Purification and Characterization of Extracellular Caseinolytic Enzyme of Micrococcus Sp. MCC-315 Isolated from Cheddar Cheese

Abstract

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.

Extracellular proteinases from Micrococcus GF : II. Isolation and characterization

“…Zn 2 + or Cd 2 +, which partiaily inhibited the proteinases of Micrococcus GF, also partially inhibited the proteinase of M caseolyticus (Desmazeaud and Hermier, 1968). Mn 2 + stimulated the extracellular proteinase of Micrococcus MCC-315 (Prasad et al, 1986) but inhibited the proteinase of M caseolyticus (Desmazeaud and Hermier, 1968) and inhibited the proteinases of Micrococcus GF. Cu 2 + has been reported to inhibit ail the proteinases discussed here.…”

“…The proteinase of Micrococcus MCC-315 was also irreversibly inactivated by EDTA (Prasad et al, 1986). The proteinase from M caseolyticus could be partially reactivated by 8r2+ and Ca2+ (Desmazeaud and Hermier, 1968).…”

“…The optimum temperature for the activity of bath extracellular proteinases of Micrococcus GF was 45 -c. The extracellular proteinases from M freudenreichii (Husain and McDonald, 1958) and M caseolyticus (Desmazeaud and Hermier, 1968) were optimally active at 50 oC. Prasad et al (1986) (Desmazeaud and Hermier, 1968), 10.6 for Micrococcus MCC-315 (Prasad et al, 1986) (Desmazeaud and Hermier, 1968), but stimulated the extracellular proteinase of Micrococcus MCC-315 (Prasad et al, 1986). Zn 2 + or Cd 2 +, which partiaily inhibited the proteinases of Micrococcus GF, also partially inhibited the proteinase of M caseolyticus (Desmazeaud and Hermier, 1968).…”

“…Nath and Ledford (1972) detected extracellular proteolytic activity in 3 of 18 Micrococcus strain isolated from Cheddar cheese. Prasad et al (1986) isolated a metalloproteinase from Micrococcus MCC-315.…”

Purification and Characterization of a Metalloprotease fromChryseobacterium indologenesIx9a and Determination of the Amino Acid Specificity with Electrospray Mass Spectrometry

“…Proteolysis by these enzymes leads to the formation of short peptides, which cause off flavors and volume reductions in cheese, yoghurt, and other dairy products. Much work has already been carried out on the proteases from Pseudomonas species (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) and to a lesser extent on the proteases from other psychrotrophic organisms such as Aeromonas (14), Micrococcus (15), Serratia (16), Bacillus (17), and Flavobacterium (18,19) species. No purification data are yet available for the extracellular proteases from Chryseobacterium species.…”