Study On The Cultural Condition Of The Cardiovascular Drugs Canrenone Into C₁₁α-hydroxylated Product By Aspergillus Ochraceus

The introduction of an oxygen atom in the 11α-site of the steroid molecule would enhance the ability of anti-inflammatory effectively.However,owing to the presence of multiple chiral centers,the hydroxylation of steroids in a chemical route must be realized in a multiple step.In addition,the drawbacks of complicated process,high cost,presence of side reactions and toxic organic solvents would limit the wide use in the industry.Up to now,the hydroxylation of steroids by microorganisms has become a key technology in the biotransformation.11α-hydroxyl-canrenone is a diuretics and mineralocorticoid receptor antagonists,which has been used for therapy of edematous states such as congestive heart failure and for treatment of essential hypertension.Aspergillus ochraceus is an important strain to produce hydroxylcanrenone,as the role of cytochrome P-450 monooxygenase in the cell.In the present study,shake flask level was selected to investigate the hydroxylation of canrenone catalyzed by Aspergillus ochraceus,and then the conditions were optimized by combining single factor test,Plackett-Burman and response surface method.The Aspergillus ochraceus was first cultured in 250 mL shake flask at 28℃and 200 rpm.The wet mycelium was obtained by filtration in vacuo after the fermentation,and then 50 mL of ethyl acetate was added and shaked for 1-2 h.The crystallization and recrystallisation were preformed to obtain the product,the purity of which was 96.4%detected by HPLC.IR and NMR spectra revealed that the hydroxylation site was C₁₁α.UV-visuable spectrum showed that theλ_maxwas at 286 nm. Furthermore,the optimization of the cultural condition was performed to increase the conversion of canrenone.Through single factor test, Plackett-Burman and response surface method,we got the optimum conditions. The optimal cultural and bioconversion media were(g/L):glucose 26,solid corn liquid 30,yeast extract 6,peptone 4,ammonium sulfate 0.5,ammonium nitrate 0.5,dipotassium phosphate 6,magnesium sulfate 0.15,distilled water 1000 mL.The optimal reaction conditions were as follows:the optimal reaction system was 1 media in 5 Erlenmeyer flask;the optimal temperature of the cultivante stage was 28℃and the temperature of the biotransformation stage was 25℃;the optimal rate of rotation was from 160 to 200 rpm;the optimal inoculum size was 4×10⁷ spore in 50 mL media;the initial pH was 6.7.The investigations of substrate induction and the feeding fermentation indicated that conversation was dramatically raised by the substrate induction and feeding fermentation.In all,the conversation of canrenone exceeded 90% under the optimal conditions,above than using the basic medium.As our research showed,the inexpensive medium components and the fermentation conditions were achieved by biology optimization design.These provide integrated theoretic foundation for the industrial production of 11α-hydroxyl-canrenone.