Mechanistic Study Into The Epoxidation Of Alkenes In Penicillium Camembertii Lipase

Lipases are a group of enzymes which catalyze hydrolysis of lipids. Owing to their thermal stability and tolerance to organic solvent, lipases have wide application in food processing, pharmaceutical and chemical industries. Lipases are able to catalyze epoxidation of alkenes. In recent years, many studies have utilized lipases to synthesize valuable epoxides. However, the mechanism of lipase-catalyzed epoxidation of alkenes has not been clearly stated. In this study, we chose Penicillium camembertii lipase(PCL) as the researched object and investigated the mechanism of PCL-catalyzed epoxidation of alkenes through biochemical experiments, protein structure study and molecular docking. The results are as follows:1.Cloning, expression and purification of PCL and its mutants: we have synthesized the whole gene of PCL and constructed mutants S145 A and H259 A of catalytic triad in the active site of PCL. Both PCL and its mutants had high-level expression in Pichia pastoris. Purified proteins were obtained by anionic exchange chromatography.2.Studies of epoxidation of alkenes catalyzed by PCL and its mutants: PCL displayed high epoxidation activities towards oleic acid and 1-octadecylene. S145 A showed much lower activities toward these substrates compared to PCL, but a fraction of activities were still retained. H259 A did not exhibit any epoxidation activity. ESI-MS analysis showed that both PCL and S145 A catalyzed epoxidation of alkenes through a chemo-enzymatic process. In this process, enzymes firstly catalyzed perhydrolysis of carboxylic acid and forms peracid, after which the released peracid epoxidized alkenes automatically. Results showed that the epoxidation activities of PCL were not affected by pH, while activities of S145 A were much more susceptible to pH changes. Besides, the epoxidation activities of S145 A were more sensitive to changes of hydrogen peroxide concentration, compared to PCL.3.Structural biological studies of PCL: We have determined crystal structure of PCL in high-resolution by protein crystallization experiments. Crystal structure of PCL has been deposited to Protein Data Bank with ID 5CH8. Based on this structure, we have constructed modelled structure of PCL-hydrogen peroxide-octanoic acid complex by molecular docking of computer simulation.4. Mechanistic studies into PCL-catalyzed epoxidation of alkenes: Taken all the biochemical results and the structural model together, we proposed that lipase-driven production of peracid is a two-stage synergistic process. One stage is the formation of acyl-enzyme complex by the active site Ser145, which is a canonical mechanism. Another stage is a mechanism mediated by the active site His259, which is proposed and proved by us. His259 is a critical residue to the epoxidation activity of PCL. It either strip proton from Ser145 or hydrogen peroxide in the two stages. Although Ser145 is not the critical residue to enzyme function, it dramatically improves epoxidation activity of PCL.In this study, we proposed the mechanism of PCL-catalyzed epoxidation in detail at the molecular level through biochemical experiments, structural biological studies and molecular docking. These results are of great significance to the engineering and selecting potential enzymes in epoxidation industry.