Hydration of 3-cyanopyridine to nicotinamide by crude extract nitrile hydratase

Summary The kinetic and stability characteristics of crude extract nitrile hydratase fromBrevibacterium R-312 were studied for the hydration of 3-cyanopyridine to nicotinamide. The enzyme was substrate and product inhibited and had the following kinetic constants:K _m =28 mM;K _p =36 mM;K _s =155 mM;V _m =5.8 mol/min/mg protein (25°C). Itsmaximum temperature and pH (phosphate buffer) were 35°C and 8.0, respectively and it had half-lives of 50 days, 10 days and 1 day at 4°C, 10°C and 25°C, respectively. The crude extract also exhibited amidase activity on nicotinamide, but it became significant only at nicotinamide concentrations greater than 300 mM. Mathematical models for batch and fed-batch hydrations were developed to account for substrate and product inhibitions and for enzyme decay. They predicted to within 10% experimental results for initial substrate and final product concentrations up to 300 mM; the accuracies decreased at higher concentrations primarily because of the relatively rapid hydrolysis of nicotinamide.