abstractbook lisboa oxizimes

3rd European Meeting in Oxizymes

Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa

OXIZYMES IN OEIRAS 3rd European Meeting in Oxizymes Abstract Book Oxizymes in Oeiras – 3rd 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

4

WELCOME TO OEIRAS

The Organizing Committee of the 3rd 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

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OXIZymes in Oeiras SPONSORS

Oxizymes in Oeiras, 3rd European Meeting in Oxizymes

6 September 7-9, 2006 Oeiras, Portugal

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



OXIZymes in Oeiras TIMETABLE

THURSDAY SEPTEMBER, 7

FRIDAY SEPTEMBER, 8

SATURDAY SEPTEMBER, 9

8.00-9.00 REGISTRATION

9.00-9.30

OPENING 9.30-11.00 S1 MICROBIAL PHYSIOLOGY I

9.00-10.30 S5 STRUCTURE-FUNCTION RELATIONSHIPS I

9.00-10.30 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 7th FP?”

lunch poster attendance

lunch poster attendance

15.00-16.30 S3 ENZYMOLOGY I

14.30-16.00 S7 APPLICATIONS I

coffee-break coffee-break 17.00 -18.30 S4 ENZYMOLOGY II

16.30 -18.00 S8 APPLICATIONS II

18.30-19.00 Welcome Drink

“Porto de Honra”

20.30 Oxizymes dinner



Thursday, September 7, 2006

8.00-9.00 Registration

9.00-9.30 Opening

9.30-11.00

9.30-9.55

9.55-10.20

10.20-10.40

10.40-11.00

S1 - MICROBIAL PHYSIOLOGYI

CHAIRPERSON: TAJALLI KESHAVARZ

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

11.30-11.55

11.55-12.20

12.20-12.40

12.40-13.00

S2 - MICROBIAL PHYSIOLOGY II

CHAIRPERSON: ANNELE HATAKKA

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



Thursday, September 7, 2006

15.00-16.30

15.00-15.25

15.25-15.50

15.50-16.10

16.10-16.30

S3 – ENZYMOLOGY I

CHAIRPERSON: THIERRY TRON

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

17.00-17.25

17.25-17.50

17.50-18.10

18.10-18.30

S4 – ENZYMOLOGY II

CHAIRPERSON: STEFFEN DANIELSEN

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”



Friday, September 8, 2006

9.00-10.30

9.00-9.25

9.25-9.50

9.50-10.10

10.10-10.30

S5 - STRUCTURE-FUNCTION RELATIONSHIPS I

CHAIRPERSON: CLÁUDIO M. SOARES

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

11.00-11.25

11.25-11.50

11.50-12.10

12.10-12.30

S6 - STRUCTURE-FUNCTION RELATIONSHIPS II

CHAIRPERSON: CLÁUDIO M. SOARES

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



Friday, September 8, 2006

14.30-16.00

14.30-14.55

14.55-15.20

15.20-15.40

15.40-16.00

S7 – APPLICATIONS I

CHAIRPERSON: LIISA VIIKARI

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

16.30-16.55

16.55-17.20

17.20-17.40

17.40-18.00

S8 – APPLICATIONS II

CHAIRPERSON: PAUL ANDER

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



Saturday, September 9, 2006

9.00-10.30

9.00-9.25

9.25-9.50

9.50-10.10

10.10-10.30

S9 – APPLICATIONS III

CHAIRPERSON: CHRISTIAN-MARIE BOLS

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 7th FP?”

Chairpersons: Liisa Viikari and Giovanni Sannia

Angel T Martínez

Georg M. Gübitz

Christian-Marie Bols

ORAL PRESENTATIONS



L1

Antimicrobial and Biochemical Properties of a Novel type of Lysine Oxidase Expressed by Marinomonas

mediterranea. Daniel Gómeza, Patricia Lucas-Elíoa, Francisco Solanob, Antonio Sanchez-Amata a Department of Genetics and Microbiology, Faculty of Biology; bDepartment of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Campus de Espinardo, Murcia 30100, Spain E-mail: [email protected] 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 activity1. 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 activity2. 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 H2O2. 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.



L2

Lignin-Modifying Peroxidases and Laccases of the White Rot Basidiomycete Phlebia radiata

Taina Lundella, Kristiina S. Hildéna, Miia R. Mäkeläa, Annele Hatakkaa

aDepartment of Applied Chemistry and Microbiology, Division of Microbiology, University of Helsinki, Finland; E-mail: [email protected] 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 intron-exon 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. Structure-function 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



L3

Essential Role of the LPR1 Family of Metallo-Oxidases in the Arabidopsis thaliana Root Growth Response to Low-

Phosphate Media Sergio Svistoonoffa,1, Cécile Sigoillot-Claudea, Matthieu Reymonda,2, Audrey Creffa, Lilian Ricauda, Aline Blancheta, Laurent Nussaumea and Thierry Desnosa

aLaboratoire de Biologie du Développement des Plantes, DEVM, CEA cadarache, 13108 St Paul-lez-Durance cedex, France; 1Present address: Federal Institute of Technology (ETH) Zurich, Institute of Plant Sciences, Experimental Station Eschikon 33, CH-8315 Lindau, Switzerland. 2Present 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: [email protected] 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.



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: [email protected] 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).



L5

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: [email protected] 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.



L6

Oxizymes in Hardwood Forest Soil: Production of Oxidases and Peroxidases by Exploratory Mycelium of Saprotrophic

Soil Basidiomycetes

Jaroslav Šnajdra, Vendula Valáškováa, Tomáš Cajthamla, Věra Merhautováa, Petr Baldriana

aInstitute of Microbiology ASCR, Vídeňská 1083, 14220 Prague 4, Czech Republic E-mail: [email protected] 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 (soil-lower) 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).



L7

Novel Efficient Producers of Blue Laccases A. Chernykha, L. Golovlevaa, N. Myasoedovaa, N. Psurtsevab, N. Belovab, M. Ferraronic, A. Scozzafavac, F. Brigantic

aG.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, Russia; bKomarov Botanical Institute RAS, Russia; cUniversitá degli Studi di Firenze, Italy E-mail: [email protected] 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, CuSO4, 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 CuSO4, 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 Cu2+ 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 Cu2+ 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.



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: [email protected] 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 monooxygenase1. 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

O2 OH2

FADH2 FAD

StyA

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.



L9

Production and Characterization of a Secreted C-terminally Processed Tyrosinase from the Filamentous

Fungus Trichoderma reesei

Kristiina Kruusa, Emilia Selinheimoa, Markku Saloheimoa, Elina Aholab, Ann Westerholm-Parvinena, Nisse Kalkkinenb and Johanna Bucherta

aVTT Technical Research Centre of Finland, P.O. Box 1500, Espoo FIN-02044 VTT, Finland; bProtein Chemistry Research Group and Core Facility, Institute of Biotechnology, P.O. Box 65, FIN-00014 University of Helsinki, Finland E-mail: [email protected] 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.



L10

Understanding the Selection of Arene Dioxygenase Enzymes for Optimal Chemo-, Stereo- and Regio-Selectivity

of Biotransformation Processes Christopher CR Allena, Derek R Boydb, Leonid L Kulakova,c, Narain D Sharmab. aSchool of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland. bSchool of Chemistry & Chemical Engineering, Queen’s University Belfast, David keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland. cThe QUESTOR Centre, Queen’s University Belfast, David keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland. E-mail: [email protected] 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 industries1. 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 azaarenes2. 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 process-relevant 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.



L11

Redesign of AtGALDH, a Flavoprotein Involved in Vitamin C Biosynthesis

Nicole G. H. Leferinka, Yu Lua, Willy A. M. van den Berga, Willem J. H. van Berkela

aLaboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands E-mail: [email protected] 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 (Km = 80 µM, kcat = 87 s-1) and cytochrome c (Km = 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 FAD-binding 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



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: [email protected] 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 (<10%) and inactivation rate constants were at least two orders of magnitudes higher. In conclusion, citrate and particularly succinate may be recommended as suitable buffer substances for use in laccase assays, while acetate should be avoided. However, the non-uniform behaviour of the various carboxylic acids tested did not allow proposing a common mechanism for the inhibition of laccase activity.



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: [email protected]

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.



L14

A Flavin-Dependent Tryptophan 6-Halogenase and Its Use In Combinatorial Biosynthesis

C. Schmida, H. Schnerra J. Rumpfa, A. Kunzendorfa, C. Hatschera, T. Wagea, A. Ernyeia, C. Dongb, J. H. Naismithb, K.-H. van Péea

aBiochemie, Technische Universität Dresden, D-01062 Dresden, Germany, and bCentre for Biomolecular Sciences, EaStchem, University of St. Andrews, St. Andrews KY16 9ST, UK E-mail: [email protected] 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 two-component 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.



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

aDepartamento de Ingeniería Celular y Biocatálisis , bDepartamento de Medicina Molecular y Bioprocesos, cUnidad de Proteómica. Instituto de Biotecnología, Universidad Nacional Autónoma de México. AP 510-3 Cuernavaca, Morelos 62250, México E-mail [email protected] 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 redox-based 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



L16

Functional Hybrids of Haloperoxidases and Cytochrome P450 Monooxygenases from Alkaliphilic Mushrooms

René Ullricha, Dau Hung Anha,b, Martin Klugea, Matthias Kinnea, Katrin Scheibnerc, Martin Hofrichtera

aInt. Graduate School of Zittau, Environ. Biotech. Unit, Markt 23, 02763 Zittau Germany; bVietnamese Acad of Sci. & Techn., Dept of Biotechnology,18-Hoang Quoc Viet Rd., Hanoi, Vietnam; cLausitz Univ of Appl. Sci.s, Dept of Biotechnology, 01958 Senftenberg, Germany E-mail: [email protected] 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 P450nor, 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 H2O2 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



L17

Laccase Engineering by Rational and Random Mutagenesis Giovanna Festa1, Alessandra Piscitelli1, Vincenza Faraco1, Paola Giardina1, Flavia Autore1, Franca Fraternali2 and Giovanni Sannia1

1Department of Organic Chemistry and Biochemistry, Complesso Universitario Monte S.Angelo, via Cintia 4, 80126 Naples, Italy 2Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, SE1 1UL London UK E-mail: [email protected] 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 error-prone 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



L18

Structure-Function Studies of Pleurotus Versatile Peroxidase, a Model Ligninolytic Enzyme

F.J. Ruiz-Dueñasa, M. Pérez-Boadaa, M. Moralesa, R. Pognib, R. Basosib, T. Choinowskic, M.J. Martíneza, K. Piontekc, Á.T. Martíneza

aCentro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain; bDepartment of Chemistry, University of Siena, via Aldo Moro, I-53100 Siena, Italy; cInstitute of Biochemistry, ETH, Schafmattstrasse 18, CH-8093 Zürich, Switzerland E-mail: [email protected]

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]. Mn2+ oxidation to Mn3+, 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 H2O2-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



L19

Structure-Activity Relationship of the Laccase-Mediator System

R. Pognia, B. Brogionia, A. Sinicropia, M.C. Barattoa, P. Giardinab, G. Sanniab, R. Basosia

aChemistry Department, University of Siena, Via A. Moro, Italy; bOrganic Chemistry and Biochemistry Department, University of Naples, via Cinthia 4, Italy. E-mail: [email protected] 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 H2O 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.



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: [email protected]

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 electron-withdrawing 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.



L21

A Near-Atomic Resolution Crystal Structure of Melanocarpus albomyces Laccase

Nina Hakulinena, Martina Andbergb, Anu Koivulab, Kristiina Kruusb, Juha Rouvinena

aDept. Of Chemistry, University of Joensuu, PO Box 111, FIN-80101 Joensuu, Finland bVTT Technical Research Centre of Finland, PO Box 1000, FIN-02044 VTT, Finland E-mail: [email protected] 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 non-aromatic 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 CuCl2 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 Rfree = 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 Rfree= 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.



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: [email protected] 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 Mn2+, Fe2+ or Cu1+, 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 CotA-laccase 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 (kcat/Km) towards aromatic substrates but a remarkable high for cuprous and ferrous ions, close to 3 x 106 s-1 M-1. We provide evidences for the in vivo involvement of McoA in copper and iron homeostasis.



L23

Crystal Structures of Three New Fungal Laccases: Implications on the Catalytic Mechanism and on the

Dynamics of the Copper Sites Redox States Irene Materaa, Antonella Gullottoa, Marta Ferraronia, Silvia Tillia, A.Chernykhb, Nina M. Myasoedovab, Alexey A. Leontievskyb, Ludmila Golovlevab Andrea Scozzafavaa, Fabrizio Brigantia

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: [email protected] 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 one-electron 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.



L24

Construction and Characterisation of Horseradish Peroxidase Mutants that Mimic Some of the Properties of

Cytochromes P450

Emile Ngoa, Wendy Doylea, Anabella Ivancichb, Andrew T. Smitha

aBiochemistry Department, School of Life Sciences, University of Sussex. UK; bCentre d'Etudes de Saclay, Gif-sur-Yvette. France Email: [email protected]

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.



L25

Immobilisation of Laccases for Biotransformations in Environmental and Food-Technology

M. Schroedera, A. Kandelbauera, G. Nyanhongoa, B. Poellinger-Zierlerb, A. Cavaco-Pauloc, G.M. Guebitza aGraz Universty of Technology, Dept.of Environmental Biotechnolog, bDept. of Food Technology, Petersgasse 12, 8010 Graz Austria, cDept. of Textile Engineering, University of Minho, 4800 Guimaraes, Portugal E-mail: [email protected] 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



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: [email protected] 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 cost-efficient 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



L27

Potential of White-Rot Fungi for Decolourisation and Detoxification of Dyes

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: [email protected] 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).



L28

Biotransformation of Environmental Pollutants by Aquatic Fungi – the Role of Laccases

Dietmar Schlossera, Claudia Martina, Charles Junghannsa, Monika Moederb, Magali Soléa, Gudrun Kraussa

aDepartment of Environmental Microbiology, and bDepartment of Analytical Chemistry, UFZ Centre for Environmental Research Leipzig-Halle, Permoserstrasse 15, D-04318 Leipzig, Germany E-mail: [email protected] 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.



L29

Transformation of Textile Dyes by Oxidoreductases Feng Xu Novozymes Inc, 1445 Drew Ave., Davis, CA 95616, USA E-mail: [email protected] 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 H2O2, m-nitrobenzenesulfonate, perborate, hypochlorite, iodate, bromate, or dichromate), harsh conditions (high pH or temperature), or expensive/unsafe catalysts (such as NaVO3). 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 O2 and H2O2, 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.



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 packed-bed 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.



L31

Olive Mill Wastewater Transformation and Detoxification by White-Rot Fungi: Role of the Laccase in the Process

G. Iamarinoa, J.M. Barrasab, L. Gianfredac, A.T. Martíneza and M.J. Martíneza

aCentro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain; bUniversidad de Alcalá, Alcalá de Henares, E-28871, Madrid, Spain. cDiSSPA, Università degli Studi di Napoli, Via Università 100, 80055 Portici, Na Italy ([email protected]) 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 matter1. 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 properties2. 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 species3;4, laccase appears as the sole ligninolytic enzyme in other fungal species5;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 treatment7.

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-peptone8. 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.



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: [email protected] 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 H2O2 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

peroxidase

colored compound

oxidized, colorless compound

glucose

gluconic acid

H2O

H2O

O2

H2O2

Bleaching

Figure 1: Use of oxidoreductases in bleaching processes.



L33

Laccase-Mediator System: the Definitive Solution to Pitch Problems in the Pulp and Paper Industry?

Ana Gutiérreza, Jorge Rencoreta, David Ibarrab, Ángel T. Martínezb, José C. del Ríoa

aInstituto de Recursos Naturales y Agrobiología, CSIC, PO Box 1052, E-41080, Seville, Spain; bCentro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain E-mail: [email protected] 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.



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: [email protected]

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) R2-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 oC and ca. 10% consistency, maintaining the strength properties.



L35

Studies on the Effect of the Laccase Mediator System on Ageing Properties of Hand Sheets of Different Origin Jorge Gominhoa, Ana Lourençoa, Helena Pereiraa, Cristina Máximob, Maria Costa-Ferreirab aCentro de Estudos Florestais, Departamento de Engenharia Florestal, Instituto Superior de Agronomia; 1349-017 Lisboa, Portugal; bDepartment of Biotechnology, National Institute for Engineering, Technology & Innovation - INETI; 1649-038 Lisboa, Portugal E-mai : [email protected] 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/m2. 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.



L36

Laccase in Pulp Activation and Functionalisation Anna Suurnäkkia, Marco Orlandib, Stina Grönqvista, Hannu Mikkonena, Liisa Viikaria

aVTT, Tietotie 2, 02044 VTT, Finland bDipartimento di Scienze dell’Ambiente e del Territorio, Universitá degli Studi di Milano-Bicocca, Piazza della Scienza 1, I-20126 Milan, Italy E-mail: [email protected] 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.

POSTER PRESENTATIONS



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 Valtakaric, Jarno Kallio, Richard Fagerström, and Jari Vehmaanperä



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 H2O2 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



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



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



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



P1 High Concentrations of Cell Wall Redox Enzymes Lichens

in Suborder Peltigerineae Richard P. Becketta, Zsanett Laufera, Farida V Minibayevab 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: [email protected] 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 multi-copper 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.



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: [email protected] 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 Mn2+ 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 Mn2+ and veratryl alcohol. The transcription of mnpA was induced by the addition of veratryl alcohol but not by Mn2+. 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 Mn2+ 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.



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 Institute of Biochemistry and Biophysics, Russian Academy of Science. P.O.Box 30, Kazan 420111, Russia E-mail: [email protected] 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.



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: [email protected]

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 (ale-wort 4oB, 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 ale-agar. 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.



P5

Characteristics of Laccase in the Biopulping Fungus Physisporinus rivulosus

T. Hakalaa,b, K. Hildéna, P. Maijalaa, A. Hatakkaa

aDepartment of Applied Chemistry and Microbiology, Viikki Biocenter, University of Helsinki, Helsinki, Finland; bpresent address: KCL Science and Consulting, Espoo, Finland E-mail: [email protected] 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.



P6

Laccase Production by Basidiomycetes under Various Fermentation Conditions

N. Belovaa, N. Psurtsevaa, N. Yakovlevaa, A. Kiyashkoa, T. Lundellb, A. Hatakkab

aKomarov Botanical Institute RAS, Prof. Popov Str., 2, St. Petersburg. 197376 Russia. bUniversity of Helsinki, P.O. Box 56 (Biocenter 1, Viikinkaari 9) 00014, Finland E-mail: [email protected] 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.



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: [email protected] 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.



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: [email protected] 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.



P9

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: [email protected]

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 (Pphe). [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.



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: [email protected] 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’.



P11

Heterologous Expression of Pycnoporus sanguineus UCL38531 lcc1 cDNA in Pichia pastoris

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: [email protected]

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”



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-Rincóna, A. Suarezb, T. de la Rubiac , M. Lucasc, J. Martínezc aDepartment of Microbiology Faculty of Basic Sciences, University of Pamplona, Pamplona, Colombia. bDepartment, of Biochemistry and Molecular Biology, and cDepartment of Microbiology Faculty of Pharmacy, University of Granada. Granada. Spain. E-mail: [email protected] 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). 13th Int. biodeterioration and Biodegradation Symposium. Sept.2005. Madrid, Spain. [3] Kumar ket al. (2003). Biotechnology and Bioengineering 83: 386-394



P13

Purification and Properties of a Non-Blue Fungal Laccase Isoenzyme

Albino A. Diasa, Rui M.F. Bezerraa, Irene Fragaa, António N. Pereirab

aCETAV - Dep. Engenharia Biológica e Ambiental, UTAD, Apartado 1013, 5001-801 Vila Real, Portugal; bDepartamento de Indústrias Alimentares, UTAD, Apartado 1013, 5001-801 Vila Real, Portugal E-mail: [email protected] 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 low-glycosylated (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



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: [email protected] 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’.



P15

Directed Evolution of Pleurotus ostreatus Laccases 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: [email protected] 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



P16

Production, Purification and Characterization of Laccase Enzymes from Thielavia arenaria

Kristiina Kruusa, Marja Paloheimob, Terhi Puranenb, Leena Valtakaric, Jarno Kalliob, Richard Fagerströma, and Jari Vehmaanperäb

aVTT Technical Research Centre of Finland, Espoo, Finland; bRoal Oy, Rajamäki, Finland; cAB Enzymes Oy, Rajamäki, Finland E-mail: [email protected] 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.



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.

Lorenzinic, 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 Cu2+-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 Km 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.



P18

Production of Cerrena unicolor Manganese Peroxidase and Laccase in Solid-state on Oat Husks

Ulla Moilanena, Erika Winquista, Aila Mettäläb, Pekka Maijalab, Ossi Pastinena, Annele Hatakkab

aLaboratory of Bioprocess Engineering, Helsinki University of Technology, Kemistintie 1, Espoo, Finland; bDepartment of Applied Chemistry and Microbiology, University of Helsinki, Biocenter 1, Viikinkaari 9, Helsinki, Finland E-mail: [email protected] 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.



P19

Preparation and Characterization of Crossed-Linked Laccase Aggregates from the White-Rot Fungus

Coriolopsis polyzona Hubert Cabanaa,b, J. Peter Jonesb, Spiros N. Agathosa

aUnit of Bioengineering, Catholic University of Louvain, Croix du Sud 2, 1348 Louvain-la-Neuve, Belgium; bDepartment of Chemical Engineering, University of Sherbrooke, 2 500 boulevard de l’Université, Sherbrooke (Qc), Canada E-mail: [email protected] 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 cross-linking 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 Vmax of CLEAs (0.021 µM/min) were improved comparatively to free laccase (0.0042 µM/min) using ABTS as substrate. The affinity constant, Km, 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.



P20

Enhanced Stability of Laccase by Xylitol Andre Zillea, Diego Moldesa, Ramona Irgoliçb, Artur Cavaco-Pauloa

aDepartment of Textile Engineering, University of Minho, Campus de Azurém, P-4800 Guimarães, Portugal. bTextile Department, Faculty of Mechanical Engineering, University of Maribor, SI-2000 Maribor, Slovenia. E-mail: [email protected] 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



P21

Influence of Static Magnetic Field on Laccase Activity and Stability

V. Kokola, M. Schroedera, G. M. Guebitzb

aFaculty of Mechanical Engineering, Institute of Textiles, University of Maribor, Smetanova ul. 17, SI-2000 Maribor, Slovenia; bInstitute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12,G-8010 Graz, Austria E-mail: [email protected] 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 oC 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/m2. The ABTS-activity and redox potential of laccase at different concentrations (cE = 0.05 and 0.1 g/L) and pH media (pH 3 - 9) were monitored at different temperatures (T = 20 - 70 oC) and compared to the results without the treatment. In addition, the stability of SMF exposed solutions was determined. Furthermore, the kinetic KM kcat 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



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: [email protected] 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 yellow-brown 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



P23

Ralstonia solanacearum Expresses a Unique Tyrosinase with a High Tyrosine Hydroxylase/DOPA Oxidase Ratio

Diana Hernández-Romeroa, Antonio Sanchez-Amata, Francisco Solanob aDepartment of Genetics and Microbiology, Faculty of Biology; bDepartment of Biochemistry and Moleuclar Biology, School of Medicine, University of Murcia, Campus de Espinardo, Murcia 30100, Spain E-mail: [email protected] 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 expressed1. 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 ratio2. 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.



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: [email protected]

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



P25

Spectroscopic Characterization of a Novel Naphthalene Dioxygenase from Rhodococcus sp.

Maria Camilla Barattoa, David A Lipscombb, Christopher CR Allenb, Michael J Larkinb, Riccardo Basosia , Rebecca Pognia

aDipartimento di Chimica, Università di Siena, Via Aldo Moro 2, 53100, Siena, Italia, [email protected] bSchool of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland [email protected] 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 α3β3 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 O2-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



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: [email protected] 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 O2 to H2O2. 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 peptides1. 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.



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: [email protected]

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 ortho-hydroxylation 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).



P28

Cellobiose Dehydrogenases from Ascomycetes and Basidiomycetes: Phylogenetic and Kinetic Comparison

Roland Ludwiga,b, Marcel Zámockya,b, Clemens Peterbauerb, and Dietmar Haltrichb

aResearch Centre Applied Biocatalysis; Petersgasse 14, 8010 Graz, Austria bDept. of Food Sciences and Technology, University of Natural Resources and Applied Life Sciences, Vienna; Muthgasse 18, 1190 Vienna, Austria E-mail: [email protected] 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 Km value for the electron donors than the basidiomycetous enzymes and a surprisingly low Km for maltose. In contrast to this, the Km 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.



P29

Oxalate Oxidase as a Potential Enzyme Responsible for H2O2 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: [email protected] 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 H2O2 is essential as a substrate for both peroxidases. In this group of fungi there are different possible enzymatic mechanisms for H2O2 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 Mn3+ 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 H2O2 by enzymatic degradation via OXO in Abortiporus biennis cultures is proposed. Correlation between MnP, Lac activity, H2O2 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



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: [email protected] 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.



P31

Laccase Activity Measurements in Turbid or Coloured Liquids with a Novel Optical Oxygen Biosensor

Christian-Marie Bols, Rob C .A. Onderwater Wetlands Engineering sprl, Rue du laid Burniat 5, B-1348 Ottignies-Louvain-la-Neuve, Belgium E-mail: [email protected] 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 Clark-type 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.



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: [email protected] 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 c4 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;



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: [email protected] 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].

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

NO

NO

+

R1R2

H OH

R1

R2O

NOH + H+

NO

NO

+

R1R2

H OH

R1

R2O

NOH + H+

NONO

NO

+

R1R2

H OH

R1

R2O

NOH + H+

NO

+

R1R2

H OH

R1

R2O

NOH + H+

NONO

NO

+

R1R2

H OH

R1

R2O

NOH + H+

NO

+

R1R2

H OH

R1

R2O

NOH + H+



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: [email protected] 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.



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: [email protected] 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).



P32

The Role of the C-terminal Amino Acids of Melanocarpus albomyces Laccase

Martina Andberga, Sanna Auera, Anu Koivulaa, Nina Hakulinenb, Juha Rouvinenb, Kristiina Kruusa

a VTT Technical Research Centre of Finland, P.O. Box 1500, Espoo FIN-02044 VTT, Finland; bDepartment of Chemistry, University of Joensuu, PO BOX 111, FIN-80101 Joensuu, Finland E-mail: [email protected] 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



P33

Shifting the Optimal pH of Activity for a Laccase from the Fungus Trametes Versicolor by Structure-Based

Mutagenesis C. Madzaka, M.C. Mimmib, E. Caminadec, A. Braultc, S. Baumbergerb, P. Briozzob, C. Mouginc, C. Jolivaltd aUMR 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: [email protected] 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



P34

Axial Perturbations of the T1 copper in the CotA-Laccase from Bacillus subtilis: Structural, Biochemical and Stability

Studies

Paulo Durãoa, Isabel Bentoa, André T. Fernandesa, Eduardo P. Melob, Peter F. Lindleya and Lígia O. Martinsa

a Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. Da República, , 2784-505 Oeiras, Portugal, bCenter of Molecular and Structural Biomedicine, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal E-mail: [email protected]

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 Eo 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 (Eo = 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 kcat 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 CotA-laccase activity.



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: [email protected]

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



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 aG.K. Skryabin Institute of biochemistry and physiology of microorganisms RAS, Pushchino, Russia and bLaboratorio di Chimica Bioinorganica, Universita degli Studi di Firenze, Italy, E-mail: [email protected]

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, hydroxy- and 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 Fe3+ 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.



P37

Surface-Enhanced Vibrational Spectroelectrochemistry of Immobilized Proteins

Smilja Todorovica, Peter Hildebrandtb and Daniel Murgidab

aInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República (EAN, 2784 - 505 Oeiras, Portugal; bTechnical University of Berlin, Strasse des 17. Juni 135, D-10623 Berlin, Germany E-mail: [email protected] 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 P450cam. 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 potential-dependent 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 cbb3 terminal oxidase from B. japonicum, require immobilization conditions that mimic more closely the membrane-like environment. SERRS of cbb3, 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.



P38

Enzymatic Properties, Stability And Model Structure of a Metallo-Oxidase from the Hyperthermophile Aquifex

aeolicus André T. Fernandesa, Cláudio M. Soaresa, Manuela M. Pereiraa Robert Huberb, Gregor Grassc, Eduardo P. Melod and Lígia O. Martinsa

a Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. Da República, , 2784-505 Oeiras, Portugal, bLehrstuhl fur Mikrobiologie und Archaeenzentrum, Universitat Regensburg, Germany, , cInstitute for Microbiology, Marthin Luther University, Halle, Germany and the dCenter of Molecular and Structural Biomedicine, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal E-mail: [email protected] 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 (kcat/Km) towards aromatic substrates but a remarkable high for cuprous and ferrous ions, close to 3 x 106 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 non-homologous 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∆P321-V363. 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.



P39

Degradation of Azo Dyes by Trametes villosa Laccase under Long Time Oxidative Conditions Andrea Zillea, Barbara Górnackab, Astrid Rehorekb Artur Cavaco-Pauloa

aUniversity of Minho, Department of Textile Engineering, 4800-058 Guimarães, Portugal; bUniversity of Applied Sciences Cologne, Institute of Chemical Engineering and Plant Design, Betzdorfer Str. 2, D-50679 Cologne, Germany E-mail: [email protected] 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 13C -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 13C-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.



P40

Enzymatic Decolorization of Azo and Anthraquinonic Dyes with the CotA-Laccase from Bacillus subtilis

Luciana Pereiraa, Lígia O. Martinsa a Instituto de Tecnologia Química e Biológica (ITQB),Universidade Nova de Lisboa, Av da República, 2784-505 Oeiras, Portugal E-mail: [email protected] 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).



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: [email protected] 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.



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: [email protected] 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’.



P43

Laccase from Trametes versicolor Immobilised on Novel Composite Magnetic Particles

K.-H. van Péea, A. Maturaa, T. Wagea, A. Pichb, U. Böhmerc

aBiochemie; bMakromolekulare Chemie und Textilchemie; cLebensmittel und Bioverfahrenstechnik, TU Dresden, D-01062 Dresden, Germany E-mail:[email protected]

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.



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: [email protected] 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% H2O2. Results obtained showed a 10% decrease in Kappa number, a 3.5 % increase in ISO brightness and a remarkable saving in H2O2 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.



P45

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 Departamento de Engenharia Bioquímica, Escola de Química/UFRJ, Centro de Tecnologia, Bl.E, lab.113, Rio de Janeiro - RJ, 21949-900, Brasil. E-mail: [email protected] 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, coagulation-flocculation, 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 organo-pollutants 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: O2 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 (H2O2 + Fe2+) was accomplished by two experimental design techniques, observing three variables (reaction time, Iron II concentration, and H2O2 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).



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: [email protected] 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 m3/1000 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 non-enzymatic 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.



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: [email protected] 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.



P48

Relationship between Non-Protein Fraction and Laccase Isoenzymes from Cultures of Trametes versicolor: Effect

on Dye Decolorization Diego Moldesa, Alberto Domínguezb, Mª Angeles Sanrománb

aDepartment of Textile Engineering. University of Minho. 4800 Guimarães. Portugal, bDepartment of Chemical Engineering. Isaac Newton Building. University of Vigo. 36310 Vigo. Spain E-mail: [email protected] 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 sulfate-polyacrylamide 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.



P49

Degradation of Synthetic Dyes by Coriolopsis rigida J. Gómez-Sieiro, D. Rodríguez-Solar, D. Moldes, M.A. Sanromán Department of Chemical Engineering. Isaac Newton Building. University of Vigo. 36310 Vigo. SPAIN E-mail: [email protected] 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 9th day (0.279 kU/l), and then, it rapidly increased reaching a maximum value of 26.177 kU/l on the 25th 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.



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: [email protected] 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



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: [email protected]

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



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: [email protected]

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-copper-containing 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.



P53

Biodegradation Cycles of Industrial Dyes By Immobilised Basidiomycetes

L.Casieria, G.C. Varesea, A. Anastasia, V. Prigionea, K. Svobodováb, V. Filipello Marchisioa and Č. Novotnýb. aDepartment of Plant Biology, University of Turin, Viale Mattioli 25, Turin, Italy; bLaboratory of Exp. Mycology, Institute of Microbiology ASCR, Vídeňská 1083, Prague 4, Czech Republic. E-mail: [email protected] 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.



P54

Catalytic Activity of Versatile Peroxidase from Bjerkandera fumosa and its use in Dyes Decolourization

Anna Jarosz-Wilkołazkaa, Anna Olszewskaa, Janina Rodakiewicz-Nowakb, Jolanta Lutereka

aDepartment of Biochemistry, Maria Curie-Skłodowska University, Skłodowska Place 3, 20-031 Lublin, Poland, bInstitute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Kraków, Poland E-mail: [email protected] 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 manganese-lignin 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<n-propanol<DMSO<ethanol. Finally, the ability of VP for decolourization of simple textile dyes and model colour wastewater was analyzed. 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] Martínez A.T. (2002) Enzyme Microb Technol 30, 425-444. [2] Heinfling A., Martínez M.J., Martínez A.T., Bergbauer M., Szewzyk U. (1998) Appl Environ Microbiol 64,

2788. [3] Ruiz-Dueñas F.J., Camarero S., Pérez-Boada M., Martínez M.J., Martínez A.T. (2001) Biochem Soc Trans

29, 116. [4] Moreira P.R., Dueaz C., Dehareng D., Antunes A., Almeida-Vara E., Frère J.M., Malcata F.X., Duarte J.C.

(2005) J Biotechnol 118, 339. [5] Pogni R., Baratto M.C., Giansanti S., Teutloff C., Verdin J., Valderrama B., Lendzian F., Lubitz W.,

Vazquez-Duhalt R., Basosi R. (2005) Biochemistry 44, 4267.



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.Xaviera

aCOPNA, bCICECO – Departamento de Química, Universidade de Aveiro, 3810–193 Aveiro, Portugal E-mail: [email protected] 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 [SiW11MnIII(H2O)O39]5- and [SiW11VVO40]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 SiW11MnII and SiW11VIV 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 ([SiW11VVO40]5-) at

90ºC in the bleaching reactor A, which was coupled with bioreactor B, where the reduced POM was continuously re-oxidised 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% ClO2 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.



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: [email protected]

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.



P57

Laccase-Mediated Oxidation of Natural Compounds Mattia Marzorati, Daniela Monti, Francesca Sagui, Sergio Riva Istituto di Chimica del Riconoscimento Molecolare, C.N.R..,Via Mario Bianco 9, 20131 Milano, Italy E-mail: [email protected] 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

OOHO

OH OH

R

NHAc

SMeO

MeO

MeO

O

OHOH

OOH

OOH

ROOHO OHOH

OOH

OH

OHOH

O1 : R = CH2OH 1a : R = COOH

2 : R = CH2OH 2a : R = COOH

[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.



P58

Laccase induced coating of lingocellulosic surfaces with functional phenolics

M. Schroedera, G. M. Guebitzb, V. Kokola

aFaculty of Mechanical Engineering, Institute of Textile, University of Maribor, Slovenia, Smetanova ul 17, 2000 Maribor, Slovenia; bInstitute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria E-mail: [email protected] 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 KM of 16.7 ± 0.2 µM for guaiacol and KM 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



P59

Decolourization and Detoxification of Kraft Effluent Streams by Lignolitic Enzymes of Trametes versicolor

M.S.M. Agapitoa, D. Evtuguinb, A.M.R.B. Xaviera

aCOPNA, bCICECO – Departamento de Química, Universidade de Aveiro, 3810–193 Aveiro, Portugal E-mail: [email protected] 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 problems1. 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 plant2. 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 lignin3. Trametes versicolor is one of the white-rot basidiomycetes that produce ligninolytic enzymes, such as lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase1. 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 Medium4, 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



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: [email protected] 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



P61

Involvement of the Laccase Produced by Streptomyces sp. in the Biotransformation of Coffee Pulp Residues

A.L. Orozcoa, O. Polvillo c, J.Rodríguezb, J.M. Molinab, O. Guevaraa, M.E.Ariasb, M.I. Pérezb aDepartamento de Biología. UNAN-León. Nicaragua bDepartamento de Microbiología y Parasitología. Universidad de Alcalá. 28871 Alcalá de Henares (Madrid). Spain cIRNAS-CSIC. P.O. Box 1052, 41080 Sevilla, Spain E-mail: [email protected] 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 CO2 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.



P62

Elimination of the Endocrine Disrupting Chemical Bisphenol A by using Laccase from the Ligninolytic fungus

Lentinus crinitus Carolina Arboledaa,d, Hubert Cabanab,c, J. Peter Jonesc, Amanda I. Mejíaa, Spiros N. Agathosb, Gloria A Jimenezd, Michel J. Penninckd

aLaboratorio Ciencia de Los Materiales, Instituto de Química y Facultad de Química Farmacéutifca, Universidad de Antioquia, Medellin, Colombia; bBioengineering Unit, Université Catholique de Louvain, Louvain-la-Neuve, Belgium ; cDepartment of Chemical Engineering, University of Sherbrooke, Sherbrooke (Qc), Canada; dLaboratory of Microbial Physiology and Ecology, Faculty of Sciences, Université libre de Bruxelles, Pasteur institute, Brussels, Belgium. E-mail: [email protected] 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.



P63

Tyrosinase-Catalyzed Modification of Bombyx mori Silk Proteins

Giuliano Freddia, Anna Anghileria, Sandra Sampaioa, Johanna Buchertb, Raija Lanttob, Kristiina Kruusb, Patrizia Montic, Paola Taddeic

aStazione Sperimentale per la Seta, via Giuseppe Colombo 83, Milano 20133, Italy; bVTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT, Finland; cDept. of Biochemistry, University of Bologna, via Belmeloro 8/2, Bologna 40126, Italy E-mail: [email protected] 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 end-uses. 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 copper-containing 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 self-condensation 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.



P64

Kinetics of Laccase Mediator System Delignification of an Eucalyptus globulus Kraft Pulp

Sílvia Guilhermea, Ofélia Anjosa, Rogério Simõesb

aEscola Superior Agrária, Instituto Politécnico de Castelo Branco, Quinta da Senhora de Mércules, Apartado 119, 6001 Castelo Branco, Portugal; bUnidade de Materiais Têxteis e Papeleiros, Universidade da Beira Interior, Convento de Santo António, 6201-001, Covilhã, Portugal E-mail: [email protected] 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.



P65 Model Wastewaters Decolouristion by Pseudomonas

putida MET94 Bruno Mateusa, Diana Mateusb,c, Luciana Pereirab,c, Orfeu Floresa, Lígia O. Martinsb, c

aSTAB VIDA, Av da República, 2781-901 Oeiras, Portugal, bInstituto de Tecnologia Química e Biológica (ITQB),Universidade Nova de Lisboa, Av da República, 2784-505 Oeiras, Portugal, cInstituto de Biologia Experimental e Tecnológica (IBET), Av da República, 2784-505 Oeiras, Portugal E-mail: [email protected] 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 exponential-stationary 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 (OD600nm=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).



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: [email protected] 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.

PARTICIPANTS



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: [email protected] 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: [email protected] 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: [email protected] Andberg, Martina Tietotie 2 P.O. Box 1500 FIN-02044 VTT Finland Tel: +358207225124 e-mail: [email protected] Ander, Paul WURC, Dept. of Wood Science, SLU, PO Box 7008 SE-75007 Uppsala Sweden Tel: 46-18-67 34 34 e-mail: [email protected] Anjos, Ofélia Escola Superior Agrária, Quinta da Senhora de Mércules, Apartado 119, 6001-909 Castelo Branco Portugal Tel: 272339900 e-mail: [email protected]

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: [email protected] Arias, Maria Enriqueta Departamento de Microbiologia y Parasitologia Universidad de Alcalá Alcalá de Henares Spain Tel: +34918854633 e-mail: [email protected] Baldrian, Petr Institute of Microbiology ASCR Videnska 1083 14220 Praha 4 Czech Republic Tel: +420723770570 e-mail: [email protected] Basosi, Riccardo Department of Chemistry University of Siena Via Aldo Moro 53100 Siena, Italy Tel: +39577234240 e-mail: [email protected] Beckett, Richard School of Biological Sciences University of KwaZulu-Natal PBag X01 Scottsville 3209, South Africa Tel: +27 33 260 5141 e-mail: [email protected] Behar, Candan Tamerler Istanbul Technical University Department of Molecular biology and Genetics Maslak/Istanbul Turkey Tel: 0090 212 286 22 51 e-mail: [email protected]



Belova, Nina V. Komarov Botanical Institute RAS, Prof. Popov Str., 2, St. Petersburg 197376 Russia Tel: +7(812)3464442 e-mail: [email protected] Bento, Isabel Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Av da República 2781-901 Oeiras Portugal Tel: +351 214469662 e-mail: [email protected] Bermek, Hakan Istanbul Technical University Department of Molecular biology and Genetics Maslak/Istanbul Turkey Tel: 0090 212 286 22 51 e-mail: [email protected] Bezerra, Rui Manuel Furtado Dep. Engenharia Biológica e Ambiental, UTAD, Apartado 1013, 5001-801 Vila Real, Portugal Tel: 259350465 e-mail: [email protected]. Boehmer, Ulrike Technische Universität Dresden, Institute for Food Technology and Bioprocess Engineering, Bergstraße 120 01069 Dresden Germany Tel: +49 351 46334882 e-mail: [email protected] Bols, Christian-Marie Wetlands Engineering SPRL Parc Scientific Fleming 5 Rue du Laid Burniat BE-1348 Louvain-la-Neuve Belgium Tel : +32478421647 e-mail : [email protected]

Briganti, Fabrizio Department of Chemistry University of Florence Via della Lastruccia 3 Sesto Fiorentino 50019 Florence, Italy Tel: +39 055 4573343 e-mail: [email protected] Brissos, Vânia Instituto Superior Técnico Centro de Engenharia Biológica e Química Portugal Tel: 218419132 e-mail: [email protected] Cabana, Hubert Department of Chemical engineering University of Sherbrooke 2500 Boulevard de l’Université Sherbrooke (Qc) J1K 2R1 Canada Tel : +18198217171 e-mail : [email protected] Call, Hans-Peter Bioscreen e.K. 52531 Uebach-Palenberg Heinsberger Strasse 15 Germany Tel: 0049 2451 952814 e-mail: [email protected] Caruso, Carla Dipartimento di Agrobiologia e Agrochimica Via S. Camillo de Lellis 01100 Viterbo Italy Tel:+390761357330 e-mail: [email protected] Casieri, Leonardo Department of Plant Biology, University of Turin. Viale Mattioli 25, 10125 Turin Italy Tel: 00390116705964 e-mail: [email protected]



Cavaco-Paulo, Artur Departamento de Engenharia Têxtil Universidade do Minho Campus de Azúrem 4800-058 Guimarães Portugal Tel: +351 253510280 e-mail: [email protected]

Chen, Zhenjia Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Av da República 2781-901 Oeiras Portugal Tel: +351 2144697653 e-mail: [email protected] 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: [email protected] Coelho, Rui Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Av da República 2781-901 Oeiras Portugal Tel: +351 214469535 e-mail: [email protected] Colao, Maria Chiara Dept. Agrobiology and Agrochemistry, Tuscia University Via C. de Lellis snc, 01100 Viterbo Italy Tel: +390761357236 e-mail: [email protected] Cortez, João Nottingham Trent University, Erasmus Darwin Clifton Campus, Nottingham, NG11 8NS England Tel: 0115 848 3089 e-mail: [email protected]

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: [email protected] Danielsen, Steffen Protein Design Building 1U1.20 Brudelysvej 26 Novozymes A/S Denmark Tel: +4544427761 e-mail: [email protected] de la Rubia Nieto, Teresa Dpto. Microbiology Faculty of Pharmacy Univ. of Granada Campus Cartuja 18071 Granada (Spain) Spain Tel: 38 958243875 e-mail: [email protected] de Wildeman, Stefaan DSM-Research Dept. LS-ASC&D PO Box 18 6160 MD Geleen The Netherlands Tel: +31 46 4760138 e-mail: [email protected] 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: [email protected] Dernalowicz-Malarczyk, Elzbieta Biochemistry Department, M.Curie-Sklodowska University, Sklodowska Square 3, 20-031 Lublin Poland Tel: +4881 5375770 e-mail: [email protected]



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: [email protected] Dias, José Albino Gomes Alves Dep. Engenharia Biológica e Ambiental, UTAD, Apartado 1013, 5001-801 Vila Real, Portugal Tel: 259350725 e-mail: [email protected]

Domínguez Represas, Alberto Department of Chemical Engineering. Isaac Newton Building. University of Vigo. 36310 Vigo. Spain Tel: 34986812304 e-mail: [email protected] Durão, Paulo Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Av da República 2781-901 Oeiras Portugal Tel: +351 214469535 e-mail: [email protected] Elisashivili, Vladimir Inst of Biochemistry and Biotechnology 10km Agmashenebeli Kheivani 0159 Tblisi Georgia Tel: +97248249653 e-mail: [email protected]

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: [email protected]

Ergun, Aisle Istanbul Technical University Molecular Biology and Genetics Dept. 34469 Maslak/Istanbul Turkey Tel: +90 212 286 22 51 e-mail: [email protected] Evtuguin, Dmitry V. Department of Chemistry University of Aveiro 3810-193 Aveiro Portugal Tel: +351 234 370693 e-mail: [email protected] 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: [email protected] 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: [email protected] 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: [email protected] Fillat, Amanda Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Av da República 2781-901 Oeiras Portugal Tel: +351 214469535 e-mail: [email protected]



Fonseca, Bruno Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Av da República 2781-901 Oeiras Portugal Tel: 937515800 e-mail: [email protected] Freddi, Giuliano Stazione Sperimentale per la Seta via Giuseppe Colombo, 83 20133 Milano Italy Tel: +39 02 2665990 e-mail: [email protected]

Galli, Carlo Dipartamento de Chimica Universita “La Sapienza” Roma Italy Tel: +390649913386 e-mail: [email protected] Garzillo, Anna Maria Vittoria Dipt. of Agrobiology and Agrochemistry Via S. Camillo de Lellis, snc 01100 - Viterbo Italy Tel: +390761357316 e-mail: [email protected]

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: [email protected]

Golovleva, Ludmila G.K. Sryabin Institute of Biochemistry and Physiology of Microorganisms RAS Moscow Russian Federation Tel: +4967732564 e-mail: [email protected]

Gómez Sieiro, José Department of Chemical Engineering. Isaac Newton Building. University of Vigo. 36310 Vigo. Spain Spain Tel: 34986812304 e-mail: [email protected] Gravitis, Janis SA Latvian State Institute of Wood Chemistry Dzerbenes St.27 Riga LV-1006 Latvia Tel: ++371 7553137 e-mail: [email protected] Graz, Marcin Department of Biochemistry, Maria Curie-Sklodowska University, Sklodowska Square 3, 20-031 Lublin Poland Tel: +4881 5375735 e-mail: [email protected] Güebitz, Georg Graz University of Technology, Dept of Environmental Biotechnology Petersgrasse 12, 8010 Graz Austria Tel: +43 316 8738312 e-mail: [email protected]

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: [email protected] Hadar, Yitzhak Department of Microbiology and Plant Pathology, Faculty of Agriculture, Rehovot, Israel Israel Tel: 972-8-9489935 e-mail: [email protected]



Hakala, Terhi IKCL Science & Consulting Oy Keskuslaboratorio - Centrallaboratorium Ab P.O. Box 70,02151 Espoo Finland Tel: + 358 (0) 20 7477 352 e-mail: [email protected] Hakulinen, Nina Dept. of Chemistry University of Joensuu PO Box 111, 80101 Joensuu Finland Tel: +358132512243 e-mail: [email protected] Hatakka, Annele Department of Applied Chemistry and Microbiology, PO Box 56 (Viikki Biocenter), 00014 University of Helsinki Finland Tel: +358-9-19159314 e-mail: [email protected] 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: [email protected] 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: [email protected] Hiltunen, Jaakko KCL Science and Consulting P.O.Box 70, FI-02151 Espoo, Finland Tel: +358(0)207477529 e-mail: [email protected]

Hofrichter, Martin International Graduate School of Zittau Environmental Biotechnology Unit Markt 23 02763 Zittau Germany Tel: +493583771521 e-mail: [email protected] Jarosz-Wilkolazka, Anna Biochemistry Department Maria Curie-Sklodowska University Sklodowska Place 3 20-031 Lublin, Poland Tel: 48 81 537 56 65 e-mail: [email protected] 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 : [email protected] Joosten, Vivi Laboratory of Biochemistry Wageningen University Dreijenlaan 3 6703 HA Wageningen The Netherlands Tel: +31-317-484468 e-mail: [email protected] Junghanns, Charles UFZ Centre for Environmental Research Leipzig-Halle Department of Environmental Microbiology Permoserstraße 15 D-04318 Leipzig Germany Tel: +493412352547 e-mail: [email protected] Keshavarz, Tajalli Department of Applied and Molecular Biosciences University of Westminster London W1W 6UW Tel 44-(0)20 79115000 ext 3562 e-mail: [email protected]



Kiekens, Paul University of Ghent Department of Textiles Technologiepark 907 B-9052 Gent (Zwijnaarde) Belgium Tel: + 329 2645735 e-mail: [email protected] 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: [email protected] 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: [email protected] Kruus, Kristiina VTT Technical Research Centre of Finland P.O. Box 1500 Espoo FIN-02044 VTT Finland Tel: + 358-20-722 5143 e-mail: [email protected] Kurt, Gunseli Istanbul Technical University Department of Molecular Biology Maslak / Istanbul Turkey Tel: 0090 212 286 22 51 e-mail: [email protected] Lamarino, Giseppina Università degli Studi di Napoli, Facoltà di Agraria Italia Tel: +390812539166 e-mail: [email protected]

Leferink, Nicole Laboratory of Biochemistry Wageningen University Dreijenlaan 3 6703 HA Wageningen The Netherlands Tel: +0317-484468 e-mail: [email protected] 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: [email protected] Lundell, Taina University of Helsinki, Department of Applied Chemistry and Microbiology Finland Tel: +358 9 19159316 e-mail: [email protected], 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: [email protected] Marino, Gennaro Department of Organic Chemistry and Biochemistry Federico II University of Naples, Via Cinthia 80126 Napoli Italy Tel: +39-081674312 e-mail: [email protected] Martin, Claudia UFZ Centre for Environmental Research Leipzig-Halle Permoserstr. 15; 04318 Leipzig Germany Tel: 0049 341 235 2547 e-mail: [email protected]



Martínez, Angel T. CIB, CSIC Ramiro de Maeztu 9 E-28040 Madrid Spain Phone: +34 918373112 e-mail: [email protected] Martínez, María J. CIB, CSIC Ramiro de Maeztu 9 E-28040 Madrid Spain Tel: +34 918373112 e-mail: [email protected] 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: [email protected] Mateus, Bruno STAB Vida Av da República 2781-901 Oeiras Portugal Tel: +351 214469763 e-mail: [email protected] Matijosyte, Inga Delft University of Technology Julianalaan 136 2628 BL Delft The Netherlands Tel: + 310152782693 e-mail: [email protected]

Matura, Anke Professur Allgemeine Biochemie, TU Dresden, D-01062 Dresden Germany Tel: +49 351 4633 5505 e-mail: [email protected]

Maximo, Cristina UBB-DB INETI Est Paço do Lumiar, 22 1649-038 Lisboa Portugal Tel: +351 210924729 e-mail: [email protected] Minibayeva, Farida Institute of Biochemistry and Biophysics Russian Academy of Science 2/31 Lobachevsky Street Kazan 420111, Tatarstan Russia Tel: +7 8432386320 e-mail: [email protected] Mink, Daniel DSM Research Dept. LS-ASC&D PO Box 18 6160 MD Geleen The Netherlands Tel: +31 46 60869 e-mail: [email protected] Moilanen, Ulla Laboratory of Bioprocess Engineering, Helsinki University of Technology, P.O. Box 6100, FI-02015 TKK Finland Tel: +358 45 6753925 e-mail: [email protected] Moldes Moreira, Diego Departamento de Engenharia Textil Universidade do Minho Campus Azurem. 4800-Guimaraes Portugal Tel: +351 253510280 e-mail: [email protected] Monti, Daniela Istituto di Chimica del Riconoscimento Molecolare -CNR, Via Mario Bianco, 9, 20131 Milano Italy tel: ++39 02285 00038 e-mail: [email protected]



Munõz-Dorado, Jose Departamento de Microbiologia Facultad de Ciencias Universidad de Granada Spain Tel: +34958243183 e-mail: [email protected] Nikolov, Alexandre Novozymes A/S Krogshøejvej 2880 Bagsvaerd Denmark Tel: + 45 44492212 e-mail: [email protected] Olszewska, Anna Department of Biochemistry, Maria Curie-Sklodowska University, Sklodowska Place 3, 20-031 Lublin Polan Tel: +48815375705 e-mail: [email protected] Opwis, Klaus Deutsches Textilforschungszentrum Nord-West e.V. Adlerstr. 1 D-47798 Krefeld Germany Tel: +49-2151-843-205 e-mail: [email protected] Ostergaard, Lars Novozymes A/S Dept. of Protein Diversity Brudelysvej 26 DK-2880 Bagsvaerd Denmark Tel: +45 444 60271 Email: [email protected] Oudia, Atika Unidade de Têxteis e Materiais de Papel UBI - Universidade da Beira Interior 6201-001 Covilhã Portugal Tel: +3519692165 e-mail: [email protected]

Papa, Rosanna Department of Organic Chemistry and Biochemistry Federico II University of Naples Napoli Italy Tel: +39 081674320 e-mail: [email protected] Pereira, Luciana Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Av da República 2781-901 Oeiras Portugal Tel: +351 214469535 e-mail: [email protected] 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: [email protected] 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: [email protected] Pérez-Torres, Juana Departamento de Microbiologia Facultad de Ciencias Universidad de Granada Spain Tel: +34958243183 e-mail: [email protected] 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: [email protected]



Pogni, Rebecca Department of Chemistry University of Siena Via Aldo Moro 53100 Siena Italy Tel: +39577234258 e-mail: [email protected] Polak, Jolanta Department of Biochemistry, Maria Curie-Sklodowska University, Sklodowska Place 3, 20-031 Lublin Poland Tel: +48815375705 e-mail: [email protected]

Psurtseva, Nadya V. Komarov Botanical Institute RAS, Prof. Popov Str., 2, St. Petersburg 197376 Russia Tel: +7(812)3464442 e-mail: [email protected]

Rebhun, Moti MycoEnzyme, Ltd. Institute of Evolution, University of Haifa Mount Carmel, Haifa 31905 Israel Tel: +972503231065 e-mail: [email protected]

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: [email protected] 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: [email protected]

Sanchez-Amat, Antonio Facultad de Biologia Dep de Microbiologia y Genetica Universidad de Murcia 30071 Murcia Spain Tel: +34 968364955 e-mail: [email protected]

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: [email protected]

Sanromán, Mª Angeles Braga Department of Chemical Engineering. Isaac Newton Building. University of Vigo. 36310 Vigo, Spain Tel: 34986812383 e-mail: [email protected] 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: [email protected] 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: [email protected] Scozzafava, Andrea Department of Chemistry University of Florence Via della Lastruccia 3 Sesto Fiorentino 50019 Florence, Italy Tel: +39 055 4573273 e-mail: [email protected]~



Seifert, Jana Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg Germany Tel: +49-3731-394015 e-mail: [email protected]

Sigoillot-Claude, Cécile LBDP/DEVM CEA Cadarache 13108 St Paul lez Durance Cedex France Tel: +33 (0)4 42 25 31 45 e-mail: [email protected] Smith, Andrew T. Biochemisty Department School of Life Sciences University of Sussex UK e-mail: [email protected] 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: [email protected] Songulashvili, Giorgi Institute of Evolution University of Haifa Mt Carmel Haifa 31905 Israel Tel: +97248249653 e-mail: [email protected] 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: [email protected]

Suurnäkki, Anna VTT P.O. Box 1000 02044 VTT Finland Tel: +358 20 722 7178 e-mail: [email protected] Todorovic, Smilja Instituto de Tecnologia Química e Biológica Av. da Republica EAN 2780-157 Oeiras Portugal Tel: 351-214469321 e-mail: [email protected] Tranchimand, Sylvain Laboratoire de Bioinorganique Structurale Faculté des Sciences de St Jérôme case 432, 13397 Marseille cedex 20 France Tel: +33491282856 e-mail: [email protected] Tron, Thierry Laboratoire of Bioinorganique Structurale CNRS UMR 6517 Case 432, Faculté des Sciences Saint Jérôme 13397 Marseille France Tel : +33491282856 e-mail: [email protected] 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: [email protected] Tzanov, Tzanko Technical University of Catalonia Colom 108223 Terrasa, Barcelona Spain Tel: +34628081722 e-mail: [email protected]



Valderrama, Brenda Biotechnology Institute University of Mexico Av. Universidad 2001 Col. Chamilpa CP62210 Cuernavaca, Mor. Mexico Tel: +527773291610 e-mail: [email protected] van Berkel, Willem J.H. Laboratory of Biochemistry Wageningen University The Netherlands Tel: 31-317-482861 e-mail: [email protected]

van Dijk, Alard DSM Food Specialties 699-0330 PO Box 1 2600 MA Delft The Netherlands Tel: +31-152793661 e-mail: [email protected] van Hellemond, Erik Laboratory of Biochemistry Nijenborgh 4 9747 AG Groningen Netherlands Tel: +31-50-3633540 e-mail: [email protected] van Pée, Karl-Heinz Biochemie TU Dresden D-01062 Dresden Germany Tel: +49351-463-34494 e-mail: [email protected] 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: [email protected]

Varesse, Cristina Giovanna Dept. Plant Biology University of Turin viale Mattioli, 25 10125 Turin Italy Tel: +39 011 6705964 e-mail: [email protected] 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: [email protected] Viikari, Liisa VTT PO BOX 1000 02044 VTT Espoo Finland Tel: +358207225140 e-mail: [email protected] 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: [email protected] Xavier, Ana M.R.B. Department of Chemistry University of Aveiro 3810-193 Aveiro Portugal Tel: +351 234 370716 e-mail: [email protected] Xu, Feng Novozymes Inc 1445 Drew Ave Davis, CA 95616 USA Tel: 530-757-8100 e-mail: [email protected]



Yesiladali, S. Koray Istanbul Technical University Molecular Biology and Genetics Department Maslak/Istanbul Turkey Tel: 0090 212 286 22 51 e-mail: [email protected] Zille, Andrea Departamento de Engenharia Têxtil Universidade do Minho Campus de Azúrem 4800-058 Guimarães Portugal Tel: +351253510280 e-mail: [email protected] Zimmermann, Wolfgang Institute of Biochemistry Department of Microbiology and Bioprocess Technology University of Leipzig Johannisallee 21-23 D-04103 Tel: + 49 3419736781 e-mail: [email protected]

AUTHOR INDEX



Agapito, M. S. M. P59

Agathos, S. N. L30, P19, P62

Ahola, E. L9

Allen, C. L10, P25

Amaral, P. F. F. P45

Anastasi, A. P53

Andberg, M. P32, L21

Anghileri, A. P63

Anh, D. H. L16

Anjos, O. P64

Arboleda, C. P62

Arends, W. C. E. P68

Arias, M. E. P44, P61

Asimgil, H. P14

Auer, S. P32

Autore, F. L17, P15

Baldrian, P. L6

Baratto, M. C. P25, L19

Barrasa, J. M. L31

Basar, F. P42

Basosi, R. L18, L19, P25

Basto, C. L26

Batista, C. F. L15

Baumberger, S. P33

Bebrone, C. P17

Beckett, R. P1

Behar, T. P42

Belova, N. V. P4, P6, L7

Bento, I. P34, P35

Bermek, H. P14

Bertini, L. P9

Bezerra, R. M. F. P13, P51

Blanchet, A. L3

Böhmer, U. P43

Bols, C. M. P17, P31

Boyd, D. R. L10

Brault, A. P33

Briganti, F. L23, L7, P36

Briozzo, P. P33

Brogioni, B. L19

Bruckman, T. P66

Buchert, J. L9, P63

Buonocore, V. P9, P47

Cabana, H. P19, P62

Cajthaml, T. L6

Calafell, M. P66

Call, H.-P. L34

Caminade, E. P33

Cammarota, M. C. P45, P46

Caporale, C. P9

Caruso, C. P9

Casieri, L. P53

Cavaco-Paulo, A. L26, L25, P20, P39

Cestone, R. P15

Chasov, A. V. P3

Chernykh, A. L7, L23

Choinowski, T. L18

Coelho, Mª A. Z. P45, P46

Colao, M. C. P47

Corbisier, A. M. L27, P17, P41

Costa-Ferreira, M. L35

Creff, A. L3

D'Annibale, A. L20

de la Rubia Nieto, T. P12

de Vries, S. P26

del Rio, J. C. L33



Dernalowicz-Malarczyk, E. P7, P29, P52

Desnos, T. L3

Di Berardino, I. P9

Dias, J. A. G. A. P13, P51

Domínguez Represas, A. P48, P60

Dong, C. L14

Doyle, W. L24

Durão, P. P34, P35

Elisashvili, V. L4

Enaud, E. L27, P17, P41

Ergun, A. P42

Ernyei, A. L14

Evtuguin, D. V. P55, P59

Fagerström, R. P16

Faraco, V. L17

Fernandes, A. T. P34, P35, P38

Ferranoni, M. L7, L23, P36

Festa, G. L17, P15

Flores, O. P65

Fonseca, B. P67

Fraaije, M. W. L8

Fraga, I. P13, P51

Fraternali, F. L17

Freddi, G. P63

Frère, J.-M. P17

Galli, C. L20

Gamelas, J. A .F. P55

Garzillo, A. M. V. P47

Gaudin, C. P30

Gaydou, V. P30

Gentili, P. L20

Gianfreda, L. L31, P70

Giardina, P. L17, L19, P15, P24

Gil, P. L15

Golovleva, L. L7, L23, P36

Gómez, D. L1

Gómez-Santos, N. L5, P8

Gómez-Sieiro, J. P49

Gominho, J. L35

Gordon, L. K. P3

Górnacka, B. P39

Grąz, M. P7, P29, P52

Grönqvist, S. L36

Guebitz, G. M. L25, P21, P58

Guevara, O. P61

Guilherme, S. P64

Guillén, F. P44

Gullotto, A. L23

Gutiérrez, A. L33

Hakala, T. K. P2, P5

Hakulinen, N. L21, P32

Haltrich, D. P28

Hatakka, A. L2, P2, P5, P6, P18

Hatscher, C. L14

Hernández, M. P44

Hernandéz-Romero, D. P23

Hildebrandt, P. P37

Hildén, K. L2, P2, P5

Hofrichter, M. L16

Huber, R. P38

Hubert, S. P17

Hüner, P. P14

Iacazio, G. P30

Iamarino, G. L31, P70

Ibarra, D. L33

Irgoliç, R. P20



Ivancich, A. L24

Janssen, D. B. L8

Jarosz-Wilkolazka, A. P7, P29, P50, P52, P54

Jimenez, G. A. P62

Jolivalt, C. P33

Jones, J. P. P19, P62

Joosten, V. P26

Junghanns, C. L28, P69

Kachlishvili, E. L4

Kalkkinen, N. L9

Kallio, J. P16

Kandelbauer, A. L25

Karagüler, N. G. P10, P11

Karatas, A. P10, P11

Kaschabek, S. P27

Kavieva, A. A. P3

Kim, S.-Y. L26

Kinne, M. L16

Kiyashko, A. P4, P6

Kluge, M. L16

Knittel, D. L32

Kochmanska-Rdest, J. P7, P50

Koivula, A. L21, P32

Kokol, V. P21, P58

Kolesnikov, O. P. P3

Kolomytseva, M. P. P36

Kooij. R. P68

Krauss, G. L28

Kruus, K. L9, L21, P16, P32, P63

Kulakov, L. L. L10

Kuncinger, T. L12

Kunzendorf, A. L14

Kurt, G. P10, P11

Kvesitadze, G. L4

Lantto, R. P63

Larkin, M. J. P25

Laufer, Z. P1

Leferink, N. L11

Leontievsky, A. A. L23

Lindley, P. F. P34, P35

Lipscomb, D. A. P25

Longo, M. A. P60

Lorenzini, B. P17

Lourenço, A. L35

Louro, R. O. P67

Lu, Y. L11

Lucas, M. P12

Lucas-Elío, P. L1

Ludwig, R. P28

Lundell, T. L1, L2, P6

Luterek, J. P54

Madzak, C. P33

Magali, C. P46

Maijala, P. P2,P5, P18

Makela, M. R. L2

Marchisio, V. F. P53

Martin, C. L28

Martínez, A. T. L18, L31, L33

Martínez, J. P12

Martínez, M. J. L31, L18, P2

Martins, L. O. L22, P34, P35, P38, P40, P65

Marzorati, M. P57

Matera, I. L23

Mateus, B. P65

Mateus, D. P65

Matijosyte, I, P68



Matura, A. P22, P43

Máximo, C. L35

Mejía, A. I. P62

Melo, E. P. P34, P38

Merhautová, V. L6

Mertens, V. L27

Metreveli, E. L4

Mettälä, A. P18

Mikiasshvili, N. L4

Mikkonen, H. L36

Mimmi, M. C. P33

Minibayeva, F. V. P1, P3

Mityashina, S. Y. P3

Moeder, M. L28

Moilanen, U. P18

Moldes, D. P20, P48, P49, P60

Molina, J. M. P44, P61

Monti, D. P57

Monti, P. P63

Moraleda-Muñoz, A. L5, P8

Morales, M. L18

Mougin, C. P33

Moya, R. P44

Muñoz-Dorado, J. L5, P8

Murgida, D. P37

Myasoedova, N. M. L7, L23

Naismith, J. H. L14

Ngo, E. L24

Nouaimeh, N. P17

Novotný, C. P53

Nussaume, L. L3

Nyanhongo, G. L25

Olsson C. P2

Olszewska, A. P7, P50, P54

Onderwater, R. C. A. P31

Opwis, K. L32

Orlandi, M. L36

Orozco, A. L. P61

Oudia, A. P56

Öztemel, Z. P. Ç. P42

Pacheco, I. P67

Paloheimo, M. P16

Pamplona-Aparicio, M. P17, P41

Papa, R. P24

Parrilli, E. P24

Pastinen, O. P18

Penninck, M. J. P62

Pereira, A. N. P13

Pereira, H. L35

Pereira, L. P40, P65

Pereira, M. P38

Pereira, P. M. P67

Pérez, M. I. P61

Pérez-Boada, M. L18

Pérez-Torres, J. L5, P8

Peterbauer, C. P28

Pich, A. P43

Pinto, F. V. P45

Piontek, K. L18

Piscitelli, A. L17, P15

Poellinger-Zierler, B. L25

Pogni, R. L19, L18, P25

Polak, J. P7, P50, P52

Polvillo, O. P61

Pontes, A. S. N. L20, P55

Prigione, V. P53



Psurtseva, N. V. L7, P4, P6

Puranen, T. P16

Queiroz, J. P56

Rao, M. A. P70

Rehorek, A. P39

Rencoret, J. L33

Reymond, M. L3

Ricaud, L. L3

Riva, S. P57

Rodakiewicz-Nowack, J. P54

Rodríguez, J. P61

Rodríguez-Rincon, F. P12

Rodríguez-Solar, D. P49

Rouvinen, J. L21, P32

Ruiz-Dueñas, F. J. L18

Rumpf, J. L14

Russo, F. P70

Ruzzi, M. P47

Sagui, F. P57

Saloheimo, M. L9

Sampaio, S. P63

Sanchez-Amat, A. L1, P23

Sánchez-Sutil, M. C. L5, P8

Sannia, G. L17, L19, P15, P24

Sanromán, M. A. P48, P49, P60

Scelza, R. P70

Scheibner, K. L16

Schlömann, M. P27

Schlosser, D. L28, P69

Schmid, C. L14

Schnerr, H. L14

Schollmeyer, E. L32

Schroeder, M. L25, P21, P58

Scozzafava, A. L7, L23, P36

Seifert, J. P27

Selinheimo, E. L9

Sergi, F. L20

Sheldon, R. A. P68

Sharma, N. D. L10

Sigoillot-Claude, C. L3

Silvestri, F. P47

Simeonov, P. P27

Simões, R. P56, P64

Sinicropi, A. L19

Smith, Andrew T. L24

Snajdr, J. L6

Soares, C. M. P38

Solano, F. L1, P23

Solé, M. L28

Srebotnik, E. L12

Stancarone, V. P9

Suarez, A. P12

Suurnäkki, A. L36

Svistoonoff, S. L3

Svobodová, K. P53

Taddei, P. P63

Tadesse, M. A. L20

Tamerler, C. P10, P11, P14

Ters, T. L12

Tilli, S. L23

Todorovic, S. P37

Tranchimand, S. P30

Tron, T. P30

Trovaslet, M. L27, P17, P41

Tsiklauri, N. L4

Tutino, M. L. P24



Tzanov, T. P66

Ullrich, R. L16

Valásková, V. L6

Valderrama, B. L15

Valtakari, L. P16

van Berkel, W. J.H. L13, L11, P26

van den Berg, W. A. M. L11, P26

van Hellemond, E. L8

van Pée, K. H. L14, P22, P43

Vanhulle, S. L27, P17, P41

Varese, G. C. P53

Vazquez-Duhalt, R. L15

Vehmaanperä, J. P16

Viikari, L. L36

De Vries, S. P68

Wage, T. L14, P43

Westerholm-Parvinen, A. L9

Winquist, E. P18

Xavier, Ana M.R.B. P59, P55

Xu, F. L29

Yakovleva, N. P4, P6

Yesiladali, S. K. P10, P42

Zámocky, M. P28

Zille, A. L26, P39, P20

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