The Study Of Transfer Pathway And The Process Of Converting Lovastatin Into Wuxistatin By Amycolatopsis Sp.ST2710

Wuxistatin was a inhibitor of 3-hydroxy-3-methylglutary(HMG)-CoA reductase founded by our laboratory which was the rate-limiting enzyme in the process of cholesterol synthesis. Amycolatopsis sp.ST2710 could convert lovastatin into wuxistatin. According to our research, the first product named productâ… was with the group of 3-CH₂OH which had a–OH added. Then wuxistatin, which was isomeric with the first product, appeared. There were also some relevance in production between them. According to these phenomena, the transfer pathway of wuxistatin were inferred. The primary target of this reseasch is to prove the transferpathway of wuxistatin and improve the process of fermentation.For further studies of transfer pathway, pure product was indispensable. Therefore, we optimized the method of sepration: HP 20 macroporous resin was employed to extract the intermediate productâ… from the fermentation broth. The results of static experiments showed that HP 20 macroporous resin had the highest adsorption capacity of 28 mg(the intermediate productâ… )/g(dry resin). The optimum adsorption time was 7 h. Dynamic experiments showed that within 50%-60% methanol solutio, the elution contained only the intermediate productâ… . And the suitable loading concentration was about 1 g/L. The optimal flow rate of loading sample was 1.5 BV/h. The optimal flow rate of elution was 1.0 BV/h.By methanol concentration gradient, colorless bioconvension products was collected. The relative purity of productâ… could reach 95%.In order to find out the transfer pathway, we preliminarily proved those assumptions by fermentation test and found that wuxistatin could be producted by productâ… . On this basis, we introduced the stable oxygen isotope tracer technique to find that the oxygen atom of hydroxy on wuxistatin came from productâ… which further proved the hypothesis.According to the results mentioned above and the characteristic of fermentation, a new model of fermentation was advanced- two-step strategy: Firstly, productâ… was formed from lovastatin by hydroxylation, pH 7.5; then the conditions of fermentation were changed to be more suitable for transferring productâ… to wuxistatin, pH 5.5. In order to achieve more wuxistatin, the fermentation conditions of transferring productâ… to wuxistatin was researched. The optimum conditions such as inoculum size, temperature, pH and shaking frequency were 10%, 30℃, 5.5, 120 r/min separately. Furthermore, some metal ions'effects were studied. We found out that Mg2+,Fe2+ and Cu2+ were conducive to the process. By orthogonal experiment, the optimum fermentation conditions were determined: Fe2+,Cu2+,Mg2+ were separately added 1.429,0.040,0.428 mmol/L into the culture-medium. By this way, the convertion would increase, the time was shorten by a big margin and the final product would be more easily to purify. The fermentation pattern that based on one strain could provide reference for the study of fermentation with intermediate product and long-time fermentation.