Professional Documents
Culture Documents
Abstract Book
Oxizymes in Oeiras – 3rd European Meeting in Oxizymes
September, 7-9, 2006
Oeiras, Portugal
This book of abstracts was carefully produced. Nevertheless we do not warrant the information
contained therein to be free of errors
WELCOME TO OEIRAS
The Organizing Committee of the 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.
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.
4
OXIZymes in Oeiras SPONSORS
5
Oxizymes in Oeiras, 3rd European Meeting in Oxizymes
Scientific Committee
Lígia O. Martins
André T Fernandes
Paulo Durão
Luciana Pereira
Cláudio M. Soares
Isabel Bento
Manuela M. Pereira
lunch lunch
poster attendance poster attendance
15.00-16.30 14.30-16.00
S3 S7
ENZYMOLOGY I APPLICATIONS I
coffee-break coffee-break
17.00 -18.30 16.30 -18.00
S4 S8
ENZYMOLOGY II APPLICATIONS II
18.30-19.00 20.30
Welcome Drink Oxizymes dinner
“Porto de Honra”
8.00-9.00 Registration
9.00-9.30 Opening
11.00-11.30 Coffee
11.30-11.55 L5 - Biological Functions and Regulation of the Multicopper Oxidase LcsA from
Myxococcus xanthus
Juana Pérez-Torres, Granada Univ, Spain
11.55-12.20 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
12.20-12.40 L7 – Novel Efficient Producers of Blue Laccases
Ludmila Golovleva, GKS Inst Biochem Physiolo Microorg, Moscow,
Russia
12.40-13.00 L8 – Discovery of an Epoxide Forming Monooxygenase from the Metagenome
Erik van Hellemond, Groningen Univ, The Netherlands
15.00-16.30 S3 – ENZYMOLOGY I
CHAIRPERSON: THIERRY TRON
16.30-17.00 Coffee
17.00-18.30 S4 – ENZYMOLOGY II
CHAIRPERSON: STEFFEN DANIELSEN
10.30-11.00 Coffee
14.30-16.00 S7 – APPLICATIONS I
CHAIRPERSON: LIISA VIIKARI
16.00-16.30 Coffee
16.30-18.00 S8 – APPLICATIONS II
CHAIRPERSON: PAUL ANDER
10.30-11.00 Coffee
L1
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.
[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
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.
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
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
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
L6
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
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.
L8
StyA
O2 H2O
FADH2 FAD
StyB
NAD+ NADH, H+
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
Kristiina Kruusa, Emilia Selinheimoa, Markku Saloheimoa, Elina Aholab, Ann Westerholm-
Parvinena, Nisse Kalkkinenb and Johanna Bucherta
a
VTT Technical Research Centre of Finland, P.O. Box 1500, Espoo FIN-02044 VTT, Finland;
b
Protein Chemistry Research Group and Core Facility, Institute of Biotechnology, P.O. Box
65, FIN-00014 University of Helsinki, Finland
E-mail: kristiina.kruus@vtt.fi
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
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.
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
Nicole G. H. Leferinka, Yu Lua, Willy A. M. van den Berga, Willem J. H. van Berkela
a
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen,
The Netherlands
E-mail: nicole.leferink@wur.nl
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.
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
Competence Centre for Wood Composites and Wood Chemistry, St.-Peter-Strasse 25, 4021
Linz, Austria; and Institute of Chemical Engineering, Vienna University of Technology,
Getreidemarkt 9, 1060 Wien, Austria
E-mail: ewald.srebotnik@tuwien.ac.at
We have observed unsatisfactory linearity and reproducibility in routine assays for fungal
laccase activity. It was found that these problems were due to a slight but significant
inhibition of laccase by acetate, the most commonly used buffering substance in laccase
assays.
Kinetic measurements performed with recombinant laccase from Trametes villosa (44008,
Novo Nordisk) and ABTS as a substrate revealed an s-linear, i-parabolic mixed inhibition
type for acetate at pH 5.0 with calculated Ki and Ki’ values of 38.8 mM and 117.5 mM,
respectively. Thus the affinity of acetate for laccase was very low compared to the classical
inhibitor azide which exhibited Ki and Ki’ values of 0.0176 mM and 0.0106 mM,
respectively. Similar effects were observed at pH 4.0 and also for wild-type laccases from
several other Trametes species such as T. pubescens CBS 696.94. However, due to the
relatively high concentrations used in routine assays, inhibition levels were substantial
ranging from 15% to 50% of initial activity at acetate concentrations from 10 mM to 100 mM,
respectively. The first order inactivation rate constant was rather low (k ~0.1 min-1). In
practice this means that upon contact with acetate, 90% of the final (stable) inactivation level
is reached only after ~23 min.
No correlation was found between the size of the carboxyl anion and the extent of inhibition -
formiate, propionate as well as butyrate were stronger inhibitors than acetate. Moreover, the
results indicated a simple linear non-competitive type for formiate in contrast to acetate.
Several α-hydroxycarboxylic, di- and tricarboxylic acids were also tested at pH values from
3.0 to 5.0. While inhibition characteristics of lactate and glycolate were similar to those of
acetate, the inhibition characteristics of citrate and succinate were not: generally the extent of
inhibition by citrate and succinate was much lower (<10%) and inactivation rate constants
were at least two orders of magnitudes higher.
L13
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?
L14
[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
Paloma Gil a,b, Cesar Ferreira Batista c, Rafael Vazquez-Duhalt a and Brenda Valderrama a,b
a
Departamento 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 brenda@ibt.unam.mx
Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and
are thus potentially useful for multiple applications. However, hemeperoxidases are unusually
susceptible to self-inflicted oxidative damage [1]. The search for more stable
hemeperoxidases has been actively pursued by different methods in the past, including 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
L17
Laccase Engineering by Rational and Random Mutagenesis
Giovanna Festa1, Alessandra Piscitelli1, Vincenza Faraco1, Paola Giardina1, Flavia Autore1,
Franca Fraternali2 and Giovanni Sannia1
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.
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
a
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain;
b
Department of Chemistry, University of Siena, via Aldo Moro, I-53100 Siena, Italy; cInstitute
of Biochemistry, ETH, Schafmattstrasse 18, CH-8093 Zürich, Switzerland
E-mail: ATMartinez@cib.csic.es
Versatile peroxidase (VP) represents a third type of fungal ligninolytic peroxidase together
with lignin peroxidase and manganese peroxidase [1]. Ligninolytic peroxidases differ from
peroxidases from saprophytic basidiomycetes (e.g. Coprinopsis cinerea) and plant
peroxidases involved in monolignol polymerization, by their high redox potential enabling
oxidative degradation of lignin. This property makes them the biocatalyst of choice for
industrial applications requiring enzymatic oxidation of recalcitrant aromatic compounds,
including simple and complex/polymeric substrates. However, the native enzymes are far
from optimally operating under industrial conditions. Therefore, enzyme structure-function
studies are required as a first step for designing new tailor-made biocatalysts. VP has been
described in fungi from the genera Pleurotus and Bjerkandera. The VP from Pleurotus
eryngii, a species of biotechnological interest, is the most extensively investigated [2,3].
Using structure-function studies the catalytic properties of this new enzyme can be explained,
and provide general information on peroxidases. VP versatility is related to different substrate
oxidation sites that have been identified in high-resolution crystal structures, and were
confirmed by site-directed mutagenesis in combination with spectroscopic techniques [4-6].
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
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
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.
L21
Nina Hakulinena, Martina Andbergb, Anu Koivulab, Kristiina Kruusb, Juha Rouvinena
a
Dept. 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: nina.hakulinen@joensuu.fi
Laccases (E.C. 1.10.3.2, p-diphenol dioxygen oxidoreductases) are redox enzymes that use
molecular oxygen to oxidize various phenolic compounds, anilines and even some 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.
[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
Lígia O. Martins
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.
L23
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: fabrizio.briganti@unifi.it
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
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
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
L27
Potential of White-Rot Fungi for Decolourisation and
Detoxification of Dyes
Microbiology Unit (MBLA), catholic University of Louvain, Croix du Sud 3 boîte 6, B 1348
Louvain-la-Neuve, Belgium,
e-mail: vanhullesophie@hotmail.com
The use of white rot fungi (WRF) appears to be a promising alternative to treat dyes
containing wastewater. Based on a previous screening of 300 WRF, six strains belonging to
the species Coriolopsis polyzona, Perenniporia ochroleuca, Pycnoporus sanguineus,
Perenniporia tephropora and Trametes versicolor were selected for an extensive search on
decolourisation and detoxification of dyes.
The major metabolites resulting of the biotransformation of the blue anthraquinonic dye Acid
Blue 62 (ABu62, previously called NY3) were isolated, characterized and a mechanism of
decolourisation was proposed. A first rapid step leading to red intermediates, was mainly due
to a dimerization of the initial molecule and was followed by a slower step leading to
uncoloured products formed by degradation of this main dimer into smaller fragments. As
laccase was the main ligninolytic activity of these strains, LAC-1 from Pycnoporus
sanguineus MUCL 41582 (PS7) was selected as a model for kinetic studies. While displaying
a traditional Michaelis-Menten kinetic behaviour with ABTS as substrate, LAC-1 presented
an atypical behaviour when ABu62 was used as substrate. In addition, LAC-1 only catalysed
the first step of Abu62 biotransformation. Therefore, Pycnoporus strains were used as model
to understand the role of laccases in the in vivo decolourisation of three anthraquinonic dyes:
Abu62, Acid Blue 281 and Reactive Blue 19. All three dyes caused an increase in laccase
activity. In vitro, oxidation of thel three anthraquinones by a laccase preparation was obtained
to a lesser extend than the whole cell process; suggesting that other factor(s) could be required
for a complete decolourisation. The activity of cellobiose dehydrogenase (CDH) was
therefore monitored. Present early in the broth during the growth of the fungi, CDH displayed
in vitro a synergism with laccases in the decolourisation of ABu62, and an antagonism
with laccases in the decolourisation of ABu281 and RBu19.
Nevertheless, decolourisation does not imply that the resulting metabolites are less toxic than
the parent molecules. Toxicity assays were previously developped on human Caco-2 cells,
which are considered as a validated model for the human intestinal epithelium. Depending on
the strain used, a cytoxicity reduction between 25 % and 85 % was observed after two
weeks of culture. No mutagenic character appeared during the biotransformation, as verified
through VITOTOX TM assays. Enzymatic treatment of ABu 62 with purified laccase (EC
1.10.3.2) from Pycnoporus sanguineus allowed in one day a cytotoxicity reduction
comparable to that obtained in 7 days by a complete culture. PS7 was further used to treat an
industrial effluent and compared to the effectiveness of ozonolysis. The effluent toxicity was
reduced by only 10% through ozonolysis, whereas the fungal treatment reached a 35%
reduction. Moreover, a mixed treatment (ozone, then PS7) caused a 70% cytotoxicity
reduction. Raw effluent presented mutagenic character. Ozonized effluent was still
mutagenic, while the genotoxic effect was completely removed after fungal treatment
(patent WO2002EP10077 20020909).
L28
Dietmar Schlossera, Claudia Martina, Charles Junghannsa, Monika Moederb, Magali Soléa,
Gudrun Kraussa
a
Department of Environmental Microbiology, and bDepartment of Analytical Chemistry, UFZ
Centre for Environmental Research Leipzig-Halle, Permoserstrasse 15, D-04318 Leipzig,
Germany
E-mail: dietmar.schlosser@ufz.de
Fungi occuring in freshwater environments and their laccases have gained considerably less
attention than terrestrial fungi, with respect to their possible contribution to the natural
attenuation of organic pollutants in the environment and their potential biotechnological
application in the removal of hazardous pollutants from wastewater.
Endocrine disrupting chemicals and ingredients of personal care products found in
aqueous environments led to increasing concerns regarding their potentially hazardous effects
on human health and the environment, but the knowledge about their biodegradability by
microorganisms of aquatic environments is still limited. Technical nonylphenol, a mixture of
mainly para-substituted nonylphenol isomers with variously branched side chains, is known
to act as a xenoestrogen. HHCB (galaxolide®) and AHTN (tonalide®) are polycyclic musk
fragrances used in personal care products and were reported to inhibit multixenobiotic
resistance transporters in aquatic organisms. We investigated the bioconversion of HHCB,
AHTN, and nonylphenol, the latter being applied as an isomeric mixture and also in the form
of single nonyl chain-branched isomers, by freshwater-derived, laccase-producing mitosporic
fungi. Degradation studies involved both fungal cultures and isolated laccases. Fungal
cultures removed nonylphenol more efficiently under conditions where high extracellular
laccase activities were expressed, as compared to conditions where laccase activities were low
or negligible. Nonylphenol conversion by isolated laccases led to the formation of oxidative
coupling products. This is in favour of an extracellular attack on nonylphenol catalyzed by
laccase. In addition, as yet unknown intracellular enzymes attack nonylphenol at the alkyl
chain as implied by certain biotransformation metabolites. In fungal cultures, several
metabolites detected during the removal of HHCB and AHTN indicated biotransformation
initiated by intracellular hydroxylation. Moreover, isolated laccases were also able to convert
both, HHCB as well as AHTN. Laccase treatment of HHCB strongly increased the
concentration of the known HHCB metabolite HHCB-lactone, suggesting that laccase
catalyzes the oxidation of HHCB into HHCB-lactone.
Textile dyes left unconsumed in textile industry effluents represent another example for
potentially hazardous environmental contaminants. We investigated the ability of aquatic
fungi to decolourise synthetic dyes and addressed the potential involvement of the laccases of
these organisms in decolourisation.
All together, these results demonstrate a potential of aquatic fungi and their laccases to
affect the environmental fate of organic contaminants in freshwater ecosystems and their
possible application in technical processes aiming at the removal of organic pollutants
wastewater.
L29
Feng Xu
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
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
a
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain;
b
Universidad de Alcalá, Alcalá de Henares, E-28871, Madrid, Spain. cDiSSPA, Università
degli Studi di Napoli, Via Università 100, 80055 Portici, Na Italy (mjmartinez@cib.csic.es)
Large amounts of dark effluents, called olive oil mill wastewaters (OMW), are produced
during oil extraction from olives. These effluents are characterized by low pH, intense dark
brown colour, high organic load including lipids, pectin, polysaccharides and phenols, and
high content of residual oil and solid 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
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
glucose
O2
H2O
oxidized, colorless
compound
H2O
Bleaching
L33
Ana Gutiérreza, Jorge Rencoreta, David Ibarrab, Ángel T. Martínezb, José C. del Ríoa
a
Instituto 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: anagu@irnase.csic.es
Lipophilic extractives in wood and other lignocellulosic materials exert a highly negative
impact in pulp and paper manufacturing causing the so-called pitch problems that affect both
paper machine runnability and product quality, among others. Some biotechnological
products have been developed and enzymes (lipases) have been successfully applied to
softwood mechanical pulping at mill scale [1]. However, the enzymes and microbial inocula
used till present are only effective on specific raw materials and processes. Recently, we have
shown for the first time the effectiveness of the laccase-mediator system (LMS) in removing
pulp lipids regardless the pulping process and the raw material used [2,3]. The results have
been included in a patent recently deposited [4]. In these studies, three pulps representative
for different pulping processes and raw materials - including eucalypt kraft pulping, spruce
thermomechanical pulping (TMP), and flax soda-anthraquinone (AQ) pulping - were treated
with laccase in the presence of 1-hydroxybenzotriazole (HBT) as a redox mediator. The gas
chromatography and gas chromatography/mass spectrometry analyses of the acetone extracts
from the enzymatically-treated pulps revealed that most of the lipophilic compounds present
in the different pulps were efficiently removed using the LMS. Free and conjugated (as esters
and glycosides) sitosterol, the main compounds responsible of pitch deposits in the
manufacturing of eucalypt kraft pulp, were completely removed. In spruce TMP pulp, LMS
degraded most of the resin acids, as well as sterol esters and triglycerides. In the flax soda-AQ
pulp, the main lipophilic compounds present including sterols and long chain fatty alcohols
were almost completely removed. Small amounts of oxidation products (including 7-
oxositosterol, stigmasta-3,5-dien-7-one and 7-oxositosteryl 3β-D-glucopiranoside) were
identified confirming the oxidative nature of lipid removal. Pulp and papermaking properties
of the enzymatically-treated pulps were also evaluated. In conclusion, LMS treatment is an
efficient method to remove pitch-causing lipophilic compounds from hardwood, softwood as
well as nonwood paper pulps (at the same time that lignin content is reduced).
[1] Gutiérrez, A.; del Río, J.C.; Martínez, M.J.; Martínez, A.T. The biotechnological control of pitch in paper
pulp manufacturing. Trends Biotechnol. 2001, 19, 340.
[2] Gutiérrez, A.; del Río, J.C.; Ibarra, D.; Rencoret, J.; Romero, J.; Speranza, M.; Camarero, S.; Martínez,
M.J.; Martínez, A.T. Enzymatic removal of free and conjugated sterols forming pitch deposits in
environmentally sound bleaching of eucalypt paper pulp. Environ. Sci. Technol 2006, (in press).
[3] Gutiérrez, A.; del Río, J.C.; Rencoret, J.; Ibarra, D.; Martínez, A.T. Main lipophilic extractives in different
paper pulp types can be removed using the laccase-mediator system. Appl. Microbiol. Biotechnol. 2006,
(in press) DOI: 10.1007/s00253-006-0346-1.
[4] Gutiérrez, A.; del Río, J.C.; Rencoret, J.; Ibarra, D.; Speranza, A. M.; Camarero, S.; Martínez, M. J.;
Martínez, A.T. Sistema enzima-mediador para el control de los depósitos de pitch en la fabricación de
pasta y papel. Patent (Spain) 2005, 200501648.
L34
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
Jorge Gominhoa, Ana Lourençoa, Helena Pereiraa, Cristina Máximob, Maria Costa-Ferreirab
a
Centro 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 : Maria.ferreira@ineti.pt
Microbial agents, either whole cells or enzymes from these, have been applied to the different stages of pulp
and paper processing. The present work describes a study on the effect of applying ligninolytic enzymes,
such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and
subsequently subjecting these to different ageing processes.
The starting material was industrial pulps obtained from different Portuguese pulp and paper companies. The
pulps used were 1) unbleached pine pulp from Portucel Tejo; 2) unbleached eucalyptus pulp from Portucel
Setúbal; 3) bleached eucalyptus pulp from Portucel Setúbal; and 4) pulp made from recycled paper from
Renova S.A. Several types of handsheets were produced with 2 different grammage namely, 60 and 180
g/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
Anna Suurnäkkia, Marco Orlandib, Stina Grönqvista, Hannu Mikkonena, Liisa Viikaria
a
VTT, Tietotie 2, 02044 VTT, Finland
b
Dipartimento di Scienze dell’Ambiente e del Territorio, Universitá degli Studi di Milano-
Bicocca, Piazza della Scienza 1, I-20126 Milan, Italy
E-mail: anna.suurnakki@vtt.fi
The presence of surface lignin in pulp fibres offers possibilities to enhance the existing pulp
properties or even to create completely new pulp properties by enzymatic means. Improving
the properties of wood fibres is a constant interest of pulp, paper and board manufacturing
industry. New methods for targeted modification of wood materials could also reveal
completely new application areas for wood fibres.
Oxidative enzymes such as laccases can be used to activate the surface lignin of lignin-rich
pulps by radicalisation. The primary reaction of laccase catalysed oxidation is the formation
of phenolic radicals to the substrate. Due to the high reactivity of these radicals (either with
each other or with a secondary substrate), reactions such as polymerisation, depolymerisation,
co-polymerisation and grafting can occur. The size of laccases limits the action of the enzyme
on the fibre surface, which can be considered both as a limitation or an opportunity when
applying laccases in fibre applications. Enzymatic activation of fibre surfaces can be exploited
after further functionalisation of fibres with specific chemical components in tailoring fibre
properties.
In this work the laccase catalysed activation and functionalisation of lignin-rich pulps was
studied. The radical formation in pulps during oxidation with different laccases was analysed
by oxygen consumption measurement and EPR spectroscopy. Changes in the pulp lignin
stucture by laccase activation were determined by NMR. The factors affecting the activation
and further functionalisation of pulps were elucidated. A novel chemo-enzymatic
functionalisation method developed for lignin-rich pulps and its potential in modification of
fibre properties will be discussed in the presentation.
MICROBIAL PHYSIOLOGY
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
P22 Novel Laccases and Peroxidases for Dye Decolourisation and Bleaching
Processes
Matura,A. and K.-H. van Pée
P67 Preliminary study of soluble heme proteins from Shewanella oneidensis MR1
Bruno Fonseca, Patrícia M. Pereira, Isabel Pacheco, Ricardo O. Louro
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
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
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
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
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
P1
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
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: minibayeva@mail.knc.ru
Production of reactive oxygen species (ROS) is one of the widely reported stress responses of
plants. However, the nature of enzymes responsible for ROS production in the apoplast and
their regulation are still open questions. We studied intra- and extracellular redox activities of
wheat (Triticum aestivum L.) roots. It was found that wheat roots and leachates derived from
these roots possess redox activities, which were strongly stimulated following wounding or
heavy metal stresses. Plant cell wall has an enormous capacity to accumulate redox enzymes
in different cell wall fractions. Although total intracellular peroxidase, tyrosinase and ROS
producing activities were much higher compared to those in the leachates and cell wall
fractions, this was mainly due to the high intracellular protein content. Treatment of isolated
cell walls with digitonin and NaCl doubled protein release compared to that of the buffer
soluble fraction. Interestingly the specific ROS producing and peroxidase activities were
highest in the weakly bound enzymes and enzymes bound to the cell wall by strong
electrostatic forces. Treating the cell wall with detergent did not increase tyrosinase activity as
has been shown for fungal tyrosinases, suggesting that catalytical properties of these enzymes
of different origin can vary. Analysis of the phenolics in the cell wall fractions revealed that
the fraction containing redox enzymes bound by strong electrostatic forces is characterized by
the highest diversity of phenolic acids, with syringic acid being abundant. However, the
substrate for the weakly bound phenoloxidases and peroxidases is probably caffeic acid, the
concentration of which was 10 times higher in this fraction than in others.
Our results suggest that enzymes weakly bound to the cell wall have higher redox activity
compared to those more tightly bound. We assume that ROS production by free or readily
released from the cell wall redox enzymes is a part of the universal stress response and signal
transduction in plant cells.
P4
Evaluation of Oxidase Potential and Growth Rate of
Saprotrophic Basidiomycetes Cultures
N. Psurtseva, A. Kiyashko, N. Yakovleva, N. Belova
V.L. Komarov Botanical Institute RAS, St. Petersburg, Russia
E-mail: NadyaPsu@NP1512.spb.edu
Basidiomycetes fungi are well-known oxidoreductases producers. Wide screening in
Basidiomycetes for active species and strains can reveal new perspective source of oxidases.
Evaluation of oxidase potential and growth rate of Basidiomycetes cultures from various
taxonomic groups belonging to xylotrophic and litter-decomposing fungi was the aim of the
present study. About 300 strains with a broad taxonomical, ecological and geographical
diversity were involved in the experiment. The cultures were collected in different
geographical regions (mainly in Russia and former USSR) and maintained in the LE (BIN)
Basidiomycetes Culture Collection. All the strains were grown on ale-wort agar plates (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
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
a
Komarov Botanical Institute RAS, Prof. Popov Str., 2, St. Petersburg. 197376 Russia.
b
University of Helsinki, P.O. Box 56 (Biocenter 1, Viikinkaari 9) 00014, Finland
E-mail: cultures@mail.ru
Fermentation conditions are essential to productive capacity of Basidiomycetes strains.
Cultures of various taxonomical and ecological groups having high natural ligninolytic
potential may be different in requirements regarding medium compounds and fermentation
methods during their cultivation. To reveal the most favorable cultivation conditions for
growth and laccase production for a number of selected strains have been initiated this study.
29 strains of 25 Basidiomycetes species from families Strophariaceae and Tricholomataceae
(Agaricales), Crepidotaceae (Cortinariales), Lentinellaceae (Hericiales), Coriolaceae,
Lentinaceae, and Polyporaceae (Poriales), and Steccherinaceae (Stereales) were studied as
stationary and submerged cultures using several liquid nutritional media. The fungal strains
were selected as a result of screening on laccase activity by rapid assay methods and by
cultural characters. Some of the fungal isolates were fruited in culture. A number of the
selected strains included species well-known as laccase producers belonging to the genera
Lentinus, Hypholoma, and Trametes. On the contrary, several of the fungal isolates belonged
to genera such as Lentinellus, Lenzites, Oudemansiella, Polyporus, Steccherinum, and
Tubaria that have not been studied yet for laccase production. Liquid ale-wort, malt extract
and two glucose-peptone media with different mineral components were used for stationary
and submerged cultivations. Laccase activity was estimated by using syringaldazine and
pyrocatechol as enzyme substrates. The experiments showed that the selected fungal strains
had different capacity for growth on used media. Moreover, each isolate had individual
priorities for nutritional media and cultivation method regarding its laccase production.
Growth on malt extract showed high laccase activity in Lenzites betulina, Oudemansiella
mucida, Tubaria sp., and Polyporus squamosus. Lenzites betulina revealed high laccase
production under both stationary and submerged cultivation on liquid malt extract, but not on
glucose-peptone LN-AS medium. High level of laccase activity and biomass production
during submerged cultivation on glucose-peptone medium was found in Trametes gibbosa
strains. Selected cultures of Lentinellus ursinus f. robustus and Steccherinum ochraceum
produced laccase under both cultivation conditions but showed difficulties in growth. It was
found that S. ochraceum produced not only high laccase activity but manganese peroxidase
also. The selected isolates of the genus Polyporus had a high potential for laccase production
under submerged cultivation but active production of some mucilaginous substance
(presumably polysaccharides) caused problems with measuring of laccase activity. Cultures
of Hypholoma fasciculare and H. sublateritium showed very low laccase production together
with poor growth on liquid media. Absence of any phenol oxidase or laccase activity was
observed with various cultivation methods for the isolates identified as Armillaria borealis,
Conocybe vexans, Marasmius rotula, Microporus luteus, and Macrolepiota procera while
they revealed very high laccase activity when rapid assay methods were used. As a result of
the experiments, several new Basidiomycetes isolates from various fungal genera that were
not studied in this regard before can be proposed as promising new producers of laccases.
This work was supported by the INTAS grant 03-51-5889.
P7
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
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
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
S. Koray Yesiladali, Gunseli Kurt, Ayten Karatas, Nevin Gül Karagüler, Candan Tamerler
This study is funded by EU 6th Framework Integrated Project (IP), ‘SOPHIED - Novel
sustainable bioprocesses for the European colour industries’.
P11
Günseli Kurt, Nevin Gül Karagüler, Ayten Yazgan Karataş, Candan Tamerler
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
[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
[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
This study is funded by EU 6th Framework Integrated Project (IP), ‘SOPHIED - Novel
sustainable bioprocesses for the European colour industries’.
P15
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
Kristiina Kruusa, Marja Paloheimob, Terhi Puranenb, Leena Valtakaric, Jarno Kalliob, Richard
Fagerströma, and Jari Vehmaanperäb
a
VTT Technical Research Centre of Finland, Espoo, Finland; bRoal Oy, Rajamäki, Finland;
c
AB Enzymes Oy, Rajamäki, Finland
E-mail: kristiina.kruus@vtt.fi
P17
Production, Purification and Kinetic Characterisation of a
Thermostable Pycnoporus sanguineus Laccase (LAC-1)
a b a b a a
M. Trovaslet , C. Bebrone , E. Enaud , S. Hubert , N. Nouaimeh , M. Pamplona-Aparicio , B.
c c b a a
Lorenzini , Ch.-M. Bols , J-M. Frère , A-M. Corbisier , S. Vanhulle
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
P19
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
[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
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
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
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.
[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
Rosanna Papa, Ermenegilda Parrilli, Paola Giardina, Maria Luisa Tutino and Giovanni Sannia
Department of Organic Chemistry and Biochemistry, University Federico II, Naples – Italy
E-mail: rosapapa@unina.it
Microbial degradation of aromatic hydrocarbons has been extensively studied with the aim of
developing applications for the removal of toxic compounds from contaminated
environments. Although many pollution problems occur in sea waters and in effluents of
industrial processes which are characterised by low temperatures, considerable effort has been
directed toward the genetic manipulation of mesophilic bacteria to create or improve their
ability to degrade various pollutants.
With the aim to investigate the degradation of aromatic compounds at low temperatures the
Antarctic psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125)
was efficiently used for the production of the recombinant aromatic oxidative activity
encoded by the Toluene-o-Xylene Monooxygenase gene from the mesophilic bacterium
Pseudomonas spp. OX1 [1]. Catalytic performances of PhTAC125 cells expressing ToMO
have been already characterized on different aromatic substrates in various conditions [1].
The genome of PhTAC125 was recently sequenced [2]. Analysis of the annotation of this
genome revealed the presence of a CDS coding for a putative laccase-like protein. Bacterial
laccases have also been reported to be able to oxidize dioxygenated aromatic compounds such
as catechols [3].
The gene coding for PhTAC125 laccase belongs to a gene cluster possibly involved in copper
homeostasis. Preliminary studies demonstrated that this gene is expressed in PhTAC125 cells
only in the presence of copper, as reported for other bacterial species [4].
By using the recombinant capabilities conferred from ToMO enzyme to PhTAC125 and the
endogenous activity due to the presence of the laccase protein we analyzed the catabolic
features of this engineered microorganism. Results prospect the possibility of developing
specific degradative capabilities using this psychrophilic bacterium for the bioremediation of
chemically contaminated marine environments and/or of cold effluents.
[1] Siani, L., Papa, R., Di Donato, A. and Sannia, G. (2006) Recombinant expression of Toluene o-Xylene
monooxygenase (ToMO) from Pseudomonas stutzeri OX1 in the marine Antarctic bacterium
Pseudoalteromonas haloplanktis TAC125. J. Biotechnol. in press
[2] Medigue, C., Krin, E., Pascal, G., Barbe, V., Bernsel, A., Bertin, P.N., Cheung, F., Cruveiller, S., D’Amico,
S., Duilio, A., Fang, G., Feller, G., Ho, C., Mangenot, S., Marino, G., Nilsson, J., Parrilli, E., Rocha, E.P.C.,
Rouy, Z., Sekowska, A., Tutino, M.L., Vallenet, D., von Heijne, G . and Danchin A. (2005) Coping with cold:
the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. Genome
Res. 10, 1325-1335.
[3] Grass, G., Thakali, K., Klebba, P.E., Thieme, D., Muller, A., Wildner, G.F. and Rensing ,C. (2004) Linkage
between catecholate siderophores and the multicopper oxidase CueO in Escherichia coli. J. Bacteriol. 186,
5826-5833.
[4] Brown, N.L., Barrett, S.R., Camakaris, J., Lee, B.T. and Rouch, D.A. (1995) Molecular genetics and
transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004. Mol.
Microbiol. 17, 1153-1166
P25
Spectroscopic Characterization of a Novel Naphthalene
Dioxygenase from Rhodococcus sp.
[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
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.
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
P28
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.
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
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].
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
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
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
[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
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].
R1 R2
H OH
N
O
N+ N
+
O OH + H
R1
O
R2
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.
P69
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
Maria Antonietta Rao, Giuseppina Iammarino, , Rosalia Scelza, Fabio Russo, Liliana
Gianfreda
E-mail: giusiam@hotmail.com
The environment is continuously enriched by organic substances differing in their chemical
and structural complexity and deriving from both natural and anthropogenic sources. Several
of them having toxic properties may behave as harmful pollutants. Adverse, negative effects
on the environmental and human health may derive.
One of the most effective mechanisms in remediating environments polluted by organic
pollutants is the oxidation by biotic and abiotic catalysts which may occur in natural
attenuation processes or in engineered remediation processes.
Oxidative enzymes such as laccases, tyrosinases and peroxidases are the main effectors of
biotic processes. They differ for some molecular and catalytic characteristics, but all have
been proved to be active towards several organic pollutants.
The main purpose of this paper was to evaluate the catalytic behavour of some of these
catalysts, with particular attention to laccases, when applied to different polluted systems.
Laccase from plant origins showed differentiated efficiencies in transforming polluting
phenolic compounds under various experimental conditions. In particular, the effect of the
initial concentration of the phenolic substance, the repeated addition of fresh enzyme amounts
as well as the presence of more than one phenol and/or pollutant of different chemical nature
(like phenanthrene) in the reaction mixture strongly affected the efficiency of laccase action.
Comparative studies were also performed with fungal laccases and with tyrosinase in both
synthetic and natural phenolic waste waters.
Moreover, the catalytic performance of a peroxidase was assessed in a complex system
simulating a natural situation occurring in soil and rhizosphere soil. A mixture of pyrogallol
or tannic acid, both representative of humic precursors and very abundant in soil and
rhizosphere, were incubated with a plant peroxidase, and the oxidation of the phenolic
compounds (pyrogallol or tannic acid) and the formation and properties of polymeric products
obtained under different experimental conditions, i.e. initial substrate concentration, amount
of peroxidase, incubation time, etc. were evaluated. Further studies were performed in the
presence of phosphatase, a key enzyme very often released extracellularly by plant roots and
catalyzying the hydrolysis of organic phospho-esters in inorganic orthophosphate, the only
form available to plant roots and soil microorganisms. The involvement of the phosphatase in
the process and its residual catalytic efficiency towards a synthetic phosphoric substrate was
assessed as well.
Acknowledgements
This research was supported by Ministero dell’Università e della Ricerca, Italy. Programmi di
Interesse Nazionale PRIN 2004-2005 and by the INCO-MED Program (Contract ICA3-CT-
2002-10033).
P32
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: martina.andberg@vtt.fi
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
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.
P34
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: pdurao@itqb.unl.pt
The catalytic rate-limiting step in laccases is considered to be the oxidation of substrate at the
T1 copper site, most probably controlled by the redox potential difference between this site
and the trinuclear site. Redox potentials exhibited by laccases span a broad range of values
from 400 mV for plant laccases to 790 mV for some fungal laccases. The conserved
coordinating amino acids for the T1 copper site are two histidines and a cysteine, and the
natural variations occur in the so-called axial position with a single interaction from a Met
being the most common arrangement. Fungal laccases have the non-coordinating Phe or Leu
at this position and these may contribute, at least partially, for the higher 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
Isabel Bento, Paulo Durão, André T. Fernandes, Lígia O.Martins and Peter F. Lindley
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa,
Av. da República, EAN, 2784 - 505 Oeiras, Portugal
E-mail: bento@itqb.unl.pt
Laccases are enzymes that are able to couple substrate oxidation with the reduction of
dioxygen to water. They belong to the multicopper oxidase family and show at least two
different types of copper centre; a mononuclear T1 centre and a trinuclear centre that
comprises two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear
centre whereas reduction of molecular oxygen to water occurs at the tri-nuclear centre. Using
the CotA laccase as a model system, we have recently proposed a putative mechanism for
oxygen reduction for this type of enzyme [1]. In the present work we have tried to increase
our understanding of such a mechanism and have determined the three dimensional structure
of three different mutants of glutamate 498. This residue interacts indirectly, through a water
molecule, with a dioxygen moiety bound in between the two T3 copper atoms. It has been
proposed to play a key role in the protonation events that occur during the mechanism.
Indeed, this study not only shows the relevance of this residue in protonation but also the
importance of its presence in the stabilisation of the whole trinuclear centre.
[1] Bento, I. Martins, L.O., Gato, G.L., Carrondo, M.A., and Lindley, P.F. (2005) Dalton Transactions
21, 3507-3513
P36
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
[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
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: andref@itqb.unl.pt
The Aquifex aeolicus AAC07157.1 gene encoding a multicopper oxidase (McoA) and
localized on the genome as part of a putative copper-resistance determinant, was cloned,
overexpressed in Escherichia coli, and purified to homogeneity. The isolated enzyme shows
spectroscopic and biochemical characteristics of well-characterized multicopper oxidases.
McoA presents poor catalytic efficiency (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
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
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
Microbiology Unit, Université catholique de Louvain, Place Croix du Sud 3 bte 6, B-1348
Louvain-la-Neuve, BELGIUM,
E-mail: vanhullesophie@hotmail.com
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
Aisle Ergun, Firuze Basar, S. Koray Yesiladalı, Z. Petek Çakar Öztemel, Candan Tamerler
Behar
This study is funded by EU 6th Framework Integrated Project (IP), ‘SOPHIED - Novel
sustainable bioprocesses for the European colour industries’.
P43
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
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
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
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
Anna Maria V. Garzillo, Federica Silvestri, M. Chiara Colao, Maurizio Ruzzi, Vincenzo
Buonocore
Dpt. of Agrobiology and Agrochemistry, Via S. Camillo de Lellis s.n.c., Viterbo (Italy)
E-mail: amg@unitus.it
Laccases (E.C. 1.10.3.2) are multicopper oxidases which oxidize a variety of phenolic and
non-phenolic compounds with the simultaneous reduction of molecular oxygen to water. The
broad substrate specificity makes these enzymes very attractive for a number of applications,
including bioremediation processes. In these cases, the use of laccases in immobilized form
may result in increased enzyme stability, multiple use and easy separation from the reaction
mixture.
The main laccase (Lcc1) from Trametes trogii has been immobilized both covalently
(Eupergit C) and non-covalently (polyacrylamide gel intrapment, Sepharose ConA
adsorption) ; the last technique gave the highest binding yields (≈ 100%) and capacity of
substrate (2,6-dimethoxyphenol, DMP) degradation. Thus, this study was conducted with
Lcc1 adsorbed at pH 4 on Sepharose ConA; phenol and dye degradation was monitored by
HPLC. In a first group of experiments, phenolic compounds (0.4 g/l each, 100 ml) were
passed separately in continuous through immobilized laccase (50 I.U.) packed in a small
column; after 20 h flowing (flow rate 1 ml/min), caffeic acid (CA) was degraded by 100%, p-
coumaric acid (pCA) by a 20% and 4-hydroxyphenylacetic acid (HPA) by a 10%. When a
mixture of the three phenols was passed through the column, to mimic real situation of waste
waters, degradation after 20 h was 55% (CA), 20% (pCA) and 10% (HPA). Even lower
degradation rates (e.g. 20% for CA) were observed with a mixture of seven phenols
containing also recalcitrant products (3-hydroxyphenylic acid). These data indicate that a
strong substrate competition for the enzyme will affect compound degradation in complex
mixtures. A similar set of experiments was carried out by challenging immobilized laccase
and phenols in batch; in these conditions, the degradation was more effective as compared
with the column system: CA was completely degraded in 1 h, whereas after 20 h pCA and
HPA were degraded by 95 and 50 %, respectively. When applied in a mixture, a competition
effect was observed: CA disappeared after 3 h, pCA and HPA were degraded after 20 h by 75
and 30%, respectively.
The batch system has also been used to assess degradation of some synthetic dyes,
extensively used in industrial processes. The three dyes chosen have typical chromophoric
groups: alizarin (AL, anthraquinone), amaranth (AM, azo) and indigo carmine (IC).
Degradation rate by immobilized laccase was different depending on dye structure: after 20 h
AL was degraded by 90%, IC by 45% and AM by 5%. When violuric acid (VA) was added as
a mediator, the degradation of the three dyes was completed in less than 5 h; 1-
hydroxybenzotriazole was less effective then VA in mediating the interaction between the
enzyme and the dyes.
These preliminar data indicate that immobilized laccase from T. trogii can efficiently promote
decolorization of industrial effluents; further investigations are needed to clarify competition
effects among substrates and the role of mediators in the degradation process.
P48
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.
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.
P49
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
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
CETAV - Dep. Engenharia Biológica e Ambiental, UTAD, Apartado 1013, 5001-801 Vila
Real, Portugal
E-mail: bezerra@utad.pt
White-rot fungi can degrade a wide variety of recalcitrant compounds like lignin, dyestuffs
and other xenobiotic compounds by their extracellular ligninolytic enzyme systems. Several
studies in vitro have shown that fungal laccases are able to decolorize and detoxify industrial
dyes [1]. The objective of the present work was to evaluate the potential of laccase-based
treatment for removing of coloured azo solutions and associated toxicity. Mixtures of seven
azo dyes treated with laccases were degraded nevertheless with the production of other more
polar compounds. It is remarkable that when they were studied individually, acid red 337,
acid red 57, orange II and methyl orange need a mediator such as ABTS to be degraded.
Otherwise when these dyes were in mixtures with others that were degraded without any
mediator (acid black 194 and acid blue 113) the results showed no differences between assays
carried out with or without mediators (ABTS, acetovanillone, acetoseringone and carminic
acid) suggesting that acid black 194 and acid blue 113 exhibit a mediator effect.
Germinability experiments with water cress (Lepidium sativum) were conduced in the
presence of different dilutions of enzyme–treated azo compounds. Our results showed
significant toxicity abatement after laccase treatment as assessed by germination index which
increase from 50% to 94%.
[1] A. A. Dias, R. M. Bezerra, P. M. Lemos and A. N. Pereira. 2003. In vivo and laccase-catalysed
decolourization of xenobiotic azo dyes by a basidiomycetous fungud: charactersation of its ligninolytic system.
World J Micriobiol Biotecnol 19:969-975
P52
Transformation of Simple Phenolic Compounds by Fungal
Laccase to Produce Colour Compounds
[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
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).
P54
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).
P55
José A.F. Gamelas,b Ana S.N. Pontes,a Dmitry V. Evtuguin,b Ana M.R.B.Xaviera
a
COPNA, bCICECO – Departamento de Química, Universidade de Aveiro, 3810–193 Aveiro,
Portugal
E-mail: Dmitryr@dq.ua.pt
The pulp and paper industry is facing an increasing pressure from environmentally concerned
institutions to replace the conventional chorine-based bleaching techniques by
environmentally friendly technologies. Oxygen delignification catalysed by polyoxometalates
(POM) has been proposed a nice alternative to pulp bleaching [1, 2]. Applied as catalysts
under aerobic conditions, POM oxidise selectively the residual lignin in kraft pulp, and the
reduced form of POM should be re-oxidised by molecular oxygen at the same process stage.
Unfortunately, the most selective polyoxometalates for bleaching purposes such as
[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.
P56
Research & Development Unit of Textile and Paper Materials, University of Beira Interior
6201-001 Covilhã – Portugal
E-mail: atika@ubi.pt
Environmental awareness and concerns during the recent years have led to an increased interest in
using biotechnology in pulp and paper industry. Eucalyptus globulus is of great economical
importance for the Portuguese pulp and paper industry, since eucalyptus pulp represents about 85% of
pulp production. Laccase ([EC 1.10.3.2], p-diphenol: dioxygen oxidoreductase) is a member of the
blue multicopper protein family, which also includes the plant enzyme ascorbate oxidase and the
mammalian plasma protein ceruloplasmin [1]. Trametes versicolor laccase can catalyse
depolymerisation of kraft pulp lignin in presence of a mediator [2].
Two wood samples of Eucalyptus globulus (one industrial chip sample and another obtained
from a clone tree) were submitted to the kraft cooking processes in order to evaluate its pulping
potential. The purpose of pulping is to remove lignin from wood celluloses. Traditionally, kappa
number is regarded as a parameter that proportional to the residual lignin in the pulps. A recent study
has shown that the hexenuronic acid (HexA) groups in pulps are responsible for a significant
percentage of the kappa number [3], especially from hardwood pulps due to there higher content of
xylan.
Therefore, in this work we used the Klason lignin content in pulps, instead of kappa number,
to evaluate the pulpability, at the given pulping conditions: Active Alkali Charge [%] on wood = 19;
Sulfidity [%] = 30; Liquor: Wood Ratio [L/Kg] = 4:1; Cooking temperature [ºC] = 160, Time to
temperature [min] = 90, Time at temperature [min] = 60. The Klason lignin kappa number content in
brownstocks from clone eucalyptus wood species is much lower than that of the industrial wood
species. Laccase mediator system (LMS) process was applied for the further biodelignification of the
pulps from the conventional kraft pulping process. It was observed that roughly 49% of Klason lignin
has been removed from the clone eucalyptus pulps at LMS. However, it only removed approximately
42% of Klason lignin from the industrial eucalyptus pulps at the same LMS conditions. The Laccase
mediator system diminishes the pulp contents of lignin and hexenuronic acids (HexA). The data shows
that the amount of HexA is quite high in the unbleached Eucalyptus globulus (clone) contrast to
industrial Eucalyptus globulus kraft pulps, 64 mmol/kg and 52.1mmol/kg respectively. However, the
LMS (E) treatments only have a small effect on these compounds.
In view of the results obtained in this study, it can be concluded that LMS treatment can be
applied as a pretreatment in the bleaching sequences in order to reduce the use of chlorine dioxide.
Besides, it indicates that the cloned eucalyptus globulus is an easy to be pulped and bleached wood
species. Moreover, it has a significant importance to the pulping industry economics, particularly on
energy cost savings and production capacity improvement.
Acknowledgements: This research was supported by FCT (Science and Technology Foundation),
SFRH/BD/10893/2002.
References
[1] Mayer, A.M. and Staples, R.C. (2002) Laccase: new functions for an old enzyme. Phytochem.60,
551-565.
[2] Bourbonnais, R., Paice, M.G., Freiermuth, B., Bodie, E. and Borneman, S. (1997) Reactivities of
various mediators and laccase with kraft pulp and lignin model compounds. Appl. Environ.
Microbial.63, 4627-4632.
[3] J. Li, G. Gellerstedt, Carbohyd. Res. 302 (1997) 213.
P57
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
R O
HO OH
O O
HO OH
OH OH OH
HO OH OH
MeO O O HO
NHAc O O O
HO R
MeO O
1 : R = CH2OH 2 : R = CH2OH
HO 2a : R = COOH
1a : R = COOH O
OH
SMe
P58
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
P59
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
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).
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
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
Carolina Arboledaa,d, Hubert Cabanab,c, J. Peter Jonesc, Amanda I. Mejíaa, Spiros N. Agathosb,
Gloria A Jimenezd, Michel J. Penninckd
a
Laboratorio 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: carboled@farmacia.udea.edu.co
Bisphenol A (BPA) is used as raw material for the production of polycarbonates and epoxy
resins. Its discharge in the environment can occur from factories producing BPA or
incorporating it into plastics from leaching of plastic wastes and landfill sites. Recent research
has demonstrated that this chemical can mimic or interfere with the action of animal
endogenous hormones by acting as estrogen agonists, binding to the estrogen receptor or
eliminating a normal biological response; consequently, they may pose a risk to human health
and an environmental impact as they end up in nature as waste through several anthropogenic
activities.
Laccase, that has been shown to catalyze the oxidation of various phenols, aromatic amines
and some dyes, may constitute a good way to treat BPA which is a good substrate for laccases
because of its phenolic structure. A few studies have used fungi and ligninolytic enzymes to
eliminate BPA. In this project, we used Lentinus crinitus, one WRF scanty studied, to remove
BPA from aqueous solutions.
Experiments were carried out in order to test several parameters such as the range of pH,
temperature and contact time, and the presence of mediators, like 2,2’-azino-bis-(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) on the elimination of BPA. A Box–Behnken
type design was used, in order to evaluate the impact of the three parameters (temperature, pH
and processing time) and their potential interactions upon the degradation of BPA. This
statistical procedure makes it possible to reduce the number of experiments required. In this
case T, pH and time of contact, were statistically significant model terms.
Our results demonstrate that using 200 mU ml-1 of laccase, 97.8% of a 22 µM solution of
BPA was eliminated within 6 hours at pH 3 and 40°C. The yeast estrogen test (YES) was used
to measure the elimination of the estrogenic activity of BPA, which is associated with the
elimination of this substance. After 6 hours of treatment, up to 90 % of the estrogenic activity
of BPA was lost. Finally, we demonstrate that the use of ABTS in the laccase/mediator
system significantly improves the laccase catalyzed elimination of BPA.
P63
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
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
a
STAB 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: bamateus@gmail.com
Azo aromatic dyes are the major group of textile dyestuff. These are chemically stable
structures to meet various colouring requirements and often are not degraded and/or removed
by conventional physical and chemical processes. Moreover, many of these compounds are
highly resistant to microbial attack and therefore, hardly removed from effluents by
conventional biological processes such as activated sludge treatment. Over the last decades,
considerable work has been done with the goal of using microorganisms as bioremediation
agents in the treatment of wastewater containing textile dyes.
Pseudomonas putida strain MET94 was selected among 84 bacterial strains has the most
active textile dye degrader. This strain showed significant decolourization improvement on 6
different azo dyes but no effect on anthraquinonic dyes. This strain was able to decolorize up
to 70-85% of Reactive R4 (RR4), Reactive black 5 (RB5), Direct Blue 1 (CSB), Acid Red
299 (NY1), Direct Black 38 (CB) and Direct Red 28 (CR) out of 11 different dyes tested,
after 24h of growth in complex liquid culture media. Higher degradation rates as well as
higher extent of decolourization were obtained in anaerobic when compared with aerobic
conditions. In the absence of oxygen (i) degradation is growth associated, (ii) specific growth
rates were higher in the presence of dyes, suggesting that these could be used as electron
acceptors in anaerobic respiration. In the presence of oxygen (i) growth rates as well as
biomass yields were lower in the presence of dyes, suggesting that dyes could be exerting
toxicity over cells, (ii) maximum decolourization activity occurred at the late 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
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.
Yesiladali, S. Koray
Istanbul Technical University Molecular
Biology and Genetics Department
Maslak/Istanbul
Turkey
Tel: 0090 212 286 22 51
e-mail: yesiladali@itu.edu.tr
Zille, Andrea
Departamento de Engenharia Têxtil
Universidade do Minho
Campus de Azúrem
4800-058 Guimarães
Portugal
Tel: +351253510280
e-mail: azille@det.uminho.pt
Zimmermann, Wolfgang
Institute of Biochemistry
Department of Microbiology and Bioprocess
Technology
University of Leipzig
Johannisallee 21-23
D-04103
Tel: + 49 3419736781
e-mail: wolfgang.zimmermann@uni-leipzig.de
Baldrian, P. L6 Caporale, C. P9
Di Berardino, I. P9 Gómez, D. L1
Reymond, M. L3 Sigoillot-Claude, C. L3
van den Berg, W. A. M. L11, P26 Xavier, Ana M.R.B. P59, P55