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.