Recombinant Escherichia Coli For Production Of Human Collagen, High-density Fermentation Regulation And Plasmid Stability

Recombinant Escherichia coli BL21 was used to express human-like collagen in high-density fed-batch cultivation. The effect of ammonia and nitrogen addition on the biomass and Human-Like Collagen was observed; Exhaust gas analyzer was utilized to research into the fermentation gas and its related parameters; the plasmid stability of Esherichia.coli BL21 was primarily discussed by the method of plasmid replicating. The results are as the follows:1. The relation between the consumption of ammonia and the biomass growth was y = 0.0205x² - 0.7350x + 11.3078; the relation between the consumption of ammonia and the formation of Human-like collagen was y=-1.7643x²+18.4958x +9.5015, where the consumption of ammonia reached its climax when the concentration of Human-like collagen was 5.2415g/L. The consumption of ammonia and the biomass concentration had similar trend,so we could assume the biomass according to the consumption of ammonia, and perfect Human-Like Collagen output could be got by adjusting the consumption of ammonia.2. The prior nitrogen resource was (NH₄)₂SO₄ during the delayed phase; during the exponential phase organic nitrogen resource was utilized; the utilization of organic nitrogen resource slowed during the equilibrium phase ,and we inferred that the nitrogen resource needed to produce Human-Like Collagen were more likely from ammonia.3. The consumption of O₂ and the expiration of CO₂ accelerated in the delayed phase, and the expiration of O₂ and CO₂ were 4.15% and 5.47% respectively before the exponential phase; during the exponential phase the expiration of O₂ and CO₂ stabled to a steady range, O₂: 16.01%-16.77%, CO₂: 4.58%-5.67%; The consumption of O₂ and the expiration of CO₂ accelerated again, and the expiration of O₂ and CO₂ stabled to another steady range O₂: 13.85%-14.00%, CO₂: 6.85%-7.53%.4. OUR was comparatively low(less than 0.05mol/L·2h), and CER rose rapidly during the delayed phase; during the exponential phase, CER and OUR decreased respectively from their climax 0.3501 mol/L·h and 0.209 mol/L·h to 0.331 mol/L·h and 0.208 mol/L·h; a transition period could be seen after the nitrogen addition , CER and OUR rose to 0.338mol/L·h and 0.229 mol/L·h before induction; during the induction stage, CER and OUR stayed around 0.35 mol/L·h and 0.32 mol/L·h.5. The relation between RQ and the consumption of ammonia was Y=-0.46224 exp (-x/12.08825)+0.94072, the inflection point of which was 20mmol/L(the consumption of ammonia). Both RQ and the consumption of ammonia rose when the latter was below the inflection point, and RQ ranged between 0.5 and 0.85; when the latter was above the inflection point, RQ would be above 0.9.Thus we could estimate the metabolic of Escherichia coli BL21 according to the consumption of ammonia.6. We could get an exponential equation between the kanamycin concen- tration and the percentage of plasmid bearing cells Y=-37.1663 exp (-x/2.28407) + 89.6690. The inflection point was 0.00606 g/L (the kanamycin concentration).So, the addition concentration of kanamycin should not be below 0.006 g/L to insure the plasmid.stability7. From the angle of the plasmid stability,μ=0.15 in the exponential stage would be the best choice, Y_x/G and Y_p/G in the circumstance ofμ=0.10 would be lower than that ofμ=0.15; and the plasmid bearing cells lost fast in the circumstance ofμ=0.20 andμ=0.25.