甘油对毕赤酵母高密度发酵表达重组水蛭素Ⅱ的影响

Effects of glycerol on recombinant hirudin Ⅱ expression in high density fermentation by Pichia pastoris

目的:考察甘油对毕赤酵母生长和重组水蛭素Ⅱ表达的影响。方法:在摇瓶和10L发酵罐中用含有不同浓度甘油的培养基培养毕赤酵母,测定毕赤酵母的生长和重组水蛭素Ⅱ的表达。结果:发酵培养基中初始甘油浓度为5 g/L时,毕赤酵母生长速度明显加快;补料过程中甘油浓度大于70 g/L时,毕赤酵母生长受到抑制。与不加甘油相比较,诱导表达期维持培养基中甘油浓度在5 g/L以下,重组水蛭素表达可提前4 h,诱导30 h其活性达13 000 ATU/mL,比仅用甲醇诱导时效价提高了7.7%。结论:降低初始培养基中甘油浓度有利于毕赤酵母生长,补料过程中培养基甘油浓度应维持在抑制毕赤酵母生长的浓度之下,诱导阶段培养基中少量的甘油有利于水蛭素的表达。

Aim：To investigate the effects of glycerol on Pichia pastoris growth and recombinant hirudin Ⅱ （Hir Ⅱ ） expression. Methods： Pichia pastoris was cultured in the medium containing different concentrations of glycerol in flasks and 10 L fermentor. Cell density and Hir Ⅱ activity were detected. Results： Cell growth was increased obviously at 5 g/L glycerol in the initial medium and inhibited at glycerol of 70 g/L. When glycerol was maintained less than 5 g/L at induction phase, Hir Ⅱ expression was found to be accomplished 4 hours earlier than that without addition of glycerol, and 30-hour induction resulted in Hir Ⅱof 13 000 ATU/mL which was only increased by 7.7 % of compared to that following methanol induction. Conclusion： Low glycerol concentration （5 g/L） at lag phase and induction phase is helpful for Pichia pastoris growth and Hir Ⅱexpression, and fed-batch glycerol should be kept under inhibited concentration of 70 g/L.