Directed Evolution Of Cytochrome P450 119 And Catalytic The Asymmetric Epoxidation Of Styrene

Objective:In the human body cytochrome P450 enzyme is mainly found in liver which participate in the numerous types reaction of life,including drugs,alcohol,other xenobiotics' metabolism,steroid fatty acid,prostaglandins and other end ogenous compounds metabolism and synthesis.P450 monooxygenase is the candidate in organic synthesis which is the potential biological catalyst.CYP119 is an acid heat resistant P450 enzyme.The gene sequence and the crystal structure of CYP119 have been confirmed.Although the endogenous substrate for CYP119 is not known,it has been confirmed that the enzyme can catalyze the hydroxylate of lauric acid.The wild type CYP119 can catalyze epoxidation reaction of styrene in the presence of hydrogen peroxide.Technology of directed evolution is good for changing the structure and the activity of the enzyme.In the present study,the mutant library of CYP 119 though error prone PCR was constructed.After expression and purification,the mutants T213G/T214V,T214V,T213G were used for catalysising epoxidation of styrene and styrene derivatives Dynamic properties,conversion rate and enantioselectivity of the mutants T213G/T214V,T214V and T213G were described.Methods:5 groups of error prone PCR products with conditions of Mn2+ concentration 0 mmol/L,0.05 mmol/L,0.1 mmol/L,0.2 mmol/L and 0.4 mmol/L were connected with the PMD18-T vector and transformed into DH5a and sequenced.The rate of mutation were collected and the optimal condition for constructing the mutation library were getted.The purification products of error prone PCR were also connected whith PET30a vector,transformed into E.coli BL21(DE3)cells,then constructed the expression library.The mutant enzymes T213G/T214V,T214V,T213G were expressed and purified,ancd catalyzed the epoxidation of styrene and its derivatives.The products were analysised by high performance liquid chromatography to test these enzymes,kinetic properties and conversion.GC-MS with chiral column was used to test the enantioselectivity.Results:The best condition for constructing the library is dATP 0.2 mmol/L,dGTP 0.2 mmol/L,dTTP 0.8 mmol/L,dCTP 0.8 mmol/L,Mn2+ 0.2 mmol/L.The base mutation rate is 1.4725%,and sense mutation rate is 30%.From the sequencing results we known thal S40A,S221T,Y16C,I208T,Y16C,I208T,R279Q E296A,R328G,T214V,T213G,T214V/T213G sense mutation have been earned,and these mutations may have certain effects on enzyme's properties.Products were analysed by HPLC and GC-MS.Three mutants can keep good thermal stability,and its activity in high temperature is highe than which in room temperature,but the enantioselectivity is decreased when in high temperature.The mutants of T213G,T214V's Vm,kcat and the enantioselectivity were better than wild type enzyme in 35? and 70 ?.The substrate affinity and catalytic activity of T213G/T214V is lower than that of wild type enzyme both at room temperature and high temperature,but the enantioselectivity is slightly better than that of the wild type enzyme.The results suggested that T213,T214 site are important for the enzyme's catalytic activity and the enantioselectivity.T214V,T213G both increasec the enzyme's activity,but when the two site mutation at the same time,catalytic activity decreased,which indicated that 213 and 214 sites threonine must keep one of them in order to ensure the enzyme activity.Conclusion:When we construced CYP119 mutant library the best condition is the concentration of Mn2+0.2 mmol/L.Catalytic activity of T213G is higher than the wide type and T214V.Catalytic activity of T213G/T214V is decreased but enhanced the enantioselective.