毕赤酵母基因工程菌胞内AOX酶的检测方法

巴斯德毕赤酵母(Pichia pastoris)作为外源基因的表达宿主,已成功表达出一系列胞内和胞外蛋白1～6],并已建立起了一套较成熟的发酵工艺.巴斯德毕赤酵母基因工程菌的外源基因,由胞内AOX酶(乙醇氧化酶)基因启动子调控.在非甲醇碳源条件下(如甘油或葡萄糖),AOX酶基因表达被抑制,外源基因也处于不表达状态.而以甲醇为唯一碳源时,AOX酶在胞内大量合成,同时外源基因被调控表达.在一般情况下,AOX酶的变化直接反映了外源基因的表达状况,因此通过分析检测胞内AOX酶的含量和变化速率,就可以确定外源基因所处的状态.

The Measurement of Intra-cellular AOX in Recombinant Pichia pastoris

The activities of intracellular alcohol oxidase(AOX) in recombinant P. pastoris expressing Pro-UK were determined by a self-designed dissolved oxygen measuring equipment. The enzyme vitality and specific enzyme vitality were defined nd the condition for detecting the enzyme vitality was also established. The experimental results showed that with a certain quantity of biomass in a phosphate buffer containing methanol, the consuming rate of dissolved-oxygen reflected the enzyme vitality of intracellular AOX. It was also found that the pH of the buffer could be very freely between 4.7 and 7.4 and the suitable optical dersity of cell concentration at 600 nm was between 0.5 and 2.0. Furthermore, the values of qo2max and Km of AOX versus oxygen consumption, which were 0.409 s-1 and 0.16 respectively, were calculated. It is a simple and sensitive and feasible method for quick measuring of AOX.