ALTERED SPECIFICITY OF LACTOCOCCAL PROTEINASE P-I (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 a lpha(s1)-, beta-, and kappa-caseins by the cell envelope-associated pr oteinase (lactocepin; EC 3.4.21.96) with type P-1 specificity (i.e., l actocepin I) from Lactococcus lactis subsp. lactis BN1 was investigate d at water activities (a(w)) and salt concentrations reflecting those in cheddar type cheese. In the presence of polyethylene glycol 20000 ( PEG 20000)-NaCl (a(w) = 0.95), hydrolysis of beta-casein resulted in p roduction of the peptides comprising residues 1 to 6 and 47 to 52, whi ch are characteristic of type P-III enzyme activity (lactocepin III) i n buffer. The fragment comprising residues 1 through 166, inclusive (f ragment 1-166), which is typical of lactocepin I activity in buffer sy stems, was not produced. Similarly, peptide 152-160 from kappa-casein, which is usually produced in aqueous buffers exclusively by lactocepi n III, was a major product of lactocepin I. Most of the specificity di fferences obtained in the presence of PEG 20000-NaCl were also obtaine d in the presence of PEG 20000 alone (a(w) = 0.99). In addition, alpha (s1)-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 a n a(w) of 0.95) was generally as expected for lactocepin I activity ex cept that beta-casein peptide 47-52 and kappa-casein fragment 1-160 we re produced; both of these are normally formed by lactocepin III in bu ffer. The differences in lactocepin specificity obtained in the humect ant systems can he attributed to a combination of a(w) and humectant h ydrophobicity, both of which are parameters that are potentially relev ant to the cheese-ripening environment.