Chaperone-assisted expression, purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni.
Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.
Stevens
Julie M
JM
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR 8601 CNRS), Université Paris V, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France.
Rao Saroja
Narsing
N
Jaouen
Maryse
M
Belghazi
Maya
M
Schmitter
Jean Marie
JM
Mansuy
Daniel
D
Artaud
Isabelle
I
Sari
Marie Agnès
MA
D002384:Catalysis; D018835:Chaperonin 10 / Q000737:chemistry; D018834:Chaperonin 60 / Q000378:metabolism; D020604:Comamonas testosteroni / Q000201:enzymology*; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry*
D018835:Chaperonin 10; D018834:Chaperonin 60; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C048831:nitrile hydratase; D003545:Cysteine; D010100:Oxygen