DOI: 10.6084/m9.figshare.7326458
title: Raw dataset for manuscript on Enhancing the stability of Geobacillus zalihae T1 lipase in organic solvents and insights into the stability of its variants

description: The functioning of enzymes as biocatalysts in non-aqueous organic solvents has important implications for their catalytic and structural behavior. Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from <i>Geobacillus zalihae</i> T1 lipase was developed using random mutagenesis (error-prone PCR). High-throughput screening of the variants in organic solvents (methanol, ethanol, propanol, butanol, and pentanol) at a temperature of 60 <sup>o</sup>C resulted in the selection of six (6) mutants (A83D/K251E, R21C, G35D/S195N, K84R/R103C/M121I/T272M, and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195N/T272M, both of which had improved stability in methanol, ethanol, and propanol. Kinetic investigation showed that the <i>k<sub>m</sub></i><sub> </sub>and <i>k<sub>cat</sub></i><sub> </sub>values of these mutants were not significantly different from those of the wild-type enzyme, in contrast to those in the methanol/substrate mixture. Greater half-life was achieved for all mutant proteins in methanol with better exponential decay constants. The biophysical properties in 50 % and 60 % methanol showed that on the whole, thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. Molecular Dynamic simulations were relied upon to provide insight into perturbations of the protein matrix which triggered a reorganization of the secondary structural elements. This revealed that changes in the structural organization are intertwined with the conformational interplay of the protein backbone with the organic solvents. The solvent simulations were found to induce conformational changes on the protein surface resulting in induced changes between structural features and the conformational stability of the protein. Correlations were seen between solvent mobility and residue motions as indicated by the state of the root mean square deviations (RMSD) and B-factor. Spatially-exposed charged residues of the mutants showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. The newly formed structural interactions between the mutant residues and the other surrounding residues enhanced the native conformation of the secondary structural characteristics of the quadruple mutants A83D/M121I/S195N/T272M and A83D/M121I/K251E/G375S. The dynamic changes revealed by MD insights demonstrates the engineered mutants although manifest an appreciable amount of stability to methanol, if further improved upon are promising candidates for future application as biocatalysts for non-aqueous processes. As newly formed structural interactions could be targeted and incorporated experimentally based on the mobility of the solvents and residues of the mutants.