Enhancing the stability of Geobacillus zalihae T1 lipase in organic solvents and insights into the structural stability of its variants

Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics relation solvent polarity. A pool mutants derived from Geobacillus zalihae T1 lipase was screened organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting selection six at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M R106H/G327S). Site-directed mutagenesis further yielded quadruple A83D/M121I/K251E/G327S A83D/M121I/S195 N/T272 M, both which had improved activity after incubation methanol. The km kcat values these vary marginally with wild-type enzyme methanol/substrate mixture. Thermally induced unfolding accompanied some loss secondary structure content. root mean square deviations (RMSD) B-factors revealed that changes organization are intertwined an interplay protein backbone solvents. Spatially exposed charged residues showed correlations between solvation methanol hydrophobicity residues. short distances radial distribution function provided required for hydrogen bond formation hydrophobic interactions. These dynamic demonstrate newly formed interactions could be targeted incorporated experimentally on basis mobility mutant