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

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 (BpIA) 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 BpIA. 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. (C) 2010 Elsevier B.V. All rights reserved.