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

Authors :
Yao Lan
Xiaohuan Zhang
Zhongmei Liu
Li Zhou
Ruihua Shen
Xianping Zhong
Wenjing Cui
Zhemin Zhou
Source :
PLoS ONE, Vol 12, Iss 6, p e0179833 (2017)
Publication Year :
2017
Publisher :
Public Library of Science (PLoS), 2017.

Abstract

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.

Subjects

Subjects :
Medicine
Science

Details

Language :
English
ISSN :
19326203
Volume :
12
Issue :
6
Database :
Directory of Open Access Journals
Journal :
PLoS ONE
Publication Type :
Academic Journal
Accession number :
edsdoj.918ea969b19438a984236a19367f0de
Document Type :
article
Full Text :
https://doi.org/10.1371/journal.pone.0179833