Biosci. Biotechnol. Biochem., 75 (6), 1055–1060, 2011

Purification and Characterization of an NADH-Dependent Dehydrogenase from Candida maris for the Synthesis of Optically Active 1-(Pyridyl)ethanol Derivatives

y Shigeru KAWANO, Miho YANO, Junzo HASEGAWA, and Yoshihiko YASOHARA

Frontier Biochemical and Medicinal Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho, Takasago, Hyogo 676-8688, Japan

Received July 23, 2010; Accepted March 28, 2011; Online Publication, June 13, 2011 [doi:10.1271/bbb.100528]

A novel (R)-specific alcohol dehydrogenase (AFPDH) tor.6) We found that Candida maris IFO10003 has a (R)- produced by Candida maris IFO10003 was purified to specific reducing enzyme that reduces acetylpyridine homogeneity by ammonium sulfate fractionation, derivatives to (R)-1-(pyridyl)ethanol derivatives with DEAE-Toyopearl, and Phenyl-Toyopearl, and charac- more than 99% e.e.7) Here we describe the purification terized. The relative molecular mass of the native and characterization of that (R)-specific enzyme. enzyme was found to be 59,900 by gel filtration, and that of the subunit was estimated to be 28,900 on SDS- Materials and Methods polyacrylamide gel electrophoresis. These results sug- gest that the enzyme is a homodimer. It required NADH Chemicals. 5-Acetylfuro[2,3-c]pyridine (AFP) and 5-acetyl-7- as a cofactor and reduced various kinds of carbonyl chlorofuro[2,3-c]pyridine were prepared following previous reports.8,9) compounds, including and aldehydes. AFPDH Racemic were prepared by NaBH4 reduction. Glucose dehydrogenase was purchased from Amano Enzyme (Aichi, Japan). reduced acetylpyridine derivatives, -keto esters, and All the other chemicals used in this study were of analytical grade and some compounds with high enantioselectivity. were commercially available. This is the first report of an NADH-dependent, highly enantioselective (R)-specific alcohol dehydrogenase iso- Enzyme assays and protein determination. The reductive activity of lated from a yeast. AFPDH is a very useful enzyme for the enzyme was assayed spectrophotometrically at 30 C by the the preparation of various kinds of chiral alcohols. decrease in absorbance of NADH at 340 nm. The reaction mixture contained 100 mM potassium phosphate buffer (pH 6.5), 0.3 mM Key words: purification; alcohol dehydrogenase; Candida NADH, and 1 mM AFP as substrate, 0.3% dimethyl sulfoxide (DMSO), and the enzyme solution in a total volume of 3 mL. One unit of the maris; 1-(pyridyl)ethanol derivative enzyme was defined as the amount catalyzing the oxidation of 1 mmol of coenzyme per min. The oxidative reaction of the enzyme was also In the pharmaceutical industry, the optical purities of measured at 340 nm in 3 mL of reaction mixture containing 100 mM drugs are a very important factor and an essential potassium phosphate buffer (pH 8.0), 0.3 mM NADþ, and 1 mM AFP as problem.1) The application of chiral raw materials such substrate and 0.3% DMSO. One unit of the enzyme was defined as the as optically active alcohols in the syntheses of optically amount catalyzing the reduction of 1 mmol of coenzyme per min. active pharmaceuticals and agrochemicals facilitates the Protein was measured by the protein-dye binding method using bovine serum albumin as standard.10) production processes for these compounds. Many procedures have been developed for the preparation of Microorganisms and cultivation. Candida maris IFO10003 was 2) optically active alcohol compounds. One of the most obtained as Candida maris NBRC10003 from the NITE Biological convenient of these methods is enzyme-assisted asym- Resource Center, Japan (Chiba, Japan). The medium was composed of 3) . metric reduction of carbonyl compounds. In many 0.7% KH. 2PO4, 1.3% (NH4)2.HPO4, 0.08% MgSO4 .7H2O, 0.007% cases, enzymatic reduction of carbonyl compounds ZnSO4 .7H2O, 0.009% FeSO4 7H2O, 0.0005% CuSO4 5H2O, 0.001% under appropriate conditions affords chiral alcohol at MnSO4 4H2O, 0.01% NaCl, 0.3% yeast extract, and 7% glucose, pH 7.0. C. maris IFO10003 was inoculated into a 500-mL shaking high optical purity. flask containing 100 mL of the medium, and the mixture was then Oxidoreductases such as alcohol dehydrogenases incubated for 24 h at 30 C with shaking. The cultures were then (ADH) and carbonyl reductases are available for chiral transferred to a 5-L fermenter (Marubishi, Tokyo, Japan) containing alcohol production from the corresponding carbonyl 3.5 L of the same medium. The cultures were thermostatted at 30 C, compounds. Many alcohol dehydrogenases reduce and the pH was set at 5.5 by automatic titration with aqueous sodium ketones to (S)-alcohols under the Prelog’s rule process.4) hydroxide. Constant stirring was maintained at 300 rpm, and the air- On the other hand, only a few enzymes follow the anti- flow rate was 1.05 L/min during cultivation over 6 d. Prelog’s rule.5) Purification of the AFP-reducing enzyme. All purification proce- (R)-5-(1-Hydroxyethyl)-furo[2,3-c]pyridine (FPH) dures were performed at 4 C. C. maris cells were collected by (Fig. 1) is an important intermediate in the synthesis centrifugation from 10 L of the cultured broth, and were washed with of a non-nucleoside HIV reverse-transcriptase inhibi- 5 L of physiological saline. The cells were suspended in 2 L of 100 mM

y To whom correspondence should be addressed. Fax: +81-79-445-2668; E-mail: [email protected] Abbreviations: e.e., enantiomeric excess; AFP, 5-acetylfuro[2,3-c]pyridine; FPH, 5-(1-hydroxyethyl)-furo[2,3-c]pyridine; ADH, alcohol dehydrogenase; SDS–PAGE, SDS-polyacrylamide gel electrophoresis 1056 S. KAWANO et al. Table 1. Purification of AFPDH from Candida maris IFO10003a O O Total Total Specific Purifi- Yield N N Step protein activity activity cation (%) O OH (mg) (units) (units/mg) (fold) AFP (R)-FPH Cell-free extract 11,700 8,630 0.738 100 1 Ammonium sulfate 11,970 8,800 0.735 100 1 Fig. 1. Enantioselective Reduction of a 2-Acetylpyridine Derivative. DEAE-Toyopearl 274 2,720 9.950 32 13 Phenyl-Toyopearl 48 1,333 27.600 15 37

aThe assay conditions are given in ‘‘Materials and Methods.’’ Tris–HCl buffer (pH 7.5) containing 5 mM -mercaptoethanol, and disrupted with 0.25-mm glass beads for 1.5 h. After centrifugation, 3 g of protamine sulfate was added and the mixture was stirred for 1 h. The A B precipitate was discarded after centrifugation. Maintaining the pH of the crude enzyme solution at 7.5 with aqueous ammonia, solid KDa ammonium sulfate was added to attain 35% saturation, and the resulting precipitate was removed by centrifugation. More solid ammonium sulfate was added to the supernatant to attain 55% saturation, and the resulting precipitate was collected by centrifuga- 94 tion. The precipitate was dissolved in 200 mL of 20 mM Tris–HCl 67 buffer (pH 7.5) containing 5 mM -mercaptoethanol, and the solution was then dialyzed overnight with the same buffer. The dialyzed solution was applied to a DEAE-Toyopearl 650M column (Tosoh, 43 Tokyo, Japan) and equilibrated with 20 mM Tris–HCl buffer (pH 7.5) containing 5 mM -mercaptoethanol. The enzyme was eluted with a sodium chloride linear gradient solution (0–0.3 M) in the same buffer. 30 The active fractions were collected, and solid ammonium sulfate was dissolved in them to a final concentration of 0.5 M. The solution was applied to a Phenyl-Toyopearl 650M column (Tosoh) equilibrated with 20.1 20 mM Tris–HCl buffer (pH 7.5) containing 5 mM -mercaptoethanol and 0.5 M ammonium sulfate. The enzyme was eluted with an ammonium sulfate linear gradient solution (0.5–0 M). The active Fig. 2. SDS–PAGE of the AFPDH from Candida maris IFO10003. fractions were collected and dialyzed against 20 mM Tris–HCl buffer Lane A, purified enzyme; lane B, molecular mass marker pro- (pH 7.5) containing 5 mM -mercaptoethanol. The enzyme preparation teins: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), purified in this way showed a single spot on SDS-polyacrylamide gel ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor electrophoresis (SDS–PAGE) (Fig. 2). (20.1 kDa). The gel was stained with Coomassie Brilliant Blue.

Determination of enzyme molecular mass. The molecular mass of Results the native enzyme was estimated by column chromatography using a TSKgel G3000 SWXL column (Tosoh) and a standard molecular Purification of the AFP-reducing enzyme marker with 100 mM potassium phosphate buffer (pH 7.0) containing The purification of the AFP-reducing enzyme 100 mM sodium sulfate. The molecular mass of the subunit was (AFPDH) is summarized in Table 1. AFPDH was estimated by SDS–PAGE (10%) with an SDS–PAGE marker as standard. purified 37-fold to homogeneity from the cell-free extract by two column chromatography without using other Determination of substrate specificity. The substrate specificity of affinity column chromatography such as Blue-Sepharose. the enzyme was determined spectrophotometrically by measuring the The purified AFPDH showed that 27.6 U/mg of protein decrease in absorbance of NADH at 340 nm. The reaction conditions was obtained from the cells, with an overall recovery of were the same as for the enzyme assay system, except for changes in 15%. The purified enzyme gave a single band on SDS- the substrate and enzyme concentration. polyacrylamide gel electrophoresis (Fig. 2). The relative molecular mass of AFPDH was found to be 59,900 by gel Reduction of various carbonyl compounds. The various reaction mixtures (0.5 mL each), comprising 0.5 units of AFPDH, 5 mg of filtration. The relative molecular mass of the subunit was substrate, 20 mg of glucose, 0.5 mg of NADþ, and 4 units of glucose estimated to be approximately 28,900 on SDS-polyacryl- dehydrogenase in 0.5 mL of 0.1 M potassium phosphate buffer (pH 6.5) amide gel electrophoresis (Fig. 2). were stirred at 30 C for 17 h. Ethyl acetate (5 mL) was added, and the reaction mixture was centrifuged. The organic layer was used to N-Terminal amino acid sequence analysis determine the optical purity of the product. Automated Edman degradation of the purified AFPDH protein with a pulsed liquid phase sequencer Analysis. The optical purities of the FPH and 1-(pyridyl)ethanol was unsuccessful, suggesting that the N-terminal of the derivatives were determined by HPLC, as described previously.7) The optical purities of 1-phenylethanol and 20,20,20-trifluoro-1-phenyetha- enzyme is blocked. nol were measured with an HPLC-equipped Chiralcel OD-H (4:6mm 250 mm) column (Daicel Chemicals, Osaka, Japan). The Effects of pH and temperature HPLC conditions included n-hexane/2-propanol ¼ 92.5/7.5 (v/v). The effects of pH on reductive activity were measured The optical purities of ethyl 4-chloro-3-hydroxybutyrate and 2-chloro- in 0.1 M sodium acetate buffer (pH 3.5–5.5), 0.1 M (m-chlorophenyl)ethanol were determined by HPLC, as described 11) potassium phosphate buffer (pH 5.0–8.0), and 0.1 M previously. The optical purity of methyl 3-hydroxybutanoate was Tris–HCl buffer (pH 7.0–9.0). The enzyme showed determined by analysis of the optical purity of the phenylurethane derivative with an HPLC-equipped Chiralcel AS-H (4:6mm maximum reductive activity at pH 5.5–6.0 (Fig. 3). 250 mm) column (Daicel Chemicals). The HPLC conditions included The effects of pH on oxidative activity were measured in n-hexane/2-propanol ¼ 90/10 (v/v). potassium phosphate buffer (pH 5.0–8.0) and Tris–HCl Alcohol Dehydrogenase of Candida maris 1057

Fig. 3. Effects of pH on the Activity of AFPDH in the Reduction of Fig. 5. Effects of Temperature on the Stability of AFPDH. AFP and Oxidation of (R)-FPH. The assay conditions are given in ‘‘Materials and Methods.’’ Activity was measured in the following 100 mM buffers: CH3COONa-CH3COOH (pH 3.5 to 5.5) ( ), potassium phosphate Table 2. Effects of Various Chemicals on Enzyme Activity buffer (pH 4.5 to 8.0) ( ), Tris–HCl (pH 7.0 to 9.0) ( ) in the reductive reaction, potassium phosphate buffer (pH 5.0 to 8.0) ( ), Relative Concentration Tris–HCl (pH 7.0 to 9.0) ( ) in the oxidative reaction. Compound activitya (mM) (%) None 100 Metal ions MgSO4 1.00 81 MnCl2 1.00 76 ZnSO4 1.00 74 CuSO4 1.00 103 CoCl2 1.00 85 HgCl2 1.00 0 Chelating agents EDTA 2Na 1.00 98 1,10-Phenanthroline 1.00 98 Reducing agents 2-Mercaptoethanol 1.00 94 Dithiothreitol 1.00 93 Sulfhydryl reagents N-Ethylmaleimide 1.00 83 Iodoacetate 1.00 80 p-Chloromercuribenzoic acid 0.10 100 5,50-Dithiobis-2-nitrobenzoic acid 0.01 98 Others Quercetin 0.01 78 Fig. 4. Effects of Temperature on the Activity of AFPDH in the Reduction of AFP. aEnzyme activity was measured spectrophotometrically in the presence of The assay conditions are given in ‘‘Materials and Methods.’’ the test substance (1 mM) under standard conditions. buffer (pH 7.0–9.0). The enzyme showed maximum compound individually to the reaction mixture. Among oxidative activity at pH 8.5 (Fig. 3). The pH stability of the metal ions tested, mercury ions strongly inhibited the AFPDH was also measured after incubation at 30 C for reaction, while magnesium ions, manganese ions, zinc 0.5 h in one of the buffers described above. It was stable ions, and cobalt ions caused slight inhibition. Chelating in a pH range from 7.0 to 9.0. agents, reducing agents, sulfhydryl reagents such as The reductive activity of AFPDH was measured at p-chloromercuribenzoic acid, and 5,50-dithiobis-2-nitro- various temperatures, and the optimum temperature was had no influence on enzyme activity. The observed at 50 C (Fig. 4). The thermal stability of use of sulfhydryl reagents, including N-ethylmaleimide, AFPDH was also examined by pre-incubation at various iodoacetate, and quercetine, a nonspecific inhibitor of temperatures for 0.5 h in 0.1 M potassium phosphate human brain carbonyl reductase,12) also caused slight buffer (pH 7.8), after which its residual activity was inhibition of the reaction. measured. It was stable at lower than 25 C and retained 80% activity (Fig. 5). Substrate specificity NADH was absolutely required as a cofactor. When it Effects of chemicals was replaced with NADPH, no decrease in absorbance The effects of various compounds, listed in Table 2, at 340 nm due to reduction of AFP was observed. on enzyme activity were examined by adding each Kinetic parameters were measured by Lineweaver-Burk 1058 S. KAWANO et al. Table 3. Substrate Specificities of AFPDH from Candida maris IFO10003 for Ketonesa

Relative Relative Substrateb activityc Substrateb activityc (%) (%) 5-Acetylfuro[2,3-c]pyridine (AFP) 100 2-Acetylcyclopentanone 86 1-Chrolo-5-acetylfuro[2,3-c]pyridine 66 2-Acetylcyclohexanone 68 2-Acetylpyridine 111 Methyl pyruvate 132 3-Acetylpyridine 107 Ethyl pyruvate 133 4-Acetylpyridine 130 Methyl acetoacetate 128 Acetylpyrazine 133 Ethyl acetoacetate 141 2-Acetylpyrrole 100 Ethyl 4-chloroacetoacetate 88 2-Acetylthiophene 77 Pyruvic acid 3 2-Acetylfuran 43 2-Ketobutyric acid 5 2-Acetylthiazole 86 2-Ketopentanoic acid 7 Acetophenone 88 Oxalacetic acid 4 m-Nitroacetophenone 136 Cyclopentanone 3 p-Nitroacetophenone 116 Cyclohexanone 4 o-Chloroacetophenone 9 Cycloheptanone 4 m-Chloroacetophenone 120 1-Tetralone 2 p-Chloroacetophenone 88 2-Tetralone 0 p-Fluoroacetophenone 88 Pyridine-2-aldehyde 54 2-Chloroacetophenone 54 Pyridine-3-aldehyde 47 2,30-Dichloroacetophenone 19 Pyridine-4-aldehyde 64 Benzylacetone 96 Benzaldehyde 94 40 o-Nitrobenzaldehyde 9 2-Butanone 94 m-Nitrobenzaldehyde 103 2-Pentanone 67 p-Nitrobenzaldehyde 162 2-Hexanone 38 o-Chlorobenzaldehyde 0 2-Octanone 38 m-Chlorobenzaldehyde 111 Methyl iso-propyl ketone 43 p-Chlorobenzaldehyde 104 Methyl iso-butyl ketone 14 Acetaldehyde 36 Acetoin 43 Propionaldehyde 70 Diacetyl 107 1-Butylaldehyde 123 Acetylacetone 123 1-Hexylaldehyde 71 Diethylketone 22 Dipropylketone 2 Chloroacetone 99

aEnzyme activity was measured as described in ‘‘Materials and Methods.’’ bThe substrate concentration was 1 mM. cTo calculate relative activity, the activity for AFP was taken to be 100%.

plots. For AFP, the Km and Vmax values were 0.53 mM industry were also examined. Table 5 shows the and 45.9 mmol/min/mg respectively. As shown in enantioselectivity of the enzymes. Table 3, a broad range of carbonyl compounds was used to investigate substrate specificities. The enzyme Discussion reduced various aromatic and aliphatic ketone and aldehyde compounds. In particular, methylketones such Optically active 1-(pyridyl)ethanol derivatives are as acetylpyridine derivatives, and -or-keto esters useful chemicals, functioning as intermediates for such as pyruvic acid esters and acetoacetic acid esters pharmaceuticals and ligands for catalysts of asymmetric were good substrates for the enzyme. Despite these synthesis.8,13) To synthesize these derivatives, enzymatic results, cyclic ketones such as cyclopentanone and enantioselective reduction of their corresponding car- 2-tetralone, -keto acids such as pyruvic acid, and bonyl compounds, acetylpyridine derivatives, is one of ortho-substituted aromatic compounds such as o-chlor- the most practical and economical strategies. To obtain oacetophenone, o-nitrobenzaldehyde, and o-chloroben- 1-(pyridyl)ethanol derivatives at high optical purity, a zaldehyde, were not good substrates as compared to the reductase showing high enantioselectivity is necessary. chemicals described above. The enzyme also oxidized We found a suitable reductase for this purpose in the secondary alcohols such as 1-(pyridyl)ethanol deriva- yeast Candida maris IFO10003.7) AFPDH was purified tives and 2-propanol in the presence of NADþ (Table 4). to homogeneity on SDS–PAGE from cell-free extract of The oxidative activity for (R)-FPH of AFPDH was Candida maris IFO10003 by sequential column chro- 5.1 U/mg. Primary alcohols were not oxidized well. matography. The relative molecular mass of the native enzyme was found to be 59,900 by gel filtration, and the Stereoselectivity relative molecular mass of the subunit was estimated to The stereospecificities of AFPDH for some acetyl- be 28,900 by SDS–PAGE. These results suggest that it is pyridine derivatives were studied. As shown in Table 5, a homodimer. It showed high reducing activity on a acetylpyridine derivatives were reduced with high wide range of carbonyl compounds, including aromatic enantioselectivity. The stereoselectivities of AFPDH ketones and aldehydes and aliphatic ones. In particular, for several versatile carbonyl compounds on chiral 4-acetylpyridine, acetylpyrazine, methyl pyruvate, ethyl Alcohol Dehydrogenase of Candida maris 1059 Table 4. Substrate Specificities of AFPDH from Candida maris IFO10003 for Alcoholsa

Relative Relative Substrateb activityc Substrateb activityc (%) (%) (R)-5-(1-Hydroxyethyl)-furo[2,3-c]pyridine((R)-FPH) 100 1-Butanol 5 1-(1-Pyridyl)ethanol 23 (R)-2-Butanol 113 1-(2-Pyridyl)ethanol 110 1-Pentanol 1 1-(3-Pyridyl)ethanol 24 4-Phenyl-2-butanol 219 (R)-1-Phenylethanol 225 1,2-Propanediol 2 4 Glycerol 2 Ethanol 6 3-Chloro-1,2-propanediol 1 1-Propanol 8 3-Bromo-1,2-propanediol 0 2-Propanol 226

aEnzyme activity was measured as described in ‘‘Materials and Methods.’’ bThe substrate concentration was 1 mM. cTo calculate relative activity, the activity for AFP was taken to be 100%.

Table 5. Stereoselectivity of AFPDH for Various Carbonyl Compoundsa

Optical purity Optical purity Substrate Product Substrate Product (%e.e.) (%e.e.)

O O > 99 (R) > 99 (R ) N N O OH O OH O O > 99 (R) CF3 CF3 > 99 (S ) Cl N Cl N O OH O OH Cl Cl

N N > 99 (R) > 99 (S ) O OH Cl Cl O OH

O OH N N > 99 (R) Cl COOEt Cl COOEt > 99 (S ) O OH

N N O OH > 99 (R) COOMe COOMe > 99 (R ) O OH

aThe reaction conditions were as described in ‘‘Materials and Methods.’’ pyruvate, and p-nitrobenzaldehyde are good substrates. phenyl-1,2-ethanediol. The requirement of a coenzyme, It showed high oxidative activity on secondary alcohols, the subunit structure, and the oxidative activity toward including 2-propanol, 4-phenyl-2-butanol, and (R)-2- an alcohol of the AFPDH were different from the butanol. On the other hand, oxidative activity for C. parapsilosis enzyme. In comparison with the (R)-1- primary alcohols, such as methanol, ethanol, 1-propanol, phenyl-1,3-propanediol-producing enzyme from Tricho- and 1-butanol, was very low, similarly to NADþ- sporon fermentans, the subunit structure and inhibition dependent secondary alcohol dehydrogenases from by metal ions were similar to AFPDH. On the other bacteria.14,15) Furthermore, AFPDH affords various hand, the requirement for a coenzyme, molecular mass, versatile chiral alcohols at high optical purity from optimum temperature, and pH were different from those the corresponding carbonyl compounds. The purified of the T. fermentans enzyme. Bacterial (R)-specific enzyme reduced not only acetylpyridine derivatives but enzymes were isolated from Leifsonia sp.,20,21) Lacto- also acetophenone derivatives and -keto esters at very bacillus kefir,22,23) Lactobacillus brevis,5) and Pseudo- high stereoselectivity, of more than 99% e.e. Of those monas sp.24,25) These were examined closely. These chiral alcohols, (S)-ethyl 4-chloro-3-hydroxybutyrate were tetrameric enzymes with molecular mass of and (S)-2-chloro-1-(30-chlorophenyl)ethanol are also approximately 105–110 kDa, while AFPDH was a useful building blocks for the synthesis of optically dimeric enzyme with a molecular mass of 59.9 kDa. active pharmaceuticals.16,17) The substrate specificity of AFPDH appeared to be Many secondary alcohol dehydrogenases were found, similar to those of Leifsonia sp. ADH and Lactobacillus and the stereoselectivities of the enzymes were closely kefir ADH, but differed from that of Pseudomonas sp. examined, but the majority of the enzymes were found ADH. In terms of coenzyme dependency and the Mg2þ to be (S)-specific alcohol dehydrogenases, the (R)- requirement, AFPDH differed from L. kefir ADH specific ones being uncommon. NADPH-dependent (Table 6). (R)-specific enzymes of yeast have been isolated from Several examples of the effective enzymatic reduction Candida parapsilosis18) and Trichosporon fermentans.19) system coupled with enzymatic regeneration of the The enzyme from C. parapsilosis was a monomer reduced coenzyme through the use of glucose dehydro- enzyme that showed no oxidation activity toward 1- genase,4,26) formate dehydrogenase,27) or phosphite 1060 S. KAWANO et al. Table 6. Comparison of Biochemical Properties of (R)-Specific Alcohol Dehydrogenases

Leifsonia sp. Lactobacillus kefir Lactobacillus brevis Pseudomonas sp. Property AFPDH ADHa ADHb ADHc ADHd Classification Short-chain ADH Short-chain ADH Short-chain ADH Short-chain ADH Coenzyme NADH NADH NADPH NADPH NADH Molecular mass 59.9 kDa, 110 kDa, 105 kDa, 105 kDa, 162 kDa, homodimer homotetramer homotetramer homotetramer homotetramer Metal requirement none none Mg2þ (Mn2þ)Mg2þ (Mn2þ)NDe Temperature optimum 50 C50C37C65CND pH-optimum reduction pH 5.0–5.5 pH 6.0 pH 7.0 pH 7.0 pH 6.5 oxidation pH 8.5 pH 9.5 pH 8.0 ND >pH 10 Substrate specificity (R)-1-(2-Pyridyl)-ethanol >99%e.e. ND >97%e.e. ND ND (R)-1-Phenylethanol >99%e.e. 99%e.e. >99%e.e. ND 94%e.e. (S)-1-Phenyl-2,2,2-trifluoroethanol >99%e.e. >99%e.e. >99%e.e. ND 92%e.e.

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