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KMID : 0380219940270040323
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Journal of Biochemistry and Molecular Biology
1994 Volume.27 No. 4 p.323 ~ p.329
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Purification and Characterization of and Extracellular Alkaline Protease from Bacillus subtilis RM615
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Abstract
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Abstract:
@EN Bacillus subtilis RM615, isolated from the house roach, produced extracellular proteases on a skim milk plate. The major protease(AprA) was purified by 80% actone precipitation and DE and CM-cellulose chromatography. The purified enzyme had a
molecular eight of 28,000 daltons on SDS-PAGE. The enzyme was inhibited by phenylmethane-sulphonyl fluoride (PMSF) and soybean trypsin inhibitor(STI), but not by ethylene diamine tetra-acetic acid (EDTA), 0-phenanthroline, elastatinal, histidine,
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leupeptin. These results indicated that it is a serine protease. This protease preferentially cleaved Leu or Arg as the P1 site. The proteolytic activity of AprA was reduced to 75% of maximum activity by treatment with 5 M urea for 1 h at 25% by
treatment with 1% sodium dodecyl sulfate (SDS). The protease showed high enzyme activity in a wide pH range from 6.0 to 12.0, and was stable in alkali, retaining 85% of its initial activity at pH 12.0 after 24 h incubation period at 25℃. Enzyme
activity reached a maximum at approximately 60℃. When the enzyme was incubated for 30 min at pH 11.0(50 mM CAPS buffer, 10 mM CaCl2) at 70℃, the residual activity of the enzyme was 71%.
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