title: Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a.
abstract: A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg(-1). The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5-11.5) and temperature (40-90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co(2+), Hg(2+), Cu(2+), Ni(2+), and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K m and V max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min(-1) mg(-1) protein by analyzing Michaelis-Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.
authors.affiliation: Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171005, India.
authors.forename: Praveen Kumar
authors.initials: PK
authors.lastname: Mehta
authors.forename: Shashi Kant
authors.initials: SK
authors.lastname: Bhatia
authors.forename: Ravi Kant
authors.initials: RK
authors.lastname: Bhatia
authors.forename: Tek Chand
authors.initials: TC
authors.lastname: Bhalla
mesh_terms: D000577:Amides / Q000737:chemistry; D000581:Amidohydrolases / Q000737:chemistry* / Q000302:isolation & purification; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D008670:Metals / Q000737:chemistry
chemical_list: D000577:Amides; D008670:Metals; D000581:Amidohydrolases; C019665:amidase