Induction of mutation in Bacillus subtilis lipase gene using error-prone PCR

Introduction and objective: Directed evolution is an incredibly powerful tool for altering the properties of enzymes. This approach uses a "sloppy" version of PCR, in which the polymerase has a fairly high error rate to amplify the wild-type sequence. The aim of this study was to produce the Bacillus subtilis mutant lipase which can be active at low temperatures using the error-prone PCR method. Materials and methods: The pGEM-T vector containing B. subtilis lipase gene was transformed into the Escherichia coli DH5 alpha. The lipase gene was mutated using the error-prone PCR technique. Screening of the mutants was carried out using both microtiter and Luria-Bertani plates containing 1% tributyrin and 100mg/ml ampicillin. Ten out of 1300 clones which showed the lipase activity at 10 C were isolated. Among them, three desirable mutants named pFJK1, pFJK2 and pFJK3 were selected. Clones containing the wild type and the mutated lipase gene were both purified and characterized. The enzyme assay at different temperatures was carried out. Results: The optimum temperature for the activity of lipase was achieved at 10 degrees C and 30 degrees C for the mutants and control, respectively. The relative enzyme activity of pFJK1, pFJK2 and pFJK3 at 10 degrees C was more than two times of their activity at 30 degrees C. The enzyme retained 70% of its activity at 10 degrees C and there was a drastic decline in enzyme activity at 50 degrees C. Conclusion: Based on the present findings, the enzyme was characterized as a cold adapted lipase which showed the highest activity at 10 degrees C. Significance and impact of the study: Cold adapted lipase can be developed for industrial applications such as additives in detergents.