Study On Performance Enhancement Of Chemically Modified Lipase Guided By Molecular Simulation

Lipase is one of the most important biocatalysts because of its stereoselectivity towards chiral substrates and reasonable adaptiveness of non-conventional reaction media, including hydrophobic organic solvents. Increasing the selectivity of lipase has been a continuous hotspot. This paper attempted to study on the lipase’s performance enhancement of the resolution of chiral secondary alcohols and chiral acids by means of computer molecular dynamics simulations (MD), binding MALDI-TOF-MS and chemical modification methods independent of molecular biology. The main contents of this work were as follows:Stereoselective acylation of chiral secondary alcohols catalyzed by lipase from Pseudomonas cepacia (PcL) in n-hexane was taken as the model reaction. Compared to the native PcL, binding energy of Tyr29-acetylated PcL and secondary alcohols was larger. According to the results from molecular dynamics simulation, when the Tyr29 inside the PcL catalytic cavity was modified by N-acetylimidazole (NAI), the enantioselectivity of the lipase was significantly increased. The simulation result was agreed with the experimental data. MALDI-TOF-MS confirmed the Tyr29 modified position, and kinetic resolution of chiral secondary alcohols by NAI-modified PcL displayed an increase of enantioselectivity ratio from 21.6 to 83.7 and higher activity.Lipase from Candida rugosa (CrL) were chemically modified with a wide range of hydrophobic groups such as lauroyl,t-butoxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, and acetyl moieties. Stereoselective hydrolysis of chiral n-butyl 2-phenoxy propionate (BPP) catalyzed by CrL in an aqueous buffer solution (pH=7.0) was taken as the model reaction. Compared to the native CrL, binding energy of modified CrL and BPP was larger in addition to CrL modified with p-nitrobenzyloxycarbonyl. According to the results from molecular dynamics simulation, when the Lys residues of the CrL surface were chemically modified with benzyloxycarbonyl, the enantioselectivity of the lipase was significantly increased. By further experiments, kinetic resolution of BPP by CrL modified with benzyloxycarbonyl displayed an increase of enantioselectivity ratio from 2 to 5, although the enzymatic activity was decreased.