采用混合策略提高葡萄糖氧化酶在毕赤酵母中的表达水平

High-level production of glucose oxidase by recombinant pichia pastoris using a combined strategy

摘要:

为了提高葡萄糖氧化酶 (GOD) 在毕赤酵母中的表达水平，提出了甲醇/山梨醇混合碳源诱导和共表达分子伴侣二硫键异构酶 (PDI) 和透明颤菌血红蛋白 (VHb) 两种策略。利用对照菌株X33/pPIC9k–GOD 在5 L发酵罐放大培养时，采用甲醇/山梨醇混合碳源诱导，GOD最终酶活为456 U/mL，比只采用甲醇作为单一碳源诱导时GOD最终酶活提高了20%。利用整合伴侣蛋白菌株X33/pPIC9k-GOD/pPICZ-PDI-VHb在5 L发酵罐进行高密度发酵，采用甲醇/山梨醇混合碳源诱导，GOD最终酶活达到716 U/mL，蛋白浓度为7.4 g/L。研究结果对提高外源蛋白在毕赤酵母中的表达有重要参考价值。

Abstract:

To enhance the production of glucose oxidase by recombinant Pichia pastoris, two strategies were developed, which were namely co-feeding of methanol and sorbitol and co-expressing of the protein disulfide isomerase (PDI) and Vitreoscialla hemoglobin (VHb). The volumetric activity reached 456 U/mL by using the strain X33/pPIC9k-GOD, in 5 liter fermentator, with the co-feeding of methanol and sorbitol, it was 0.2 fold higher than that only feeding by methanol. The improved strain was obtained by co-expressing PDI-VHb with GOD. While fermented in a 5 liter fermentator by feeding methanol and sorbitol, the activity of the improved strain reached 716 U/mL with a yield of 7 400 mg/L total soluble protein concentration. These results indicated that heterologous protein expression level can be enhanced by optimizing fermentation condition and co-expression molecular chaperon in Pichia pastoris.