Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1.
Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1.
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红红球菌 J1 中两种腈水合酶的过表达和表征
DOI：
10.1371/journal.pone.0179833
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Zhou Z
中科院分区：
文献类型：
--
作者：
Lan Y;Zhang X;Liu Z;Zhou L;Shen R;Zhong X;Cui W;Zhou Z
Nitrile hydratase (NHase) from Rhodococcus rhodochrous J1 is widely used for industrial production of acrylamide and nicotinamide. However, the two types of NHases (L-NHase and H-NHase) from R. rhodochrous J1 were only slightly expressed in E. coli by routine methods, which limits the comprehensive and systematic characterization of the enzyme properties. We successfully expressed the two types of recombinant NHases in E. coli by codon-optimization, engineering of Ribosome Binding Site (RBS) and spacer sequences. The specific activity of the purified L-NHase and H-NHase were 400 U/mg and 234 U/mg, respectively. The molecular mass of L-NHase and H-NHase was identified to be 94 kDa and 504 kDa, respectively, indicating that the quaternary structure of the two types of NHases was the same as those in R. rhodochrous J1. H-NHase exhibited higher substrate and product tolerance than L-NHase. Moreover, higher activity and shorter culture time were achieved in recombinant E. coli, and the whole cell catalyst of recombinant E. coli harboring H-NHase has equivalent efficiency in tolerance to the high-concentration product relative to that in R. rhodochrous J1. These results indicate that biotransformation of nitrile by R. rhodochrous J1 represents a potential alternative to NHase-producing E. coli.
