Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus

摘要: Enzymatic production of biodiesel by transesterification triglycerides and alcohol, catalyzed lipases, offers an environmentally friendly efficient alternative to the chemically process while using low-grade feedstocks. Methanol is utilized frequently as alcohol in reaction due its reactivity low cost. However, one major drawbacks enzymatic system presence high methanol concentrations which leads methanol-induced unfolding inactivation biocatalyst. Therefore, a methanol-stable lipase great interest for industry. In this study, protein engineering was applied substitute charged surface residues with hydrophobic ones enhance stability from Geobacillus stearothermophilus T6. We identified variant, R374W, combined it variant found previously, H86Y/A269T. The triple mutant, H86Y/A269T/R374W, had half-life value at 70 % 324 min reflects 87-fold enhanced compared wild type together elevated thermostability buffer 50 methanol. This also exhibited improved yield waste chicken oil commercial Lipolase 100L® Novozyme® CALB. Crystal structures variants provided insights regarding structure-stability correlations. most prominent features were extensive formation new hydrogen bonds between directly or mediated structural water molecules stabilization Zn Ca binding sites. Mutation sites characterized lower B-factor values calculated X-ray indicating rigidity.