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3 Biotech (1)
AMB Express (1)
Indian Journal of Microbiology (1)
Bhalla, Tek Chand (3)
Sharma, Monica (2)
Sharma, Nitya Nand (2)
Pratush, Amit (1)
Seth, Amit (1)
Year of Publication
Biotransformation of Acetamide to Acetohydroxamic Acid at Bench Scale Using Acyl Transferase Activity of Amidase of Geobacillus pallidus BTP-5x MTCC 9225
Sharma, Nitya Nand
Indian Journal of Microbiology
The bioprocess employing acyl transferase activity of intracellular amidase of Geobacillus pallidus BTP-5x MTCC 9225 was harnessed for the synthesis of pharmaceutically important acetohydroxamic acid. G. pallidus BTP-5x exhibited highest acyl transferase activity with acetamide: hydroxylamine in ratio of 1:5 in 0.1 M NaH2PO4/Na2HPO4 buffer (pH 7.5) at 65°C. In one liter fed-batch reaction containing 1:5 ratio of two substrates total of eight feedings of 0.05 M/20 min of acetamide were made and it was found that maximum acetohydroxamic production was achieved at 3:5 ratios of substrate and cosubstrate. In 1 l bench scale batch reaction containing 0.3 M acetamide, 0.5 M hydroxylamine in 0.1 M NaH2PO4/Na2HPO4 buffer (pH 7.5, 50°C, 400 rpm) and 0.5 mg/ml (dry cell weight) of whole cells of G. pallidus BTP-5x (as biocatalyst) resulted in an yield of 0.28 M of acetohydroxamic acid after 20 min reaction time at 50°C. The acetamide bioconversion rate was 90–95% (mol mol−1) and 51 g powder containing 40% (w/w) acetohydroxamic acid was recovered after lyophilization.
Geobacillus pallidus BTP-5x MTCC 9225; Acetohydroxamic acid; Thermophilic amidase; Acyl transferase activity; Hydroxamic acid
Purification and characterization of nitrile hydratase of mutant 4D of Rhodococcus rhodochrous PA-34
Nitrile hydratase (NHase; E.C. 220.127.116.11) has been purified and characterized using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography from the mutant 4D of Rhodococcus rhodochrous PA-34. The SDS-PAGE and MALDI-TOF analysis of the purified enzyme revealed that it is dimmer consisting of α- and β-subunits with a molecular mass of 25 and 30 kDa, respectively. The Km and Vmax values were 102 mM and 350.8 μmol/min/mg using 3-cyanopyridine as substrate. The purified NHase was stable in higher concentration of potassium ions and in acidic pH 5.5 as compared to NHase of the wild R. rhodochrous PA-34. The analysis of the N-terminal amino acid sequence of this enzyme revealed that this enzyme has 90 % homology with the high molecular weight nitrile hydratase of R. rhodochrous J1.
Characterization; Mutant; Purification; Rhodococcus rhodochrous PA-34
Nocardia globerula NHB-2 nitrilase catalysed biotransformation of 4-cyanopyridine to isonicotinic acid
Sharma, Nitya Nand
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.
4-Cyanopyridine; Isonicotinic acid; Isobutyronitrile; Bacterial nitrilase; Biotransformation; Substrate inhibition; Fed batch
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