The Preliminary Study On Bioconversion Of Lovastatin To Wuxistatin By Amycolatopsis Sp. ST2710

Statins, which were inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase, were the prime drugs of cardiovascular diseases. They were the focus of most research at home and abroad. Wuxistatin, discovered by our research group, was a novel inhibitor of 3-hydroxy-3-methyl-glutaryl(HMG)-CoA reductase, which was a rate-limiting enzyme of cholesterol biosynthesis. Lovastatin could be converted into wuxistatin by Amycolatopsis sp.ST 2710. This paper mainly presents the process of bioconversion of lovastatin into wuxistatin by Amycolatopsis sp.ST 2710 and the enzymes involved in the bioconversion. Besides, the optimization of the fermentation conditions of lovastatin bioconverting to the intermediate product was also studied.According to the comparison between the structures of the metabolic products, three hypotheses of mmtabolic mechanisms were brought forward to test the possible metabolic pathways. During the further study of the metabolic pathway, the concentrations of lovastatin, the productâ… and wuxistatin during the fermentation process were investigated. The hypothesis of metabolic mechanism of wuxistatin was initiately determined according to the change of concentration of the intermediate product. Inhibitors of enzymes were used to block the bioconversion of lovastatin into wuxistatin. The hypothesis of metabolic mechanism of wuxistatin by Amycolatopsis sp. ST2710 was inferred as follows: lovastatin was hydroxylated into the productâ… , and then the intermediate productâ… was isomerized into wuxistatin.The bioconversion of lovastatin into the intermediate productâ… was the first step of the whole process and was catalyzed by hydroxylase. The method for detemining hydroxylase was to establish by cell-free extract. The results showed that NADH, acted as coenzyme, provided H+ necessary for the reaction. The optimal pH and the temperature were 7.5 and 30℃, 150 r/min, reaction time was 6h.. The hydroxylase which catalyzed lovastatin into the intermediate product was inferred as cytocrome P450 based on the facts that the hydroxylase in the cell extraction was intra-cellular inducible enzyme and could be obviously inhibited by CO. Respectively,ATP, Fe²⁺ and ascorbic acid were active to the reaction.High purity of the productâ… was necessary for further study of the bioconversion of the intermediate product into wuxistatin. To obtain more productâ… , the optimization of the fermentation conditions was also studied. The optimal inoculum size, temperature, pH, shaking frequency and the concentration of lovastain were 10%, 30℃, 7.5, 120 r/min and 0.8 g/L, respectively. The effects of metal ions to the bioconversion were carried out. The results showed that Mg²⁺,Fe²⁺,Cu²⁺ were positive to the bioconversion. Besieds, the effects of the three metal ions to the fermentation were studied further by orthogonal experiment and the optimal fermentation conditions were determined. The optimal fermentation conditions can been received when Mg²⁺,Fe²⁺,Cu²⁺ were respectively added 0.857,0.040,0.257 mmol/L.