Optimal Expression,Purification And The Primary Study On Characterization Of CYP119:A Cytochrome P450 From The Sulfolobus Solfataricus

Objective:To optimal expression and purification conditions of cytochrome CYP119 from the Sulfolobus Solfataricus,identified the activity and research the kinetics of CYP119 enzyme,which oxidize styrene in a H2O2-denpenden at 35? or 70? system,respectivly.Methods:The two kinds of recombinant strain,which is constructed by recombinant plasmid PET-30a-CYP119 and host strain BL21(DE3)plysS or Rosetta repeatability by heat shock transformation andidentified by genetic engineering,were expressed in small culture medium.By using the 12%SDS-PAGE method to analysis the expression products,we had selected the better positive colonies containing the correspondding host strain and the best expression yield single colony as a starting strain.Then,we had determined the best culture medium,induction starting time,induction temperature,inducer concentration and induction time.The expressed product was purified by comparing the method of pH gradient elution and Imidazole gradient elution.Under the optimal condition to induce 1L engineering bcteria,the target protein was purified and concentrated and identified by UV spectrophotometer from 250 nm to 700 nm,as well as the reduced CYP119 enzyme.The result of CYP119 enzyme-catalyze the epoxidation of different concentrations of styrene in H2O2-dependent,for the same time,at 35? or 70?,respectively,were identified by HPLC.And the enzyme kinetic parameters were determined by Lineweaver-Burk polt.Results:When using TB containing trace element as the medium and adding final concentration of 0.4 mmol/L of IPTG into the growth bacteria for 3.5 hours to induce for 45 h at 32?,the expression product,with a relative molecular mass of about 43 kDa,was purified by Imidazole gradient elution,which had the less other protein and more quickly than the pH gradient elution.The yield of the target protein was 25.14 mg/L.Identified by UV spectropHotometer,the peak was 415 nm,and A415/A280=1.34.the characteristic peak drift to 450 nm when reduced CYP119 enzyme was subjected to CO.The values of vmax= 0.196 mmol·min-1,km= 8.38 mM and kcat=15.69 min-1 at 35? system,while the vmax= 0.978 mmol·min-1,km= 9.07 mM,kcat=78.24 min-1 at 35 ? system were found,in H2O2-dependent.Conclusion:We had established the high level expression and purification condition of CYP119.By establishing the excellent system of CYP119 enzyme-catalyze the epoxidation of styrene in dependent H2O2 at 35? or 70? system,the enzyme kinetic parameters is obtained.The 70? is better.