基于长链底物亲和的脂肪酶 MAS1 理性设计改造

The optimum temperature, pH, and pH stability of lipase MAS1 for the catalysis of the long-chain substrate 4-nitrophenol myristate (pNP-C14) were determined. Two single point mutations, G145W and T141L, were initially screened out according to the decrease of binding free energy by molecular dynamics simulation (Molecular Dynamics, MD). Experiments showed that K_m of the mutant G145W with a significant decrease in binding free energy was reduced by 11% compared with the wild-type MAS1, and k_cat /K_m was 1.29 times that of the wild-type MAS1. Compared with the wild-type MAS1, K_m of the mutant T141L with a smaller decrease in binding free energy was increased by 22%, and k_cat /K_m was 0.88 times that of the wild-type MAS1. Compared with the wild type, the affinity and catalytic efficiency of G145W for long-chain substrates were improved, while those of T141L were lower than the wild type. It showed that the absolute zero value of the binding energy difference was not an accurate mutation screening criterion. In-depth analysis was performed by Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) residue disassembly. It was concluded that residues T38, F39, L149, F153, V202 and V233 contributed more to the binding stability of the lipase active pocket and long-chain substrates than other residues; Residues T38, G40, N41, N45, and T237 showed important contributions to the improvement of lipase affinity for long-chain substrates and were predicted to be hotspot residues whose reduced binding free energy can serve as a reference standard for mutation screening.