Separation And Purification Of Brefeldin A From The Fermentation Broth Of Eupenicillium Brefeldianum CCTCC M 208113

Brefeldin A(BFA)is a natural macrolide lactone antibiotic.Due to its antifungal activity and potential as a lead compound for anticancer drug development,brefeldin A production through microbial fermentation and chemical synthesis attracted great attentions.However,few documents concerning brefeldin A separation and pruifiication were available.In this thesis,brefeldin A adsorption on macroporous resin and brefeldin A crystallization were studied in detail,followed by the process development of brefeldin A separation from Eupenicillium brefeldianum CCTCC M 208113 fermentation broth.Thermodynamic and kinetic characterization of brefeldin A adsorption on macroporous resin HZ830 was conducted.Results indicated that the adsorption process was spontaneous,endothermic,and Freundlich model fitted the static equilibrium adsorption data well.Kinetic study revealed that the adsorption process followed the pseudo-second order model,and the adsorption rate was controlled by the liquid film diffusion resistance and pore diffusion resistance.In terms of the recovery and the shape of brefeldin A crystals,anti-solvent crystallization was preferred among the three crystallization methods,in which ethanol was chosen as the original solvent and water was used as the anti-solvent.Solubility and super-solubility of brefeldin A in different solvent systems was determined.After analysis of the metastable zone,it was found that increasing the stirring speed,seed loading size and anti-solvent imput rate decreased the metastable zone width.Kinetics study showed that raising stirring speed significantly increased nucleation rate and crystal growth rate.Based on the above mentioned outcomes,an efficient purification process of brefeldin A from E.brefeldianum CCTCC M 208113 fermentation broth was developed,consisting of four operations:(1)Solid-liquid separation of the fermentation broth;(2)Extraction of brefeldin A from the precipitate of fermentation broth.Ethyl acetate was used as the extractant,yielding a net extraction rate of 37.0 g bredelidn A per kg mycelial precipitate.(3)Macroporous resin adsorption chromatography.Under the optimized chromatography conditions,brefeldin A with a purity of 90.4%(w/w)was prepared in a yield of 92.1%(w/w)by a one-step adsorption chromatography using the stepwise elution protocol.(4)Anti-solvent crystallization.Under the optimal anti-solvent crystallization conditions,colorless crystals with large size and well distribution had been prepared in a yield of 90.5%(w/w).In this way,high quality brefelidn A with purity over 99.0%(w/w)was prepared by this four-step separation process,in a yield of 63.1%(w/w).In summary,a separation strategy involving one-step macroporous resin adsorption chromatography and crystallization was successfully established for brefeldin A purification from E.brefeldianum CCTCC M 208113 fermentation broths.It has great potential in brefeldin A production.