A Novel Thermal Stable Carbonyl Reductase from Bacillus cereus by Gene Mining as Biocatalyst for β-Carbonyl Ester Asymmetric Reduction Reaction

Abstract

Carbonyl reductase, as biocatalyst, is very important to chiral alcohols production through asymmetric reduction of carbonyl compound. A novel thermal stable carbonyl reductase from Bacillus cereus (BcCR) dependant on NADPH was obtained through a new genome mining strategy proposed in this work. By analyzing its amino acid sequence and structure, the BcCR should be a thermal stability and wide pH tolerance carbonyl reductase. Its gene was cloned by PCR with B. cereus genomic DNA as template. Its heterologous expression system, E. coli BL21 (DE3) plysS/pET28a-bccr, was constructed, and BcCR was successfully expressed. Enzymatic properties show that at 57.5 °C and pH 7.0 it can reach maximum reaction rate. Its Km and Vmax to ethyl 4-chloroacetoacetate is 1.85 mmol/L and 0.22 µmol/(min·mgprotein), respectively. It can catalyze the asymmetric reduction of the β-carbonyl compound, such as ethyl 4-chloroacetoacetate to ethyl S-4-chloro-3-hydroxybutyrate. This paper proposes a practical method for discovery of new carbonyl reductases, and provides a novel enzyme as biocatalyst for asymmetric reduction of β-carbonyl compound.