Semirational Design of Methanol-Resistant Lipase Mutants: A Pathway to Efficient Biodiesel Production.

The development of a methanol-resistant thermostable lipase is of significant importance in biodiesel synthesis. In this study, a semirational design approach was employed to combine targeted mutagenesis and the introduction of N-glycosylation modifications to enhance the activity and stability of Thermomyces lanuginosus lipase. The activity of mutant A113G was 46% higher than that of the wild type. The double mutant A113G/L74N exhibited 33% residual activity after 24 h of incubation in 50% (v/v) methanol at 50 °C. The favorable temperature stability and methanol tolerance permitted a one-step synthesis of biodiesel; A113G/L74N achieved 91.2% and 80.6% biodiesel yield with soybean oil and waste frying oil, respectively. Molecular dynamics simulations demonstrated that hydrogen bonding between branched glycan and the main chain protein plays a pivotal role in stabilizing the protein structure, with the potential to extend over the surface of the main chain protein. Consequently, this study offers a promising and environmentally friendly strategy for biodiesel production.