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

(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 °C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 °C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24-30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27…E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability.

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

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.