Enhanced enzyme thermostability of a family I.3 lipase LipSR1 by T118A mutation at the calcium-binding site

ObjectivesThe lipase gene lipSR1 isolated from oil-contaminated soil exhibits high hydrolytic activity for short-chain fatty acid substrates. A single calcium ion is required to anchor the lid of LipSR1 in an open conformation by coordination with two aspartate residues and three other residues in the lid. The lid of LipSR1 is anchored by Ca2+, which is coordinated by side-chain carboxyl oxygens of Asp153 and Asp157, carbonyl oxygens of Thr118 and Ser144, and the side chain of Gln120.ResultsD157A, D153R, Q120A, S144A, and T118A mutants were produced by site-directed mutagenesis in this study. Analyses of hydrolytic activity and thermostability showed that the properties of D157A, D153R, Q120A, and S144A were almost lost, suggesting that Asp157, Asp153, Gln120, and Ser144 are important residues for LipSR1. However, the catalytic performance of T118A was clearly maintained. Moreover, the thermostability of mutant T118A was higher than that of wild-type LipSR1.ConclusionsThese results indicated that mutation of threonine at position 118 improved the stability of the enzyme at high temperature.

Automated summary

In this study, mutants D157A, D153R, Q120A, and S144A were obtained by site-directed mutagenesis. It was found that the hydrolytic activity and thermostability of these mutants were almost lost, indicating the importance of Asp157, Asp153, Gln120, and Ser144 residues in LipSR1. However, the catalytic performance of T118A mutant was clearly maintained, and the thermostability of T118A mutant was higher than that of wild-type LipSR1. These results suggest that mutation of threonine at position 118 improves the stability of the enzyme at high temperature.