Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine kappa-casein.
The specificity of the cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine kappa-casein was studied. A 4-h digest (pH 6.2, 15 degrees C) of kappa-casein 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 and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The 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 primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160-161 and 151-152 peptide bonds. Two minor LMM products were identified as the fragments 96-104 and 103-106, indicating 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 electrophoretic and identification data it is concluded that the lactococcal CEPI, CEPIII and several mixed-type proteinases all act on the peptide bonds at positions 79-80 and 95-96.(ABSTRACT TRUNCATED AT 250 WORDS)
Visser
S
S
Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), BA Ede.
Slangen
C J
CJ
Robben
A J
AJ
van Dongen
W D
WD
Heerma
W
W
Haverkamp
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D013294:Lactococcus lactis / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis
D002364:Caseins; D010446:Peptide Fragments; D010450:Endopeptidases