Improving Pseudomonas alcaligenes lipase’s diastereopreference in hydrolysis of diastereomeric mixture of menthyl propionate by site-directed mutagenesis

摘要: The resolutions of racemic diastereomeric mixtures menthyl propionate was performed by Pseudomonas alcaligenes lipase (PaL) to produce (2S, 5R) L-menthol. Because the inherently low diastereopreference PaL, covalent docking and molecular dynamic (MD) simulations were used investigate possible avenues improvement. Rational site-directed mutagenesis PaL revealed residues V180 A272 be hotspots for diastereopreference. double V180L/A272F mutant exhibited highest degree diastereopreference, as ratio L-menthol increased towards both (2R, 5S) L-neomenthol (dr1) D-isoneomenthol (dr2) (diastereomeric ratios dr1 dr2 4.65 2.13 times that wild-type PaL). MD simulation analysis indicated these mutations decrease flexibility surrounding protein regions. combination steric exclusion decreased results in less favorable binding non-target substrates, L-neomenthyl D-isoneomenthyl propionate, mutant. These confirmed further improved our previously proposed model diastereomer recognition mechanism based on combined effect regional flexibility.