ACTION OF A CELL-ENVELOPE PROTEINASE (CEPIII-TYPE) FROM LACTOCOCCUS-LACTIS SUBSP CREMORIS AM(1) ON BOVINE KAPPA-CASEIN

The specificity of the cell-envelope proteinase (CEP(III)-type) from L actococcus lactis subsp. cremoris AM(1) in its action on bovine kappa- casein was studied. A 4-h digest (pH 6.2, 15 degrees C) of kappa-casei n was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic an d chromatographic techniques. Isolated HMM and LMM products were ident ified by amino acid analysis, end-group determination and mass spectro metry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. T he HMM products formed were the fragments 1-160, 1-151, 1-95 and 1-79 of kappa-casein, whereas the main LMM products found were the 161-169 and 152-160 fragments. The enzyme specificity was concluded to be prim arily directed towards the C-terminal region of the substrate molecule by cleavage of the 160-161 and 151-152 peptide bonds. Two minor LMM p roducts were identified as the fragments 96-104 and 103-106, indicatin g additional cleavage at positions 102-103, 104-105 and 106-107 of the sequence. Also several peptide bonds within the 161-169 sequence were found to be subject to secondary cleavage by the proteinase. From ele ctrophoretic and identification data it is concluded that the lactococ cal CEP(I), CEP(III) and several mixed-type proteinases all act on the peptide bonds at positions 79-80 and 95-96. However, the C-terminal r egion of the kappa-casein sequence is the exclusive target of the CEP( III-) and, to variable extents, of the mixed-type enzymes.