Purification and Characterization of Heat-Stable Proteases from Bacillus stearothermophilus RM-67

Abstract 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°C. Protease I and II, when together, retained 100% activity at 60°C for 30 min. Manganese imparted 100% stability to the pooled proteases at 65°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 13,620 and 10,470 cal/mol.