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. 1995 Jun;177(12):3472–3478. doi: 10.1128/jb.177.12.3472-3478.1995

The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides.

V Juillard 1, H Laan 1, E R Kunji 1, C M Jeronimus-Stratingh 1, A P Bruins 1, W N Konings 1
PMCID: PMC177051  PMID: 7768856

Abstract

The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide. All peptides corresponding to each of the MH+ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from beta-casein by proteinase only.

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Selected References

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  1. Chopin A. Organization and regulation of genes for amino acid biosynthesis in lactic acid bacteria. FEMS Microbiol Rev. 1993 Sep;12(1-3):21–37. doi: 10.1111/j.1574-6976.1993.tb00011.x. [DOI] [PubMed] [Google Scholar]
  2. Covey T. R., Huang E. C., Henion J. D. Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions. Anal Chem. 1991 Jul 1;63(13):1193–1200. doi: 10.1021/ac00013a003. [DOI] [PubMed] [Google Scholar]
  3. Creamer L. K., Richardson T. Anomalous behavior of bovine alpha s1- and beta-caseins on gel electrophoresis in sodium dodecyl sulfate buffers. Arch Biochem Biophys. 1984 Nov 1;234(2):476–486. doi: 10.1016/0003-9861(84)90295-9. [DOI] [PubMed] [Google Scholar]
  4. Exterkate F. A. Differences in short peptide-substrate cleavage by two cell-envelope-located serine proteinases of Lactococcus lactis subsp. cremoris are related to secondary binding specificity. Appl Microbiol Biotechnol. 1990 Jul;33(4):401–406. doi: 10.1007/BF00176654. [DOI] [PubMed] [Google Scholar]
  5. Katta V., Chowdhury S. K., Chait B. T. Use of a single-quadrupole mass spectrometer for collision-induced dissociation studies of multiply charged peptide ions produced by electrospray ionization. Anal Chem. 1991 Jan 15;63(2):174–178. doi: 10.1021/ac00002a016. [DOI] [PubMed] [Google Scholar]
  6. Kunji E. R., Hagting A., De Vries C. J., Juillard V., Haandrikman A. J., Poolman B., Konings W. N. Transport of beta-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis. J Biol Chem. 1995 Jan 27;270(4):1569–1574. doi: 10.1074/jbc.270.4.1569. [DOI] [PubMed] [Google Scholar]
  7. Kunji E. R., Smid E. J., Plapp R., Poolman B., Konings W. N. Di-tripeptides and oligopeptides are taken up via distinct transport mechanisms in Lactococcus lactis. J Bacteriol. 1993 Apr;175(7):2052–2059. doi: 10.1128/jb.175.7.2052-2059.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. Laan H., Konings W. N. Autoproteolysis of the Extracellular Serine Proteinase of Lactococcus lactis subsp. cremoris Wg2. Appl Environ Microbiol. 1991 Sep;57(9):2586–2590. doi: 10.1128/aem.57.9.2586-2590.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Laan H., Konings W. N. Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2. Appl Environ Microbiol. 1989 Dec;55(12):3101–3106. doi: 10.1128/aem.55.12.3101-3106.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laan H., Smid E. J., de Leij L., Schwander E., Konings W. N. Monoclonal Antibodies to the Cell-Wall-Associated Proteinase of Lactococcus lactis subsp. cremoris Wg2. Appl Environ Microbiol. 1988 Sep;54(9):2250–2256. doi: 10.1128/aem.54.9.2250-2256.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lee T. D., Vemuri S. MacProMass: a computer program to correlate mass spectral data to peptide and protein structures. Biomed Environ Mass Spectrom. 1990 Nov;19(11):639–645. doi: 10.1002/bms.1200191103. [DOI] [PubMed] [Google Scholar]
  14. Leenhouts K. J., Gietema J., Kok J., Venema G. Chromosomal stabilization of the proteinase genes in Lactococcus lactis. Appl Environ Microbiol. 1991 Sep;57(9):2568–2575. doi: 10.1128/aem.57.9.2568-2575.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mierau I., Tan P. S., Haandrikman A. J., Mayo B., Kok J., Leenhouts K. J., Konings W. N., Venema G. Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase. J Bacteriol. 1993 Apr;175(7):2087–2096. doi: 10.1128/jb.175.7.2087-2096.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Monnet V., Ley J. P., Gonzàlez S. Substrate specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCDO 763. Int J Biochem. 1992 May;24(5):707–718. doi: 10.1016/0020-711x(92)90004-k. [DOI] [PubMed] [Google Scholar]
  17. Pritchard G. G., Coolbear T. The physiology and biochemistry of the proteolytic system in lactic acid bacteria. FEMS Microbiol Rev. 1993 Sep;12(1-3):179–206. doi: 10.1111/j.1574-6976.1993.tb00018.x. [DOI] [PubMed] [Google Scholar]
  18. Reid J. R., Ng K. H., Moore C. H., Coolbear T., Pritchard G. G. Comparison of bovine beta-casein hydrolysis by PI and PIII-type proteinases from Lactococcus lactis subsp. cremoris [corrected]. Appl Microbiol Biotechnol. 1991 Dec;36(3):344–351. doi: 10.1007/BF00208154. [DOI] [PubMed] [Google Scholar]
  19. Roepstorff P., Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 1984 Nov;11(11):601–601. doi: 10.1002/bms.1200111109. [DOI] [PubMed] [Google Scholar]
  20. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  21. Smid E. J., Driessen A. J., Konings W. N. Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis. J Bacteriol. 1989 Jan;171(1):292–298. doi: 10.1128/jb.171.1.292-298.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smid E. J., Poolman B., Konings W. N. Casein utilization by lactococci. Appl Environ Microbiol. 1991 Sep;57(9):2447–2452. doi: 10.1128/aem.57.9.2447-2452.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tan P. S., Chapot-Chartier M. P., Pos K. M., Rousseau M., Boquien C. Y., Gripon J. C., Konings W. N. Localization of peptidases in lactococci. Appl Environ Microbiol. 1992 Jan;58(1):285–290. doi: 10.1128/aem.58.1.285-290.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tan P. S., Poolman B., Konings W. N. Proteolytic enzymes of Lactococcus lactis. J Dairy Res. 1993 May;60(2):269–286. doi: 10.1017/s0022029900027606. [DOI] [PubMed] [Google Scholar]
  25. Tan P. S., van Kessel T. A., van de Veerdonk F. L., Zuurendonk P. F., Bruins A. P., Konings W. N. Degradation and debittering of a tryptic digest from beta-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2. Appl Environ Microbiol. 1993 May;59(5):1430–1436. doi: 10.1128/aem.59.5.1430-1436.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tynkkynen S., Buist G., Kunji E., Kok J., Poolman B., Venema G., Haandrikman A. Genetic and biochemical characterization of the oligopeptide transport system of Lactococcus lactis. J Bacteriol. 1993 Dec;175(23):7523–7532. doi: 10.1128/jb.175.23.7523-7532.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Visser S., Robben A. J., Slangen C. J. Specificity of a cell-envelope-located proteinase (PIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine beta-casein. Appl Microbiol Biotechnol. 1991 Jul;35(4):477–483. doi: 10.1007/BF00169753. [DOI] [PubMed] [Google Scholar]
  28. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]

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