The Study Of Asymmetric Epoxidation To Cis-anethole Derivatives Catalyzed By P450 Enzyme

Objective:Anethole occurs naturally allylbenzene derivatives as a major component of essential oils from fennel and staranise and is also present in numerous plants such as dill,basil,and tarragon.And it is often used as a natural flavoring and aromatic compounds because of its special aroma.Recent studies show that it has cytotoxic effect on many tumor cells.In addition,its oxide not only produced a positive result in inducing tumors in mice,but also have cytotoxicity to rat hepatocytes which is due to its epoxide.It is showed that the epoxide may have some important biological activities,so it is necessary to synthesize a series of epoxide and study their biological activities.At present,the chemical methodthe is main for this kind products,but it is limited because of many problems such as by-products,low yield,heavy environmental pollution,heavy metal residues.The biocatalytic reaction that has high substrate specificity and mild reaction conditions is becoming an ideal green catalytic reaction.CYP119 enzyme is a kind of P450 enzyme which has the ability to asymmetric epoxidation of styrene and its analogues.And there is no report for the study on the epoxidation of these compounds by CYP119,so we tried to use the CYP119 enzyme and its mutants to catalyze the epoxidation of anethole and its analogues.Methods:Obtaining a series of mutants by rational design and fixed point mutation technology and the enzyme was isolated and purified.Next,the asymmetric epoxidation was catalyzed by the wild-type CYP119 enzyme and the mutants T213G,T214V that has been constructed by our laboratory and the new mutants T213M and T213Y,and studying the kinetic properties of this reaction and the catalytic ability of the mutants.Results:In this study,the mutants T213M and T213Y were successfully constructed.Comparing the ability of the asymmetric epoxidation of a series of mutants to cis-anethole analogues and the mutants T213M and T213Y has high stericselectivity.The cis-?-methyl styrene was catalyzed by the mutants T213M and T213Y,the kinetic constant of the T213M K_m=5.23±1.2,K_cat=32.93±3.9,the kinetic constant of T213Y K_m=7.33±0.670.K_cat=04067±0.002;In addition,The products catalyzed by this two mutants were 1S,2R configuration,in which the T213M ee is up to 90.6%,the conversion reached 74.7%,the cis-anethole ee is 83.5%,the conversion is 74.7%.Conclusion:It is beneficial to improve the selectivity of CYP119enzyme on the asymmetric epoxidation of cis-anethole by introduce two large polar amino acids to the 213 site.It is the highest ee that the epoxidation reaction of these substrates catalyzed by the P450 enzyme.And it is important for the asymmetric epoxidation of cis-anethole,it also provides a better evolutionary template for improving the catalytic performance of CYP119,and it is helpful for us to further study the reaction mechanism of CYP119 and the relationship between the substrate and the structure of the enzyme.