INTRODUCTION
Aryl-alcohol oxidase (AAO; EC 1.1.3.7) is a flavoenzyme of the GMC (glucose-methanol-choline) oxidoreductase superfamily, the members of which share a N-terminal FAD-binding domain containing the canonical ADP-binding motif. Secreted by several white-rot fungi, this monomeric flavoprotein plays an essential role in natural lignin degradation (
1). Accordingly, AAO oxidizes lignin-derived compounds and aromatic fungal metabolites, releasing H
2O
2 that is required by ligninolytic peroxidases to attack the plant cell wall (
2). Moreover, the H
2O
2 produced by AAO is an efficient vehicle to generate highly reactive hydroxyl radicals through the Fenton reaction (Fe
2+ + H
2O
2 → OH˙ + OH
− + Fe
3+), such that OH˙ can act as a diffusible electron carrier to depolymerize plant polymers. AAO oxidizes a variety of aromatic benzyl (and some aliphatic polyunsaturated) alcohols to the corresponding aldehydes. In addition, AAO participates in the oxidation of aromatic aldehydes to the corresponding acids and also has activity on furfural derivatives (
3).
The past few years have witnessed an intense effort to discern the basis and mechanism of action underlying AAO catalysis (
3–10). The AAO catalytic cycle involves dehydrogenative oxidation mediated by two half-reactions: (i) the reductive half-reaction in which the donor alcohol is two-electron oxidized by the FAD cofactor, the latter receiving one of the alcohol's α-Hs through a hydride transfer process that yields the aldehyde product and the reduced flavin, and (ii) the oxidative half-reaction, in which O
2 is two-electron reduced by the FAD, releasing H
2O
2 and the reoxidized flavin (
5).
Directed molecular evolution is by far the best strategy currently available to design enzymes to industrial standards (
11–14). However, AAO has not been subjected to directed evolution, probably due to the lack of appropriate functional expression systems. Indeed, AAO has only been heterologous expressed in
Aspergillus nidulans (
15), an unsuitable host for directed evolution experiments (
16), and in
Escherichia coli after the
in vitro refolding of inclusion bodies, an approach incompatible with directed evolution campaigns (
17).
In the present study, the native signal peptide of AAO was replaced by two different signal sequences to drive its functional expression in
Saccharomyces cerevisiae: (i) the signal prepro-leader of the mating α-factor of
S. cerevisiae, which has been used widely to evolve different ligninases (
18–23), and (ii) the signal prepro(δ)-leader and the γ-spacer segment of the K
1 killer toxin, which have been seen to be useful in boosting β-lactamase secretion in yeast (
24,
25). For the first time, chimeric versions of these leaders were designed by combining the different pre- and pro-regions, and these constructs were subjected to conventional and focused-directed evolution using a very sensitive dual high-throughput screening (HTS) assay based on the Fenton reaction. The best mutant identified dramatically improved the total activity and stability being readily secreted by yeast. Indeed, this active, highly stable and soluble AAO variant is a promising point of departure for new engineering goals.
MATERIALS AND METHODS
All chemicals were of reagent-grade purity. Ferrous ammonium sulfate, xylenol orange, sorbitol, benzyl alcohol, p-methoxybenzyl alcohol, veratryl (3,4-dimethoxybenzyl) alcohol, 2,4-hexadien-1-ol, ABTS [2,2′azinobis(3-ethylbenzthiazolinesulfonic acid)], horseradish peroxidase (HRP), Taq polymerase, and a yeast transformation kit were purchased from Sigma (Madrid, Spain). Zymoprep yeast plasmid miniprep, yeast plasmid miniprep kit I, and a Zymoclean gel DNA recovery kit were obtained from Zymo Research (Orange, CA). Restriction enzymes BamHI and XhoI were from New England BioLabs (Hertfordshire, United Kingdom). I-Proof high-fidelity DNA polymerase was from Bio-Rad (USA). The episomal shuttle vector pJRoC30 was from the California Institute of Technology (Caltech) and plasmids pRE1219 and pJRoC30-δγN2C1 were kindly donated by S. Camarero (CIB-CSIC, Madrid, Spain). E. coli XL2-Blue competent cells were from Stratagene (La Jolla, CA), whereas the protease-deficient S. cerevisiae strain BJ5465 (MATa ura3-52 trp1 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL) was obtained from LGC Promochem (Barcelona, Spain).
Culture media.
Minimal medium SC contained 100 ml of 6.7% (wt/vol) sterile yeast nitrogen base, 100 ml of a 19.2-g/liter sterile yeast synthetic dropout medium supplement without uracil, 100 ml of sterile 20% (wt/vol) raffinose, 700 ml of sterile double-distilled H2O (sddH2O), and 1 ml of chloramphenicol at 25 g/liter. YP medium contained 10 g of yeast extract, 20 g of peptone, and ddH2O to 650 ml, whereas YPD medium also contained 20% (wt/vol) glucose. AAO expression medium contained 144 ml of 1.55× YP, 13.4 ml of 1 M KH2PO4 (pH 6.0) buffer, 22.2 ml of 2% (wt/vol) galactose, 0.222 ml of chloramphenicol at 25 g/liter, and ddH2O to 200 ml. Luria broth (LB) medium contained 10 g of sodium chloride, 5 g of yeast extract, 10 g of peptone, 1 ml of ampicillin at 100 mg/ml, and ddH2O to 1 liter.
Fusion genes and signal chimeric leaders.
AAO mature protein was fused both to the α-factor prepro-leader (preproα-AAO, construct i) and to the prepro(δ)-γ regions of the prepro-toxin K1 killer (preproK-AAO, construct ii). In addition, two chimeric signal peptides were constructed and attached to the AAO: the α-factor pre-leader fused to the γ segment of the K1 killer toxin (preαproK-AAO, construct iii) and the prepro(δ)-signal sequence of the K1 killer toxin fused to the α-factor pro-leader (preKproα-AAO, construct iv). The design of overlapping areas of ∼40 bp between adjacent fragments allowed the in vivo fusion of the different genetic elements using the S. cerevisiae homologous recombination machinery.
AAO was amplified from pflag1AAO vector (
17) using oligonucleotide sense AAO/N-ter primer (5′-GCCGATTTTGACTACGTTGTCGTCG-3′) and oligonucleotide antisense AAO/C-ter/pJRo-overhang primer (5′-
CATAACTAATTACATGATGCGGCCCTCTAGATGCATGCTCGAGCGGCCGCCTACTGATCAGCCTTGATAAGATCGGCT-3′; the overhang for pJRoC30 is underlined). The α-factor prepro-leader (89 residues, including the STE13 cleavage site EAEA) was obtained from pJRoC30-αVP (
20) using oligonucleotide sense RMLN primer (5′-CCTCTATACTTTAACGTCAAGG-3′) and oligonucleotide antisense αC-ter/AAO-overhang primer (5′-CCGCGTTCCCCGCCCCGACGACAACGTAGTCAAAATCGGC
AGCTTCAGCCTCTCTTTTCTC-3′; the overhang for AAO is underlined). The preα fragment, used to create preαproK-AAO, was amplified from pJRoC30-αVP with oligonucleotide sense RMLN and oligonucleotide antisense preαC-ter/proK-overhang (5′-
AGTCGTTAGCTGGGAGTATACTAATACCATGTTCATTTAAGTTGACTGGAGCAGCTAATG-3′; the overhang for the proK is underlined) primers; this fragment was designed to include the 19 residues of the α-pre-leader plus the first four residues—APVN—of the α-pro-leader. The proα fragment (66 residues and the STE13 cleavage site) used to create preKproα-AAO was obtained from pJRoC30-αVP with the oligonucleotide sense proα/N-ter primer (5′-GCTCCAGTCAACACTACAAC-3′) and the oligonucleotide antisense αC-ter/AAO-overhang. Prepro(δ)-leader from the K
1 killer toxin, used as the preK-leader (47 residues long, including the C-terminal EAP acid environment) with different overhangs (for subsequent assembly in yeast to give rise to different chimeras), was obtained from two independent PCRs from plasmid pRE1219, which contains the prepro(δ) region (1 to 44 residues) and the alpha-toxin subunit (45 to 149 residues): (i) to be fused in preproK-AAO with the oligonucleotide sense primer preKN-ter/pJRo-overhang (5′-
TATACTTTAACGTCAAGGAGAAAAAACTATAGGATCATAGGATCCATGACGAAGCCAACCCAAGTATTA-3′; the overhang for pJRoC30 is underlined) and the oligonucleotide antisense primer preKC-ter/proK-overhang (5′-
AGTCGTTAGCTGGGAGTATACTAATACCATGTTCATTTAACGGCGCTTCACGTTTTAGTAATGACACTGGT-3′; the overhang for proK is underlined) and (ii) with preKN-ter/pJRo-overhang as the oligonucleotide sense primer and preKC-ter/proα-overhang (5′-
TTTGTGCCGTTTCATCTTCTGTTGTAGTGTTGACTGGAGCCGGCGCTTCACGTTTTAGTAATGACACTGGT-3′; the overhang for proα is underlined) as the antisense primer to be part of the preKproα-AAO. The truncated γ-spacer-segment from the K
1 killer toxin (64 residues), the proK segment in both preproK-AAO and preαproK-AAO, was obtained from pJRoC30-δγN2C1 with the oligonucleotide sense proKN-ter primer (5′-TTAAATGAACATGGTATTAGTATACTCCCA-3′) and the antisense primer proKC-ter/AAO-overhang (5′-
CCGCGTTCCCCGCCCCGACGACAACGTAGTCAAAATCGGCACGCTTGGCCACTGCTGGAAT-3′; the overhang for AAO is underlined).
pJRoC30 was linearized with BamHI and XhoI. PCRs were performed in a final volume of 50 μl containing a 250 nM concentration of each primer, 10 ng of template, deoxynucleoside triphosphates (dNTPs) at 200 μM each, 3% (vol/vol) dimethyl sulfoxide (DMSO), and 0.02 U of iProof high-fidelity polymerase/liter. The amplification reactions were carried out in a thermal cycler Mycycler (Bio-Rad). The PCR cycles were as follows: 98°C for 30 s (1 cycle); 98°C for 10 s, 50°C for 25 s, and 72°C for 60 s (28 cycles); and 72°C for 10 min (1 cycle). PCR fragments and the linearized vector were loaded onto a preparative agarose gel (0.75% [wt/vol]) and purified using a Zymoclean gel DNA recovery kit. PCR products (400 ng of each) were mixed with the linearized vector (100 ng; PCR product/vector ratio of 4:1) and transformed in yeast (yeast transformation kit), promoting the recombination and cloning in vivo. Transformed cells were plated in SC (synthetic complete) dropout plates, followed by incubation for 3 days at 30°C; individual clones were fermented in 96-well plates and screened for AAO activity. For each positive construct, the plasmids were extracted and sequenced. Fusions were retransformed into yeast and fermented in 100-ml flasks while monitoring cell growth and activity (using HRP-ABTS and FOX [ferrous oxidation by xylenol orange] assays [see below]) over time.
Focused-directed AAO evolution.
All of the PCR products were cleaned, concentrated, loaded onto a low melting-point preparative agarose gel (0.75% [wt/vol]) and purified using a Zymoclean gel DNA recovery kit before being cloned into pJRoC30. The plasmid was linearized with BamHI and XhoI. pJRoC30-preαproK-AAO variant was used as DNA template for focused random mutagenesis. The preαproK-AAO fusion was split into three different segments for MORPHING (
Mutagenic
Organized
Recombination
Process by
Homologous
IN vivo Grouping) (
26). Amplified by PCR, each fragment included homologous overlapping overhangs of ∼50 bp so that the whole gene could be reassembled
in vivo by transformation into
S. cerevisiae. Mutagenic regions M-I and M-II (590 and 528 bp, respectively, excluding the recombination areas) were subjected to
Taq/MnCl
2 amplification, and the remaining segment (844 bp) was amplified by high-fidelity PCR. To adjust the mutagenic loads, small mutant libraries (around 500 clones each) were created with equal concentrations of DNA template and different MnCl
2 concentrations (0.025, 0.05, and 0.01 mM combining both segments and with 0.05 mM in segment M-I and 0.025 mM in segment M-II). The percentage of inactive clones (with <10% of the parent activity) was calculated to estimate mutational loads. Four mutant libraries were created. Two mutagenic libraries (∼1,000 clones each) were prepared targeting segment M-I or segment M-II independently for random mutagenesis. The third library (∼1,000 clones, library M-I-II) was constructed by assembling mutagenic segments (M-I and M-II) flanking a nonmutagenic amplification in the middle of the gene. Finally, the whole preαproK-AAO fusion was subjected to
Taq/MnCl
2 amplification (library M-IV), adjusting the mutational rate to 1 to 3 mutations per gene (∼2,000 clones). Concentrations of 0.05 and 0.01 mM MnCl
2 were used for MORPHING and full gene random mutagenesis, respectively.
(i) Mutagenic PCR of targeted segments.
Reaction mixtures were prepared in a final volume of 50 μl containing DNA template (0.92 ng/μl), 90 nM oligonucleotide sense primer (RMLN for segment M-I and AAOMBP [5′-AACTCTGCTCATTGGGAGACCATCT-3′] for segment M-II), 90 nM reverse primer (AAO92C [5′-CCCAGTTCCATCCTTCATCGCCA-3′] for segment M-I and RMLC [5′-GGGAGGGCGTGAATGTAAGC-3′] for segment M-II), 0.3 mM dNTPs (0.075 mM each), 3% (vol/vol) DMSO, 1.5 mM MgCl2, increasing concentrations of MnCl2 (0.025, 0.05, and 0.1 mM), and 0.05 U of Taq DNA polymerase/μl. Mutagenic PCRs parameters were as follows: 95°C for 2 min (1 cycle); 95°C for 45 s, 50°C for 45 s, and 74°C for 45 s (28 cycles); and 74°C for 10 min (1 cycle).
(ii) High-fidelity PCR.
Reaction mixtures were prepared in a final volume of 50 μl containing DNA template (0.2 ng/μl), 250 nM oligonucleotide sense HFF (5′-TTCGATCGCTATGCGGCTGTCAC-3′), and 250 nM oligonucleotide antisense HFR (5′-GGGTGGAACCATTGGTTGGAAAAG-3′). High-fidelity PCRs were performed using the following parameters: 98°C for 30 s (1 cycle); 98°C for 10 s, 55°C for 25 s, and 72°C for 45 s (28 cycles); and 72°C for 10 min (1 cycle).
(iii) Whole-gene reassembly.
The whole gene was cloned and recombined in vivo by transformation into S. cerevisiae. PCR products were mixed in equimolar amounts (400 ng) and transformed with linearized plasmid (200 ng) into chemically competent cells. Transformed cells were plated on SC dropout plates and incubated for 3 days at 30°C. Colonies containing the whole autonomously replicating vector were picked and screened for activity.
HTS assay.
Individual clones were picked and cultured in sterile 96-well plates containing 50 μl of minimal medium (SC). In each plate, column 6 was inoculated with the parental type (internal standard) and well H1 with URA3− S. cerevisiae cells (negative control). Plates were sealed to prevent evaporation and incubated at 30°C, 225 rpm, and 80% relative humidity in a humidity shaker (Minitron-INFORS; Biogen, Spain). After 48 h, 160 μl of expression medium was added to each well, followed by culture for an additional 48 h. Finally, 20-μl portions of the supernatants were screened for activity with the FOX and HRP-ABTS assays using veratryl or p-methoxybenzyl alcohol as the substrate as described below. One unit of AAO activity is defined as the amount of enzyme that converts 1 μmol of alcohol to aldehyde with the stoichiometric formation of H2O2 per min under the reaction conditions.
Chemical (direct) FOX assay.
Aliquots of 20 μl of yeast supernatants were transferred with liquid handler robotic station Freedom EVO (Tecan, Männedorf, Switzerland) and incubated with 20 μl of substrate (2 mM
p-methoxybenzyl alcohol or 10 mM veratryl alcohol in 100 mM phosphate buffer [pH 6.0]) for 30 min at room temperature, and then 160 μl of FOX reagent was added with a Multidrop Combi-Reagent dispenser (Thermo Scientific, Waltham, MA) to assess the AAO H
2O
2 production [final concentration of FOX mixture in the well: 100 μM xylenol orange, 250 μM Fe(NH
4)
2(SO
4)
2, and 25 mM H
2SO
4] (
27). Plates were recorded in endpoint mode at 560 nm using a spectrophotometer SpectraMax 384 Plus (Molecular Devices, Sunnyvale, CA); it required ∼20 min of incubation to develop an intense and stable colorimetric response. The relative activities were calculated from the difference between the absorbance value after incubation to that of the initial measurement normalized to the parental type for each plate. To enhance method sensitivity, several additives may be added to the reagent, such as organic cosolvents (DMSO, ethanol, and methanol) or sorbitol (
28). In our case, the response was amplified by adding a final concentration of 100 mM sorbitol, which acts as chain amplifier generating additional ferric ions to increase the response of the method (
29). The assay was validated by determining the coefficient of variance, the linearity of the response and the detection limit. The detection limit was calculated by the blank determination method on a 96-well plate with triplicate standards (0, 0.5, 1, 1.5, 2, 2.5, 3, and 4 μM H
2O
2) and several portions of supernatants from
S. cerevisiae URA3
− lacking plasmid (
30). FOX signal stability was tested with different H
2O
2 concentrations (0, 2, 4, 6, 8, 10, 15, and 18 μM) for 300 min at 24°C.
Enzymatic (indirect) HRP-ABTS assay.
Aliquots of 20 μl of yeast supernatants were added to 180 μl of HRP-ABTS reagent (final concentrations of HRP-ABTS reagent in the well: 1 mM p-methoxybenzyl alcohol or 5 mM veratryl alcohol, 2.5 mM ABTS, 1 μg of HRP/ml in 100 mM phosphate buffer [pH 6.0]) dispensed with a Multidrop Combi-Reagent dispenser. The plates were incubated at room temperature and measured in endpoint or kinetic mode at 418 nm (εABTS˙+ = 36,000 M−1 cm−1).
The dual HTS assay incorporated two consecutive rescreenings to rule out the selection of false positives.
(i) First rescreening.
Aliquots of 5 μl of the best clones of the screening were transferred to new sterile 96-well plates with 50 μl of minimal medium per well. Columns 1 and 12 plus rows A and H were not used to prevent the appearance of false positives. After 24 h of incubation at 30°C and 225 rpm, 5-μl portions were transferred to the adjacent wells, followed by further incubation for 24 h. Finally, 160 μl of expression medium was added, and the plates were incubated for 48 h. Accordingly, each mutant was grown in four independent wells. The parental type was subjected to the same procedure (lane D, wells 7 to 11). Plates were assessed according to the same HTS protocol of the screening described above.
(ii) Second rescreening.
An aliquot from the best clones from the first rescreening was inoculated in 3 ml of YPD medium, followed by incubation at 30°C for 24 h at 225 rpm. The plasmids from these cultures were recovered with a Zymoprep yeast plasmid miniprep kit I. Since the product of the Zymoprep was impure and the DNA extracted was very low concentrated, the shuttle vectors were transformed into supercompetent E. coli XL2-Blue cells and plated onto LB-ampicillin (LB-amp) plates. Single colonies were selected to inoculate 5 ml of LB-amp medium and incubated overnight at 37°C and 225 rpm. The plasmids from the best mutants and the parental type were extracted (NucleoSpin plasmid kit) and transformed into S. cerevisiae. Five colonies for each mutant were picked and rescreened as described above.
AAO production and purification. (i) Production of recombinant AAO variants in S. cerevisiae.
A single colony from the S. cerevisiae clone containing the AAO fusion gene was picked from a SC dropout plate, inoculated in SC medium (20 ml) and incubated for 48 h at 30°C and 220 rpm (Minitron-INFORS, Biogen Spain). An aliquot of cells was removed and used to inoculate minimal medium (100 ml) in a 500-ml flask (optical density at 600 nm [OD600] = 0.25). The cells completed two growth phases (6 to 8 h; OD600 = 1), and then expression medium (900 ml) was inoculated with the preculture (100 ml; OD600 of 0.1). After incubation for 72 h at 25°C and 220 rpm (maximal AAO activity; OD600 = 25 to 30), the cells were recovered by centrifugation at 4,500 rpm and 4°C (Avanti J-E centrifuge; Beckman Coulter, Inc., Brea, CA), and the supernatant was double filtered (using both a glass membrane filter and a nitrocellulose membrane [0.45-μm pore size]).
(ii) Purification of AAO mutant.
AAO (FX7 variant) was purified by FPLC (ÄKTA purifier; GE Healthcare, United Kingdom). The crude extract was concentrated and dialyzed in 20 mM piperazine buffer (buffer P [pH 5.5]) by tangential ultrafiltration (Pellicon; Millipore, Temecula, CA) through a 10-kDa-pore-size membrane (Millipore) by means of a peristaltic pump (Masterflex Easy Load; Cole-Parmer, Vernon Hills, IL). The sample was filtered and loaded onto a weak anion-exchange column (HiTrap Q FF; GE Healthcare) preequilibrated with buffer P and coupled to the ÄKTA purifier system. The proteins were eluted with a linear gradient of buffer P + 1 M NaCl in two phases at a flow rate of 1 ml/min: from 0 to 50% in 15 min and from 50 to 100% in 2 min. Fractions with AAO activity were pooled, dialyzed against buffer P, concentrated, and loaded onto a high-resolution resin, strong-anion-exchange column (Biosuite MonoQ 10 cm; Waters, Milford, MA) preequilibrated in buffer P. The proteins were eluted with a linear gradient from 0 to 0.5 M NaCl in two phases at a flow rate of 1 ml/min: from 0 to 50% in 20 min and from 50 to 100% in 2 min. Fractions with AAO activity were pooled, dialyzed against buffer 20 mM phosphate buffer (pH 6.0), concentrated, and further purified by high-pressure liquid chromatography with a Superose 12 HR 10/30 molecular exclusion column (Amersham Bioscience) preequilibrated with 150 mM NaCl in phosphate buffer (pH 6.0) at a flow rate of 0.5 ml/min. The fractions with AAO activity were pooled, dialyzed against buffer (20 mM phosphate buffer [pH 6.0]), concentrated, and stored at −20°C. Throughout the purification protocol the fractions were analyzed by SDS-PAGE on 10% gels in which the proteins were stained with Protoblue Safe (National Diagnostics, USA). All protein concentrations were calculated using Bio-Rad protein assay reagent and bovine serum albumin (BSA) as the standard for the protein concentration.
(iii) Production and purification of native AAO from E. coli.
Native heterologous AAO expressed in
E. coli after
in vitro refolding (
EcAAO) was produced and purified as described elsewhere (
17).
Biochemical characterization. (i) MALDI-TOF-MS analysis and pI determination.
The matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS) experiments were performed on an Autoflex III MALDI-TOF-TOF instrument with a Smartbeam laser (Bruker Daltonics). The spectra were acquired using a laser power just above the ionization threshold, and the samples were analyzed in the positive-ion detection and delayed extraction linear mode. Typically, 1,000 laser shots were summed into a single mass spectrum. External calibration was performed using the BSA from Bruker, covering the range from 15,000 to 70,000 Da. Purified FX7 (8 μg) was subjected to two-dimensional electrophoresis gel in order to determine the pI.
(ii) N-terminal analysis.
Purified AAO was subjected to SDS-PAGE, and the protein band was blotted onto polyvinylidene difluoride (PVDF) membranes. The PVDF membrane was stained with Coomassie brilliant blue R-250, and then the enzyme band was excised and processed for N-terminal amino acid sequencing on a precise sequencer at the core facilities of the Helmholtz Centre for Infection Research, Germany.
(iii) Determination of kinetic thermostability (T50).
Appropriate dilutions of purified FX7 and EcAAO were prepared for the assay, while the samples of parental preαproK-AAO were obtained from the crude supernatants. A temperature gradient scale ranging from 30 to 80°C was established as follows: 30.0, 31.4, 34.8, 39.3, 45.3, 49.9, 53, 55, 56.8, 59.9, 64.3, 70.3, 75, 78.1, and 80°C. This gradient profile was achieved using a thermocycler (Mycycler). After 10 min of incubation, FX7 and EcAAO samples were removed and chilled on ice for 10 min, followed by further incubation at room temperature for 5 min. Finally, 20-μl samples were added to 180-μl volumes of 100 mM sodium phosphate buffer (pH 6.0) containing 1 mM p-methoxybenzyl alcohol, and the activity was measured as anisaldehyde production by determining the absorption at 285 nm (ε285 = 16,950 M−1 cm−1). In the case of parental preαproK-AAO supernatants, the samples were subjected to an HRP-ABTS assay described above for the screening. Thermostability values were calculated from the ratio between the residual activities incubated at different temperature points and the initial activity at room temperature. The T50 value was determined by the transition midpoint of the inactivation curve of the protein as a function of temperature, which in our case was defined as the temperature at which the enzyme lost 50% of its activity after an incubation of 10 min. All reactions were performed by triplicate.
(iv) Thermoactivity (Ta).
Enzyme dilutions of purified FX7 (33 nM, final concentration) and EcAAO (18 nM, final concentration) were prepared in such a way that aliquots of 20 μl gave rise to a linear response in kinetic mode. The optimum temperature for activity was estimated in prewarmed 96-well reading plates (Labnet VorTemp 56 Shaking Incubator; Labnet International, USA) with 100 mM sodium phosphate (pH 6.0) containing 1 mM p-methoxybenzyl alcohol at various corresponding temperatures (25, 30, 40, 50, 60, 70, 80, 90, and 99°C), followed by incubation in an Eppendorf Thermomixer Comfort apparatus (Thermo Fisher Scientific). Reactions were performed by triplicate and p-methoxybenzyl alcohol oxidation, followed by aldehyde production at 285 nm.
(v) Kinetic parameters.
Kinetic constants for AAO were estimated in 100 mM sodium phosphate (pH 6.0). The final enzyme concentrations used were as follows: with p-methoxybenzyl alcohol, 33 and 18 nM for FX7 and EcAAO, respectively; with veratryl alcohol, 38 and 32 nM for FX7 and EcAAO, respectively; with benzyl alcohol, 62 and 47 nM for FX7 and EcAAO, respectively; and with 2,4-hexadien-1-ol, 15 and 4 nM for FX7 and EcAAO, respectively. Reactions were performed in triplicate, and substrate oxidations were monitored by measuring the absorption at 285 nm for p-methoxybenzyl alcohol (ε285 = 16,950 M−1 cm−1), 310 nm for veratryl alcohol (ε310 = 9,300 M−1 cm−1), 250 nm for benzyl alcohol (ε250 = 13,800 M−1 cm−1), and 280 nm for 2,4-hexadien-1-ol (ε280 = 30,140 M−1 cm−1). Steady-state kinetic parameters were determined by fitting the initial reactions rates at different substrate concentrations to the Michaelis-Menten equation for one substrate, v/e = kcat·S/(Km + S), where e represents the enzyme concentration, kcat is the maximal turnover rate, S is the substrate concentration, and Km is the Michaelis constant. The data were fit using SigmaPlot 10.0 (Systat Software, Inc., Richmond, CA).
(vi) pH activity and stability profiles.
Appropriate dilutions of enzyme samples were prepared in such a way that aliquots of 20 μl yielded a linear response in kinetic mode. The optimum pH activity was determined using 100 mM citrate-phosphate-borate buffer at different pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) containing the corresponding alcohol concentration (0.3, 5, 9, and 1.2 mM for p-methoxybenzyl alcohol, veratryl alcohol, benzyl alcohol, and 2,4-hexadien-1-ol, respectively). To measure pH stability, enzyme samples were incubated at different times over a range of pH values. The residual activity was deduced from the activity before and after incubation with 0.3 mM p-methoxybenzyl alcohol in 100 mM phosphate buffer (pH 6.0).
(vii) Protein modeling.
The crystal structure of the AAO from
P. eryngii at a resolution of 2.55 Å (Protein Data Bank Europe [PDB] accession number 3FIM [
31]) was used for the FX7 mutant homology model, obtained by PyMol (Schrodinger, LLC [
http://www.pymol.org]).
(viii) DNA sequencing.
All genes were verified by DNA sequencing (using a BigDye Terminator v3.1 cycle sequencing kit). The primers used were common to the four constructions: sense primers RMLN and AAOsec1F (5′-GTGGATCAACAGAAGATTTCGATCG-3′) and antisense primers RMLC (5′-GCTTACATTCACGCCCTCCC-3′), AAOsec2R (5′-GTGGTTAGCAATGAGCGCGG-3′), and AAOsec3R (5′-GGAGTCGAGCCTCTGCCCCT-3′).