Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1.
A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.
Nagasawa
T
T
Department of Agricultural Chemistry, Kyoto University, Japan.
Takeuchi
K
K
Yamada
H
H
D000595:Amino Acid Sequence; D003035:Cobalt / Q000032:analysis*; D004795:Enzyme Stability; D006836:Hydro-Lyases / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012240:Rhodococcus / Q000201:enzymology*; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013696:Temperature; D014508:Urea / Q000494:pharmacology*
D003035:Cobalt; D014508:Urea; D006836:Hydro-Lyases; C048831:nitrile hydratase