A novel molecular chaperone GroEL2 from Rhodococcus ruber and its fusion chimera with nitrile hydratase for co-enhanced activity and stability
A novel molecular chaperone GroEL2 from Rhodococcus ruber and its fusion chimera with nitrile hydratase for co-enhanced activity and stability
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来自红球菌的新型分子伴侣 GroEL2 及其与腈水合酶的融合嵌合体，共同增强活性和稳定性
DOI：
10.1016/j.ces.2018.07.045
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发表时间：
2018-12
影响因子：
4.7
通讯作者：
Huimin Yu
中科院分区：
文献类型：
--
作者：
Yangzi Chen;Song Jiao;Miaomiao Wang;Jie Chen;Huimin Yu
橡胶红球菌携带胞内腈水合酶（nase），广泛应用于丙烯酰胺的大规模生产。转录组分析。在尿素诱导和热休克条件下，发现了新的伴侣蛋白GroEL2和GroES.M。以文献报道的在有机溶剂中具有官能性的黄芪甲酮rTHS为对照。体外实验（70 °C/90 °C孵育）表明，GroEL2从mr。橡胶具有很高的热稳定性，也可以稳定其他蛋白质。GroEL2与nase inE共表达。coliin有三种表达方式：(a)单顺子表达一个T7启动子，(b)双顺子表达两个T7启动子，(c)融合表达一个T7启动子驱动nase - groel2嵌合体。实验结果表明，NHase-GroEL2嵌合体是最成功的表达策略。与单酶对照相比，最大酶活性提高了63.6%。稳定性评价表明，热休克和丙烯酰胺（AM）浸泡后的残留活性水平分别提高了2.9倍和1.1倍。融合嵌合体NHase-GroES和NHase-rTHS的热稳定性也显著提高了1.6倍和64.0%；但对AM的抗性均降低（分别为36.2%和29.0%）。体内热失活曲线进一步证实了伴侣蛋白融合增强了nase的热稳定性，其中nase - groel2是最稳定的。其失活常数仅为单个nase的1/4。细胞内nase - groel2的kcat也显著高于nase对照。
Rhodococcus ruberharboring intracellular nitrile hydratase (NHase) is widely used in large-scale acrylamide production. Transcriptome analyses ofR. ruberunder urea induction and heat shock revealed the novel chaperones GroEL2 and GroES.M. jannaschiichaperone rTHS, functional in organic solvent as reported in literature, was selected as control. In vitro experiments (70 °C/90 °C incubation) showed that GroEL2 fromR. ruberwas highly thermostable and can stabilize other proteins as well. GroEL2 was co-expressed with NHase inE. coliin three ways: (a) monocistronic expression with one T7 promoter, (b) bicistronic expression with double T7 promoters, and (c) fusion expression with one T7 promoter driving the NHase-GroEL2 chimera. Experimental results showed that the NHase-GroEL2 chimera was the most successful expression strategy. Maximal NHase activity was enhanced by 63.6% compared with the single NHase control. A stability assessment showed that the residual activity levels after heat shock and acrylamide (AM) immersion increased by 2.9× and 1.1×, respectively. For the fusion chimeras NHase-GroES and NHase-rTHS, their thermal stability also significantly enhanced by 1.6× and 64.0%, respectively; but their AM resistance both reduced (36.2% and 29.0%, respectively). In vivo heat inactivation curves further confirmed thermal stability enhancement of NHase by chaperone fusion, in which NHase-GroEL2 was the most stable one. Its inactivation constantkdwas only ∼1/4 that of the single NHase. TheKcatof the intracellular NHase-GroEL2 was also remarkably higher than that of the NHase control.
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影响因子：
2.2
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
11.4
作者：
Ma, Yuchao;Yu, Huimin;Shen, Zhongyao
通讯作者：
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