| 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 grounp, was a novel inhibitor of 3-hydroxy-3-methyl-glutaryl(HMG)-CoA reductase, which was a rate-limiting enzyme of cholesterol biosynthesis. Amycolatopsis sp.ST2710 could convert lovastatin into wuxistatin. In this paper, we made search about the optimization of the fermentation conditions of lovastatin bioconverting into wuxistatin by Amycolatopsis sp.ST2710, and purification of the bioconversion product (wuxistatin) by macroperous adsorption resins.The effects of all kinds of fermention conditions on its myceliuim morphology in shake flask were studied. In the flask conditions, the initial pH of the medium, medium volume, inoculating mode, the inoculation concentration, the shear rate were the factors affecting myceliuim morphology. The result showed that uniform mycelium pellets(pellet diameter of 0.5 to 0.7 mm)enhanced the transformation of lovastatin. And we obtained that the shear was the key factor affecting the bioconversion rate of lovastatin.The concentration of lovastatin for Amycolatopsis sp.ST2710 was studied, the critical inhibitory concentration of lovastatin for Amycolatopsis sp.ST2710 was 1.5 g/L, and the suitable concentration of lovastatin was 1 g/L. The best temperature for the period of transforming lovastatin was 28℃, consisting with the optimum temperature of myceliuim growing. Based on the myceliuim morphology, the best optimized fermentation conditions were as follows: initial pH 7.0~8.0, medium volume 25 mL/250 mL shake flask, the inoculum in the form of spore, inoculum 10%, shaking rate 120 r/min. On the best optimized flask conditions, the yield could reach 0.48 g/L.To enhance the yield of wuxistatin by Amycolatopsis sp.ST2710, several fermentation parameters of batch fermentation at different agitation rates from 100 to 400 r/min in the 5 L fermentor were investigated. It was found that the dissolved oxygen increased and the lag stage of cell growth could be shortened with controlling at the agitation rate of 300 r/min. On the other hand, at the lower agitation rate of 200 r/min, the foam was decreased at the stage of bioconversion and the bioconversion rate of lovastatin was promoted. Based on the parameters analysis, a two-stage agitation rate controlling strategy, in which agitation rate was controlled at 300 r/min in the stage of cell growth and then shifted to 200 r/min, was suggested and experimentally verified. Following this strategy, the following results were obtained that the biomass was 4.02 g/L at the end of the stage of cell growth, the concentration of wuxistatin reached 0.50 g/L in culture filtrate and the whole fermentation time in 5 L fermentor was 80 h, which was shorter 16 h than in shake flask. In fed-batch culture, we effectively affected mycelial shape and increase yields of wuxistatin by controlling the concentration of lovastatin. While using fed-batch process with lovastatin, the yield could be increased to 1.2 g/L, which was up to 2.4 times higher than that of normal batch mode.Five kinds of macroporous adsorption resins were used to extract wuxistatin from fermented broth. The static experiments showed that HP20 macroporous resin had the highest adsorption capacity of 89.89 mg(wuxistatin)/g(dry resin), the optimal pH of adsorption was 7.0, and the optimum adsorption time was 3 h. Dynamic experiments showed that in the optimal flow rate of fermented broth of 1 BV/h, the adsorption ability of HP20 was 60 mg wuxistatin/ml wet resin, and the suitable concentration was 0.9 to 1.1 g/L. By acetone concentration gradient, we got colorless bioconvension products, which recovery and concentration could reach 75% and 95%. |
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