Title
Point mutation Arg153-His at surface of Bacillus lipase contributing towards increased thermostability and ester synthesis: insight into molecular network
Author
Nisha Chopra, Jagdeep Kaur
Year
2017
Journal
Molecular and Cellular Biochemistry
Abstract
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 (t1/2 = 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.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Thermodynamics, Thermal stability, Protein folding, Biochemistry