Recombinant E. Coli High-density Fermentation For Production Of Human Collagen Protein Ii Fermentation Process Optimization,

To achieve large-scale production and improve the yield of HLCⅡ, the key parameters of pH and temperature and the feeding strategy of production process of human-like collagen by recombinant Escherichia coli BL21 3.7 was investigated systematically in a 30 L fermentation tank with triplex turbine impeller.In the process of optimizing pH during the fermentation, the effects of pH ranging from 6.0 to 7.8 on cell growth and HLCⅡproduction during fermentation process were studied, and an optimal pH strategy was determined to increase HLCⅡyield. From experimental data, the cell growth and collagen synthesis were affected significantly by pH. At pH of 7.5, the highest cell density of 68.5 g/L was obtained with HLCⅡconcentration of 8.5 g/L; while at pH of 6.8, the highest HLCⅡconcentration of 10.5 g/L was obtained, but the cell density was only 60.9 g/L. At the same time, the total concentration of organic acid during the fermentation was also monitored. The total concentration of organic acid increased as the pH raised, and the highest concentration of organic acid of 15.61 g/L was reached when pH kept constantly at 7.8 in the process of fermentation. As a result, a pH shift method was developed to gain higher profitability of mediums and production of HCLⅡ. Finally the concentration of HCLⅡcould reach 11.3 g/L which increased by 7.61% as compared with constant pH fermentation.In the process of optimizing temperature in fermentation, three stage of temperature culturing mode was taken:during the period of 3 h before induction, the temperature was set at 36℃,37℃and 38℃, respectively, then the temperature was shifted to 42℃for induction. The influences of different temperature culturing mode on yield were studied. From experimental data, the yield of 36℃and 38℃culturing mode was relatively low. As a result, in the fermentation process, the temperature was kept at 34℃then 37℃3 h before induction, then shifted to 42℃for induction. In the 37℃temperature culturing mode, a comparatively high cell density of 69.4g/L was obtained, and a highest concentration of HLCⅡof 11.93g/L was also obtained, which were increased by 4.9% and 5.5%, respectively, when compared with pH shift fermentation method.In the process of optimizing feeding strategy in fermentation, the effects of different feeding strategy on cell density and HLCⅡproduction during the fermentation process were studied. From experimental data, an optimal specific growth rate was 0.15h-1 before induction, the cell density could reach 73.4g/L and the concentration of HLCⅡwas 12.43g/L. After induction, different feeding strategy such as pH-stat, exponentially increasing feed, intermittent feed and step decreasing feed were used to control the fermentation and the parameters during fermentation were monitored. The result showed that the step decreaseing feeding strategy was suitable for cell growth and production of HLCⅡ:the cell density could reach 78.2g/L and the concentration of HLCⅡwas 13.28g/L. The production of HLCⅡhad increased by 11.3% compared with previous studies, In conclusion, feeding strategy was very important for the production in the fermentation process and choosing an optimal feeding strategy can optimize the control of fermentation and effectively improve the production of HLCⅡ.