Study On The Optimization Of Fermentation Process And Separation With High-speed Contercurrent Chromatography For Rapamycin Production

Rapamycin is, also called as sirolimus, macrolide antibiotics produced by Streptomyces hygroscopicus,which was isolated by Veniza in1975. Rapamycin has been employed as a potential immunosuppressant for clinical treatment. Some of novel derivatives from the chemical modification of its structure has also been found that have new therapeutics roles on the immunosuppressive, anticancer, anti-parkinson’s disease and AIDS. However, due to the low yield, and the complexity of the downstream process, there still faced a tremendous challenge for efficient rapamycin production.In this work, the process for fermentaion and downstream for rapamycin production was investigated with the strain S.hygroscopicus FMT11. From the view of the biosynthesis pathway of rapamycin, different nutrient components were screened in shake flasks. The results showed that feeding CoC12, L-lysine hydrochloride, citric acid, L-lysine hydrochloride, L-tyrosine, and L-Glutamine in the initial medium can increase the titer of rapamycin, respectively. The optimal concentrations of amino acids were determined with the response surface method (RSM), which were L-lysine hydrochloride2%, L-tyrosine0.4%, and L-Gutamine0.3%, respectively. Moreover, in order to strengthen the metabolic flux in rapamycin biosynthesis phase, feeding corn steep liquor0.6%or ammonium sulfate0.2%at72h was also confirmed to increase the rapamycin production. Based on the above results, a novel two-stage feeding strategy was developed successfully to increase the flux of rapamycin biosynthesis, in which the optimized amino acid components were fed at the initial phase of fermentation, and then switched to feed2g/1ammonium sulphate at72h. The maximal rapamycin production and productivity reached860.6mg/1and4.21mg/1h in71fermentor, which was182%and131.3%higher than that of the control, respectively.On the other hand, the method of high speed countercurrent chromatography was attempted to employ for the rapid rapamycin separation from the fermentation broth in the downstream process. The crude rapamycin with acetone and ethyl acetate extracted from mycelium of fermentation broth was separated by HSCCC. The mobile phase was screened, and showed that a solvent system of hexane, ethyl acetate, ethanol and water (7:5:5:5, v/v) with upper phase as the stationary phase, and the lower phase as mobile phase could achieve the better separation for rapamycin. Under the optimized conditions, the recovery ratio was over90%. The purity of rapamycin from the ethyl acetate extract from mycelium was89.97%, and the inpurity was further confirmed by ESI/MS analysis and belonged to its isomeride. The above results showed that the method of high-speed countercurrent chromatography can shorten the separation process of rapamycin and improve the yield of rapamycin.