A thermostable variant of Bacillus subtilis esterase: Characterization and application for resolving dl-menthyl acetate

Abstract

The thermostability of an esterase from Bacillus subtilis was enhanced by random mutagenesis and combination of beneficial mutations. The hydrolysis of racemic menthyl acetate by BSE V4 at elevated temperature indicates that better thermostability could reduce the enzyme dosage or reaction time. The modeled structure demonstrates the addition of ionic bonds, hydrogen bonds and/or hydrophobic interactions will contribute mostly to the structure stability. • Thermostability of BSE was successfully enhanced by random mutagenesis. • Increased T 50 15 value and optimum temperature of up to 4.5 and 5.0 °C. • The total turn-over number of BSE V4 was 1.1–1.5 folds higher with 1.0 M of substrate loading for each batch. • Increased ionic bonds, hydrogen bonds and hydrophobic interactions contribute to stabilize the protein. Bacillus subtilis esterase (BSE) exhibits high activity, extraordinary substrate/product tolerance and excellent enantioselectivity in the production of l -menthol through enantioselective hydrolysis of dl -menthyl acetate. However, rapid inactivation of wild-type BSE at elevated temperatures often hampers its applications. In this work, directed evolution was used to create thermostable mutants of BSE. After screening and recombination of beneficial mutations, BSE V4 was chose for the best mutant. The BSE V4 had half-lives of 462, 248 and 0.34 h at 30, 40 and 50 °C, respectively, which were 5.6, 4.1 and 2.0 folds longer than those of BSE WT . Moreover, BSE V4 showed an increase of 4.5 °C in T 50 15 and a higher temperature optimum compared with the wild-type enzyme. In the kinetic resolution of dl -menthyl acetate at 1.0 M substrate loading, BSE V4 displayed improvements in operational stability than BSE WT , leading to a 1.5-fold higher total turnover number at 45 °C. The model structure of BSE V4 with four mutations, built with a highly homologous p -nitrobenzyl esterase (PDB ID: 1QE3 ) as the template, revealed that the newly formed hydrogen bonds and ionic bonds were beneficial for enhancing the thermostability of BSE.