Tài liệu Báo cáo khoa học: Plasticity of laccase generated by homeologous recombination in yeast docx

Plasticity of laccase generated by homeologous
recombination in yeast
Angela M. Cusano*
,
, Yasmina Mekmouche*, Emese Megleczà and Thierry Tron
Laboratoire Biosciences, Institut des Sciences Mole
´
culaires de Marseille, Universite
´
Aix-Marseille, ISM2 CNRS UMR 6263, Marseille Cedex
20, France
Keywords
cupredoxin domains; functional hybrids;
heterologous expression; multicopper
enzyme; recombination
Correspondence
T. Tron, Laboratoire Biosciences, Institut
des Sciences Mole
´
culaires de Marseille,
Universite
´
Aix-Marseille, ISM2 CNRS UMR
6263, Avenue Escadrille Normandie
Niemen, case 342, F-13397 Marseille Cedex
20, France
Fax: +33 491 288440
Tel: +33 491 289196
E-mail:
*These authors contributed equally to this
work

Present addresses
INRA NANCY, UMR 1136
Interactions Arbres–Micro-organismes,
Equipe de Pathologie Forestie
`
re, Route
d’amance, 54280 Champenoux,
France
àIMEP, Case 36, Universite
´
de Provence,
3 Place Victor Hugo, 13331 Marseille
Cedex 3, France
Database
The sequences of the laccase hybrid cDNAs
lac131, lac232 and lac 535 have been sub-
mitted to the GenBank database under the
accession numbers FJ817449, FJ817450
and FJ817451, respectively
(Received 28 May 2009, revised 9 July
2009, accepted 23 July 2009)
doi:10.1111/j.1742-4658.2009.07231.x
Laccase-encoding sequences sharing 65–71% identity were shuffled in vivo
by homeologous recombination. Yeast efficiently repaired linearized plas-
mids containing clac1, clac2 or clac5 Trametes sp. C30 cDNAs using a
clac3 PCR fragment. From transformants secreting active variants, three
chimeric laccases (LAC131, LAC232 and LAC535), each resulting from
double crossovers, were purified, and their apparent kinetic parameters
were determined using 2,2¢-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
and syringaldazine (SGZ) as substrates. At acidic pH, the apparent kinetic

parameters of the chimera were not distinguishable from each other or
from those obtained for the LAC3 enzyme used as reference. On the other
hand, the pH tolerance of the variants was visibly extended towards alka-
line pH values. Compared to the parental LAC3, a 31-fold increase in
apparent k
cat
was observed for LAC131 at pH 8. This factor is one of the
highest ever observed for laccase in a single mutagenesis step.
Abbreviations
ABTS, 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); BCBD, blue copper binding domain; bp, base pair; SGZ, syringaldazine
(4-hydroxy-3,5-dimethoxybenzaldehyde azine).
FEBS Journal 276 (2009) 5471–5480 ª 2009 The Authors Journal compilation ª 2009 FEBS 5471
Introduction
Laccases (p-diphenol oxidase, EC 1.10.3.2) are polyphe-
nol oxidases that catalyse the reduction of dioxygen to
water, with a concomitant oxidation of phenolic com-
pounds. The enzyme active site comprises four copper
atoms classified into types T1, T2 or T3, according to
their spectroscopic characteristics. Substrate oxidation
occurs at the T1 copper site, while the T2–T3 tri-atomic
cluster is responsible for O
2
reduction [1]. The overall
outcome of the catalytic cycle is reduction of one mole-
cule of dioxygen into two molecules of water, coupled
with oxidation of four substrate molecules (phenols or
anilines) into four radicals that can form dimers, oligo-
mers and polymers. These enzymes are common in
plants, fungi, insects and bacteria [2,3].
Laccases are intensely studied for their potential uses

in industrial processes. They generally work under
mild conditions: room temperature and atmospheric
pressure, with water as solvent [4–7]. Over the past
decade, a significant number of reports focusing on
applications of this eco-friendly enzyme in technologi-
cal and bioremediation processes, in addition to their
use in organic synthesis, have been published [5]. For
industrial use, the current challenge is to obtain both
enhanced expression levels and improved laccases with
desirable physicochemical characters such as a higher
redox potential, optimal activity at neutral or alkaline
pH, and thermostability [8]. Strategies to obtain such
variants include natural biodiversity screening and
optimization of nature-derived scaffolds.
Mutagenesis (rational or random) is often used to
generate laccase variants. In their pioneering work, Xu
et al. [9] have reported significant changes in pH opti-
mum, K
M
and k
cat
for triply mutated fungal laccases.
Replacement of the aspartic acid D206 by alanine in a
Trametes versicolor laccase resulted in a threefold
increase in k
cat
[10]. A similar improvement factor was
also reported for variants found in simple libraries of
in vitro randomly generated mutants from Fomes ligno-
sus [11] or Pleurotus ostreatus [12]. On the other hand,

combination of in vitro mutation and in vivo recombi-
nation strategies to evolve a Myceliophthora thermo-
phila laccase led to a 170-fold increase in total laccase
activity, corresponding to a 22-fold improvement in
k
cat
[13]. In a recent report, a similar approach allowed
authors to isolate a variant of a M. thermophila laccase
capable of resisting a wide array of co-solvents at con-
centrations as high as 50% v ⁄ v [14]. In all available
examples of molecular evolution of laccase, variants
with improved properties have been derived from lac-
case sequences from a single origin at a time. Com-
pared to the shuffling of randomly mutated sequences,
recombination of distantly related sequences allows
large distances in sequence space to be travelled with-
out disturbing the function and ⁄ or structure, but this
method has yet to be applied to laccase. In a model
organism such as Saccharomyces cerevisiae, homolo-
gous recombination properties have largely been used
for gene targeting and allele cloning. Utilizing free
DNA ends as efficient substrates for homologous
recombination, the gap repair methodology allow effi-
cient rescue of a replicative linearized plasmid by inter-
molecular recombination within co-introduced
sequence-related DNA. On the other hand, it has been
shown that recombination involving similar but not
identical DNA sequences (homeologous DNA) can
occurs at rates proportional to the length of homology
[15,16]. Thus, some groups have used in vivo homeolo-

gous recombination to yield low-complexity chimeric
enzymes [15–17]. Usually, a chimera generated in vivo
results from the shuffling of large blocks of sequence
corresponding to one or more structural domains.
When high-complexity chimeric enzymes are desired,
in vitro recombination methods, either random [18,19]
or structure-oriented [20], are preferred.
The scaffold of laccases and related copper-contain-
ing proteins of various functions (e.g. bacterial nitrite
reductase, plant ascorbate oxidase, the E. coli metallo-
oxidase CueO, human ceruloplasmin etc.) consist of
repeats of a homologous sequence domain (blue cop-
per-binding domain) that shares distant homology to
the single-domain cupredoxins [21,22]. The evolution-
ary path from a single-domain cupredoxin to a three-
domain laccase (D1, D2, D3) is thought to involve a
duplication of genes and recruitment of a domain [22].
During evolution from an electron transfer protein to
an oxidase, proto-laccase lost unnecessary blue copper-
binding sites (in D1 and D2), acquired a T2–T3 cluster
binding site (the dioxygen reduction site mapping at
the boundary of D1 and D3) and substrate-binding
sites (one for the electron donor and one for O
2
)in
neo-formed clefts [21,22] (see Fig. 1).
Taking inspiration from evolutionary pathways
within the blue copper binding domain (BCBD)
protein family, we aim to evolve laccases into artificial
catalysts performing new activities. In a first approach,

basic protein engineering techniques – such as fusion
of laccase with an interacting domain [23] – were used
to explore properties of simple artificial laccases
expressed in heterologous hosts. Here, we report on
the construction of laccase chimeras through yeast-
mediated homeologous recombination of Trametes sp.
strain C30 laccase cDNAs sharing 65–71% identity.
Functional hybrids of laccases from Trametes C30 sp. A. M. Cusano et al.
5472 FEBS Journal 276 (2009) 5471–5480 ª 2009 The Authors Journal compilation ª 2009 FEBS
Active variants of laccase were selected directly on
transformation plates. Expression, purification and
analysis of the pH activity profile allowed the charac-
terization of a variant of laccase presenting unusual
oxidation activity at pH 8 corresponding to a substan-
tial increase in k
cat
.
Results
Homeologous recombination of laccase-encoding
sequences
Chimeric laccase-encoding sequences were obtained in
three independent homeologous intermolecular recom-
bination experiments. In each experiment, two parental
laccase-encoding cDNAs were introduced in yeast by
co-transformation of a linearized expression vector,
containing either clac1, clac2 or clac5, in the presence
of an overlapping double-stranded PCR fragment of
clac3. Upon transformation, intermolecular recombina-
tion within the homeologous sequences led to re-circu-
larization of the replicative plasmid, and yeast

transformants were selected on the plasmid-borne
URA3 marker without selection for the point of
recombination between the homeologous genes. The
frequency of recombination ranged from 10
2
(cla535)
to 10
4
(cla131 and cla232) transformants per lgof
DNA. The frequency of recombination depends on the
homology of the sequences that are being recombined,
and therefore frequencies one to two orders of magni-
tude lower than the frequencies reported for the
recombination of homologous sequences [15] probably
reflect the level of identity between the sequences we
used (cla1 versus cla c3, 68.4%; cl a2 versus clac3,71%;
cla5 versus clac3, 65.3%).
Among the transformants, active laccase-secreting
clones were detected as those able to oxidize the 2-meth-
oxyphenol present in the selective medium. Plasmids
recovered from these transformants were first analysed
by restriction mapping in order to confirm their hybrid
nature (results not shown) and then sequenced. Hybrid
laccase-encoding genes lac131, lac232 and lac535
obtained by recombination were all found to contain a
clac3 central sequence (700–800 bp) (Fig. 1). In recom-
binant sequences, junctions were found to map within
short stretches of identity varying from 5–45 bp. Similar
lengths for 5¢ and 3¢ recombination zones were found in
other randomly picked clones (data not shown).

Deduced amino acid sequences of LAC131, LAC232
and LAC535 hybrids were found to resemble more clo-
sely that of LAC3 (89, 94 and 85% identity, respec-
tively) than that of either LAC1 (81%), LAC2 (83%)
or LAC5 (83%). All together, recombination induced
swapping of amino acids for 94 positions (19% of the
residues) in the original LAC3 sequence. The C- and
N-termini of the hybrids were 33–37% and 9–26%
different, respectively, from that of LAC3.
Expression, purification and characterization of
the laccase hybrids
LAC3 and the LAC131, LAC232 and LAC535 hybrids
were heterologously expressed in Saccharomyces cerevi-
siae W303-1A, and extracellular laccase production was
analysed. The production levels in the hybrids were on
average six times lower than observed for LAC3
(300 UÆL
)1
versus 2000 U ÆL
)1
using SGZ). This may
be due either to differences in the activity of the
enzymes or differences in expression conditions (differ-
ent plasmid context, glycosylation level etc.; see below
and Discussion). Recombinant laccases were purified
from 10 L fermentor cultures in three steps according to
our previous protocol [24]. For all these enzymes, we
obtained a yield of 20% of pure enzyme, with a specific
activity of 300 UÆmg
)1

determined in acetate buffer
(0.1 m, pH 5.5) using SGZ as the substrate.
A B
Fig. 1. Schematic representation of gene structures obtained in intermolecular recombination assays. (A) Parental (lac1, lac2, lac3 and lac5)
and hybrid (lac131, lac232 and lac535) sequences are represented by rectangles of variable lengths. Recombinant junctions are indicated by
vertical bars. (B) Representation of cupredoxin domain organization in the laccase structure. Black diamond, T1 copper atom; black circle, T2
copper atom; white circle, T3 copper atoms.
A. M. Cusano et al. Functional hybrids of laccases from Trametes C30 sp.
FEBS Journal 276 (2009) 5471–5480 ª 2009 The Authors Journal compilation ª 2009 FEBS 5473
The apparent molecular mass of LAC3 and hybrids
estimated by SDS–PAGE was found to be substantially
higher than that expected from the amino acid sequences
(Fig. 2). Previous analysis on LAC3 has suggested that
these differences are due to N-hyperglycosylation, a
well-known process occurring during expression of for-
eign proteins in S. cerevisiae [25]. Among the laccases
studied, the apparent molecular mass of LAC535
(approximately 79 kDa) was slightly lower than that
observed for the three other proteins (approximately
87 kDa), probably because of the replacement of N-gly-
cosylation sites during the recombination process. This
is supported by an in silico analysis of the hybrid amino
acid sequences, which indicated that the LAC535
sequence contains two potential N-glycosylation sites
fewer than the other sequences (data not shown).
Kinetic results
The apparent kinetic parameters measured for LAC3
and hybrids using 2,2¢-azino-bis(3-ethylbenzthiazoline-
6-sulphonic acid) (ABTS) and syringaldazine as
substrates are reported in Table 1. At pH 5.7 (in Mes

buffer), a pH at which oxidation of both phenolic
(SGZ) and non-phenolic (ABTS) substrates was found
to be maximal for the original LAC3 enzyme, the cata-
lytic efficiency of hybrids was not distinguishable from
that of LAC3. Apparent K
M
values determined for
SGZ were in the micromolar range for all the enzymes,
but were in the millimolar range for ABTS.
We quickly checked the pH tolerance of the hybrids
in Britton–Robinson buffer using ABTS as the colori-
metric substrate, as ABTS is known to be stable at
4.0 £ pH £ 11.0 [26] (Fig. 3). Various patterns of activ-
ity were observed from pH 6.0–8.0, with LAC131 and
LAC232 having substantial activity at neutral to alka-
line pH (Fig. 3). Similar behaviour was observed with
SGZ as substrate, but precipitation and reversible
transformation of SGZ at neutral pH led us to discon-
tinue this experiment with this substrate. Based on
these initial observations, we recorded the kinetics of
ABTS oxidation for LAC3 and the three hybrids in
Britton–Robinson buffer at various pH within a pH
range of 4.5–8.0. As expected from previous reports on
laccase kinetics, the catalytic efficiency of the tested
enzymes towards ABTS decreased rapidly as pH
increased, reaching values < 10% of the original (i.e.
at pH 4.5) between pH 7.5 and 8.0. Variations in the
apparent K
M
, k

cat
and k
cat
⁄ K
M
values as function of
pH were plotted. Below pH 6.0, all enzymes behaved
almost identically. Above pH 6.0, the apparent K
M
value for LAC131 was almost stable (a threefold
decrease was observed at pH 8.0), whereas the values
for LAC3, LAC232 and LAC535 were 10–20 times
lower than those observed at acidic pH. Apparent k
cat
values decreased rapidly, but the enzymes appeared to
be differently affected: the apparent k
cat
values for
LAC3 and LAC535 were three orders of magnitude
lower than the corresponding values at pH 5.0,
whereas LAC232 and LAC131 values were reduced by
factors of 500 and 50, respectively (Table 2). Thus,
between pH 4.5 and 8.0, the LAC3 and LAC535
enzymes appear undistinguishable from a kinetic point
of view. On the other hand, the LAC131 and LAC232
enzymes appear to be more tolerant to alkaline pH, as
their activity profiles were found to be shifted by at
Fig. 2. Coomassie staining of 8% SDS–PAGE of purified laccase
enzymes. Lane 1, LAC3; lane 2, LAC131; lane 3, LAC232; lane 4,
LAC535; lane M, molecular mass standards (kDa). Each well

contained 4 lg of protein.
Table 1. Apparent kinetic parameter values for SGZ and ABTS in 50 mM MES buffer, pH 5.7, at 30 °C.
Enzyme
SGZ ABTS
k
cat
(min
)1
) K
M
(lM) k
cat
⁄ K
M
(min
)1
ÆlM
)1
) k
cat
(min
)1
) K
M
(lM) k
cat
⁄ K
M
(min
)1

ÆlM
)1
)
LAC3 38 784 23.75 1633 82 348 2754 29.9
LAC131 22 777 11.87 1919 50 713 1599 31.7
LAC232 31 140 17.72 1757 56 851 1978 28.9
LAC535 37 419 14.70 2546 64 746 1769 36.6
Functional hybrids of laccases from Trametes C30 sp. A. M. Cusano et al.
5474 FEBS Journal 276 (2009) 5471–5480 ª 2009 The Authors Journal compilation ª 2009 FEBS
least one pH unit toward alkalinity. Relative to the
LAC3 parental enzyme, if one considers a higher
activity at pH 8.0 as an improvement, the apparent
k
cat
values for the LAC232 and LAC131 enzymes
improved 5- and 31-fold, respectively (Fig. 4). In terms
of catalytic efficiency, this corresponds to improve-
ments of 5- and 12-fold, respectively.
Discussion
Basidiomycete genomes contain multiple genes encod-
ing laccase isoenzymes, with large variations in identity
(for example ranging from 38–86% in Coprinopsis cine-
rea [27]). This natural diversity within in a single
organism can be used for protein engineering purpose.
In Trametes sp. C30, we previously characterized five
genes [24,28–31], four of which encoded expressed pro-
teins and were used here for molecular breeding experi-
ments. The lac131, lac232 and lac535 hybrid genes
contain more than half of the lac3 gene sequence,
flanked by the 5¢ and 3¢ lac1, lac2 and lac5 regions,

respectively. In all three chimeric genes, the recombina-
tion points between parental sequences more than
1500 nucleotides long involve less than 50 nucleotides.
However, in the donor sequence (lac3), the 5¢ recombi-
nation zone (279 nucleotides long) is about five times
larger than the 3¢ one (58 nucleotides long). In the 5¢
recombination zone, blocks of identical nucleotides are
short and spread over the entire segment (66% overall
identity within the four sequences), whereas in a win-
dow of comparable size (about 280 nucleotides) cen-
tred on the 3¢ recombination zone, the highest identity
is found in the central 58 nucleotide block (87% over-
all identity within the four sequences). Studying ho-
meologous recombination of P450 sequences, Me
´
zard
et al. [16] concluded that the preferred points of
recombination could be those corresponding to maxi-
mal identity in the overall alignment of the parental
sequences. However, the short window of recombina-
tion found in the 3¢ zone suggests a bias in the selec-
tion of recombination points. It has been suggested
that optimal recombination points allow swapping of
structural blocks [19], and combination of large pro-
tein fragments in our chimera led to fully functional
enzymes. Moreover, as recombination of nature-
selected sequences is conservative [32], crossovers lead-
ing to functional hybrids occur at positions that mini-
mize disruption of interactions [18]. In the chimera,
recombination preserved the integrity of domain D1

and the very end part of domain D3, two regions that
interact precisely in the natural laccase fold (Fig. 5).
Because of the bias introduced by linearization of the
receptor fragment at restriction sites, it is difficult to
interpret the position of the recombination points rela-
tive to domain D2. However, it seems that D2 ⁄ D3
interactions are favoured in the chimera (all LAC3),
whereas D1 ⁄ D2 interactions are favoured only in
Fig. 3. Variations in ABTS oxidation rates as function of pH for
LAC3 and the hybrids LAC131, LAC232 and LAC535. ABTS
(5.5 m
M final concentration) was added to the appropriate enzyme
solution (0.6 nM) at the desired pH. Oxidation rates are proportional
to variations in the absorbance at 410 nm per minute and are indi-
cated as DA ⁄ min. Inset: microtitre plate with enzyme ⁄ substrate
mixtures at various pH values; the photograph was taken after
3 min of incubation at 30 °C.
Table 2. Apparent kinetic parameter values for ABTS at various pH
in Britton–Robinson buffer adjusted to the relevant pH at 30 °C.
Enzyme
k
cat
(min
)1
) K
M
(lM)
k
cat
⁄ K

M
(min
)1
ÆlM
)1
)
pH 5.0 pH 8.0 pH 5.0 pH 8.0 pH 5.0 pH 8.0
LAC3 18 052 10 573 57 31.5 0.18
LAC131 16 082 316 410 143 39.2 2.2
LAC232 23 336 46 466 60 50 0.78
LAC535 22 728 12 473 109 48 0.11
Fig. 4. Relative increase in apparent k
cat
for laccase hybrids as a
function of pH.
A. M. Cusano et al. Functional hybrids of laccases from Trametes C30 sp.
FEBS Journal 276 (2009) 5471–5480 ª 2009 The Authors Journal compilation ª 2009 FEBS 5475
LAC535 (all LAC5). Nevertheless, the 5¢ recombina-
tion points apparently match structural block limits in
chimeras as junctions were found: at the limit of
domain D2 in LAC131, at the limit of domain D1 in
LAC232, and at the position (or nearby) of the cyste-
ine residue C228 (LAC3 numbering) that is involved in
a disulfur bridge with C140 (D1) in LAC535. Based on
the present observations, a better knowledge on toler-
ance to block exchange in the laccase enzyme should
be obtained by in vitro sequence permutation experi-
ments and swapping of cupredoxin domains (D1, D2,
D3). Such experiments are in progress.
One of the beneficial effects of production of the

present Trametes sp. C30 laccase chimeras was to
create hybrid sequences that are better expressed in the
host than the parental sequences clac1, clac2 and clac5.
Among the parental sequences used for this recombi-
nation study, only constructions bearing the sequence
encoding the LAC3 isoenzyme have previously been
found to lead to substantial production of recombinant
enzyme in yeast [29,30]. Whereas LAC1 and LAC2
have been purified and fully characterized from Tra-
metes sp. C30 [28,31], their recombinant counterparts
produced in yeast are barely detectable on activity
plates (T. Tron, unpublished results). These differences
in expression of recombinant enzyme coding sequences
are likely largely related to inappropriate codon usage
by the heterologous host, as low-frequency codons can
cause translation pauses depending on their position
and abundance. Upon recombination with clac3
Fig. 5. Molecular models of LAC3 and hybrids. Models were constructed using the structure of the laccase 2HRG from T. trogii as template.
A ribbon representation is used for the LAC3 model; copper atoms are represented as grey spheres; the p-methylbenzoate present in the
structure 2HRG is used in the models to indicate a potential substrate-interacting zone. A surface representation is used for the hybrids; the
surface of the parts of hybrids originating from LAC3 is coloured in light blue; the surface of the parts of hybrids originating from either
LAC1, LAC2 or LAC5 but identical to LAC3 is coloured in dark blue; the surfaces of the parts of hybrids corresponding to LAC1, LAC2 or
LAC5 substitutions are coloured in yellow, red and magenta, respectively.
Functional hybrids of laccases from Trametes C30 sp. A. M. Cusano et al.
5476 FEBS Journal 276 (2009) 5471–5480 ª 2009 The Authors Journal compilation ª 2009 FEBS
sequence, the clac1-, clac2- and clac5-based construc-
tions led to functional expression of the hybrid
sequences clac131, clac232 and clac535. A simple
inspection of coding preference plots [33] for the
parental and chimeric sequences confirmed that

exchanging large sequence segments with the clac3
sequence substantially reduces the number of codons
that potentially cause translation pauses. During func-
tional expression of a M. thermoplila laccase gene in
S. cerevisiae by directed evolution, synonymous muta-
tions to more frequently used codons improved pro-
duction of the recombinant laccase up to eightfold
[13]. For our laccase chimera, it is difficult to calculate
a fold improvement in production relative to LAC1,
LAC2 or LAC5 because of the absence of a reference
level for these parental enzymes. On the other hand,
compared to LAC3, as the steady-state kinetic parame-
ters for all the enzymes are of the same order of mag-
nitude, the ratio of the total volumetric activities
reflects a decrease in production by the hybrids of
approximately fivefold. This suggests that the codon
usage can probably be improved further, for example
through design of synthetic sequences.
LAC3 is representative of a class of laccases found
in basidiomycetes: it is an acidic enzyme that works
best in a pH window from 4.5–6.0. Under catalysis
conditions previously established for LAC3 (buffer,
pH, temperature), all three variants are as active as
LAC3. This is remarkable as a 60–90% decrease in
activity has been reported for P450 chimera (similar to
our laccases in sequence size, identity, recombination
area), although, in this case, chimera activities may
account both for intrinsic kinetic differences in sub-
strate oxidation and differences in interaction with a
reductase [16]. In our case, as discussed above, recom-

bination essentially preserved domain interactions as
well as the architecture of coordination sites. More-
over, the substrate-interacting zone, as defined by the
location of substrate analogues in the crystal structures
of Coriolaceae laccases 1KIA [34] and 2HRG (http://
www.rcsb.org), is identical to that of the LAC3
enzyme, either because it is entirely composed of
LAC3 sequence (LAC131, LAC232) or because residue
variations in that zone are conservative (LAC535)
(Fig. 5). These may be major reasons why the kinetic
behaviour of the hybrids is closer to that of LAC3
rather than that of the other parental enzymes [28,31].
Like other basidiomycetous laccases, LAC3 variant
activities are progressively inhibited by an increasing
concentration of OH
)
[35], which binds the T2 copper,
but significant differences distinguish them from each
other. Thus, the LAC3 and LAC535 pH profiles are
superimposable, suggesting strong conservation of the
original LAC3 properties upon recombination,
although the protein sequence of this hybrid is the least
related to that of LAC3 (Fig. 5). On the other hand,
LAC131 and LAC232 hybrids oxidize ABTS 31- and 5-
fold faster, respectively, than the parental LAC3
enzyme at pH 8.0. As the kinetic behaviour of all of
our enzymes is very similar below pH 6.0, these results
probably reflect a significant improvement in the stabil-
ity of LAC131 and LAC232 hybrids at alkaline pH.
Further studies on this type of mutants should help to

deepen our knowledge on protein regions modulating
laccase activity in response to pH changes.
In conclusion, recombination of large fragments of
sequence coding for laccase isoenzymes leads to the
exchange of structural blocks, allowing synthesis of
hybrid enzymes with properties that distinguish them
from the parental enzymes. Differences in laccase
activity observed at pH 8.0 do not reflect an enhance-
ment in k
cat
but rather reflect an enhancement of the
enzyme stability at alkaline pH. Nevertheless, the cata-
lytic efficiency of the best-performing hybrid (LAC131)
is more than 12 times that of the parental enzyme
(LAC3). Compared to studies involving mutagenesis,
such a factor is one of the highest ever observed in a
single step. Thus, hybrids obtained by homeologous
recombination constitute a valuable tool set to study
the plasticity of the enzyme.
Experimental procedures
Materials and reagents
Chemicals were purchased from Sigma-Aldrich (St Louis,
MO, USA) and were of the highest available grade. The
Britton–Robinson buffer was produced by mixing 0.1 m
boric acid, 0.1 m acetic acid and 0.1 m phosphoric acid with
45% NaOH to the desired pH. 2-(N-morpholino)ethane-
sulfonic acid (Mes) buffer was adjusted to pH 5.7 with
NaOH. Spectroscopic measurements were performed using
either a CARRY 50 spectrophotometer (Varian, Palo Alto,
CA, USA) or a KC4 microtitre plate reader (BioTek,

Winooski, VT, USA). A DuoFlow FPLC apparatus (Bio-
Rad, Hercules, CA, USA) was used for chromatographic
separations.
Strains and vectors used for cloning and
expression
S. cerevisiae W303-1A (MATD, ade2-1, his3-11, 15, leu2-
3/112, trp1-1, ura3-1, can1-100) was used for expression of
laccase. Yeast expression vector pDP51 (2l, Amp
r
, URA3,
GAL10 ⁄ CYC1) pBM258 (GAL1 ⁄ GAL10, CEN4 ⁄ ARS1,
Amp
r
, URA3) and pSAL4 (2l, Amp
r
, URA3, CUP1) were
A. M. Cusano et al. Functional hybrids of laccases from Trametes C30 sp.
FEBS Journal 276 (2009) 5471–5480 ª 2009 The Authors Journal compilation ª 2009 FEBS 5477
respectively obtained from Dr D. Pompon (Centre de
Ge
´
ne
´
tique Mole
´
culaire, Gif ⁄ Yvette, France), Dr D. Bot-
stein (Princeton University, NJ, USA) and Dr R. Gaxiola
(Departamento de Biologı
´
a Molecular de Plantas, UNAM,

Cuernavaca, Morelos, Mexico). Standard techniques were
used for cloning, transformation and analysis [36].
Construction of chimera and gap repair
The four parental laccase-encoding sequences clac1, clac2,
clac3 and clac5 have been previously isolated from Trametes
sp. strain C30 and heterologously expressed in S. cerevisiae
in our laboratory [24,29–31]. Expression vectors bearing
clac1 (AKY160), clac2 (EMY162) or clac5 (EMY164)
sequences were linearized at the SmaI, Kpn2I and ClaI
restriction sites, respectively, located in the laccase-coding
region. The clac3 sequence was amplified by PCR from the
construct pAKY145 [29,30] using EM53 (5¢-TTCCTTTTG
GCTGGTTTTGC-3¢) and EM54 (5¢-CAGTTATTACCC
TATGCGGTGTGA-3¢), respectively, as forward and
reverse primers. The resulting 2015 bp amplicon was
gel-purified and further used in co-transformation assays (lg
donor DNA/lg vector DNA=4) with various linearized
laccase-encoding vectors. Transformants were plated on
selective medium (per litre: yeast nitrogen base without
amino acids and ammonium sulfate, 6.7 g; casaminoacids,
5 g; adenine sulfate, 30 mg; CuSO
4
100 lm; succinate buffer
50 mm, pH 5.3; 1.5% agar) containing 2% galactose as the
carbon source and 0.05% v ⁄ v guaı
¨
acol as the laccase
substrate. Laccase-active transformants were picked and
further studied.
Enzyme production

Yeasts were cultivated at 28 °C. Pre-cultures were obtained
in two stages from a single colony freshly grown on a selec-
tive plate. Cells were first grown in 15 mL tubes containing
5 mL of selective medium for 24 h on a rotating wheel. A
volume of suspension sufficient to reach a final attenuance
at 600 nm of 0.1 was then used to inoculate 250 mL Erlen-
meyer flasks containing 50 mL of selective medium, and cells
were then grown for 24 h on a reciprocal shaker (150 rpm).
Bio-reactor cultivations (batch) were performed in a 15 L
fermentor vessel (B. Braun Biotech International GmbH,
Melsungen, Germany) containing 10 L of selective medium.
The inoculum was added to a final attenuance at 600 nm of
0.1, and yeasts were grown under stirring (220 rpm) and
with an air flow of 16 LÆh
)1
. Samples (1 mL) to be used
for laccase activity and cell density determination were
withdrawn and analysed regularly throughout cultivation.
Purification
Cells were sedimented by centrifugation at 1600 g and 4 °C
for 10 min. Culture supernatant (10 L) was successively
filtered through Whatman paper glass (porosity 1.5 lm),
0.45 and 0.22 lm poly(vinylidene difluoride) membranes.
Filtrate was then concentrated 50-fold by ultrafiltration
using a Prep ⁄ Scale cartridge (approximately 0.23 m
2
YM10
membrane, Amicon ⁄ Millipore, Bedford, MA, USA), and
buffer-exchanged with 20 mm phosphate, pH 6.0 (buffer
A). The sample was further concentrated to 50 mL on

76 mm diameter YM10 membrane and applied to an ion-
exchange DEAE-Sepharose column (2.5 · 20 cm, Amer-
sham Pharmacia Biotech Europe GmbH, Freiburg, Ger-
many) pre-equilibrated with the same buffer. Proteins were
eluted at a flow rate of 4 mLÆmin
)1
with a step gradient of
NaCl: 0.1, 0.15, 0.2, 0.25, 0.3 and 1 m. Fractions containing
laccase activity were pooled and concentrated to a volume
of 600 lL by ultrafiltration on a 25 mm diameter YM10
membrane, and loaded on a Superdex S200 column (Amer-
sham Pharmacia) equilibrated with 20 mm phosphate, pH
6.0, 200 mm NaCl. Fractions containing laccase activity
were pooled and concentrated. Exchange with buffer con-
taining no salt, concentration and addition of 15% glycerol
were undertaken for long-term storage of the protein
()20 °C). Enzyme purity in active fractions was then con-
firmed by SDS–PAGE.
Standard enzyme assay
Protein concentration was determined by the Bradford
method using BSA as standard, or by UV-vis spectroscopy
(e
600 nm
=5· 10
3
m
)1
Æcm
)1
for the T1 copper) [37]. Lac-

case activity was routinely assayed at 30 °C using SGZ as
the substrate. Oxidation of SGZ was detected by measuring
the absorbance increase at 525 nm (e
525nm
= 6.5 · 10
4
m
)1
Æcm
)1
) after 2 min using a spectrometer (Carry 50 UV-
vis spectrophotometer) [38]. The reaction mixture (1 mL)
contained 10 lL of appropriately diluted enzyme sample
and 980 lL of Mes buffer (50 mm, pH 5.7), and 10 lLof
0.8 mgÆmL
)1
SGZ in MeOH was added to initiate the reac-
tion. One unit (U) of laccase oxidizes one micromole of
substrate per minute.
Kinetic parameter determination and effect of pH
Determination of kinetics parameters was undertaken using
two substrates: SGZ and ABTS. For SGZ, the same condi-
tions were used as those for the standard enzyme assay.
ABTS oxidation was determined in both MES and Britton–
Robinson buffers by monitoring the absorbance change at
414 nm with an extinction coefficient of 3.5 · 10
4
m
)1
Æcm

)1
[39]. Variation of the ABTS oxidation rate as function of
pH was assayed in a 96-well plate at 30 °C for 2 min using
Britton–Robinson buffer adjusted to a pH from 4.5–8.0.
Apparent K
M
and k
cat
values were obtained from the initial
rate (v), enzyme concentration (E) and substrate concentra-
tion (S) according to the equation v = k
cat
ES⁄ (K
M
+ S)
(non-linear regression fitting using prizm program, Graph-
Functional hybrids of laccases from Trametes C30 sp. A. M. Cusano et al.
5478 FEBS Journal 276 (2009) 5471–5480 ª 2009 The Authors Journal compilation ª 2009 FEBS
pad, San Diego, CA). Because laccase catalysis involves
two substrates and the [O
2
] was invariant and assumed to
be saturating in this study, the measured K
M
for the vari-
ous substrates used should be considered apparent. Because
of the assumption that 100% of the laccase participated in
the catalysis as active enzyme, the measured k
cat
should

also be considered apparent.
Molecular models
3D models were obtained from the Swiss Model Server
(swissmodel.expasy.org) using the crystallographic coordi-
nates from Trametes trogii laccase 2HRG obtained from
the Research Collaboratory for Structural Bioinformatics
(RCSB) Protein Data Bank ().
Acknowledgements
This work was partly supported by the European
Commission, Sixth Framework Program (NMP2-
CT2004-505899, SOPHIED). Angela Cusano was the
recipient of a Re
´
gion Provence Alpes Coˆ te d’Azur
Postdoc fellowship. Emese Meglecz was the recipient
of a Ministe
´
re de le Recherche Postdoc fellowship. We
thank Marius Re
´
glier, Jalila Simaan, Erin Wallace-
Bomati and Gilles Iacazio (Laboratoire Biosciences,
Institut des Sciences Mole
´
culaires de Marseille, Univer-
site
´
Aix-Marseille, France) for helpful discussions.
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