Directed evolution of Bacillus licheniformis lipase for improvement of thermostability

Lipases catalyze the hydrolysis of carboxylic acid esters and owing to their vast substrate specificity, they have many industrial applications. Due to the demand of thermostable lipases in industrial applications, we have enhanced the thermostability of lipase from Bacillus licheniformis RSP-09. The thermostable mutant lipases of Bacillus licheniformis RSP-09 were isolated following two rounds of directed evolution using error-prone PCR. The best mutant lipases obtained after first and second round of error-prone PCR were purified and characterized. The mutant lipases showed increased thermostability and retained catalytic function. The best mutant lipase (eP-231-51) showed 13.5-fold increase in percentage thermal stability (% remaining activity after incubation of purified enzyme at 60 C for I h) than wild-type lipase. Also, this mutant lipase (ep-231-51) showed 30% improved catalytic efficiency compared with the wildtype which is due to significant decrease in K-m and marginal increase in k(cat). In addition, the thermostable mutant lipases have shown resistance to hydrophobic organic solvents. The role of mutations in the best mutant lipases of second round i.e. eP-231-51 (Asp72Gly, Asp61Gly, Tyr129His, and Thr101Pro) and eP-231-137 (Leu49Arg, Thr101Pro, Asp72Gly), that led to thermostability have been postulated after the comparison of molecular models of wild-type and mutated enzymes. (C) 2014 Elsevier B.V. All rights reserved.