Improvement of a newly cloned carbonyl reductase and its application to biosynthesize chiral intermediate of duloxetine

摘要

In this study, a carbonyl reductase RtSCR9 from Rhodosporidium toruloides was engineered to improve its activity for asymmetric reduction of N,N-dimethyl-3-keto-3-(2-Thienyl)-1-keto propanamine (DMAK) to (S)-3-(dimethylamino)-1-(2-Thienyl)-1-propanol (S-DMAA). Error-prone polymerase chain reaction (epPCR) combined with megaprimer PCR of whole plasmid (MEGAWHOP) method and site saturation mutagenesis was used to construct random mutant libraries. The positive mutant was screened by measuring the decrease in the absorbance of NADPH at 340 nm and high performance liquid chromatography (HPLC). The best Mut-A89N/F154Y was obtained and compared with wild type RtSCR9, the V-max and k(cat) of Mut-A89N/F154Y were enhanced by 2.08 and 3.86-fold, respectively, while the K-m decreased by 2.36-fold. The reduction of DMAK by Mut-A89N/F154Y afforded S-DMAA with a yield of 91.2% and enantiomeric excess of 99.9% at 2 M DMAK, which was highest ever reported. This mutant could be a potential candidate for the upscale production of S-DMAA.