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Improvement of a newly cloned carbonyl reductase and its application to biosynthesize chiral intermediate of duloxetine
- Source :
- Process Biochemistry. 70:124-128
- Publication Year :
- 2018
- Publisher :
- Elsevier BV, 2018.
-
Abstract
- 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 Vmax 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.
- Subjects :
- 0106 biological sciences
Carbonyl Reductase
biology
010405 organic chemistry
Chemistry
Stereochemistry
Mutant
Wild type
Rhodosporidium toruloides
Bioengineering
biology.organism_classification
01 natural sciences
Applied Microbiology and Biotechnology
Biochemistry
High-performance liquid chromatography
0104 chemical sciences
010608 biotechnology
Enzyme kinetics
Enantiomeric excess
Saturated mutagenesis
Subjects
Details
- ISSN :
- 13595113
- Volume :
- 70
- Database :
- OpenAIRE
- Journal :
- Process Biochemistry
- Accession number :
- edsair.doi...........286d48b00a3b328c3a46970c29c4b8d9