Purification and characterization of extracellular caseinolytic enzyme of Micrococcus sp. MCC-315 isolated from...

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.