Kinetic studies of Gly28:Ser mutant form of Bacillus pumilus lipase: Changes in kcat and thermal dependence

Abstract

Lipases are useful catalysts for a wide variety of industrial purposes. Herein we report the stability and thermal dependence of the activity of wild-type Bacillus pumilus lipase (BplA) and four site-directed mutants designed to improve its thermal stability. The Gly28:Ser mutation produces a dramatic four-fold increase in its k cat and a remarkable increase in its stability. While the increase in k cat is temperature-independent, the increase in stability shows that the resultant interactions of this mutation have a strong enthalpic component. Thermal dependence of stability, k cat, K M and k cat/ K M were analysed to gain insight on the structural effects of mutations on BplA. Our results are consistent with a gain in enzyme mobility for those mutants displaying enhanced catalytic properties; the analysis of thermal dependence of kinetic parameters indicates that the mutations did not change either the catalytic mechanism or the rate-limiting step of catalysis.