Bioprocess development for nicotinic acid hydroxamate synthesis by acyltransferase activity of Bacillus smithii strain IITR6b2

Abstract

In this work, acyltransferase activity of a new bacterial isolate Bacillus smithii strain IITR6b2 was utilized for the synthesis of nicotinic acid hydroxamate (NAH), a heterocyclic class of hydroxamic acid. NAH is an important pyridine derivative and has found its role as bioligand, urease inhibitor, antityrosinase, antioxidant, antimetastatic, and vasodilating agents. Amidase having acyltransferase activity with nicotinamide is suitable for nicotinic acid hydroxamate production. However, amidase can also simultaneously hydrolyze nicotinamide and nicotinic acid hydroxamate to nicotinic acid. Nicotinic acid is an undesirable by-product and thus any biocatalytic process involving amidase for nicotinic acid hydroxamate production needs to have high ratios of acyltransferase to amide hydrolase and acyltransferase to nicotinic acid hydroxamate hydrolase activity. Isolate Bacillus smithii strain IITR6b2 was found to have 28- and 12.3-fold higher acyltransferase to amide and hydroxamic acid hydrolase activities, respectively. This higher ratio resulted in a limited undesirable by-product, nicotinic acid (NA) synthesis. The optimal substrate/co-substrate ratio, pH, temperature, incubation time, and resting cells concentration were 200/250 mM, 7, 30 °C, 40 min, and 0.7 mg(DCW) ml(-1), respectively, and 94.5 % molar conversion of nicotinamide to nicotinic acid hydroxamate was achieved under these reaction conditions. To avoid substrate inhibition effect, a fed-batch process based on the optimized parameters with two feedings of substrates (200/200 mM) at 40-min intervals was developed and a molar conversion yield of 89.4 % with the productivity of 52.9 g h(-1) g (DCW) (-1) was achieved at laboratory scale. Finally, 6.4 g of powder containing 58.5 % (w/w) nicotinic acid hydroxamate was recovered after lyophilization and further purification resulted in 95 % pure product.