Aliphatic nitrile hydratase from Arthrobacter sp. J-1 purification and characterization.

Abstract

A new enzyme, aliphatic nitrile hydratase, which hydrates acetonitrile to form acetamide was purified from the cell-free extract of acetonitrile-grown Arthrobacter sp. J-l. The overall purification was about 290-fold with a yield of 10%. The purified enzyme was homogeneous as judged by ultracentrifugation and disc gel electrophoresis. The enzyme catalyzed the stoichiometric hydration of acetonitrile to form acetamide according to the following scheme: CH3CN+H2O →CH3CONH2. The enzyme was inducibly formed and then amidase which hydrolyzed acetamide was formed. The molecular weight of the enzyme was determined to be about 420, 000 by gel filtration. The enzyme was composed of two kinds of subunits, of which the molecular weights were 24, 000 and 27, 000. The isoelectric point was 3.6. The enzyme was active toward low molecular weight aliphatic nitriles of 2 to 5 carbon atoms. The Km value for acetonitrile was determined to be 5.78mM. The enzyme was inactivated by sulfhydryl reagents. The enzyme was competitively inhibited by potassium cyanide: the Ki value was 1.5 μm.