| Human-like collagenⅡ(HLCⅡ)is one derivative of collagen family,which has perfect bioactiveproperties,has promoted epithelial cell and cartilage cell regenerated,hemostatic function andmechanical character also well.The texture of HLCⅡis more hard aftervacuum freeze drying,gelatinized under room temperature,and colloid chacharacter is not changed. Therefore,it can be used to make medical materials,such as artificial skin,artificial blood vessel,suture for surgery,artificial bone,and so on.To study and optimize the fermentation parameters for expressing HLCⅡduring E.coli fed-batch fermentation.The effects of temperature,pH,control of dissolved oxygen,specific growth rate and induction condition on the cell growth and human-like collagenⅡproduction were investigated to optimize the fermentation conditions.The resultsdemonstrated:1 The optimum pH of E.coli growth and human-like collagenⅡexpression was 6.5 and 6.8 respectively,compared to constant pH,adopting step-control pH was not only qualified to cell growth,but also to human-like collagenⅡexpression.Controlling pH in phase of induction at 6.8 and initial pH at 6.5,cell density and human-like collagenⅡconcentration could reach 82.3 g.L-1 and 10.20 g·L-1.2 when initial temperature was 34℃,human-like collagenⅡconcentration reached 7.89 g·L-1. Properly cutting down the temperature is beneficia for engineering bacterium to promote protein expression.3 Controlling dissolved oxygen concentration at 20%,the yield of human-like collagen II was more,which won't cause oxygen toxicity for bacteria.On this basis,two-stage oxygen-supply control mode,controlling DO in phase of induction at 40%and initial DO at 20%,was proposed and experimently proved to increase the human-like collagenⅡproduction,cell density and human-like collagenⅡyield reached 83.5 g·L-1 and 12.2 g.L-1, respectively.4 The influences of specific growth rate on the cell growth and human-like collagenⅡ expression in the phases of before and after induction were different,controlling different specific growth rate in different stage was available in order to yield higher cell and human-like collagen.The results proved:the optimum specific growth rate before induction was 0.20-0.25 h-1,and the optimum specific growth rate after induction was 0.05-0.06 h-1. At the optimum specific growth rate,the final cell density and human-like collagenⅡconcentration could reach 13.2 g·L-1 and 83.9 g·L-1,respectively.5 compared to the middle of logarithmic phase and middle-end of logarithmic phase, implementing induction at the later of logarithmic phase could get more than biomass and human-like collagenⅡ,finally both reached 86.0 g·L-1 and 14.5 g·L-1;adopting a novel induction mode,which cultivated to the later logarithmic growth phase at 30℃after at 34℃for 10h,after up to 42℃for 3h,lowing down to 39℃,the cell density and human-like collagenⅡyield concentration could reach 84.5 g·L-1 and 15.7 g·L-1,respectively.
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