Improvement of Thermal Stability via Outer-Loop Ion Pair Interaction of Mutated T1 Lipase from Geobacillus zalihae Strain T1

Abstract

Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The Tm for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher Tm as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.

Recent advances in production and biotechnological applications of thermostable and alkaline bacterial lipases

“…So, these protein engineering strategies have been established as efficient tools to successfully improve biotechnologically relevant properties of enzymes . Moreover, in recent years, computational protein design is gaining more and more attention as a novel strategy to predict the effects of mutations on protein structure, function or stability of libraries of enzyme variants generated by means of in silico approaches . Thermal stability is a major requirement for commercial enzymes, being critical for industrial applications, as thermal denaturation leads to enzyme inactivation .…”

Cold active lipases – an update

“…Thermal denaturation is a common cause of enzyme inactivation in industrial applications; therefore an industrial enzyme necessarily needs to be thermostable (Ruslan et al 2012). The psychrophilic yeast, Candida antarctica expresses two lipases, namely C.antarctica lipase-A and C. antarctica lipase-B (CAL-A and CAL-B) with different physiochemical properties.…”

“…CAL-B and Geobacillus zalihae T1 lipase were successfully engineered by mutating five amino acid pairs to cysteine and by introducing an ion-pair in the inter-loop (Le et al 2012;Ruslan et al 2012). …”

Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study

“…Results of a study showed that the introduction of an additional ion pair in the lipase structure increased the stability of the protein at high temperatures. The enhanced stability of D311E lipase was due to additional inter-loop interactions, which were indicated by the atomic details of D311E lipase with the observed additional ion pair and hydrogen bond interactions (Ruslan et al, 2012). Mutations located in or contacting flexible regions of the haloalkane dehalogenase LinB had a larger stabilizing effect than mutations outside such regions (Floor et al, 2014).…”