Gene cloning and expression of a novel (S)-specific carbonyl reductase

Abstract:

Abstract：[Objective] A novel (S)-specific carbonyl reductase gene (scrⅡ) was cloned from the genome of Candida parapsilosis CCTCC M203011, and its catalytic function for the biotransformation of chiral alcohol was verified. [Methods] The possible carbonyl reductase gene scrⅡ was amplified by PCR method from the C. parapsilosis genome. Using the recombinant Escherichia coli BL21/pET28a-scrⅡ as the catalyst and 2-hydroxyacetophenone as the substrate, the biotransformation was carried out. The optical purity and yield of the final product were investigated by HPLC analysis. The optimal pH and temperature of the reaction were also determined. [Results] The gene scrⅡ coded 279 amino acids with an open reading frame of 837 bp. It shared 85% identity with the reported gene scr. By analysis, SCRⅡ contained two typical motifs of the short-chain carbonyl reductase including a Rossmann-fold Thr40-Gly41-(X)3-Gly45-X-Gly47 and a conserved catalytic triad Ser172-(X)n-Tyr187-(X)3-Lys191. SDS-PAGE results showed that SCRⅡ was overexpressed at 30℃ after the induction of 0.1 mmol/L IPTG. When the concentration of 2-hydroxyacetophenone was 6 g/L, 10% (w/v) wet recombinant E. coli cells showed excellent performance to give (S)-1-phenyl-1,2-ethanediol with high optical purity of 99.1% enantiomeric excess in a yield of 89.6% under the optimal conditions of pH5.5 and 35℃. SCRⅡ catalyzed the tansformation of (S)-1-phenyl-1,2-ethanediol more efficiently than SCR. When compared with SCR, its substrate concentration was increased by two-fold, and the optical purity and yield of (S)-1-phenyl-1,2-ethanediol were improved by 10% and 28%, respectively. [Conclusion] The gene coding for novel carbonyl reductase SCRⅡ was isolated using the molecular cloning technique and its discovery supplied a solid research foundation for chiral alcohol preparation efficiently.