双碳源流加对重组毕赤酵母高效表达葡萄糖氧化酶的影响

Effects of mixed carbon sources on glucose oxidase production by recombinant Pichia pastoris

葡萄糖氧化酶(GOD)是一种具有广泛应用前景的工业酶。为了实现葡萄糖氧化酶的高效生产,提高重组毕赤酵母生产GOD的产量和增强生产强度,对重组毕赤酵母诱导阶段的初始菌体浓度和甲醇浓度进行了优化。在此基础上,诱导期采用了双碳源(甘油、山梨醇和甘露醇)与甲醇混合流加的模式。研究发现,最佳诱导前初始菌体浓度和甲醇浓度分别为100 g/L和18 g/L,此时GOD产量为427.6 U/mL。在诱导阶段采用甘油、山梨醇和甘露醇与甲醇的混合添加均可以提高GOD产量,其中甘露醇与甲醇的混合流加效果最为显著。当甲醇与甘露醇混合流加的比例为20∶1(W/W)时,诱导156 h GOD产量和生产强度分别可达711.3 U/mL和4.60 U/(mL.h),比甲醇单一流加策略结果分别提高了66.3%和67.9%。此外采用合适的甘露醇混合流加策略不但不会抑制AOX1启动子的表达,甚至有一定促进作用,AOX酶活性为8.8 U/g(对照为5.2 U/g)。双碳源流加方式还能推广到毕赤酵母其他表型中,为该系统高效表达外源蛋白提供一种新策略。

Glucose oxidase（GOD） is an important industrial enzyme with many potential applications.In order to increase the production and productivity of GOD by recombinant Pichia pastoris GS115,we investigated the feeding strategies of mixed carbon sources during induction phase,based on results of the optimization of initial cell and methanol concentration on GOD production.The optimal initial cell and methanol concentration were 100 g/L and 18 g/L.During induction phase,the mixed-carbon-sources strategies showed that glycerol,sorbitol or mannitol co-feeding with methanol could enhance GOD production.With mannitol co-feeding（20：1（W/W））,the maximum GOD production and maximum GOD productivity reached 711.3 U/mL and 4.60 U/（mL.h） after an induction period of 156 h.Compared to the control,the enhancements of GOD production and productivity were 66.3% and 67.9%,respectively.Meanwhile,we found an appropriate mannitol co-feeding strategy that would not inhibit the expression of promote.The activity of alcohol oxidase was 8.8 U/g,which was enhanced by 69.2% compared to the control（5.2 U/g）.We can use the same optimization process to improve the production of other proteins from recombinant Pichia pastoris by changing the fermentation parameters.