Studies On The 11β-Hydroxylation Of Steroids And The Cytochrome P450 Enzyme Of Curvularia Lunata

The dissertation focused on the relationship of the cytochrome P450 enzyme and the 11 -hydroxylation of steroids by Curvularia lunata, the mutation and screening of high production strains, the fermentation process optimization, the optimal RSA addition method and the effect of hydrogen peroxide on the steroids conversion. The main research contents and results were as follows:[1] The HPLC method for the determination of hydrocortisone and relational constituents in the culture broth was improved. The HPLC was performed on a Phenomenex Prodigy Silica (5 m, 250mm 4.6mm i.d.) column, by dichloromethane-ether-methanol-water (385:60:30:2, V/V) as mobile phase. The UV detection wavelength was 242nm. The flow rate was 0.8mL/min. This method was accuracy, delicacy and reproducible. The determination period was 15min.[2] The optimal conditions for protoplasts formation and regeneration of Curvularia Lunata were established: mycelia were harvested at 22h and suspended in 0.3mol/L DTT for 20min. And then treated with 0.5% novozym 234 and 1% cellulase (pH5.6) using 0.6mol/L KC1 as osmotic stabilizer at 30 C for 3h, protoplasts were harvested at the concentration of 7.76 106/mL. The regeneration rate of the protoplasts was 23.07%. The strains regenerated remained the ability of 11 -hydroxylation of steroids.[3] The cytochrome P450 was determinated in the protoplasts of Curvularia lunata by means of CO-difference spectrum method Cytochrome P450 could be induced by SPB and MEHP and be inhibited by ketoconazle. Cytochrome P450 was demonstrated as an intracellular enzyme. The cytochrome P450 content had positive relationship with hydrocortisone formation rate.[4] A hydrocortisone high-producing mutant KA-91, which carried about the markers of ketoconazle resistance, was obtained by NTG and UV mutation. Cultured at the original fermentation conditions for 72h, the hydrocortisone conversion rate and yield ratio were 63.05% and 53.3%, respectively, with the RSA addition content 1.0g/L. The genetic markers had been very stable after subcultured 5 times. According to the results, the mechanism of mutation was analyzed. It was probably that the ketoconazle resistance of C. lunata was due to the excess expression of CYP51.[5] RSA could induce the expression of cytochrome P450. Based on the induction of RSA to cytochrome P450 and the effect of pH on the production of hydrocortisone, a novel fermentation process was established. After inoculation, 0.3g/L RSA was added at 16h, followed by the addition of 0.7g/L RSA at 24h. pH was adjusted to 6.5 at 32h and kept at this level by adjusting every 12h. The new fermentation process improved 11.16% expression of cytochrome P450 and 12.67% production of hydrocortisone within 72h.[6] The effect of organic solvent dissolution, surface active medical Tween80 dispersion, polymer silicone milking, ultrasonic wave dispersion and -cyclodextrins inclusion on the dissolutionABSTRACTof RSA were studied. The optimal RSA addition method was performed. P -cyclodextrin (P -cyclodextrins/RSA 1:1, mole ratio) was added to RSA blended with 1.2% TweenSO, followed by a 20 minutes sterilization.[7] The optimal fermentation conditions of KA-91 in shake flask were established. Fermentation medium was composed with glucose at 22.5 g/L, beef extract at 5.5 g/L, yeast extract at 5.0 g/L and KH2PO4 at 3.0 g/L. 30mL medium was cultured in 250mL flask, with the shake speed at 160 r/min from the beginning to 6h and at 200 r/min from 6h to 72h. The fermentation time was 72h. Based on the optimized fermentation conditions and the optimal RSA addition method, the hydrocortisone conversion rate was 74.88% with the RSA addition content at l.Og/L.Based on the optimal fed-batch fermentation conditions above, the fed-batch fermentation in 7L fermentor was also studied. The hydrocortisone conversion rate reached 77.92% within 68h, with the RSA addition content at l.Og/L.[8] The effect of addition hydrogen peroxide on the production of hydrocortisone during IIP -hydroxylation of steroids was studied. The expe...