Study On The Purification And Characterization Of P450 Oxidase CYP-c13 From Colletotrichum Lini

3?,7?,15?-Trihydroxy-5-androsten-17-one?7?,15?-diOH-DHEA?is a key precursor of the novel oral contraceptive“Yasmin”,which can be obtained by microbial dihydroxylation in the C7?and C15?positions of dehydroepiandrosterone?DHEA?.In the early stage of this laboratory,a P450 oxidase CYP-c13 that can dihydroxylate DHEA to produce7?,15?-diOH-DHEA was found from Colletotrichum lini ST-1,and initially realized heterologous expression in Pichia pastoris.However,there are still problems such as the low enzyme activity and unclear dihydroxylation mechanism of CYP-c13.In this paper,the recombinant Pichia pastoris was used as the starting strain,and the enzyme activity of CYP-c13 was further improved through optimizing the fermentation conditions.On this basis,the pure enzyme of CYP-c13 was obtained by purification,and the enzymatic properties of P450 oxidase CYP-c13 were systematically studied.The main findings are as follows:?1?Optimization of fermentation conditions of recombinant Pichia pastoris.The fermentation conditions of recombinant Pichia pastoris were optimized through single factor experiments and orthogonal array design method.The optimal fermentation conditions were determined as follows:induction pH 8.0,glycerol concentration 1.5%,methanol concentration 1.5%,YNB content 1.6%,100 mL/500 mL of the liquid volume and 72 h of the induction time.Under this best fermentation conditions,the enzyme activity of CYP-c13increased from 4811 U·mL^-1 to 7392 U·mL^-1,which was 1.5 times before optimization On this basis,the transformation curve of the optimized recombinant P.pastoris for DHEA was studied.The results showed that the C7?position of DHEA was first hydroxylated by the recombinant P.pastoris to generate the monohydroxy product 7?-OH-DHEA,and then the C15?position of 7?-OH-DHEA was hydroxylated to produce the dihydoxylation product7?,15?-diOH-DHEA.?2?Purification of P450 oxidase CYP-c13.The recombinant Pichia pastoris crude enzyme was subjected to purification steps such as ammonium sulfate fractionation,Ni-NTA affinity chromatography and Superdex G-75 gel filtration chromatography,and successfully obtained pure CYP-c13.The specific enzyme activity of CYP-c13 increased from 88.4 U·mg^-1to 447.6 U·mg^-1,the purification fold was 5.1 times,and the recovery rate was 2.8%.?3?Studies on characterization of P450 oxidase CYP-c13.First,the effects of temperature,pH and metal ions on the activity of CYP-c13 were studied.The results showed that the optimal temperature of CYP-c13 was 30oC and the optimal pH was 7.0.At low concentrations,metal ions Na⁺,K⁺,Mg²⁺and Mn²⁺slightly promoted the CYP-c13;at high concentrations,all metal ions have different degrees of inhibition on the CYP-c13.The substrate specificity study of CYP-c13 found that it could hydroxylated five steroids including progesterone,androstenedione,epoxyprogesterone,testosterone and canrenone.Among them,the androstenedione,epoxyprogesterone and canrenone could be dihydroxylated by CYP-c13.?4?The kinetic parameters study of CYP-c13 with substrate DHEA and by-product7?-OH-DHEA.The K_m and k_cat values of CYP-c13 for DHEA and 7?-OH-DHEA were determined.The results showed that CYP-c13 had a greater affinity and higher catalytic efficiency for DHEA than 7?-OH-DHEA On this basis,the reason for the difference in the affinity between CYP-c13 with DHEA and 7?-OH-DHEA was preliminarily analyzed through calculation and simulation.