Improvement of a newly cloned carbonyl reductase and its application to biosynthesize chiral intermediate of duloxetine. (July 2018)

• Record Type: Journal Article
• Authors: Wang, Xin-Xin; Lin, Chao-Ping; Zhang, Xiao-Jian; Liu, Zhi-Qiang; Zheng, Yu-Guo
• Is Part Of: Process biochemistry. Volume 70(2018)
• Page Start: 124
• Page End: 128
• Publication Date: 2018-07
• Subjects: Carbonyl reductase -- Asymmetric reduction -- Two steps screening methodology -- (S)-3-(Dimethylamino)-1-(2-Thienyl)-1-propanol; Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2018.04.010
• Languages: English
• ISSNs: 1359-5113

Abstract

Graphical abstract: Highlights: A carbonyl reductase was used to synthesize intermediate of duloxetine. The Mut-A89N/F154Y with highest activity was obtained after screened. 2 M of substrate was bioreduced to prepare product with yeild >91.2% and e.e. >99.9%. Abstract: In this study, a carbonyl reductase Rt SCR9 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 Rt SCR9, the V max and kcat of Mut-A89N/F154Y were enhanced by 2.08 and 3.86-fold, respectively, while the Km 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.