Directed Evolution of Lipase to Have Enhanced Activity toward Short Chain Fatty Acids and Elucidation of Structural Features of Mutant
2023
Shin, J.Y. | Seo, J.H.
Development of lipase having substrate specificity toward short chain fatty acids have industrial importance. Among the various lipase, LipC12 has substrate specificity to long chain fatty acids, good thermostability and tolerance in high concentration of aqueous solutions of polar organic solvents, thereby showing good evolvability. In this study, LipC12 was subjected to random mutation and mutant having activity to short chain fatty acids was identified using p-nitrophenol derivative of various length of fatty acids. Among the mutants, S4T mutant showed similar activity to 4-nitrophenyl acetate and 2-fold increased activity to 4-nitrophenyl butyrate when compared to the activity of wild type. Interestingly, S4T mutant showed 10% reduced activity to 4-nitrophenyl octanoate, 20% reduced activity to 4-nitrophenyl decanoate and 21% reduced activity to 4-nitrophenyl palmitate. To elucidate the reason for changed activities, molecular dynamics simulation was used. As a result, S4T showed higher rigidity in loop close to lid domain and C-terminal region than wild type. In addition, overall movement of S4T mutant showed higher compactness and lower solvent accessible surface area than wild type. All of these analysis results indicates the increased overall rigidity of S4T mutant structure in spite of small increase in some local structures such as lid domain and Cterminal region. Structural rigidity of S4T may be one reason for the increased activity to short chain fatty acids and decreased activity to long chain fatty acids.
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