Thermostable nitrilase catalysed production of nicotinic acid from 3-cyanopyridine

A thermostable nitrilase produced by the thermophilic bacterium Bacillus pa llidus Dac521 catalyzed the direct hydrolysis of 3-cyanopyridine to nicotin ic acid without detectable formation of nicotinamide. The reaction conditio ns for nicotinic acid production were optimized by using free bacterial cel ls. 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 conver ted to nicotinic acid at a conversion rate of 76 nmol/min/mg dry cell weigh t. Free bacterial cells were effective in converting 3-cyanopyridine at con centrations of up to 0.3 M and the intracellular 3-cyanopyridinase stabilit y was increased in the presence of the substrate at concentrations of 0.2 a nd 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 re sistant 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 continuousl y supplied with 0.1 M 3-cyanopyridine at 50 degrees C and 60 degrees C, res pectively. The conversion efficiencies of the bioreactors operated at 50 de grees 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.