Purification and Cloning of a Ketoreductase used for the Preparation of Chiral Alcohols

摘要: The synthesis of the leading candidate compound in an anticancer program required (S)-2-chloro-1-(3-chlorophenyl)-ethanol as intermediate. Other possible compounds used analogues S-alcohol. Of 119 microbial cultures screened for reduction corresponding ketone to S-alcohol, Hansenula polymorpha ATCC 58401 (73.8% ee) and Rhodococcus globerulus 21505 (71.8% had highest enantioselectivity producing desired alcohol. A ketoreductase from polymorpha, after purification homogeneity, gave S-alcohol with 100% ee. Amino acid sequences purified enzyme were design PCR primers cloning ketoreductase. cloned NADP(H), a subunit molecular weight 29,220 native 88,000. was expressed E. coli together glucose 6-phosphate dehydrogenase Saccharomyces cerevisiae allow regeneration NADPH by An extract containing two recombinant enzymes reduce 2-chloro-1-(3-chloro-4-fluorophenyl)-ethanone related ketones S-alcohols. Intact cells provided prepare (S)-2-chloro-1-(3-chloro-4-fluorophenyl)-ethanol 89% yield