【摘要】 目的 :为了探索用酵母菌表达乙型干扰素 (倍菲隆 )的大批生产的发酵工艺流程及优化的重要参数。选用 1 5L不锈钢发酵罐作为乙型干扰素发酵工艺研究系统。方法 :探索了种细胞密度对初始发酵期 (甘油利用期 )的影响 ,启动甘油补充期的溶解氧浓度 ,诱导目的蛋白表达时添加甲醇的速率条件控制 ,pH对酵母菌生长与蛋白表达的影响以及发酵时间对乙型干扰素表达量影响等重要发酵工艺参数。结果 :接种高密度的种菌 (OD60 0为 9)时酵母菌增殖快 ,湿细胞重量于1 8h即达到 1 5 7g L。当DO恢复至 67%时即开始补充甘油可使酵母菌在 5h内增殖达到 2 1 9g L。添加甲醇速率从 2 %(甲醇终浓度为 0 0 3%)开始 ,逐步增加 ,在 2 4h内达到 33%的添加速率 (甲醇终浓度为 0 5 %)。甲醇诱导前pH为 6时最有利于酵母菌生长 ,在蛋白表达时期 ,控制发酵罐内pH值在 3 0 ,可得最高蛋白表达量。发酵 70h乙型干扰素表达量达到高峰。结论 :接种高密度种菌可促进工程菌生长 ,当DO恢复至 67%时开始补充甘油可加快酵母菌生长 ,在诱导期中缓慢添加甲醇并维持发酵罐pH为 3 0 ,发酵 70h可获得最佳蛋白生产量。更多还原

【Abstract】 Aim.To study on procession and optimization of large scale expression of human β interferon in 15 liter bioreactors with yeast pichia system.Method:How long the glycerol using phase will take with different of density of seed yeast was studied by inoculating two density of yeast.The best time of starting glycerol feed phase was identified by dissolved oxygen(DO).The methanol concentration was controlled by different feed speed.Optimized pH in yeast growth phase and protein expression phase were confirmed by compared β IFN yield in different pH.The collecting time of high yield of protein was demonstrated by comparing protein concentration in different time after induction phase.Result:The weight of wet cells could reach 157g/L in 18 hours fermentation.The 219g/L of wet cells could be gotten in 5 hours when starting another glycerol feed by returning DO to 67%.The protein expression was higher using very low Methanol feed starting speed(methanol concentration was 0 03%).The ideal β IFN yield was gotten using 0 5% of final methanol concentration.pH6 0 was much good for yeast growth during growth phase but high β IFN will get when pH3 0 was kept in all of the process of protein expression phase.The high yield of β IFN was reached after 70 hours from starting feed methanol.Conclusion:High density seed cells could enhance the growth of yeast.Yeast growth was faster in glycerol feed phase when starting to add glycerol at 67% of DO.High protein yield will get by feeding methanol slowly,kept pH3 0 after methanol feeding.The protein concentration will reach highest peak at 70 hours after feeding methanol.;更多还原