Isolation, purification and characterization of a surfactants-, laundry detergents- and organic solvents-resistant alkaline protease from Bacillus sp. HR-08.
Bacillus sp. HR-08 screened from soil samples of Iran, is capable of producing proteolytic enzymes. 16S rDNA analysis showed that this strain is closely related to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, and Bacillus atrophaeus. The zymogram analysis of the crude extract revealed the presence of five extracellular proteases. One of the proteases was purified in three steps procedure involving ammonium sulfate precipitation, DEAE-Sepharose ionic exchange and Sephacryl S-200 gel filtration chromatography. The molecular mass of the enzyme on SDS-PAGE was estimated to be 29 kDa. The protease exhibited maximum activity at pH 10.0 and 60 degrees C and was inhibited by PMSF but it was not affected by cysteine inhibitors, suggesting that the enzyme is a serine alkaline protease. Irreversible thermoinactivation of enzyme was examined at 50, 60, and 70 degrees C in the presence of 10 mM CaCl(2). Results showed that the protease activity retains more than 80% and 50% of its initial activity after incubation for 30 min at 60 and 70 degrees C, respectively. This enzyme had good stability in the presence of H(2)O(2), nonionic surfactant, and local detergents and its activity was enhanced in the presence of 20% of dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and isopropanol. The enzyme retained more than 90% of its initial activity after pre-incubation 1 h at room temperature in the presence of 20% of these solvents. Also, activation can be seen for the enzyme at high concentration (50%, v/v) of DMF and DMSO.
Moradian
Fatemeh
F
Department of Biochemistry and Biophysics, Faculty of Sciences, Tarbiat Modares University, Tehran, Iran.
Khajeh
Khosro
K
Naderi-Manesh
Hossein
H
Sadeghizadeh
Majid
M
D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D003902:Detergents / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D004789:Enzyme Activation; D004795:Enzyme Stability; D009930:Organic Chemicals / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry*; D013501:Surface-Active Agents / Q000737:chemistry*
D001426:Bacterial Proteins; D003902:Detergents; D009930:Organic Chemicals; D012997:Solvents; D013501:Surface-Active Agents; D010450:Endopeptidases; C087822:alkaline protease