Cloning, purification, crystallization and preliminary X-ray diffraction analysis of nitrile hydratase from the themophilic Bacillus smithii SC-J05-1.
Nitrile hydratase (NHase) converts nitriles to the corresponding amides and is recognized as having important industrial applications. Purification, cloning, crystallization and initial crystallographic studies of the NHase from Bacillus smithii SC-J05-1 (Bs NHase) were conducted to analyze the activity, specificity and thermal stability of this hydrolytic enzyme. Bs NHase was purified to homogeneity from microbial cells of B. smithii SC-J05-1 and the nucleotide sequences of both the alpha- and beta-subunits were determined. Purified Bs NHase was used for crystallization and several crystal forms were obtained by the vapour-diffusion method. Microseeding and the addition of magnesium ions were essential components to obtain crystals suitable for X-ray diffraction analysis.
Hourai
Shinji
S
Sumitomo Chemical Co. Ltd Environmental Health Science Laboratory, 3-1-98 Kasugade-naka, Konohanaku, Osaka 554-8558, Japan. shinji-horai@ds-pharma.co.jp
Ishii
Takeshi
T
Miki
Misao
M
Takashima
Yoshiki
Y
Mitsuda
Satoshi
S
Yanagi
Kazunori
K
D001407:Bacillus / Q000201:enzymology*; D002845:Chromatography; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006836:Hydro-Lyases / Q000737:chemistry*; D008027:Light; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D012685:Sepharose / Q000737:chemistry; D014961:X-Ray Diffraction
D012685:Sepharose; D006836:Hydro-Lyases; C048831:nitrile hydratase