| 11α-Hydroxycanrenone is a sort of steroids, which is the pharmic intermediate for the use of synthesizing eplerenone-a new selective aldosterone receptor antagonist with cardiovascular protective effect. Aldosterone receptor antagonist has effect of treating cardiovascular disease; decreasing the frequency and death rate by blocking process of Renin-Angiotensin-Aldosterone System (RAAS) and the selective one is more effective than the unselective one. In the process of semi-chemical synthesizing eplerenone from canrenone, 11α-hydroxylation catalyzed by microorganism is the first step and one of the most key steps. Therefore the first key step of synthesizing eplerenone is the 11α-hydroxylation of canrenone by microbial transformation. Study on preperation of 11α-hydroxylation by microbial transformation was performed in this dissertation, including isolation of strains for the enzymatic reaction, preperation of 11α-hydroxylation of canrenone by microbial transformation and structural characterization of the product, optimization of the technological factors of preperation process of 11α-hydroxylation by Aspergillus ochraceus NG0216, and purification of 11α-hydroxycanrenone. Furthermore, 11α-hydroxy-transformation of canrenone has been studied using immobilized whole Aspergillus ochraceus NG0216 cells too. Results of study on the 11α-hydroxylation showed that rate of transformation by Aspergillus ochraceus NG0216 was better than Rhizopus stolonifer NG0305 and Trichothecium roseum NG0207. Structural characterization by HPLC-MS, 1H-NMR,13C-NMR,HMBC 和COSY verified the product as 11α-hydroxycanrenone Optimal culture medium of Aspergillus ochraceus NG0216 for 11α-hydroxylation was as follows: glucose 25 g/L, C.S.L 28 g/L, yeast extract 3 g/L, MgSO4·7H2O 0.9 g/L, pH 6.0. In addition, some operations could enhance the rate of transformation remarkably, such as, substrate canrenone was made into suspension with water and treated with ultrasonic, before the transformation of canrenone 5 h, low concentration of canrenone (0.25 g/L medium) was added for inducement. Optimal temperature of cultivation is 28 ℃, optimal temperature of transformation is 26 ℃. Especially it was necessary for hydroxylation of canrenone that glucose was supplied intermittently for keeping constant content in the medium. Transformation of canrenone by NG0216 on the scale of 5 L and 50 L fermenter were performed with optimal medium and optimal operational condition. Concentration of canrenone in 5 L and 50 L fermenter was 20 and 15(g/L medium) respectively, the rate of transformation reached 98.9 % and 97.6 % respectively after transforming for 72 h, the cycle of transformation was shortened about 24 h than that in shaking flask. Investigation of hydroxylation of canrenone was catalyzed by immobilized whole cell of aspergillus ochraceus NG0216 showed that the optimal carrier was 4% sodium alginate loading 15% wet cells and solidified in 4% calcium chloride for 1 h. Optimal concentration of transformed canrenone was 8 g/L. Optimal pH and temperature were 6.0 and 28 ℃. The immobilized cells could transform three time for the batch hydroxylation of canrenone under the optimal condition and the rate of transformation could exceed 85% every time. Isolation and purification of 11α-hydroxycanrenone were studied at last. After solid-liqued separation, the mycelium of Aspergillus ochraceus NG0216 was subjected to extraction with ethyl acetate, and the ethyl acetate mixture was boiled with circumfluence for twice for condensation (Dosage of ethyl acetate is 100 mL per 1.5 g canrenone added, 50 mL every time). Time of extraction was 60 min each time; rotary velocity of the stir was 300 r/min. Then the raw product was obtained through evaporation, the yield rate of product was 73.6 %. The purity of 11α-hydroxycanrenone was gained from 93.4 % to 99.7 % after raw product purified by third re-crystallization in ethyl acetate, the purified crystals appeared canary acicular, its melting point was 240 ℃, its [α]2D0 in ethyl acetate is -12.825°whenthe concentration was 5.000 g/L. |
