作　　者： ; ; ; ;

机构地区： 河南科技学院生命科技学院

出　　处： 《生物技术通讯》 2007年第1期60-63,共4页

摘　　要： 目的:对毕赤嗜甲醇酵母工程菌inu-26高密度培养表达黑曲霉菊粉内切酶的条件进行优化,找出最佳的外源蛋白表达条件。方法:在摇瓶优化培养的基础上进行发酵罐高密度培养,优化最佳产酶条件。结果:以葡萄糖为碳源、微量元素添加量100～200mL/L、甲醇浓度1g/L、pH6.0～7.0、诱导时间96h时酶的表达量最高;摇瓶模拟高密度培养表明影响酵母生长的最主要因素葡萄糖和硫酸铵的最佳浓度分别为20～45和11.5g/L;利用培养基F1进行高密度培养优于其他培养基,工程菌生长符合指数生长曲线,细胞生长延迟期为1.36h,比生长速率μ为0.4846h-1。结论:以葡萄糖为碳源,采用葡萄糖-甲醇混合诱导和100%甲醇单一诱导相结合,在菌体鲜重约为280g/L时连续诱导96h,菌体生长良好,不会出现自溶,且酶的表达量最高,为摇瓶培养的3倍多,酶活最高可达540 U/mL。 Objective： To optimize the expression conditions of endo-inulinase from Aspergillus niger in high-density cultivation of recombinant Pichia methanolica inu-26. Methods： On the foundation of cultivation by shake flask experiments, the optimum conditions that express enzyme by high-density cultivation in fermentor were obtained. Results ＆ Conclusion： The transformant utilized the carbon sources glucose better than the others, and the best concentration of the trace elements was 100- 200 ml/L, the optimum range of pH was from 6.0 to 7.0, the optimum methanol concentration as sole was 0.1%. The fermentation period through methanol inducement was about 96 hours. The orthogonal experiments showed that the optimum conditions for yeast growthing was glucose concentration 20-45 g/L and （NH4）2SO4 concentration 11.5 g/L. On the base of above experimental, the growth with the culture medium F1 as the fermentation medium fit the exponential model,the lag phase of cell growth was 1.36 h, the average specific growth rate of the fed-batch culture was 0.4846 h-1. Adopt the glucose-methanol mixture induces to combine together with 100% inducements with single methyl alcohol, at the cell mass（FW） was or so for 280 g/L, continue to induce 96 h, the cell was not autolyze, the enzyme activity could reach to 540 U/mL, which was more than three times the shake flask culture＇s.

关 键 词： 毕赤嗜甲醇酵母 黑曲霉菊粉内切酶 高密度培养

领　　域： [生物学]