Yoshiki Takashima

Factors affecting the production of nitrile hydratase by thermophilic Bacillus smithii SC-J05-1.

Ammonium present in the medium was found to control the production of nitrile hydratases (NHase) by a moderate thermophile, Bacillus smithii SC-J05-1. The enzyme production is initiated in the absence of ammonium and suppressed in the presence of ammonium. In addition to cobalt ions as the coordinated metal ion, manganese ions are also required for NHase production.

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.