ISSN:
1432-0614
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Abstract The specificity of the cell-envelope protein-ase (CEPIII–type) from Lactococcus lactis subsp. cre-moris AM1 in its action on bovine κ-casein was studied. A 4-h digest (pH 6.2, 15° C) of κ-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 electropho-retic and chromatographic techniques. Isolated HMMand LMM products were identified by amino acid anal-ysis, end-group determination and mass spectrometry.On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identi-fication of its components. The HMM products formedwere the fragments 1–160, 1–151, 1–95 and 1–79 of κ-casein, whereas the main LMM products found werethe 161–169 and 152–160 fragments. The enzyme spec-ificity was concluded to be primarily directed towardsthe 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. However, the C-terminal region of the κ-casein sequence is the exclusive target of the CEPIII– and, to variable extents, of the mixed-type enzymes.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00167279
