Increasing Activity of Lipase from Bacillus subtilis by Directed Evolution

The specific activity of a Bacillus subtilis lipase was increased by directed evolution. Through two cycles of error prone PCR, coupled with a sensitive high-throughput screening method, a mutant named 3-1B2 was obtained. Its catalytic activity was 4.5-fold compared to that of wild lipase BSL2. DNA sequencing revealed that two amino acids were changed. Further experiments showed that the thermostability and pH stability of 3-1B2 were slightly increased than those of BSL2, and the optimum temperature and pH of the mutant lipase were similar to those of wild lipase. The molecular structures of BSL2 and 3-1B2 were homologously modeled based on the crystal structure of BSLA and docked with the substrate. The results showed that the binding energy of 3-1B2 with substrate was 1.29 kcal/mol lower than that of BSL2, and the distance between catalytic residue Ser77 of 3-1B2 and substrate was 0.278 nm, lower than that of BSL2 (0.319 nm). This implied that small distance between Ser77 and substrate might increase reaction rate and then cause an increase in activity.