Breeding Of Resistance Mutants And Analysis Of P450 Enzyme By Curvularia Lunata

In this paper, the relationship between the cytochrome P450 enzyme and the 11 -hydroxylation of steroids of Curvularia lunata,the screening of high production strain,optimum fermentation process and conditions were studied. The main research contents and results are as follows:The cytochrome P450 was determinated in the protoplasts of Curvularia lunata by means of CO-difference spectrum method SPB , MEHP and RSA could induce the expression of cytochrome P450. Cytochrome P450 was inhibited by ketoconazle. The hydrocortisone formation rate was belonging to the cytochrome P450 content.The optimal conditions for protoplasts formation and regeneration of Curvularia Lunata were observed: mycelia of Curvularia lunata cultured for 22h were harvested and suspended in 0.3mol/L DTT for 20min. The protoplasts yield achieved 7.76 106/mL under 30 C with 0.5% novozym 234 and 1% cellulase (pH5.6) using 0.6mol/L KC1 as osmotic stabilizer in 3h. About 23.07% of protoplasts were capable of regeneration, meanwhile the morphology and the 11 -hydroxylation ability of steroids by colonies regenerated were the same with the parent strain.The protoplasts from Curvularia lunata CL-114 were treated with NTG and ultraviolet ray to improve the efficiency of 11 -hydroxylation. Mutant KA-91 which carried about the markers of ketoconazle resistance was obtained. The hydrocortisone conversion rate and yield ratio were 63.05%. The genetic markers had been very stable after subcultured 5 times.The optimal fermentation condition of KA-91 in shake flask was established: fermentation medium(g/L): glucose 22, beef extract 5, yeast 4; growth and hydroxylation pH 6.5; 30mL medium in 250mL flask; shaker speed 0~6h:160 r/min and 6~68h:200 r/min; fermentation period 72h. The hydrocortisone conversion rate was 74.88% based on the improved fermentation process and the optimal RSA adding method while the RSA addition content 1 .0g/L.Based on the optimal fed-batch fermentation condition in shake flask, the fed-batch fermentation in 7L fermentor was also observed. The hydrocortisoneconversion rate was 77.92% within 68h.