Caratterizzazione di laccasi native e modificate e loro uso nella degradazione di coloranti
Author(s)
Silvestri, Federica
Date Issued
January 8, 2009
Type
Doctoral Thesis
Abstract
Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) belong to the family of
“multicopper oxidases”. Laccases were discovered in plants, then have been described in
fungi, insects and, more recently, in prokaryotes. They are thought to be nearly ubiquitous
among fungi, mainly in the wood-rotting basidiomycetes causing white-rot, where they are
usually produced in multiple isoforms as extracellular proteins. Laccases are involved in
several physiological functions, such as lignin biosynthesis, plant pathogenesis, insect
sclerotisation, and degradation of lignocellulosic materials. The cultural broth of laccaseproducing
fungi contains generally a main laccase and a number of isoforms some of which
closely related, others differing for structural and catalytic properties. The production of
different isoenzymes is due to the occurrence of multiple laccase genes; it is known that the
expression of some of these genes is regulated up by the presence in the culture medium of
specific inducers such as copper, ferulic acid or 2,5-xylidine.
Most fungal laccases are monomeric glycoproteins with molecular masses ranging
between 60,000 and 70,000 Da and the extent of glycosylation between 10 and 25%; they
catalyze the one-electron oxidation of a large variety of substrates (usually diphenols or
aromatic amines) coupled with the reduction of dioxygen to two molecules of water. In the
blue laccases, the redox process is brought about by four copper ions arranged in three
different centres. One type-1 (T1) copper ion is characterized by a strong absorption nearly
600 nm which is responsible of the intense blue colour of these proteins; T1 copper shows a
trigonal coordination, with two histidine and a cysteine residues as conserved ligands; either a
leucine or a phenylalanine residue occupies the fourth ligand position in fungal laccases. The
coordination geometry and ligand nature of T1 copper might be responsible of the high redox
potential of fungal laccases, as compared to plant laccases and other blue copper oxidases. A
second copper ion, coded as type-2 (T2), has a weak absorption in the visible region, is
electron paramagnetic resonance (EPR)-active and is coordinated by two histidine residues.
The last two copper ions [type-3, (T3)] form a binuclear centre characterized by an absorption
at 330 nm; they are EPR-silent due to an antiferromagnetic coupling mediated by a hydroxyl
bridge. The two T3 copper ions are coordinated by six histidine residues and are positioned
close to the T2 copper ion to form a trinuclear cluster. T1 copper is the site where the
substrate oxidation occurs; the extracted electrons are transferred, probably through a strongly
conserved HisCysHis tripeptide motif, to the T2T3 trinuclear cluster, where dioxygen is
reduced to water.
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Laccase are very versatile enzymes, being able to oxidize an extensive list of aromatic
compounds containing hydroxy or amino groups, including pesticides, polycyclic aromatic
hydrocarbons and dyes. Further, the presence of small molecular weight redox mediators such
as 2,2_-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), violuric acid (VA) and 1-
hydroxybenzotriazole (1-HBT), enhances the range and the rates of compounds to be oxidized
(including recalcitrant dyes). These properties make laccases good candidates for applications
in the pulp and paper industry, textile industry, biosensor development, bioremediation of
polluted water and soil. The great interest that laccases aroused for biotechnological uses has
promoted intense investigations for clarifying their oxidative mechanism and for discovering
new enzymes with desirable characteristics and the molecular determinants able to modulate
enzyme properties for specific applications. For many applications it is important to find an
enzyme that is thermostable and works at relatively high pH-value.
The objective of this thesis is the characterization of native and modified laccases for
biotechnological applications, as dye decolourization. To such aim, three main ways have
been continuations: characterization of native laccases secreted from the fungus
basidiomycete Trametes trogii; isolation and expression of new T. trogii laccase genes;
production and characterization of mutants of the main T. trogii laccase.
Trametes trogii, a typical basidiomycete, produces at least five laccase isoforms; the
main isozyme, coded as Lcc1, has been purified and characterized. It could be of interest to
analyze role and properties of the other isoforms, but their purification and characterization is
time-consuming since they account altogether for less than 10% of the total laccase activity.
However, two T. trogii laccase isoforms (coded as P5 and P6) have been partially purified and
characterized in comparison to Lcc1; in particular the profile pH/activity show that P6 has an
optimal activity shifted toward neutrality for phenolic substrates (about 0.5 and 1.0 pH units
for DMP and guaiacol, respectively, as compared to Lcc1)
To find easier ways to produce laccase isoforms, we have looked for new laccaseencoding
genes in the T. trogii cDNA using degenerate primers designed on conserved
regions of basidiomycete laccases; a laccase gene, coded as lcc2, has been isolated, cloned,
sequenced and expressed in the heterologous host Pichia pastoris. The product of lcc2 (Lcc2)
has an interesting behaviour towards aromatic substrates exhibiting an optimal pH closer to
the neutrality than Lcc1; in particular Lcc2 shows a shift towards higher pH values, as
compared to Lcc1, of 2.0 and 1.0 units for DMP and guaiacol, respectively. It is worthwhile
to note that the whole pH/activity profile of Lcc2 with phenolic substrates appears shifted
towards values closer to neutrality, so that the enzyme conserves about 40% of its activity at
7
pH 6. Finally, at the optimum pH, Lcc2 shows a higher affinity (lower Km values) towards
the phenolic substrates, as compared to Lcc1.
The three-dimensional structure of Lcc1 laccase from T. trogii was used as a template
for the construction of the Lcc2 model. Lcc2 shows a substrate cavity very similar to those of
the so far laccases crystallized; the aspartic acid residue is conserved and is very close to the
histidine residue. A main difference is observed between Lcc1 and Lcc2 with regard to two
spatially close residues surrounding the substrate cavity: Thr 164 and Ser 264 of Lcc1 are
replaced in Lcc2 by the two hydrophobic residues Phe 163 and Ile 265, respectively. This
variation affects in same way the capacity of Lcc2 to interact with ligands.
The catalytic efficiency of Lcc2 towards bulky ligands carrying polar groups (e.g. ABTS)
decreases significantly due to both an increase in Km and a decrease in kcat, indicating a
concomitant decrease in the affinity for the substrate and in the kinetics of electron transfer.
Conversely, the catalytic efficiency of Lcc2 towards small hydrophobic substrates (e.g.
guaiacol, DMP) shows a moderate decrease as compared to Lcc1, due to an about 5-fold
increase in the affinity for the substrates and to a more sensible decrease in kcat (Table 1). The
increase in affinity of Lcc2, as compared to Lcc1, towards hydrophobic ligands which are
embedded in the substrate cavity could be due to an induced-fit closing movement of the
cavity led by the amino acid residues of the border loops closer to the ligand. The decrease in
kcat observed in Lcc2, as compared to Lcc1, indicates that the electron transfer from the
substrate to the T1 copper, being the rate-limiting step in laccase kinetics, must have been
affected by the structural differences existing between the two laccase isoforms; however, it
cannot be excluded that the internal electron transfer from the T1 copper to the T2T3
trinuclear cluster and then to the dioxygen molecule and the product release have been
affected.
Laccase biotransformation of xenobiotics in natural media suffers from two main
limitations of the enzyme: an acidic optimal pH for activity and the requirement in several
cases for a redox mediator. The modification of these laccase properties should be achieved
through a site-directed mutagenesis strategy. On the basis of previous structural analyses of
the amino acid residues surrounding the active site of the laccase IIIb from T. versicolor, it
was suggested that the histidine in position 458, that also coordinate the T1 copper, acts as the
primary electron acceptor from the substrate, and the aspartate in position 205 is hydrogen
bonded via the terminal oxygen of its side chain to the reducing substrate and influence the
dependence of the activity towards pH. In addition it was observed that this acidic residue is
highly conserved among fungal laccases and is expected to play an important role in the
structure and function of the enzyme. In the current study the Asp205 of Lcc1 from T. trogii
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was mutated in amino acids with different chemical properties: Ser or Cys, non charged polar
residues, Lys, a positively charged residue. The pH/activity profiles of the mutated laccases
with phenolic substrates indicated a shift of the optimal pH towards neutrality while
maintaining the characteristic bell shape curve. However, it is important to note that the
specific activity of the tested mutants was significantly lower than that of the wild type at all
pH levels and the catalytic improvement at pH closer to neutrality could be associated with a
much reduced efficiency at acidic values of pH. The presence of a negatively charged residue
close to the substrate binding pocket could stabilize the radical cation formed following
electron subtraction and the absence of the Asp205 residue determined a significant decrease
in the specific activity of the mutated enzymes.
Degradation experiments of phenolic compounds and of a number of dyes with free and
immobilized Lcc1 have shown that this laccase has a wide oxidizing capacity; many
recalcitrant compounds may be degraded in the presence of an appropriate mediator.
Preliminary screening experiments of decolourization on microtiter plates have been carried
out on various structurally different dyes: azo (amaranth, carmoisine, new coccine, sunset
yellow), anthraquinonic (remazol brilliant blue R), triarylmethane (patent blue) and indigoid
(indigo). Finally, the process of decolourization has been optimized for amaranth and remazol
brilliant blue R by the response surface methodology (RSM) which allows the monitoring at
the same time of a number of variables (enzyme, substrate and mediator concentration, time,
pH). These experiments have shown that it is possible to modulate experimental conditions as
suitable to reach optimal results; in particular, it is possible to operate at pH closer to
neutrality at appropriate mediator concentrations or at low mediator levels by modulating
enzyme concentration and/or the time of the process.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
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