Enhancement of extracellular pullulanase production from recombinant Escherichia coli by combined strategy involving auto-induction and temperature control
Enhancement of extracellular pullulanase production from recombinant Escherichia coli by combined strategy involving auto-induction and temperature control
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DOI：
10.1007/s00449-013-1026-z
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发表时间：
2014-04-01
影响因子：
3.8
通讯作者：
Xiao, Rong
中科院分区：
文献类型：
--
作者：
Chen, Wen-Bo;Nie, Yao;Xiao, Rong
采用组合策略，利用工程菌胞外生产普鲁兰酶。当采用自诱导代替异丙基β-D-1-硫代半乳糖苷（IPTG）诱导方法时，我们观察到细胞外活性（4.2 U ml（-1））和细胞生物量（7.95 g DCW l（-1））增加。随后的温度对发酵的影响的研究表明，在25 ℃下进行的培养呈现出最高的胞外滴度和细胞生物量。为了缩短延长生产周期，我们开发了一个两阶段的温度控制策略。其应用不仅将生产周期从72 h缩短到36 h，而且进一步提高了胞外普鲁兰酶的产量。最后，为了释放更多的细胞内普鲁兰酶，我们改变了细胞膜的通透性与各种介质添加剂。经IPTG诱导后，胞外滴度可达68.23 U ml（-1），是IPTG诱导法的近35倍。该组合策略可用于重组大肠杆菌生产其他胞外蛋白。杆菌
Pullulanase was extracellularly produced with an engineered Escherichia coli with a combined strategy. When auto-induction instead of isopropyl beta-D-1-thiogalactopyranoside (IPTG) induction method was implemented, we observed increased extracellular activity (4.2 U ml(-1)) and cell biomass (7.95 g DCW l(-1)). Subsequent investigation of temperature effect on fermentation showed cultivation performed at 25 degrees C presented the highest extracellular titer and cell biomass. In order to reduce the extended production period, we developed a two-stage temperature control strategy. Its application not only reduced the production period from 72 to 36 h, but also further enhanced the yield of extracellular pullulanase. Finally, with a view to releasing more intracellular pullulanase, we altered cell membrane permeability with various medium additives. As a result, extracellular titer was elevated to 68.23 U ml(-1), nearly 35-fold higher than that with IPTG induction method. The combined strategy developed here may be useful for the production of other extracellular proteins by recombinant E. coli.