VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

orange juice industry and its utilization in the PL production leads to anincrease in yield with a reduction in the process cost; moreover, it adds valueto the waste from the orange juice industry. The goal of this work was todetermine an efficient methodology to isolate the major extracellular pectinlyase produced by Aspergillus giganteus and to characterize its enzymaticactivity. The main PL was purified after three steps: 1. DEAE-Sephadex A-50 column equilibrated with imidazole-NaOH buffer pH 6.0, proteins wereeluted with a linear gradient from 0 to 1.0 M NaCl; 2. CM-Sephadex C-50column equilibrated with sodium acetate buffer pH 5.5, proteins were elutedwith a linear gradient from 0 to 0.5 M NaCl; 3. Sephadex G-100 columnequilibrated with ammonium acetate pH 6.8. After the three steps the PLpresented eletrophoretic homogeneity and purification fold of 47.8 withrecovery of 12.4%. The purified PL has a molecular weight of 55 kDa. Theenzyme presented its higher activity when incubated in pH 8.5 and 50ºC. Inthe absence of substrate the PL is reasonably stable at 40ºC, keeping 70% ofits activity after 15 min, but at 50ºC the enzyme loses its activity fast with ahalf-live of 9 min, although the A. giganteus PL are more stable than thecommercially available enzymes Rapidase C80 ® (DSM) and PectinaseCCM ® (Novozymes). The best condition to stock the enzyme at 4ºC is inacid and neutral solution. The PL degrades better citrus pectins with 72 and34% of esterification, but is also able to degrade apple pectin and highesterified citrus pectin. The kinetics parameters, measured on citrus pectin72% esterified, were K m 4.8 mg.mL -1 , V max 1,129.8 U.mg -1 .min -1 and K cat770 s -1 .[1] Hoondal, G.S. et al (2002): Appl Microbiol. Biotechnol., 59, 409-418.[2] Ortega, N. et al (2004): Int. J. Food. Sci. Technol., 39, 631-639.FBP026The role of BEM-1 as possible regulator of NOX-1 andNOX-2inNeurospora crassaN. Cano*, J. AguirreInstitute of Cellular Physiology, Department of Cell and DevelopmentalBiology, National Autonomous University, MexicoReactive oxygen species (ROS) play essential roles in cell differentiation inmicrobial eukaryotes (Lara-Ortíz et al., 2003, Aguirre et al., 2005, Cano-Domínguez et al., 2008). The NADPH oxidase (NOX) enzyme complexcatalyzes the production of superoxide by transferring electrons fromNADPH to O 2. The phagocytic NOX consists of the membrane-associatedcatalytic core gp91 phox and p22 phox subunits (cytochrome b558). Theassembly of the cytosolic regulatory proteins p47 phox , p40 phox , p67 phox andRac2 with cytochrome b 558 results in NOX activation. Neurospora crassacontains two NADPH oxidase genes (nox-1 and nox-2), which encodeproteins that are homologous to phagocyte Nox2 (gp91 phox ). We reportedthat deletion of nox-1 results in mutants unable to differentiate sexualfruiting bodies and show reduction of growth and asexual development. Theinactivation of nox-2 only affects the germination of the sexual spores(ascospores). N. crassa NOX activity requires other proteins like the p67 phoxortologue NOR-1, and possibly other proteins like BEM-1 which wasproposed as a functional homologue of p40 phox (Kawahara and Lambeth,2007)). BEM-1 contains two amino-terminal Src homology 3 (SH3)domains and carboxy-terminal PX and PB1 domains. N. crassa mutantslacking BEM-1 (bud emergence protein in S. cerevisiae) show a decrease inradial growth but were are able to develop normal sexual fruiting bodies,while most ascospores from Δbem-1 homozygous crosses failed togerminate. This results suggests that BEM-1 is required for full NOX-1activity during vegetative growth and for NOX-2 activity during ascosporegermination but is fully dispensable for NOX-1 function during sexualdevelopment. To test whether NOR-1 and BEM-1 interact and regulate polargrowth, we have generated nor-1::gfp and bem-1::rfp fusions and willdetermine their localization.used temperate producers. An important component of this potential arecold-active (CA) enzymes, which remain active at low temperatures andhave correspondingly low temperature activity optima. Catalase (CAT, EC1.11.1.6) is one of the key antioxidant enzymes involved in aerobic cellresponse against oxidative stress by scavenging of H2O2. Moreover, CATcan be very useful in medicine, food, pharmaceutical and textile industry.In the present study, psychrophilic and mesophilic filamentous fungi wereisolated from samples collected in the vicinity of the permanent BulgarianAntarctic base „St. Kliment Ohridski” on Livingston Island. The purecultures from 55 isolates were identified at least to genus level and screenedfor ability to produce CA CAT. All tested strains demonstrated enzymaticpotential. Among screened isolates 25 strains produced high level of CAT.The best producers belong to genera Penicillium, Cladosporium,Aspergillus, Geomyces, Lecanicillium, Epicoccum.. Psychrotrophic strainsCladosporium oxysporum 251, Penicilium dierckxii 246, Penicilliumitalicum 232 and Aspergillus sp. 266 gave the highest activity. Thephysiological characteristics of flask and bioreactor cultures were assessedto understand optimal growth conditions. The results indicated that variousfactors including carbon and nitrogen source, air, pH, and inoculum sizeinfluence enzyme synthesis. Optimum pH and temperature for crude CATweredetermined.Acknowledgments: This work was supported by the National ScientiWcFund of the Ministry of Education and Science, Bulgaria (grants DO02-172/08 and BG051PO001-3.3.04/32), which is greatly acknowledged.FBP028Analysis of the diversity and biodegradation possibilitiesof fungi in wastewater biocoenosesB. Herzog* 1 , H. Horn 1 , E. Müller 1 , H. Lemmer 21 Institute of Water Quality Control, Technical University Munich,Garching, Germany2 Bavarian Environment Agency, Munich, GermanyFungi are ubiquitous in the environment and play an important role in avariety of different ecosystems, e.g. wastewater biocoenoses. Asdecomposers of many micro- as well as macro-pollutants, they represent anessential component in the „living” part of activated sludge. Nevertheless,our understanding of the wastewater fungal diversity and their exactfunctions in these biocoenoses remains uncertain. An attempt to gain insightinto the abundance and biodegradation abilities of wastewater fungi was theaim of this work. To shed light on some of these questions, culture-basedmethods were combined with the following molecular techniques:denaturing gradient gel electrophoresis (DGGE), PCR, DNA sequencing andthe fluorescent-in-situ-hybridization-method (FISH). Different wastewatertreatment plant (WWTP) samples were collected, enriched with the desiredcompound (the antibiotic Sulfamethoxazole) and tested for the occurrence offungi. The result was 12 different species of fungi in pure cultures able togrow on agar plates containing Sulfamethoxazole as the sole C and Nsource. Visible growth occurred within 3-5 days after inoculation, which,even compared to bacteria, is quite fast concerning that the nutrient source isan antibiotic. Chemical analysis, carried out by GC-MS/MS, will hopefullyprovide information about the extend to which Sulfamethoxazole is used andthus degraded, and if this compound is not fully mineralized, what„byproducts” are formed. Another approach that will be taken is PCR-DGGE. This cultivation-independent method will allow for thecharacterization and comparison of wastewater fungal biocoenoses withoutthe „great plate count anomaly” problem. This method reveals thecommunity’s diversity and allows for comparison of the „original” fungalspecies from activated sludge with the cultured ones. The final task will beto identify the metabolic (end)-products and hopefully link them with theproducing species.FBP027Antarctic fungi as a potential bioresource of cold-activeantioxidant enzyme catalaseE. Krumova* 1 , V. Dishlijska 1 , S. Pashova 1 , M. Angelova 1 , S. Tosi 21 The Stephan Angeloff Institute of Microbiology, Bulgarian Academy ofSciences, Mycology, Sofia, Bulgaria2 Department of Soil Ecology, University of Pavia, Pavia, ItalyIn recent years, a lot of evidence has accumulates revealing that the coldadaptedmicroorganisms possess enormous biotechnological potential,offering a range of economic and environmental advantages over previouslyFBP029Exploration of rCciAPO1 from Coprinopsis cinerea:First recombinant aromatic peroxygenaseC. Dolge*, A. Saß, R. Ullrich, M. HofrichterUnit of Environmental Biotechnology, International Graduate School (IHI)Zittau, GermanyThe gene CC1G_08427, heterologously expressed in A. oryzae byNovozymes A/S codes for a aromatic peroxygenase (APO): rCciAPO1.Spectroscopic studies with CO-bound protein indicate the constituation of aheme-thiolate enzyme. The observed ability to hydroxylate naphthalene to 1-naphthol as major reaction product and the N-oxygenation of pyridine arespektrum | Tagungsband 2011