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Volume 142, Issue 4

A new, broad-substrate-specificity aminopeptidase from the dairy organism SBT 2171 Free

Abstract

An aminopeptidase with a very broad substrate specificity was purified to homogeneity from SBT 2171 by FPLC. The enzyme was purified 144-fold from a cell-free extract with a yield of 16%. The purified enzyme appeared as a single band on an SDS-PAGE gel. It had a molecular mass of 95 kDa and an isoelectric point of 4.9. The enzyme hydrolysed a large range of naphthylamide- and nitroanilide-substituted amino acids, as well as several di-, tri- and oligopeptides. It also exhibited significant prolineiminopeptidase-like activity, since it hydrolysed several proline-containing peptides. Prolyl--nitroanilide was hydrolysed with a low affinity (Michaelis-Menten constant 0.6 mM) and a of 2.5 μmol min (mg protein) while lysyl--nitroanilide was hydrolysed with a high affinity 0.003 mM; 37.5 μmol min (mg protein)]. The aminopeptidase activity, which was optimal between pH 6.0 and 8.0 and at 50 °, was very stable at 30 ° for more than 7 d. The activity lost by treatment with the thiol-blocking reagents could be restored with ß-mercaptoethanol, while Co and Mn restored the activity of the EDTA-treated enzyme. Immunological experiments with antibodies raised against the aminopeptidases from and clearly showed that both aminopeptidases are at least immunologically different from each other.

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Keyword(s): aminopeptidase , lactic acid bacteria , Lactobacillus, helveticus and proteolytic system
© Society for General Microbiology 1996
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1996-04-01
2023-03-29
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References

  1. Arora G., Lee B.H. Purification and characterization of aminopeptidase from Lactobacillus casei subsp casei. J Dairy Sci 1992; 75:700–710
     [Google Scholar]
  2. Baankreis R., Exterkate F.A. Characterization of a peptidase from Lactococcus lactis subsp cremoris HP that hydrolyses di- and tripeptides containing proline or hydrophobic residues as the aminoterminal amino acid. Syst Appl Microbiol 1991; 14:317–323
     [Google Scholar]
  3. Blanc B., Laloi P., Atlan D., Gilbert C., Portalier R. Two cell-wall-associated aminopeptidases from Lactobacillus helveticus and the purification and characterization of APII from strain ITGL1. J Gen Microbiol 1993; 139:1441–1448
     [Google Scholar]
  4. Bockelmann W., Schultz Y., Teuber M. Purification and characterization of an aminopeptidase from Lactobacillus delbrueckii subsp bulgaricus. Int Dairy J 1992; 2:95–107
     [Google Scholar]
  5. Chopin A. Organisation and regulation of genes for amino acid biosynthesis in lactic acid bacteria. FEMS Microbiol Rev 1993; 12:21–38
     [Google Scholar]
  6. Dudley E.G., Steele J. Nucleotide sequence and distribution of thepepPN gene from Lactobacillus helveticus CNRZ32. FEMS Microbiol Lett 1994; 119:41–46
     [Google Scholar]
  7. Eggimann B., Bachmann M. Purification and partial characterization of an aminopeptidase from Lactobacillus lactis. Appl Environ Microbiol 1980; 40:876–882
     [Google Scholar]
  8. Exterkate F.A. Comparison of strains of Streptococcus cremoris for proteolytic activities associated with the cell wall. Neth Milk Dairy J 1976; 30:95–105
     [Google Scholar]
  9. Exterkate F.A., De Veer G.J.C.M. Purification and some properties of a membrane-bound aminopeptidase A from Streptococcus cremoris. Appl Environ Microbiol 1987; 53:577–583
     [Google Scholar]
  10. Geis A., Bockeimann W., Teuber M. Simultaneous extraction and purification of a cell wall-associated peptidase and ß-casein specific protease from Streptococcus cremoris AC1. Appl Microbiol Biotechnol 1985; 23:79–84
     [Google Scholar]
  11. Gilbert C., Atlan D., Blanc B., Portalier R. Proline iminopeptidase from Lactobacillus delbrueckii subsp bulgaricus CNRZ 397. Microbiology 1994; 140:537–542
     [Google Scholar]
  12. Habibi-Najafi M.B., Lee B.H. Purification and characterization of proline iminopeptidase from Lactobacillus casei ssp. casei LLG. J Dairy Sei 1995; 78:251–259
     [Google Scholar]
  13. Khalid N.M., Marth E.H. Partial purification and characterization of an aminopeptidase from Lactobacillus helveticus CNRZ 32. Syst Appl Microbiol 1990; 13:311–319
     [Google Scholar]
  14. Kok J., De Vos W.M. The proteolytic system of lactic acid bacteria. In Genetics and Biotechnology of Lactic Acid Bacteria 1994 Edited by Gasson M.J., De Vos W.M. Glasgow: Blackie Academic and Professional; pp 169–210
     [Google Scholar]
  15. Kunji E.R.S., Hagting A., De Vries C.J., Juillard V., Haan-drikman A.J., Poolman B., Konings W.N. Transport of β-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis. Biol Chem 1995; 270:1569–1574
     [Google Scholar]
  16. Law B. Flavour development in cheese. In Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk 1984 Edited by Davis F.L., Law B.A. New York: Elsevier Applied Science; pp 187–208
     [Google Scholar]
  17. Lee C., Levin A., Branton D. Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrykmide gels. Anal Biochem 1987; 166:308–312
     [Google Scholar]
  18. Machuga E.J., 81 Ives D.H. Isolation and characterization of an aminopeptidase from Lactobacillus acidophilus R-26. Biochim Biophys Acta 1984; 789:26–36
     [Google Scholar]
  19. Miyakawa H., Kobayashi S., Shimamura S., Tomita M. Purification and characterization of an aminopeptidase from Lactobacillus helveticus LHE-511. J Dairy Sei 1992; 75:27–35
     [Google Scholar]
  20. Miyakawa H., Hashimoto I., Nakamura T., Ishibashi N., Shimamura S. Purification and characterization of a prolyl aminopeptidase from Lactobacillus helveticus LHE-511. Milchwissenschaft 1994; 49:615–619
     [Google Scholar]
  21. Morishita T., Deguchi Y., Yajima M., Sakurai T., Yura T. Multiple nutritional requirements of lactobacilli: genetic lesions affecting amino acid biosynthetic pathways. J Bacteriol 1981; 148:64–71
     [Google Scholar]
  22. Neviani E., Boquien C.Y., Monnet V., Phan Thanh L., Gripon J.-C. Purification and characterization of an aminopeptidase from Lactococcus lactis subsp cremoris AM2. Appl Environ Microbiol 1989; 55:2308–2314
     [Google Scholar]
  23. Niven G.W. Purification and characterization of aminopeptidase A from Lactococcus lactis subsp lactis NCDO 712. J Gen Microbiol 1991; 137:1207–1212
     [Google Scholar]
  24. Nowakowski C.M., Bhowmik T.K., Steele J.L. Cloning of peptidase genes from Lactobacillus helveticus CNRZ. Appl Microbiol Biotechnol 1993; 39:204–210
     [Google Scholar]
  25. Pritchard G.G., Coolbear T. The physiology and biochemistry of the proteolytic system in lactic acid bacteria. FEMS Microbiol Rev 1993; 12:179–206
     [Google Scholar]
  26. Sahlstrbm S., Chrzanowska J., Sbrhaug T. Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp cremoris IMN-C12. Appl Environ Microbiol 1993; 59:3076–3082
     [Google Scholar]
  27. Sasaki M., Bosman B.W., Tan P.S.T. Comparison of proteolytic activities in various lactobacilli. J Dairy Res 1995a; 62:601–610
     [Google Scholar]
  28. Sasaki M., Bosman B.W., Tan P.S.T. Immunological and electrophoretic study of the proteolytic enzymes from various Lactococcus and Lactobacillus strains. J Dairy Res 1995b; 62:611–620
     [Google Scholar]
  29. Tan P.S., T. & Konings W.N. Purification and characterization of an aminopeptidase from Lactococcus lactis subsp. cremoris Wg2. Appl Environ Microbiol 1990; 56:526–532
     [Google Scholar]
  30. Tan P.S.T., 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 1992a; 58:285–290
     [Google Scholar]
  31. Tan P.S.T., Van Kessel T., Van De Veerdonk F., Zuurendonk P., Bruins A.P., Konings W.N. Degradation and debit-tering of a tryptic digest of z-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris WG2. Appl Environ Microbiol 1992b; 59:285–290
     [Google Scholar]
  32. Tan P.S.T., Poolman B., Konings W.N. The proteolytic enzymes of Lactococcus lactis. J Dairy Res 1993; 60:269–286
     [Google Scholar]
  33. Tan P.S., T., Sasaki M., Bosman B.W., Iwasaki T. Purification and characterization of a dipeptidase from Lactobacillus helveticus SBT2171. Appl Environ Microbiol 1995; 61:3430–3435
     [Google Scholar]
  34. Wohlrab Y., Bockelmann W. Purification and characterization of a second aminopeptidase (PepC-like) from Lactobacillus delbrueckii subsp. bulgaricus. Inf Dairy J 1993; 3:685–701
     [Google Scholar]
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A new, broad-substrate-specificity aminopeptidase from the dairy organism Lactobacillus helveticus SBT 2171
Microbiology 142, 799 (1996); https://doi.org/10.1099/00221287-142-4-799
/content/journal/micro/10.1099/00221287-142-4-799
/content/journal/micro/10.1099/00221287-142-4-799
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