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Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1

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...

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Autores principales: Lan, Yao, Zhang, Xiaohuan, Liu, Zhongmei, Zhou, Li, Shen, Ruihua, Zhong, Xianping, Cui, Wenjing, Zhou, Zhemin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482477/
https://www.ncbi.nlm.nih.gov/pubmed/28644864
http://dx.doi.org/10.1371/journal.pone.0179833
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author Lan, Yao
Zhang, Xiaohuan
Liu, Zhongmei
Zhou, Li
Shen, Ruihua
Zhong, Xianping
Cui, Wenjing
Zhou, Zhemin
author_facet Lan, Yao
Zhang, Xiaohuan
Liu, Zhongmei
Zhou, Li
Shen, Ruihua
Zhong, Xianping
Cui, Wenjing
Zhou, Zhemin
author_sort Lan, Yao
collection PubMed
description 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.
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spelling pubmed-54824772017-07-06 Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1 Lan, Yao Zhang, Xiaohuan Liu, Zhongmei Zhou, Li Shen, Ruihua Zhong, Xianping Cui, Wenjing Zhou, Zhemin PLoS One Research Article 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. Public Library of Science 2017-06-23 /pmc/articles/PMC5482477/ /pubmed/28644864 http://dx.doi.org/10.1371/journal.pone.0179833 Text en © 2017 Lan et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Lan, Yao
Zhang, Xiaohuan
Liu, Zhongmei
Zhou, Li
Shen, Ruihua
Zhong, Xianping
Cui, Wenjing
Zhou, Zhemin
Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1
title Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1
title_full Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1
title_fullStr Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1
title_full_unstemmed Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1
title_short Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1
title_sort overexpression and characterization of two types of nitrile hydratases from rhodococcus rhodochrous j1
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482477/
https://www.ncbi.nlm.nih.gov/pubmed/28644864
http://dx.doi.org/10.1371/journal.pone.0179833
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