Discovery of the Key Mutation Site Influencing the Thermostability of Thermomyces lanuginosus Lipase by Rosetta Design Programs

Abstract

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications.

Improving the Thermostability of Thermomyces lanuginosus Lipase by Restricting the Flexibility of N-Terminus and C-Terminus Simultaneously via the 25-Loop Substitutions

“…To prevent the denaturation of TLL, it is necessary to improve its thermostability. Comparing lipases from other thermophilic organisms, TLL showed a slightly lower optimal reaction temperature (40 • C) [21]. In contrast, the optimum temperatures of lipases from Enterobacter sp.…”

“…Qu et al combined MD simulation and in silico mutation prediction to provide TLL variants with improved thermostability, and finally obtained variant G91C with both improved residual activity and a 5 • C increment in the optimal temperature without losing its specific and catalytic activity [25]. Han et al substituted a surface-charged residue, R209, in TLL and finally obtained three single variants (R209M, R209H, and R209I) with both an increased optimal temperature and melting temperature directed by the ∆∆G calculation [21]. Gupta et al reported the binary immobilization of TLL using chitosan as the support and resulted in increased thermostability against the crude enzyme [26].…”