivula b , Kristiina Kruus b , Juha Rouvinen a
a Dept. Of Chemistry, University of Joensuu, PO Box 111, FIN-80101 Joensuu, Finland b VTT
Technical Research Centre of Finland, PO Box 1000, FIN-02044 VTT, Finland
E-mail: nina.hakulinen@joensuu.fi
Laccases (E.C. 1.10.3.2, p-diphenol dioxygen oxidoreductases) are redox enzymes that use
molecular oxygen to oxidize various phenolic compounds, anilines and even some nonaromatic
compounds by a radical-catalyzed reaction mechanism. Oxidization of reducing
substrates occurs concomitantly with the reduction of molecular oxygen to water. Laccases
share the arrangement of the catalytic sites with other blue multi-copper oxidases including
ascorbate oxidase, ceruloplasmin, CueO, and Fet3p. For catalytic activity, four copper atoms
are needed: one type-1 (T1) copper forming a mononuclear site, one type-2 (T2) copper and
two type-3 (T3 and T3´) coppers forming a trinuclear site. Reducing substrates are oxidized
near the mononuclear site and then electrons are transferred to the trinuclear site, where
dioxygen is reduced to water.
Several three-dimensional structures of laccases have been solved today. Many of the laccases
have been reported to have one oxygen atom, most likely hydroxyl group, between the two T3
coppers, but Melanoarpus albomyces laccase (MaL) shows a di-oxygen molecule amidst
coppers in the trinuclear site [1] . Recently, three-dimensional structure of CuCl 2 soaked form of
Bacillus subtilis laccase has also been reported to have the dioxygen molecule inside its
trinuclear site [2] . In addition, MaL has a unique feature that the C-terminus of the enzyme
penetrates to the tunnel leading to trinuclear site. This tunnel has been postulated to form
access route for dioxygen to enter to the trinuclear site.
We have now determined the three-dimensional structure of recombinant M. albomyces
laccase (rMaL) at 1.3 Å resolution (R = 17.9 % and R free = 20.5 %). In addition, we have
determined the three-dimensional structure of DSGA mutant (last residue Leu559 mutated to
Ala) at 2.5 Å resolution. Specific activity of the DSGA mutant on ABTS is about 5-fold lower
than specific activity of the wild-type enzyme. Refinement of the mutant structure is currently
underway (R = 22.3 % and R free = 28.2 %). In both structures, the dioxygen is refined amidst
the coppers in the trinuclear site and the C-terminus plugs the tunnel leading to trinuclear
copper site as observed in the MaL. Details of the near-atomic resolution structure of rMaL
will be presented and the structural reasons for the lowered activity of the DSGA mutant will
be discussed.
[1] Hakulinen, N., Kiiskinen, L.-L., Kruus, K., Saloheimo, M., Paananen, A., Koivula, A. and Rouvinen J.
(2002) Nature Structural Biology 9, 601.
[2] Bento I, Martins, L.O., Lopes, G.G., Carrondo, M.A. and Lindley, P.F. Dalton Trans. (2005) Dalton Trans.
3507.
36 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L22
Structure-Function Relationships in Bacterial Multicopper
Oxidases
Lígia O. Martins
Instituto de Tecnologia Química e Biológica (ITQB),Universidade Nova de Lisboa, Av da
República, 2784-505 Oeiras, Portugal
E-mail: lmartins@itqb.unl.pt
The multi-copper oxidases constitute a family of enzymes whose principal members are
ceruloplasmin (Fe(II) oxygen oxidoreductase, EC 1.16.3.1), ascorbate oxidase (L-ascorbate
oxygen oxidoreductase, EC 1.10.3.3) and laccase (benzenediol oxygen oxidoreductase, EC
1.10.3.2) (1, 2). This family of enzymes is widely distributed throughout nature and members
are encoded in the genomes of organisms in all three domains of life – Bacteria, Archaea and
Eukarya. Multi-copper oxidases have broad substrate specificity; laccases, with a function in
intermediary metabolism, presents relative high substrate specificity for bulky aromatic (poly)
phenols and amines. A few members present higher efficiency to lower valent metal ions such
as Mn 2+ , Fe 2+ or Cu 1+ , being thus broadly designated as metallo-oxidases. These have been
suggested to play an in vivo catalytic role in the maintenance of both copper and iron
homeostasis in their respective organisms.
We have settled a multidisciplinary research approach focused on the study of bacterial
multicopper oxidases. As a model bacterial laccase system the CotA-laccase from Bacillus
subtilis has been extensively studied. Recent results that have been undertaken on the CotAlaccase
after site-directed mutagenesis on the catalytic mononuclear T1 copper site will be
presented. It has been shown that subtle rearrangements in the coordination sphere of the T1
copper result in major loss of function regarding the catalytic as well as the overall stability of
the enzyme, launching new questions regarding our understanding of the structure and
function of the oxidative copper site of the blue multicopper oxidases. This information will
assist the development of strategies targeted at the improvement of laccases as biocatalysts.
The results on a robust and hyperthermostable recombinant metallo-oxidase (McoA) from
Aquifex aeolicus will also be discussed. McoA presents poor catalytic efficiency (k cat /K m )
towards aromatic substrates but a remarkable high for cuprous and ferrous ions, close to 3 x
10 6 s -1 M -1 . We provide evidences for the in vivo involvement of McoA in copper and iron
homeostasis.
37 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Crystal Structures of Three New Fungal Laccases:
Implications on the Catalytic Mechanism and on the
Dynamics of the Copper Sites Redox States
L23
Irene Matera a , Antonella Gullotto a , Marta Ferraroni a , Silvia Tilli a , A.Chernykh b , Nina M.
Myasoedova b , Alexey A. Leontievsky b , Ludmila Golovleva b Andrea Scozzafava a , Fabrizio
Briganti a
a
Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, I-50019 Sesto
Fiorentino (FI), Italy. b Skryabin Institute of Biochemistry and Physiology of Microorganisms,
Russian Academy of Sciences, Nauka Prospect 5, 142290 Pushchino Moscow region, Russia.
E-mail: fabrizio.briganti@unifi.it
Laccases (benzenediol oxygen oxidoreductase, EC 1.10.3.2) are polyphenol oxidases
belonging to the family of multicopper oxidases. These multi-copper enzymes contain four
copper atoms per molecule, organized into three different copper sites which catalyze the oneelectron
oxidation of four reducing-substrate molecules concomitant with the four-electron
reduction of molecular oxygen to water molecules. Blue copper oxidases contain at least one
type-1 copper, which is presumably the primary oxidation site whereas blue multicopper
oxidases typically employ at least three additional coppers: one type-2 and two type-3 copper
ions arranged in a trinuclear cluster, the latter being the site where the reduction of molecular
oxygen occurs.
Biotechnological researches on laccases, aiming at the development of various industrial
processes such as pulp delignification and removal of environmental pollutants, for instance
pesticides and textile dyes, from contaminated soil and water, are currently performed.
In order to optimize such promising processes the complete comprehension of the catalytic
mechanism of laccases, and in particular of their redox potential and substrate selectivity
control are needed and a detailed characterization of the high resolution molecular structure of
such enzymes will surely help in achieving such aims.
Three new structures of blue laccases from the white-rot basidiomycetes fungi Panus tigrinus,
Trametes trogii, and Steccherinum ochraceum, enzymes involved in lignin biodegradation
have been recently solved at high resolution in our laboratory.
The details revealed by these new structures and their implications on the electronic structure
dynamics of the copper sites, on substrates and substrates analogues binding and on the
overall catalytic mechanism are analyzed and discussed.
38 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L24
Construction and Characterisation of Horseradish
Peroxidase Mutants that Mimic Some of the Properties of
Cytochromes P450
Emile Ngo a , Wendy Doyle a , Anabella Ivancich b , Andrew T. Smith a
a Biochemistry Department, School of Life Sciences, University of Sussex. UK; b Centre
d'Etudes de Saclay, Gif-sur-Yvette. France
Email: A.T.Smith@sussex.ac.uk
Plant peroxidases cannot normally transfer an oxygen atom stereoselectively to a substrate but
catalyse the production of aromatic radicals at a haem edge site. The acid base residues
required for highly efficient O-O bond cleavage in peroxidases sterically restrict direct access
of substrates to the ferryl intermediate and the enzyme which has a somewhat closed haem
architecture. In part, by mimicking the more open hydrophobic architecture of
chloroperoxidase, variants of a horseradish peroxidase have been engineered which have at
least some of the key functional properties of a cytochrome P450. Several variants are very
efficient peroxygenases, with rates exceeding ~ 17 s -1 and are highly effective in producing
enantiomerically pure sulphoxides (100% pure), strongly implying an oxene transfer
mechanism. They undergo a low spin (LS) to high spin transition on substrate binding (with
sub micro molar Kd's). Their optical features in combination with EPR studies have revealed
that all variants remain high-spin from pH 5 to 9, unless the haem pocket is both very open
and a His residue was located in a strained loop region at the Asn70 position. These variants
in particular showed evidence of a concerted mechanism in which prior binding of substrate
activates (by removing the low spin ligand) the enzyme for reaction with hydrogen peroxide.
These and other observations lead us to hypothesise that the mutations collectively allow a
rearrangement of the B-C loop region, which permits stabilisation of a labile LS ligand at the
haem centre of the resting state. The tight binding of substrates to the engineered cavity can in
turn then displace the labile ligand. The LS variants which showed this behaviour were much
more resistant to inactivation by hydrogen peroxide than chloroperoxidase or P450’s under
the same conditions.
39 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Immobilisation of Laccases for Biotransformations in
Environmental and Food-Technology
L25
M. Schroeder a , A. Kandelbauer a , G. Nyanhongo a , B. Poellinger-Zierler b , A. Cavaco-Paulo c ,
G.M. Guebitz a
a Graz Universty of Technology, Dept.of Environmental Biotechnolog, b Dept. of Food
Technology, Petersgasse 12, 8010 Graz Austria, c Dept. of Textile Engineering, University of
Minho, 4800 Guimaraes, Portugal
E-mail: guebitz@tugraz.at
The immobilisation of laccases from bacterial and fungal sourced onto water-soluble and
insoluble carriers was investigated for various biotransformations. Laccases from Trametes
modesta were immobilised on γ-aluminum oxide pellets and biotransformations of
systematically substituted model substrates were studied in an enzyme-reactor. The reactor
was equipped with various UV/Vis spectroscopic sensors allowing the continuous online
monitoring (immersion transmission probe, diffuse reflectance measurements of the solid
carrier material). Immobilisation of the laccase did not sterically affect oxidation while
electron donating substitutents on the aromatic ring generally enhanced reaction rates [1]. The
immobilised laccases were successfully applied in continuous degradation of phenolic
compounds such a textile dyes. Microbial off-flavours in fruit juices such as 2,6-
dibromophenol, borneol, guaiacol can also be eliminated by immobilised laccase treatment.
This was shown by chemical analysis (solid phase micro extraction / GC-MS) combined with
evaluation by a certified test panel. Besides immobilisation of fungal laccases the potential of
naturally immobilised spore laccases is discussed.
Laccases could prevent fabrics and garments from re-deposition of dyes during washing and
finishing processes by degrading the solubilized dye. However, laccase action must be
restricted to solubilized dye molecules thereby avoiding decolorization of fabrics. Here we
show that covalent immobilisation of laccases with polyethylene glycol (PEG) can drastically
reduce the activity of the modified laccases on fibre bound dye decreasing the adsorption of
the enzyme on fabrics. PEG modification of a laccase from T. hirsuta resulted in enhanced
enzyme stability while with increasing molecular weight of attached PEG the substrate
affinity for the laccase conjugate decreased [2].
Immobilisation of laccases onto polysaccharide based carriers was investigated with regard to
the production of biodegradable explosives. During microbial degradation of TNT laccase
catalysed binding onto humic monomers (200mM) prevented the accumulation of all major
stable TNT metabolites (aminodinitrotoluenes [AMDNT]) by at least 92 %. Complete
enzymatic elimination was seen for 4-HADNT (4-hydroxylaminodinitrotoluene) and 2-
HADNT with a concurrent decrease of toxicity [3].
[1] Kandelbauer,A., Maute,O., Kessler,R., Erlacher,A., Guebitz,G.M., 2004. Biotechnol. Bioeng. 87, 552-563.
[2] Schroeder,M., Heumann,S., Silva,C., Cavaco-Paulo,A., Guebitz,G.M., 2005. Biotechnol Lett. in press
[3] Nyanhongo,G.S., Rodriguez Couto,S., Guebitz,G.M., 2006. Chemosphere, in press
40 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L26
Laccase-catalyzed Polymerization for Coating and Material
Modification
Andrea Zille, Carlos Basto, Su-Yeon Kim, Artur Cavaco-Paulo
University of Minho, Department of Textile Engineering, 4800-058 Guimarães, Portugal
E-mail: artur@det.uminho.pt
The enzymatic polymerization and material modification with laccases is a promising
technology especially for the coating of the natural and synthetic materials at mild conditions
of temperature and pH [1]. The “in situ” enzymatic coating of several natural materials as
sisal, linum, cotton and wood were performed, in a batchwise process at different temperature
and pH. Small colorless aromatic compounds such as diamines, aminophenols,
aminonaphtols, and phenols, were oxidized by laccase resulting in dimeric, oligomeric, and
polymeric molecules [2]. The coupling and polymerizing ability of laccase was used for
colored and non-colored surface modifications of the materials in order to obtain coating with
water-proof, flame retardant, strength and adhesive properties. Sisal and wood were
enzymatic coated with laccase using several phenols and amines. Interesting waterproof
properties as well as different hues and depth of shades in the color pallet were observed.
Enzymatic coating with catechol of amized cellulose fibers was also performed in the
presence of laccase [3]. The LC/MS analysis of the hydrolyzed coated-cellulose confirming
the presence of functionalized glucose and cellobiose units coupled to poly(catechol)
molecules (m/z 580 and m/z 633). Furthermore, laccase was tested in combination with
ultrasound to improve coloration of wool by “in situ” radical polymerization of catechol [4].
In the sonicated laccase/catechol system a large polymerization was observed even more than
the laccase/catechol stirring system. The ultrasonic waves produce hydroxyl radicals, improve
the diffusion processes and may also have positive effect on the laccase active center structure
[5]. Extension of these methods to other laccase substrates, using appropriate and costefficient
functionalization techniques, may provide a new route to environmentally friendly
materials with predefined structures and properties.
[1] Mayer, A.M.; Staples, R.C. Phytochemistry 2002, 60, 551
[2] Pilz, R.; Hammer, E.; Schauer, F.; Kragl, U. App. Microbiol. Biotechnol. 2003, 60, 708
[3] Chhagani, R. R.; Iyer, V.; Shenai, V. A. Colourage 2000, 47, 27
[4] Mahamuni, N. N.; Pandit, A. B. Ultrason. Sonochem. 2006, 13,165
[5] Entezari, M. H.; Pétrier, C. App. Catal. B: Environ. 2004, 53, 257
41 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Potential of White-Rot Fungi for Decolourisation and
Detoxification of Dyes
L27
S. Vanhulle, E. Enaud, Lucas, Naveau, V. Mertens, M. Trovaslet, A.M. Corbisier
Microbiology Unit (MBLA), catholic University of Louvain, Croix du Sud 3 boîte 6, B 1348
Louvain-la-Neuve, Belgium,
e-mail: vanhullesophie@hotmail.com
The use of white rot fungi (WRF) appears to be a promising alternative to treat dyes
containing wastewater. Based on a previous screening of 300 WRF, six strains belonging to
the species Coriolopsis polyzona, Perenniporia ochroleuca, Pycnoporus sanguineus,
Perenniporia tephropora and Trametes versicolor were selected for an extensive search on
decolourisation and detoxification of dyes.
The major metabolites resulting of the biotransformation of the blue anthraquinonic dye Acid
Blue 62 (ABu62, previously called NY3) were isolated, characterized and a mechanism of
decolourisation was proposed. A first rapid step leading to red intermediates, was mainly due
to a dimerization of the initial molecule and was followed by a slower step leading to
uncoloured products formed by degradation of this main dimer into smaller fragments. As
laccase was the main ligninolytic activity of these strains, LAC-1 from Pycnoporus
sanguineus MUCL 41582 (PS7) was selected as a model for kinetic studies. While displaying
a traditional Michaelis-Menten kinetic behaviour with ABTS as substrate, LAC-1 presented
an atypical behaviour when ABu62 was used as substrate. In addition, LAC-1 only catalysed
the first step of Abu62 biotransformation. Therefore, Pycnoporus strains were used as model
to understand the role of laccases in the in vivo decolourisation of three anthraquinonic dyes:
Abu62, Acid Blue 281 and Reactive Blue 19. All three dyes caused an increase in laccase
activity. In vitro, oxidation of thel three anthraquinones by a laccase preparation was obtained
to a lesser extend than the whole cell process; suggesting that other factor(s) could be required
for a complete decolourisation. The activity of cellobiose dehydrogenase (CDH) was
therefore monitored. Present early in the broth during the growth of the fungi, CDH displayed
in vitro a synergism with laccases in the decolourisation of ABu62, and an antagonism
with laccases in the decolourisation of ABu281 and RBu19.
Nevertheless, decolourisation does not imply that the resulting metabolites are less toxic than
the parent molecules. Toxicity assays were previously developped on human Caco-2 cells,
which are considered as a validated model for the human intestinal epithelium. Depending on
the strain used, a cytoxicity reduction between 25 % and 85 % was observed after two
weeks of culture. No mutagenic character appeared during the biotransformation, as verified
through VITOTOX TM assays. Enzymatic treatment of ABu 62 with purified laccase (EC
1.10.3.2) from Pycnoporus sanguineus allowed in one day a cytotoxicity reduction
comparable to that obtained in 7 days by a complete culture. PS7 was further used to treat an
industrial effluent and compared to the effectiveness of ozonolysis. The effluent toxicity was
reduced by only 10% through ozonolysis, whereas the fungal treatment reached a 35%
reduction. Moreover, a mixed treatment (ozone, then PS7) caused a 70% cytotoxicity
reduction. Raw effluent presented mutagenic character. Ozonized effluent was still
mutagenic, while the genotoxic effect was completely removed after fungal treatment
(patent WO2002EP10077 20020909).
42 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L28
Biotransformation of Environmental Pollutants by Aquatic
Fungi – the Role of Laccases
Dietmar Schlosser a , Claudia Martin a , Charles Junghanns a , Monika Moeder b , Magali Solé a ,
Gudrun Krauss a
a Department of Environmental Microbiology, and b Department of Analytical Chemistry, UFZ
Centre for Environmental Research Leipzig-Halle, Permoserstrasse 15, D-04318 Leipzig,
Germany
E-mail: dietmar.schlosser@ufz.de
Fungi occuring in freshwater environments and their laccases have gained considerably less
attention than terrestrial fungi, with respect to their possible contribution to the natural
attenuation of organic pollutants in the environment and their potential biotechnological
application in the removal of hazardous pollutants from wastewater.
Endocrine disrupting chemicals and ingredients of personal care products found in
aqueous environments led to increasing concerns regarding their potentially hazardous effects
on human health and the environment, but the knowledge about their biodegradability by
microorganisms of aquatic environments is still limited. Technical nonylphenol, a mixture of
mainly para-substituted nonylphenol isomers with variously branched side chains, is known
to act as a xenoestrogen. HHCB (galaxolide®) and AHTN (tonalide®) are polycyclic musk
fragrances used in personal care products and were reported to inhibit multixenobiotic
resistance transporters in aquatic organisms. We investigated the bioconversion of HHCB,
AHTN, and nonylphenol, the latter being applied as an isomeric mixture and also in the form
of single nonyl chain-branched isomers, by freshwater-derived, laccase-producing mitosporic
fungi. Degradation studies involved both fungal cultures and isolated laccases. Fungal
cultures removed nonylphenol more efficiently under conditions where high extracellular
laccase activities were expressed, as compared to conditions where laccase activities were low
or negligible. Nonylphenol conversion by isolated laccases led to the formation of oxidative
coupling products. This is in favour of an extracellular attack on nonylphenol catalyzed by
laccase. In addition, as yet unknown intracellular enzymes attack nonylphenol at the alkyl
chain as implied by certain biotransformation metabolites. In fungal cultures, several
metabolites detected during the removal of HHCB and AHTN indicated biotransformation
initiated by intracellular hydroxylation. Moreover, isolated laccases were also able to convert
both, HHCB as well as AHTN. Laccase treatment of HHCB strongly increased the
concentration of the known HHCB metabolite HHCB-lactone, suggesting that laccase
catalyzes the oxidation of HHCB into HHCB-lactone.
Textile dyes left unconsumed in textile industry effluents represent another example for
potentially hazardous environmental contaminants. We investigated the ability of aquatic
fungi to decolourise synthetic dyes and addressed the potential involvement of the laccases of
these organisms in decolourisation.
All together, these results demonstrate a potential of aquatic fungi and their laccases to
affect the environmental fate of organic contaminants in freshwater ecosystems and their
possible application in technical processes aiming at the removal of organic pollutants
wastewater.
43 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Feng Xu
Transformation of Textile Dyes by Oxidoreductases
Novozymes Inc, 1445 Drew Ave., Davis, CA 95616, USA
E-mail: fxu@novozymes.com
L29
During the dyeing of fabrics, most vat and sulfur dyes have to undergo sequentially a
reduction (to increase the solubility), an adsorption (by fabric), and a re-oxidation (to enhance
the fastness) step. The reduction can be made with various chemical reductants (such as
sodium dithionite). The re-oxidation can be made either by simply exposing to air or more
often by complex processings involving chemical oxidants (such as H 2 O 2 , m-
nitrobenzenesulfonate, perborate, hypochlorite, iodate, bromate, or dichromate), harsh
conditions (high pH or temperature), or expensive/unsafe catalysts (such as NaVO 3 ).
Modifying the chemical re-oxidation step with an enzymatic technology could be of
significant interest in terms of production economy as well as waste or hazardous chemicals
handling. The concept was tested on several representative vat and sulfur dyes with a laccase
and a heme peroxidase. It was shown that the enzymes could catalyze the re-oxidation of
reduced dyes by O 2 and H 2 O 2 , respectively. Small redox-active mediators facilitated the
enzymatic re-oxidation. An enzymatic dye-reducing system was also tested. Mediated by
redox mediator, a carbohydrate oxidase could reduce several representative dyes, leading to
decolorization. Thus oxidoreductase could replace chemical oxidizing or reducing agents in
transforming dyes.
44 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L30
Free, Supported and Insolubilized Laccases: Novel
Biocatalysts for the Elimination of Micropollutants and
Xenoestrogens
Spiros N. Agathos
Unit of Bioengineering, Catholic University of Louvain,
Croix du Sud 2, B-1348 Louvain-la-Neuve, Belgium
Several emerging pollutants occur in relatively low concentrations (micropollutants) and may
include active ingredients in personal care products and known or suspected endocrine
disrupting substances (EDS, xenoestrogens). These contaminants constitute a major
preoccupation in the water quality and treatment field because of their potential risk to human
health and their environmental impact, since they resist conventional treatment and they tend
to accumulate in hydrophobic matrices. The biocatalytic elimination of established or
suspected xenoestrogens including nonylphenol (NP), bisphenol A (BPA) and triclosan (TCS)
is currently investigated in our laboratory, using a variety of enzyme preparations based on
fungal laccases. These polyphenoloxidases (EC 1.10.3.2) are multicopper oxidases
particularly adapted to the oxidation of phenol-like compounds and aromatic amines, i.e.,
molecules sharing several structural features with the above xenoestrogens. Initial studies
have focused on EDS elimination using crude laccase preparations from the white rot fungi
Coriolopsis polyzona, Lentinus critinus or Ganoderma japonicum. Statistical experimental
design was used to establish optimal ranges of pH, temperature and time of contact for the
removal of NP, BPA and TCS. The use of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS) as a mediator has been found to enhance the efficiency of the enzymatic
treatment. The removal of NP and BPA was accompanied by the disappearance of estrogenic
activity, as demonstrated by the yeast estrogen test (YES). Mass spectrometry analysis
showed that the enzymatic treatment produces high molecular weight metabolites through
radical polymerization of NP, BPA and TCS leading to C-C or C-O bond formation. The
polymerization of these contaminants produces a range of oligomers (from dimers up to
pentamers) which are inert. In an effort to overcome the limitations of free laccases, in terms
of re-usability and stability against denaturants in an industrial setting, we have studied their
immobilization. Laccase from Coriolopsis polyzona was covalently immobilized on
diatomaceous earth supports (Celite ® ), whose surface had been activated with
aminopropyltriethoxysilane and then cross-linked with gluteraldehyde. Despite the relatively
low enzyme/support ratio (w/w), the supported biocatalyst displayed improved stability
against thermal inactivation and denaturation by salts and proteases. When used in a packedbed
reactor, the immobilized laccase was able to eliminate BPA from aqueous solutions under
different operational conditions, including several consecutive treatment cycles, with
sustained removal performance. Finally, in a further simplification of biocatalyst preparation
and in order to enhance the specific activity with retention of stability and re-usability
features, the same crude laccase was insolubilized in the form of cross-linked enzyme
aggregates (CLEAs). The optimal biocatalyst preparation involved the precipitation of laccase
with polyethylene glycol, cross-linking with glutaraldehyde and recovery of active CLEAs
upon removal of the precipitant. Characterization of this new insolubilized biocatalyst and
initial tests with BPA have shown that, both from a kinetic and from a stability point of view,
laccase CLEAs have strong potential not only in the sustainable elimination of
micropollutants but also in a variety of other biotechnological applications.
45 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L31
Olive Mill Wastewater Transformation and Detoxification by
White-Rot Fungi: Role of the Laccase in the Process
G. Iamarino a , J.M. Barrasa b , L. Gianfreda c , A.T. Martínez a and M.J. Martínez a
a Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain;
b Universidad de Alcalá, Alcalá de Henares, E-28871, Madrid, Spain. c DiSSPA, Università
degli Studi di Napoli, Via Università 100, 80055 Portici, Na Italy (mjmartinez@cib.csic.es)
Large amounts of dark effluents, called olive oil mill wastewaters (OMW), are produced
during oil extraction from olives. These effluents are characterized by low pH, intense dark
brown colour, high organic load including lipids, pectin, polysaccharides and phenols, and
high content of residual oil and solid matter 1 . Due to its low cost and high mineral content,
OMW could be used as organic fertilizer and irrigation waters of agricultural lands but a
previous detoxification is necessary, since they show phytotoxic and antimicrobial
properties 2 . Because the phenolic compounds present in OMW, which are the main
responsible of its toxicity, show similar structure that those derived from lignin
biodegradation, the use of ligninolytic fungi or their enzymes to treat this industrial effluent is
being studied. The presence during the lignin biodegradation process of different extracellular
ligninolytic enzymes (laccases and peroxidases) depends on the fungal species studied.
Whereas peroxidases seem to play an important role in OMW degradation by Phanerochaete
species 3;4 , laccase appears as the sole ligninolytic enzyme in other fungal species 5;6 . In this
work we studied the transformation and detoxification of OMW by the white-rot fungi
Pycnoporus coccineus, Coriolopsis rigida, Polyporus alveolaris and Calocera cornea, the first
species as a reference since its use has already been reported in OMW treatment 7 .
The results in solid and liquid medium with OMW from Morocco (7.5, 15 and 30%), as
sole carbon source, showed a strong decolourisation by C. cornea, whereas the other fungi
decreased the colour at lower concentration but increased it at the highest ones. In the liquid
medium all the fungi were able to growth and reduce the content of phenolic compounds,
although the reduction was much higher in C. rigida cultures, which produced the highest
laccase levels. Peroxidases were not detected in any case. OMW samples at different
concentrations (7.5, 15, 30 and 50%) were also treated with the C. rigida laccase produced in
a basal medium with glucose-yeast extract-peptone 8 . At all the dilutions assayed, the enzyme
was stable after 24 h of incubation and a strong decrease of phenol content was observed. To
confirm the potential of C. rigida and its laccase to degrade and detoxify OMW, phenolic
compound identification (by HPLC/GC-MS) and toxicity test of the treated industrial effluent
(using Microtox and germination tests) are currently in course.
[1] C.Paredes, J.Cegarra, A.Roig, M.A.Sánchez-Monedero, and M.P.Bernal, Bioresour. Technol. 67 (1999) 111-115.
[2] R.Capasso, G.Cristinzio, A.Evidente, and F.Scognamiglio, Phytochemistry 31 (1992) 4125-4128.
[3] S.Sayadi and R.Ellouz, Appl. Environ. Microbiol. 61 (1995) 1098-1103.
[4] O.Ben Hamman, T.de la Rubia, and J.Martínez, Environ. Toxicol. Chem. 18 (1999) 2410-2415.
[5] A.Jaouani, F.Guillén, M.J.Penninckx, A.T.Martínez, and M.J.Martínez, Enzyme Microb. Technol. 36 (2005) 478-486.
[6] G.Aggelis, D.Iconomou, M.Christou, D.Bokas, S.Kotzailias, G.Christou, V.Tsagou, and S.Papanikolaou, Water Res.
37 (2003) 3897-3904.
[7] A.Jaouani, S.Sayadi, M.Vanthournhout, and M.Penninckx, Enzyme Microb. Technol. 33 (2003) 802-809.
[8] M.C.N.Saparrat, F.Guillén, A.M.Arambarri, A.T.Martínez, and M.J.Martínez, Appl. Environ. Microbiol. 68 (2002)
1534-1540.
46 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L32
Combined Application of Glucose Oxidases and
Peroxidases in Bleaching Processes
Klaus Opwis, Dierk Knittel, Eckhard Schollmeyer
Deutsches Textilforschungszentrum Nord-West e.V., Adlerstr. 1, D-47798 Krefeld, Germany
E-mail: opwis@dtnw.de
Peroxidases (POD) are used in textile decolorization and bleaching processes, but their
activity is limited by the hydrogen peroxide concentration, which attack the POD during the
reactions. A new concept for a simultaneous use of glucose oxidase and peroxidase was
developed. Figure 1 illustrates the combined application of both enzymes. Starting with
glucose as substrate for the glucose oxidase (GOD) hydrogen peroxide is generated in situ.
The fresh built substrate H 2 O 2 is used immediately by the POD to oxidize colored compounds
in dyeing baths. Therefore the stationary peroxide concentration is nearly zero during the
whole reaction time and the enzymes are not degraded by the substrate. Moreover
experiments are done to check the possibility to use this two compound system for textile
bleaching of natural fibres like cotton or hemp. First results are of great promise for further
investigations in future.
glucose oxidase
glucose
peroxidase
O 2
gluconic acid
H 2 O 2
colored compound
H 2 O
H 2 O
oxidized, colorless
compound
Bleaching
Figure 1: Use of oxidoreductases in bleaching processes.
47 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L33
Laccase-Mediator System: the Definitive Solution to Pitch
Problems in the Pulp and Paper Industry?
Ana Gutiérrez a , Jorge Rencoret a , David Ibarra b , Ángel T. Martínez b , José C. del Río a
a Instituto de Recursos Naturales y Agrobiología, CSIC, PO Box 1052, E-41080, Seville,
Spain; b Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid,
Spain
E-mail: anagu@irnase.csic.es
Lipophilic extractives in wood and other lignocellulosic materials exert a highly negative
impact in pulp and paper manufacturing causing the so-called pitch problems that affect both
paper machine runnability and product quality, among others. Some biotechnological
products have been developed and enzymes (lipases) have been successfully applied to
softwood mechanical pulping at mill scale [1]. However, the enzymes and microbial inocula
used till present are only effective on specific raw materials and processes. Recently, we have
shown for the first time the effectiveness of the laccase-mediator system (LMS) in removing
pulp lipids regardless the pulping process and the raw material used [2,3]. The results have
been included in a patent recently deposited [4]. In these studies, three pulps representative
for different pulping processes and raw materials - including eucalypt kraft pulping, spruce
thermomechanical pulping (TMP), and flax soda-anthraquinone (AQ) pulping - were treated
with laccase in the presence of 1-hydroxybenzotriazole (HBT) as a redox mediator. The gas
chromatography and gas chromatography/mass spectrometry analyses of the acetone extracts
from the enzymatically-treated pulps revealed that most of the lipophilic compounds present
in the different pulps were efficiently removed using the LMS. Free and conjugated (as esters
and glycosides) sitosterol, the main compounds responsible of pitch deposits in the
manufacturing of eucalypt kraft pulp, were completely removed. In spruce TMP pulp, LMS
degraded most of the resin acids, as well as sterol esters and triglycerides. In the flax soda-AQ
pulp, the main lipophilic compounds present including sterols and long chain fatty alcohols
were almost completely removed. Small amounts of oxidation products (including 7-
oxositosterol, stigmasta-3,5-dien-7-one and 7-oxositosteryl 3β-D-glucopiranoside) were
identified confirming the oxidative nature of lipid removal. Pulp and papermaking properties
of the enzymatically-treated pulps were also evaluated. In conclusion, LMS treatment is an
efficient method to remove pitch-causing lipophilic compounds from hardwood, softwood as
well as nonwood paper pulps (at the same time that lignin content is reduced).
[1] Gutiérrez, A.; del Río, J.C.; Martínez, M.J.; Martínez, A.T. The biotechnological control of pitch in paper
pulp manufacturing. Trends Biotechnol. 2001, 19, 340.
[2] Gutiérrez, A.; del Río, J.C.; Ibarra, D.; Rencoret, J.; Romero, J.; Speranza, M.; Camarero, S.; Martínez,
M.J.; Martínez, A.T. Enzymatic removal of free and conjugated sterols forming pitch deposits in
environmentally sound bleaching of eucalypt paper pulp. Environ. Sci. Technol 2006, (in press).
[3] Gutiérrez, A.; del Río, J.C.; Rencoret, J.; Ibarra, D.; Martínez, A.T. Main lipophilic extractives in different
paper pulp types can be removed using the laccase-mediator system. Appl. Microbiol. Biotechnol. 2006,
(in press) DOI: 10.1007/s00253-006-0346-1.
[4] Gutiérrez, A.; del Río, J.C.; Rencoret, J.; Ibarra, D.; Speranza, A. M.; Camarero, S.; Martínez, M. J.;
Martínez, A.T. Sistema enzima-mediador para el control de los depósitos de pitch en la fabricación de
pasta y papel. Patent (Spain) 2005, 200501648.
48 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L34
Optimization of a Laccase-based Delignification System
which uses as Mediators Fatty Hydroxamic Acids in situ
Generated by Lipases
Hans-Peter Call, Simon Call
Bioscreen e.K.,Heinsberger Strasse 15,
D-52531 Uebach-Palenberg, Germany
E-mail: Bioscreen@t-online.de
Based on a general concept using enzymatically -mainly laccase- generated reactive oxygen
species (ROS) or reactive nitrogen species (RNS) we have recently developed and published
different new approaches for delignificaton of pulp or for other applications.
One of the most promising methods is a laccase-based concept which uses fatty hydroxamic
acids as mediators formed in situ by special lipases.
This system consists of an optimal combination of
1) Laccase
2) Hydrolases (Lipases)
3) Fatty acid/fats (or corresponding esters)
4) R 2 -NOH compounds
This special mixture of system components causes a continuous and slow generation of
fatty hydroxamic acids (R-C=O-NHOH), i.e. siderophore like compounds as substrate for
laccase.
The fatty acid hydroxamic acids are released by the reaction of (particularly) lipases with
special NO-containing precursor compounds and fatty acid/fats (or corresponding esters).
We will summarize new results referring to further optimization of the mentioned new
delignification method mainly in respect to better performance and environmentally safer
NO-precursor compounds.
The obtained results indicate a good performance in respect to the delignification rates, i.e. it
could be demonstrated that in most cases [with different pulps such as sulfate (SW, HW)
sulfite (SW, HW)] delignification rates up to 40% and more could be reached during a 2-4
hours treatment at pH 4-8, at 50- 60 o C and ca. 10% consistency, maintaining the strength
properties.
49 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L35
Studies on the Effect of the Laccase Mediator System on
Ageing Properties of Hand Sheets of Different Origin
Jorge Gominho a , Ana Lourenço a , Helena Pereira a , Cristina Máximo b , Maria Costa-Ferreira b
a Centro de Estudos Florestais, Departamento de Engenharia Florestal, Instituto Superior de
Agronomia; 1349-017 Lisboa, Portugal; b Department of Biotechnology, National Institute for
Engineering, Technology & Innovation - INETI; 1649-038 Lisboa, Portugal
E-mai : Maria.ferreira@ineti.pt
Microbial agents, either whole cells or enzymes from these, have been applied to the different stages of pulp
and paper processing. The present work describes a study on the effect of applying ligninolytic enzymes,
such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and
subsequently subjecting these to different ageing processes.
The starting material was industrial pulps obtained from different Portuguese pulp and paper companies. The
pulps used were 1) unbleached pine pulp from Portucel Tejo; 2) unbleached eucalyptus pulp from Portucel
Setúbal; 3) bleached eucalyptus pulp from Portucel Setúbal; and 4) pulp made from recycled paper from
Renova S.A. Several types of handsheets were produced with 2 different grammage namely, 60 and 180
g/m 2 . The prepared handsheets were subject to an aging sequence in three different chambers: ultraviolet
radiation (wavelength of 280 nm), temperature (19ºC) and moisture (70%); and thick saline fog at a
concentration of 1% and temperature of 35ºC.
In order to evaluate the effect of moisture cycles and temperature, two aging sequences were used for each
type of handsheet. In the first, the moisture varied (60, 80 and 100 %), while the temperature was held
constant (25ºC); in the second the temperature varied (60, 70 and 80ºC) and the moisture was held constant
(50%). Following the aging phase, the handsheets were subject to several chemical (viscosity and index
Kappa) and physico-mechanical (colour, tensile breaking strength, stretch and the bursting strength) tests in
order to characterize the effect of the aging conditions. Results will be presented describing the effect of
application of different enzymatic treatments on the ageing phenomenon.
We gratefully acknowledge funding from the Fundação para a Ciência e a Tecnologia, for a
project entitled “Enzymatic modification of E. globulus pulp fibres” POCTI/AGR/47309/02.
50 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
L36
Laccase in Pulp Activation and Functionalisation
Anna Suurnäkki a , Marco Orlandi b , Stina Grönqvist a , Hannu Mikkonen a , Liisa Viikari a
a VTT, Tietotie 2, 02044 VTT, Finland
b Dipartimento di Scienze dell’Ambiente e del Territorio, Universitá degli Studi di Milano-
Bicocca, Piazza della Scienza 1, I-20126 Milan, Italy
E-mail: anna.suurnakki@vtt.fi
The presence of surface lignin in pulp fibres offers possibilities to enhance the existing pulp
properties or even to create completely new pulp properties by enzymatic means. Improving
the properties of wood fibres is a constant interest of pulp, paper and board manufacturing
industry. New methods for targeted modification of wood materials could also reveal
completely new application areas for wood fibres.
Oxidative enzymes such as laccases can be used to activate the surface lignin of lignin-rich
pulps by radicalisation. The primary reaction of laccase catalysed oxidation is the formation
of phenolic radicals to the substrate. Due to the high reactivity of these radicals (either with
each other or with a secondary substrate), reactions such as polymerisation, depolymerisation,
co-polymerisation and grafting can occur. The size of laccases limits the action of the enzyme
on the fibre surface, which can be considered both as a limitation or an opportunity when
applying laccases in fibre applications. Enzymatic activation of fibre surfaces can be exploited
after further functionalisation of fibres with specific chemical components in tailoring fibre
properties.
In this work the laccase catalysed activation and functionalisation of lignin-rich pulps was
studied. The radical formation in pulps during oxidation with different laccases was analysed
by oxygen consumption measurement and EPR spectroscopy. Changes in the pulp lignin
stucture by laccase activation were determined by NMR. The factors affecting the activation
and further functionalisation of pulps were elucidated. A novel chemo-enzymatic
functionalisation method developed for lignin-rich pulps and its potential in modification of
fibre properties will be discussed in the presentation.
51 September 7-9, 2006
Oeiras, Portugal

POSTER PRESENTATIONS

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
MICROBIAL PHYSIOLOGY
P1 High concentrations of cell wall redox enzymes lichens in Suborder Peltigerineae
Richard P. Beckett, Zsanett Laufer, Farida V Minibayeva
P2 Characterization and differential regulation of variable manganese peroxidase
genes in the white-rot fungus Physisporinus rivulosus
Kristiina Hildén, Terhi K. Hakala, Pekka Maijala, Cia Olsson and Annele Hatakka
P3 Peroxidase and phenoloxidase activities in the cell walls of wheat roots
Farida V Minibayeva, Oleg P. Kolesnikov, Albina A. Kavieva, Svetlana Y. Mityashina, Andrei V. Chasov, Lev K.
Gordon
P4 Evaluation of oxidase potential and growth rate of saprotrophic Basidiomycetes
cultures
N. Psurtseva, A. Kiyashko, N. Yakovleva, N. Belova
P5 Characteristics of laccase in the biopulping fungus Physisporinus rivulosus
T. Hakala, K. Hildén, P. Maijala, A. Hatakka
P6 Laccase Production by Basidiomycetes under Various Fermentation Conditions
N. Belova, N. Psurtseva, N. Yakovleva, A. Kiyashko, T. Lundell, A. Hatakka
P7 Effect of various phenolics in agar medium on pattern of fungal mycelium
Malarczyk E., Jarosz-Wilkolazka A., Polak J., Olszewska A., Graz M., Kochmanska-Rdest J
P8 Multicopper oxidases from Myxococcus xanthus: a model for applications,
functions and regulation
Nuria Gómez-Santos, Aurelio Moraleda-Muñoz, María Celestina Sánchez-Sutil, Juana Pérez-Torres and José
Muñoz-Dorado
P9 Characterisation of Pseudomonas sp. ox1 phe operon
Bertini L., Stancarone V., Di Berardino I., Caporale C., Buonocore V. and Caruso C.
P10 Cloning of Laccase Gene from Coriolopsis polyzona MUCL 38443
S. Koray Yesiladali, Gunseli Kurt, Ayten Karatas, Nevin Gül Karagüler, Candan Tamerler
P11 Heterologous Expression of Pycnoporus sanguineus MUCL38531 lcc1 cDNA in
Pichia pastoris
Günseli Kurt, Nevin Gül Karagüler, Ayten Yazgan Karataş, Candan Tamerler
ENZYMOLOGY
P12 The Deduced Amino Acid Sequence and the Substrate Oxidation Profile of the
Phanerochaete Flavido-Alba Laccase Identifies the Enzyme as “Ferroxidase-Laccase”
Rodríguez-Rincón F, Suarez, A., de la Rubia, T., Lucas, M. and Martínez, J
P13 Purification and properties of a non-blue fungal laccase isoenzyme
Albino A. Dias, Rui M.F. Bezerra, Irene Fraga, António N. Pereira
P14 Laccase purification from Coriolopsis polyzona MUCL 38443
Pınar Hüner, Hande Asımgil, Koray Yeşiladalı, Hakan Bermek, Candan Tamerler
P15 Directed evolution of Pleurotus ostreatus laccases
Giovanna Festa, Paola Giardina, Alessandra Piscitelli, Flavia Autore, Rosa Cestone and Giovanni Sannia
P16 Production, Purification and Characterization of Laccase Enzymes from Thielavia
arenaria
Kristiina Kruus, Marja Paloheimo, Terhi Puranen, Leena Valtakari c , Jarno Kallio, Richard Fagerström, and Jari
Vehmaanperä
54 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P17 Production, Purification and Kinetic Characterisation of a Thermostable
Pycnoporus sanguineus Laccase (LAC-1)
M. Trovaslet, C. Bebrone, E. Enaud, S. Hubert, N. Nouaimeh, M. Pamplona-Aparicio, B. Lorenzini, Ch.-M. Bols, J-
M. Frère, A-M. Corbisier, S. Vanhulle
P18 Production of Cerrena unicolor Manganese Peroxidase and Laccase in Solid-state
on Oat Husks
Ulla Moilanen, Erika Winquist, Aila Mettälä, Pekka Maijala, Ossi Pastinen, Annele Hatakka
P19 Preparation and Characterization of Crossed-Linked Laccase Aggregates from the
White-Rot Fungus Coriolopsis polyzona
Hubert Cabana, J. Peter Jones, Spiros N. Agathos
P20 Enhanced Stability of Laccase by Xylitol
Andre Zille, Diego Moldes, Ramona Irgoliç, Artur Cavaco-Paulo
P21 Influence of Static Magnetic Field on Laccase Activity and Stability
V. Kokol, M. Schroeder, G. M. Guebitz
P22 Novel Laccases and Peroxidases for Dye Decolourisation and Bleaching
Processes
Matura,A. and K.-H. van Pée
P23 Ralstonia solanacearum Expresses a Unique Tyrosinase with a High Tyrosine
Hydroxylase/DOPA Oxidase Ratio
Hernández-Romero, D., Sanchez-Amat, A., Solano, F.
P24 Engineering of a Psychrophilic Microorganism for the Oxidation of Aromatic
Compounds
Rosanna Papa, Ermenegilda Parrilli, Paola Giardina, Maria Luisa Tutino and Giovanni Sannia
P25 Spectroscopic Characterization of a Novel Naphthalene Dioxygenase from
Rhodococcus sp.
Maria Camilla Baratto, David A Lipscomb, Christopher CR Allen, Michael J Larkin, Riccardo Basosi , Rebecca
Pogni
P26 Identification of Novel Sulfhydryl Oxidases
Vivi Joosten, Willy van den Berg, Sacco de Vries, Willem van Berkel
P27 Chlorohydroquinone Monooxygenase - a Novel Enzyme in the 2,4-
dichlorophenoxyacetate Biodegradation Pathway of Nocardioides simplex 3E –
Enzymatic and Genetic Aspects
Jana Seifert, Peter Simeonov, Stefan Kaschabek and Michael Schlömann
P28 Cellobiose Dehydrogenases from Ascomycetes and Basidiomycetes:
Phylogenetic and Kinetic Comparison
Roland Ludwig, Marcel Zámocky, Clemens Peterbauer, and Dietmar Haltrich
P29 Oxalate Oxidase as a Potential Enzyme Responsible
for H 2 O 2 Generation in Abortiporus biennis
Marcin Grąz, Anna Jarosz-Wilkołazka, Elżbieta Malarczyk
P30 Production, Purification and Molecular Characterisation of a Quercetinase from
Penicillium olsonii
S. Tranchimand, V. Gaydou, T. Tron, C. Gaudin , G. Iacazio
P31 Laccase Activity Measurements in Turbid or Coloured Liquids with a Novel
Optical Oxygen Biosensor
Christian-Marie Bols and Rob C .A. Onderwater
55 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P67 Preliminary study of soluble heme proteins from Shewanella oneidensis MR1
Bruno Fonseca, Patrícia M. Pereira, Isabel Pacheco, Ricardo O. Louro
P68 Aerobic Oxidation of Alcohols Catalyzed by Laccase from Trametes versicolor
and Mediated by TEMPO
Inga Matijosyte, R.van Kooij, l W.C.E. Arends, S. de Vries, R. A. Sheldon
P69 Role of Laccases in the Decolourisation of Synthetic Dyes by Aquatic Fungi
Charles Junghanns, Dietmar Schlosser
P70 Application of Oxidative Enzymes for the Detoxification of Xenobiotic Pollutants
Maria Antonietta Rao, Giuseppina Iammarino, , Rosalia Scelza, Fabio Russo, Liliana
Gianfreda
STRUCTURE-FUNCTION RELATIONSHIPS
P32 The Role of the C-terminal Amino Acids of Melanocarpus albomyces Laccase
Martina Andberg, Sanna Auer, Anu Koivula, Nina Hakulinen, Juha Rouvinen, Kristiina Kruus
P33 Shifting the optimal pH of activity for a laccase from the fungus Trametes
versicolor by structure-based mutagenesis
C. Madzak, M.C. Mimmi, E. Caminade, A. Brault, S. Baumberger, P. Briozzo, C. Mougin, C. Jolivalt
P34 Axial perturbations of the T1 copper in the CotA-laccase from Bacillus subtilis:
Structural, Biochemical and Stability Studies
Paulo Durão, Isabel Bento, André T. Fernandes, Eduardo P. Melo, Peter F. Lindley, Lígia O. Martins
P35 Structural Studies in CotA Mutants: Understanding of the Protonation Events that
occur during Oxygen Reduction to Water
Isabel Bento, Paulo Durão, André T. Fernandes, Lígia O.Martins, Peter F. Lindley
P36 Relationship of Substrate and Enzyme Structures as a Basis for Intradiol
Dioxygenases Functioning
Kolomytseva M.P., Ferraroni M., Scozzafava A., Briganti F., Golovleva L.
P37 Surface-enhanced Vibrational Spectroelectrochemistry of Immobilized Proteins
Smilja Todorovic, Peter Hildebrandt and Daniel Murgida
P38 Enzymatic Properties, Conformational Stability and Model Structure of a Metallo-
Oxidase from the Hyperthermophile Aquifex aeolicus
André T. Fernandes, Cláudio M. Soares, Manuela Pereira, Robert Huber, Gregor Grass, Eduardo P. Melo and
Lígia O. Martins
APPLIED
P39 Degradation of Azo Dyes by Trametes villosa Laccase under Long Time Oxidative
Conditions
Andrea Zille, Barbara Górnacka, Astrid Rehorek Artur Cavaco-Paulo
P40 Enzymatic Decolorization of Azo and Anthraquinonic Dyes with the CotA-Laccase
from Bacillus subtilis
Luciana Pereira, Lígia O. Martins
P41 Selection of Laccases with Potential for Decolourisation of Wastewater Issued
from Textile Industry
E. Enaud, M. Trovaslet, M. Pamplona-Aparicio, A-M. Corbisier, S. Vanhulle
P42 Decolorization of Textile Dyes by the White-Rot Fungus Coriolopsis polyzona
MUCL 38443
Aisle Ergun, Firuze Basar, S. Koray Yesiladalı, Z. Petek Çakar Öztemel, Candan Tamerler Behar
56 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P43 Laccase from Trametes versicolor Immobilised on Novel Composite Magnetic
Particles
K.-H. van Pée, A. Matura, T. Wage, A. Pich, U. Böhmer
P44 Biotechnological Applications of a pH-Versatile Laccase from Streptomyces
ipomoea CECT 3341
Molina, J.M., Moya, R. Guillén, F., Hernández, M. and Arias, M.E.
P45 Oxidative Reactions for the Decolorization of Synthetic Dyes – Laccase versus
Fenton’s Reagent
Amaral, P.F.F., Pinto, F.V., Cammarota, M.C., Coelho, M.A.Z.
P46 Application of Tyrosinase Obtained from Agaricus bispora for Color Removal
from Textile Effluents
Magali C. Cammarota, Maria Alice Z. Coelho
P47 Phenols and Dyes Degradation by an Immobilized Laccase from Trametes trogii
Anna Maria V. Garzillo, Federica Silvestri, M. Chiara Colao, Maurizio Ruzzi, Vincenzo Buonocore
P48 Relationship between Non-Protein Fraction and Laccase Isoenzymes from
Cultures of Trametes versicolor: Effect on Dye Decolorization
Diego Moldes, Alberto Domínguez, Mª Angeles Sanromán
P49 Degradation of Synthetic Dyes by Coriolopsis rigida
J. Gómez-Sieiro, D. Rodríguez-Solar, D. Moldes, M.A. Sanromán
P50 Immobilization of Laccase and Versatile Peroxidase Considering Their Further
Application
Anna Olszewska, Jolanta Polak, Anna Jarosz-Wilkołazka, Janina Kochmańska-Rdest
P51 Removal of Several Azo Dyes by Trametes sp. Crude Laccase: Reaction
Increment in the Presence of Azo Dye Mixtures
Rui M.F. Bezerra, Irene Fraga, Albino A. Dias
P52 Transformation of Simple Phenolic Compounds by Fungal Laccase to Produce
Colour Compounds
Jolanta Polak, Anna Jarosz-Wilkołazka, Marcin Grąz, Elżbieta Dernałowicz-Malarczyk
P53 Biodegradation cycles of industrial dyes by immobilised basidiomycetes
L.Casieri, G.C. Varese, A. Anastasi, V. Prigione, K. Svobodová, V. Filipello Marchisio and Č. Novotný.
P54 Catalytic Activity of Versatile Peroxidase from Bjerkandera fumosa and its use in
Dyes Decolourization
Anna Jarosz-Wilkołazka, Anna Olszewska, Janina Rodakiewicz-Nowak, Jolanta Luterek
P55 Bleaching of Kraft Pulp Employing Polyoxometalates and Laccase
José A.F. Gamelas, Ana S.N. Pontes, Dmitry V. Evtuguin, Ana M.R.B.Xavier
P56 Influence of Trametes versicolor laccase on the contents of hexenuronic acids in
two Eucalyptus globules kraft pulp
Atika Oudia, Rogério Simões, João Queiroz
P57 Laccase-Mediated Oxidation of Natural Compounds
Mattia Marzorati, Daniela Monti, Francesca Sagui, Sergio Riva
P58 Laccase induced coating of lingocellulosic surfaces with functional phenolics
M. Schroeder, G. M. Guebitz, V. Kokol
57 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P59 Decolourization and Detoxification of Kraft Effluent Streams by Lignolitic
Enzymes of Trametes versicolor
M.S.M. Agapito, D. Evtuguin, A.M.R.B. Xavier
P60 Effect of Medium Composition on Laccase Production by Trametes versicolor
Immobilized in Alginate Beads
A. Domínguez, D. Moldes, M. A. Longo and M. A. Sanromán
P61 Involvement of the Laccase Produced by Streptomyces sp. in the
Biotransformation of Coffee Pulp Residues
Orozco, A.L., Polvillo O., Rodríguez, J., Molina, J.M., Guevara, O., Arias, M.E., Pérez, M.I.
P62 Elimination of the Endocrine Disrupting Chemical Bisphenol A by using Laccase
from the Ligninolytic fungus Lentinus crinitus
Carolina Arboleda, Hubert Cabana, J. Peter Jones, Amanda I. Mejía, Spiros N. Agathos, Gloria A Jimenez, Michel
J. Penninck
P63 Tyrosinase-catalyzed modification of Bombyx mori silk proteins
Giuliano Freddi, Anna Anghileri, Sandra Sampaio, Johanna Buchert, Raija Lantto, Kristiina Kruus, Patrizia Monti,
Paola Taddei
P64 Kinetics of Laccase Mediator System Delignification of a Eucalyptus globulus
Kraft Pulp
Sílvia Guilherme, Ofélia Anjos, Rogério Simões
P65 Model Wastewaters Decolourisation by Pseudomonas putida MET 94
Bruno Mateus, Diana Mateus, Luciana Pereira, Orfeu Flores, Lígia O. Martins
P66 Cellulose-Based Agglomerates from Enzymatically Recycled Paper Wastes
Tina Bruckman, Margarita Calafell, Tzanko Tzanov
58 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
High Concentrations of Cell Wall Redox Enzymes Lichens
in Suborder Peltigerineae
Richard P. Beckett a , Zsanett Laufer a , Farida V Minibayeva b
a
School of Biological and Conservation Sciences, University of KwaZulu Natal, PBag X01,
Scottsville 3209, South Africa, b Institute of Biochemistry and Biophysics, Russian Academy of
Science. P.O.Box 30, Kazan 420111, Russia
E-mail: rpbeckett@gmail.com
In this study, we tested for the presence of extracellular redox enzymes in a range of 40
species of lichens. Two main types of enzymes were detected, laccases and tyrosinases,
although small amounts of a catalase-peroxidase were also found. Identification of laccases
was based on ability of lichens and lichen leachates to readily metabolize substrates such as
2,2’-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in
the absence of hydrogen peroxide, sensitivity of the enzymes to cyanide and azide, the
enzymes having typical pH and temperature optima, and an absorption spectrum with a peak
at 614 nm. Electrophoresis showed that the active form of laccase from Peltigera was a
tetramer with a molecular mass of 340 kD and a pI of 4.7. Further testing showed that lichens
can also readily metabolize substrates such as tyrosine, 3,4 dihydroxyphenylalanine (DOPA),
epinephrine, and m-cresol, substrates more usually associated with another group of multicopper
oxidases, the tyrosinases. Electrophoresis confirmed the presence of tyrosinases. In
Peltigera, the active form had a molecular mass of 60 kD. Detergents strongly activated
tyrosinase activity. Laccase and tyrosinase activities were detected in almost all lichens in the
Suborder Peltigerineae, but not in other species. Within the Peltigerineae, the activities of the
enzymes were significantly correlated to each other, but a fractionation technique showed that
they are bound to different cell wall components. Wounding stress strongly stimulated both
laccase and tyrosinase activities, while desiccation stress increased laccase but not tyrosinase
activity. Possible roles of theses enzymes in lichens are discussed.
P1
60 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P2
Characterization and Differential Regulation of Variable
Manganese Peroxidase Genes In The White-Rot Fungus
Physisporinus rivulosus
Kristiina Hildén, Terhi K. Hakala, Pekka Maijala, Cia Olsson and Annele Hatakka
Department of Applied Chemistry and Microbiology, University of Helsinki, Finland
Email: Kristiina.S.Hilden@helsinki.fi
Physisporinus rivulosus strain T241i is a lignin-degrading basidiomycete that is able to
selectively remove lignin from wood and is one of the most promising fungi for the use in
biopulping. During growth in wood chips, the fungus produces manganese peroxidase (MnP),
which is considered as the main ligninolytic enzyme in the lignin degradation. Present study
provides the primary structure of two MnP encoding genes mnpA and mnpB of Physisporinus
rivulosus T241i. Surprisingly, the mnp genes are significantly divergent in sequence, length
and intron-exon structure. The mnpA gene of P. rivulosus could be classified to the typical
MnP –group, whereas mnpB shared characteristics with the lignin peroxidase-type MnP –
group. Such diversity of mnp genes appears to be rare among white-rot fungi, and merits
further investigation.
The complex structure of wood makes it difficult to investigate enzyme regulation
under natural growth conditions. Thus, to study the expression of two different MnP encoding
genes of P. rivulosus and their regulation by different chemical compounds, we cultivated the
fungus on defined media under nutrient limited or sufficient conditions supplemented with
Mn 2+ or a non-phenolic aromatic compound veratryl alcohol. The expression of the two mnp
genes in agitated liquid cultures implicated quantitative variation and differential regulation in
response to Mn 2+ and veratryl alcohol. The transcription of mnpA was induced by the addition
of veratryl alcohol but not by Mn 2+ . In the cultures with sawdust a clear induction of mnpA
was observed. On the contrary, the transcription of mnpB was induced by addition of either
veratryl alcohol or Mn 2+ and only slightly by sawdust. This study suggests that the regulation
of MnP production in P. rivulosus is obviously multifactorial. Genes encoding enzyme
isoforms are expressed differentially and the inducers act both separately and in conjunction.
61 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Peroxidase and Phenoloxidase Activities in the Cell Walls
of Wheat Roots
Farida V Minibayeva, Oleg P. Kolesnikov, Albina A. Kavieva, Svetlana Y. Mityashina,
Andrei V. Chasov, Lev K. Gordon
Institute of Biochemistry and Biophysics, Russian Academy of Science. P.O.Box 30, Kazan
420111, Russia
E-mail: minibayeva@mail.knc.ru
Production of reactive oxygen species (ROS) is one of the widely reported stress responses of
plants. However, the nature of enzymes responsible for ROS production in the apoplast and
their regulation are still open questions. We studied intra- and extracellular redox activities of
wheat (Triticum aestivum L.) roots. It was found that wheat roots and leachates derived from
these roots possess redox activities, which were strongly stimulated following wounding or
heavy metal stresses. Plant cell wall has an enormous capacity to accumulate redox enzymes
in different cell wall fractions. Although total intracellular peroxidase, tyrosinase and ROS
producing activities were much higher compared to those in the leachates and cell wall
fractions, this was mainly due to the high intracellular protein content. Treatment of isolated
cell walls with digitonin and NaCl doubled protein release compared to that of the buffer
soluble fraction. Interestingly the specific ROS producing and peroxidase activities were
highest in the weakly bound enzymes and enzymes bound to the cell wall by strong
electrostatic forces. Treating the cell wall with detergent did not increase tyrosinase activity as
has been shown for fungal tyrosinases, suggesting that catalytical properties of these enzymes
of different origin can vary. Analysis of the phenolics in the cell wall fractions revealed that
the fraction containing redox enzymes bound by strong electrostatic forces is characterized by
the highest diversity of phenolic acids, with syringic acid being abundant. However, the
substrate for the weakly bound phenoloxidases and peroxidases is probably caffeic acid, the
concentration of which was 10 times higher in this fraction than in others.
Our results suggest that enzymes weakly bound to the cell wall have higher redox activity
compared to those more tightly bound. We assume that ROS production by free or readily
released from the cell wall redox enzymes is a part of the universal stress response and signal
transduction in plant cells.
P3
62 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P4
Evaluation of Oxidase Potential and Growth Rate of
Saprotrophic Basidiomycetes Cultures
N. Psurtseva, A. Kiyashko, N. Yakovleva, N. Belova
V.L. Komarov Botanical Institute RAS, St. Petersburg, Russia
E-mail: NadyaPsu@NP1512.spb.edu
Basidiomycetes fungi are well-known oxidoreductases producers. Wide screening in
Basidiomycetes for active species and strains can reveal new perspective source of oxidases.
Evaluation of oxidase potential and growth rate of Basidiomycetes cultures from various
taxonomic groups belonging to xylotrophic and litter-decomposing fungi was the aim of the
present study. About 300 strains with a broad taxonomical, ecological and geographical
diversity were involved in the experiment. The cultures were collected in different
geographical regions (mainly in Russia and former USSR) and maintained in the LE (BIN)
Basidiomycetes Culture Collection. All the strains were grown on ale-wort agar plates (alewort
4 o B, agar 20g/l). Laccase and tyrosinase activities were determined at 1, 2, 3 and 4
weeks of cultivation using rapid assay methods (reagents: tannic acid, syringaldazine,
guaiacol and L-tyrosine). Growth rate was expressed as a number of weeks that cultures
required to cover 90 mm plates. Various rate of laccase activity was found in 235 strains of
118 species from 70 genera of Basidiomycetes. Over 70 cultures belonging mainly to
xylotrophic fungi but to litter-decomposers too were considered as fast growing with intense
laccase reaction. High activity was detected for collection strains of species well known in the
world as laccase producers – Cerrena unicolor, Lentinus tigrinus, Trametes spp, Pleurotus
spp, Pycnoporus cinnabarinus, and Phlebia radiata. Some other species of Lentinus and
Trametes maintained in the LE (BIN) Collection also possessed high laccase activity.
Besides, new active strains of xylotrophic fungi from genera which were not well investigated
in the world on oxidase enzymes – Antrodiella, Byssomerulius, Hericium, Irpex, Irpicodon,
Junghuhnia, Lenzites, Lindtneria, Meripilus, Steccherinum, Treshispora, and Trichaptum
were found. All studied Polyporus species revealed high laccase activity. Several of them can
be of sufficient interest for further investigation. Traditionally oxidases producers are
considered to be mostly polypores fungi but some agaricoid fungi also have a great oxidases
potential. Publications on Pleurotus and Lentinus confirm this statement very well. It was
shown in our study that such xylotrophic agarics as Flammulaster limulatoides,
Hohenbuehelia fluxilis, Lentinellus ursinus, Marasmiellus omphaliiformis, Marasmius rotula,
Oudemansiella mucida, O. orientalis, and Xeromphalina campanella could also produce a
high level of laccase activity. Besides xylotrophic, other groups of saprotrophic fungi were
studied on laccase activity: fungi on buried wood, bark, cone, humus, dung, grass, coal,
mushrooms remains, died insects and fungi from grassland communities. High laccase
activity was found in Clavicorona pyxydata, Coprinus atramentarius, Macrolepiota procera,
Strobilurus tenacellus, Xerula radicata, and some other. However not all mentioned fungi
could be proposed as perspective laccase producers because of relatively slow growth on aleagar.
On the other hand cultures of some species possessed moderate activity but fast growth
could be perspective laccase producers. All strains involved into the experiment were studied
on their cultural characters. Over 20 cultures formed primordia or developed fruit bodies in
plates. As a result of the research a number of perspective strains were selected for further
investigations on laccase production.
This work was supported by the following grants: INTAS 03-51-5889 and Russian
Fund of Fundamental Researches 04-04-49813 and 06-04-49043.
63 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P5
Characteristics of Laccase in the Biopulping Fungus
Physisporinus rivulosus
T. Hakala a,b , K. Hildén a , P. Maijala a , A. Hatakka a
a Department of Applied Chemistry and Microbiology, Viikki Biocenter, University of
Helsinki, Helsinki, Finland; b present address: KCL Science and Consulting, Espoo, Finland
E-mail: pekka.maijala@helsinki.fi
Physisporinus rivulosus strain T241i is a lignin-degrading basidiomycete that is able to
selectively remove lignin from wood [1] and is one of the most promising fungi for the use in
biopulping. It degrades softwood lignin efficiently, grows in a wide temperature range, and
decreases the energy consumption in wood chip refining stage. In wood chip cultures P.
rivulosus began to secrete laccase already after 5-7 days, prior to substantial manganese
peroxidase (MnP) production [2]. This suggests that laccase may have an important role in
initiating lignin degradation or in colonization of wood, whereas MnP appears to be the main
lignin-degrading enzyme in subsequent lignin degradation in this fungus. In wood chip
cultures, laccase was secreted as four closely related acidic isoforms (pI-values between 3.1-
3.3). Identical N-terminal peptide sequences of the isoforms indicate that a single gene
encodes these isoforms. We have cloned and sequenced and characterized lac1 gene. The
inferred amino acid sequence of lac1 differs only at the first amino acid in the amino terminus
from the N-terminal peptide sequence obtained from laccase isoforms in wood chip cultures.
In liquid cultures the highest amounts of laccase were produced in the presence of peptone,
wood sawdust and charcoal. High content of glucose and veratryl alcohol also improved
laccase production in P. rivulosus. In contrast to laccase, MnP was highly secreted when
ammonium nitrate and asparagine were used as nitrogen sources. Peptone addition clearly
suppressed MnP production. In liquid cultures an additional laccase isoform with pI 4.5 was
efficiently produced when culture medium was supplemented with the lignocellulose
substrate. Both laccases possessed their maximal activity against phenolic substrates at pH
3.0, but laccase with pI 4.5 retained its activity better at alkaline pH region when compared to
laccase with pI 3.5. The pI 4.5 laccase showed also good thermostability. It showed half-life
of one hour at 70ºC.
[1] Hakala, T.K., Maijala, P., Konn, J., Hatakka, A. Enzyme Microb. Technol. 2004, 34:255-263.
[2] Hakala, T., Lundell, T., Galkin, S., Maijala, P., Kalkkinen, N., Hatakka, A. Enzyme Microb.
Technol. 2005, 36: 461-468.
64 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P6
Laccase Production by Basidiomycetes under Various
Fermentation Conditions
N. Belova a , N. Psurtseva a , N. Yakovleva a , A. Kiyashko a , T. Lundell b , A. Hatakka b
a Komarov Botanical Institute RAS, Prof. Popov Str., 2, St. Petersburg. 197376 Russia.
b University of Helsinki, P.O. Box 56 (Biocenter 1, Viikinkaari 9) 00014, Finland
E-mail: cultures@mail.ru
Fermentation conditions are essential to productive capacity of Basidiomycetes strains.
Cultures of various taxonomical and ecological groups having high natural ligninolytic
potential may be different in requirements regarding medium compounds and fermentation
methods during their cultivation. To reveal the most favorable cultivation conditions for
growth and laccase production for a number of selected strains have been initiated this study.
29 strains of 25 Basidiomycetes species from families Strophariaceae and Tricholomataceae
(Agaricales), Crepidotaceae (Cortinariales), Lentinellaceae (Hericiales), Coriolaceae,
Lentinaceae, and Polyporaceae (Poriales), and Steccherinaceae (Stereales) were studied as
stationary and submerged cultures using several liquid nutritional media. The fungal strains
were selected as a result of screening on laccase activity by rapid assay methods and by
cultural characters. Some of the fungal isolates were fruited in culture. A number of the
selected strains included species well-known as laccase producers belonging to the genera
Lentinus, Hypholoma, and Trametes. On the contrary, several of the fungal isolates belonged
to genera such as Lentinellus, Lenzites, Oudemansiella, Polyporus, Steccherinum, and
Tubaria that have not been studied yet for laccase production. Liquid ale-wort, malt extract
and two glucose-peptone media with different mineral components were used for stationary
and submerged cultivations. Laccase activity was estimated by using syringaldazine and
pyrocatechol as enzyme substrates. The experiments showed that the selected fungal strains
had different capacity for growth on used media. Moreover, each isolate had individual
priorities for nutritional media and cultivation method regarding its laccase production.
Growth on malt extract showed high laccase activity in Lenzites betulina, Oudemansiella
mucida, Tubaria sp., and Polyporus squamosus. Lenzites betulina revealed high laccase
production under both stationary and submerged cultivation on liquid malt extract, but not on
glucose-peptone LN-AS medium. High level of laccase activity and biomass production
during submerged cultivation on glucose-peptone medium was found in Trametes gibbosa
strains. Selected cultures of Lentinellus ursinus f. robustus and Steccherinum ochraceum
produced laccase under both cultivation conditions but showed difficulties in growth. It was
found that S. ochraceum produced not only high laccase activity but manganese peroxidase
also. The selected isolates of the genus Polyporus had a high potential for laccase production
under submerged cultivation but active production of some mucilaginous substance
(presumably polysaccharides) caused problems with measuring of laccase activity. Cultures
of Hypholoma fasciculare and H. sublateritium showed very low laccase production together
with poor growth on liquid media. Absence of any phenol oxidase or laccase activity was
observed with various cultivation methods for the isolates identified as Armillaria borealis,
Conocybe vexans, Marasmius rotula, Microporus luteus, and Macrolepiota procera while
they revealed very high laccase activity when rapid assay methods were used. As a result of
the experiments, several new Basidiomycetes isolates from various fungal genera that were
not studied in this regard before can be proposed as promising new producers of laccases.
This work was supported by the INTAS grant 03-51-5889.
65 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P7
Effect of Various Phenolics in Agar Medium
on Pattern of Fungal Mycelium
E. Malarczyk, A. Jarosz-Wilkolazka, J. Polak, A., Olszewska, M. Graz, J. Kochmanska-Rdest
Biochemistry Departament, University of M. Curie-Sklodowska, Lublin, Poland
E-mail: malar@hermes.umcs.lublin.pl
In the process of cultivation of fungi on agar plates, enriched in different kinds of aromatics,
the radial pictures was observed during the hyphal growth. It was compare to natural fruiting
of mushrooms where the combination of generally radial growth was observed according to
mycelium exploration of a new area, with branching hyphae growing out from behind the
leading hyphae. Expansion of the mycelium on these new terrains joint with the utilization of
earlier created hyphea as the source of energy. Around natural fruiting mycelium the so colled
“fairy rings” are very common as the result of maturation of hyphe spores mating for
production of fruit bodies. In natural environment the fruit bodies of some strains, example
Trametes versicolor, also are grown with creation of characteristic well visible colored rings.
Our observation was connected with growth of some strains of Basidiomycetes on separate
agar media, enriched in many kinds of aromatic compounds, mainly phenolic origin. Many of
these substances provoke the mycelium to radial growth with production of distinct circles,
laying in the definite distances, characteristic for type of phenolics. For fungal strains, fruiting
in laboratory conditions, the rings are also the places where fruit body are produced, looked
like as miniaturized “fairy rings”. The patterns and numbers of artificial rings were
categorized according to kind of phenolics and enzymatic set of tested fungus. The confocal
and scanning microscopy showed the deep differences between the mycelium taken from
rings or around. These results seem to have the practical aspect in the ring patterns for
additional characterization of fungal strains. The mechanism of phenolic respond during
cultivation of Basidiomycetes in the presence of various aromatic substrates is discussed.
66 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P8
Multicopper Oxidases from Myxococcus xanthus: a Model
for Applications, Functions and Regulation
Nuria Gómez-Santos, Aurelio Moraleda-Muñoz, María Celestina Sánchez-Sutil, Juana Pérez-
Torres and José Muñoz-Dorado
Departamento de Microbiología. Facultad de Ciencias. Universidad de Granada. Avda.
Fuentenueva s/n. E-18071 Granada. Spain.
E-mail: jdorado@ugr.es
The genome of the soil bacterium Myxococcus xanthus has revealed a 26.5 Kb region that
code for twenty proteins with conserved domains implicated in copper handling and
trafficking. Three of them enc Olszewska,de periplasmic multicopper oxidases that we have
named LcsA, LcsB and LcsC, respectively. The three genes are structurally organized in three
different operons named as curA, curB and curC. For details about curA, please attend the talk
of Sanchez-Sutil et al. Sequence analysis of LcsA, LcsB and LcsC has revealed interesting
differences, such as the presence of a histidine rich region between domains II and III in LcsA
and metionine rich motifs in the C-terminal portions of LcsB and LcsC. The three MCOs
exhibit different translocation motifs. While, LcsA seems to be secreted by Sec system, LcsB
and LcsC contain twin-arginine motifs within the leader sequences recognized by the Tat
translocation system. Probably they will be translocated by the Tat pathway with copper
bound to its active sites. The transcriptional regulation profiles of the three operons have
shown that time, copper concentration and maximum levels of expression are different for
each operon, indicating that they might be adapted to different mechanisms of detoxification.
The operons are transcriptionally controlled by at least two different regulators, which seem
to sense copper concentrations at different subcellular locations, the periplasmic and the
cytoplasmic spaces. All this interesting features, along with the fact that M. xanthus
undergoes an unique cell cycle and induces carotenogenesis by copper, give us the
opportunity to use this delta-proteobacterium as model to study copper resistance and
homeostasis in a very wide perspective.
67 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Characterisation of Pseudomonas sp. ox1 phe Operon
L. Bertini, V. Stancarone, I. Di Berardino, C. Caporale, V. Buonocore, C. Caruso
Dip. di Agrobiologia e Agrochimica, Università della Tuscia, Viterbo 01100, Italy
E-mail: caruso@unitus.it
Human activities have brought about the release into the environment of a plethora of
aromatic chemicals. Among them are aromatic hydrocarbons which are important component
of petroleum and its refined products; they are extensively used as solvent in the production
of several chemical compounds as well as in their synthesis. These aromatic compounds have
also deleterious effects on human health due to their toxic, mutagenic and carcinogenic
properties. Since their distribution in the environment is ubiquitous and the effects on human
being highly dangerous, studies on the xenobiotic biodegradation are receiving significant
attention.
Many genera of microorganisms degrade aromatic compounds, Pseudomonas being the most
extensively analysed. The interest in discovering how bacteria are dealing with hazardous
environmental pollutants has driven a large research community and has resulted in important
biochemical, genetic, and physiological knowledge about the degradation capacities of
microorganisms (1,2). In addition, regulation of catabolic pathway expression has attracted
the interest of several groups, who have tried to unravel the molecular partners in the
regulatory process, the signals triggering pathway expression, and the mechanisms of
activation and repression. Moreover, the knowledge of the regulatory mechanisms of aromatic
molecules biodegradation is particularly attractive in the development of biosensors for
phenolic compounds, which have been of major concern as one of priority pollutants due to
their toxicity (3).
Pseudomonas sp. OX1 is able to growth on toluene, o-xylene, 2,3 and 3,4 dimethylphenol and
cresol as the sole carbon and energy source due to the presence of two characteristic
hydroxylating enzymes: the multienzymatic complexes of Toluene/o-xylene Monoxygenase
(ToMO), coded by the tou operon, and Phenol Hydroxylase (PH), coded by a different
catabolic operon (phe cluster) (4). Data concerning the genetic organization and regulation of
lower pathway genes are available for some Pseudomonas strains which indicate that the gene
order within the catabolic operon is not constant.
In this comunication we report the nucleotide sequence of the last genes characteristic of the
phe meta operon of Pseudomonas sp. OX1. The genomic organization of the lower pathway
has been compared to the ones available on data banks in order to highlight common
filogenetic relationships. Moreover, the 5’ untranslated region of Pseudomonas sp. OX1 phe
cluster has been isolated and sequenced in order to carry out structural-functional
characterisation of the phe promoter (P phe ).
[1] Gibson, J., and C. S. Harwood. 2002. Annu. Rev. Microbiol. 56: 345–369.
[2] Mishra, V., R. Lal, and Srinivasan. 2001. Rev. Microbiol. 27: 133–166.
[3] Park, S. M., Park, H. H., Lim W. K. and Shin, H. J. 2003. J. Biotechnol. 103: 227-236.
[4] Cafaro, V., Izzo, V., Scognamiglio R., Notomista E., Capasso P., Casbarra, A., Pucci P. and Di Donato A:
2004. Appl. Environ. Microbiol. 70: 2211-2219.
P9
68 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P10
Cloning of Laccase Gene from Coriolopsis polyzona MUCL
38443
S. Koray Yesiladali, Gunseli Kurt, Ayten Karatas, Nevin Gül Karagüler, Candan Tamerler
Istanbul Technical University, Department of Molecular Biology and Genetics, Maslak-
Istanbul, 34469, Turkey
E-mail: yesiladali@itu.edu.tr
Coriolopsis polyzona MUCL 38443 is a fast-growing, laccase producing white-rot fungus
which belongs to basidiomycete family. The microorganism was previously investigated for
its ability in detoxification processes. High laccase levels produced by the microorganism
found to be promising for industrial applicability. Laccase production of Coriolopsis polyzona
MUCL 38443 was optimized starting from shake flask cultures up to 2L stirred tank
bioreactors. Results indicate that fermentation time in bioreactors were considerably long for
an industrial application which could be as long as 20 days. Besides microbial physiological
studies that are performed, recombinant production is a major way to shorten the time length.
Therefore, we isolate and characterize a full-length cDNA coding for major laccase, from
Coriolopsis polyzona MUCL 38443 and to produce heterologous expression of laccases in
yeast for large scale production of the enzyme and shorten the fermentation time of the
production to an acceptable level.
This study is funded by EU 6th Framework Integrated Project (IP), ‘SOPHIED - Novel
sustainable bioprocesses for the European colour industries’.
69 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Heterologous Expression of Pycnoporus sanguineus
UCL38531 lcc1 cDNA in Pichia pastoris
P11
Günseli Kurt, Nevin Gül Karagüler, Ayten Yazgan Karataş, Candan Tamerler
İstanbul Technical University, Department of Molecular Biology and Genetics, Maslak-
İstanbul, 34469, Turkey
E-mail: gunselik@yahoo.com
The orange red compound, cinnabarin, produced by Pycnoporus sanguineus MUCL 38531 is
a promising candidate for new dyes. Laccases, which are able to degrade lignin and also
polymerize phenolic compounds, play an important role in the production of cinnabarin by
coupling of 3-hydroxyanthanilate. Ligninolytic enzymes are generally difficult to overexpress
in heterologous organisms in their active form. However, the expression of active
recombinant laccases has been reported in the filamentous fungus Aspergillus oryzae and the
yeasts Sacchharomyces cerevisiae and Pichia pastoris. Here, we isolated and characterized a
full-length cDNA coding for major laccase in Pycnoporus sanguineus MUCL 38531. Next,
heterologous expression of laccase was performed in methylotropic yeast Pichia pastoris,
which is a more suitable host for large scale production of the enzyme. The lcc1 cDNA was
cloned into the yeast shuttle expression vector pPICZB with its own signal peptide for
expression in Pichia pastoris under the control of the alcohol oxidase (Aox1) promoter.
Following the transformation into the P. pastoris strain X-33, transformants were selected on
the minimal methanol plates supplemented with substrate ABTS (0.2mM). Laccase-producing
transformants oxidized the ABTS and are identified by the presence a green zone around the
Pichia colonies. Characterization of recombinant laccase was performed and the identity of
the product was also confirmed by native gel electrophoresis. Protein engineering studies will
be further integrated into the recombinant laccase production to improve the properties of the
enzyme to enhance its industrial applicability.
This study is funded by EU 6th Frame Integrated Project (IP), “SOPHIED-Novel Sustainable
Bioprocesses for European Colour Industries”
70 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P12
The Deduced Amino Acid Sequence and the Substrate
Oxidation Profile of the Phanerochaete Flavido-Alba
Laccase Identifies the Enzyme as “Ferroxidase-Laccase”
F. Rodríguez-Rincón a , A. Suarez b , T. de la Rubia c , M. Lucas c , J. Martínez c
a Department of Microbiology Faculty of Basic Sciences, University of Pamplona, Pamplona,
Colombia. b Department, of Biochemistry and Molecular Biology, and c Department of
Microbiology Faculty of Pharmacy, University of Granada. Granada. Spain.
E-mail: dlrubia@ugr.es
Laccases, ferroxidases, ascorbate oxidase, and ceruloplasmin belong to the Multicopper
Oxidase (MCOs) family of enzymes. Basidiomicetous laccases have been the most
thoroughly studied because of their involvement in biological processes and because of their
promising biotechnological applications.
This communication summarizes the results of a study on the deduced amino acid sequence
(PfaL) of the recently identified Phanerochaete flavido-alba laccase gene [1] and a
comparative phylogenetic analysis with other nulticopper oxidases. Compared with the
recombinant Phanerochaete chrysosporum MCO1 and a commercial T. versicolor laccase,
the purified P. flavido-alba laccase showed a substrate range typical of a laccase and different
to that exhibited by the P. chrysosporium MCO1 [ferroxidase] [2]. The deduced amino acid
sequence of PfaL conserved the L1-L4 signature copper sequences described by Kumar et al
[3] in fungal laccases.
P. flavido-alba being a basidiomycete, the PfaL amino acid sequence was not
phylogenetically aligned with typical basidiomycetous laccases, but with the ascomycetous
laccases.
In contrast to the ascomycetous laccases, the PfaL aminoacid sequence (as well as the four P.
chrysosporium MCO sequences) conserved the position of one of the three aminoacids
(E185) involved in iron binding in the best know ferroxidase (the Fet3 Saccharomyces
cerevisiae ferroxidase). None of the compared ascomycetous laccases conserved this
position.
After comparing the PfaL amino acid sequence with prokaryotic and eukaryotic ferroxidases,
PfaL was aligned with a subbranch of the most numerous group of proteins apart from the
group of animal ferroxidases. This group contained three basidiomycetous proteins (PfaL, P.
chrysosporium MCO1 and a Cryptococcus neoformans protein) as well as two putative
proteins from an ascomycete (Yaworria lipolytica). When PfaL was aligned against the most
closely related laccases and ferroxidases PfaL was grouped as a differentiated group from
typical laccases and ferroxidases. In summary the MCOs from P. chrysosporium and P.
flavido-alba laccase form a phylogenetic group different from laccases and ferroxidases.
These proteins share conserved residues and enzymatic properties of both laccases and
ferroxidases.
[1] Rodríguez Rincón et al. (2005). XX Congreso Nacional de Microbiología Sept. 2005. Cáceres, Spain.
[2] Lucas et al. (2005). 13 th Int. biodeterioration and Biodegradation Symposium. Sept.2005. Madrid, Spain.
[3] Kumar ket al. (2003). Biotechnology and Bioengineering 83: 386-394
71 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P13
Purification and Properties of a Non-Blue Fungal Laccase
Isoenzyme
Albino A. Dias a , Rui M.F. Bezerra a , Irene Fraga a , António N. Pereira b
a CETAV - Dep. Engenharia Biológica e Ambiental, UTAD, Apartado 1013, 5001-801 Vila
Real, Portugal; b Departamento de Indústrias Alimentares, UTAD, Apartado 1013, 5001-801
Vila Real, Portugal
E-mail: jdias@utad.pt
Laccase (E.C. 1.10.3.2; benzediol: oxygen oxidoreductase) is a member of the multi-copper
glycoproteins which includes ceruloplasmin, ascorbate oxidase and the yeast protein Fet3.
The preferred substrates are p-diphenols, but o-diphenols, aminophenols, N-hydroxi
compounds and aryl diamines are also acceptable, as well as certain inorganic ions (notably
iodide). Laccase performs two concomitant reactions: (i) non-specific oxidation of
appropriated substrates to give cation radicals and/or quinones and (ii) reduction of molecular
oxygen to water. Nowadays, laccase has received increased attention due to its potential for
several biotechnological applications. Previously [1], we reported that basidiomycetous strain
Euc-1 growing in defined liquid medium (without aromatic inducers) exhibit laccase activity.
Crude laccase was resolved by anion-exchange chromatography into two peaks, the most
abundant accounting for 95% of total laccase activity. In this work we report the purification
and characterisation of Lac 1, a native laccase isoenzyme. Purified Lac 1 is a lowglycosylated
(6%) monomeric protein with 65.7 kDa (59.0 kDa using gel filtration) and
pI=6.0. The UV-Vis spectrum of purified Lac 1 showed a poor-resolved shoulder at around
330 nm but typical T1 copper peak at 610 nm was absent. The optimum activity temperature
was 50ºC while optimum pH was bellow 3.0 for ABTS (Km = 18 µM) and respectively 3.5,
4.0, 4.5 for the phenolic substrates 2,6-dimethoxyphenol (Km = 268 µM), guaiacol (Km =
587 µM) and syringaldazine (Km = 2.7 µM). Both substrate affinity and catalytic efficiency
(kcat/Km) increased in the order: guaiacol < 2,6-dimethoxyphenol < ABTS < syringaldazine.
As observed with other laccases Lac 1 was severely inhibited by azide and fluoride.
[1] A.A. Dias, R. M. Bezerra, P. M. Lemos and A. N. Pereira (2003). In vivo and laccase-catalysed
decolourization of xenobiotic azo dyes by basidiomycetous fungus: characterization of its ligninolytic system.
World J Microbiol Biotechnol 19: 969-975
72 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P14
Laccase Purification from Coriolopsis polyzona MUCL
38443
Pınar Hüner, Hande Asımgil, Koray Yeşiladalı, Hakan Bermek, Candan Tamerler
İstanbul Technical University, Department of Molecular Biology and Genetics, Maslak-
İstanbul, 34469, Turkey
E-mail: bermek@itu.edu.tr
Production, purification and characterization of laccase enzyme of C. polyzona are under
investigation for their potential applications in xenobiotic degradation. The organism was
grown in liquid shake flasks and was found to produce the enzyme. Several different
approaches including precipitation, ion exchange chromatography, hydrophobic interaction
chromatography, and gel filtration for purification were utilized. The best result was obtained
using the Q-Sepharose ion-exchange chromatography. The enzyme eluted in deep blue
colored fractions. The gel filtration chromatography was applied using Sephadex G100 resin.
The spectral characteristics of the enzyme was similar to the standards, i.e., the 610 nm peak
which is the characteristic of the blue copper center of the laccase was observed and the
A280/A610 was around 20. The characterization studies will now be undertaken.
This study is funded by EU 6th Framework Integrated Project (IP), ‘SOPHIED - Novel
sustainable bioprocesses for the European colour industries’.
73 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Directed Evolution of Pleurotus ostreatus Laccases
P15
Giovanna Festa, Paola Giardina, Alessandra Piscitelli, Flavia Autore, Rosa Cestone and
Giovanni Sannia
Department of Organic Chemistry and Biochemistry, Complesso Universitario Monte
S.Angelo, via Cintia 4, 80126 Naples, Italy
E-mail: festa@unina.it
During the last few years, directed evolution has emerged as method of choice for engineering
functions and properties of enzymes. This approach mimics in vitro the natural process of
molecular evolution that is able to generate a potentially infinite plethora of proteins with new
function and properties, such as stability to temperature and solvents, improved catalytic
performance and substrate specificity [1]. Two cDNAs encoding Pleurotus ostreatus laccases,
POXC [2] and POXA1b [3], were selected as “parent molecules” to guide the evolution of
laccases with higher specific activity and different substrate specificities. Genetic variants
were created by random mutagenesis and DNA shuffling. poxc was mutated with low
frequency (0÷3 mutations/kbase) and poxa1b with low, medium (3÷7 mutations/kbase) and
high frequency (more than 7 mut/kbase) by errore-prone PCR; furthermore a library from
poxc and poxa1b shuffling was produced. Two hosts, Kluyveromyces lactis and
Saccharomyces cerevisiae, were available to express genetic variants [4], experimental data
induced to prefer S. cerevisiae on the basis of its transformation efficiency and stability of
plasmid DNA. To screen yeast colonies for the ability to express high levels of laccase
activity, three sequential selections were performed. One clone, 1M9B, was selected showing
a 1.6 fold increase of laccase activity production compared with the wild type. 1M9B clone
was further characterised: nucleotidic sequence of the cDNA revealed two point mutation
which resulted in a single amino acid substitution (L112F). Thermodynamic and catalytic
characterization of this mutant is in progress. 1M9B clone was used as template for
production of new mutant collection by errore-prone PCR (with low and medium frequency
of mutation). Structural and catalytic characterization of these mutants is still in progress.
[1] Farinas ET, et al., 2001, Curr. Opin. Biotechnol., 12, 545-55.
[2] Palmieri G., et al., 1993, Appl. Microbiol. Biotechnol., 39,632-636
[3] Giardina P. et al., 1999, Biochem. J., 34,655-663
[4] Piscitelli A., et al., 2005,. Appl. Microbiol. Biotechnol., 69,428-39
74 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P16
Production, Purification and Characterization of Laccase
Enzymes from Thielavia arenaria
Kristiina Kruus a , Marja Paloheimo b , Terhi Puranen b , Leena Valtakari c , Jarno Kallio b , Richard
Fagerström a , and Jari Vehmaanperä b
a VTT Technical Research Centre of Finland, Espoo, Finland; b Roal Oy, Rajamäki, Finland;
c AB Enzymes Oy, Rajamäki, Finland
E-mail: kristiina.kruus@vtt.fi
A thermophilic ascomycete fungi T. arenaria was shown to be an interesting laccase
producer. Four functional laccase genes were isolated and heterologously expressed in a
filamentous fungi Trichoderma reesei. Characterization of the purified recombinant enzymes
indicated that the T. arenaria laccases are clearly distinct proteins from each other having
unique catalytic properties. The enzymes were also tested in denim bleaching. The
predominant T. arenaria laccase, referred as TaLcc1 was found to be superior in
decolorization of Indigo dye being, thus, a promising candidate for textile applications.
Heterologous expression of the laccases as well as their characteristics will be discussed in
detail.
75 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P17
Production, Purification and Kinetic Characterisation of a
Thermostable Pycnoporus sanguineus Laccase (LAC-1)
M. Trovaslet a , C. Bebrone b , E. Enaud a , S. Hubert b , N. Nouaimeh a , M. Pamplona-Aparicio a , B.
Lorenzini c , Ch.-M. Bols c , J-M. Frère b , A-M. Corbisier a , S. Vanhulle a
a Microbiology Unit, Université catholique de Louvain, Place Croix du Sud 3 bte 6, B-1348
Louvain-la-Neuve, Belgium, b Center for Protein Engineering, Université de Liège, Allée du 6
Août B6, Sart-Tilman, 4000 Liège, Belgium c Wetlands Engineering, Parc Scientifique
Fleming, Rue du Laid Burniat 5, 1348 Louvain-la-Neuve, Belgium,
Laccases have demonstrated good potential for applications in various industrial and
environmental processes. To our knowledge, only few data describing potential cooperative,
concerted, or feed back inhibition laccase behaviour were studied. However, it seems clear
that the development of an effective biotechnological application using a laccase requires the
study of its kinetic properties against the target substrates: it is one of the objectives of this
work.
A thermostable laccase (LAC-1) from Pycnoporus sanguineus MUCL 41582 (PS7) was
produced in a 10-liters bubble column fermentor and purified in three steps. First, the medium
was concentrated by an ammonium sulphate precipitation, then the resulting laccase was
loaded on an ion-exchange QAE-Sepharose HP column and finally, homogeneity was
obtained by a Cu 2+ -affinity chromatography.
Molecular mass, isoelectric point, specific activity and some kinetic parameters of LAC-1
were determined. This enzyme was very similar to some other laccases produced by White
Rot Fungi. However, (i). its half-life at high temperatures (between 70 and 85°C) suggested a
high thermostability of this laccase; (ii). it displayed a Michaelis-Menten behaviour with 2,2’-
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and presented a low K m value with
this substrate; (iii). on anthraquinonic acid dye (ABu62), Hanes-Woolf plot ([S]/v vs. [S])
clearly showed a non-Michaelis-Menten kinetic behaviour and a Hill equation was proposed
to explain the relationship between the initial velocity (v) and the substrate concentration
([S]); (iv). when both ABTS and ABu62 were present, ABTS oxidation catalysed by LAC-1
was alternatively favoured and disfavoured when ABu62 concentration increased.
Our results, especially the rather good thermostability of PS7 laccase, its relatively easy
production and concentration, combined with its high potential of decolourisation suggest that
LAC-1 may be efficiently exploited in a variety of biotechnological applications including the
wastewater treatment.
76 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P18
Production of Cerrena unicolor Manganese Peroxidase and
Laccase in Solid-state on Oat Husks
Ulla Moilanen a , Erika Winquist a , Aila Mettälä b , Pekka Maijala b , Ossi Pastinen a ,
Annele Hatakka b
a Laboratory of Bioprocess Engineering, Helsinki University of Technology, Kemistintie 1,
Espoo, Finland; b Department of Applied Chemistry and Microbiology, University of Helsinki,
Biocenter 1, Viikinkaari 9, Helsinki, Finland
E-mail: ulla.moilanen@tkk.fi
In this study we cultivated the white-rot fungus Cerrena unicolor in solid-state on oat husks
and water. The aim was to study the production of lignin degrading enzymes, manganese
peroxidases (MnP) and laccases in different solid-state conditions. Laccase production by C.
unicolor has earlier been described by e.g. Elisashvili et al. [1]. C. unicolor has been reported
to produce also MnP but not in substantial amounts.
Oat husks are side products from food industry. They contain lignocellulose of plant cell
walls and mainly starch-containing residual oat meal. First we studied the effect of the fine
fraction (FF) in the oat husks media with the strain C. unicolor T71. The fine fraction was
obtained by sieving oat husks through 2-mm sieve and it composed mainly of oat meal and
finely ground oat husks. The fungus was cultivated in 15 g scale and the dry weight of the
medium was 33 %. Zero to 50 % fines was added to the sieved oat husks. Originally unsieved
oat husks contain approximately 50 % w/w of fines. The production of both MnP and laccase
activities was substantially improved after fines addition and the highest activities were
obtained with the highest amount fines added. Apparently oat meal provides an easily
exploitable carbon source for the fungus.
Three additional C. unicolor strains (373, 316 and PM170798) were cultivated on unsieved
oat husks. The strain PM170798 was found to be clearly the best enzyme producer among the
tested strains. Compared with the strain T71, approximately 20 % higher laccase and 60 %
higher MnP activities were obtained with the strain PM170798.
Based on these results we chose the strain PM170798 for further studies where we compared
the effects of different inducing compounds on the enzyme production. The cultivation scale
was increased to 100 g. Sieved oat husks and water were used as the basic medium and the
added compounds were FF from unsieved husks (50 % of DW), copper (500 µM), manganese
(200 µM), veratryl alcohol (2mM) and ethanol (2 % v/w). Oat meal in FF accelerated the
growth of fungi and also enzyme production. We found that MnP production was improved
only by FF addition. The highest MnP activity (227 nkat/g DW on day 12) was three times
higher than with the sieved oat husks. Laccase activity was increased when FF, copper or
manganese was added to the medium. The highest laccase activities with FF were obtained
already on day 9. This was twice as high as on sole oat husks. Laccase activity was tripled (to
425 nkat/g DW) with Cu addition. Also Mn clearly increased laccase production. Highest
activities with Cu and Mn were reached on day 14, which was later than with FF.
Finally we tested the applicability of the process in a bigger scale. We made experiments in a
solid-state bioreactor with 4 kg of cultivation media. The enzyme activities were
approximately 50 % higher than in 100 g scale because the cultivation conditions in the
reactor can be better controlled. This proves that the solid-state oat husk cultivation process
can be scaled up.
[1] Elisashvili, V., Kachlishvili, E. and Bakradze, M., Appl. Biochem. Microbiol. 38 (2002) 210-213.
77 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Preparation and Characterization of Crossed-Linked
Laccase Aggregates from the White-Rot Fungus
Coriolopsis polyzona
Hubert Cabana a,b , J. Peter Jones b , Spiros N. Agathos a
P19
a Unit of Bioengineering, Catholic University of Louvain, Croix du Sud 2, 1348 Louvain-la-
Neuve, Belgium; b Department of Chemical Engineering, University of Sherbrooke, 2 500
boulevard de l’Université, Sherbrooke (Qc), Canada
E-mail: Hubert.Cabana@Usherbrooke.ca
Substantial efforts have been made to immobilize laccase on solid supports (1). These
immobilization procedures result in laccase stabilization against thermal and chemical
denaturation, in kinetic behaviour modifications and in reusability of the enzymes. All of
these characteristics make immobilization a step forward for the utilisation of laccase in
environmental biotechnology. A disadvantage of these immobilization procedures on a solid
support is the low enzyme/support mass ratio. The immobilization of laccase through crosslinking
of the lignin modifying enzyme is a simple alternative to produce insolubilized
laccase with high volume activity (2). Cross-linked enzyme aggregates (CLEAs) have been
proposed as an alternative to conventional immobilization procedures using solid supports
and to cross-linked crystals of enzymes (3). This kind of immobilisation involves the
precipitation of the enzyme and the chemical cross-linking of the protein using an appropriate
bi-functional reagent. The cross-linking procedure prevents the solubilisation of the aggregate
after the elimination of the precipitation agent.
CLEAs were prepared using laccase from the white-rot fungus Coriolopsis polyzona. The
preparation procedure was optimized by examining various precipitants and various
concentrations of these precipitants and varying the cross-linking agent. The use of 1 g
polyethylene glycol as a precipitant for 1 mL of laccase solution and of 200 µM
glutaraldehyde as the cross-linking agent helped to obtain a solid biocatalyst with a laccase
activity of 148 U g -1 and an activity recovery of 60%. The optimal pH and temperature of the
laccase CLEAs were respectively 70°C and 3 compared to 70°C and 2.5 for the free laccase.
The half-life at pH 3 and a temperature of 40°C was 8 hours for the CLEAs and 2 hours for
the free laccase. The addition of bovine serum albumin (BSA) significantly improved the
storage stability of the CLEAs formed. The addition of 1 mg of BSA per unit of laccase
activity improves the storage stability by a factor of 3 after 50 hours comparatively to CLEAs
without BSA. The stability of laccase CLEAs against several denaturants (chelators,
proteases, solvents and salts) was higher than the stability of free laccase. Furthermore, the
Michaelis-Menten kinetic parameters V max of CLEAs (0.021 µM/min) were improved
comparatively to free laccase (0.0042 µM/min) using ABTS as substrate. The affinity
constant, K m , remained the same for CLEAs and free laccase (30 µM).
[1] Duran, N.; Rosa, M. A.; D'Annibale, A.; Gianfreda, L. Applications of laccases and tyrosinases
(phenoloxidases) immobilized on different supports: a review. Enz Microbial Technol2002, 31, 907-931.
[2] Cao, L. Immobilised enzymes: science or art? Curr Opin Chem Biol 2005, 9, 217-226.
[3] Mateo, C.; Palomo, J. M.; van Langen, L. M.; van Rantwijk, F.; Sheldon, R. A. A new, mild cross-linking
methodology to prepare cross-linked enzyme aggregates. Biotechnol Bioeng 2004, 86, 273-276.
78 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P20
Enhanced Stability of Laccase by Xylitol
Andre Zille a , Diego Moldes a , Ramona Irgoliç b , Artur Cavaco-Paulo a
a Department of Textile Engineering, University of Minho, Campus de Azurém, P-4800
Guimarães, Portugal. b Textile Department, Faculty of Mechanical Engineering, University of
Maribor, SI-2000 Maribor, Slovenia.
E-mail: diego@det.uminho.pt
Laccase is a multicopper oxidase able to perform one-electron oxidation of several aromatic
substrates.
The application of laccase on wood delignification, drug analysis, biosensor, wine
clarification, bioremediation, etc., was proposed [1].
As every enzymatic system, laccase has some limitations due to the reaction conditions,
mainly temperature and pH.
Deactivation of laccase at pH values over 6 and lower 3 are undesirable properties that must
be improved. The addition of some compounds is an easy and conventional way to get the
stabilization of laccase [2].
In this work laccase from Trametes hirsuta was studied in order to get its stabilization
towards different pH values by addition of xylitol, a polyol used in food industry with optimal
characteristics with respect to its prize and non-toxical properties.
[1] Mayer, A.M., Staples R.C. Laccase: new functions for an ols enzyme. Photochemistry 60 2002 551-565.
[2] E. V. Stepanova, O.V. Koroleva, V.P. Gvrilova, E.O. Landesman, A. Makower. Comparative stability
assessment of laccases from basidiomycetes Coriolus hirsutus and Coriolus zonatus in the presence of effectors.
Applied Biochemistry and Microbiology 39(5) 2003 482-487
79 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P21
Influence of Static Magnetic Field on Laccase Activity and
Stability
V. Kokol a , M. Schroeder a , G. M. Guebitz b
a Faculty of Mechanical Engineering, Institute of Textiles, University of Maribor, Smetanova
ul. 17, SI-2000 Maribor, Slovenia; b Institute of Environmental Biotechnology, Graz
University of Technology, Petersgasse 12,G-8010 Graz, Austria
E-mail: vanja.kokol@uni-mb.si
Environmental and economical considerations are strong motivation for developing
alternative methods which would intensify redox processes and reduce the consumption of
chemicals. Accordingly, some attempts have been done using physical methods, such as
direct current, ultrasound and electromagnetic treatment. Magnetic water treatment is another
method, which has been beneficially used for scale control in industry water processing for
last two decades [1]. An improvement in the efficiency of microbial growth, i.e. the biological
kinetic parameters, for wastewater treatment by the application of magnetic field was already
shown [2]. In addition, the effect of magnetic field on the catalase and peroxidase activity in
some mixed cultures of cellulolytic fungi was established [3].
In the present work the effect of static magnetic field (SMF) on the activity and stability of
laccases from various Trametes species was investigated. Samples of buffered solutions were
passed (at T = 20 o C and the flow velocity of 1 m/s by various cycles) through a magnetic
device of alternately arranged permanent magnets with magnetic-flux maximums of 0.7 and
0.9 Vs/m 2 . The ABTS-activity and redox potential of laccase at different concentrations (c E =
0.05 and 0.1 g/L) and pH media (pH 3 - 9) were monitored at different temperatures (T = 20 -
70 o C) and compared to the results without the treatment. In addition, the stability of SMF
exposed solutions was determined. Furthermore, the kinetic K M k cat properties on phenolic
substrates guaiacol and dimethoxyphenol were calculated.
[1] Baker JS, Judd SJ: Magnetic Amelioration of Scale Formation. Water Res, 30/2 (1996), 247-260.
[2] Yavuz H, Celebi SS: Influence of magnetic field on the kinetics of activated sludge, Environ Technol, 25/1,
(2004), 7-13.
[3] Manoliu A, Oprica L, et all: Peroxidase activity in magnetically exposed cellulolytic fungi, J Magn Magn
Mater, 300/1 (2006), 323-326
80 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P22
Novel Laccases and Peroxidases for Dye Decolourisation
and Bleaching Processes
A. Matura, K.-H. van Pée
Biochemie, TU Dresden, D-01062 Dresden, Germany
E-mail: anke.matura@chemie.tu-dresden.de
The enzymatic bleaching of cotton by different white rot fungi was investigated and a search
for enzymes participating in the bleaching process was performed [1].
Some of the fungi were found to bleach raw cotton material up to a whiteness of 60
(according to BERGER). Lignin is believed to be predominantly responsible for the yellowbrown
colour of raw cotton material and must therefore be removed during enzymatic
bleaching. Due to the structural similarity of lignin with different industrial dyes, enzymes
from fungi found to have cotton bleaching activity were analysed for the degradation of dyes
like Poly R-478, soluble lignin, remazolic dyes and triphenylmethan dyes as model
compounds [2]. Some of the detected enzymes are able to bleach many of these compounds
and are thus interesting candidates for decolourisation of dying waste water.
Different fungal ligninolytic enzymes e.g. laccases, manganese peroxidases and non- specific
peroxidases were detected in dependence of the growth conditions used. The work was
focussed on laccases. Enzyme production was found to be influenced by the addition of
mediators to the growth medium and various growth conditions such as light, temperature or
oxygen concentration. Purification strategies were developed for the enzymes including ion
exchange, hydrophobic interaction and size exclusion chromatography.
[1] Heine, E., Schuh, E., Daâloul, N., Höcker, H., Breier, R., Schimdt, M., Apitz, A., Brunner, A., van Pée, K.-
H., Scheibner, K. Oxidative Enzyme in der Textilindustrie, Biokatalyse, Sonderausgabe der DBU, Hrsg. S.
Heiden, R. Erb, Spektrum Akad. Verlag, BIOSpektrum, 2001, 49-53
[2] Schuh, E., Heine, E., Daâloul, N., Höcker, H., Breier, R., Mondschein, A., Apitz, A., van Pée, K.-H.,
Scheibner, K. Oxidative Enzyme in der Textilindustrie, transkript Sonderband Biokatalyse, 2003, 119-121
[3] Apitz, A., van Pée, K.-H. Isolation and characterization of a thermostable intracellular enzyme with
peroxidase activity from Bacillus sphaericus. Arch. Microbiol 175, 2001, 405-412
81 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Ralstonia solanacearum Expresses a Unique Tyrosinase
with a High Tyrosine Hydroxylase/DOPA Oxidase Ratio
Diana Hernández-Romero a , Antonio Sanchez-Amat a , Francisco Solano b
P23
a Department of Genetics and Microbiology, Faculty of Biology; b Department of Biochemistry
and Moleuclar Biology, School of Medicine, University of Murcia, Campus de Espinardo,
Murcia 30100, Spain
E-mail: antonio@um.es
Ralstonia solanacearum is a plant pathogenic bacterium infecting solanaceous plants such as
tomato and potato causing wilting and death. Using the available sequence of the genome of
this microorganism, several genes coding putative polyphenol oxidases (PPO) have been
detected. The characterization of the PPO system of R. solanacearum has revealed that at
least three different PPOs are expressed 1 . Using site directed mutagenesis it has been possible
to correlate the genes with the enzymatic activities detected. The products of genes RSc0337
and RSc1501 are enzymes with the typical signatures of tyrosinases including the CuA and
CuB copper binding sites to ligand the type-3 copper pair. On the other hand, gene RSp1530
encodes a laccase.
The PPO system of R. solanacearum has been characterized in terms of biochemical and
molecular properties of the enzymes detected, as well as in relation to its physiological
relevance. Regarding the enzymes similar to tyrosinases, it has been observed that in spite of
a high conservation of the copper-binding sites, they differ in terms of substrate specificity.
For instance, the product of gene RSc1501 encodes an enzyme that oxidizes more efficiently
L-dopa that L-tyrosine, a characteristic typical of most of the tyrosinases described so far. On
the contrary, the product of the gene RSc0337 encodes an unusual tyrosinase with a high
tyrosine hydroxylase/dopa oxidase ratio 2 . The unique catalytic characteristics of this enzyme
will be discussed in relation to other residues present in the active centres, apart from the six
conserved histidines involved in copper binding. First, the relevance of the residue isosteric
with the aromatic F261 present in sweet potato catechol oxidase that may determine the
accessibility to the active site. Second, the presence of a seventh histidine which may interacts
with the carboxylic group on the substrate, hence determining the preference for carboxylated
or non-carboxylated substrates.
The unusual tyrosinase expressed by Ralstonia solanacearum, is an enzyme of interest in
biotechnological processes in which it may be required the oxidation of monophenols to o-
diphenols, since the products generated are not good substrates for a subsequent oxidation to
o-quinones
[1] Hernández-Romero, D., Solano, F. & Sanchez-Amat, A. 2005. Polyphenol oxidase activity expression in
Ralstonia solanacearum. Appl. Environ. Microbiol 71: 6808-6815.
[2] Hernández-Romero, D., Sanchez-Amat, A., & Solano, F. 2006. A tyrosinase with an abnormally high
tyrosine hydroxylase/dopa oxidase ratio. Role of the seventh histidine and accessibility to the active site. FEBS
J. 273: 257-270.
82 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P24
Engineering of a Psychrophilic Microorganism for the
Oxidation of Aromatic Compounds
Rosanna Papa, Ermenegilda Parrilli, Paola Giardina, Maria Luisa Tutino and Giovanni Sannia
Department of Organic Chemistry and Biochemistry, University Federico II, Naples – Italy
E-mail: rosapapa@unina.it
Microbial degradation of aromatic hydrocarbons has been extensively studied with the aim of
developing applications for the removal of toxic compounds from contaminated
environments. Although many pollution problems occur in sea waters and in effluents of
industrial processes which are characterised by low temperatures, considerable effort has been
directed toward the genetic manipulation of mesophilic bacteria to create or improve their
ability to degrade various pollutants.
With the aim to investigate the degradation of aromatic compounds at low temperatures the
Antarctic psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125)
was efficiently used for the production of the recombinant aromatic oxidative activity
encoded by the Toluene-o-Xylene Monooxygenase gene from the mesophilic bacterium
Pseudomonas spp. OX1 [1]. Catalytic performances of PhTAC125 cells expressing ToMO
have been already characterized on different aromatic substrates in various conditions [1].
The genome of PhTAC125 was recently sequenced [2]. Analysis of the annotation of this
genome revealed the presence of a CDS coding for a putative laccase-like protein. Bacterial
laccases have also been reported to be able to oxidize dioxygenated aromatic compounds such
as catechols [3].
The gene coding for PhTAC125 laccase belongs to a gene cluster possibly involved in copper
homeostasis. Preliminary studies demonstrated that this gene is expressed in PhTAC125 cells
only in the presence of copper, as reported for other bacterial species [4].
By using the recombinant capabilities conferred from ToMO enzyme to PhTAC125 and the
endogenous activity due to the presence of the laccase protein we analyzed the catabolic
features of this engineered microorganism. Results prospect the possibility of developing
specific degradative capabilities using this psychrophilic bacterium for the bioremediation of
chemically contaminated marine environments and/or of cold effluents.
[1] Siani, L., Papa, R., Di Donato, A. and Sannia, G. (2006) Recombinant expression of Toluene o-Xylene
monooxygenase (ToMO) from Pseudomonas stutzeri OX1 in the marine Antarctic bacterium
Pseudoalteromonas haloplanktis TAC125. J. Biotechnol. in press
[2] Medigue, C., Krin, E., Pascal, G., Barbe, V., Bernsel, A., Bertin, P.N., Cheung, F., Cruveiller, S., D’Amico,
S., Duilio, A., Fang, G., Feller, G., Ho, C., Mangenot, S., Marino, G., Nilsson, J., Parrilli, E., Rocha, E.P.C.,
Rouy, Z., Sekowska, A., Tutino, M.L., Vallenet, D., von Heijne, G . and Danchin A. (2005) Coping with cold:
the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. Genome
Res. 10, 1325-1335.
[3] Grass, G., Thakali, K., Klebba, P.E., Thieme, D., Muller, A., Wildner, G.F. and Rensing ,C. (2004) Linkage
between catecholate siderophores and the multicopper oxidase CueO in Escherichia coli. J. Bacteriol. 186,
5826-5833.
[4] Brown, N.L., Barrett, S.R., Camakaris, J., Lee, B.T. and Rouch, D.A. (1995) Molecular genetics and
transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004. Mol.
Microbiol. 17, 1153-1166
83 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Spectroscopic Characterization of a Novel Naphthalene
Dioxygenase from Rhodococcus sp.
P25
Maria Camilla Baratto a , David A Lipscomb b , Christopher CR Allen b , Michael J Larkin b ,
Riccardo Basosi a , Rebecca Pogni a
a Dipartimento di Chimica, Università di Siena, Via Aldo Moro 2, 53100, Siena, Italia,
baratto@unisi.it b School of Biological Sciences, Queen’s University Belfast, Medical
Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland c.allen@qub.ac.uk
Polycyclic aromatic hydrocarbons (PAHs), among which naphthalene is an example, are
considered to be potential health risks because of their possible carcinogenic and mutagenic
activities. PAHs have been originated by using fossil and raw materials during the last
century, producing some widespread environmental pollution.
Rhodococcus sp. has been demonstrated to play a significant role in the degradation of PAHs.
The process is catalysed by a class of enzymes called Rieske nonheme iron oxygenases
(ROs), such as 1,2-dioxygenase (NDO) and these enzymes catalyse cis-dihydroxylation
reaction of the substrate. The ability to biodegrade recalcitrant aromatic compounds makes the
system of great importance for bioremediation practices [1].
The catalytic site of dioxygenases consists of two metal centers with a α 3 β 3 structure. The α
subunit is formed of Rieske-type iron sulphur center [2Fe-2S] and one mononuclear iron
center. The amminoacidic residues that ligate the Rieske cluster are two cysteines and two
histidines, while the ligands of the mononuclear ion are two histidines and one bidentate
aspartic acid and a water molecule in a distorted bipyramidal geometry [2,3]. To perform the
catalytic cycle the system requires a NAD(P)H reductase (NDR), containing FAD and a [2Fe-
2S] cluster, an electron transfer protein NDF, containing a Rieske-type cluster and an
oxygenase component (NDO). The overall reaction stoichiometry requires two electrons and
an oxygen molecule to hydroxylate the substrate with an enantio- and regiospecificity
manner.
In this work a novel naphthalene dioxygenases from Rhodococcus sp. [4] has been studied
with EPR spectroscopy at 20K, in order to characterize different iron contributions of the
enzyme in the native state and during the catalytic process in the presence of substrate. NDO
has been analysed in the native state, under reducing conditions in the presence of sodium
dithionite, after the addition of O 2 -saturated naphthalene solution, in order to identify and
assign the different role and involvement of iron centres during the catalytic process. The
peroxide shunt was also tested. These data have been compared to spectrophotometric results.
[1] Wolfe, M.D.; Parales, J.V.; Gibson, D.T.; Lipscomb, J.D.; J. Biol. Chem. 2001, 276, 3, 1945-1953.
[2] Karlsoon, A.; Parales, J.V.; Parales, R.E.; Gibson, D.T.; Eklund, H.; Ramaswamy, S.; Science 2003, 299,
1039-1042.
[3] Gakhar, L.; Malik, Z.A.; Allen, C.C.R.; Lipscomb, D.A.; Larkin, M.J.; Ramaswamy, S.; J. Bacteriology
2005, 187(21), 7222-7231.
[4] Larkin, M.J.; Allen, C.C.R.; Kulakov L.A.; Lipscomb, D.A.; J. Bacteriology, 1999, 181(19), 6200-6204
84 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P26
Identification of Novel Sulfhydryl Oxidases
Vivi Joosten, Willy van den Berg, Sacco de Vries, Willem van Berkel
Laboratory of Biochemistry, Wageningen University,
Dreijenlaan 3, 6703 HA Wageningen, the Netherlands
E-mail: vivi.joosten@wur.nl
Sulfhydryl oxidases (SOX) were recently discovered as being crucially involved in the
generation of disulfide bonds and insertion of these bonds into nascent proteins. They catalyse
the oxidation of (protein) sulfhydryl groups to disulfides with reduction of O 2 to H 2 O 2 . SOX
enzymes are ubiquitously present in eukaryotic species and localized in different cellular
compartments. The FAD cofactor of SOX is non-covalently bound to an unique four-helix
domain that is present as a single-domain in the ERV/ALR family or fused to a thioredoxin
domain in the QSOX family. Enzymes of the ERV1/ALR family can be found within the
inner mitochondrial space (Erv1p or Alr1p) or the fungal and yeast ER (Erv2p). They
generate disulfide bonds de novo and transfer these bonds to their substrate proteins (e.g. PDI
in case of Erv2p), which subsequently transfer these bonds to the next protein substrate to aid
folding. Less information is available about the subcellular location and function of proteins
from the QSOX family, although it was shown that they introduce disulfide bonds directly in
a wide range of unfolded reduced proteins and peptides 1 .
There is a growing interest of industries for the development of biocatalysts aimed at
cross-linking of proteins in food and non-food applications. SOX enzymes are envisaged as
potential candidates for the cross-linking of protein substrates. Aim of our research is to
identify new SOX proteins from plants and fungi that are of interest for applications in food
and pharmaceutical industries. For this aim three putative sox genes present in the
Arabidopsis thaliana genome were cloned and expressed in E. coli Rosetta (DE3)pLysS.
Transformants were analyzed for SOX production.
SOX1 (ERV1/ALR family) was found both in the soluble and in the pellet fraction.
Expression of the full-length protein (~ 22 kDa) was confirmed by LC-MS and
immunodetection of the C-terminal His-tag. The purified SOX1 was redox-active and showed
activity with DTT and thioredoxin. SOX2 and SOX3 (QSOX family) were found as inclusion
bodies. Inclusion bodies were solubilised and about 20mg/L purified protein was obtained.
Refolding of the solubilised proteins will be investigated and Pichia pastoris will be
evaluated as heterologous host. Cross-linking properties of the SOX enzymes will be
evaluated.
[1] Thorpe, C., K. L. Hoober, S. Raje, N. M. Glynn, J. Burnside, G. K. Turi and D. L. Coppock (2002).
"Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes." Arch Biochem
Biophys 405(1): 1-12.
85 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P27
Chlorohydroquinone Monooxygenase - a Novel Enzyme in
the 2,4-dichlorophenoxyacetate Biodegradation Pathway of
Nocardioides simplex 3E – Enzymatic and Genetic Aspects
Jana Seifert, Peter Simeonov, Stefan Kaschabek and Michael Schlömann
Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg,
Germany
E-mail: jana.seifert@ioez.tu-freiberg.de
The herbicide 2,4-dichlorophenoxyacetate (2,4-D) is utilized by the Gram-positive N. simplex
3E as the sole carbon source. Numerous bacteria are known to degrade 2,4-D via orthohydroxylation
of the 2,4-dichlorophenol intermediate to 3,5-dichlorocatechol, which is then
funnelled into an ortho-cleavage pathway.
In N. simplex 3E, 2,4-dichlorophenol is obviously converted by a para-hydroxylating
chlorophenol monooxygenase, which brings about dechlorination of the 2,4-dichlorophenol.
The genes of this two-component enzyme were sequenced and the gene of the oxygenase
compound showed about 60% similarity to TcpA, TftD and HadA. The highly induced
oxygenase was purified and showed relatively low specificity converting 2,6-dichlorophenol,
2,4,5- and 2,4,6-trichlorophenol with high and phenol and 3,4-dichlorophenol with lower
relative activity. The activity could be measured with the addition of a flavin reductase of
Rhodococcus opacus 1CP and FAD.
Chlorohydroquinone (CHQ), which is formed from 2,4-dichlorophenol is ortho-hydroxylated
without dechlorination to 6-chlorohydroxyhydroquinone (6-CHHQ) by a novel
chlorohydroquinone monooxygenase (ChqA). This enzyme is highly specific towards (chloro-
)hydroquinones and converts them to (chloro-) hydroxyhydroquinones. The respective gene,
chqA, is part of the chqRACB gene cluster (acc. No. AY822041), encoding for the already
described hydroxyhydroquinone 1,2-dioxygenase (chqB) as well as for a maleylacetate
reductase (chqC) and a putative AraC-type regulator (chqR).
86 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P28
Cellobiose Dehydrogenases from Ascomycetes and
Basidiomycetes: Phylogenetic and Kinetic Comparison
Roland Ludwig a,b , Marcel Zámocky a,b , Clemens Peterbauer b , and Dietmar Haltrich b
a Research Centre Applied Biocatalysis; Petersgasse 14, 8010 Graz, Austria b Dept. of Food
Sciences and Technology, University of Natural Resources and Applied Life Sciences,
Vienna; Muthgasse 18, 1190 Vienna, Austria
E-mail: clemens.peterbauer@boku.ac.at
The extracellular enzyme cellobiose dehydrogenase (CDH) is involved in fungal cellulose
and/or lignin degradation, albeit with an in vivo function that is not yet fully elucidated. The
enzyme generally consists of a smaller N-terminal domain with heme b as cofactor, a flexible
linker, and a flavin domain containing FAD as cofactor. CDH oxidizes cellobiose and higher
cellooligosaccharides at the anomeric carbon atom to the lactone, which hydrolyzes in an
aqueous environment to the corresponding aldonic acid. Concomitant reduction of a wide
range of different electron acceptors (variously substituted quinones, complexed metal ions,
redox dyes, or even oxygen) in the oxidative catalytic cycle is observed.
Based on currently accessible sequences, CDHs were divided into Class-1, representing CDH
sequences from basidiomycetes, and Class-2 sequences from ascomycetes. Major differences
are a shorter linker sequence and the presence of a carbohydrate-binding module in
ascomycetous CDH.
We screened a number of wood- and lignocellulose-degrading basidiomycetes and
ascomycetes for additional, not yet described enzymes. When using appropriate culture
conditions (induction by cellulose) most of the fungal species tested formed CDH activity.
The widespread occurence of CDH in both wood-rotting and phytopathogenic fungi indicates
an important role of CDH in lignocellulose degradation. CDH from Sclerotium rolfsii,
Trametes spp., Corynascus thermophilus and Myriococcum thermophilum was purified and
characterized to some extent.
Enzymes from these sources are quite comparable with respect to substrate specificity,
molecular mass (86000 – 103000 Da), isoelectric point (3.8 – 4.3), spectral properties, and
post-translational modification (ca. 10 - 15% glycosylation). Significant differences between
ascomycetous and basidiomycetous CDHs were found in the kinetic behaviour and stability.
Generally, ascomycetous CDHs have a tenfold lower K m value for the electron donors than
the basidiomycetous enzymes and a surprisingly low K m for maltose. In contrast to this, the
K m values for some electron acceptors are significantly higher. The pH-optima of
ascomycetous CDHs for some electron acceptors are shifted to the less acidic region, and
temperature stability is generally higher for ascomycetous enzymes, which are mostly
produced by thermophilic/thermotolerant fungal species.
87 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Oxalate Oxidase as a Potential Enzyme Responsible
for H 2 O 2 Generation in Abortiporus biennis
Marcin Grąz, Anna Jarosz-Wilkołazka, Elżbieta Malarczyk
Department of Biochemistry, Maria Curie-Sklodowska University, Sklodowska Square 3,
20-031 Lublin, Poland.
E-mail: mgraz@biotop.umcs.lublin.pl
P29
Fungi classified to group causing white rot are the most efficient wood decomposers. They
secrete an array of oxidases and peroxidases for lignin degradation. The three of them, laccase
(Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP) are considered as the main
enzymes which take a part in this process and extracellular H 2 O 2 is essential as a substrate for
both peroxidases. In this group of fungi there are different possible enzymatic mechanisms for
H 2 O 2 generation in which e.g. glucose oxidase, pyranose oxidase, aryl alcohol oxidase,
methanol oxidase may be involved [1].
Among different low molecular weight compounds involved in initiation of ligninolytic
process, organic acids are important factors. It has been reported that oxalic acid is a
predominant organic acid in wood-rotting fungi cultures. In wood degradation system oxalate
can play role as a proton and electron source, strong metal chelator, factor which stabilize
osmotic potential and pH of fungal growth environment. Oxalic acid can also facilitate
catalitc cycle of MnP by chelating Mn 3+ ions [2]. Oxalate oxidase (OXO) with/or oxalate
decarboxylase (ODC) are responsible for regulation of oxalic acid concentration in fungal
cultures [3].
In the present work novel role for oxalic acid as a factor providing initial concentration of
H 2 O 2 by enzymatic degradation via OXO in Abortiporus biennis cultures is proposed.
Correlation between MnP, Lac activity, H 2 O 2 concentration and secretion and enzymatic
degradation of oxalic acid in Abortiporus biennis liquid cultures are investigated in this study.
[1] Shah and Nerud (2002) Can. J. Microbiol. 48: 857 - 870
[2] Dutton and Evans (1996) Can. J. Microbiol. 42: 881 – 895
[3] Svedruzic et al. (2005) Arch. Biochem. Biophys. 433: 176 – 192
88 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P30
Production, Purification and Molecular Characterisation of
a Quercetinase from Penicillium olsonii
S. Tranchimand, V. Gaydou, T. Tron, C. Gaudin , G. Iacazio
Laboratoire de Bioinorganique Structurale, UMR-CNRS 6517, case 432, Université Paul
Cézanne, Faculté des Sciences de Saint Jérôme, Av. Escadrille Normandie-Niemen, 13397
Marseille Cedex 20, France
E-mail: s.tranchimand@univ-cezanne.fr
Quercetinase is produced by various filamentous fungi when grown on rutin as sole carbon
and energy source. We first investigated on the effect of several phenolics and sugars,
structurally related to substrates and products of the rutin catabolic pathway, on the induction
of a quercetinase activity in Penicillium olsonii. Then we managed the purification of the
extracellular quercetinase and determined physicochemical and kinetic properties. And
finally, we identified the mRNA encoding for the quercetinase using the RACE technology,
based on a previous study on genomic DNA using degenerate PCR.
89 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Laccase Activity Measurements in Turbid or Coloured
Liquids with a Novel Optical Oxygen Biosensor
Christian-Marie Bols, Rob C .A. Onderwater
P31
Wetlands Engineering sprl, Rue du laid Burniat 5, B-1348 Ottignies-Louvain-la-Neuve,
Belgium
E-mail: ch.bols@wetlands.be
We have developed a novel system for measurement of Laccase activity in turbid or coloured
liquids in which the standard colourimetric methods can not be employed.
During its catalytic cycle the Laccase enzyme consumes molecular oxygen. In the past Clarktype
electrodes have been used to monitor oxygen consumption by Laccase, but this requires
complex electronics, is only possible in relatively large sample volumes and has a low
throughput. We have developed an optical oxygen biosensor system that can be used in small
sample volumes and has a high throughput. The system makes use of an oxygen sensitive
fluorophore in an oxygen, but not water of colourant, permeable matrix. The fluorophore in
its matrix can be applied as a coating on the inside of a transparent vessel such as a vial or
microplate well. Upon excitation with blue light the fluorophore emits red light in function of
the presence of oxygen molecules. A decrease in oxygen in the liquid is reflected by a
decrease in oxygen near the fluorophore and results in an increase in fluorescence intensity
and lifetime. Thus the activity of the Laccase enzyme can be measured from the change in
fluorescence of the coating.
90 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P67
Preliminary Study of Soluble Heme Proteins from
Shewanella oneidensis MR1
Bruno Fonseca, Patrícia M. Pereira, Isabel Pacheco, Ricardo O. Louro
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127
Av. da República (EAN), 2781-901 Oeiras, Portugal.
E-mail: bfonseca@itqb.pt
The gram negative bacterium Shewanella is perhaps incomparable in its respiratory
versatility, being able to combine its metabolism to the respiration of a variety of different
electron acceptors, making this genus a potential candidate for application in bioremediation.
To support this versatility Shewanella has an enormous diversity of electron transfer proteins,
having been identified 42 possible cytochrome c genes in its genome sequence, 27 of which
should be soluble [1]. In this work, an engineered strain of Shewanella oneidensis MR-1 was
used. This strain harbours a plasmid (pCS21a) that expresses a soluble derivative of CymA.
The bacteria were grown under two different conditions, aerobic and microaerobic. Of the
several possible soluble cytochromes c produced by the bacteria, four of them have been
accurately identified by N-terminal protein sequence: a small tetraheme cytochrome c, a
monoheme cytochrome c 5, a diheme cytochrome c 4 and a diheme bacterial cytochrome c
peroxidase (bccp). The small tetraheme cytochrome c was also identified using NMR
spectroscopy. Current work involves the purification of these cytochromes for further study
and characterization by UV-Visible and NMR spectroscopy. Detailed knowledge on where
and how these proteins participate in the branched respiratory chain of Shewanella will permit
an enhanced exploitation of these bacteria in bioremediation.
[1] Meyer, T.E., Tsapin, A.I., Vandenberghe, I., de Smet, L., Frishman, D., Nealson K.H.,
Cusanovich, M.A. and van Beeumen, J.J. (2004), Identification of 42 possible cytochrome c genes in
the Shewanella oneidensis genome and characterization of six soluble cytochromes, OMICS 8, 57-77;
91 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P68
Aerobic Oxidation of Alcohols Catalyzed by Laccase from
Trametes versicolor and Mediated by TEMPO
Inga Matijosyte, R.van Kooij, W.C.E. Arends, S. de Vries, R. A. Sheldon
Biocatalysis and Organic Chemistry, Delft University of Technology,
Julianalaan 136, 2628 BL , Delft, The Netherlands
E-mail: i.matijosyte@tnw.tudelft.nl
Laccase-mediator systems that catalyze oxidation of alcohols have drawn increasing attention
in organic synthesis. Nitroxyl radical 2,2,6,6-tetramethylpiperidinyloxy (TEMPO) was shown
to be the most effective mediator of laccase catalyzed oxidation of alcohols. [1, 2] It seems
likely that oxoammonium ions, which can be formed in-situ, are the actual oxidants.
Disadvantage of the laccase-TEMPO system are the long reaction time and the large amounts
of TEMPO (up to 30 mol%) required [3].
R 1 R 2
H
OH
N
O
N
+
O
N
OH + H +
R 1
R 2
O
In order to understand and optimize the system pure enzyme was needed. Therefore, we
performed purification of the fungal laccase from Trametes versicolor in a yield of 73% of
the total units of laccase activity and in a 10-fold purification. This enzyme was used in EPR
studies to monitor the direct sequential electron transfer of TEMPO to laccase. Furthermore,
CLEA’s (cross-linked enzyme aggregates) from laccase were prepared. The results showed
that CLEA could be used as recyclable catalyst for the aerobic oxidation of alcohols.
[1] Viikari L., Kruus K. and Buchert J., (1999) WO 9923117
[2] Baiocco P., Barreca A.M., Fabbrini M., Galli C. and GentiliP. (2003) Org.Biomol.Chemistry, 1,
191-197
[3] Yu-Xin Li, Thesis, Delft, 2004
92 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P69
Role of Laccases in the Decolourisation of Synthetic Dyes
by Aquatic Fungi
Charles Junghanns, Dietmar Schlosser
Department of Environmental Microbiology, UFZ Centre for Environmental Research
Leipzig-Halle, Permoserstrasse 15, D-04318 Leipzig, Germany
E-mail: charles.junghanns@ufz.de
The persistence of most synthetic dyes left unconsumed in textile industry effluents, their
potentially hazardous effects on human health and the environment, and consequently public
demands led to strict environmental regulations, thus enforcing the development of efficient
and cost-effective technologies to cope the problems of effluent treatment. White rot
basidiomycetes represent the group of organisms most frequently considered for oxidative
dye treatment, due to their outstanding capabilities in breaking down a great variety of
different coloured pollutants including synthetic dyes. Fungi other than white rot
basidiomycetes have gained considerably less attention, although bleaching of synthetic dyes
was demonstrated for filamentous ascomycetes, ascomycetous yeasts, and mitosporic fungi,
and also for isolated laccases from filamentous ascomycetes. Aquatic ecosystems represent an
as yet only scarcely explored source of new fungi that are possibly more suitable than other
organisms for the treatment of certain waste waters since the living conditions and hence
possible organismic adaptions found there may better fit to unfavourable characteristics of
process effluents. The ability of fungi derived from aquatic ecosystems to act on recalcitrant
compounds is only rarely explored. We have isolated non-basidiomyceteous fungi from
different surface waters and investigated their ability to decolourise several azo and
anthraquinone type dyes. Concomitantly, laccase activities in fungal liquid cultures were
assessed. Different dyes were found to differentially affect extracellular laccase titers, with
the highest enzyme activities found during decolourisation of the anthraquinone type dye C.I.
Reactive Blue 19. Dye decolourisation was also investigated with isolated laccases. Using
high performance liquid chromatography, profiles of metabolites arising from dye
decolourisation by whole fungal cultures and isolated laccases were recorded and will be
discussed with respect to the contribution of laccases to dye decolourisation by aquatic fungi.
93 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P70
Application of Oxidative Enzymes for the Detoxification of
Xenobiotic Pollutants
Maria Antonietta Rao, Giuseppina Iammarino, , Rosalia Scelza, Fabio Russo, Liliana
Gianfreda
Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università di Napoli
Federico II, Via Università 100. 80055 Portici, Napoli, Italy
E-mail: giusiam@hotmail.com
The environment is continuously enriched by organic substances differing in their chemical
and structural complexity and deriving from both natural and anthropogenic sources. Several
of them having toxic properties may behave as harmful pollutants. Adverse, negative effects
on the environmental and human health may derive.
One of the most effective mechanisms in remediating environments polluted by organic
pollutants is the oxidation by biotic and abiotic catalysts which may occur in natural
attenuation processes or in engineered remediation processes.
Oxidative enzymes such as laccases, tyrosinases and peroxidases are the main effectors of
biotic processes. They differ for some molecular and catalytic characteristics, but all have
been proved to be active towards several organic pollutants.
The main purpose of this paper was to evaluate the catalytic behavour of some of these
catalysts, with particular attention to laccases, when applied to different polluted systems.
Laccase from plant origins showed differentiated efficiencies in transforming polluting
phenolic compounds under various experimental conditions. In particular, the effect of the
initial concentration of the phenolic substance, the repeated addition of fresh enzyme amounts
as well as the presence of more than one phenol and/or pollutant of different chemical nature
(like phenanthrene) in the reaction mixture strongly affected the efficiency of laccase action.
Comparative studies were also performed with fungal laccases and with tyrosinase in both
synthetic and natural phenolic waste waters.
Moreover, the catalytic performance of a peroxidase was assessed in a complex system
simulating a natural situation occurring in soil and rhizosphere soil. A mixture of pyrogallol
or tannic acid, both representative of humic precursors and very abundant in soil and
rhizosphere, were incubated with a plant peroxidase, and the oxidation of the phenolic
compounds (pyrogallol or tannic acid) and the formation and properties of polymeric products
obtained under different experimental conditions, i.e. initial substrate concentration, amount
of peroxidase, incubation time, etc. were evaluated. Further studies were performed in the
presence of phosphatase, a key enzyme very often released extracellularly by plant roots and
catalyzying the hydrolysis of organic phospho-esters in inorganic orthophosphate, the only
form available to plant roots and soil microorganisms. The involvement of the phosphatase in
the process and its residual catalytic efficiency towards a synthetic phosphoric substrate was
assessed as well.
Acknowledgements
This research was supported by Ministero dell’Università e della Ricerca, Italy. Programmi di
Interesse Nazionale PRIN 2004-2005 and by the INCO-MED Program (Contract ICA3-CT-
2002-10033).
94 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P32
The Role of the C-terminal Amino Acids of Melanocarpus
albomyces Laccase
Martina Andberg a , Sanna Auer a , Anu Koivula a , Nina Hakulinen b , Juha Rouvinen b , Kristiina
Kruus a
a
VTT Technical Research Centre of Finland, P.O. Box 1500, Espoo FIN-02044 VTT,
Finland; b Department of Chemistry, University of Joensuu, PO BOX 111, FIN-80101
Joensuu, Finland
E-mail: martina.andberg@vtt.fi
Melanocarpus albomyces is a thermophilic fungus expressing a thermostable laccase with a
pH optimum in a neutral pH region with phenolic substrates. These properties make the M.
albomyces laccase (MaL) an interesting enzyme for many applications. The three-dimensional
structure of MaL has been solved as one of the first complete laccase structures [1].
The C-terminus of the secreted M. albomyces laccase is processed after an amino acid
sequence DSGL. The processing site is conserved among some ascomycete type of laccases
and the cleavage take place between the leucine and the following lysine residue. According
to the crystal structure of MaL, the four C-terminal amino acids of the mature protein
penetrate into a tunnel in the protein [1]. The C-terminal carboxylate group makes a hydrogen
bond to a side chain of His 140, which also coordinates to the T3 type copper in the trinuclear
center. In order to analyse the role of the processed C-terminus, site-directed mutagenesis of
the M. albomyces laccase cDNA was performed, and the mutated proteins were expressed in
Saccharomyces cerevisiae.
The mutated enzymes were purified to homogeneity from the yeast culture supernatant and
the effect of the C-terminal mutations on the protein properties of the enzyme e.g. the specific
activity and kinetic parameters were analyzed. Moreover, the three-dimensional structure of
one of the mutants was determined. The biochemical characterization of the mutant protein
will be presented as well as the structural data of the mutant laccase.
[1] Hakulinen, N., Kiiskinen, L. L., Kruus, K., Saloheimo, M., Paananen, A., Koivula, A. & Rouvinen, J. 2002.
Nature Struct. Biol. 9, 601-605
95 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P33
Shifting the Optimal pH of Activity for a Laccase from the
Fungus Trametes Versicolor by Structure-Based
Mutagenesis
C. Madzak a , M.C. Mimmi b , E. Caminade c , A. Brault c , S. Baumberger b , P. Briozzo b , C.
Mougin c , C. Jolivalt d
a UMR Microbiologie et Génétique Moléculaire, INRA / CNRS / INA-PG, CBAI, 78850
Thiverval-Grignon, France. b UMR INRA-INAPG 206 de Chimie Biologique, 78850
Thiverval-Grignon, France. c Unité de Phytopharmacie et Médiateurs Chimiques, INRA, route
de Saint-Cyr, 78026 Versailles Cedex, France. d Laboratoire de Synthèse sélective organique
et produits naturels, UMR CNRS 7573, ENSCP, 11, rue Pierre et Marie Curie, 75231 Paris
Cedex 05, France.
E-mail: claude-jolivalt@enscp.fr
Laccases are multicopper oxidases used in industrial oxidative processes, with potential
applications in depollution (Mougin 2003). The design of recombinant laccases fully adapted
to industrial applications will be possible using genetic engineering. Y. lipolytica expression
system enables high transformation efficiency, as well as control of both copy number and
integration locus of transformants. The successful production of active Trametes versicolor
laccase (Jolivalt 2005) has been a preliminary step towards engineering this enzyme for
environmental applications.
Crystal structure of T. versicolor laccase (Bertrand 2002) enlighted the interaction of amino
acid 206 (Aspartate) with the substrate. This Aspartate is conserved among laccases from
basidiomycetes. We tested the effects of its replacement by Glutamate (conserved among
ascomycetes), Asparagine (conserved among plants), or Alanine. Mutated recombinant
laccases were expressed in Y. lipolytica, using an expression/secretion vector (Madzak 2000),
which allows the precise targeting of monocopy integration events at a docking platform into
the recipient strain genome. This system reproducibly provides transformants carrying a
unique expression cassette, integrated at a precisely known site. We were thus able to analyze
the consequences of each mutation on laccase activity on various substrates.
[1] Mougin, Environ.Chem.Lett. 2003, 1, p.145
[2] Jolivalt, PEDS 2006, 19(2), p.77
[3] Bertrand, Biochem. 2002, 41, p.7325
[4] Madzak, JMMB 2000, 2, p.207
96 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P34
Axial Perturbations of the T1 copper in the CotA-Laccase
from Bacillus subtilis: Structural, Biochemical and Stability
Studies
Paulo Durão a , Isabel Bento a , André T. Fernandes a , Eduardo P. Melo b , Peter F. Lindley a and
Lígia O. Martins a
a Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. Da
República, , 2784-505 Oeiras, Portugal, b Center of Molecular and Structural Biomedicine,
Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
E-mail: pdurao@itqb.unl.pt
The catalytic rate-limiting step in laccases is considered to be the oxidation of substrate at the
T1 copper site, most probably controlled by the redox potential difference between this site
and the trinuclear site. Redox potentials exhibited by laccases span a broad range of values
from 400 mV for plant laccases to 790 mV for some fungal laccases. The conserved
coordinating amino acids for the T1 copper site are two histidines and a cysteine, and the
natural variations occur in the so-called axial position with a single interaction from a Met
being the most common arrangement. Fungal laccases have the non-coordinating Phe or Leu
at this position and these may contribute, at least partially, for the higher E o observed in these
enzymes, although other elements of the protein matrix are known to affect this important
parameter of the T1 Cu center.
Site-directed mutagenesis has been used to replace Met-502 in CotA-laccase by the residues
leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with
the Wt CotA shows that the geometry of the T1 copper site is maintained as well as the
overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site
leads to an increase in the redox potential by ~100 mV relative to the Wt enzyme (E o =
455mV). No direct correlation was found between the redox potentials calculated for the
mutant enzymes and the oxidation rates of the substrates tested. The M502L mutant exhibits a
2-4 fold decrease in the k cat values for all substrates tested and the catalytic activity in M502F
is even more severely compromised; 10% activity and 0.15-0.05% for the non-phenolic
substrates and for the phenolic substrates tested, when compared with the Wt enzyme. T1
copper depletion is a key event in the inactivation and thus it is a determinant of the
thermodynamic stability of Wt and mutant proteins. However, whilst the unfolding of the
tertiary structure in the Wt enzyme is a two state process displaying a mid point at a
guanidinium hydrochloride concentration of 4.6M and a free energy exchange in water of
10kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9M
guanidinium hydrochloride, half of the molecules are at an intermediate conformation, only
slightly less stable than the native state (~ 1.4 kcal/mol). The T1 copper center clearly plays a
key role, from the structural, catalytic and stability viewpoints in the regulation of CotAlaccase
activity.
97 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P35
Structural Studies in CotA Mutants: Understanding of the
Protonation Events that occur during Oxygen Reduction to
Water
Isabel Bento, Paulo Durão, André T. Fernandes, Lígia O.Martins and Peter F. Lindley
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa,
Av. da República, EAN, 2784 - 505 Oeiras, Portugal
E-mail: bento@itqb.unl.pt
Laccases are enzymes that are able to couple substrate oxidation with the reduction of
dioxygen to water. They belong to the multicopper oxidase family and show at least two
different types of copper centre; a mononuclear T1 centre and a trinuclear centre that
comprises two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear
centre whereas reduction of molecular oxygen to water occurs at the tri-nuclear centre. Using
the CotA laccase as a model system, we have recently proposed a putative mechanism for
oxygen reduction for this type of enzyme [1]. In the present work we have tried to increase
our understanding of such a mechanism and have determined the three dimensional structure
of three different mutants of glutamate 498. This residue interacts indirectly, through a water
molecule, with a dioxygen moiety bound in between the two T3 copper atoms. It has been
proposed to play a key role in the protonation events that occur during the mechanism.
Indeed, this study not only shows the relevance of this residue in protonation but also the
importance of its presence in the stabilisation of the whole trinuclear centre.
[1] Bento, I. Martins, L.O., Gato, G.L., Carrondo, M.A., and Lindley, P.F. (2005) Dalton Transactions
21, 3507-3513
98 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P36
Relationship of Substrate and Enzyme Structures as a Basis
for Intradiol Dioxygenases Functioning
Kolomytseva M.P. a , Ferraroni M. b , Scozzafava A. b , Briganti F. b , Golovleva L. a
a G.K. Skryabin Institute of biochemistry and physiology of microorganisms RAS, Pushchino,
Russia and b Laboratorio di Chimica Bioinorganica, Universita degli Studi di Firenze, Italy,
E-mail: golovleva@ibpm.pushchino.ru
During the biodegradation a large variety of the natural compounds and xenobiotics is
converted into a small number of central intermediates, containing two adjacent hydroxylic
groups in the aromatic ring: protocatechate, catechol, chloro- and dichlorocatechols, hydroxyand
chlorohydroxyquinols. One of the ways of the following degradation of such
intermediates is intradiol cleaving of the aromatic ring with incorporation of both atoms of
molecular oxygen into substrate catalyzed by non-heme Fe(III)-dependent decyclizing
intradiol dioxygenases. According to physiological substrate, intradiol dioxygenases differ in
physicochemical properties. At this time 3D-structures for seven intradiol dioxygenases are
reported [1-6].
More detailed investigation of R. opacus 1CP chlorocatechol 1,2-dioxygenases
(CCDOs) kinetic data was performed using variable substrate analogs modeling different
ways of substrate binding in the active site that achieved by modification of nature and
quantity of the reaction groups and additional insertion into substrate aromatic ring of various
nature and quantity of substituents. Structure properties and reactivity of used substrates and
substrate analogs and their influence on the enzymes functioning were studied using
computational methods in quantum chemistry. Based on the enzyme kinetic properties and the
substrate analogs reactivity it is shown that the binding of the last ones in the active sites of
the enzymes is determined by the character of interactions resulting between substituent in
substrate analog molecule and interior surface of active center. It is determined that catalytic
process directly depends on the value of oxygen charge of the first hydroxylic group of
substrate. Calculated order of deprotonation of adjacent hydroxylic groups of substrate agrees
with earlier known binding order of substrate molecule with Fe 3+ of intradiol dioxygenases
active site. Performed comparative structure/function analysis of CCDOs and other known
structure intradiol dioxygenases showed that the differences in the substrate specificity of
enzymes can be caused by corresponding changes in aminoacid composition of enzyme active
centers and their entrances.
This work was supported by grants RFBR 050449659 and Naukograd-RFBR 040497266.
[1] Orville A.M., Lipscomb J.D., Ohlendorf D.H. 1997 Biochemistry, V.36, pp. 10052-10066.
[2] Vetting M.W., D’Argenio D.A., Ornston L.N., Ohlendorf D.H. 2000 Biochemistry, V.39, N27, pp. 7943-
7955.
[3] Vetting M.W., Ohlendorf D.H. 2000 Structure, V.8, pp. 429-440.
[4] Earhart C.A., Vetting M.W., Gosu R., Michaud-Soret I., Que L.Jr., Ohlendorf D.H. 2005 Biochem. Biophys.
Res. Commun., V.338, pp. 198-205
[5] Ferraroni M., Seifert J., Travkin V.M., Thiel M., Kaschabek S., Scozzafava A., Golovleva L., Schlömann M.,
Briganti F. 2005 J.Biol.Chem., V.280, pp. 21144-21154.
[6] Ferraroni M, Solyanikova IP, Kolomytseva MP, Scozzafava A, Golovleva LA, Briganti F. 2004 J Biol Chem.,
V. 279, pp. 27646-27655.
99 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P37
Surface-Enhanced Vibrational Spectroelectrochemistry of
Immobilized Proteins
Smilja Todorovic a , Peter Hildebrandt b and Daniel Murgida b
a Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa,
Av. da República (EAN, 2784 - 505 Oeiras, Portugal; b Technical University of Berlin,
Strasse des 17. Juni 135, D-10623 Berlin, Germany
E-mail: smilja@itqb.unl.pt
The combination of surface-enhanced vibrational spectroscopy with electrochemical
techniques provides a set of powerful tools for the investigation of structural, thermodynamic
and kinetic aspects of metalloproteins.
Surface-enhanced resonance Raman spectroscopy (SERRS) of heme proteins probes the
redox active sites with high selectivity and sensitivity, yielding detailed information on
coordination, redox and spin state. This method requires an immobilized sample on
nanoscopically roughened metal surface coated with biocompatible material in order to
preserve the protein native structure upon adsorption.
One of the most versatile approaches for generation of biocompatible coatings, particularly
suitable for soluble proteins, is based on the self-assembly of ω-functionalized alkanethiols on
Ag and Au surfaces [1]. We have employed this strategy for studying electric field effects on
the structure and redox potential of cytochrome P450 cam . Potential-dependent SERR
measurements revealed modulation of the redox potential of the adsorbed enzyme by
interplay of two opposing effects.
Immobilization of membrane proteins requires a different strategy in order to preserve the
physiological hydrophobic environment. In some cases, solubilized proteins can be directly
adsorbed on a “bare” Ag electrode without displacement of detergent which thus provides a
biocompatible interface [2]. Using this strategy, we were able to determine, by potentialdependent
SERR, the individual midpoint potentials and Coulombic interactions in the
multiheme proteins such as quinol oxidase from A. ambivalens and succinate dehydrogenase
from R. marinus.
Some other membrane complexes, like the cbb 3 terminal oxidase from B. japonicum, require
immobilization conditions that mimic more closely the membrane-like environment. SERRS
of cbb 3 , attached via a His-tag to an electrode coated with Ni (or Zn) nitrilo triacetate (Ni-
NTA), show reversible electrochemistry. The high affinity of the Ni-NTA monolayer towards
the His-tag guarantees a large surface coverage of uniformly oriented proteins even at
relatively high ionic strengths similar to physiological conditions. The anchored enzyme is
then incubated in the presence of lipids and biobeads in order to remove the solubilizing
detergent and allow the formation of a lipid bilayer [3].
[1] Murgida, D. and Hildebrandt, P. (2004) Acc. Chem Res. 37, 854-61.
[2] Todorovic, S., Pereira, M., Bandeiras, T., Teixeira, M., Hildeebrandt, P., Murgida, D. (2005) J. Am. Chem.
Soc. 127, 13561.
[3] Friedrich, M., Giess, F., Naumann, R., Knoll, W., Ataka, J., Heberle, J., Hrabakova, J., Murgida, D.,
Hildebrandt, P. (2004) Chem. Comm. 21, 2376.
100 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P38
Enzymatic Properties, Stability And Model Structure of a
Metallo-Oxidase from the Hyperthermophile Aquifex
aeolicus
André T. Fernandes a , Cláudio M. Soares a , Manuela M. Pereira a Robert Huber b , Gregor Grass c ,
Eduardo P. Melo d and Lígia O. Martins a
a Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. Da
República, , 2784-505 Oeiras, Portugal, b Lehrstuhl fur Mikrobiologie und Archaeenzentrum,
Universitat Regensburg, Germany, , c Institute for Microbiology, Marthin Luther University,
Halle, Germany and the d Center of Molecular and Structural Biomedicine, Universidade do
Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
E-mail: andref@itqb.unl.pt
The Aquifex aeolicus AAC07157.1 gene encoding a multicopper oxidase (McoA) and
localized on the genome as part of a putative copper-resistance determinant, was cloned,
overexpressed in Escherichia coli, and purified to homogeneity. The isolated enzyme shows
spectroscopic and biochemical characteristics of well-characterized multicopper oxidases.
McoA presents poor catalytic efficiency (k cat /K m ) towards aromatic substrates but a
remarkable high for cuprous and ferrous ions, close to 3 x 10 6 s -1 M -1 . This robust activity is
30- to 100-fold higher than that of metallo-oxidases CueO from E. coli, yeast Fet3p or human
ceruloplasmin. Addition of copper is required for maximal catalytic efficiency. A striking
structural feature in the McoA comparative model structure is the presence of a nonhomologous
methionine-rich segment comparable to ones present in copper homeostasis
proteins. The role of this segment in the McoA catalytic mechanism has been examined using
deletion mutagenesis to obtain recombinant McoA∆P 321 -V 363 . The kinetic properties of this
mutant enzyme when compared to the wild type provide evidence for the key role of this
region in the modulation of the catalytic mechanism, presumably through copper binding.
McoA is a thermoactive (optimal temperature of 75ºC) and hyperthermostable enzyme with a
three-domain thermal unfolding characterized by temperatures values at the mid-point of 105,
110 and 114ºC. Interestingly, the stability of McoA at room temperature is very low (2.8
kcal/mol) showing that the mechanism of thermostability relies on a flat dependence of
stability on temperature. McoA probably plays a crucial in vivo role in copper and iron
homeostasis.
101 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P39
Degradation of Azo Dyes by Trametes villosa Laccase
under Long Time Oxidative Conditions
Andrea Zille a , Barbara Górnacka b , Astrid Rehorek b Artur Cavaco-Paulo a
a University of Minho, Department of Textile Engineering, 4800-058 Guimarães, Portugal;
b University of Applied Sciences Cologne, Institute of Chemical Engineering and Plant
Design, Betzdorfer Str. 2, D-50679 Cologne, Germany
E-mail: azille@det.uminho.pt
Trametes villosa laccase was used for direct azo dye degradation for which the reaction
products were analyzed over long periods of time. Laccases have been extensively studied for
the degradation of azo dyes [1-6].These enzymes are multi-copper phenol oxidases that
decolorize azo dyes through a highly non-specific free radical mechanism forming phenolic
type compounds, thereby avoiding the formation of toxic aromatic amines [7,8].In the
literature, there are a large number of papers reporting on decolorization of azo dyes however
the fate of the products of azo dye laccase reactions is ignored [9-12]. Therefore, the purpose
of this work is the study of the azo dye degradation products in the presence of laccase. Direct
azo dye laccase degradation and amino-phenols polymerization was performed for several
days. The formed soluble products were studied by LC-MS while the polymerized insoluble
products were studied by 13 C -NMR. LC-MS analysis shows the formation of phenolic
compounds in the dye oxidation process as well as a large amount of polymerized products
that retain the azo group integrity. The amino-phenols reactions were also investigated by 13 C-
NMR and LC-MS analysis and the real polymerization character of laccase enzymes was
shown. This study highlights the fact that laccases polymerize the reaction products obtained
in long time batch decolorization processes of the azo dyes. These polymerized products
provide unacceptable color levels in effluents limiting the application of laccases as
bioremediation agents.
[1] Adosinda, M., M. Martins, N. Lima, A. J. D. Silvestre, and M. J. Queiroz. 2003. Chemosphere. 52:967-973.
[2] Blanquez, P., N. Casas, X. Font, X. Gabarrell, M. Sarra, G. Caminal, and T. Vicent. 2004. Water Res.
38:2166-2172.
[3] Maximo, C., and M. Costa-Ferreira. 2004. Proc. Biochem. 39:1475-1479.
[4] Novotny, C., K. Svobodova, A. Kasinath and P. Erbanova. 2004. Int. Biodeterior. Biodegrad. 54:215-223.
[5] Peralta-Zamora, P., C. M. Pereira, E. R. L. Tiburtius, S. G. Moraes, M. A. Rosa, R. C. Minussi, and N. [1] [1]
[6] Duran. 2003. Appl. Catal. B: Environ. 42:131-144.
[7] Wesenberg, D., I. Kyriakides, and S. N. Agathos. 2003. Biotechnol. Adv. 22:161-187.
[8] Wong, Y., and J. Yu. 1999. Wat. Res. 33:3512-3520.
[9] Chivukula, M., and V. Renganathan. 1995. Appl. Environ. Microbiol. 61: 4347-4377.
[10] Chagas, P. E., and R. L. Durrant. 2001. Enzyme Microb. Technol. 29:473-477.
[11] Jarosz-Wilkolazka, A., J. Kochmanska, E. Malarczyk, W. Wardas, and A. Leonowicz. 2002. Enzyme [1]
Microb. Technol. 30:566-572.
[12] Robinson, T., B. Chandran, and P. Nigam. 2001. Enzyme Microb. Technol. 29:575-579.
102 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P40
Enzymatic Decolorization of Azo and Anthraquinonic Dyes
with the CotA-Laccase from Bacillus subtilis
Luciana Pereira a , Lígia O. Martins a
a
Instituto de Tecnologia Química e Biológica (ITQB),Universidade Nova de Lisboa, Av da
República, 2784-505 Oeiras, Portugal
E-mail: luciana@itqb.unl.pt
Purified recombinant CotA-laccase from Bacillus subtilis was tested on its ability to degrade
azo and antraquinonic dyes in the absence and presence of redox mediators (ABTS, VA and
HBT). Eleven different dyes were tested, three anthraquinone and eight azo dyes. All dyes
tested were, at a different extent, oxidatively bleached by 1U.mL -1 of CotA-laccase in the
absence of mediators. Decolourisation was shown to be pH-dependent, being maximal at the
alkaline range of pH (pH 7-9). Reactive Black 5 (RB5), Acid Blue 62 (NY3), Direct Black 38
(DB38) and Reactive Red 4 (RR4) were selected for detailed studies. The time course for
degradation of these dyes was followed in the presence and absence of mediators.
Decolourisation proceeds following a first order kinetics presenting a maximal rate of
degradation in the presence of ABTS, with an increase of 4.5 fold for RB5, 2.5 for DB38 and
2 for NY3 and RR4 comparatively with the reaction in absence of mediators. The level of dye
decolourisation at the equilibrium was found to be independent of the presence of mediators
(90, 80, 60 and 40% degradation for RB5, NY3, CB and RR4, respectively).
This work has been done in the frame of EC-F6P SOPHIED project - “Novel Sustainable Processes for the
European Colour Industries” (FP6-NMP2-CT-2004-505899).
103 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P41
Selection of Laccases with Potential for Decolourisation of
Wastewater Issued from Textile Industry
E. Enaud, M. Trovaslet, M. Pamplona-Aparicio, A-M. Corbisier, S. Vanhulle
Microbiology Unit, Université catholique de Louvain, Place Croix du Sud 3 bte 6, B-1348
Louvain-la-Neuve, BELGIUM,
E-mail: vanhullesophie@hotmail.com
Development of a bioreactor for wastewater treatment requires the selection of an adapted
biocatalyst. Laccases proved efficient against dyes present in wastewaters issued from textile
industry. However, they may be sensitive to several denaturing agents found in dye-baths
such as high salt concentrations, temperature, high or low pH. In the perspective of an
industrial application of fungal laccases, the influence of these parameters was studied on
activity and stability of 3 laccases concentrates from different white rot fungi (PT32, PO33
and PS7).
PT32 and PO33 laccases were not stable at ambient temperature as well as in presence of
NaCl concentrations higher than 128 mM, while PS7 laccase showed promising results and
interesting potential of decolourisation. This laccase was further studied.
In partnership with numerous textile industry, a survey of the effluent compositions was made
and model dye baths were designed to mimic acid-, reactive- and direct-dye wastewaters.
Both model dyes and model wastewaters were treated by isolated laccases. Amongst model
dyes, acid dyes were the most sensitive to PS7 laccase activity. Model acid effluents were
also efficiently decolourised. The influence of individual parameters on laccase activity was
investigated in the simulated wastewater conditions. Dyes showed a strong effect on laccase
stability while pH and salt concentration showed less influence.
The rather good stability of PS7 laccase combined with its high potential of decolourisation
suggest that PS7 laccase may be efficiently exploited in a variety of biotechnological
applications including the wastewater treatment.
104 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P42
Decolorization of Textile Dyes by the White-Rot Fungus
Coriolopsis polyzona MUCL 38443
Aisle Ergun, Firuze Basar, S. Koray Yesiladalı, Z. Petek Çakar Öztemel, Candan Tamerler
Behar
İstanbul Technical University, Department of Molecular Biology and Genetics, Maslak-
İstanbul, 34469, Turkey
E-mail: asl_ergun@yahoo.com
Ligninolytic enzyme-producing white-rot fungus Coriolopsis polyzona was investigated for
its textile dye decolorization potential. C. polyzona is a fast growing and laccase-producing
white-rot fungus. Laccase is an extracellular oxidoreductase produced abundantly by C.
polyzona, which can be exploited for decolorization of dyes.
Decolorization effect of C. polyzona was investigated for azo dyes which are the largest class
of dyes in textile industry. Similar to many other aromatic pollutants, neither the activated
sludge nor aerobic bacterial isolates can fully degrade azo dyes and thus effluent treatment
becomes a serious issue because of their negative impact on water ecosystems and human
health. Here we investigated four different azo dyes, Remazol Brilliant Blue and Remazol
Black 5, Reactive Red 195 and Remazol Turquoise. Based on spectrophotometric
measurements of culture supernatants at the beginning and the end of the cultivations (7
days), C. polyzona was able to decolorize 90% of Remazol Black 5, 95% of Remazol Brilliant
Blue, 82% of Remazol Turquoise and 73% of Reactive Red 195 where the initial
concentration of each dye in the liquid culture was 50 mg/L. Results indicate that C. polyzona
has a potential for exploitation in industrial dye decolorization studies.
This study is funded by EU 6th Framework Integrated Project (IP), ‘SOPHIED - Novel
sustainable bioprocesses for the European colour industries’.
105 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Laccase from Trametes versicolor Immobilised on Novel
Composite Magnetic Particles
P43
K.-H. van Pée a , A. Matura a , T. Wage a , A. Pich b , U. Böhmer c
a Biochemie;
b Makromolekulare Chemie und Textilchemie;
Bioverfahrenstechnik, TU Dresden, D-01062 Dresden, Germany
E-mail:karl-heinz.vanpee@chemie.tu-dresden.de
c Lebensmittel
und
For use of enzymes in bioremediation, it is of great importance to keep costs for the enzymes
as low as possible. This can be achieved by stabilisation and reuse of the enzyme. For this
purpose, immobilisation of the enzyme is of great advantage. Polymeric particles which are
used as carriers can be produced in a number of different sizes and morphologies.
Additionally, the surface layer can be modified by a variety of functional groups located on
certain distances from the particle core. This provides sufficient flexibility in terms of enzyme
immobilisation and further technical applications. We report on the study of laccase
immobilisation on different kinds of carrier particles. The immobilisation of the enzyme on
the particle surface with respect to the immobilisation efficiency and properties of the
immobilised enzyme is discussed. The immobilisation of laccase on polystyrene particles
bearing reactive β-diketone groups is characterised by high efficiency, but grafting of the
enzyme increases the stability of the colloidal system which has a negative influence on the
separation/purification procedure. Additionally, the extreme colloidal stability of the
immobilisates makes the application of such particles impossible when recycling of enzyme
should be performed. It has been found that hybrid polystyrene-acetoacetoxyethyl
methacrylate (PS-AAEM) particles equipped with magnetite show similar immobilisation
efficiency as their analogues without magnetite and additionally can be manipulated in a
magnetic field. The activity of the immobilised laccase is much higher in the pH region 5 - 7
and temperature range of 50 - 70° C when compared with free enzyme. Additionally,
immobilised enzymes exhibit also much better storage stability. In future work, porous
microgels will be used. They have the same properties as the compact polystyrene particles,
but in addition they provide a structure-based enzyme stabilisation, especially against
mechanical stress. Hybrid carriers with immobilised laccase were used for the biobleaching of
dyes used in the textile industry. The efficiency of the immobilised enzyme in bleaching of
different dye molecules was examined by means of UV-vis spectroscopy with samples of
waste-water from textile industry. Further candidates for biobleaching of dyes were found in
other fungi.
106 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P44
Biotechnological Applications of a pH-Versatile Laccase
from Streptomyces ipomoea CECT 3341
J.M. Molina, R. Moya, F. Guillén, M. Hernández, M.E.Arias
Departamento de Microbiología y Parasitología. Universidad de Alcalá (Madrid). Spain.
E-mail: enriqueta.arias@uah.es
During last years, fungal laccases have received great attention in biotechnological
applications. In fact, the enhancement of its oxidation capability throughout the action of
redox mediators allows the development of new strategies for degradation of xenobiotics
compounds, pulp delignification, textile dyes bleaching, etc (1, 2, 3). Although several
bacterial laccases have been recently described (4, 5) its biotechnological usefulness has not
been established yet. In this sense, our group has described the potential application of a
laccase produced by Streptomyces cyaneus CECT 3335 for biobleaching of eucalyptus kraft
pulp (6). Recently, we have purified a new laccase produced by S. ipomoea CECT 3341
which shows some different physico-chemical characteristics compared with that produced by
S. cyaneus. In fact, substrate specificity of this laccase depends on the pH, (i.e. optimal pH for
ABTS or phenolic compounds are 4,5 or 8, respectively). We suggest this pH versatile laccase
enlarges the range of biotechnological applications of these enzymes preventing the limitation
of some other laccases which are active only at low pH.
In the present work we screen the potential application of the laccase produced by S. ipomoea
and different mediators for the biobleaching of eucalptus kraft pulp and for decolourisation
and detoxification of a textile azo-type dye.
The treatment of eucalyptus kraft pulp was carried out with 300 mU laccase per gram of pulp
in the presence of 1 mM ABTS as mediator in acetate buffer pH 4.5 to get a 10% (w/v)
consistency. Enzyme treatment was maintained at 60°C for 1 hour followed by a bleaching
step with 2% H 2 O 2 . Results obtained showed a 10% decrease in Kappa number, a 3.5 %
increase in ISO brightness and a remarkable saving in H 2 O 2 consumption.
On the other hand, application of LMS to decolourise textile dyes requires non-chromogenic
mediators and up to date best results were obtained with phenolic compounds related with
lignin. For this study, best results to decolourise an azo-type dye (Reactive Green) were
obtained with 300 mU laccase and 0.1 mM acetosyringone as mediator. With this LMS
system, a 90% decolourization was achieved. Analysis of toxicity after the treatment
(Microtox ® System) also showed a high degree of detoxification (more than 50 % increase in
EC).
[1] Collins, P.J., Kotterman, M.J.J., Field, J.A. and Dobson, A.D.W. (1996). Appl. Environ. Microbiol. 62: 4563-
4567.
[2] Bourbonnais, R and Paice, M.G. (1996). TAPPI J. 79: 199-204.
[3] Camarero, S. Ibarra, Martinez, M.J. and Martinez, A.T. (2005). Appl. Environ. Microbiol. 71: 1775-1784.
[4] Martins, L.O., Soares, C.M., Pereira, M.M., Texeira, M., Costa, T., Jones, G.H. and Henriques, A.O. (2002).
J. Biol. Biochem. 277: 18849-18859.
[5] Solano, F., Lucas-Elio, P., Lopez-Serrano, D,. Fernandez, E., and Sanchez-Amat, A. (2001). FEMS
Microbiol. Lett. 204: 175-181.
[6] Arias, M.E., Arenas, M., Rodriguez, J., Soliveri, J., Ball, A.S. and Hernández, M. (2003). Appl. Environ.
Microbiol. 69: 1953-1958.
107 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Oxidative Reactions for the Decolorization of Synthetic
Dyes – Laccase versus Fenton’s Reagent
P.F.F.Amaral, F.V. Pinto, M.C. Cammarota, M.A.Z. Coelho
P45
Departamento de Engenharia Bioquímica, Escola de Química/UFRJ, Centro de Tecnologia,
Bl.E, lab.113, Rio de Janeiro - RJ, 21949-900, Brasil.
E-mail: alice@eq.ufrj.br
The wastewater from the textile industry is known to be strongly colored, presenting large
amounts of suspended solids, pH broadly fluctuating, high temperature, besides high chemical
oxygen demand (COD) [1]. Physical and chemical methods such as adsorption, coagulationflocculation,
oxidation, filtration, and electrochemical methods may be used for wastewater
decolorization. Chemical oxidation methods can result in almost complete mineralization of
organic pollutants and are effective for a broad range of organics. The oxidation with
Fenton’s reagent based on ferrous ion and hydrogen peroxide is a proven and effective
technology for destruction of a large number of hazardous and organic pollutants. Over the
past decade, white rot fungi have been studied for their ability to degrade recalcitrant organopollutants
such as polyaromatic hydrocarbons, chlorophenols, and polychlorinated biphenyls
[2]. The low specificity of the lignin-degrading enzymes produced by these fungi suggests
that they may be suitable for the degradation of textile dyeing wastewater. Trametes
versicolor releases laccase as its major extracellular enzyme, a copper-containing polyphenol
oxidase (benzenediol: O 2 oxidoreductase, EC 1.10.3.2) which catalyses the oxidation of
phenolic compounds [3]. Laccase can also catalyses the oxidation of organic pollutants
through molecular oxygen reduction, even in the absence of hydrogen peroxide [4]. In the
present work two different oxidation approaches were investigated for the decolorization of
synthetic wastewater, the chemical oxidation with Fenton’s reagent and an enzymatic
oxidation with laccase produced by T. versicolor. The utilization of Fenton (H 2 O 2 + Fe 2+ ) was
accomplished by two experimental design techniques, observing three variables (reaction
time, Iron II concentration, and H 2 O 2 concentration) under two levels, keeping stable
conditions of pH, temperature of 30ºC as well as the dye concentration of 167 mg/L. So the
variables were optimized till the color removal efficient achieved 96%. For the enzymatic
treatment, it was studied not only the decolorization of a synthetic wastewater but also faces
the problem of dealing with a real dyeing wastewater [5]. Decolorization of synthetic and real
wastewaters were performed by Trametes versicolor. A decolorization of 97% was achieved
for initial dye concentrations up to 100 mg/L. The pH and the presence of glucose were
identified as important parameters for an adequate decolorization performance. For a real
wastewater, decolorization reached efficiencies of about 92% in a diluted system
(approximately 50 mg dye/L). The results reported in this study showed that both treatments
were efficient for decolorization and the choice for industrial applications may consider
economic and safety aspects.
[1] Robinson, T., Chandran, B. and Nigam, P. Water Res., 36, 2824–2830 (2002).
[2] Reddy, C.A. Curr. Opin. Biotechnol., 6, 320–328 (1995).
[3] Swamy, J. and Ramsay, J.A. Enzyme Microbiol. Technol., 24, 130–137 (1999).
[4] Thurston, C. F. Microbiol., 140, 19-26 (1994).
[5] Amaral, P.F.F., Tavares, A.P.M., Xavier A.B.M.R., Cammarota, M.C., Coutinho, J.A.P. and Coelho, M.A.Z.
Environ. Technol., 25, 1313-1320 (2004).
108 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P46
Application of Tyrosinase Obtained from Agaricus bispora
for Color Removal from Textile Effluents
Magali C. Cammarota, Maria Alice Z. Coelho
Escola de Química, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro
de Tecnologia, Bloco E, Sl. E-203, 21949-900, Rio de Janeiro – RJ, Brasil
E-mail: alice@eq.ufrj.br
The textile industry has contributed significantly for the pollution of rivers in some regions of
Brazil, once it generates large volumes of effluents (120 - 380 m 3 /1000 m of manufactured
fabric) containing varied amounts of contaminating agents among which pigments stand out.
Besides the high volume and variability, typical characteristics of effluents generated from
textile industries are the reduced biodegradability potential (low BOD/COD ratios), presence
of heavy metals and toxic compounds and high pigment contents. Several color removal
methods such as chemical oxidation processes, coagulation/flocculation, adsorption, ionic
exchange and separation with membranes have been tested. These processes, however,
present economic limitations, low removal efficiency, formation of intermediate compounds
and toxic sludge and cannot be used with some types of pigments at high concentrations. In
conventional biological processes, the color removal rate is low, once most pigment
molecules are not biodegradable, being therefore removed through precipitation or adsorption
to the sludge flocs. In the last decades, the use of enzymes in the treatment of effluents has
been object of several scientific works. Enzymes may act on specific recalcitrant compounds
increasing their biodegradability or removing them through precipitation. Tyrosinase enzyme
catalyzes the o-hydroxylation of monophenols into catechols and the dehydrogenation of
catechols into o-quinones that once being unstable in aqueous solution, undergo nonenzymatic
polymerization through oxidative and nucleophilic reactions and precipitate, being
removed from the aqueous solution. The present work assesses the color removal from textile
effluents with the use the tyrosinase enzyme. To do so, a raw enzymatic extract obtained from
Agaricus bispora mushrooms and synthetic solutions of reactive pigments widely employed
in the textile industry (Procion Orange MX-2R, Remazol Red 3B and Remazol Black GF)
was used. Different enzyme (activity): pigment (type and concentration) combinations were
evaluated. Previous results indicate a technical feasibility of the treatment, once color
removals of 80%, 78% and 56% have been obtained for pigments Remazol Black GF,
Remazol Red 3B and Procion Orange MX-2R, respectively after 24 h of treatment with
enzymatic activity of 85 U/mL and initial pigment concentration of 83 mg/L.
[1] Correia V.M., Stephenson T., Judd J.S. (1994). Characterization of textile wastewaters – a review. Environ.
Technol. 15:917.
[2] O’Neill C.O., Wawkes F.R., Hawkes D.L., Lourenço N.D., Pinheiro H.M., Delée W. (1999). Colour in
textile effluents – sources, measurement, discharge consents and simulation: a review. J. Chem. Technol.
Biotechnol. 74:1009.
[3] Wada s., Ichikawa H., Tatsumi K. (1993). Removal os phenols from wastewater by soluble and immobilized
tyrosinase. Biotechnol. Bioeng. 42:854.
[4] Atlow S.C., Bonadonna-Aparo L., Klibanov A.M. (1983). Dephenolization of industrial wastewaters
catalysed by polyphenol oxidase. Biotechnol. Bioeng. 26:599.
109 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P47
Phenols and Dyes Degradation by an Immobilized Laccase
from Trametes trogii
Anna Maria V. Garzillo, Federica Silvestri, M. Chiara Colao, Maurizio Ruzzi, Vincenzo
Buonocore
Dpt. of Agrobiology and Agrochemistry, Via S. Camillo de Lellis s.n.c., Viterbo (Italy)
E-mail: amg@unitus.it
Laccases (E.C. 1.10.3.2) are multicopper oxidases which oxidize a variety of phenolic and
non-phenolic compounds with the simultaneous reduction of molecular oxygen to water. The
broad substrate specificity makes these enzymes very attractive for a number of applications,
including bioremediation processes. In these cases, the use of laccases in immobilized form
may result in increased enzyme stability, multiple use and easy separation from the reaction
mixture.
The main laccase (Lcc1) from Trametes trogii has been immobilized both covalently
(Eupergit C) and non-covalently (polyacrylamide gel intrapment, Sepharose ConA
adsorption) ; the last technique gave the highest binding yields (≈ 100%) and capacity of
substrate (2,6-dimethoxyphenol, DMP) degradation. Thus, this study was conducted with
Lcc1 adsorbed at pH 4 on Sepharose ConA; phenol and dye degradation was monitored by
HPLC. In a first group of experiments, phenolic compounds (0.4 g/l each, 100 ml) were
passed separately in continuous through immobilized laccase (50 I.U.) packed in a small
column; after 20 h flowing (flow rate 1 ml/min), caffeic acid (CA) was degraded by 100%, p-
coumaric acid (pCA) by a 20% and 4-hydroxyphenylacetic acid (HPA) by a 10%. When a
mixture of the three phenols was passed through the column, to mimic real situation of waste
waters, degradation after 20 h was 55% (CA), 20% (pCA) and 10% (HPA). Even lower
degradation rates (e.g. 20% for CA) were observed with a mixture of seven phenols
containing also recalcitrant products (3-hydroxyphenylic acid). These data indicate that a
strong substrate competition for the enzyme will affect compound degradation in complex
mixtures. A similar set of experiments was carried out by challenging immobilized laccase
and phenols in batch; in these conditions, the degradation was more effective as compared
with the column system: CA was completely degraded in 1 h, whereas after 20 h pCA and
HPA were degraded by 95 and 50 %, respectively. When applied in a mixture, a competition
effect was observed: CA disappeared after 3 h, pCA and HPA were degraded after 20 h by 75
and 30%, respectively.
The batch system has also been used to assess degradation of some synthetic dyes,
extensively used in industrial processes. The three dyes chosen have typical chromophoric
groups: alizarin (AL, anthraquinone), amaranth (AM, azo) and indigo carmine (IC).
Degradation rate by immobilized laccase was different depending on dye structure: after 20 h
AL was degraded by 90%, IC by 45% and AM by 5%. When violuric acid (VA) was added as
a mediator, the degradation of the three dyes was completed in less than 5 h; 1-
hydroxybenzotriazole was less effective then VA in mediating the interaction between the
enzyme and the dyes.
These preliminar data indicate that immobilized laccase from T. trogii can efficiently promote
decolorization of industrial effluents; further investigations are needed to clarify competition
effects among substrates and the role of mediators in the degradation process.
110 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P48
Relationship between Non-Protein Fraction and Laccase
Isoenzymes from Cultures of Trametes versicolor: Effect
on Dye Decolorization
Diego Moldes a , Alberto Domínguez b , Mª Angeles Sanromán b
a Department of Textile Engineering. University of Minho. 4800 Guimarães. Portugal,
b Department of Chemical Engineering. Isaac Newton Building. University of Vigo. 36310
Vigo. Spain
E-mail: sanroman@uvigo.es
Lignocellulosic materials comprise a broad range of wastes from agricultural, food and forest
industry, which are mainly composed of polysaccharides (cellulose and hemicellulose) and
lignin. Several works determined that the lignocellulosic materials can stimulate laccase
production on white rot fungi. Moreover, these materials can also provide some of the
necessary nutrients to the fungi, which imply a considerable reduction in production costs [1-
2]. The lignocellulosic materials employed to perform the present study were grape seeds,
grape stalks, barley straw, corn cob and barley bran and the white rot fungus selected
Trametes versicolor. The selection was made taking into account previous studies and that all
materials are agricultural-industrial wastes with different composition.
The cultures of Trametes versicolor growing in presence of these lignocellulosic wastes
produce enzymatic complexes with different dye decolorization activity. In order to explain
these differences, the separation of two fractions (protein and non-protein) from the
extracellular liquid was carried out. Moreover, decolorization capability of both fractions was
tested. In this preliminary study was detected that there is an enzymatic (laccase)
decolorization factor and a non-enzymatic one.
In an attempt to quantify the enzymatic factor on the dye decolorization, the isolation and
purification of laccase from the extracellular liquids were required. Two laccases isoenzymes
named Lac I and Lac II were detected, showed a clear band in sodium dodecyl sulfatepolyacrylamide
gel electrophoresis (SDS-PAGE) at ~65 and ~60 KDa, respectively. The
decolorization capability is higher as the activity of LacI increase, although the assays were
carried out with the same total laccase activity. Thus, the decolorization obtained by laccase
depends on the level of enzymatic activity and the laccase isoenzymes (Lac I and Lac II)
proportion forming the enzymatic complex.
The decolorization produced by the non-enzymatic factor shows us that there is a parallel
degradation mechanism on these cultures able to produce decolorization of dyes. This
decolorization activity is due to small and relatively stable metabolites, which probably react
under a radical formation mechanism.
This research was financed by Xunta de Galicia (PGIDIT04TAM314003PR).
[1] Rodríguez E, Pickard M.A., Vázquez-Duhalt R. (1999) Current Microbiol 38:27-32.
[2] Lorenzo M., Moldes D., Rodríguez Couto S., Sanromán A. (2002) Bioresour. Technol. 82:109-113.
111 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Degradation of Synthetic Dyes by Coriolopsis rigida
J. Gómez-Sieiro, D. Rodríguez-Solar, D. Moldes, M.A. Sanromán
P49
Department of Chemical Engineering. Isaac Newton Building. University of Vigo. 36310
Vigo. SPAIN
E-mail: josegomez@uvigo.es
Dye effluents are poorly decolourised by conventional biological wastewater treatment and
may be toxic to the microorganisms present in the treatment plants due to the complex
aromatic structures of these dyes. As an alternative method, biological decolourisation with
white-rot fungi is a feasible method. The ligninolytic system of the white-rot Basidiomycete
Coriolopsis rigida has recently been described by Saparrat [1]. They found that C. rigida
produced extracellular laccase as the sole ligninolytic enzyme even if peptone is present in the
culture medium. For this reason, this fungus is particularly suitable for the study of
xenobiotics degradation by laccase.
In the present work several wastes of the food processing industry such as chestnut shell,
grape seeds, grape stalks, barley straw, corn cob and barley bran were evaluated as potential
substrates for laccase production by Coriolopsis rigida under solid-state conditions with a
basal medium [2]. Amongst them, the use of barley bran was particularly suitable for the
laccase formation and it was strongly stimulated by the addition of copper. In the barley bran
copper-supplemented cultures, laccase first appeared on the 9 th day (0.279 kU/l), and then, it
rapidly increased reaching a maximum value of 26.177 kU/l on the 25 th day of cultivation.
In addition, the ability to degrade structurally different dyes, by C. rigida was analysed. The
dyes tested were Indigo Carmine (indigoid), Bromophenol Blue (sulphonephthaleine),
Lissamine Green (acid diphenylnaphthylmethane) and two dyes from a leather factory: Sella
Solid Red and Sella Solid Blue, manufactured by TFL (Germany) and their chemical structure
have not yet been disclosed. In solid-state cultures the in vivo decolourisation of structurally
these dyes was monitored. The percentage of biological decolourisation of Indigo Carmine
and Bromophenol Blue attained was around 100% in only 24 h, whereas it was rather low in
the leather factory dyes at the same time. Moreover, in vitro decolourization was carried out
in spectrophotometer cuvettes at 30ºC and the reaction mixture contained succinic buffer (25
mM, pH 4.5), dye and extracellular liquid containing mainly laccase (1.5 U). The dyes Indigo
Carmine and Bromophenol Blue were easily decolourised by the extracellular liquid obtained
in such cultures, whereas Lissamine Green and especially Sella Solid Red showed much more
resistance to degradation. This shows the specificity of laccase towards different dye
structures.
This research was financed by xunta de galicia (pgidit04tam314003pr). The authors wish to thank Dr. M.J.
Martínez (cib, csic, Madrid, spain) for providing Coriolopsis igida (cect 20449).
[1] Saparrat, M.C.N., Guillen, F., Arambarri, A.M., Martinez, A.T., & Martinez, M.J. (2002). Applied and
Environmental Microbiology, 68, 1534-1540.
[2] Moldes, D., Gallego, P.P., Rodríguez Couto, S., & Sanromán, A. (2003). Biotechnol Letters, 25, 491-495.
112 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P50
Immobilization of Laccase and Versatile Peroxidase
Considering Their Further Application
Anna Olszewska, Jolanta Polak, Anna Jarosz-Wilkołazka, Janina Kochmańska-Rdest
Department of Biochemistry, Maria Curie-Sklodowska University, Sklodowska Place 3, 20-
031 Lublin, Poland.
E-mail: aolszewska@wp.pl
Enzyme immobilization provides easy recovery and reuse of the enzyme and many other
advantages, including easy in product separation and continuous operation. For successful
development and application of immobilized biocatalysts, the enzyme support is generally
considered as the most important component contributing to the performance of the reactor
system (1). There is a variety of methods by which enzymes can be localized on/into support,
ranging from covalent chemical bonding to physical entrapment but no single method and
support is the best for all enzymes and their different applications (2). This is because of the
widely different chemical characteristics and composition of enzymes, the different properties
of substrates and products, and the different uses to which the product can be applied (3). The
ideal support is cheap, inert, physically strong and stable. However, in many cases,
immobilization affects the diffusion of the substrate towards the active site of the enzyme. For
example the immobilized enzymes can be inactivated by the interactions with products
formed in the reactions.
Versatile peroxidase (VP) from Bjerkandera sp. and laccase (Lac) from Cerrena unicolor
were immobilized using different carriers. Different strategies were considered concerning the
type of supports and their activation. Different carriers were tested during experiments:
alginate beads, polyacrylamide hydrogel, gelatin, Sipernat, controlled porosity glass (CPG),
grit, and alumina. Among physical methods the best were alginate beads, among covalent
chemical bonding method – Sipernat and CPG. Immobilized Lac was used in both
decolourization processes and coupling reaction using different phenolic precursors.
Immobilized VP was used in decolourization of simple model dyes and colour wastewaters.
[1] N. Munjal, S. K., Sawhney. 2001. Enzyme Microb Technol 30, 613-619
[2] P. J. Worsfold. 1995. Pure & Appl Chem 67, 597-600
[3] B. Krajewska. 2004. Enzyme Microb Technol 35, 126-139
113 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P51
Removal of Several Azo Dyes by Trametes sp. Crude
Laccase: Reaction Increment in the Presence of Azo Dye
Mixtures
Rui M.F. Bezerra, Irene Fraga, Albino A. Dias
CETAV - Dep. Engenharia Biológica e Ambiental, UTAD, Apartado 1013, 5001-801 Vila
Real, Portugal
E-mail: bezerra@utad.pt
White-rot fungi can degrade a wide variety of recalcitrant compounds like lignin, dyestuffs
and other xenobiotic compounds by their extracellular ligninolytic enzyme systems. Several
studies in vitro have shown that fungal laccases are able to decolorize and detoxify industrial
dyes [1]. The objective of the present work was to evaluate the potential of laccase-based
treatment for removing of coloured azo solutions and associated toxicity. Mixtures of seven
azo dyes treated with laccases were degraded nevertheless with the production of other more
polar compounds. It is remarkable that when they were studied individually, acid red 337,
acid red 57, orange II and methyl orange need a mediator such as ABTS to be degraded.
Otherwise when these dyes were in mixtures with others that were degraded without any
mediator (acid black 194 and acid blue 113) the results showed no differences between assays
carried out with or without mediators (ABTS, acetovanillone, acetoseringone and carminic
acid) suggesting that acid black 194 and acid blue 113 exhibit a mediator effect.
Germinability experiments with water cress (Lepidium sativum) were conduced in the
presence of different dilutions of enzyme–treated azo compounds. Our results showed
significant toxicity abatement after laccase treatment as assessed by germination index which
increase from 50% to 94%.
[1] A. A. Dias, R. M. Bezerra, P. M. Lemos and A. N. Pereira. 2003. In vivo and laccase-catalysed
decolourization of xenobiotic azo dyes by a basidiomycetous fungud: charactersation of its ligninolytic system.
World J Micriobiol Biotecnol 19:969-975
114 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P52
Transformation of Simple Phenolic Compounds by Fungal
Laccase to Produce Colour Compounds
Jolanta Polak, Anna Jarosz-Wilkołazka, Marcin Grąz, Elżbieta Dernałowicz-Malarczyk
Department of Biochemistry, Maria Curie-Sklodowska University, Sklodowska Place 3,
20-031 Lublin, Poland.
E-mail: jolanta_polak@wp.pl
Carotenoids, melanins, flavonoids, quinones and more specifically monascins, violacein or
indigo there are coloured compounds synthesize by fungi in natural environment [1, 2].
However, there is a long way from Petri dishes to the industrial scale. Isolation of natural
pigments and/or bioconversions of precursors to obtained natural pigments are innovative
biotechnological techniques for the more environmentally friendly synthesis of different
commercially valuable processes. A number of specific or selective reactions have been
reported where laccases, the extracellular enzymes produced by many fungal strains, have
been used to synthesize products of commercial importance (pharmaceutics, food ingredients,
polymers). Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are multi-coppercontaining
enzymes, widely distributed in plants and fungi, that catalyze oxidative conversion
of a broad range of substrates such as phenols or lignin-derivatives [7]. Laccase from
Pycnoporus cinnabarinus catalyzed coupling of 3-(3,4-dihydroxyphenyl) propionic acid with
4-aminobenzoic acid [3]. The synthesis of the actinocin from 4-methyl-3-hydroxyanthranilic
acid gave the chromophore of actinomycin antibiotics, conversion of alkaloids or the
production of mithramicine there are examples of using laccase to yield biologically active
products [4, 5, 6].
Laccases has also ability to induce oxidative coupling reactions of the chemicals, such as
phenol derivatives to other phenolic structures, producing intensely coloured products. The
uses of laccases in dyes synthesis processes represent a promising alternative to chemical
synthesis of existing or new dyes.
The aim of this study was to examine the ability of an extracellular laccase produced by a
commonly occuring wood-degrading fungus Cerrena unicolor to form coloured compounds
from simple organic precursors. Screening of 30 different phenolic derivatives such as o-, m-,
and p-methoxy, -hydroxy, -sulfonic and aromatic amines were studied in the presence of
laccase in liquid media. The findings show that laccase catalyzes the oxidative coupling
reaction between selected substrates producing coloured compounds (from yellow by brown
to red and blue). Coloured compounds were isolated and analysed firstly by
spectrophotometer and secondly by capillary electrophoresis. To check participation of
substrates in product formation substrates were added to incubation mixtures in various ratios.
This work was partially supported by EC FP6 Project SOPHIED (NMP2-CT-2004-505899) and the State
Committee for Scientific Research (139/E-339/SPB/6. PR UE/DIE 450/2004-2007).
[1] Dufosse et al. (2005) Trends Food Sci Technol 16, 384-406.
[2] Duran et al. (2002) Crit Rev Food Sci Nutr 42 (1) 53-66.
[3] Pilz et al. (2003) Appl Microbiol Biotechnol 60, 708-712.
[4] Burton S.G. (2003) Curr Opin Chem 7 (13), 1317-1331.
[5] Manda et al. (2005) J Mol Cat B: Enzym 35, 86-92.
[6] Osiadacz et al. 72, 141-149.
115 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P53
Biodegradation Cycles of Industrial Dyes By Immobilised
Basidiomycetes
L.Casieri a , G.C. Varese a , A. Anastasi a , V. Prigione a , K. Svobodová b , V. Filipello Marchisio a
and Č. Novotný b .
a Department of Plant Biology, University of Turin, Viale Mattioli 25, Turin, Italy;
b Laboratory of Exp. Mycology, Institute of Microbiology ASCR, Vídeňská 1083, Prague 4,
Czech Republic.
E-mail: leonardo.casieri@unito.it
Treatment of recalcitrant and toxic dyes with traditional technologies is not always effective
or may not be environmental-friendly. Alternative technologies, such as biodegradation, have
been explored to demonstrate that various fungi are able to degrade a broad spectrum of
structurally different synthetic dyes. In particular, ligninolytic fungi and their non-specific,
oxidative enzymes have been reported to be responsible for decolourisation of a number of
dyes. Although many studies have been made to assess the dye-decolourisation capabilities of
fungi, only a few reported a reduction of the effluent toxicity as the effect of the treatment.
The decolourisation capabilities of Trametes pubescens (MUT 2295) and Pleurotus ostreatus
(MUT 2976), previously evaluated in industrial dye decolourisation screenings, have been
employed to degrade azo and anthraquinone industrial dyes, R243 and B49, and the model
anthraquinone dye RBBR. Fungi were immobilised on polyurethane foam cubes and used in
bioreactors. Low nitrogen mineral medium (LNMM) to which various dyes were added at
different concentrations was circulated by means of a peristaltic pump. Five sequential cycles
were run for each dye and fungus (3 at 200 ppm, 1 at 1000 ppm and 1 at 2000 ppm
concentrations).
Laccase (Lac), Mn dependent peroxidase (MnP), Mn independent peroxidase (MiP), Lignin
peroxidase (LiP) and Aryl alcohol oxidase (AAO) were daily monitored during all cycles.
Besides the toxicity of LNMM containing 1000 and 2000 ppm of a dye was assessed by the
ecotoxicity test using Lemna minor (duckweed) before and after the dye decolourisation. Each
fungus was able to decolourise efficiently all the dyes during the cycles at increasing
concentrations. Best results were obtained with the anthraquinone dyes, but a good removal of
the azo dye was also achieved. During all Pleurotus ostreatus decolourisation cycles a high
Lac activity was observed and the presence of industrial dyes enhanced the production of this
enzyme. On the contrary, the enzyme activity of Trametes pubescens varied greatly during
cycles and no clear correlation between decolourisation and the enzyme activities was
observed. Duckweed ecotoxicity test showed a significant reduction (P≤0,05) of the toxicity
after the treatment with both fungi.
116 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P54
Catalytic Activity of Versatile Peroxidase from Bjerkandera
fumosa and its use in Dyes Decolourization
Anna Jarosz-Wilkołazka a , Anna Olszewska a , Janina Rodakiewicz-Nowak b , Jolanta Luterek a
a Department of Biochemistry, Maria Curie-Skłodowska University, Skłodowska Place 3, 20-
031 Lublin, Poland, b Institute of Catalysis and Surface Chemistry, Polish Academy of
Sciences, Niezapominajek 8, 30-239 Kraków, Poland
E-mail: aolszewska@wp.pl
The extracellular ligninolytic system from white rot fungi consists mainly of oxidative
enzymes: laccases (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP).
However, during last years, a novel class of ligninolytic peroxidase, named versatile
peroxidase (VP), has been described. VP can both efficiently oxidize Mn(II) to Mn(III) (like
MnP) and carry out Mn(II)-independent activity on aromatic substrates (like LiP). Until
today, VP was described only in various strains of two fungal species – Pleurotus and
Bjerkandera. In the case of Bjerkandera sp. BOS55, versatile peroxidase it is manganeselignin
peroxidase hybrid enzyme, which is able to oxidize various phenolic and non-phenolic
substrates, such as veratryl alcohol, in the absence of Mn(II) ions. VP from Bjerkandera
adusta described by Pogni and coworkers, it is a structural hybrid between LiP and MnP and
this hybrid combines the catalytic properties of two above peroxidases, being able to oxidize
typical LiP and MnP substrates [1-5].
Versatile peroxidase (VP) from the white rot fungus Bjerkandera fumosa was isolated and
purified by ion exchange and gel filtration chromatography. Its catalytic activity was studied
taking into account substrate range, pH, ionic strength, temperature and presence of organic
solvents. Its primary catalytic activity in oxidation of Mn(II) was studied in aqueous solutions
in the presence of varying concentrations (up to 8 M) of acetonitrile (MeCN),
dimethylsulfoxide (DMSO), ethanol, and n-propanol. The observed maximum reaction rate
values decreased with the addition of organic solvents in the order: MeCN

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P55
Bleaching of Kraft Pulp Employing Polyoxometalates and
Laccase
José A.F. Gamelas, b Ana S.N. Pontes, a Dmitry V. Evtuguin, b Ana M.R.B.Xavier a
a COPNA, b CICECO – Departamento de Química, Universidade de Aveiro, 3810–193 Aveiro,
Portugal
E-mail: Dmitryr@dq.ua.pt
The pulp and paper industry is facing an increasing pressure from environmentally concerned
institutions to replace the conventional chorine-based bleaching techniques by
environmentally friendly technologies. Oxygen delignification catalysed by polyoxometalates
(POM) has been proposed a nice alternative to pulp bleaching [1, 2]. Applied as catalysts
under aerobic conditions, POM oxidise selectively the residual lignin in kraft pulp, and the
reduced form of POM should be re-oxidised by molecular oxygen at the same process stage.
Unfortunately, the most selective polyoxometalates for bleaching purposes such as
[SiW 11 Mn III (H 2 O)O 39 ] 5- and [SiW 11 V V O 40 ] 5- are slowly re-oxidised by dioxygen (even at high
temperatures), which hinders their practical application [3]. A solution to break the
thermodynamic barrier in the oxidation of SiW 11 Mn II and SiW 11 V IV was found employing
laccase. A multi-stage process was proposed using an alterative treatment of kraft pulp with
polyoxometalate at high temperature (110 ºC) followed by the polyoxometalate re-oxidation
with laccase (45-60 ºC) in a separate L stage [4]. More than 50 % of removal of the residual
lignin was achieved. The main loss of pulp viscosity occurred in L stage. It was proposed that
the pulp delignification with POM separated from POM re-oxidation with laccase should give
better delignification selectivity.
In this work unbleached E. globulus kraft pulp was delignified with POM ([SiW 11 V V O 40 ] 5- ) at
90ºC in the bleaching reactor A, which was coupled with
bioreactor B, where the reduced POM was continuously reoxidised
by laccase at 45ºC under aerobic conditions (Fig.).
After separation from laccase on the ultrafiltration ceramic
membrane C, re-oxidised POM was pumped back to the
bleaching reactor. This allowed sustainable pulp
delignification with minimal pulp viscosity loss. Thus, about
70 % pulp delignification was reached with only 15 %
viscosity loss (6h of treatment). The kinetic of pulp
delignification in new POM(L) system was investigated. The implementation of POM(L)
stage instead the first chlorine dioxide stage (D) in DEDED bleaching allowed about 60%
ClO 2 savings for the same final pulp brightness (90% ISO) and similar pulp strength
properties.
[1] I. A. Weinstock, R. H. Atalla, R. S. Reiner, M. A. Moen, K. E. Hammel, C. J. Houtman, C. L. Hill,
M. K. Harrup, J. Mol. Cat., 1997, 116, 59-84.
[2] D. V. Evtuguin, C. Pascoal Neto, Holzforschung, 1997, 51, 338-342.
[3] J. A. F. Gamelas, A. R. Gaspar, D. V. Evtuguin, C. Pascoal Neto, Appl. Catal. A, 2005, 295, 134-
141.
[4] A. Tavares, J. Gamelas, A. Gaspar, D. V. Evtuguin, A. Xavier, Cat. Commun., 2004, 5, 485-489.
118 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P56
Influence of Trametes versicolor laccase on the contents of
xexenuronic acids in two Eucalyptus globules Kraft Pulp
Atika Oudia, Rogério Simões, João Queiroz
Research & Development Unit of Textile and Paper Materials, University of Beira Interior
6201-001 Covilhã – Portugal
E-mail: atika@ubi.pt
Environmental awareness and concerns during the recent years have led to an increased interest in
using biotechnology in pulp and paper industry. Eucalyptus globulus is of great economical
importance for the Portuguese pulp and paper industry, since eucalyptus pulp represents about 85% of
pulp production. Laccase ([EC 1.10.3.2], p-diphenol: dioxygen oxidoreductase) is a member of the
blue multicopper protein family, which also includes the plant enzyme ascorbate oxidase and the
mammalian plasma protein ceruloplasmin [1]. Trametes versicolor laccase can catalyse
depolymerisation of kraft pulp lignin in presence of a mediator [2].
Two wood samples of Eucalyptus globulus (one industrial chip sample and another obtained
from a clone tree) were submitted to the kraft cooking processes in order to evaluate its pulping
potential. The purpose of pulping is to remove lignin from wood celluloses. Traditionally, kappa
number is regarded as a parameter that proportional to the residual lignin in the pulps. A recent study
has shown that the hexenuronic acid (HexA) groups in pulps are responsible for a significant
percentage of the kappa number [3], especially from hardwood pulps due to there higher content of
xylan.
Therefore, in this work we used the Klason lignin content in pulps, instead of kappa number,
to evaluate the pulpability, at the given pulping conditions: Active Alkali Charge [%] on wood = 19;
Sulfidity [%] = 30; Liquor: Wood Ratio [L/Kg] = 4:1; Cooking temperature [ºC] = 160, Time to
temperature [min] = 90, Time at temperature [min] = 60. The Klason lignin kappa number content in
brownstocks from clone eucalyptus wood species is much lower than that of the industrial wood
species. Laccase mediator system (LMS) process was applied for the further biodelignification of the
pulps from the conventional kraft pulping process. It was observed that roughly 49% of Klason lignin
has been removed from the clone eucalyptus pulps at LMS. However, it only removed approximately
42% of Klason lignin from the industrial eucalyptus pulps at the same LMS conditions. The Laccase
mediator system diminishes the pulp contents of lignin and hexenuronic acids (HexA). The data shows
that the amount of HexA is quite high in the unbleached Eucalyptus globulus (clone) contrast to
industrial Eucalyptus globulus kraft pulps, 64 mmol/kg and 52.1mmol/kg respectively. However, the
LMS (E) treatments only have a small effect on these compounds.
In view of the results obtained in this study, it can be concluded that LMS treatment can be
applied as a pretreatment in the bleaching sequences in order to reduce the use of chlorine dioxide.
Besides, it indicates that the cloned eucalyptus globulus is an easy to be pulped and bleached wood
species. Moreover, it has a significant importance to the pulping industry economics, particularly on
energy cost savings and production capacity improvement.
Acknowledgements: This research was supported by FCT (Science and Technology Foundation),
SFRH/BD/10893/2002.
References
[1] Mayer, A.M. and Staples, R.C. (2002) Laccase: new functions for an old enzyme. Phytochem.60,
551-565.
[2] Bourbonnais, R., Paice, M.G., Freiermuth, B., Bodie, E. and Borneman, S. (1997) Reactivities of
various mediators and laccase with kraft pulp and lignin model compounds. Appl. Environ.
Microbial.63, 4627-4632.
[3] J. Li, G. Gellerstedt, Carbohyd. Res. 302 (1997) 213.
119 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Laccase-Mediated Oxidation of Natural Compounds
Mattia Marzorati, Daniela Monti, Francesca Sagui, Sergio Riva
P57
Istituto di Chimica del Riconoscimento Molecolare, C.N.R..,Via Mario Bianco 9, 20131
Milano, Italy
E-mail: daniela.monti@icrm.cnr.it
Laccases are a group of oxidative enzymes whose exploitation as biocatalysts in organic
synthesis has been neglected in the past, probably because they were not commercially
available. The search for new, efficient and environmentally benign processes for the textile
and pulp and paper industries has increased interest in these essentially ‘green’ catalysts,
which work with air and produce water as the only by-product, making them more generally
available to the scientific community. 1
Typical substrates of laccases are phenols and aliphatic or aromatic amines, the reaction
products being mixtures of dimers or oligomers derived by the coupling of the reactive radical
intermediates. For instance, we have recently exploited these biotransformations to isolate
new dimeric derivatives of the phytoalexin resveratrol 2 and of the hormone β-estradiol. 3 In
these studies we have also observed a significant influence of the solvent on the reaction
outcomes. 4
Additionally, laccases oxidation of non-phenolic groups, particularly benzyl and – more
generally – primary alcohols, is also possible thanks to the ancillary action of the so-called
“mediators” (i.e., TEMPO, HBT, ABTS): the oxidation step is performed by the oxidized
form of a suitable mediator, generated by its interaction with the laccase. Accordingly, we
have oxidized a series of sugar derivatives (mono- and disaccharides, cyclodextrins, water
soluble cellulose) 5 and of natural glycosides (i.e., thiocolchicoside, 1 to 1a, and asiaticoside, 2
to 2a). 6, 7
R
HO
HO
O
OH
1 : R = CH 2
OH
1a : R = COOH
MeO
O
MeO
SMe
O
NHAc
HO
HO
OH
O
HO
O
O
OH
OH
O HO
O
OH
O
R
2 : R = CH 2
OH
2a : R = COOH
OH
O
OH
OH
[1] S. Riva, Trends Biotechnol. 2006, 24, 219-226.
[2] S. Nicotra, M.R. Cramarossa, A. Mucci, U. Pagnoni, S. Riva, L. Forti, Tetrahedron 2004, 60, 595 − 600.
[3] S. Nicotra, A. Intra, G. Ottolina, S. Riva, B. Danieli, Tetrahedron: Asymmetry 2004, 15, 2927 - 2931.
[4] A. Intra, S. Nicotra, S. Riva, B. Danieli, Adv. Synth. Catal. 2005, 347, 973 – 977.
[5] M. Marzorati, B. Danieli, D. Haltrich, S. Riva, Green Chem., 2005, 7, 310 – 315.
[6] L. Baratto, A. Candido, M. Marzorati, F. Sagui, S. Riva, B. Danieli, J. Mol. Catal. B-Enzymatic, 2006, 39,
3-8.
[7] D. Monti, A. Candido, M. Cruz Silva, V. Kren, S. Riva, B. Danieli, Adv. Synth. Catal., 2005, 347, 1168 –
1174.
120 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P58
Laccase induced coating of lingocellulosic surfaces with
functional phenolics
M. Schroeder a , G. M. Guebitz b , V. Kokol a
a Faculty of Mechanical Engineering, Institute of Textile, University of Maribor, Slovenia,
Smetanova ul 17, 2000 Maribor, Slovenia; b Institute of Environmental Biotechnology, Graz
University of Technology, Petersgasse 12, 8010 Graz, Austria
E-mail: marc.schroeder@uni-mb.si
Enzymatic induced coupling of functional groups could improve fibre properties such as wet
ability, hydrophobicity, or effects like better dye ability. Also surface functionalisation for
special application could be achieved by coupling e.g. flame retardants or antimicrobial
agents onto the surface enhancing the bulk properties of existing products for better
performance [1].
For this purpose a laccase from T. hirsuta was purified and characterised. Preliminary studies
showed optimal conditions for enzyme activity at 50°C and pH 5.0. Kinetic properties on
model substrates were calculated K M of 16.7 ± 0.2 µM for guaiacol and K M of 21.0 ± 0.9 µM
for dimethoxyphenol in aqueous solutions. Different phenols, e.g. hydroxyquinone, guaiacol,
vanillin, ferulic acid, and catechol, were screened for their potential as and antibacterial
performance. While oxidative of guaiacol showed strong colouration (∆K/S 9.3) with weak
fastness, bacterial growth of Staphylococcus aureus and Klebsiella pneumoniae could be
reduced using ferulic acid for coating.
Enzymatic treatment of natural fibres is affected by different factors such as nature and ionic
strength of the treatment buffer, as well as enzyme activity and incubation time [2].
Furthermore, the process depends on adsorption and de-sorption of the enzymes which can
result in a non-uniform treatment. In order to determine optimum incubation conditions, an
experimental design with three factors (molar ratio reactant, enzyme activity, and incubation
time) at five different levels, varying from 0 to 50 mM (reactant), from 0 to 20 Units
(activity), and from 0 to 240 min (time) based on a central composite statistical design was
followed [3].
This research has been supported by a Marie Curie Transfer of Knowledge Fellowships of the
EC 6FP under contract no MTKD-CT-2005-029540
[1] M. Lund, A. J. Ragauskas, (2001) Appl. Microbiol. Biotechnol., 55: 699-703
[2] J. Shen et al, (1999) J. Textile Inst. 90:404-411
[3] T. Tzanov et al, (2003) Appl. Biochem. Biotechnol, 111:1-13
121 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Decolourization and Detoxification of Kraft Effluent
Streams by Lignolitic Enzymes of Trametes versicolor
M.S.M. Agapito a , D. Evtuguin b , A.M.R.B. Xavier a
P59
a COPNA, b CICECO – Departamento de Química, Universidade de Aveiro, 3810–193 Aveiro,
Portugal
E-mail: abx@dq.ua.pt
The pulp industry deals mainly with delignification of wood to produce cellulosic pulp (pulping
process) and with pulp bleaching to fulfil the brightness of fibrous material necessary for the
papermaking. Both technologic processes produce a large amount of liquid effluents, which
cause serious pollution problems 1 . The wastewater colour and toxicity are determined primarily
by lignin and its derivates, which are discharged in the effluents from pulping, bleaching and
chemical recovery stages in the pulp plant 2 . Current bio-purification of effluent streams involving
activated sludge frequently faces serious problems to control the activity of wild microorganisms
due to their biodiversity and unpredictability. In this context the use of specific targeting
microorganisms deserves attention. White-rot fungi produce non-specific extracellular oxidative
enzymes to initiate the degradation of lignin 3 . Trametes versicolor is one of the white-rot
basidiomycetes that produce ligninolytic enzymes, such as lignin peroxidase (LiP), manganese
peroxidase (MnP) and laccase 1 . Distinct laccase and MnP oxidative activities can be obtained
under different specific experimental conditions.
The aim of this work was to study the capacity of white-rot fungi Trametes versicolor, to reduce
the chemical oxygen demand (COD) and to decolourise the effluent of kraft pulp mill using E.
globulus wood as a basic row material. The fermentation of this fungus on Trametes Defined
Medium 4 , water, industrial effluent or their mixtures was carried out and compared. Laccase and
Manganese Peroxidase oxidative activity were analysed in relation to colour degradation and to
reduction of chemical oxygen demand. The obtained results show that enzymatic activities of
laccase and MnP on industrial effluent were higher than those obtained without any effluent. The
maximum decolourization, of 60%, was attained at the tenth day of fermentation, and a reduction
of the chemical oxygen demand higher than 60% was attained on the end of fermentation.
This fungus has shown an excellent capacity of development in toxic environments once its cell
growth was observed and oxidative enzymatic activity was remarkably increased in presence of
effluent and both high decolourization and detoxification parameters were attained.
[1] Manzanares, P.; Fajardo, S.; Martín C, Journal of Biotechnology, 43:125-132, 1995
[2] Selvam,K.; Swaminathan,K.; Song,Myung Hoon; Chae, Keon-Sang, World Journal Microbiology &
Biotechnology, 18:523-526,2002
[3] Toh, Yi-Chin; Yen, Jocelyn Jia Lin; Obbard, Jeffrey Philip; Ting, Yen-Peng, Enzyme and Microbial
Technology, 33:569-575, 2003
[4] Tien, M.; Kirk, T. K., Methods Enzymology,161:238-247,1998
122 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P60
Effect of Medium Composition on Laccase Production by
Trametes versicolor Immobilized in Alginate Beads
A. Domínguez, D. Moldes, M. A. Longo and M. A. Sanromán
Department of Chemical Engineering. Isaac Newton Building. University of Vigo. 36310
Vigo. SPAIN
E-mail: alberdom@uvigo.es
The main limitation for the extensive industrial application of microbial enzymes is their high
cost. In industrial operations, immobilized microbial cell systems could provide additional
advantages over freely suspended cells such as ease of regeneration and reuse of the biomass,
easier liquid-solid separation and minimal clogging in continuous-flow systems. Therefore, a
good strategy to increase the productivity of the fermentation processes would be the
operation with the fungus immobilised in alginate beads and the optimization of the culture
conditions [1].
In the present study, the effect of adding veratryl alcohol and copper sulphate on laccase
activity production by calcium alginate-immobilized Trametes versicolor has been
investigated. Employing copper sulphate as laccase inducer or supplementing the culture
medium with veratryl alcohol, led to maximum values of laccase activity. However, the
highest laccase activity (around 4000 U l -1 ) was obtained in cultures simultaneously
supplemented with copper sulphate (3 mM) and veratryl alcohol (20 mM). These values
implied a considerable enhancement in relation to control cultures without any inducer
(around 200 U l -1 ).
The production of laccase by immobilized T. versicolor in a 2 litre-airlift bioreactor with the
optimized inducer has been evaluated. Laccase activities around 1500 U l -1 were attained. The
bioreactor operated for 44 days without operational problems and the bioparticles maintained
their shape throughout the fermentation. Moreover, the extracellular liquid obtained was
studied in terms of optimum pH and temperature for activity and stability. On the other hand,
anthracene was added in two-repeated batches in order to determine the efficiency of this
process to degrade pollutants. Near complete degradation was reached in both batches.
Moreover, in vitro degradation of several PAHs by crude laccase was also performed.
This work was financed by the Spanish Ministry of Science and Technology and European
FEDER (Project CTM2004-01539/TECNO)
[1] Domínguez A., Rodríguez S. and Sanroman M. (2005). Dye decolorization by Trametes hirsuta immobilized
into alginate beads. World Journal of Microbiology & Biotechnology. 21: 405-409
123 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P61
Involvement of the Laccase Produced by Streptomyces sp.
in the Biotransformation of Coffee Pulp Residues
A.L. Orozco a , O. Polvillo c , J.Rodríguez b , J.M. Molina b , O. Guevara a , M.E.Arias b , M.I. Pérez b
a Departamento de Biología. UNAN-León. Nicaragua
b Departamento de Microbiología y Parasitología. Universidad de Alcalá. 28871 Alcalá de
Henares (Madrid). Spain
c IRNAS-CSIC. P.O. Box 1052, 41080 Sevilla, Spain
E-mail: misabel.perez@uah.es
Coffee pulp and coffee husk are toxic residues containing caffeine, tannins and polyphenols.
Their disposal is a problem for the processing industries as it leads to serious environmental
problems. SSF is a process which has been applied to detoxify coffee residues for improved
application in several biotechnological processes [1, 2].
Streptomyces sp., a thermophylic strain isolated from volcanic soil of Nicaragua, has been
used in our laboratory to transform under SSF conditions coffee pulp residues obtained from
Coffea arabica berries. The main objective of this work is to study the production of oxidative
enzymes such as laccases and peroxidases during the transformation process and to analyse
the chemical modifications performed by the microorganism in the fermented substrate.
The microorganism was grown at 45ºC for 10 days on the coffee pulp residue supplemented
with cassava (65% humidity). The growth of the strain was estimated as the CO 2 released
during the incubation period. The oxidative enzymes, laccase and peroxidase, were obtained
from the fermented substrate [3] and assayed according the methods previously described [4].
Chemical modifications of the residue were examined through Pyrolysis-GC/MS [5].
The strain which produced an optimal colonization of the substrate, reached the maximum growth after three
days of incubation. In SSF conditions, higher levels of laccase activity were obtained than those achieved
when the microorganism was grown in a soya-manitol liquid medium. It is remarkable that the optimal
temperature of this enzyme was 70ºC.
Results obtained by Py-GC/MS of the fermented substrate showed a clear decrease in the
lignin-derived compounds by the action of the microorganism, from both syringyl and
guaiacyl units. In addition, the increase in the relative abundance of the most of syringyl and
guaiacyl units with a higher oxidation degree suggests an oxidative action of the strain on the
lignin molecule. These transformations could be attributed to the laccase activity, the unique
oxidative enzyme produced by the microorganism in SSF conditions.
[1] Pandey, A. Soccol, C.R. and Mitchell, D. Process Biochemistry, 35 (2000) 1153.
[2] Ulloa, J.B., Verreth, J.A.J. Amato, S. and Huisman, E.A. Bioresource Technology, 89 (2003) 267.
[3] Ferraz, A., Baeza, J., Rodríguez, J. and Freer, J. Bioresource Technology, 74 (2000) 201.
[4] Hernández, M., Hernández-Coronado, M.J., Montiel, M.D., Rodríguez, J., Pérez, M.I., Bocchini, P., Galletti,
G.C. and Arias, M.E. J. Annal. Appl. Pyrolysis, 58-59 (2001) 539.
[5] Arias, M.E., Polvillo, O., Rodríguez, J., Hernández, M., Molina, J.M., González, J.A. and González-Vila, F.J.
J. Annal. Appl. Pyrolysis, 74 (2005) 138.
124 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P62
Elimination of the Endocrine Disrupting Chemical
Bisphenol A by using Laccase from the Ligninolytic fungus
Lentinus crinitus
Carolina Arboleda a,d , Hubert Cabana b,c , J. Peter Jones c , Amanda I. Mejía a , Spiros N. Agathos b ,
Gloria A Jimenez d , Michel J. Penninck d
a Laboratorio Ciencia de Los Materiales, Instituto de Química y Facultad de Química
Farmacéutifca, Universidad de Antioquia, Medellin, Colombia; b Bioengineering Unit,
Université Catholique de Louvain, Louvain-la-Neuve, Belgium ; c Department of Chemical
Engineering, University of Sherbrooke, Sherbrooke (Qc), Canada; d Laboratory of Microbial
Physiology and Ecology, Faculty of Sciences, Université libre de Bruxelles, Pasteur institute,
Brussels, Belgium.
E-mail: carboled@farmacia.udea.edu.co
Bisphenol A (BPA) is used as raw material for the production of polycarbonates and epoxy
resins. Its discharge in the environment can occur from factories producing BPA or
incorporating it into plastics from leaching of plastic wastes and landfill sites. Recent research
has demonstrated that this chemical can mimic or interfere with the action of animal
endogenous hormones by acting as estrogen agonists, binding to the estrogen receptor or
eliminating a normal biological response; consequently, they may pose a risk to human health
and an environmental impact as they end up in nature as waste through several anthropogenic
activities.
Laccase, that has been shown to catalyze the oxidation of various phenols, aromatic amines
and some dyes, may constitute a good way to treat BPA which is a good substrate for laccases
because of its phenolic structure. A few studies have used fungi and ligninolytic enzymes to
eliminate BPA. In this project, we used Lentinus crinitus, one WRF scanty studied, to remove
BPA from aqueous solutions.
Experiments were carried out in order to test several parameters such as the range of pH,
temperature and contact time, and the presence of mediators, like 2,2’-azino-bis-(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) on the elimination of BPA. A Box–Behnken
type design was used, in order to evaluate the impact of the three parameters (temperature, pH
and processing time) and their potential interactions upon the degradation of BPA. This
statistical procedure makes it possible to reduce the number of experiments required. In this
case T, pH and time of contact, were statistically significant model terms.
Our results demonstrate that using 200 mU ml -1 of laccase, 97.8% of a 22 µM solution of
BPA was eliminated within 6 hours at pH 3 and 40°C. The yeast estrogen test (YES) was used
to measure the elimination of the estrogenic activity of BPA, which is associated with the
elimination of this substance. After 6 hours of treatment, up to 90 % of the estrogenic activity
of BPA was lost. Finally, we demonstrate that the use of ABTS in the laccase/mediator
system significantly improves the laccase catalyzed elimination of BPA.
125 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Tyrosinase-Catalyzed Modification of Bombyx mori Silk
Proteins
P63
Giuliano Freddi a , Anna Anghileri a , Sandra Sampaio a , Johanna Buchert b , Raija Lantto b ,
Kristiina Kruus b , Patrizia Monti c , Paola Taddei c
a Stazione Sperimentale per la Seta, via Giuseppe Colombo 83, Milano 20133, Italy; b VTT
Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT, Finland; c Dept. of
Biochemistry, University of Bologna, via Belmeloro 8/2, Bologna 40126, Italy
E-mail: freddi@ssiseta.it
In recent years, the interest on new biobased, high-performing, and environmentally friendly
polymers is growing rapidly. Silk proteins, i.e. fibroin and sericin produced by the silkworm
species Bombyx mori, are not only a valuable starting material for the textile industry but also
renewable biopolymers suitable for a range of applications, from cosmetic to medical enduses.
Biological properties of silk proteins, in particular silk fibroin which is endowed with
excellent biocompatibility, strongly recommend their use as a mean to develop innovative
biomaterials. In order to increase the application potential of silk proteins, chemical
modification and/or functionalization may be needed. To this aim, enzymes are expected to
offer cleaner and safer alternatives to current chemical practices. Oxidases seem the most
promising enzymes for protein modification. Of the oxidative enzymes, tyrosinase, a coppercontaining
enzyme widely distributed in nature, has proved to be useful to modify proteins by
oxidizing tyrosine residues to o-quinones, which are active species that can condense with
each other or react with nucleophiles, such as the free amine groups of protein-bound amino
acid residues or of the polysaccharide chitosan.
The capability of Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues
of silk proteins was studied under homogeneous and heterogeneous reaction conditions, by
using fibroin and sericin aqueous solutions and a series of fibroin substrates differing in
surface and bulk morphology and structure (gel, powder, and fibre). Tyrosinase was able to
oxidize about 30% and 57% of the tyrosine residues of soluble fibroin and sericin,
respectively. The yield of the reaction decreased under heterogeneous reaction conditions
(about 10–11% of tyrosine was oxidized in silk gels) owing to steric hindrance which limited
the accessibility of the aromatic side chain groups buried into the compact protein matrix. The
concentration of tyrosine in oxidized samples decreased gradually with increasing the
enzyme-to-substrate ratio. FT-IR and FT-Raman spectroscopy gave evidence of oxidation.
New bands attributable to vibrations of oxidized tyrosine species (o-quinone) appeared while
the intensity of tyrosine bands decreased. The average molecular weight of sericin
significantly increased by oxidation, indicating that cross-linking occurred via selfcondensation
of o-quinones and/or coupling with the free amine groups of lysine. When
oxidation of silk proteins was conducted in the presence of chitosan, protein-polysaccharide
bioconjugates were obtained, which were characterized by thermal analysis and FT-IR and
Raman spectroscopy. Spectral changes were interpreted in terms of reaction mechanism. The
results obtained in this study show the potential of the enzymatically initiated protein–
polysaccharide grafting for the production of a new range of environmentally friendly
polymers. Grafting with Ch may impart useful antimicrobial activity. Moreover, the use of
other functional compounds with nucleophile groups reactive towards quinones may extend
the range of performance of enzyme-modified silk proteins.
126 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P64
Kinetics of Laccase Mediator System Delignification of an
Eucalyptus globulus Kraft Pulp
Sílvia Guilherme a , Ofélia Anjos a , Rogério Simões b
a Escola Superior Agrária, Instituto Politécnico de Castelo Branco, Quinta da Senhora de
Mércules, Apartado 119, 6001 Castelo Branco, Portugal; b Unidade de Materiais Têxteis e
Papeleiros, Universidade da Beira Interior, Convento de Santo António, 6201-001, Covilhã,
Portugal
E-mail: ofelia@eesa.ipcb.pt
Laccase mediator system (LMS) was applied to one industrial Eucalyptus globulus kraft pulp
with kappa numbers 15.2, using violuric acid (VA) as mediator. The objective of the present
work is to quantify the influence of the reaction conditions on the delignification rate and
extent, establishing the kinetic equations. The effects of oxygen pressure, laccase and
mediator charges, and reaction time on delignification were evaluated. The kinetic studies
were carried out in a 1.5 L jacketed reactor with temperature control and magnetic mixer. The
experiments were carried out with 10 grams of pulp at very low consistency (0.6%) in order
to minimize inter-fibre mass transfer resistances. The oxygen pressure was varied between 1
and 7 bar and no significant differences were observed in terms of delignification rate and
extent, at a given charge of laccase and mediator. The laccase (EC 1.10.3.2) charge was
ranged between 10 and 250 IU per gram of pulp and the mediator between 10 and 70 mg per
gram of pulp. The presence of mediator is required because the enzyme cannot diffuse into
the porous structure of the fibre wall, where lignin should be oxidised. The delignification
potential of the LMS was evaluated by measuring the kappa number of the pulp, after
alkaline extraction. Control tests similar to the LMS followed by alkaline extraction, but
without enzyme, were carried out and the mean value of kappa number was 14.04. The
decrease of the kappa number of the pulp from 15.2 to 14.04 can be interpreted as the
consequence of the extraction of some fragments of lignin during the two stages. This
procedure enable us to access the real effect of laccase. The hexeneuronic acid (HexA) has,
particularly in hardwood pulps, an important contribution to the kappa number value.
However, the experimental data have shown that LMS does not remove significantly the
HexA, which is in good agreement with the literature. So, the kappa number can be used to
evaluate the potential of LMS to lignin extraction. For the levels of laccase 50 IU per gram
and 40 mg of VA per gram, the delignification was reached 37%, which is a good result. The
profile of kappa number with reaction time follows an exponential trend. In addition, the
initial rate methodology is being used to quantify the influence of laccase and mediator
concentrations on the kinetic rate. The data have shown that the delignification rate exhibits a
linear dependence on the mediator concentration, for the low range tested. The effect of
laccase charge seems to be lower. The experimental data are under exploitation.
127 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Model Wastewaters Decolouristion by Pseudomonas
putida MET94
Bruno Mateus a , Diana Mateus b,c , Luciana Pereira b,c , Orfeu Flores a , Lígia O. Martins b, c
P65
a STAB VIDA, Av da República, 2781-901 Oeiras, Portugal, b Instituto de Tecnologia Química
e Biológica (ITQB),Universidade Nova de Lisboa, Av da República, 2784-505 Oeiras,
Portugal, c Instituto de Biologia Experimental e Tecnológica (IBET), Av da República, 2784-
505 Oeiras, Portugal
E-mail: bamateus@gmail.com
Azo aromatic dyes are the major group of textile dyestuff. These are chemically stable
structures to meet various colouring requirements and often are not degraded and/or removed
by conventional physical and chemical processes. Moreover, many of these compounds are
highly resistant to microbial attack and therefore, hardly removed from effluents by
conventional biological processes such as activated sludge treatment. Over the last decades,
considerable work has been done with the goal of using microorganisms as bioremediation
agents in the treatment of wastewater containing textile dyes.
Pseudomonas putida strain MET94 was selected among 84 bacterial strains has the most
active textile dye degrader. This strain showed significant decolourization improvement on 6
different azo dyes but no effect on anthraquinonic dyes. This strain was able to decolorize up
to 70-85% of Reactive R4 (RR4), Reactive black 5 (RB5), Direct Blue 1 (CSB), Acid Red
299 (NY1), Direct Black 38 (CB) and Direct Red 28 (CR) out of 11 different dyes tested,
after 24h of growth in complex liquid culture media. Higher degradation rates as well as
higher extent of decolourization were obtained in anaerobic when compared with aerobic
conditions. In the absence of oxygen (i) degradation is growth associated, (ii) specific growth
rates were higher in the presence of dyes, suggesting that these could be used as electron
acceptors in anaerobic respiration. In the presence of oxygen (i) growth rates as well as
biomass yields were lower in the presence of dyes, suggesting that dyes could be exerting
toxicity over cells, (ii) maximum decolourization activity occurred at the late exponentialstationary
growth phase. Both in aerobic and in anaerobic conditions the enzymatic catabolic
system employed is constitutive as growth initiated by adapted and nonadapted innocula did
not present any significant difference.
The ability of bacterial strain P. putida MET94 in the decolourisation of four wastewater
models: (i) Acid dye bath for wool (ii) Acid dye bath for leather, (iii) Reactive dye bath (for
cotton) and, (iv) Direct dye bath (for cotton) was assessed. Whole-cell catalysis systems under
oxic and anoxic conditions were tested. Decolourisation was shown to be pH dependent;
higher decolourisations were found at pH 5 for the acid baths and at pH 8 for the direct and
reactive baths. After 24 days P. putida (OD 600nm =15) was able to decolourise around 70-90%
of the acid dye baths, around 80%-90% of the direct bath and around 40%-60% of reactive
bath model wastewaters tested. However, for the direct model wastewater when
decolourisation was monitored at 400 nm the highest decolorization was observed around
30%.
This work has been done in the frame of EC-F6P SOPHIED project - “Novel Sustainable Processes for the
European Colour Industries” (FP6-NMP2-CT-2004-505899).
128 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
P66
Cellulose-Based Agglomerates from Enzymatically
Recycled Paper Wastes
Tina Bruckman, Margarita Calafell, Tzanko Tzanov
Technical University of Catalonia, Colom 1, 08222 Terrassa, Spain
E-mail: tzanko.tzanov@upc.edu
This work reports on the enzymatic processing of paper wastes from the graphics industry
into useful agglomerates. These heavily loaded with inks and additives paper wastes,
normally not reusable, were submitted to treatment with an enzymatic cocktail containing
cellulases, hemicellulases and pectinases. Thereby the strength of the cellulose fibres was
preserved eliminating the defibering and deinking operations in paper recycling. In the
following step laccase was added to the enzymatically-treated paper mass, which was further
submitted to vacuum filtration to obtain the agglomerate product. In this way a complete
reuse of the paper material and included additives was achieved. The resulting panel-like
agglomerated material showed improved exploitation characteristics making it useful in
packaging, construction and other application.
129 September 7-9, 2006
Oeiras, Portugal

PARTICIPANTS

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Agathos, Spiros N.
Unit of Bioengineering (GEBI)
University of Louvain
Place Croix du Sud 2/19
B-1348 Louvain-la-Neuve
Belgium
Tel: +32 10473644
e-mail: spiros.agathos@uclouvain.be
Arboleda Echavarría, Carolina
Calle 67 No. 53- 108 A.A. 1226
Universidad de Antioquia
Facultad Química Farmacéutica
Grupo Cienica de los Materiales
Colombia
Tel: 574 2106549
e-mail: carboled@farmacia.udea.edu.co
Allen, Christopher
School of Biological Sciences,
Queen’s University Belfast,
Medical Biology Centre,
97 Lisburn Road, Belfast BT9 7BL,
Northern Ireland
Tel: +44 28 90976547
e-mail: c.allen@qub.ac.uk
Amaral, Priscilla F. F.
Escola de Química/UFRJ
Centro de Tecnologia, Bloco E, Lab. 113
Cidade Universitária, Ilha do Fundão
CEP 21949-900
Rio de Janeiro,RJ, Brasil
Tel: 00 55 21 2562 7622
e-mail: pffamaral@gmail.com
Andberg, Martina
Tietotie 2
P.O. Box 1500
FIN-02044 VTT
Finland
Tel: +358207225124
e-mail: martina.andberg@vtt.fi
Ander, Paul
WURC, Dept. of Wood Science, SLU,
PO Box 7008
SE-75007 Uppsala
Sweden
Tel: 46-18-67 34 34
e-mail: paul.ander@trv.slu.se
Anjos, Ofélia
Escola Superior Agrária,
Quinta da Senhora de Mércules, Apartado
119, 6001-909 Castelo Branco
Portugal
Tel: 272339900
e-mail: ofelia@esa.ipcb.pt
Arias, Maria Enriqueta
Departamento de Microbiologia y Parasitologia
Universidad de Alcalá
Alcalá de Henares
Spain
Tel: +34918854633
e-mail: enriquetaarias@uah.es
Baldrian, Petr
Institute of Microbiology ASCR
Videnska 1083
14220 Praha 4
Czech Republic
Tel: +420723770570
e-mail: baldrian@biomed.cas.cz
Basosi, Riccardo
Department of Chemistry
University of Siena
Via Aldo Moro
53100 Siena, Italy
Tel: +39577234240
e-mail: ribasosi1@tin.it
Beckett, Richard
School of Biological Sciences
University of KwaZulu-Natal
PBag X01
Scottsville 3209, South Africa
Tel: +27 33 260 5141
e-mail: rpbeckett@gmail.com
Behar, Candan Tamerler
Istanbul Technical University Department of
Molecular biology and Genetics
Maslak/Istanbul
Turkey
Tel: 0090 212 286 22 51
e-mail: tamerler@itu.edu.tr
132 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Belova, Nina V.
Komarov Botanical Institute RAS,
Prof. Popov Str., 2,
St. Petersburg 197376
Russia
Tel: +7(812)3464442
e-mail: cultures@mail.ru
Bento, Isabel
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469662
e-mail: bento@itqb.unl.pt
Bermek, Hakan
Istanbul Technical University Department of
Molecular biology and Genetics
Maslak/Istanbul
Turkey
Tel: 0090 212 286 22 51
e-mail: bermek@itu.edu.tr
Bezerra, Rui Manuel Furtado
Dep. Engenharia Biológica e Ambiental,
UTAD, Apartado 1013,
5001-801 Vila Real,
Portugal
Tel: 259350465
e-mail: bezerra@utad.pt.
Briganti, Fabrizio
Department of Chemistry
University of Florence
Via della Lastruccia 3
Sesto Fiorentino 50019
Florence, Italy
Tel: +39 055 4573343
e-mail: fabrizio.briganti@unifi.it
Brissos, Vânia
Instituto Superior Técnico
Centro de Engenharia Biológica e Química
Portugal
Tel: 218419132
e-mail: vaniabrissos@ist.utl.pt
Cabana, Hubert
Department of Chemical engineering
University of Sherbrooke
2500 Boulevard de l’Université
Sherbrooke (Qc) J1K 2R1
Canada
Tel : +18198217171
e-mail : hubert.cabana@usherbrooke.ca
Call, Hans-Peter
Bioscreen e.K.
52531 Uebach-Palenberg
Heinsberger Strasse 15
Germany
Tel: 0049 2451 952814
e-mail: Bioscreen@t-online.de
Boehmer, Ulrike
Technische Universität Dresden,
Institute for Food Technology and Bioprocess
Engineering,
Bergstraße 120 01069 Dresden
Germany
Tel: +49 351 46334882
e-mail: ulrike.boehmer@tu-dresden.de
Bols, Christian-Marie
Wetlands Engineering SPRL
Parc Scientific Fleming
5 Rue du Laid Burniat
BE-1348 Louvain-la-Neuve
Belgium
Tel : +32478421647
e-mail : ch.bols@wetlands.be
Caruso, Carla
Dipartimento di Agrobiologia e Agrochimica
Via S. Camillo de Lellis
01100 Viterbo
Italy
Tel:+390761357330
e-mail: caruso@unitus.it
Casieri, Leonardo
Department of Plant Biology, University of
Turin.
Viale Mattioli 25, 10125 Turin
Italy
Tel: 00390116705964
e-mail: leonardo.casieri@unito.it
133 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Cavaco-Paulo, Artur
Departamento de Engenharia Têxtil
Universidade do Minho
Campus de Azúrem
4800-058 Guimarães
Portugal
Tel: +351 253510280
e-mail: artur@det.uminho.pt
Chen, Zhenjia
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 2144697653
e-mail: chen@itqb.unl.pt
Coelho, Maria Alice Zarur
Escola de Quimica / UFRJ
Centro de Tecnologia, Bl. E, Lab. 113, Cidade
Universitaria, 21949-900 Rio de Janeiro-RJ
Brasil
Tel: 55 21 25627622
e-mail: alice@eq.ufrj.br
Costa-Ferreira, Maria
Department of Biotechnology- INETI
National Institute for Engineering, Technology
and Innovation
Estrada do Paço do Lumiar, 22
1649-038 Lisboa
Portugal
Tel: 351 21 0924720
e-mail: maria.ferreira@ineti.pt
Danielsen, Steffen
Protein Design
Building 1U1.20
Brudelysvej 26
Novozymes A/S
Denmark
Tel: +4544427761
e-mail: sted@novozymes.com
de la Rubia Nieto, Teresa
Dpto. Microbiology Faculty of Pharmacy Univ.
of Granada
Campus Cartuja 18071 Granada (Spain)
Spain
Tel: 38 958243875
e-mail: dlrubia@ugr.es
Coelho, Rui
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469535
e-mail: coelhor@itqb.unl.pt
Colao, Maria Chiara
Dept. Agrobiology and Agrochemistry,
Tuscia University
Via C. de Lellis snc, 01100 Viterbo
Italy
Tel: +390761357236
e-mail: colao@unitus.it
Cortez, João
Nottingham Trent University, Erasmus Darwin
Clifton Campus,
Nottingham, NG11 8NS
England
Tel: 0115 848 3089
e-mail: joao.cortez@ntu.ac.uk
de Wildeman, Stefaan
DSM-Research
Dept. LS-ASC&D
PO Box 18
6160 MD Geleen
The Netherlands
Tel: +31 46 4760138
e-mail: stefaan-de.wildeman@dsm.com
Del Rio, Jose C.
Instituto de Recursos Naturales y Agrobiologia
(IRNAS, CSIC)
Reina Mercedes 10, PO Box 1052; 41080
Seville
Spain
Tel: +34 95 4624711
e-mail: delrio@irnase.csic.es
Dernalowicz-Malarczyk, Elzbieta
Biochemistry Department,
M.Curie-Sklodowska University,
Sklodowska Square 3, 20-031 Lublin
Poland
Tel: +4881 5375770
e-mail: malar@hermes.umcs.lublin.pl
134 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Desnos, Thierry
Laboratoire de Biologie du Développement des
Plantes,
DEVM,CEA cadarache,
13108 St Paul-lez-Durance cedex,
France
Tel: (33) 4 42 25 31 52
e-mail: thierry.desnos@cea.fr
Dias, José Albino Gomes Alves
Dep. Engenharia Biológica e Ambiental,
UTAD, Apartado 1013,
5001-801 Vila Real,
Portugal
Tel: 259350725
e-mail: jdias@utad.pt
Ergun, Aisle
Istanbul Technical University Molecular
Biology and Genetics Dept. 34469
Maslak/Istanbul
Turkey
Tel: +90 212 286 22 51
e-mail: asl_ergun@yahoo.com
Evtuguin, Dmitry V.
Department of Chemistry
University of Aveiro
3810-193 Aveiro
Portugal
Tel: +351 234 370693
e-mail: Dmitry@dq.ua.pt
Domínguez Represas, Alberto
Department of Chemical Engineering.
Isaac Newton Building.
University of Vigo.
36310 Vigo.
Spain
Tel: 34986812304
e-mail: alberdom@uvigo.es
Faraco, Vincenza
Department of Organic Chemistry and
Biochemistry,
Universitá degli Studi di Napoli Federico II
Complesso Universitario Monte S.
Angelo80126 Napoli
Italy
Tel: +39 081674324
e-mail: vfaraco@unina.it
Durão, Paulo
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469535
e-mail: pdurao@itqb.unl.pt
Elisashivili, Vladimir
Inst of Biochemistry and Biotechnology
10km Agmashenebeli Kheivani
0159 Tblisi
Georgia
Tel: +97248249653
e-mail: velisashvili@hotmail.com
Enaud, Estelle
Unité de Microbiologie (MBLA)
Univ Catholique Louvain-la-Neuve
Place Croix du Sud 3, bte-6
1348 Louvain-La-Neuve
Tel: +32 10 47 30 84
e-mail: enaud@mbla.ucl.ac.be
Fernandes, André T.
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469535
e-mail: andref@itqb.unl.pt
Festa, Giovanna
Department of Organic Chemistry and
Biochemistry,
Universitá degli Studi di Napoli Federico II
Complesso Universitario Monte S. Angelo, via
Cintia 4
80126 Napoli, Italy
Tel: +39081674324
e-mail: giofesta@unina.it
Fillat, Amanda
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469535
e-mail: amanda@itqb.unl.pt
135 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Fonseca, Bruno
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: 937515800
e-mail: bfonseca@itqb.unl.pt
Freddi, Giuliano
Stazione Sperimentale per la Seta
via Giuseppe Colombo, 83
20133 Milano
Italy
Tel: +39 02 2665990
e-mail: freddi@ssiseta.it
Gómez Sieiro, José
Department of Chemical Engineering. Isaac
Newton Building. University of Vigo. 36310
Vigo. Spain
Spain
Tel: 34986812304
e-mail: josegomez@uvigo.es
Gravitis, Janis
SA Latvian State Institute of Wood Chemistry
Dzerbenes St.27
Riga LV-1006
Latvia
Tel: ++371 7553137
e-mail: jgravit@edi.lv
Galli, Carlo
Dipartamento de Chimica
Universita “La Sapienza”
Roma
Italy
Tel: +390649913386
e-mail: carlo.galli@uniroma1.it
Garzillo, Anna Maria Vittoria
Dipt. of Agrobiology and Agrochemistry
Via S. Camillo de Lellis, snc
01100 - Viterbo
Italy
Tel: +390761357316
e-mail: amg@unitus.it
Graz, Marcin
Department of Biochemistry,
Maria Curie-Sklodowska University,
Sklodowska Square 3,
20-031 Lublin
Poland
Tel: +4881 5375735
e-mail: mgraz@biotop.umcs.lublin.pl
Güebitz, Georg
Graz University of Technology,
Dept of Environmental Biotechnology
Petersgrasse 12, 8010 Graz
Austria
Tel: +43 316 8738312
e-mail: guebitz@tugraz.at
Giardina, Paola
Department of Organic Chemistry and
Biochemistry,
Universitá degli Studi di Napoli Federico II
Complesso Universitario Monte S. Angelo, via
Cintia 4
80126 Napoli, Italy
Tel: +39 081674319
e-mail: giardina@unina.it
Golovleva, Ludmila
G.K. Sryabin Institute of Biochemistry and
Physiology of Microorganisms RAS
Moscow
Russian Federation
Tel: +4967732564
e-mail: golovleva@ibpm.pushchino.ru
Gutierrez, Ana
Instituto de Recursos Naturales y Agrobiologia
(IRNAS, CSIC),
Reina Mercedes 10, PO Box 1052; 41080
Seville
Spain
Tel: +34 95 4624711
e-mail: anagu@irnase.csic.es
Hadar, Yitzhak
Department of Microbiology and Plant
Pathology,
Faculty of Agriculture, Rehovot, Israel
Israel
Tel: 972-8-9489935
e-mail: hadar@agri.huji.ac.il
136 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Hakala, Terhi
IKCL Science & Consulting
Oy Keskuslaboratorio - Centrallaboratorium Ab
P.O. Box 70,02151 Espoo
Finland
Tel: + 358 (0) 20 7477 352
e-mail: Terhi.hakala@kcl.fi
Hakulinen, Nina
Dept. of Chemistry
University of Joensuu
PO Box 111, 80101 Joensuu
Finland
Tel: +358132512243
e-mail: nina.hakulinen@joensuu.fi
Hatakka, Annele
Department of Applied Chemistry and
Microbiology,
PO Box 56 (Viikki Biocenter),
00014 University of Helsinki
Finland
Tel: +358-9-19159314
e-mail: annele.hatakka@helsinki.fi
Hernandez Cutuli, Manuel
Departamento Microbiología y
Parasitología.Universidad de Alcalá.Campus
Universitario. NII.Km.33.6.28871.Alcalá de
Henares. Madrid
Spain
Tel: +34-918855145
e-mail: manuel.hernandez@uah.es
Hildén, Kristiina
Dept. of Appl. Chemistry and Microbiology/
Div. Microbiology
P.O.Box 56 (Viikinkaari 9)
00014 Univ. of Helsinki
Finland
Tel: +358-9-19159319
e-mail: Kristiina.S.Hilden@helsinki.fi
Hiltunen, Jaakko
KCL Science and Consulting
P.O.Box 70, FI-02151 Espoo,
Finland
Tel: +358(0)207477529
e-mail: jaakko.hiltunen@kcl.fi
Hofrichter, Martin
International Graduate School of Zittau
Environmental Biotechnology Unit
Markt 23
02763 Zittau
Germany
Tel: +493583771521
e-mail: hofrichter@ihi-zittau.de
Jarosz-Wilkolazka, Anna
Biochemistry Department
Maria Curie-Sklodowska University
Sklodowska Place 3
20-031 Lublin,
Poland
Tel: 48 81 537 56 65
e-mail: ajarosz@biotop.umcs.lublin.pl
Jolivalt, Claude
Laboratoire de Sinthése Sélective Organique
et Produits Naturels,
UMR CNRS 7573
ENSCP, 11, Rue Pierre et Marie Curie
75231 Paris cedex 05
France
Tel: +33 (0)1 44276754
e-mail : claude-jolivalt@enscp.fr
Joosten, Vivi
Laboratory of Biochemistry
Wageningen University
Dreijenlaan 3
6703 HA Wageningen
The Netherlands
Tel: +31-317-484468
e-mail: vivi.joosten@wur.nl
Junghanns, Charles
UFZ Centre for Environmental Research
Leipzig-Halle
Department of Environmental Microbiology
Permoserstraße 15
D-04318 Leipzig
Germany
Tel: +493412352547
e-mail: charles.junghanns@ufz.de
Keshavarz, Tajalli
Department of Applied and Molecular
Biosciences
University of Westminster
London W1W 6UW
Tel 44-(0)20 79115000 ext 3562
e-mail: T.Keshavarz@westminster.ac.uk
137 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Kiekens, Paul
University of Ghent
Department of Textiles
Technologiepark 907
B-9052 Gent (Zwijnaarde)
Belgium
Tel: + 329 2645735
e-mail: Paul.Kiekens@Ugent.be
Kokol, Vanja
University of Maribor,
Faculty of Mechanical Engineering,
Textile department,
Smetanova ul 17, SI-2000 Maribor
Slovenia
Tel: 00386 (0)2 220 7896
e-mail: vanja.kokol@uni-mb.si
Leferink, Nicole
Laboratory of Biochemistry
Wageningen University
Dreijenlaan 3
6703 HA Wageningen
The Netherlands
Tel: +0317-484468
e-mail: nicole.leferink@wur.nl
Lindley, Peter F.
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469261
e-mail: lindley@itqb.unl.pt
Kolomytseva, Marina
Institute of Biochemistry and Physiology of
Microorganisms RAS,
Pushchino, Moscow region, Nauka prospect 5.
Russian Federation
Tel: 7(496)7-73-25-64
e-mail: marinak@ibpm.pushchino.ru
Lundell, Taina
University of Helsinki,
Department of Applied Chemistry and
Microbiology
Finland
Tel: +358 9 19159316
e-mail: taina.lundell@helsinki.fi,
Kruus, Kristiina
VTT Technical Research Centre of Finland
P.O. Box 1500
Espoo FIN-02044 VTT
Finland
Tel: + 358-20-722 5143
e-mail: kristiina.kruus@vtt.fi
Maijala, Pekka
Department of Applied Chemistry and
Microbiology,P.O. Box 56, FI-00014 University
of Helsinki
Finland
Tel: +358-9-1915 9320
e-mail: pekka.maijala@helsinki.fi
Kurt, Gunseli
Istanbul Technical University
Department of Molecular Biology
Maslak / Istanbul
Turkey
Tel: 0090 212 286 22 51
e-mail: gunselik@yahoo.com
Lamarino, Giseppina
Università degli Studi di Napoli,
Facoltà di Agraria
Italia
Tel: +390812539166
e-mail: giusiam@hotmail.com
Marino, Gennaro
Department of Organic Chemistry and
Biochemistry
Federico II University of Naples, Via Cinthia
80126 Napoli
Italy
Tel: +39-081674312
e-mail: gmarino@unina.it
Martin, Claudia
UFZ Centre for Environmental Research
Leipzig-Halle
Permoserstr. 15; 04318 Leipzig
Germany
Tel: 0049 341 235 2547
e-mail: claudia.martin@ufz.de
138 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Martínez, Angel T.
CIB, CSIC
Ramiro de Maeztu 9
E-28040 Madrid
Spain
Phone: +34 918373112
e-mail: ATMartinez@cib.csic.es
Martínez, María J.
CIB, CSIC
Ramiro de Maeztu 9
E-28040 Madrid
Spain
Tel: +34 918373112
e-mail: MJMartinez@cib.csic.es
Martins, Lígia O.
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469534
e-mail: lmartins@itqb.unl.pt
Mateus, Bruno
STAB Vida
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469763
e-mail: bamateus@itqb.unl.pt
Maximo, Cristina
UBB-DB
INETI
Est Paço do Lumiar, 22
1649-038 Lisboa
Portugal
Tel: +351 210924729
e-mail: cristina.maximo@ineti.pt
Minibayeva, Farida
Institute of Biochemistry and Biophysics
Russian Academy of Science
2/31 Lobachevsky Street
Kazan 420111, Tatarstan
Russia
Tel: +7 8432386320
e-mail: minibayeva@mail.knc.ru
Mink, Daniel
DSM Research
Dept. LS-ASC&D
PO Box 18
6160 MD Geleen
The Netherlands
Tel: +31 46 60869
e-mail: daniel.mink@dsm.com
Moilanen, Ulla
Laboratory of Bioprocess Engineering,
Helsinki University of Technology,
P.O. Box 6100, FI-02015 TKK
Finland
Tel: +358 45 6753925
e-mail: ulla.moilanen@tkk.fi
Matijosyte, Inga
Delft University of Technology
Julianalaan 136
2628 BL Delft
The Netherlands
Tel: + 310152782693
e-mail: i.majitosyte@tnw.tudelft.nl
Matura, Anke
Professur Allgemeine Biochemie,
TU Dresden,
D-01062 Dresden
Germany
Tel: +49 351 4633 5505
e-mail: anke.matura@chemie.tu-dresden.de
Moldes Moreira, Diego
Departamento de Engenharia Textil
Universidade do Minho
Campus Azurem.
4800-Guimaraes
Portugal
Tel: +351 253510280
e-mail: diego@det.uminho.pt
Monti, Daniela
Istituto di Chimica del Riconoscimento
Molecolare -CNR, Via Mario Bianco, 9,
20131 Milano
Italy
tel: ++39 02285 00038
e-mail: daniela.monti@icrm.cnr.it
139 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Munõz-Dorado, Jose
Departamento de Microbiologia
Facultad de Ciencias
Universidad de Granada
Spain
Tel: +34958243183
e-mail: jdorado@ugr.es
Nikolov, Alexandre
Novozymes A/S
Krogshøejvej
2880 Bagsvaerd
Denmark
Tel: + 45 44492212
e-mail: alxn@novozymes.com
Olszewska, Anna
Department of Biochemistry,
Maria Curie-Sklodowska University,
Sklodowska Place 3, 20-031 Lublin
Polan
Tel: +48815375705
e-mail: aolszewska@wp.pl
Opwis, Klaus
Deutsches Textilforschungszentrum Nord-
West e.V.
Adlerstr. 1
D-47798 Krefeld
Germany
Tel: +49-2151-843-205
e-mail: opwis@dtnw.de
Ostergaard, Lars
Novozymes A/S
Dept. of Protein Diversity
Brudelysvej 26
DK-2880 Bagsvaerd
Denmark
Tel: +45 444 60271
Email: laq@novozymes.com
Oudia, Atika
Unidade de Têxteis e Materiais de Papel
UBI - Universidade da Beira Interior
6201-001 Covilhã
Portugal
Tel: +3519692165
e-mail: atika@ubi.pt
Papa, Rosanna
Department of Organic Chemistry and
Biochemistry
Federico II University of Naples
Napoli
Italy
Tel: +39 081674320
e-mail: rosapapa@unina.it
Pereira, Luciana
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469535
e-mail: luciana@itqb.unl.pt
Pereira, Manuela M.
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469321
e-mail: mpereira@itqb.unl.pt
Pérez Leblic, Maria Isabel
Departamento Microbiología y
Parasitología.Universidad de Alcalá.Campus
Universitario. NII.Km.33.6.28871.Alcalá de
Henares. Madrid
Spain
Tel: +34-918855145
e-mail: misabel.perez@uah.es
Pérez-Torres, Juana
Departamento de Microbiologia
Facultad de Ciencias
Universidad de Granada
Spain
Tel: +34958243183
e-mail: jptorres@ugr.es
Peterbauer, Clemens
Dept. of Food Sciences & Technology
University of Natural Resources & Applied Life
Sciences
Muthgasse 18
A-1190 Vienna
Austria
Tel: +43 1 36006 6274
e-mail: clemens.peterbauer@boku.ac.at
140 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Pogni, Rebecca
Department of Chemistry
University of Siena
Via Aldo Moro
53100 Siena
Italy
Tel: +39577234258
e-mail: pogni@unisi.it
Polak, Jolanta
Department of Biochemistry,
Maria Curie-Sklodowska University,
Sklodowska
Place 3, 20-031 Lublin
Poland
Tel: +48815375705
e-mail: jolanta_polak@wp.pl
Psurtseva, Nadya V.
Komarov Botanical Institute RAS,
Prof. Popov Str., 2,
St. Petersburg 197376
Russia
Tel: +7(812)3464442
e-mail: NadyaPsu@NP1512.spb.edu
Sanchez-Amat, Antonio
Facultad de Biologia
Dep de Microbiologia y Genetica
Universidad de Murcia
30071 Murcia
Spain
Tel: +34 968364955
e-mail: antonio@um.es
Sannia, Giovanni
Universitá degli Studi di Napoli Federico II
Dipartimento di Chimica Organica e
Biochimica, via Cinthia, 4 -
I-80126 Naples
Italy
Tel: +39 081 674310
e-mail: sannia@unina.it
Sanromán, Mª Angeles Braga
Department of Chemical Engineering. Isaac
Newton Building.
University of Vigo.
36310 Vigo, Spain
Tel: 34986812383
e-mail: sanroman@uvigo.es
Rebhun, Moti
MycoEnzyme, Ltd.
Institute of Evolution,
University of Haifa
Mount Carmel, Haifa 31905
Israel
Tel: +972503231065
e-mail: mrebhun@study.haifa.ac.il
Robalo, Maria Paula
Secção de Química Inorgânica
Departamento de Engenharia Química
Instituto Superior de Engenharia de Lisboa
Rua Conselheiro Emídio Navarro, 1
1959-007 Lisboa, Portugal
Tel: +351 218317163
e-mail: mprobalo@deq.isel.ipl.pt
Rodriguez Bullido, Juana
Departamento Microbiología y
Parasitología.Universidad de Alcalá.Campus
Universitario. NII.Km.33.6.28871.Alcalá de
Henares. Madrid
Spain
Tel: +34-918855145
e-mail: juana.rodriguez@uah.es
Schlosser, Dietmar
UFZ Centre for Environmental Research
Leipzig-Halle
Department of Environmental Microbiology
Permoserstraße 15
D-04318 Leipzig, Germany
Germany
Tel: +49 341 235 3254
e-mail: dietmar.schlosser@ufz.de
Schroeder, Marc
Faculty of Mechanical Engineering
Institute of Textile, University of Maribor
Smetanova ul 17
2000 Maribor
Slovenia
Tel: +386 (0)2 220 7934
e-mail: marc.schroeder@uni-mb.si
Scozzafava, Andrea
Department of Chemistry
University of Florence
Via della Lastruccia 3
Sesto Fiorentino 50019
Florence, Italy
Tel: +39 055 4573273
e-mail: andrea.scozzafava@unifi.it~
141 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Seifert, Jana
Environmental Microbiology,
TU Bergakademie Freiberg, Leipziger Str. 29,
09599 Freiberg
Germany
Tel: +49-3731-394015
e-mail: jana.seifert@ioez.tu-freiberg.de
Suurnäkki, Anna
VTT
P.O. Box 1000
02044 VTT
Finland
Tel: +358 20 722 7178
e-mail: anna.suurnakki@vtt.fi
Sigoillot-Claude, Cécile
LBDP/DEVM
CEA Cadarache
13108 St Paul lez Durance Cedex
France
Tel: +33 (0)4 42 25 31 45
e-mail: cecile.claude@cea.fr
Smith, Andrew T.
Biochemisty Department
School of Life Sciences
University of Sussex
UK
e-mail: a.t.smith@sussex.ac.uk
Soares, Cláudio M.
Instituto de Tecnologia Química e Biológica
Universidade Nova de Lisboa
Av da República
2781-901 Oeiras
Portugal
Tel: +351 214469610
e-mail: claudio@itqb.unl.pt
Songulashvili, Giorgi
Institute of Evolution
University of Haifa
Mt Carmel
Haifa 31905
Israel
Tel: +97248249653
e-mail: gsongulashvili@yahoo.com
Srebotnik, Ewald
Kompetenzzentrum Holz GmbH
c/o Institute of Chemical Engineering, Vienna
University of Technology,
Getreidemarkt 9
A-1060 Wien
Austria
Tel: +43 1 58801-17242
e-mail: ewald.srebotnik@tuwien.ac.at
Todorovic, Smilja
Instituto de Tecnologia Química e Biológica
Av. da Republica EAN
2780-157 Oeiras
Portugal
Tel: 351-214469321
e-mail: smilja@itqb.unl.pt
Tranchimand, Sylvain
Laboratoire de Bioinorganique Structurale
Faculté des Sciences de St Jérôme
case 432,
13397 Marseille cedex 20
France
Tel: +33491282856
e-mail: s.tranchimand@univ-cezanne.fr
Tron, Thierry
Laboratoire of Bioinorganique Structurale
CNRS UMR 6517
Case 432, Faculté des Sciences Saint Jérôme
13397 Marseille
France
Tel : +33491282856
e-mail: thierry.tron@univ.u-3mrs.fr
Trovaslet, Marie
Faculté d'Ingenierie Biologique, Agronomique
Et Environnementale
Unité de Microbiologie (MBLA)
Place Croix du Sud 3, bte-6
1348 Louvain-La-Neuve
Belgium
Tel: +32 10 47 30 84
e-mail: trovaslet@mbla.ucl.ac.be
Tzanov, Tzanko
Technical University of
Catalonia Colom
108223 Terrasa, Barcelona
Spain
Tel: +34628081722
e-mail: tzanko.tzanov@upc.edu
142 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Valderrama, Brenda
Biotechnology Institute
University of Mexico
Av. Universidad 2001
Col. Chamilpa
CP62210 Cuernavaca, Mor.
Mexico
Tel: +527773291610
e-mail: brenda@ibt.unam.mx
van Berkel, Willem J.H.
Laboratory of Biochemistry
Wageningen University
The Netherlands
Tel: 31-317-482861
e-mail: willem.vanberkel@wur.nl
van Dijk, Alard
DSM Food Specialties 699-0330
PO Box 1
2600 MA Delft
The Netherlands
Tel: +31-152793661
e-mail: alard.dijk-van@dsm.com
van Hellemond, Erik
Laboratory of Biochemistry
Nijenborgh 4
9747 AG Groningen
Netherlands
Tel: +31-50-3633540
e-mail: E.W.van.Hellemond@rug.nl
Varesse, Cristina Giovanna
Dept. Plant Biology University of Turin viale
Mattioli, 25
10125 Turin
Italy
Tel: +39 011 6705964
e-mail: cristina.varese@unito.it
Vicente, Joao B.
Instituto de Tecnologia Química e Biológica /
Universidade Nova de Lisboa
Av. da República (EAN), Apt. 127
2784-505 Oeiras
Portugal
Tel: +351214469323
e-mail: jvicente@itqb.unl.pt
Viikari, Liisa
VTT
PO BOX 1000
02044 VTT
Espoo
Finland
Tel: +358207225140
e-mail: liisa.viikari@vtt.fi
Winquist, Erika
Laboratory of Bioprocess Engineering,
Helsinki University of Technology,
P.O. Box 6100, FI-02015 TKK
Finland
Tel: +358 50 573 1529
e-mail: erika.winquist@tkk.fi
van Pée, Karl-Heinz
Biochemie
TU Dresden
D-01062 Dresden
Germany
Tel: +49351-463-34494
e-mail: karl-heinz.vanpee@chemie.tudresden.de
Vanhulle, Sophie
Unité de Microbiologie
Université Catholique de Louvain
Place de l’Úniversité
Croix de Sud 3 boîte 6
Louvain La Neuve
Belgium
Tel : +3210473737
e-mail: vanhullesophie@hotmail.com
Xavier, Ana M.R.B.
Department of Chemistry
University of Aveiro
3810-193 Aveiro
Portugal
Tel: +351 234 370716
e-mail: abx@dq.ua.pt
Xu, Feng
Novozymes Inc
1445 Drew Ave
Davis, CA 95616
USA
Tel: 530-757-8100
e-mail: fxu@novozymes.com
143 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Yesiladali, S. Koray
Istanbul Technical University Molecular
Biology and Genetics Department
Maslak/Istanbul
Turkey
Tel: 0090 212 286 22 51
e-mail: yesiladali@itu.edu.tr
Zille, Andrea
Departamento de Engenharia Têxtil
Universidade do Minho
Campus de Azúrem
4800-058 Guimarães
Portugal
Tel: +351253510280
e-mail: azille@det.uminho.pt
Zimmermann, Wolfgang
Institute of Biochemistry
Department of Microbiology and Bioprocess
Technology
University of Leipzig
Johannisallee 21-23
D-04103
Tel: + 49 3419736781
e-mail: wolfgang.zimmermann@uni-leipzig.de
144 September 7-9, 2006
Oeiras, Portugal

AUTHOR INDEX

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Agapito, M. S. M.
P59
Böhmer, U.
P43
Agathos, S. N.
L30, P19, P62
Bols, C. M.
P17, P31
Ahola, E.
L9
Boyd, D. R.
L10
Allen, C.
L10, P25
Brault, A.
P33
Amaral, P. F. F.
P45
Briganti, F.
L23, L7, P36
Anastasi, A.
P53
Briozzo, P.
P33
Andberg, M.
P32, L21
Brogioni, B.
L19
Anghileri, A.
P63
Bruckman, T.
P66
Anh, D. H.
L16
Buchert, J.
L9, P63
Anjos, O.
P64
Buonocore, V.
P9, P47
Arboleda, C.
P62
Cabana, H.
P19, P62
Arends, W. C. E.
P68
Cajthaml, T.
L6
Arias, M. E.
P44, P61
Calafell, M.
P66
Asimgil, H.
P14
Call, H.-P.
L34
Auer, S.
P32
Caminade, E.
P33
Autore, F.
L17, P15
Cammarota, M. C.
P45, P46
Baldrian, P.
L6
Caporale, C.
P9
Baratto, M. C.
P25, L19
Caruso, C.
P9
Barrasa, J. M.
L31
Casieri, L.
P53
Basar, F.
P42
Cavaco-Paulo, A.
L26, L25, P20, P39
Basosi, R.
L18, L19, P25
Cestone, R.
P15
Basto, C.
L26
Chasov, A. V.
P3
Batista, C. F.
L15
Chernykh, A.
L7, L23
Baumberger, S.
P33
Choinowski, T.
L18
Bebrone, C.
P17
Coelho, Mª A. Z.
P45, P46
Beckett, R.
P1
Colao, M. C.
P47
Behar, T.
P42
Corbisier, A. M.
L27, P17, P41
Belova, N. V.
P4, P6, L7
Costa-Ferreira, M.
L35
Bento, I.
P34, P35
Creff, A.
L3
Bermek, H.
P14
D'Annibale, A.
L20
Bertini, L.
P9
de la Rubia Nieto, T.
P12
Bezerra, R. M. F.
P13, P51
de Vries, S.
P26
Blanchet, A.
L3
del Rio, J. C.
L33
148 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Dernalowicz-Malarczyk, E.
P7, P29, P52
Gil, P.
L15
Desnos, T.
L3
Golovleva, L.
L7, L23, P36
Di Berardino, I.
P9
Gómez, D.
L1
Dias, J. A. G. A.
P13, P51
Gómez-Santos, N.
L5, P8
Domínguez Represas, A.
P48, P60
Gómez-Sieiro, J.
P49
Dong, C.
L14
Gominho, J.
L35
Doyle, W.
L24
Gordon, L. K.
P3
Durão, P.
P34, P35
Górnacka, B.
P39
Elisashvili, V.
L4
Grąz, M.
P7, P29, P52
Enaud, E.
L27, P17, P41
Grönqvist, S.
L36
Ergun, A.
P42
Guebitz, G. M.
L25, P21, P58
Ernyei, A.
L14
Guevara, O.
P61
Evtuguin, D. V.
P55, P59
Guilherme, S.
P64
Fagerström, R.
P16
Guillén, F.
P44
Faraco, V.
L17
Gullotto, A.
L23
Fernandes, A. T.
P34, P35, P38
Gutiérrez, A.
L33
Ferranoni, M.
L7, L23, P36
Hakala, T. K.
P2, P5
Festa, G.
L17, P15
Hakulinen, N.
L21, P32
Flores, O.
P65
Haltrich, D.
P28
Fonseca, B.
P67
Hatakka, A.
L2, P2, P5, P6, P18
Fraaije, M. W.
L8
Hatscher, C.
L14
Fraga, I.
P13, P51
Hernández, M.
P44
Fraternali, F.
L17
Hernandéz-Romero, D.
P23
Freddi, G.
P63
Hildebrandt, P.
P37
Frère, J.-M.
P17
Hildén, K.
L2, P2, P5
Galli, C.
L20
Hofrichter, M.
L16
Gamelas, J. A .F.
P55
Huber, R.
P38
Garzillo, A. M. V.
P47
Hubert, S.
P17
Gaudin, C.
P30
Hüner, P.
P14
Gaydou, V.
P30
Iacazio, G.
P30
Gentili, P.
L20
Iamarino, G.
L31, P70
Gianfreda, L.
L31, P70
Ibarra, D.
L33
Giardina, P.
L17, L19, P15, P24
Irgoliç, R.
P20
149 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Ivancich, A.
L24
Kvesitadze, G.
L4
Janssen, D. B.
L8
Lantto, R.
P63
Jarosz-Wilkolazka, A.
P7, P29, P50, P52, P54
Larkin, M. J.
P25
Jimenez, G. A.
P62
Laufer, Z.
P1
Jolivalt, C.
P33
Leferink, N.
L11
Jones, J. P.
P19, P62
Leontievsky, A. A.
L23
Joosten, V.
P26
Lindley, P. F.
P34, P35
Junghanns, C.
L28, P69
Lipscomb, D. A.
P25
Kachlishvili, E.
L4
Longo, M. A.
P60
Kalkkinen, N.
L9
Lorenzini, B.
P17
Kallio, J.
P16
Lourenço, A.
L35
Kandelbauer, A.
L25
Louro, R. O.
P67
Karagüler, N. G.
P10, P11
Lu, Y.
L11
Karatas, A.
P10, P11
Lucas, M.
P12
Kaschabek, S.
P27
Lucas-Elío, P.
L1
Kavieva, A. A.
P3
Ludwig, R.
P28
Kim, S.-Y.
L26
Lundell, T.
L1, L2, P6
Kinne, M.
L16
Luterek, J.
P54
Kiyashko, A.
P4, P6
Madzak, C.
P33
Kluge, M.
L16
Magali, C.
P46
Knittel, D.
L32
Maijala, P.
P2,P5, P18
Kochmanska-Rdest, J.
P7, P50
Makela, M. R.
L2
Koivula, A.
L21, P32
Marchisio, V. F.
P53
Kokol, V.
P21, P58
Martin, C.
L28
Kolesnikov, O. P.
P3
Martínez, A. T.
L18, L31, L33
Kolomytseva, M. P.
P36
Martínez, J.
P12
Kooij. R.
P68
Martínez, M. J.
L31, L18, P2
Krauss, G.
L28
Martins, L. O.
L22, P34, P35, P38, P40, P65
Kruus, K.
L9, L21, P16, P32, P63
Marzorati, M.
P57
Kulakov, L. L.
L10
Matera, I.
L23
Kuncinger, T.
L12
Mateus, B.
P65
Kunzendorf, A.
L14
Mateus, D.
P65
Kurt, G.
P10, P11
Matijosyte, I,
P68
150 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Matura, A.
P22, P43
Olszewska, A.
P7, P50, P54
Máximo, C.
L35
Onderwater, R. C. A.
P31
Mejía, A. I.
P62
Opwis, K.
L32
Melo, E. P.
P34, P38
Orlandi, M.
L36
Merhautová, V.
L6
Orozco, A. L.
P61
Mertens, V.
L27
Oudia, A.
P56
Metreveli, E.
L4
Öztemel, Z. P. Ç.
P42
Mettälä, A.
P18
Pacheco, I.
P67
Mikiasshvili, N.
L4
Paloheimo, M.
P16
Mikkonen, H.
L36
Pamplona-Aparicio, M.
P17, P41
Mimmi, M. C.
P33
Papa, R.
P24
Minibayeva, F. V.
P1, P3
Parrilli, E.
P24
Mityashina, S. Y.
P3
Pastinen, O.
P18
Moeder, M.
L28
Penninck, M. J.
P62
Moilanen, U.
P18
Pereira, A. N.
P13
Moldes, D.
P20, P48, P49, P60
Pereira, H.
L35
Molina, J. M.
P44, P61
Pereira, L.
P40, P65
Monti, D.
P57
Pereira, M.
P38
Monti, P.
P63
Pereira, P. M.
P67
Moraleda-Muñoz, A.
L5, P8
Pérez, M. I.
P61
Morales, M.
L18
Pérez-Boada, M.
L18
Mougin, C.
P33
Pérez-Torres, J.
L5, P8
Moya, R.
P44
Peterbauer, C.
P28
Muñoz-Dorado, J.
L5, P8
Pich, A.
P43
Murgida, D.
P37
Pinto, F. V.
P45
Myasoedova, N. M.
L7, L23
Piontek, K.
L18
Naismith, J. H.
L14
Piscitelli, A.
L17, P15
Ngo, E.
L24
Poellinger-Zierler, B.
L25
Nouaimeh, N.
P17
Pogni, R.
L19, L18, P25
Novotný, C.
P53
Polak, J.
P7, P50, P52
Nussaume, L.
L3
Polvillo, O.
P61
Nyanhongo, G.
L25
Pontes, A. S. N.
L20, P55
Olsson C.
P2
Prigione, V.
P53
151 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Psurtseva, N. V.
L7, P4, P6
Scozzafava, A.
L7, L23, P36
Puranen, T.
P16
Seifert, J.
P27
Queiroz, J.
P56
Selinheimo, E.
L9
Rao, M. A.
P70
Sergi, F.
L20
Rehorek, A.
P39
Sheldon, R. A.
P68
Rencoret, J.
L33
Sharma, N. D.
L10
Reymond, M.
L3
Sigoillot-Claude, C.
L3
Ricaud, L.
L3
Silvestri, F.
P47
Riva, S.
P57
Simeonov, P.
P27
Rodakiewicz-Nowack, J.
P54
Simões, R.
P56, P64
Rodríguez, J.
P61
Sinicropi, A.
L19
Rodríguez-Rincon, F.
P12
Smith, Andrew T.
L24
Rodríguez-Solar, D.
P49
Snajdr, J.
L6
Rouvinen, J.
L21, P32
Soares, C. M.
P38
Ruiz-Dueñas, F. J.
L18
Solano, F.
L1, P23
Rumpf, J.
L14
Solé, M.
L28
Russo, F.
P70
Srebotnik, E.
L12
Ruzzi, M.
P47
Stancarone, V.
P9
Sagui, F.
P57
Suarez, A.
P12
Saloheimo, M.
L9
Suurnäkki, A.
L36
Sampaio, S.
P63
Svistoonoff, S.
L3
Sanchez-Amat, A.
L1, P23
Svobodová, K.
P53
Sánchez-Sutil, M. C.
L5, P8
Taddei, P.
P63
Sannia, G.
L17, L19, P15, P24
Tadesse, M. A.
L20
Sanromán, M. A.
P48, P49, P60
Tamerler, C.
P10, P11, P14
Scelza, R.
P70
Ters, T.
L12
Scheibner, K.
L16
Tilli, S.
L23
Schlömann, M.
P27
Todorovic, S.
P37
Schlosser, D.
L28, P69
Tranchimand, S.
P30
Schmid, C.
L14
Tron, T.
P30
Schnerr, H.
L14
Trovaslet, M.
L27, P17, P41
Schollmeyer, E.
L32
Tsiklauri, N.
L4
Schroeder, M.
L25, P21, P58
Tutino, M. L.
P24
152 September 7-9, 2006
Oeiras, Portugal

Oxizymes in Oeiras, 3 rd European Meeting in Oxizymes
Tzanov, T.
P66
Vehmaanperä, J.
P16
Ullrich, R.
L16
Viikari, L.
L36
Valásková, V.
L6
De Vries, S.
P68
Valderrama, B.
L15
Wage, T.
L14, P43
Valtakari, L.
P16
Westerholm-Parvinen, A.
L9
van Berkel, W. J.H.
L13, L11, P26
Winquist, E.
P18
van den Berg, W. A. M.
L11, P26
Xavier, Ana M.R.B.
P59, P55
van Hellemond, E.
L8
Xu, F.
L29
van Pée, K. H.
L14, P22, P43
Yakovleva, N.
P4, P6
Vanhulle, S.
L27, P17, P41
Yesiladali, S. K.
P10, P42
Varese, G. C.
P53
Zámocky, M.
P28
Vazquez-Duhalt, R.
L15
Zille, A.
L26, P39, P20
153 September 7-9, 2006
Oeiras, Portugal