Point mutation Arg153-His at surface of Bacillus lipase contributing towards increased thermostability and ester synthesis: insight into molecular network.

In order to design proteins with improved properties i.e. thermostability, catalytic efficiency and to understand the mechanisms underlying, a thermostable variant of Bacillus lipase was generated by site-directed mutagenesis with enhanced thermal (∆Tm = + 12 °C) and chemical (∆Cm denaturation for Gdmcl = + 1.75 M) stability as compared to WT. Arg153-His variant showed 72-fold increase in thermostability (t 1/2  = 6 h) at 60 °C as compared to WT (t 1/2  = 5 min). Increase in thermostability might be contributed by the formation of additional hydrogen bonds between His153/AO-Arg106/ANH2 as well as His153-Arg106/ANE. The variant demonstrated broad substrate specificity. A maximum conversion of 59 and 62% was obtained for methyl oleate and methyl butyrate, respectively, using immobilized variant lipase, whereas immobilized WT enzyme synthesizes 35% methyl oleate. WT enzyme was unable to synthesize methyl butyrate as it showed negligible activity with pNP-butyrate.