Thermostable nitrilase catalysed production of nicotinic acid from 3-cyanopyridine

A thermostable nitrilase produced by the thermophilic bacterium Bacillus pallidus Dac521 catalyzed the direct hydrolysis of 3-cyanopyridine to nicotinic acid without detectable formation of nicotinamide. The reaction conditions for nicotinic acid production were optimized by using free bacterial cells. Temperature and pH optima were 60 degrees C and 8.0, respectively, with no detectable mass transfer limitation at the highest cell loading. Under optimized conditions, 100% of the 3-cyanopyridine substrate could be converted to nicotinic acid at a conversion rate of 76 nmol/min/mg dry cell weight. Free bacterial cells were effective in converting 3-cyanopyridine at concentrations of up to 0.3 M and the intracellular 3-cyanopyridinase stability was increased in the presence of the substrate at concentrations of 0.2 and 0.3 M. Both 3-cyanopyridine and nicotinic acid inhibited the hydrolysis of 3-cyanopyridine at concentrations greater than 0.2 M. Cells immobilized in calcium alginate beads retained 98% of initial activity and were more resistant to inactivation/inhibition than nonimmobilized cells at 60 degrees C. Calcium alginate immobilized cells used in a column bioreactor retained 100% of 3-cyanopyridinase activity for over 100 h and 10 h when continuously supplied with 0.1 M 3-cyanopyridine at 50 degrees C and 60 degrees C, respectively. The conversion efficiencies of the bioreactors operated at 50 degrees C and 60 degrees C, at 100% 3-cyanopyridinase activity, were 104 mg (substrate)/g (cells)/h and 208 mg (substrate)/g (cells)/h, respectively. (C) 1999 Elsevier Science Inc. All rights reserved.