Chaperones-assisted soluble expression and maturation of recombinant Co-type nitrile hydratase in Escherichia coli to avoid the need for a low induction temperature.
Chaperones-assisted soluble expression and maturation of recombinant Co-type nitrile hydratase in Escherichia coli to avoid the need for a low induction temperature.
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DOI：
10.1016/j.jbiotec.2015.03.004
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发表时间：
2015-06
影响因子：
4.1
通讯作者：
Xiaolin Pei;Qiuyan Wang;Lijun Meng;Jing Li;Zhengfen Yang;Xiaopu Yin;Lirong Yang;Shao-Yun Chen;Jianping Wu
中科院分区：
文献类型：
--
作者：
Xiaolin Pei;Qiuyan Wang;Lijun Meng;Jing Li;Zhengfen Yang;Xiaopu Yin;Lirong Yang;Shao-Yun Chen;Jianping Wu
腈水合酶（Nitrile hydratase，简称NHase）是一种重要的工业酶，可生物合成高价值的酰胺类化合物。然而，除非诱导温度降低到约20 °C，否则在大肠杆菌中表达的大多数NH酶容易聚集成无活性的包涵体。在大肠杆菌中表达了氧化锰橙单胞菌的N-羟化酶。大肠杆菌，并显示出相当大的潜力，在工业应用中生产烟酰胺。本实验研究了GroEL/ES、Dnak/J-GrpE和触发因子等分子伴侣对重组Co-type NHase表达的影响。结果表明，3种分子伴侣在30 °C下均能显著促进重组NHase的活性表达。在摇瓶中，当NHase与GroEL/ES和DnaK/J-GrpE共表达时，总NHase活性分别达到263和155 U/ml，分别是无伴侣时的52倍和31倍。这种增加可能是由于分子伴侣辅助的重组腈水合酶的可溶性表达。此外，GroEL/ES和DnaK/J-GrpE被确定为促进Co型NH酶在E中的成熟。大肠杆菌在亲本激活基因缺失的情况下。这些关于分子伴侣对腈水合酶表达的影响的知识对于理解Co型腈水合酶的生物合成是有用的。
Nitrile hydratase (NHase) is an important industrial enzyme that biosynthesizes high-value amides. However, most of NHases expressed inEscherichia colieasily aggregate to inactive inclusion bodies unless the induction temperature is reduced to approximately 20 °C. The NHase fromAurantimonas manganoxydanshas been functionally expressed inE. coli, and exhibits considerable potential for the production of nicotinamide in industrial application. In this study, the effects of chaperones including GroEL/ES, Dnak/J-GrpE and trigger factor on the expression of the recombinant Co-type NHase were investigated. The results indicate that three chaperones can significantly promote the active expression of the recombinant NHase at 30 °C. The total NHase activities reached to 263 and 155 U/ml in shake flasks when the NHase was co-expressed with GroEL/ES and DnaK/J-GrpE, which were 52- and 31-fold higher than the observed activities without chaperones, respectively. This increase is possibly due to the soluble expression of the recombinant NHase assisted by molecular chaperones. Furthermore, GroEL/ES and DnaK/J-GrpE were determined to promote the maturation of the Co-type NHase inE. coliunder the absence of the parental activator gene. These knowledge regarding the chaperones effect on the NHase expression are useful for understanding the biosynthesis of Co-type NHase.