title: Characterization of a (2R,3R)-2,3-Butanediol Dehydrogenase from Rhodococcus erythropolis WZ010.
abstract: The gene encoding a (2R,3R)-2,3-butanediol dehydrogenase from Rhodococcus erythropolis WZ010 (ReBDH) was over-expressed in Escherichia coli and the resulting recombinant ReBDH was successfully purified by Ni-affinity chromatography. The purified ReBDH in the native form was found to exist as a monomer with a calculated subunit size of 37180, belonging to the family of the zinc-containing alcohol dehydrogenases. The enzyme was NAD(H)-specific and its optimal activity for acetoin reduction was observed at pH 6.5 and 55 °C. The optimal pH and temperature for 2,3-butanediol oxidation were pH 10 and 45 °C, respectively. The enzyme activity was inhibited by ethylenediaminetetraacetic acid (EDTA) or metal ions Al3+, Zn2+, Fe2+, Cu2+ and Ag+, while the addition of 10% (v/v) dimethyl sulfoxide (DMSO) in the reaction mixture increased the activity by 161.2%. Kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2R,3R)-2,3-butanediol and NAD+. The activity of acetoin reduction was 7.7 times higher than that of (2R,3R)-2,3-butanediol oxidation when ReBDH was assayed at pH 7.0, suggesting that ReBDH-catalyzed reaction in vivo might favor (2R,3R)-2,3-butanediol formation rather than (2R,3R)-2,3-butanediol oxidation. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2R,3R)-2,3-butanediol via (R)-acetoin, demonstrating its potential application on the synthesis of (R)-chiral alcohols.
authors.affiliation: Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University, Hangzhou 310018, China. meilanyu@zstu.edu.cn.
authors.forename: Meilan
authors.initials: M
authors.lastname: Yu
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, China. HuangMJroline@163.com.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Huang
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, China. Songqqbeauty@163.com.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Song
authors.affiliation: Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University, Hangzhou 310018, China. jshao@zstu.edu.cn.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Shao
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, China. yingxx@zjut.edu.cn.
authors.forename: Xiangxian
authors.initials: X
authors.lastname: Ying
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002072:Butylene Glycols / Q000378:metabolism*; D003001:Cloning, Molecular; D003931:Diacetyl / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002072:Butylene Glycols; D009243:NAD; C026978:2,3-butylene glycol; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase; D003931:Diacetyl