Recombinant Escherichia Coli For Production Of Human Collagen Iii Optimization Of Fermentation Conditions And The Separation And Purification Studies

Fermentation medium optimization,process parameters optimization,feeding strategy, separation and purification of human-like collagenⅢproduced by recombinant Escherichia coli were researched in this paper.Response surface methodology was employed to optimize the fermentation medium and process parameters.In the stage of medium optimization,mixed carbon source(mass ratio of glucose and glycerol was 1:4) and nitrogen source(yeast powder and ammonium sulfate) were screened as most the suitable carbon and nitrogen source in the single factor screening experiments.Plackett-Burman design was applied to evaluate the effects of eight medium components(mixed carbon source,yeast powder,ammonium sulfate,magnesium sulfate, EDTA,phosphate buffer,calcium chloride and trace elements) on the production of HLCⅢ, which showed that mixed carbon source,yeast powder and ammonium sulfate were the most significant ingredients(P＜0.05).The steepest ascent experiment was adopted to determine the optimal region of the medium composition.The optimum composition of the fermentation medium,as determined on the basis of a three-level three-factor Box-Behnken design(BBD), was obtained by response surface methodology(RSM),which were mixed carbon source 12.25g/L,ammonium sulfate 6.8g/L and yeast powder 5.2g/L respectively.The predicted maximum HLCⅢyield is 6.05g/L.An average 6.12g/L HLCⅢwas yielded in the validation experiments by the 12.8L fermenter.Response surface methodology was also employed in the process parameters optimization based on the optimized medium.The important process parameters identified by Plackett-Burman design were induction time,inoculum age and pH.Afterwards, Box-Behnken design was used to determine the optimal process settings of the screened factors for the HLCⅢproduction.The results indicated that when the parameters setting levels were induction time 3.3h,inoculum age 13h and pH 7.0,the predicted maximum HLCⅢyield is 9.87g/L.These predicted values were also verified by validation experiments.For feeding strategy,different carbon and nitrogen source feeding methods,specific growth rates of fermentation process were investigated and evaluated by the final dry cell weight and HLCⅢproduction.The results showed that continuous feeding of carbon and nitrogen source,controlling the specific growth rate at 0.1～0.15h^-1 are the appropriate fermentation control strategy for HLCⅢproduction.For HLCⅢseparation and purification process,total protein solution was attained after the pretreatment of fermentation broth.Orthogonal test was conducted to locate the best levels of pH and the concentration of NaCl for precipitation.Then,the dialysis membrane with a 30kD cut-off molecule weight was used to filtrate the supematant from precipitation.Finally, ion exchange chromatography was applied to purify the HLCⅢ,and a purity of 93.01%of HLCⅢwas yielded.