title: Production, optimization and purification of a novel extracellular protease from the moderately halophilic bacterium Halobacillus karajensis.
abstract: The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34 degrees C, pH 8.0-8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50 degrees C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0-10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.
authors.affiliation: Department of Biology, Faculty of Sciences, Shiraz University, Shiraz, Iran.
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Karbalaei-Heidari
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Amoozegar
authors.forename: Mahbod
authors.initials: M
authors.lastname: Hajighasemi
authors.forename: Abed-Ali
authors.initials: AA
authors.lastname: Ziaee
authors.forename: Antonio
authors.initials: A
authors.lastname: Ventosa
mesh_terms: D001406:Bacillaceae / Q000737:chemistry / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D046508:Culture Techniques; D004795:Enzyme Stability; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism*; D012988:Soil Microbiology
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D012965:Sodium Chloride; D010447:Peptide Hydrolases
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Biosci Biotechnol Biochem. 2006 Jun;70(6):1395-401
references.pmid: 16794319
references.citation: FEMS Microbiol Lett. 2000 Nov 1;192(1):67-72
references.pmid: 11040430
references.citation: Int J Syst Evol Microbiol. 2003 Jul;53(Pt 4):1059-1063
references.pmid: 12892126
references.citation: Enzyme Microb Technol. 1994 Apr;16(4):266-75
references.pmid: 7764632
references.citation: Microbiol Mol Biol Rev. 1998 Sep;62(3):597-635
references.pmid: 9729602
references.citation: Extremophiles. 2007 Mar;11(2):237-43
references.pmid: 17072685
references.citation: Anal Biochem. 1980 Feb;102(1):196-202
references.pmid: 7188842
references.citation: J Ind Microbiol Biotechnol. 2008 Feb;35(2):121-31
references.pmid: 17994257
references.citation: Res Microbiol. 2005 May;156(4):478-84
references.pmid: 15862445
references.citation: Extremophiles. 2003 Jun;7(3):221-8
references.pmid: 12768453
references.citation: J Gen Physiol. 1947 Mar 20;30(4):291-310
references.pmid: 19873496
references.citation: Extremophiles. 2000 Jun;4(3):181-8
references.pmid: 10879563