Genome Mining and Characterization of Diaryl Ketone Reductase from Kluyveromyces polysporus

Abstract:

In this study，an alcohol dehydrogenase KpADH from Kluyveromyces polysporus was identified by genome mining strategy. This newly mined KpADH belongs to extended SDRs subfamily，and catalyze the asymmetric reduction of diaryl ketones into chiral diaryl alcohols. KpADH also displays substrate-coupled NADPH recycling ability by oxidation of cosubstrate isopropanol. The recombinant KpADH was purified by Ni-NTA affinity chromatography，and the enzymatic properties of the purified KpADH were characterized. The optimum pH of oxidizing and reducing activity is 9.5 and 5.5 respectively. The Km and Vmax of KpADH towards CPMK are 0.5 mmol/L and 25 μmol/（min·mg） respectively，while the Km and Vmax of KpADH towards isopropanol are 6.36 mmol/L and 21.37 μmol/（min·mg） respectively. KpADH shows higher catalytic activity on ketone esters and 2，3-butanediol. Within 10 h，100 mmol/L CPMK was asymmetrically reduced into（R）-CPMA by recombinant E. coli BL21/pET28a-kpadh cells，giving >99% conversion，82% ee and 88.7% molar isolation yield. This study provides useful guidance for the application of this newly identified KpADH in the preparation of chiral diaryl alcohols.