Altered Specificity of Lactococcal Proteinase PI (Lactocepin I) in Humectant Systems Reflecting the Water Activity and Salt Content of Cheddar Cheese

By using various humectant systems, the specificity of hydrolysis of αs1-, β-, and κ-caseins by the cell envelope-associated proteinase (lactocepin; EC 3.4.21.96) with type P1 specificity (i.e., lactocepin I) from Lactococcus lactis subsp. lactis BN1 was investigated at water activities (aw) and salt concentrations reflecting those in cheddar type cheese. In the presence of polyethylene glycol 20000 (PEG 20000)-NaCl (aw = 0.95), hydrolysis of β-casein resulted in production of the peptides comprising residues 1 to 6 and 47 to 52, which are characteristic of type PIII enzyme activity (lactocepin III) in buffer. The fragment comprising residues 1 through 166, inclusive (fragment 1-166), which is typical of lactocepin I activity in buffer systems, was not produced. Similarly, peptide 152-160 from κ-casein, which is usually produced in aqueous buffers exclusively by lactocepin III, was a major product of lactocepin I. Most of the specificity differences obtained in the presence of PEG 20000-NaCl were also obtained in the presence of PEG 20000 alone (aw = 0.99). In addition, αs1-casein, which normally is resistant to lactocepin I activity, was rapidly hydrolyzed in the presence of PEG 20000 alone. Hydrolysis of casein in the presence of PEG 300-NaCl or glycerol-NaCl (both having an aw of 0.95) was generally as expected for lactocepin I activity except that β-casein peptide 47-52 and κ-casein fragment 1-160 were produced; both of these are normally formed by lactocepin III in buffer. The differences in lactocepin specificity obtained in the humectant systems can be attributed to a combination of aw and humectant hydrophobicity, both of which are parameters that are potentially relevant to the cheese-ripening environment.