Enhancing methanol tolerance of Thermomyces lanuginosus lipase by rational design and biodiesel production through one-step feeding of methanol

The addition of a significant quantity of methanol solution during the biodiesel production results in the denaturation of lipase. To address this issue, this study proposed a novel approach to the preparation process by designing a methanol-tolerant Thermomyces lanuginosus lipase (TLL) using a computer-aided strategy. In silico prediction yielded seven mutants, out of which two mutants, E129Y and E134W, exhibited varying degrees of improved methanol tolerance and catalytic efficiency. The methanol tolerance and stability of E129Y and E134W were observed to increase by 10–30 % and 17–35 %, respectively. Additionally, E134W exhibited a 1.13-fold increase in catalytic efficiency compared to the wild type TLL, while E129Y showed a reduction of 12.05 %. Following a one-shot addition of methanol (molar ratio of 3:1 methanol to Cornus wilsoniana oil) for 72 h in the transesterification reaction, the yields of fatty acid methyl ester (FAME) for E129Y and E134W were 69 % and 85 %, respectively, with the yield of E134W increasing by 22 %. Structural analysis indicated that the E129Y mutation formed a single hydrogen bonding interaction, while E134W produced hydrophobic interactions with neighboring amino acids, thereby augmenting the structural stability of the mutations under high methanol concentration. This study has the potential to advance the commercialization of lipases in the biodiesel production industry.