Engineering of a metagenome derived lipase toward thermal tolerance: Effect of asparagine to lysine mutation on the protein surface

A highly thermostable mutant lipase was generated and characterized. Mutant enzyme demonstrated 144 fold enhanced thermostability over the wild type enzyme at 60 °C. Interestingly, the overall catalytic efficiency (kcat/Km) of mutant was also enhanced (~ 20 folds). Circular dichroism spectroscopy, studied as function of temperature, demonstrated that the mutant lipase retained its secondary structure up to 70–80 °C, whereas wild type protein structure was completely distorted above 35 °C. Additionally, the intrinsic tryptophan fluorescence (a probe for the tertiary structure) also displayed difference in the conformation of two enzymes during temperature dependent unfolding. Furthermore, mutation N355K resulted in extensive H-bonding (Lys355 HZ1OE2 Glu284) with a distance 2.44 Å. In contrast to this, Wt enzyme has not shown such H-bonding interaction.

► This study reported characterization of highly thermostable mutant lipase. ► Mutation resulted in substitution of amino acid N355K in mature polypeptide. ► Mutation improved both thermal stability and catalytic efficiency of the lipase protein. ► Mutant protein retained its secondary structure at high temperature compared to Wt protein. ► Mutant N355K resulted in extensive H bonding, in contrast to Wt enzyme.