title: Exhaustive study of the novel hyper alkalophil, thermostable, and chelator resistant metalloprotease.
abstract: Our newly discovered metalloprotease, designated as ALP NS12 was selected using gelatin agar plates with incubation at 100 °C. Subcloning of the fragments in to pUC118 to make E. coli HB101 (pPEMP01NS) with following two-step chromatography using diethylaminoethyl sepharose (DEAE-sepharose) and Sephadex G-100 columns to purify 97-kDa expressed enzyme was performed. Although activity of immobilized ALP NS12 on glass surface was established at temperatures between 70 and 120 °C and pH ranges 4.0-13.0, the optimum temperature and pH were achieved at 100 °C and 11.0, respectively. Enhancement of enzyme activity was obtained in the presence of 5 mM MnCl2 (91 %), CaCl2 (357 %), FeCl2 (175 %), MgCl2 (94 %), ZnCl2 (412 %), NiCl (86 %), NaCl (239 %), and Na-sulfate (81 %) while inhibition was observed with EDTA (5 mM), PMSF (3 mM), urea (8 M), and SDS (1 %) at 65, 37, 33, and 42 %, respectively. Consequently, the enzyme was well analyzed using crystallography and protein modeling. ALP NS12 can be applied in industrial processes at extreme temperatures and under highly basic conditions, chelators, and detergents.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia, nimasamie@gmail.com.
authors.forename: Nima
authors.initials: N
authors.lastname: Samie
authors.forename: Batoulsadat
authors.initials: B
authors.lastname: Haerian
authors.forename: Sekaran
authors.initials: S
authors.lastname: Muniandy
authors.forename: David
authors.initials: D
authors.lastname: Green
authors.forename: Masoumeh
authors.initials: M
authors.lastname: Ashouri
mesh_terms: D000468:Alkalies / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008958:Models, Molecular; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013696:Temperature
chemical_list: D000468:Alkalies; D002614:Chelating Agents; D045726:Metalloproteases