甲醇/山梨醇共混诱导改善重组毕赤酵母表达猪α干扰素发酵过程和NADH再生效率  

Improvement of porcine interferon-α production by Pichia pastoris and NADH reutilization efficiency using methanol/sorbitol co-feeding strategy

作　　者：戴科科[1] 董世娟[2] 于瑞嵩[2] 史仲平[1] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,无锡214122 [2]上海市农业科学院畜牧兽医研究所,上海201106

出　　处：《生物加工过程》2012年第2期55-62,共8页Chinese Journal of Bioprocess Engineering

基　　金：国家重点基础研究发展计划(973计划)资助项目(2007CB714303);上海科学技术委员会农业科技重点基金资助项目(073919108)

摘　　要：在10 L发酵罐中利用重组毕赤酵母诱导表达猪α干扰素(pIFN-α),考察甲醇/山梨醇共混诱导策略对pIFN-α表达水平提高和能量(NADH)再生效率的影响。结果表明:在诱导稳定期,甲醇/山梨醇共混诱导可弱化细胞的甲醇代谢,有利于缓解毒副中间产物(过氧化氢、甲醛等)的生成积累;以0.785 g/(L.h)的速率缓慢共混流加山梨醇时,pIFN-α抗病毒活性最大,最高活性可达1.8×107IU/mL,与30℃常温甲醇单独诱导(最高活性1.0×104IU/mL)和20℃低温甲醇单独诱导(最高活性1.4×106IU/mL)相比,活性均大幅提高,且胞外pIFN-α的降解减缓;发酵体系的抗高甲醇浓度冲击能力有效提高,发酵生产的稳定性增强;能量利用效率大幅提高,NADH的再生利用效率提高了29%～84%。Abstract： The effects of methanol/sorbitol co-feeding strategy on porcine interferon-a （pIFN-a） produc- tion by recombinant Pichia pastoris and the relevant energy （NADH） regeneration efficiency during induc- tion phase were investigated in a 10-L fermentor. The results showed that methanol/sorbitol co-feeding weakened the methanol metabolism in cells and relieved accumulation of the toxic intermediate metabo- lites （formaldehyde, H202, etc. ）. Compared with 30 ℃pure methanol induction strategy （maximum pIFN-a antiviral activity 1.0 × 104 IU/mL ） and 20℃ low temperature pure methanol induction strategy （maximum pIFN-a antiviral activity 1.4 × 106 IU/mL） , methanol/sorbitol co-feeding strategy and sorbi- tol feeding rate at 0. 785 g/（L·h） could improve pIFN-a antiviral activity, and the highest pIFN-a anti- viral activity reached 1.8 × 107 IU/mL. In addition, the targeted protein degradation was also relieved when using the co-feeding strategy. With the co-feeding strategy, the resistant ability against high metha-nol concentration of the fermentation process was enhanced for ensuring the fermentation stability. Using the co-feeding strategy, energy utilization rate was improved and NADH reutilization efficiency was raised from 29% to 84%.

关 键 词：山梨醇 毕赤酵母 猪Α干扰素 

分 类 号：Q815[生物学—生物工程]