Directed evolution of lipase A from Bacillus subtilis for the preparation of enantiocomplementary sec-alcohols

Abstract

Biocatalysts with tailor-made or controllable stereoselectivity are valuable and highly desired in the precision synthesis of chiral compounds. Most lipases display excellent (R)-enantioselectivity for various sec-alcohols. In order to reverse the inherent (R)-enantioselectivity of lipase A from Bacillus subtilis (BSLA), an important amino acid position of BSLA (H76) which can reverse the (R)-enantioselectivity to (S) in the hydrolytic kinetic resolution (KR) of various esters derived from sec-alcohols was discovered, basing on the comparison with the well-known “gourd”-like pocket of Candida antarctica lipase B (CALB). After iterative mutagenesis, the best mutant H76 ​A/N18C was obtained, displaying high (S)-selectivity for the model reaction (45% conv. and 92% ee). Meanwhile, a single-point mutation of N18W was also found which can remarkably improve the inherent (R)-enantioselectivity of BSLA toward sec-alcohol. A series of enantiocomplementary sec-alcohols can be prepared through the KR catalyzed by WT BSLA or mutants, and the highest enantiomeric ratio (E) value is up to >300 for (R)-enantiomer and 178 for (S)-enantiomer, respectively.

Keywords

• Lipase A from Bacillus subtilis
• Directed evolution
• sec-Alcohols
• Hydrolysis