Production, optimization and purification of a novel extracellular protease from the moderately halophilic bacterium Halobacillus karajensis.
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.
Karbalaei-Heidari
Hamid Reza
HR
Department of Biology, Faculty of Sciences, Shiraz University, Shiraz, Iran.
Amoozegar
Mohammad Ali
MA
Hajighasemi
Mahbod
M
Ziaee
Abed-Ali
AA
Ventosa
Antonio
A
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
D001426:Bacterial Proteins; D003470:Culture Media; D012965:Sodium Chloride; D010447:Peptide Hydrolases
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