Raw dataset for manuscript PeerJ on Enhancing the stability of Geobacillus zalihae T1 lipase in organic solvents and insights into the stability of its variants

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 Geobacillus zalihae 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 oC 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 km
and kcat 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.