Computational Design of Burkholderia cepacia Lipase Mutants that Show Enhanced Stereoselectivity in the Production of l-Menthol

l-Menthol, valued for its aroma, cooling properties, and biological activity, is often produced as a mixture with d-menthol, which negatively impacts taste and odor. To improve the purity of industrial l-menthol production, we engineered Burkholderia cepacia lipase (BCL) to improve its stereoselectivity. While wild-type BCL achieves only 98% enantiomeric excess, we used molecular dynamics simulations of BCL bound to menthol acetate, combined with computational rational design method (MSPER), to identify key mutation sites. We experimentally confirmed that five mutations that can effectively improve the enantiomeric excess. In particular, Q88A increased enantiomeric excess to 99.4% and productivity from 14.5% to 49.9%. Q88G exhibited the highest productivity at 54.0% with 99.3% optical purity. These BCL mutants offer a greener, more efficient approach to high-purity l-menthol production, contributing to more sustainable chemical processes.