ELBn12 is a lipase isolated fromEnterobacter sp. Bn12 with
potential application in biotechnology. Homology modeling
and rational design were applied to improve thermal stability
of the lipase. K173A substitution introduced an AXXXA motif
on the lipase model and it may have role in dimerization and
thermostability of the protein. Site-directed mutagenesis was
performed to construct the lipase variant. The mutated lipase
was expressed inEscherichia coli pLysS and partially purified.
Thermostability of the mutated lipase after 1 h of incubation
at 70 ° C was twice that of wild-type lipase under the same
conditions. Catalytic activity of the variant was about 1.5-fold
towards tricaprylin at 60 ° C and pH 8.0; moreover, the lipase
variant preserved its stability within the pH range of 7.0–11.0.
Substitution of superficial hydrophilic Lys with hydrophobic
Ala residue increased stability of the mutated lipase in the
presence of nonionic surfactants, but this substitution caused
lower stability towards polar solvents. Analysis of circular dichroism spectroscopy showed that the K173A mutation altered the secondary structure of the lipase into a more helical
one. In conclusion, results of this study demonstrate the positive role of generation of a stabilizing protein motif through
rational protein engineering that improves the enzyme characteristics