Purification and Characterization of an NADH-Dependent Alcohol
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Biosci. Biotechnol. Biochem., 75 (6), 1055–1060, 2011 Purification and Characterization of an NADH-Dependent Alcohol 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 ketones and aldehydes. AFPDH Racemic alcohols 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 ketone 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.