title: Extracellular protease of Natrialba magadii: purification and biochemical characterization.
abstract: A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60 degrees C in the presence of 1.5M NaCl. The enzyme was stable and had a broad pH profile (6-12) with an optimum pH of 8-10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated.
authors.affiliation: Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Argentina.
authors.forename: M I
authors.initials: MI
authors.lastname: Giménez
authors.forename: C A
authors.initials: CA
authors.lastname: Studdert
authors.forename: J J
authors.initials: JJ
authors.lastname: Sánchez
authors.forename: R E
authors.initials: RE
authors.lastname: De Castro
mesh_terms: D000595:Amino Acid Sequence; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004795:Enzyme Stability; D000431:Ethanol; D006216:Halobacteriaceae / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D019634:Natronococcus / Q000201:enzymology; D009842:Oligopeptides / Q000737:chemistry; D012492:Salts; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D009842:Oligopeptides; D012492:Salts; D015842:Serine Proteinase Inhibitors; D000431:Ethanol; D012697:Serine Endopeptidases