Pleurotus ostreatus invaluable source of laccases for industrial production
A.101 - SANNIA Giovanni
A.101 - SANNIA Giovanni
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Università degli Studi di Napoli Federico II - Dipartimento di Chimica Organica e<br />
Biochimica - Biotecnologie Molecolari ed Ambientali<br />
<strong>Pleurotus</strong> <strong>ostreatus</strong>: <strong>invaluable</strong><br />
<strong>source</strong> <strong>of</strong> <strong>laccases</strong> <strong>for</strong> <strong>industrial</strong><br />
<strong>production</strong><br />
7th International Conference on Polymer ans Textile Biotechnology<br />
Milan, 1 st -4 th March 2011<br />
Giovanni Sannia
Laccases: general features<br />
Multi-copper-containing enzymes catalysing the oxidation <strong>of</strong> a wide spectrum <strong>of</strong><br />
aromatic compounds, primarily phenols and anilines, along with reducing<br />
molecular oxygen to water.<br />
The Cu1 is the primary electron acceptor site in laccase catalysed reaction. Four 1-<br />
electron oxidations <strong>of</strong> a reducing substrate occur at this site. The electron is then<br />
transferred, through the highly conserved His-Cys-His tripeptide, to the TNC,<br />
where O 2 is reduced to water.<br />
IPBT 2011, Milan Giovanni Sannia
Laccases: origin and distribution<br />
Laccase was first detected (1883) in the Japanese lac tree Toxicodendron verniciflua<br />
(<strong>for</strong>merly Rhus vernicifera).<br />
Later, it was found in certain other plants, in many insects, and in a variety <strong>of</strong> fungi.<br />
It is particularly widespread in ligninolytic basidiomycetes, and more than 125<br />
different basidiomycetous laccase genes have been described.<br />
The occurrence <strong>of</strong> laccase in prokaryotes<br />
species.<br />
seems to be restricted to certain<br />
IPBT 2011, Milan Giovanni Sannia
Laccases from white-rot fungi<br />
Glycoproteins (carbohydrate content between 10%-25%)<br />
<br />
<br />
<br />
<br />
<br />
<br />
Acidic pI<br />
Contain four copper atoms distributed into three redox sites:<br />
Type 1 (T1), Type 2 (T2) Type 3 (T3)<br />
Monomeric structure <strong>of</strong> 60-70 KDa<br />
Laccase structure is organized in three domains each with -barrel type<br />
architecture<br />
Many fungi produce several laccase isozymes differing with regard to<br />
optimum pH, substrate specificity, molecular weight, cellular localization,<br />
and quaternary structure. These enzymes are differentially expressed as<br />
function <strong>of</strong> the environmental growth conditions<br />
Laccase gene families have been found in different basidiomycetes,<br />
indicating that they may have evolved through duplication-divergence<br />
events.<br />
IPBT 2011, Milan Giovanni Sannia
Laccases: origin and distribution<br />
Due to their low substrate specifity <strong>laccases</strong> have widespread applications:<br />
• Effluent decolourisation and detoxification<br />
• Pulp bleaching;<br />
• Textile industries;<br />
• Biorefinery;<br />
• Conversion <strong>of</strong> chemical intermediates;<br />
• Removal <strong>of</strong> phenols from wines;<br />
• Fiber synthesis and grafting;<br />
• Biosensors;<br />
• Bi<strong>of</strong>uel cells;<br />
• Synthesis <strong>of</strong> drugs.<br />
IPBT 2011, Milan Giovanni Sannia
Laccases in Industry<br />
Industrial enzyme market<br />
is valued at $2 billion per<br />
annum with a potential<br />
annual growth rate <strong>of</strong> 3 to<br />
5%.<br />
Laccase stake in this market<br />
is about 4% thus making it a<br />
potential $800 million<br />
market cap.<br />
Source: BCC Research<br />
IPBT 2011, Milan Giovanni Sannia
A case study: P. <strong>ostreatus</strong> <strong>laccases</strong><br />
Task 1<br />
Structural functional<br />
characterization<br />
Laccases<br />
application<br />
Task 2<br />
Enhancing <strong>production</strong><br />
Task 3<br />
Improving enzyme<br />
per<strong>for</strong>mances<br />
IPBT 2011, Milan Giovanni Sannia
A case study: P. <strong>ostreatus</strong> <strong>laccases</strong><br />
Structural functional characterization<br />
Laccases<br />
application<br />
• Genome mining<br />
• Structure-function relationships analysis<br />
IPBT 2011, Milan Giovanni Sannia
P. <strong>ostreatus</strong> <strong>laccases</strong> gene families<br />
Investigation <strong>of</strong> the white-rot fungus P. <strong>ostreatus</strong> genome disclosed a complex MCO<br />
family composed <strong>of</strong> twelve members, ten <strong>of</strong> which correspond to <strong>laccases</strong> in sensu<br />
stricto.<br />
POX1<br />
POXC<br />
POX4<br />
LACC3<br />
LACC12<br />
LACC7<br />
POX3<br />
POXA1b<br />
LACC8<br />
POX5<br />
POXA3<br />
LACC5<br />
One putative fungal<br />
ferroxidase<br />
Seven out <strong>of</strong> twelve laccase genes<br />
and/or proteins isolated and<br />
characterized; all <strong>of</strong> them endowed<br />
with different peculiar properties<br />
One laccase<br />
pseudogene<br />
Genome mining<br />
IPBT 2011, Milan Giovanni Sannia
P. <strong>ostreatus</strong> <strong>laccases</strong><br />
POX1<br />
POXC<br />
POX4<br />
LACC3<br />
LACC12<br />
LACC7<br />
POX3<br />
POXA1b<br />
LACC8<br />
POX5<br />
POXA3<br />
LACC5<br />
A neutral blue laccase very stable at<br />
alkaline pH<br />
t 1/2 at pH 10: 100 days<br />
Giardina P., et al., Biochem. J. 1999<br />
Structure-function relationships analysis<br />
POXC is very effective in dye<br />
synthesis.<br />
Bioscreen<br />
POXC<br />
PS344<br />
CotA<br />
PO33<br />
PS7NI<br />
Remaining LAR1 ABu62produced<br />
0 500 200 1000 400 1500 600 2000 800 1000<br />
[ABu62] [LAR1] µM µM<br />
SOPHIED, European Project FP6 2004-2008<br />
An atypical heterodimeric<br />
laccase very effective in dyes<br />
decolourization.<br />
Reactive blue 19<br />
(Remazol Brilliant blue R -RBBR)<br />
Palmieri G., et al., Enz. Microb. Technol. 2005<br />
Palmieri G., et al., Biotechnol. Prog. 2005<br />
IPBT 2011, Milan Giovanni Sannia
A case study: P. <strong>ostreatus</strong> <strong>laccases</strong><br />
Enhancing <strong>production</strong><br />
Laccases<br />
application<br />
• Extracellular communication<br />
• Classical breeding<br />
• Cost reduction<br />
IPBT 2011, Milan Giovanni Sannia
Laccase activity U/ml<br />
Biomass log (g/l)<br />
Extracellular communication in P. <strong>ostreatus</strong><br />
€£≠∞<br />
?<br />
♂♫ךּשׁ<br />
…<br />
Metabolomic pr<strong>of</strong>ile <strong>of</strong> P. <strong>ostreatus</strong> cultures allowed to identify 12 putative<br />
signalling molecules.<br />
These molecules were used to condition submerged fungal cultures. Among<br />
these, 2-hydroxy-4-nitrobenzoic acid significantly increase laccase<br />
<strong>production</strong>. 40<br />
100<br />
induction<br />
35<br />
∆≥©♥<br />
30<br />
D<br />
10<br />
DAY<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
C<br />
B<br />
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21<br />
La<br />
c<br />
c<br />
as<br />
e<br />
a<br />
cti<br />
vit<br />
y<br />
U/<br />
ml<br />
1<br />
mass spectrometry<br />
Days giorni<br />
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21<br />
A<br />
Secretomic analysis by<br />
0,1<br />
0,01<br />
Conditioned<br />
Control<br />
Extracellular communication<br />
Time course in PDY medium<br />
IPBT 2011,<br />
+ 150<br />
Milan<br />
µM CuSO4 Giovanni Sannia
Extracellular communication in P. <strong>ostreatus</strong><br />
Effect <strong>of</strong> 2-hydroxy-4-nitrobenzoic acid on protein pr<strong>of</strong>iles <strong>of</strong> P. <strong>ostreatus</strong> secretome.<br />
control<br />
2-hydroxy-4-nitro<br />
benzoic cid<br />
1<br />
8<br />
6<br />
5<br />
7<br />
2<br />
4<br />
3<br />
Besides <strong>laccases</strong>, <strong>production</strong><br />
<strong>of</strong> several <strong>industrial</strong>ly useful<br />
proteins was increased in<br />
conditioned medium.<br />
Differentially produced<br />
proteins detected after 9<br />
days <strong>of</strong> growth in<br />
conditioned medium<br />
Extracellular communication<br />
IPBT 2011, Milan Giovanni Sannia
Laccase <strong>production</strong>: troubles and solutions<br />
Strain selection<br />
Classical<br />
breeding<br />
Production cost<br />
Raw material<br />
Productivity<br />
Medium<br />
conditioning<br />
IPBT 2011, Milan Giovanni Sannia
Classical breeding by two P. <strong>ostreatus</strong> varieties<br />
Dikaryon 1<br />
Dikaryon 2<br />
Meiosis<br />
New monokaryons<br />
X<br />
Crossing <strong>of</strong> compatible new<br />
monokaryons<br />
New dikaryons<br />
P. <strong>ostreatus</strong> var.<br />
florida<br />
P. <strong>ostreatus</strong> var.<br />
<strong>ostreatus</strong><br />
…<br />
3D 9D 11D<br />
strain 1D 2D 3D 4D 5D 6D 7D 8D 9D 10D 11D 12D 13D 14D<br />
5A Nd Nd + Nd Nd Nd Nd Nd + Nd + Nd Nd Nd<br />
6A Nd Nd + Nd Nd Nd Nd Nd + Nd + Nd Nd Nd<br />
18A Nd Nd + Nd Nd Nd Nd Nd - Nd + Nd Nd Nd<br />
+ compatible (visible clamp connection); - incompatible (no clamp connection); N.d. not determed<br />
Three out <strong>of</strong> eight selected dikaryons proved to be good laccase producers<br />
IPBT 2011, Milan Giovanni Sannia
laccase activity (U/L) ;<br />
Laccase <strong>production</strong>s <strong>of</strong> the new dikaryotic strains AxD<br />
Basal medium: PDY+ CuSO 4 150mm<br />
50000<br />
40000<br />
30000<br />
20000<br />
10000<br />
0<br />
DAY<br />
0 1 2 3 4 5 6 7 8 9 10 11<br />
5AxD3 , 5AxD11 and 6AxD11 are the best<br />
producers<br />
IPBT 2011, Milan Giovanni Sannia
laccase activity (U/L) ;<br />
Laccase <strong>production</strong>s <strong>of</strong> the new dikaryotic strains AxD<br />
All the strains revealed to be differentially sensitive to the addition <strong>of</strong><br />
ferulic acid<br />
120000<br />
100000<br />
5AxD3 produces more<br />
80000<br />
than 100,000 U/L <strong>of</strong><br />
laccase activity in<br />
60000<br />
presence <strong>of</strong> ferulic<br />
acid (2mM)<br />
40000<br />
20000<br />
0<br />
0 1 2 3 4 5 6 7 8 9 10 11<br />
IPBT 2011, Milan Giovanni Sannia
Laccase <strong>production</strong>: troubles and solutions<br />
Strain selection<br />
Classical<br />
breeding<br />
Production cost<br />
Raw material<br />
Rapeseed cake<br />
Productivity<br />
Medium<br />
conditioning<br />
IPBT 2011, Milan Giovanni Sannia
Rapeseed cake as raw material <strong>for</strong> fungal<br />
fermentation<br />
Rapeseed (Brassica napus), is a bright yellow flowering member <strong>of</strong> the family<br />
Brassicaceae (mustard or cabbage family).<br />
Rapeseed is grown <strong>for</strong> the <strong>production</strong> <strong>of</strong> vegetable oil <strong>for</strong> human consumption, and<br />
biodiesel; leading producers include the European Union, Canada, the United<br />
States, Australia, China and India.<br />
The seeds <strong>of</strong> the rapeseed provides a quantity <strong>of</strong> oil ranging from 40 to 53% <strong>of</strong> dry<br />
matter.<br />
Processing <strong>of</strong> rapeseed <strong>for</strong> oil <strong>production</strong> provides rapeseed cake as a by-product.<br />
Agro-<strong>industrial</strong> residues are generally considered the best substrates <strong>for</strong> the bioprocesses,<br />
and use <strong>for</strong> fungal fermentation <strong>for</strong> the <strong>production</strong> <strong>of</strong> enzymes is no<br />
exception to that.<br />
IPBT 2011, Milan Giovanni Sannia
U/mL<br />
Costs and productivity: choosing <strong>of</strong> raw materials<br />
When grown in rapeseed cake + 150 mM Cu sulphate the new hybrids<br />
display different perfomances respect to parental strains<br />
40<br />
New dikaryons show high<br />
levels <strong>of</strong> <strong>production</strong> in the<br />
basal raw material<br />
Rapeseed cake is an useful<br />
low cost medium <strong>for</strong><br />
laccase <strong>production</strong><br />
0<br />
3<br />
days<br />
4 5 6 7 9<br />
11 13 17 18<br />
ATCC 5AxD3 6AxD11<br />
30<br />
20<br />
10<br />
0<br />
IPBT 2011, Milan Giovanni Sannia
Laccase <strong>production</strong>: troubles and solutions<br />
Strain selection<br />
Classical<br />
breeding<br />
Production cost<br />
Raw material<br />
Rapeseed cake<br />
Productivity<br />
Medium<br />
conditioning<br />
Lignosulphonates,<br />
aromatics, N- and<br />
C-<strong>source</strong>s<br />
IPBT 2011, Milan Giovanni Sannia
Costs and productivity: choose <strong>of</strong> inducers and additional<br />
nutrient <strong>source</strong>s<br />
U/ml<br />
The effect <strong>of</strong> different inducers, N- and C- <strong>source</strong>s on laccase yields, was tested<br />
Inducers Concentration<br />
2,5-xilidine 1-2 mM<br />
Gallic acid 1-2 mM<br />
Ferulic acid 1-2 mM<br />
Sinapinic acid 1-2 mM<br />
p-cumaric acid 1-2 mM<br />
Caffeic acid 1-2 mM<br />
Lignosulphonate 15-30 g/L<br />
C- Sources Concentration<br />
Wheat-straw 15-30 g/L<br />
Starch<br />
15-30 g/L<br />
N-<strong>source</strong>s Concentration<br />
Yeast extract 5-15-30g/L<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
days<br />
4 5 6 7 9 10<br />
IPBT 2011, Milan Giovanni Sannia
Costs and productivity: choose <strong>of</strong> inducers and additional<br />
nutrient <strong>source</strong>s<br />
The effect <strong>of</strong> different inducers, N- and C- <strong>source</strong>s on laccase yields, was tested<br />
PDY + 150 µM CuSO 4<br />
+ 2mM Ferulic<br />
Acid<br />
€/L Umax/L €/U<br />
~4.5 ~150,000<br />
Rapeseed cake + 150 µM CuSO 4<br />
0.00615 35,000<br />
3.00<br />
E-05<br />
1.76<br />
E-07<br />
Many <strong>of</strong> them resulted in an<br />
increased laccase <strong>production</strong><br />
respect to basal conditions<br />
Rapeseed cake + 150 µM CuSO 4<br />
+<br />
2mM Ferulic acid<br />
Rapeseed cake + 150 µM CuSO 4<br />
+<br />
2mM Gallic acid<br />
0.18075 50,000<br />
0.08315 68,000<br />
3.62<br />
E-06<br />
1.22<br />
E-06<br />
How much<br />
does it cost?<br />
Rapeseed cake + 150 µM CuSO 4<br />
+<br />
1mM 2-5,xilidine<br />
0.02615 58,000<br />
4.51<br />
E-07<br />
Rapeseed cake + 150 µM CuSO 4<br />
+<br />
15g/L Starch<br />
Rapeseed cake + 150 µM CuSO 4<br />
+<br />
30g/L lignosulphonates<br />
Rapeseed cake + 150 µM CuSO 4<br />
+<br />
yeast extract<br />
1.33615 60,000<br />
0.05265 100,000<br />
0.16115 65,000<br />
2.23<br />
E-05<br />
5.27<br />
E-07<br />
2.48<br />
E-06<br />
Rapeseed cake 0,015€/kg<br />
CuSO4 ~100€/kg<br />
lignosulphonates 1,55 €/kg<br />
Starch 89 €/kg<br />
2-5 xilidine 170 €/kg<br />
Gallic acid 225 €/kg<br />
Ferulic acid 450 €/kg<br />
IPBT 2011, Milan Giovanni Sannia
Response surface methodology: factors and boundary<br />
conditions<br />
Rapeseed cake<br />
Placeholder Levels: 1X text – 0.5X<br />
Yeast extract<br />
Levels: 0 - 5g/L<br />
CuSO 4<br />
Levels: 150 – 450 µM<br />
Lignosulphonates<br />
Level: 20 – 60g/L<br />
Analysis was per<strong>for</strong>med with Minitab v.16 ®<br />
IPBT 2011, Milan Giovanni Sannia
Response surface methodology: experiments setting<br />
and computing matrix<br />
Once factors and leveles were<br />
defined, 27 experiments were<br />
planned<br />
Results were added to the<br />
computing matrix<br />
Maximum value indicated by +<br />
Minimum value indicated by -<br />
Average value indicated by 0<br />
Quadratic equation model was<br />
constructed<br />
Evaluation <strong>of</strong> media costs was<br />
per<strong>for</strong>med and added to the<br />
matrix<br />
IPBT 2011, Milan Giovanni Sannia
Responce Surface Methodology: optimization<br />
processing<br />
Maximization <strong>of</strong> laccase <strong>production</strong><br />
and minimiziation <strong>of</strong> culture<br />
medium cost<br />
Costs<br />
Productivity<br />
Company<br />
Commercial market cost<br />
Predicted Responses:<br />
U/l = 190,000<br />
€/l = 0.16<br />
€/U = 8.62E-07<br />
Novozym 51003 1,05 E-04<br />
Novo Denlite II 1,99 E-04<br />
P. <strong>ostreatus</strong> 8.00 E-04<br />
Sigma 3,30 E-03<br />
Bioscreen<br />
(0xizym)<br />
1,12 E-03<br />
Wetlands 1,20 E-03<br />
Jena<br />
Biosciences<br />
6,00 E-02<br />
IPBT 2011, Milan Giovanni Sannia
Scaling up: the final countdown!<br />
Classical<br />
Strain selection<br />
breeding<br />
Production cost<br />
Responce surface<br />
methodology<br />
Rapeseed<br />
Raw cake material<br />
Productivity<br />
Lignosulphonates<br />
Medium<br />
and N-<strong>source</strong>s<br />
conditioning<br />
Pilot-scale<br />
IPBT 2011, Milan Giovanni Sannia
A case study: P. <strong>ostreatus</strong> <strong>laccases</strong><br />
Improving enzyme per<strong>for</strong>mances<br />
Laccases<br />
application<br />
• Recombinant expression in suitable hosts<br />
• Functional evolution<br />
IPBT 2011, Milan Giovanni Sannia
P. <strong>ostreatus</strong> laccase functional evolution<br />
Mutant Generation activity<br />
pH stability<br />
pH3 pH5 pH7 pH10<br />
Mutant Generation activity<br />
T<br />
stability<br />
1M9B I 1.5x Χ Χ Χ Χ Χ<br />
pH stability<br />
pH3 pH5 pH7 pH10<br />
T stability<br />
1L2B II 2.5x Χ √ Χ √ √<br />
1M10B II 2.5x Χ √ √ √ √<br />
3M7C II 2.5x Χ √ √ √ √<br />
Three generation <strong>of</strong> libraries<br />
(3,300 variants) have been<br />
screened using different<br />
criteria, and seven variants<br />
endowed with improved<br />
features have been selected<br />
Mutant Generation activity<br />
pH stability<br />
pH3 pH5 pH7 pH10<br />
T<br />
stability<br />
R4 R4 2.5x Χ √ √ √ √<br />
Mutant Generation activity<br />
pH stability<br />
pH3 pH5 pH7 pH10<br />
T<br />
stability<br />
1H6C III 4.5x Χ √ √ √ √<br />
4M10G III 4.5x Χ √ √ √ √<br />
Functional evolution<br />
Festa G., et al., Proteins 2008<br />
Miele A., et al., Enz. Microb. Technol. 2009<br />
Miele A., et al., Mol Biotechnol. 2010<br />
IPBT 2011, Milan Giovanni Sannia
P. <strong>ostreatus</strong> laccase functional evolution<br />
Specific<br />
Activity<br />
(U/mg)<br />
Stability pH<br />
t ½ (days)<br />
Stability T<br />
t ½ (h)<br />
pH3 pH5 pH7 pH10 pH7, 60°C<br />
Wild type 155 ± 1 5.8 10.3 11.3 30.0 4.5<br />
1H6C 700 ± 1 3 35 39 63 7.2<br />
The best selected mutant, 1H6C, and the wild-type enzyme, POXA1b, were<br />
heterologously expressed in Aspergillus niger<br />
Aspergillus niger<br />
POXA1b<br />
1H6C<br />
Extracellular Activity 38,500 U L -1 55,500 U L -1<br />
IPBT 2011, Milan Giovanni Sannia
Laccases application<br />
Production and tailoiring <strong>of</strong><br />
<strong>laccases</strong> is in progress<br />
1<br />
2<br />
3<br />
4<br />
Protein characterization<br />
is being completed<br />
Industrial applications……..Coming soon!<br />
IPBT 2011, Milan Giovanni Sannia
People<br />
Giovanni Sannia<br />
Cinzia Pezzella<br />
Alessandra Piscitelli<br />
Lucia Guarino<br />
Vincenzo Lettera<br />
Paola Giardina<br />
Claudia Del Vecchio<br />
Laura Sorrentino<br />
Vincenza Faraco<br />
Gemma Macellaro<br />
IPBT 2011, Milan Giovanni Sannia
Collaboration<br />
Lucía Ramírez & Antonio G. Pisabarro<br />
Genetics and Microbiology Research Group- Public University <strong>of</strong> Navarre- Spain<br />
DOE Joint Genome Institute<br />
Riccardo Basosi & Rebecca Pogni<br />
Dipartimento di Chimica- Università degli studi di Siena- Italia<br />
Sophie Vanhulle & Alexandra Bazes<br />
Microbiology Unit - Université Catholique de Louvain- Belgium<br />
Thierry Tron<br />
CNRS, Faculté des Sciences et Techniques. Saint Jérôme Marseille- France<br />
Tajalli Keshavarz<br />
Biotechnology Department - School <strong>of</strong> Biosciences- University <strong>of</strong> Westminster- UK<br />
IPBT 2011, Milan Giovanni Sannia
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