title: Cloning and characterization of three ketoreductases from soil metagenome for preparing optically active alcohols.
abstract: OBJECTIVE
  To discover novel ketoreductases (KRED) from soil metagenome preparation of chiral alcohols.
  
  
  RESULTS
  Three putative KRED were cloned, heterologously expressed in Eschericha coli and characterized based on the sequence analysis of soil metagenome. All the three enzymes (KRED424, KRED432, and KRED433) had maximum activity at 55 °C and pH 7. KRED424 had a broader substrate spectrum compared with the other two. Three prochiral carbonyl compounds were used to evaluate the abilities of enantioselective reductions of the KRED. For N-Boc-3-pyrrolidone, all enzymes produced an (S)-type alcohol in enantiomeric excess (>99 % ee). For ethyl 2-oxo-4-phenylbutyrate, KRED424 showed a higher conversion (91.5 %) and enantioselectivity (S-type, >99 % ee) than KRED432 and KRED433. For ethyl 4-chloroacetoacetate (COBE), both of KRED424 and KRED433 completely converted 20 mM substrate and KRED433 could obtain an (R)-alcohol with 94 % ee.
  
  
  CONCLUSIONS
  The three ketoreductases have potential in the preparation of pharmaceuticals and fine chemicals.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. hlwang@ecust.edu.cn.
authors.forename: Hualei
authors.identifier: http://orcid.org/0000-0002-0111-753X
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Kai
authors.initials: K
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000438:Alcohols / Q000737:chemistry*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D054892:Metagenome; D012988:Soil Microbiology; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D000429:Alcohol Oxidoreductases
keywords: Asymmetric reduction; Ethyl 2-oxo-4-phenylbutyrate; Ethyl 4-chloroacetoacetate; Ketoreductases; N-Boc-3-pyrrolidone; Soil metagenome