Cloning And Expression Of Gene Encoding (R)-Specific Carbonyl Reductase From Candida Parapsilosis In Escherichia Coli

Optically active 1-phenyl-1,2-ethanediol is a versatile group as chiral building block for the syntheses of pharmaceuticals, agrochemicals, pheromones, and liquid crystals, etc. In the lab, Candida parapsilosis CCTCC M203011 was screened for the asymmetric conversion of racemic 1-phenyl-1,2-ethanediol to (S)-1-pheny l-1,2-ethanediol. Then the purified (R)-specific carbonyl reductase(rCR) protein which worked in the asymmetric conversion was gained. The intention of this research was gained and study the rcr gene, and be a basic to get the more efficiency enzyme.The gene which encodes rCR from Candida parapsilosis CCTCC M203011 was cloned. Then the PCR sequence was ligated to pMD18-T Vector and gained the pT-rCR plasmid. The cloned sequence includes an open reading frame (ORF) consisting of 1011bp, encoding a protein of 336 amino acids, with a molecular weight of 35.9 kD. Searched the rcr homologous gene in NCBI, then multi-sequences alignment and consensus analysis were performaced by ClustalW, the result revealed that the rCR protein has 3 domain as Adh_zinc, NAD+_Binding, GroEs_like which are coincident with the pure enzyme. Then use the amino acid sequence of rCR construct phylogenetic tree.Constructed the expression plasmid pTrc-rCR and checked it. The recombinant Escherichia coli JM109 strain harboring the expression plasmid was induced by IPTG, gained the rCR protein band by SDS-PAGE analysis, and the specific activity of the E.coli JM109/pTrc-rCR/IPTG was 0.294U/mg. The whole cell recombinant could product (R)-1-phenyl-1,2-ethanediol(97.5%e.e., 28%yield) fromα-hydroxyacetophenone.Through the shaking-flask condition research, the optimum induction conditions had determined which was as follow: 20% medium volume, temperature 30℃, IPTG concentration 1mmol/L, start time of induction 8hr, induction duration 16hr.Through research, the optimum reaction conditions in 250mL shaking-flask were obtained as pH 7.0, temperature 30℃, shaking speed 150r/min, reaction time 48hr, washed cell join reaction. To improve the reaction, 0.5% alcohol was added during reaction. The optical purity of the product reached 98%e.e. and molar yield higher than 80%.