Studies On High-cell Density Fermentation Of Recombinant Escherichia Coli Expressing Human-like Collagen And Purification Of Target Protein

The project was primarily concerned with the high-cell density fermentation of recombinant Escherichia coli, the expression of human-like collagen and the separation and purification of the target protein. Several factors that influenced the cell growth and human-like collagen expression were investigated. These factors were three oxygen-controlled methods (glucose feed-back, supplying oxygen-enriched air, increasing fermentor's pressure), four nitrogen-feeding models (1.5mL/25s, 18mL/5min, 36mL/10min, 72mL/20min interval) and inducement conditions (inducement time, inducement strength, acetic acid concentration in broth). Using the experiment data, the cultivation kinetics and cell metabolic flux analysis were studied. Furthermore, the batch ion exchange chromatography combined with gel filtration chromatography to purify human-like collagen was adopted.1. When the dissolved oxygen tension was maintained at 20%~30% by increasing the fermentor's pressure, the cell yield and human-like collagen yield reached 77.3g·L~-1 and 13.9g·L~-1 respectively, which both were the most one in the three oxygen-controlled methods.2. When the feeding nitrogen solution was fed at 36mL/10min, the cell yield and human-like collagen yield reached 84g·L~-1 and 14g·L~-1 respectively, which both were the most one in the four nitrogen-feeding models.3. When the cell was induced at the middle-end of logarithmic phase, the human-like collagen yield reached 10.3g·L~-1 , which was more than the yield of both inducement at middle of logarithmic phase and end of logarithmicphase.4. The by-product acetic acid in broth influenced the cell growth and human-like collagen expression. When the concentration of acetic acid sodium feeding in fermentor reached 3.0g·L~-1 the cell yield and human-like collagen yield was 43.3g·L~-1 and 2.0g·L~-1 respectively. The human-like collagen yield was only 20% of which no feeding of acetic acid sodium.5. The inducement strength influenced the human-like collagen yield markedly. The cell would be induced insufficiently when 42℃ was remained too shortly and the cell would lose its activity to some extent when 42℃ was remained too long. When the temperature was kept at 42℃ for 3h and then at 39℃ for 5h, the human-like collagen yield was 12.8g·L~-1, which was the most one in the four cultivations adopting different inducement strength.6. The fermentation process could be divided into three phases: batch phase, feeding phase before inducement, and inducement phase. Using the math instrument MATLAB, the cultivation kinetics models were obtained.7. Supposing the mid metabolic reaction at stable-state, the carbon metabolic flux analysis was studied.(1) At the feeding phase before inducement, the carbon metabolic flux shows different variation from the three oxygen-controlled methods. The carbon flux of Pentose Phosphate Pathway (PP) of the cultivation controlled by glucose feedback was the most one, which proved that the cell yield coefficient from the cultivation of glucose feedback was the most one from the metabolic point of view.(2) At the feeding phase before inducement, the carbon metabolic flux also shows different variation from the four nitrogen-feeding models. The carbon flux of Pentose Phosphate Pathway (PP) of the cultivation that the nitrogen solution was fed by 36mL/10min was the most one, and the carbon flux of Tricarboxylic Acid Cycle (TCA) was the least one.(3) At the inducement phase, the carbon metabolic flux of Pentose Phosphate Pathway (PP) from the three oxygen-controlled methods were all strongly decreased, in which the one from the cultivation controlled by supplying oxygen-enriched air was decreased most. Thecarbon flux of Tricarboxylic Acid Cycle (TCA) from the cultivation controlled by increasing fermentor's pressure was the most one. This may be due to the need of cell growth precursors.(4) At the inducement phase, the carbon metabolic fluxes of PentosePhosphate Pathway (PP) from the four nitrogen-feeding models alsowere all strongly decreased. The carbon flux of Pentose PhosphatePathway (PP) from the cultivation that the nitrogen solution was fed by36mL/10min still was the most one, same as that before inducement.8. Anion exchange resin DEAE52 was used as the crude separation resin andran at pH value 7.0, the NaCl concentration O^mohL"1, the feedingconcentration of protein 40 mg'mL'1, then, the Sephadex G-100 was used asgel filtration resin for further purification. Finally, the purified human-likecollagen could reach 98.2% and the purification fold multiple around 3.1.The total recovery ratio was 80.6%.