Micrococcus sp. MCC-315, an organism isolated from Cheddar cheese, produced an extracellular calcium metalloenzyme. This protease was purified to homogeneity from culture supernatant by precipitation with ammonium sulfate (50 to 70% saturation) and gel filtration through Sephadex G-100, resulting in about 82 times increase of specific activity and 53% recovery of the enzyme. The protease exhibited a pH optimum at 10.6 for both whole casein and beta-casein. It had optimum activity for whole casein in the presence and absence of calcium++ at 60 and 50 degrees C, respectively, and at 37 to 40 degrees C for beta-casein with or without calcium++. The enzyme was stable at 45 degrees C but lost activity at higher temperatures. It was inhibited by heavy metal ions but calcium++, cobalt++, manganese++, strontium++, and iron++ had a slight stimulatory effect. The enzyme was inhibited completely and irreversibly by metal chelating agents. Calcium ions were required for maintenance of an active conformation of the enzyme. The enzyme had molecular weight of 28,900 and Michaelis constants 6.66 and 5.00 mg/ml for whole casein and beta-casein. Amino acid analysis of the hydrolyzed enzyme revealed the absence of sulfhydryl groups as was indicated also by lack of inhibition by thiol reagents.

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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.

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans, five were B. circulans and four were B. licheniformis. Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65 degrees C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70 degrees C for 30 min.