Site-directed Mutagenesis Studies And Bioinformatics Analysis Of Malassezia Globosa Lipase SMG1

Lipases (E.C.3.1.1.3) are enzymes that catalyze the hydrolysis of acylglycerols toglycerol, fatty acid, mono-or diacylglycerol at oil-water interface. They have numerousapplications ranging from oils and fats processing to fine chemical synthesis.Naturallyoccurring enzymes are not directly used for industrial application. Many efforts have beenmade to improve their properties such as thermostability, activity, substrate specificity,organic solvent tolerance and enantioselectivity for their application in industry. There havetwo methods for the modify of lipase: directed evolution and rational design. It has low hitrate to get desired mutants by directed evolution. Therefore, rational design is often used tomodify proteins whose crystal structures or homologue structures are available.The structural analysis of SMG1shows that two bulky aromatic residues, W116andW229, lie at the entrance of the active site. To study the functions of these two residues in thesubstrate recognition and the catalytic reaction, they were mutated to a series of amino acids.Subsequently, biochemical properties of these mutants were investigated. Although theactivities decrease, W229L and W116A show a significant shift in substrate preference.W229L has an increased preference for short-chain substrates whereas W116A has preferencefor long-chain substrates. Besides, the half-lives of W116A and W116H at45°C are346.6min and115.5min respectively, which improve significantly compared to that of nativeenzyme. To evaluate the function of residue at the entrance of active site to enzymaticproperties of Malassezia globosa lipase (SMG1), N277D、N277V and N277F muants weredesigned. N277D mutation improved the half-life of lipase SMG1at40°C from291.7min to366.9min, and N277F mutation resulted in more preference for short-and medium-chainsubstrates. We found that the interactions between A113, W116and H111in the closedform played important roles in the conformational change of the lid as well as the lipasespecific activities. Moreover, the π-stacking interaction between W229and F278is importantin stabilizing the conformation of the lid region and the thermostability of the lipase. Thesefindings not only shed light onto the lipase structure/function relationship but also lay theframework for the potential industrial applications.