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

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

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-

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

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

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

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

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

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

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-

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,

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

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-

)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

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