title: Purification studies on a thermo-active amidase of Geobacillus pallidus BTP-5x MTCC 9225 isolated from thermal springs of Tatapani (Himachal Pradesh).
abstract: An intracellular aliphatic amide degrading inducible thermo-active amidase produced by Geobacillus pallidus BTP-5x MTCC 9225 was purified to apparent homogeneity using anion exchange and gel filtration chromatography, giving a yield of 6.7 % and a specific activity of 30.49 units mg(-1). The purified protein migrated as a single band of estimated molecular mass of 158 kDa (homo-tetramer) in 8 % polyacrylamide gel electrophoresis and ∼38.5 kDa in 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Optima of pH and temperature varied widely in broad pH range (pH 6-9) and temperature range (45-70 °C). The purified amidase was stable up to 6 h at 50 °C, with a t (1/2) of 7 h at 55 °C. The multimeric nature of the holozyme (tetramer) contributed to protection of the enzyme against thermal denaturation. The enzyme showed resistance to metal chelating agents (EDTA, 8-hydroxyquinoline, and sodium azide), explaining its non-metallic nature, and is strongly inhibited by thiol reagents that means cysteine is involved in catalysis. The amidase of G. pallidus BTP-5x preferentially hydrolyzed only small aliphatic amides and has a narrow substrate spectrum. The K (M) value for acrylamide is 10.54 mM, V (max) 45.19 μmol(-1) min(-1) mg(-1) protein, and k (cat) 4.29 min(-1). The sequence of amino acids of the purified enzyme MRHGDISSSHDTVGI appears similar to thermophilic amidases. Sequence analysis of the amidase gene showed that the enzyme is 347 amino-acid-long with a molecular weight of 38.4 kDa (as observed in SDS-PAGE), theoretical pI 5.38, and show strong similarity to thermostable amidases, possessing unique restriction sites.
authors.affiliation: Department of Biotechnology, Himachal Pradesh University, Summer Hill, 171005 Shimla, Himachal Pradesh, India. monashimla@gmail.com
authors.forename: Monica
authors.initials: M
authors.lastname: Sharma
authors.forename: Nitya Nand
authors.initials: NN
authors.lastname: Sharma
authors.forename: Tek Chand
authors.initials: TC
authors.lastname: Bhalla
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D045482:Hot Springs / Q000382:microbiology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007194:India; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D000581:Amidohydrolases; C019665:amidase