Download Screening Mutant Libraries of Fungal Laccases in the Presence of

Alcalde et Laccases
ARTICLE
10.1177/1087057105277058
Screening
al.
in Organic Cosolvents
Screening Mutant Libraries of Fungal Laccases
in the Presence of Organic Solvents
1
2
1
1
MIGUEL ALCALDE, THOMAS BULTER, MIREN ZUMÁRRAGA, HUMBERTO GARCÍA-ARELLANO,
3
1
1
MARIO MENCÍA, FRANCISCO J. PLOU, and ANTONIO BALLESTEROS
Reliable screening methods are being demanded by biocatalysts’engineers, especially when some features such as activity or
stability are targets to improve under nonnatural conditions (i.e., in the presence of organic solvents). The current work describes a protocol for the design of a fungal laccase—expressed in Saccharomyces cerevisiae—highly active in organic
cosolvents. A high-throughput screening assay based on ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) oxidation was validated. The stability of the ABTS radical cation was not significantly altered in the presence of acetonitrile, ethanol, or DMSO. With a coefficient of variance below 10% and a sensitivity limit of 15 pg laccase/µL, the assay was reproducible and sensitive. The expression system of Myceliophthora thermophila laccase variant T2 in S. cerevisiae was highly
dependent on the presence of Cu2+. Copper concentration was limited up to 10 µM CuSO4 where expression levels (~14-18
mg/L) were acceptable without compromising the reliability of the assay. A mutant library was created by error-prone PCR
with 1.1 to 3.5 mutations per kb. After only 1 generation of directed evolution, mutant 6C9 displayed about 3.5-fold higher activities than parent type in the presence of 20% acetonitrile or 30% ethanol. The method provided here should be generally
useful to improve the activity of other redox enzymes in mixtures of water/cosolvents. (Journal of Biomolecular Screening
2005:624-631)
Key words: laccase, directed molecular evolution, high-throughput screening, organic solvents, Saccharomyces cerevisiae
INTRODUCTION
L
of blue copper
enzymes that are capable of catalyzing the oxidation of phenols, polyphenols, and anilines, coupled to the 4-electron reduction of molecular oxygen to water.1 Laccases are broadly distributed in higher plants and fungi and have also been found in some
insects and bacteria.2 Although these biocatalysts are hardly active
toward nonnatural substrates, in the presence of small molecules—
called mediators—with generally higher redox potentials than the
enzyme itself, laccases become potentially useful in the transformation of nonphenolic compounds.3,4 The study of the laccase mediator system in the pulp-kraft bleaching industry or in the removal
of harmful xenobiotics (e.g., polycyclic aromatic hydrocarbons) is
well documented.5-13 Typically, the Km values of laccases for
ACCASES (EC 1.10.3.2) BELONG TO THE GROUP
1
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Madrid,
Spain.
2
Department of Chemical Engineering, University of California, Los Angeles.
3
Centro Nacional de Biotecnologia CSIC-UAM, Madrid, Spain.
Received Jan 12, 2005, and in revised form Mar 8, 2005. Accepted for publication
Mar 28, 2005.
Journal of Biomolecular Screening 10(6); 2005
DOI: 10.1177/1087057105277058
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nonnatural substrates are in the millimolar range. Thus, high turnover rates require substrate concentrations that are often beyond
the solubility in aqueous solution (e.g., the water solubility of
benzo[a]pyrene is as low as 0.012 µΜ). Water-miscible cosolvents
can increase the substrate solubility, but unfortunately, most
laccases hardly tolerate high concentrations of organic solvents,
which limits their practical use.14-18 Therefore, an optimum
cosolvent concentration constitutes a compromise between
laccase inactivation and substrate solubilization.
Directed molecular evolution of biocatalysts is a powerful tool
in enzyme engineering. Through iterative steps of random mutation, DNA recombination, and screening, improved biocatalysts
are being tailored.19,20 Laboratory evolution approaches for the improvement of enzymatic resistance toward organic solvents have
successfully been demonstrated over the years.21-25
We have recently accomplished the heterologous expression of
fungal laccase from Myceliophthora thermophila (MtL) in a functional form in Saccharomyces cerevisiae.26 After 10 rounds of directed evolution, the variant MtLT2 displayed an improved expression of 18 mg/L—the highest value reported for a laccase in yeast.
Together with a 22-fold increase in kcat, the total activity was enhanced 170-fold. In a recent work, thermophilic laccase from
Melanocarpus albomyces was cloned and expressed in S.
cerevisiae, being another suitable candidate for in vitro evolution.27
© 2005 The Society for Biomolecular Screening
Screening Laccases in Organic Cosolvents
Next to Escherichia coli, S. cerevisiae is the most commonly used
host organism in laboratory evolution. Its sophisticated cellular
machinery simplifies mutant library generation. By homologous
recombination, otherwise cumbersome ligation of mutant genes
into expression vectors or shuffling of error-prone PCR libraries
can be performed in vivo within 1 transformation step.28
Glycosylation is another exclusive feature of eukaryotic cells, allowing S. cerevisiae to secrete complex proteins to the media.
In this work, we have selected MtLT2 as a starting point for the
engineering of a laboratory evolution protocol with the main aim
of tailoring laccases highly active in organic solvents. The expression system of MtLT2 in yeast was tuned to the assay conditions. A
high-throughput screening (HTS) assay based on the oxidation of
ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) in
the presence of organic solvent was validated and used for screening a mutant library created by error-prone PCR.
MATERIAL AND METHODS
All chemicals were reagent-grade purity. ABTS (2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)) and the yeast transformation kit were purchased from Sigma (Madrid, Spain). Parent
gene (MtLT2) was obtained as reported elsewhere.26 Trametes
versicolor laccase (TvL) and plasmid pJRoC30 were from
Novozymes (Davis, CA). Yeast cells transformed with this vector
can grow on selective media (media without uracil). The shuttle
vector also includes the E. coli ampicillin resistance gene. E. coli
XL2-Blue competent cells and Genemorph PCR mutagenesis kit
were from Stratagene (La Jolla, CA). S. cerevisiae strain BJ5465
was from LGCPromochem (Barcelona, Spain). Zymoprep yeast
plasmid miniprep kit, zymoclean gel DNA recovery kit, and DNA
clean and concentrator TM-5 Kit were from Zymo Research (Orange, CA). QIAprep spin miniprep kit was from QIAGEN (West
Sussex, UK). Sepabeads FP-EP was from Resindion SRL
(Mitsubishi, Italy). BamHI and XhoI were from New England
Biolabs (Hertfordshire, UK).
Culture media and buffers
Minimal medium contained 50 mL 20% sterile raffinose, 5 mL
0.25% L-His, 5 mL 0.25% L-Trp, 5 mL 0.25% L-Leu, 10 mL 0.25%
adenine hemisulfate, 150 mL dd H2O, 25 mL 6.7% yeast nitrogen
base, and 250 µL chloramphenicol stock solution. YP medium
contained 10 g yeast extract, 20 g peptone, and dd H2O to 650 mL.
Expression medium contained 325 mL YP medium, 25 mL 1 M
KPi-buffer (pH 6.0), 4 µL sterile 1 M CuSO4, 50 mL 20% sterile
galactose, 400 µL chloramphenicol stock solution (77 µm in ethanol), and dd H2O to 400 mL. YPAD solution contained 10 g yeast
extract, 20 g peptone, 100 mL 20% sterile glucose, 100 mg adenine
hemisulfate, and dd H2O to 1000 mL. SC dropout plates contained
6.7 g yeast nitrogen base, 50 mg L-His, 50 mg L-Trp, 50 mg L-Leu,
Journal of Biomolecular Screening 10(6); 2005
50 mg adenine hemisulfate, 15 g bacto agar, 100 mL 20% sterile
glucose, 1 mL chloramphenicol stock solution, and dd H2O to
1000 mL. Britton and Robinson (B&R) buffer contained 0.1 M boric acid, 0.1 M acetic acid, and 0.1 M phosphoric acid adjusted to
the desired pH with 0.5 N NaOH.
Production of MtL in S. cerevisiae
S. cerevisiae clones carrying parent-type or mutant laccase
genes were grown in 50 mL preculture in YPAD supplemented
with 4% sterile raffinose instead of glucose as the carbon source.
After 24 h at 30 °C, 210 rpm, 200 mL expression medium containing 6 mM CuSO4 was inoculated with the preculture. After further
incubation for 24 h at 30 °C, 210 rpm, the cells were separated by
centrifugation at 1000g (4 °C), and the supernatant was filtered
(0.45 µm pore size) and concentrated to 20 mL in an ultrafiltration
cell (Amicon/Millipore, Barcelona, Spain) equipped with a 10kDa cutoff membrane. Finally, the sample was stored at –20 °C.
Purification of laccases
Native and mutant laccases were purified using fast protein liquid chromatography (FPLC) equipment (LCC-500 CI, Amersham
Bioscience, Barcelona, Spain). Sample was dialyzed in buffer T
(20 mM Tris-HCl, pH 7.8) and loaded onto a HiTrap Q sepharose
fast flow (5 mL, Amersham Pharmacia, Barcelona, Spain) preequilibrated with the same buffer. Laccase activity was eluted with
a linear gradient of 0 to 400 mM NaCl in buffer T. Active fractions
were pooled, desalted, and concentrated in an ultrafiltration cell.
The concentrate in buffer T was loaded onto a MonoQ column (1
mL, Amersham Pharmacia) preequilibrated with buffer T. The column was washed with buffer T, and laccase activity was eluted in a
linear gradient of 0 to 1 M NaCl. Active fractions were pooled, desalted, and concentrated in an ultrafiltration cell.
Laccase immobilization and generation of ABTS•+
MtLT2 was immobilized on polymethacrylate spherical beads,
Sepabeads FP-EP (pore diameter 150-300 Å, surface area 60 m2/g
dry, 110 µmol oxirane groups/g wet). Support (1 g) and MtLT2 (ratio mg enzyme/g support 1:1) were incubated for 48 h in 10 mL of
1.25 M KH2PO4 buffer, pH 10.0, with roller shaking. Thereafter,
carrier was exhaustively rinsed with 10 mL of 10 mM B&R buffer,
pH 6.0. Remaining epoxy groups were blocked by incubation in 2
M glycine, pH 8.0, for 24 h. The biocatalyst was washed again with
B&R buffer, dried, and stored at 4 °C.
For the generation of ABTS radical cation (ABTS•+), 5 mg of
immobilized laccase was incubated in 4 mL of 50 mM B&R buffer
containing 3 mM ABTS. The reaction course was followed spectrophotometrically. Once the green color was developed (ABTS
radical cation was formed), the mixture was filtered to stop the
reaction.
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Alcalde et al.
Random mutagenesis and cloning
A library of random mutants using MtLT2 as parent type was
created by error-prone PCR with the Genemorph kit (Stratagene)
according to the protocol guide for a low mutation rate (30 ng
DNA was the initial target amount, which corresponded to 1.1-3.5
introduced mutations per kb). The only exception to the manufacturer’s method was that 3% DMSO was included in the reaction
mixture. Thermal cycling parameters were 95 °C for 2 min (1 cycle), 94 °C for 0.45 min, 53 °C for 0.45 min, 74 °C for 3min(28cycles), and 74 °C for 10 min (1 cycle). The primers used for amplification
were as follows: RMLN sense (5′-CCTCTATACTTTAACGTCAAGG3′, binds at bp 5′-160-180-3′ of pJROC30) and RMLC antisense
(5′-GGGAGGGCGTGAATGTAAGC-3′, binds at bp 5′-2139-2158-3′
of pJROC30). The PCR products were cleaned and concentrated
(DNA clean and concentrator TM-5 kit, Zymo Research), loaded
into a low-melting-point preparative agarose, and purified using
the Zymoclean gel DNA recovery kit (Zymo Research). PCR
product was cloned behind the Gal 10 promoter of the expression
shuttle vector pJRoC30. Cloning was performed by replacing the
MtLT2 gene from pJRoC30. To remove parent gene, plasmid
pJRoC30 was linearized by cutting with restriction enzymes XhoI
and BamHI (New England Biolabs). Linearized plasmid was concentrated (DNA clean and concentrator TM-5 Kit) and loaded into
a preparative low-melting-point agarose followed by an agarose
gel extraction (Zymoclean gel DNA recovery kit).
PCR products (400 ng) were mixed with linearized vector (100
ng) and transformed into competent yeast cells using the yeast
transformation kit (Sigma, Madrid, Spain). Right before the thermal shock, 10% DMSO was introduced in the transformation mixture to improve the transformation efficiency (above 15,000 clones
per transformation reaction). The error rate of the library was
checked by DNA sequencing.
DNA sequencing
To locate mutations on the MtLT2 gene and estimate the mutation rate, plasmid-containing mutant genes were sequenced at the
Sequencing Core Facility of the Instituto de Investigaciones
Biomedicas, CSIC, Madrid, using an Applied Biosystems 377 automated fluorescent DNA sequencer. The primers used were as
follows: sense: RMLN and mtlsq2 (5′-GAAGGGCACCAACCTGC3′, binds at bp 5′-643-659-3′ of pJROC30); antisense: mtlsq3 (5′GCACGTAAAAGTCGTGG-3′, binds at bp 5′-1657-1673-3′ of
pJROC30) and RMLC.
Preparation of library for HTS: end-point assay
Once yeast cells were transformed with the mutagenic PCR library and linearized pJROC30 vector (ratio PCR library/linearized
vector, 4:1), cells were plated on SC dropout plates and incubated
at 30 °C for 4 days. Individual colonies were picked into 96-well
plates (Sero-well, Staffordshire, UK) containing 50 µL of minimal
medium per well. One column (column number 6) on each plate
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was inoculated with standard (parent type), and 1 well (H1) was
not inoculated (control). Plates were wrapped in parafilm (to prevent evaporation) and incubated at 30 °C and 210 rpm
(Micromagmix shaker, Ovan, Spain). After 45 h, 160 µL of expression medium was added to each well, and the plates were incubated at 30 °C and 210 rpm. After an additional 24 h, OD600 was
recorded to determine the cell density of each well using a microplate reader (Versa Max, Molecular Devices, Sunnyvale, CA).
Plates were centrifuged (Eppendorf 5810R centrifuge, Germany) for 5 min at 3000g at 4 °C, and 10 µL of supernatant was
transferred into organic solvent–resistant 96-well plates using liquid handler (Quadra 96-320, Tomtec, Hamden, CT). Then, 190 µL
of assay solution (final concentration in the well, 3 mM ABTS,
20% acetonitrile or 30% ethanol, 50 mM B&R buffer, pH 6.0) was
added to each well with the supernatant. The plates were stirred,
and the absorption at 418 nm was recorded by the plate reader
(εABTS•+ = 36,000 M–1 cm–1). Plates were incubated at room temperature until green color was developed, and the absorption was measured again. Relative activities were calculated from the difference
between initial absorption and that after the incubation divided by
the OD600 of each well. Relative activities were normalized
against the parent type in the corresponding plate. First
rescreening: aliquots of 5 µL of the best clones were used to inoculate 50 µL of minimal media in 96-well plates. Columns 1 and 12
(rows A and H) were not used. Five wells on the microplates were
inoculated with the same clone. Five wells on each plate were used
for the standard. The screening procedure then was the same as
above. Second rescreening: 3 mL of YPAD each was inoculated
with an aliquot from the wells with the most active clones of first
rescreening and incubated at 30 °C and 210 rpm for 24 h. Plasmids
from these cultures were extracted (Zymoprep yeast plasmid
miniprep kit, Zymo Research). As the product of the zymoprep
was very impure and the concentration of extracted DNA was very
low, the plasmids were transformed into super-competent E. coli
cells (XL2-Blue, Stratagene) and plated onto LB-amp plates. Single colonies were picked and used to inoculate 5 mL LB-amp media and were grown overnight. Plasmids were then extracted
(QIAprep spin miniprep kit, QIAGEN). S. cerevisiae was transformed with plasmids from the best mutants and also with the parent type and screened as described above.
RESULTS AND DISCUSSION
Selection of the chromogen for the screening
in organic solvents
A reliable HTS assay is an indispensable requisite for directed
molecular evolution of enzymes or for exploring metagenome libraries. According to one of the main paradigms in laboratory evolution (“you get what you screen for”29), a good colorimetric or
fluorimetric assay has to be sensitive to the desired function and
has to allow the isolation of lower activity clones early in evolution.
Among many alternatives, ABTS was considered as an excellent
Journal of Biomolecular Screening 10(6); 2005
Screening Laccases in Organic Cosolvents
•+
FIG. 1. Recorded spectra of ABTS (2,2′-azino-bis(3ethylbenzthiazoline-6-sulfonic acid)) in the presence of 50% acetonitrile.
Bold line, immediately after the addition of acetonitrile. Dashed line, after
•+
1 h incubation. ABTS was generated by MtLT2 immobilized on
Sepabeads FP-EP.
candidate to develop the HTS assay in organic solvents because of
its low detection limit and stability in aqueous solution. Laccase
oxidizes ABTS by abstraction of 1 electron producing the stable,
green radical cation (ABTS•+). The sensitivity limit for this reaction is ~5 nU/mL, and the green reaction product (radical cation) is
stable for several hours. Little is known about the stability of the
ABTS radical cation in water-organic solvent mixtures. Thus, a
preliminary study of ABTS•+ stability in the presence of organic
solvents was performed. ABTS•+ was generated by MtLT2 immobilized on Sepabeads FP-EP, which facilitated the separation of the
biocatalyst. An equal volume of several organic solvents was
added to ABTS•+ solution, and its UV/Vis spectrum was recorded
(Fig. 1). After 1 h of incubation, no dramatic change was detected
for acetonitrile, ethanol, or DMSO. As it was reported elsewhere,30
dioxane decreased the stability of ABTS•+; therefore, the assay was
not suitable for this solvent. Other compounds (3hydroxyantranilic acid, syringaldazine, homovanillic acid, 1naphtol, o-phenylendiamine, and diaminoanthraquinone) were
also tested as substrates in the assay. In all cases, either the signal
was not stable but decreased slowly over time or resulted in a very
strong background from growth medium (data not shown).
Preparation of the assay for directed evolution
To set up the initial organic solvent concentration to be used in
the screening, 2 fungal laccases with different redox potential
(MtLT2 and TvL) were tested in the presence of organic cosolvent.
First, laccase stability was assayed under several concentrations of
acetonitrile (Fig. 2). Both laccases behaved in similar fashion, being quite stable up to 20% organic solvent after a 4-h incubation.
However, at higher concentrations, the stability was reduced dramatically, and TvL was not able to tolerate concentrations beyond
Journal of Biomolecular Screening 10(6); 2005
FIG. 2. Stability of MtLT2 (black circles) and TvL (white circles) in
the presence of different concentrations of acetonitrile. The 96-well plates
contained either MtL2 or TvL in 0.1 M Britton and Robinson (B&R)
buffer (pH 6.0) supplemented with different amounts of acetonitrile. A
replica plate was done and its initial activity assessed by adding ABTS
(2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) to a final concentration of 3 mM. Master plates were sealed and incubated for 4 h at room
temperature. For the calculation of residual activities, activity of the master plate was assessed after incubation and refereed to that of initial activity. Each point represents the average of 8 experiments (8 wells).
30%. Unlike TvL, MtLT2 was the most stable biocatalyst, keeping
nearly 70% of initial activity after 4 h of incubation at 30%
acetonitrile.
Enzyme initial rates were further studied in the presence of
acetonitrile. Laccase activity lowered dramatically when increasing the percentage of acetonitrile (Fig. 3). A similar effect was observed in ethanol (data not shown). The screening conditions must
ensure that the expected small enhancements brought about by single amino acid substitutions can be measured. This was accomplished by adjusting the assay conditions to the point where the
parent-type activity was close to the detection limit. In 20%
acetonitrile or 30% ethanol, laccase activity was about 20% compared to the value in the absence of cosolvent, and these conditions
were chosen for the screening of the mutant library.
For the heterologous expression of MtLT2, a protease-deficient
S. cerevisiae strain BJ5465 (found to be generally suitable for secretion) was used. The laccase expression system was highly dependent on the CuSO4 concentration (Fig. 4). The Cu2+ concentration for the highest production of MtLT2 was 6 mM, and it was
used for large-scale production of protein (see Material and Methods). The difference in secreted activity between 8 µM CuSO4 and
6 mM CuSO4 was about 2.5-fold. Above 6 mM CuSO4, Cu2+ becomes toxic to the cells. One of the disadvantages of using high
concentrations of copper in the screening is that the biocatalyst expression might become dependent to it. Thus, copper concentration was limited so that there was not a selection for laccases re-
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Alcalde et al.
FIG. 3. Initial activities measured of MtLT2 (black circles) and TvL
(white circles) at several concentrations of acetonitrile. Each point represents the average of 8 experiments (8 wells).
FIG. 5. Linearity of the assay in the presence of 30% ethanol. Each
point represents the average of 8 experiments (8 wells).
FIG. 4. Relative activities of MtLT2 from cultures grown with differ2+
ent concentrations of Cu in the expression medium. Activities were normalized to the activity without addition of CuSO4, which was set to 1.
Cells were grown in 96-well plates, and each point represents the average
of 5 experiments (wells).
FIG. 6. Validation of the ABTS (2,2′-azino-bis(3ethylbenzthiazoline-6-sulfonic acid)) screening assay in the presence of
20% acetonitrile. Activities were plotted in descending order. Expression
medium with 10 µM CuSO4 was used for cell growth and expression. The
standard deviation in activity was below 10% for assays in 30% ethanol or
20% acetonitrile.
quiring higher concentrations of copper. As 10 µM CuSO4 gave us
acceptable expression levels (~14-18 mg/L), this was the
concentration chosen for the screening assay.
In developing the screening, we first evaluated the relationship
between absorbance and laccase concentration (Fig. 5). Fresh
transformants containing parent type were inoculated in a 96-well
plate, laccase was expressed, and different volumes of supernatant
from single microcultures were assessed. The detection limit was
found to be about 15 pg of laccase/µL of supernatant. To validate
the assay, a test plate containing MtLT2 in different wells was prepared. S. cerevisiae cells were transformed with the plasmid-containing MtLT2 gene and plated on SC dropout plates. The activities
of clones were evaluated from fresh supernatant preparations, as
described in the Material and Methods section. The coefficient of
variation (CV) for the ABTS endpoint assay was 9.9% in 20%
acetonitrile and 9.0% in 30% ethanol (Fig. 6).
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Screening a mutant generation created by error-prone PCR
The assay was used to characterize a library of mutants (about
1500 clones) created using error-prone PCR. Mutazyme polymerase was chosen for mutagenic PCR because its biases allow for increased variation in mutation type (GA, CT, GT, and CA changes
and also all transversion mutations). The random mutagenesis
Journal of Biomolecular Screening 10(6); 2005
Screening Laccases in Organic Cosolvents
A
B
FIG. 7. (A) Directed evolution landscape screened in the presence of
30% ethanol. The activities of clones from the library of MtLT2 mutants
prepared by error-prone PCR are plotted in descending order. Around
50% of clones were inactivated by mutagenesis. Dashed line indicates the
activity of parent type. (B) The screening of the MtLT2 library was carried
out in 20% acetonitrile and 30% ethanol. Mutants with high activities in
both solvents were chosen for further rescreening. Dashed line indicates
the activity of parent type.
conditions were set to introduce 1.1 to 3.5 mutations per kb. Errorprone PCR products were transformed into yeast together with the
linearized vector. It is worth noting that the mutagenized fragments, containing 35 bp of homology at the 5′ end and 66 bp of
homology at the 3′ end, homologously recombine with the ends of
the linearized vector to regenerate a circular, autonomously replicating vector. The screening of the mutant library was performed
both in acetonitrile 20% and ethanol 30% (Fig. 7). Several mutants
Journal of Biomolecular Screening 10(6); 2005
FIG. 8. Specific activities of MtLT2 (white bars) and 6C9 variant
(black bars) under different concentrations of organic cosolvents. Each experiment was done by triplicate.
were found to be more active both in ethanol and in acetonitrile. A
credible comparison of clones necessitates mutants that are in the
same metabolic stage, and a second rescreening with freshly transformed cells was performed. It is worth pointing out that this
screening protocol in organic solvents was very reliable because
most “winners” from the laboratory evolution round were also
highly active after 2 consecutive rescreens. Mutant 6C9 was purified and further characterized, displaying about 3.5-fold better activities in 20% acetonitrile and 30% ethanol (kcat of 1589 min–1 and
5591 min–1 for parent type and 6C9, respectively).
As one can see in Figure 8, the 6C9 variant seems to display
higher activities than the wild type, regardless of the organic solvent concentration and even in the absence of acetonitrile. It is not
surprising that 6C9 has improved its properties in both aqueous
and artificial media. In fact, very often laboratory evolution allows
us to get biocatalysts with improved features not only under the
specific screening conditions (e.g., organic cosolvents, new substrates) but also under the standard ones (aqueous media, natural
substrates).24,31
The MtL gene has a pro and a pre-pro sequence, which targets
the protein for secretion in the native organism. It also contains a Cterminal tail that is cleaved off during the processing of the protein.
The 6C9 variant showed 1 synonymous mutation, TCG/GCA
(S462S), and 2 further mutations: CTA/GTA (L476V), located in
the structural gene, and CCT/CAT (P20H), located at the signal sequence. One of the beneficial mutations that was found in our former work26 was in the same codon (A20P) and enhanced the
laccase total activity 1.5-fold. It seems to be clear that the residue in
position 20 may play an underlying role in targeting the gene for
secretion. One suitable strategy to study this function in more detail could be carried out through the construction of a small library
by saturation mutagenesis.32 Other clones with ~2-fold better ac-
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Alcalde et al.
tivities were characterized. Among them, the 3G10 variant showed
just 1 substitution, GAG/AAG, located at the C-terminal processing site of the protein introducing the change E615K. The significance of the C-terminal end for the activity of ascomycete laccases
is well known.26,27 It has been postulated that C-terminal processing may play a role in activation of laccase, being essential for early
posttranslational processing steps. Our results also support this
theory.
Functional expression is a necessary requisite for directed enzyme evolution. Fungal laccase expression in E. coli has not been
successfully reported (probably due to the requirement of
glycosylation). Among eukaryotic hosts, S. cerevisiae is the most
suitable organism for heterologous expression and HTS. Unlike
Pichia pastoris, S. cerevisiae can display higher transformation efficiencies and does not integrate the expression constructs into
genomic DNA. Moreover, secretion of mutant proteins and DNA
homologous recombination are 2 features of which one can take
advantage. Indeed, the eukaryotic machinery is able to recombine
the mutagenic products with the linearized plasmid (avoiding the
tedious task of ligating the DNA fragments to the vector) or even to
shuffle them in vivo with an exquisite performance.
The HTS in organic solvents presented in this work, along with
the expression system in S. cerevisiae for MtLT2, is suitable for exploring mutant libraries. This method can be valid not only for
screening for laccase activity in organic media but also for other redox systems (e.g., peroxidases, galactose oxidases) in complex biological mixtures.32,33
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ACKNOWLEDGMENTS
17.
This research was financed by the Comunidad Autónoma de
Madrid (Project GR/AMB/0690/2004) and Ministerio de
Educación y Ciencia, Spain (Project VEM2004-08559). We thank
Prof. V. de Lorenzo from Centro Nacional de Biotecnologia (CNB,
CSIC) for providing facilities. M. Zumárraga was supported by a
fellowship from the Gobierno Vasco, Spain.
18.
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20.
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Address reprint requests to:
Miguel Alcalde
Departamento de Biocatálisis
Instituto de Catálisis y Petroleoquímica, CSIC
Cantoblanco 28049, Madrid, Spain
E-mail: [email protected]
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