Ding_2018_Bioorg.Chem_77_330

The scissile fatty acid binding site of lipases is divided into different sub-groups and plays an important role in the catalytic properties of the enzymes. In this study, the Talaromyces thermophilus lipase was engineered by altering its crevice-like binding site for efficient synthesis of chiral intermediate of Pregablin through kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE). The substitution of residues located at the crevice-like binding site with phenylalanine (Phe) resulted in significantly increased hydrolysis activity. The variant L206F/P207F/L259F exhibited a 37.23-fold and 47.02-fold improvement in the specific activity and turnover number (kcat) toward CNDE, respectively. Simultaneously, the optimum temperature and substrate preference were both altered in the variants. The study herein successfully engineered the TTL with improved catalytic properties for efficient biosynthesis of Pregablin intermediate. The investigation of structure-functional relationship provided important guidance for further modification of lipases with crevice-like binding site domain.