Engineering of A Lipase towards Thermostability: Studies on Additive Effect of the two Thermo-Stabilising Mutations at Protein Surface

Abstract

In this study we have showed combined effect of two single point mutations S311C (LipR2) and R214C (LipR3) on the protein stability and overall change in biochemical properties. We found that both of these mutations are near the surface and individually enhanced the thermal stability of the protein (T1/2 for S311C=4.5 h & R214C=7 h at 60°C). But, their combined effect was not additive on thermostability. T1/2 of double mutant (LipR2 + LipR3) was 4 h at 60°C. Circular dichroism (CD) and fluorescence studies also supported our findings. Homology modelling studies demonstrated that in double mutant (LipR4) side chain of Cys311 is protruding towards the bulk solvent and is easily available for oxidation of sulfahydril group. This might be the reason for its low thermostability as compared to LipR3. We also observed that, side chains of Cys 214 didn’t changed. Here, one of the Cystein (Cys311) is behaving like a hydrophilic residue while the other (Cys 214) is behaving like hydrophobic residue. Keywords: Lipase; Thermostability; Mutations; Enzyme; Nucleotide sequence Introductio