Improvement of methanol tolerance and catalytic activity of Rhizomucor miehei lipase for one-step synthesis of biodiesel by semi-rational design

Exploiting highly active and methanol-resistant lipase is of great significance for biodiesel production. A semi-rational directed evolution method combined with N-glycosylation is reported, and all mutants exhibiting higher catalytic activity and methanol tolerance than the wild type (WT). Mutant N267 retained 64% activity after incubation in 50% methanol for 8 h, which was 48% greater than that of WT. The catalytic activity of mutants N267 and N167 was 30- and 71- fold higher than that of WT. Molecular dynamics simulations of N267 showed that the formation of new strong hydrogen bonds between glycan and the protein stabilized the structure of lipase and improved its methanol tolerance. N267 achieved biodiesel yields of 99.33% (colza oil) and 81.70% (waste soybean oil) for 24 h, which was much higher than WT (51.6% for rapeseed oil and 44.73% for wasted soybean oil). The engineered ProRML mutant has high potential for commercial biodiesel production.