Purification and characterization of heat-stable proteases from Bacillus stearothermophilus RM-67.
The extracellular proteases of Bacillus stearothermophilus RM-67 were purified by ammonium sulfate fractionation (40 to 70% saturation), gel filtration through Sephadex G-100, and diethylaminoethyl-Sephadex A-50 ion-exchange chromatography. Gel filtration resulted in separation of the enzyme preparation into one minor (protease I) and one major (protease II) peak. The three-step purification scheme resulted in 39.5-fold purification and an overall recovery of 8.1% of protease I and 87.8-fold purification and 59.7% recovery of protease II. Purified proteases had pH and temperature optima of 8.0 and 70 degrees C. Protease I and II, when together, retained 100% activity at 60 degrees C for 30 min. Manganese imported 100% stability to the pooled proteases at 65 degrees C for 30 min. Amino acid analysis of the major peak (protease II) revealed the absence of half cystine and methionine. Protease I and II had molecular weights of 67,610 and 19,950 and Michaelis-Menten constants (casein) of 1.33 and 2.0 mg/ml. Energy of activation was 14,300 cal/mol for protease I and 11,150 cal/mol for protease II. Corresponding heat of activation was l3,620 and 10,470 cal/mol.
D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002417:Cattle; D005110:Extracellular Space / Q000201:enzymology; D005260:Female; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008892:Milk / Q000382:microbiology*; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D013816:Thermodynamics; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D008345:Manganese; D010447:Peptide Hydrolases; D008274:Magnesium