title: Characterization of a stereospecific acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 and its application for the synthesis of (2S,3S)-2,3-butanediol.
abstract: Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K(m) values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, Zhejiang, 310014, China.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Wang
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Song
authors.forename: Meilan
authors.initials: M
authors.lastname: Yu
authors.forename: Yifang
authors.initials: Y
authors.lastname: Wang
authors.forename: Bin
authors.initials: B
authors.lastname: Xiong
authors.forename: Yinjun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jianyong
authors.initials: J
authors.lastname: Zheng
authors.forename: Xiangxian
authors.initials: X
authors.lastname: Ying
mesh_terms: D000094:Acetoin Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002072:Butylene Glycols / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002072:Butylene Glycols; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C026978:2,3-butylene glycol; D000094:Acetoin Dehydrogenase