title: Purification and biochemical characterization of a protease secreted by the Salinivibrio sp. strain AF-2004 and its behavior in organic solvents.
abstract: A metalloprotease secreted by the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 when the culture reached the stationary growth phase. This enzyme was purified to homogeneity by acetone precipitation and subsequent Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The apparent molecular mass of the protease was 31 kDa by SDS-PAGE, whereas it was estimated as approximately 29 kDa by Sephacryl S-200 gel filtration. The purified protease had a specific activity of 116.8 mumol of tyrosine/min per mg protein on casein. The optimum temperature and salinity of the enzyme were at 55 degrees C and 0-0.5 M NaCl, although at salinities up to 4 M NaCl activity still remained. The protease was stable and had a broad pH profile (5.0-10.0) with an optimum of 8.5 for casein hydrolysis. The enzyme was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), Pefabloc SC, chymostatin and also EDTA, indicating that it belongs to the class of serine metalloproteases. The protease in solutions containing water-soluble organic solvents or alcohols was more stable than that in the absence of organic solvents. These characteristics make it an ideal choice for applications in industrial processes containing organic solvents and/or salts.
authors.affiliation: Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran. heidary@ibb.ut.ac.ir
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Karbalaei-Heidari
authors.forename: Abed-Ali
authors.initials: AA
authors.lastname: Ziaee
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Amoozegar
mesh_terms: D002364:Caseins / Q000737:chemistry; D002853:Chromatography, Liquid; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000254:growth & development; D006358:Hot Temperature; D045726:Metalloproteases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry
chemical_list: D002364:Caseins; D012997:Solvents; D045726:Metalloproteases