Optimization Of Fed-batch Fermentation For Expression Of Acid And Heat Resistant Pullulanase By Recombinant Escherichia Coli

This project was mainly focused on high cell density fermentation conditions expressing recombinant heat and acid resistant pullulanase in recombinant Escherichia coli BL 21 strain.Several control factors were investigated,including: Optimization of fermentation temperature,p H and carbon-to-nitrogen molar ratio of the medium by using artificial neural network and genetic algorithm;The effect of three DO-stat control strategies,glucose feedback,shifting fermentor pressure and adding oxygen-enriched air,on cell growth and pullulanase expression;The effect of induction temperature,inducer IPTG concentration and heat shock treatments on the solubility of pullulanase.The experimental results are as follows:(1)Production of high cell density fermentation expressing pullulanase in recombinant Escherichia coli BL 21 was optimized by using artificial neural network and genetic algorithm.The effects of culture temperature,p H,and carbon-to-nitrogen molar ratio of the medium on cell growth and production were tested in a 5 L fermentor.The optimal conditions were as follows: maintain the culture temperature at 34.4 °C,p H at6.87 and C/N at 6.1 before induction,and maintain the temperature at 32.5 °C,p H at6.69 and C/N at 5.3 after induction.The maximum biomass was 56.5 g/L,and the concentration of pullulanase was 3.21 g/L under the optimal culture conditions.(2)The effect of three oxygen control strategies,glucose feedback,shifting fermentor pressure and adding oxygen-enriched air,on cell growth and pullulanase expression were tested.The oxygen transfer capacity was found to be enhanced with both increasing fermentor pressure and oxygen ratio in oxygen-enriched air.The highest pullulanase activity reached 412 U/m L in the case by adding oxygen-enriched air,which was suggested as an effective approach to enhance both cell growth and pullulanase production.But the pullulanase production was only 207 U/m L under high fermentor pressure,which might due to the cell lysis and accumulation of acetate.Although glucose feedback strategy did neither enhance the oxygen transfer capacity nor yield the highest cell density and product,but it was assessed to be an economic approach to utilize the substance and energy.(3)The induction temperature was proved to be the most significant factor that affected the pullulanase activity and biomass in induction phase.Considering the final biomass,the enzyme activity,it suggested that 32.5 °C should be an optimal choice of induction temperature in this work.IPTG concentration from 0.05 to 0.5 m M did not affect the biomass but influence the pullulanase production significantly.The pullulanase activity reached maximum 501 U/m L when induced by adding 0.2 m M IPTG and culturing at32.5 °C.All the experiments treated by heat-shock showed a positive effect on the solubility of pullulanase.However,these increases were not notable in this work,despite,the results could be references for quantitatively analyzing the behavior under inhomogeneous temperature distribution in the industrial scale fermentor.