Study On Hydroxylation Of Oleanolic Acid By Monooxygenase In Fungi

Oleanolic acid, a natural pentacyclic triterpenoid compound, possesses multi-pharmacological activities including hepatoprotection, antihyperglucemia, antivirus, and so on. But its poor bioavailability and simple pharmaceutical form limits its application and development. Molecular reforming of oleanolic acid could enhance its biological activities and efficacy, resulting in protection of environment and innovation of new drugs. The present dissertation is to obtain the C₁₁-hydroxyl oleanolic acid by microbial transformation so as to enhance the bioactivities of oleanolic acid and provide possibility of further chemical modification.First, a strain with the capacity of enzymatic biotransformation of oleanolic acid, Aspergillus ochraceus NG0216, was selected from several fungi strains preserved in our lab; after ion beam implantation on Aspergillus ochraceus NG0216, Aspergillus ochraceus NG1203 possessing good genetic stability was selected with higher ability of hydroxylation than the original strain, and the conversion rate increased from 2.29% to 6.74%. Structural characterization of the biotransformation product was conducted by HPLC, MS, ¹H-NMR and ¹³C-NMR, and the results indicated that the product was 11α-hydroxy oleanolic acid.The mechanism of oleanolic acid hydroxylation was studied by investigation of the effects of feeding glucose and H2O2. The results showed that this hydroxylation process was catalyzed by monooxygenase; feeding 10 g/L glucose each day after 48 h cultivation could gain the conversion rate; continuously feeding H2O2 solution to the broth was started as the cell's incubation 96 h upto its final content 5 mmol/L, and the conversion rate was kept at a high level.Then, the whole hydroxylation process was investigated. Optimal culture medium for hydroxylation was as follows: glucose 20 g/L, corn steep liquor 26 g/L, yeast extract 3 g/L, K₂HPO₄ 1.5 g/L. In this optimum culture medium, addition of cosolvent and inducement of hydroxylase could enhance the conversion rate. The conversion rate of hydroxylation was raised to 13.24% under condition that the content of ethanol in the medium was 3% (V/V), 2 mg/L oleanolic acid as inducer was fed after 20h incubation, and substrate was fed in batch after 24 h.Finally, enzymatic transformation in two-phase aqueous/organic system was...