Efficient preparation of the chiral intermediate of luliconazole with Lactobacillus kefir alcohol dehydrogenase through rational rearrangement of the substrate binding pocket

Abstract

Abstract Luliconazole has been approved by the FDA for fungal infection therapy because of its broad-spectrum antifungal activity. However, low efficiency and harsh reaction conditions in the current synthesis of the luliconazole chiral intermediate (S)-2-chloro-1-(2,4-dichlorophenyl) ethanol ((S)-TCPE) limits its application. In the present study, a novel biocatalytic process for producing (S)-TCPE was developed. Based on a structural analysis at the atomic level on the wild-type (WT) Lactobacillus kefir ketoreductase (L. kefir KRED), a rational design of L. kefir KRED was performed with 2,2’,4’-trichloroacetophenone (TCAP) as the substrate. An enantioselective triple mutant A94S/S96E/E145A displaying a 117-fold increase in its relative activity compared to the WT was evolved, and the catalytic efficiency was raised to 5.17 s−1mM−1 from non-detectable. With 106 kg of wet cells of the triple variant loading, 275 kg TCAP was asymmetrically reduced with the yield of (S)-TCPE achieving 91% within 12 h, thus showing great potential for industrial applications. Furthermore, the molecular mechanism supporting the promotion of activity was revealed to be related to the rearrangement of the substrate binding pocket of the L. kefir KRED. By engineering an enzyme that serves as a biocatalyst for the efficient preparation of enantiomerically pure (S)-TCPE, the current study presents a reproducible strategy for the promotion of catalytic activity based on rational design.