Study On The Production Technology Of Epothilone B Based On In-situ Separation Fermentation

Epothilones are secondary metabolites produced by the myxobacterium. They are similar to paclitaxel in the anticancer mechanism, which can inhibit the formation of the spindle in advanced cancer and block the mitosis process with broad-spectrum antineoplastic. Producted by microorganism, they can be carried out in the large-scale industrial fermentation, which is expected to become more economic anticancer drugs, and has more broader market prospects. However, the fermentation system produced epothilone is complex, such as the production strain is more difficult to operate, leading to difficulties in fermentation parameters measured, so far there is no reporting on Epothilone in the production process. In this paper, the production process and dynamics of epothilone B were studied in the 5L fermentor scale, which would lay a good foundation for industrial production of epothilones.This study had two aspects: Firstly, the fermentation parameters of Sorangium So F5-76 was optimized and the fermentation kinetics model was established in the 5L fermentor, which based on learning about the optimization test in the previous flask fermentation process; Secondly, the in-situ separation fermentation equipment was designed with macroporous adsorption resin XAD-16 for adsorbent, and the coupled process of epothilone B separation and fermentation was studied and optimized. Further more, the continuous fermentation kinetics model was established, and this model provided the basis data for scaling-up the size of epothilones fermentation technology.The main results of this paper were as follows:(1) The optimal batch fermentation condition of Sorangium cellulosum So F5-76 in 5L fermentor were as follows: inoculation size 9%, stirring speed 180 r/min, airflow rate 3.5L/min, added Antifoam B polyether defoamer. With the optimum fermentation conditions, the yield of epothilone B was 24.0mg/L.(2) On the bases of Logistic and Luedking-Piret equation, the models of cell growth, epothilone B production and substrate consumption were established, and the non-line fitting was employed to model the kinetics of fermentation by software MATLAB. The results showed that the correlation coefficients were 0.997, 0.987, 0.990, which were nearly closed to 1, and the further experimental model validation were indicated that the relative error of the cell growth model, the product formation model and the substrate consumption model were 2.47%、4.32%、4.56%,respectively.(3) The non-polar macroporous resin XAD-16 which had a high adsorption performance was determined for epothilone B. Adsorption capacity of resin XAD-16 could use for three times. In the column tests, the optimum sample concentration was 2.0mg/m L, and the flow rate was 1.0m L/min, respectively. The epothilone B would begin to leak out from the effluent, when the volume of the sample solution reached 480 m L. The eluent was methanol whose volume fraction was 75%, and the flow rate of the eluent was control at 1m L/min. Under these conditions, the recovery rate of epothilone B increased to 90%.(4) The continuous in-situ separation fermentation equipment was designed. when the Sorangium cellulosum started producting epothilone B, current pump was opened and epothilone B was adsorbed to column I, then the filtered fermentation broth returned the fermentor. When the column I saturated, closing the valve, and starting column II, Besides, the column I needed to be turned on after the column II saturated. Two columns separated epothilone B alternately, so that the fermentor could continue fermenting.(5) The in-situ separation fermentation process was studied. The coupling time and flow speed were 60 h, 50 m L/min respectively. Sorangium cellulosum could survive 2 hours in a confined environment. In the single column separation experiment, the yield of epothilone B reached 26.6mg/L through intermittent separation three times; the yield increased to 6.00mg/L.d, when using two column separation alternately.(6) The models of in-situ separation fermentation were established. The model parameters were determined when the system was in steady state. The parameters model were that specific cell growth rate was 0.350h-1; specific cutinase formation rate was 0.188h-1; specific substrate consumption rate was 0.089h-1.