title: Characterization of a newly synthesized carbonyl reductase and construction of a biocatalytic process for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate with high space-time yield.
abstract: A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg(-1). The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent K m and V max values of purified enzyme for COBE are 6.4 mM and 63.3 μmol min(-1) mg(-1), respectively. The biocatalytic process for the synthesis of (S)-CHBE was constructed by this SCR2 in an aqueous-organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, (S)-CHBE with yield of 95.3% and e.e. of 99% was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP(+) to (S)-CHBE were 26.5 mmol L(-1) h(-1) g(-1) DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works.
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang, 310014, People's Republic of China.
authors.forename: Zhong-Yu
authors.initials: ZY
authors.lastname: You
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Liu
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D000090:Acetoacetates / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000090:Acetoacetates; C111982:ethyl 4-chloro-3-oxobutanoate; D000429:Alcohol Oxidoreductases