Addition of Co2+ to culture medium decides the functional expression of a recombinant nitrile hydratase in Escherichia coli
Addition of Co2+ to culture medium decides the functional expression of a recombinant nitrile hydratase in Escherichia coli
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DOI：
10.1007/s10529-013-1215-5
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发表时间：
2013-04
影响因子：
2.7
通讯作者：
Xiaolin Pei;Qiuyan Wang;Chenglu Li;Xiaopu Yin;Rong Chen;Tian Xie
中科院分区：
文献类型：
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作者：
Xiaolin Pei;Qiuyan Wang;Chenglu Li;Xiaopu Yin;Rong Chen;Tian Xie
从氧化锰金单胞菌（Aurantimonas manganoxydans）中克隆了腈水合酶（NHase）基因，并在大肠杆菌（Escherichia coli）中进行了表达，得到了一种能有效地将3-氰基吡啶水合为烟酰胺的酶。我们现在已经发现，在LB培养基中加入0.1 mM的Co2+对于活性酶的产生是必不可少的。但当Co ~（2+）浓度≥ 0.3mM时，LB培养基中宿主细胞生长受到抑制，重组NHase产量下降。此外，β-巯基乙醇可能通过破坏关键的二硫键，从而促进Co2+掺入脱辅基酶中，从而促进体外Co2+缺陷型脱辅基酶的再生。
A nitrile hydratase (NHase) gene fromAurantimonas manganoxydans,cloned and expressed inEscherichia coli, gave an enzyme that efficiently hydrated 3-cyanopyridine to nicotinamide with high thermal stability. We have now found that adding Co2+at 0.1 mM to LB medium was essential for production of an active enzyme. However, ≥0.3 mM Co2+inhibited the growth of host cells in LB medium and decreased the production of the recombinant NHase. Furthermore, β-mercaptoethanol promoted regeneration of the Co2+-defective apoenzyme in vitro possibly by breaking a key disulfide bond thereby promoting the incorporation of Co2+into the apoenzyme.