Research Article: Nocardia globerula NHB-2 nitrilase catalysed biotransformation of 4-cyanopyridine to isonicotinic acid

Date Published: April 26, 2012

Publisher: Springer

Author(s): Nitya Nand Sharma, Monica Sharma, Tek Chand Bhalla.


Isonicotinic acid (INA) is an important pyridine derivative used in the manufacture of isoniazid (antituberculosatic drug) and other pharmaceutically important drugs. Nitrilase catalysed processes for the synthesis of pharmaceutically important acids from their corresponding nitriles are promising alternative over the cumbersome, hazardous, and energy demanding chemical processes. Nitrilase of Nocardia globerula NHB-2 (NitNHB2) is expressed in presence of isobutyronitrile in the growth medium (1.0% glucose, 0.5% peptone, 0.3% beef extract, and 0.1 % yeast extract, pH 7.5). NitNHB2 hydrolyses 4-cyanopyridine (4-CP) to INA without accumulation of isonicotinamide, which is common in the reaction catalysed via fungal nitrilases. The NitNHB2 suffers from substrate inhibition effect and hydrolysing activity up to 250 mM 4-CP was recorded. Complete conversion of 200 mM 4-CP to INA was achieved in 40 min using resting cell concentration corresponding to 10 U mL-1 nitrilase activity in the reaction. Substrate inhibition effect in the fed batch reaction (200 mM substrate feed/40min) led to formation of only 729 mM INA. In a fed batch reaction (100 mM 4-CP/20min), substrate inhibition effect was encountered after 7th feed and a total of 958 mM INA was produced in 400 min. The fed batch reaction scaled up to 1 L and 100% hydrolysis of 700 mM of 4-CP to INA at 35°C achieved in 140 min. The rate of INA production was 21.1 g h-1 mgDCW-1. This is the fastest biotransformation process ever reported for INA production with time and space productivity of 36 g L-1 h-1 using a bacterial nitrilase.

Partial Text

Isonicotinic acid (INA) is an important pyridine derivative used in the synthesis of antituberculostatic drug isoniazid (isonicotinic acid hydrazide). The conventional chemical process which utilizes 4-cyanopyridine and hydrazine hydrate as reactants is hazardous, energy demanding and expensive ([Yadav et al. 2005]). Recently ethyl isonicotinate (synthesized from INA) has been used for chemoenzymatic synthesis of isoniazid by lipase-catalyzed transesterification in non-aqueous medium onto hydrazine hydrate ([Yadav et al. 2005]). Isoniazid also inhibits the developmental stages of malarial parasites (Plasmodium gallinaceum and P. berghei) in mosquito ([Arai et al. 2004]). INA is used for the synthesis of inabenfide, a plant growth regulator. Terefenadine, an antihistamine and nialamide, an antidepressant are also derived from INA ([Scriven et al. 1998]). INA also finds application as an anticorrosion reagent, electroplating additive, and photosensitive resin stabilizer ([Wu et al. 1991]).

The bacterial nitrilase has been for the first time utilized to develop biotransformation process for the production of INA from 4-CP. NitNHB2 activity profile against various nitrilase has been published in our pervious article ([Sharma et al. 2011]). The hyperinduced resting cells of N. globerula NHB-2 showed 5.71 U mgDCW-1 (87% with respect to 3-CP) nitrilase activity for 50 mM 4-CP in reaction mixture at 35°C. The nitrilase of R. rhodochrous J1 exhibited 79% activity (0.72 U mgDCW-1) for 4-CP in comparison for activity against 3-CP ([Mathew et al. 1988]).

Nitrilase mediated conversion of nitriles to acids are gaining importance over the chemical routes, due to ease of biocatalyst production, mild reaction conditions, and formation of optically pure acids. In past decade, the search of nitrilases for optically active acids have gained importance for both academicians and industries ([Liese et al. 2000,Brady et al. 2004,DeSantis et al. 2003,Breuer et al., 2004,Wang 2005,Sheldon et al. 2007,Xue et al. 2011,Novill et al. 2011,Pandey et al. 2011]). The nitrilases also efficiently hydrolyse non chiral nitriles to acid adding advantage over the chemical routes of their synthesis ([Mathew et al. 1988], Vaughan et al. 1989, Almatawah et al. 1999, Vejvoda et al. [2006,Luo et al. 2010,Malandra et al. 2009,Prasad et al. 2007,Raj et al. 2007,Sharma et al. 2006,2011]). The current research work was an attempt to explore the potential of N. globerula NHB-2 nitrilase for the synthesis of INA from 4-CP for the first time. Further, the study was focused on improving the biotransformation process and scaling up to one liter. [Vejvoda et al. (2006]) have used a cascade of immobilized fungal nitrilase (5.5 U) and bacterial amidase (5 U) on 1 mL Butyl Sepharose column and feeding of 40 mM 4-CP (0.3 mL min-1) producing 3.102 g isonicotinic (99.8% purity) in 35 h with time and space productivity of 94 mg L-1 h-1. In CSMRs cascade loaded with nitrilase (6.5 U) and amidase (5 U) as cell-free extracts immobilized in CLEAs produced 3.36 g INA (99.9%) in 52 h with the time and space productivity of 118 mg L-1 h-1 when 50 mM 4-CP was pumped at the rate of 10.5 mL h-1 (Malandra et al. [2009]). During optimization studies for developing nitrilase mediated biotransformation process using N. globerula NHB-2, substrate inhibition effect was encountered, which was partially overcome using low concentration substrate (100 mM) feed. This improved the amount of product formation in comparison to high concentration substrate (200 mM) fed batch. The biotransformation process developed using N. globerula NHB-2 nitrilase achieved the time and space productivity of 36 g L-1 h-1 INA which is almost 38 times higher than above reports. Further, the INA produced was free from isonicotinamide due to lack of hydrating activity of N. globerula NHB-2 nitrilase, which was common with the fungal nitrilases.

The authors declare that they have no competing interests.




Leave a Reply

Your email address will not be published.