Improved thermostability of Thermomyces lanuginosus lipase by molecular dynamics simulation and in silico mutation prediction and its application in biodiesel production

• TLL was subjected to MD simulation and in silico mutation prediction. • The G91C mutant enhanced the activity and thermostability of the enzyme. • The FAME yield catalyzed by G91C mutant was as high as 91% at 43 °C. • The extra hydrogen bond formed between Cys91 and Glu87 made the α-helix more stable. To improve the thermostability of Thermomyces lanuginosus lipase (TLL) which has been widely used in biodiesel production, molecular dynamics (MD) simulation was applied to find the fluctuation regions under different temperatures. In silico mutation prediction was also applied to calculate the mutation stability of amino acid residues for site mutagenesis. Six mutants were predicted and subjected to mutagenesis and expressed in Pichia pastoris KM71H. The enzyme activity of G91C not only increased by 53.8%, but also enhanced the residual activity by about 25 ∼ 45% in thermostability. With 5 °C higher than that of the wild type in optimal temperature, the catalytic efficiency of G91C was also increased by 1.3 times. The fatty acid methyl esters (FAME) yield catalyzed by G91C was as high as 91% at 43 °C, 7% higher than that of the WT during the transesterification of Cornus wilsoniana oil to biodiesel. According to the structural comparisons, it was found that there was an extra main-side hydrogen bond between Cys91 and Glu87 separated by four amino acid residues and a more stable α-helix was obtained, which may be the key factor in the thermostability enhancement. In total, the engineered G91C mutant showed great potential in the production of biodiesel at high temperature.