Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine kappa-casein
| Autor: | W. D. van Dongen, Charles J. Slangen, W. Heerma, Arjan J. P. M. Robben, Johan Haverkamp, Servaas Visser |
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| Rok vydání: | 1994 |
| Předmět: |
Proteolysis
Molecular Sequence Data Peptide Cleavage (embryo) Applied Microbiology and Biotechnology Hydrolysate Mass Spectrometry Casein Endopeptidases medicine Peptide bond Animals Amino Acid Sequence Chromatography High Pressure Liquid chemistry.chemical_classification Chromatography medicine.diagnostic_test biology Chemistry Lactococcus lactis subsp cremoris Lactococcus lactis Cell Membrane Caseins General Medicine biology.organism_classification Peptide Fragments Biochemistry Cattle Sequence Analysis Biotechnology |
| Zdroj: | Applied microbiology and biotechnology. 41(6) |
| ISSN: | 0175-7598 |
| Popis: | The specificity of the cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris 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 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 κ-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 lactococaal 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-aand, to variable extents, of the mixed-type enzymes. |
| Databáze: | OpenAIRE |
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