1887

Abstract

The proteolytic system of lactic acid bacteria has been extensively studied over the past 10 years and peptidases from lactococci are now well known. The situation is, however, different for from which only a few peptidases have been purified and characterized. The present work was conducted to characterize an aminopeptidase of CNRZ 302, called St-PepA, with high specificity for acidic amino acids. St-PepA was purified by a three-step procedure from a spheroblast extract of CNRZ 302. Its molecular mass was estimated to be 360 kDa by gel filtration and 45 kDa by SDS-PAGE, indicating that it had an octameric structure. Its activity against aspartate-p-nitroanilide was maximal at pH 8.5 and 62 C and highly enhanced by Zn, Co and . St-PepA was inhibited by metal-chelating reagents and, to a lesser extent, by agents modifying sulfhydryl groups. It showed an activity towards p-nitroanilide derivatives, di-and tripeptides, and larger peptides such as fragment 43-58 of a-casein. It had a high substrate specificity towards N-terminal acidic amino acid residues but it could also release serine and malic acid, the -hydroxy acid homologue of aspartic acid. Kinetic studies revealed that the affinity of St-PepA was more than 18-fold higher for aspartic acid--nitroanilide ( = 0.42 mM) than for glutamic acid--nitroanilide ( = 7.65 mM) with a similar for both substrates [about 40 mol min (mg enzyme)]. St-PepA is heat stable, with a maximal loss of activity of 15% after incubation for 120 min at 50 C and 60C. It is likely to be involved in the nitrogen metabolism of and in the development of the organoleptic characteristics of cheese and yoghurt.

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1995-09-01
2021-10-18
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References

  1. Bacon C.L., Jennings P.V., Fhaolain I.N., O´Cuinn G. 1994; Purification and characterization of an aminopeptidase A from cytoplasm of Lactococcus lactis subsp. cremoris AM2.. Int Dairy J 4:503–519
    [Google Scholar]
  2. Biede S.L., Hammond E.G. 1979; Swiss cheese flavor. II. Organoleptic analysis.. J Dairy Sci 62:238–248
    [Google Scholar]
  3. Bracquart P., Lorient D., Alais C. 1978; Effet des acides aminés sur la croissance de Streptococcus thermophilus. II: Etude sur cinq souches.. Milchwissenschaft 33:341–344
    [Google Scholar]
  4. Bradford M.M. 1976; A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.. Anal Biochem 72:248–254
    [Google Scholar]
  5. Chapot-Chartier M.P., Rul F., Nardi M., Gripon J.C. 1994; Gene cloning and characterization of PepC, a cysteine aminopeptidase from Streptococcus thermophilus, with sequence similarity to the eucaryotic bleomycin hydrolase.. Eur J Biochem 224:497–506
    [Google Scholar]
  6. Cheung H.S., Cushman D.W. 1971; A soluble aspartate aminopeptidase from dog kidney.. Biochim Biophys Acta 242:190–193
    [Google Scholar]
  7. Danielson E.M., Noren O., Sjöström H., Ingram J., Kenny A. 1980; Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent and proteinase solubilized forms.. Biochem J 189:591–603
    [Google Scholar]
  8. Desmazeaud M.J. 1974; Propriétés générates et spécificité d’action d’une endopeptidase neutre intracellulaire de Streptococcus thermophilus.. Biochimie 56:1173–1181
    [Google Scholar]
  9. Desmazeaud M.J., Hermier J.H. 1972; Isolement et détermination de la composition qualitative de peptides issus de la caséine, stimulant la croissance de Streptococcus thermophilus.. Eur J Biochem 28:190–198
    [Google Scholar]
  10. Exterkate F.A., De Veer G.J.C.M. 1987; Purification and some properties of a membrane-bound aminopeptidase A from Streptococcus cremoris.. Appl Environ Microbiol 53:577–583
    [Google Scholar]
  11. Feracci H., Benajiba A., Gorvel J.P., Doumeng C., Maroux S. 1981; Enzymatic and immunological properties of the protease form of aminopeptidases N and A from pig and rabbit intestinal brush border.. Biochim Biophys Acta 658:148–157
    [Google Scholar]
  12. Kok J. 1990; Genetics of the proteolytic system of lactic acid bacteria.. FEMS Microbiol Rev 87:15–41
    [Google Scholar]
  13. Laan H., Smid E.J., Tan P.S.T., Konings W.N. 1989; Enzymes involved in the degradation and utilization of casein in Eactococcus lactis.. Neth Milk Dairy J 43:327–345
    [Google Scholar]
  14. Laemmli U.K. 1970; Cleavage of the structural proteins during the assembly of the head of bacteriophage T4.. Nature 227:680–685
    [Google Scholar]
  15. Lalu K., Lampelo S., Nummelin-Kortelainen M., Vanha-Perttula T. 1984; Purification and partial characterization of aminopeptidase A from the serum of pregnant and non-pregnant women.. Biochim Biophys Acta 789:324–333
    [Google Scholar]
  16. Law B.A., Kolstad D.J. 1983; Proteolytic systems in lactic acid bacteria.. Antonie Leeuwenhoek 49:225–245
    [Google Scholar]
  17. Meyer J., Jordi R. 1987; Purification and characterization of X-prolyl-dipeptidyl-aminopeptidase from Lactobacillus lactis and Streptococcus thermophilus.. J Dairy Sci 70:738–745
    [Google Scholar]
  18. Midwinter R.G., Pritchard G.G. 1994; Aminopeptidase N from Streptococcus salivarius subsp.thermophilus NCDO 573: purification and properties.. J Appl Bacteriol 77:288–295
    [Google Scholar]
  19. Mizutani S., Okano K., Hasegawa E., Salura H., Yamada M. 1981; Aminopeptidase A in human placenta.. Biochim Biophys Acta 678:168–170
    [Google Scholar]
  20. Monnet V., Chapot-Chartier M.-P., Gripon J.-C. 1993; Lespeptidases des lactocoques.. Lait 73:97–108
    [Google Scholar]
  21. Nagatsu I., Nagatsu T., Yamamoto T., Glenner G.G., Mehl J.W. 1970; Purification of aminopeptidase A in human serum and degradation of angiotensin II by the purified enzyme.. Biochim Biophys Acta 198:255–270
    [Google Scholar]
  22. Niven G.W. 1991; Purification and characterization of aminopeptidase A from Lactococcus lactis subsp.lactis NCDO 712.. J Gen Microbiol 137:1207–1212
    [Google Scholar]
  23. Petrovic S., Vitale L. 1990; Purification and properties of glutamyl aminopeptidase from chicken egg-white.. Comp Biochem Physiol 95B:589–595
    [Google Scholar]
  24. Rabier D., Desmazeaud M.J. 1973; Inventaire des différentes activités peptidasiques intracellulaires de Streptococcus thermophilus.. Biochimie 55:389–404
    [Google Scholar]
  25. Rul F. 1994; Contribution à l’étude du sysèeme peptidasique de Streptococcus thermophilus CNRZ 302.. PhD thesis, University of Caen, France
    [Google Scholar]
  26. Rul F., Monnet V., Gripon J.C. 1994; Purification and characterization of a general aminopeptidase (St-PepN) from Streptococcus salivarius ssp.thermophilus CNRZ 302.. J Dairy Sci 77:2880–2889
    [Google Scholar]
  27. Schechter K., Berger B. 1967; On the size of the active site of proteases. I. Papaïn.. Biochem Biophys Res Commun 27:157–162
    [Google Scholar]
  28. Shahbal S., Hemme D., Desmazeaud M. 1991; High cell wall-associated proteinase activity of some Streptococcus thermophilus strains (H-strains) correlated with high acidification rate in milk.. Lait 71:351–357
    [Google Scholar]
  29. Tan P.S.T., Konings W.N. 1990; Purification and characterization of an aminopeptidase from Lactococcus lactis subsp.cremoris Wg2.. Appl Environ Microbiol 56:526–532
    [Google Scholar]
  30. Tan P.S.T., Poolman B., Konings W.N. 1993; Proteolytic enzymes of Lactococcus lactis.. J Dairy Res 60:269–286
    [Google Scholar]
  31. Thomas T.D., Pritchard G.G. 1987; Proteolytic enzymes of dairy starter cultures.. FEMS Microbiol Rev 46:245–286
    [Google Scholar]
  32. Tobe H., Kojima F., Aoyagi T., Umezawa H. 1980; Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney.. Biochim Biophys Acta 613:459–468
    [Google Scholar]
  33. Tsakalidou E., Kalantsopoulos G. 1992; Purification and partial characterization of an intracellular aminopeptidase from Streptococcus salivarius subsp.thermophilus.. J Appl Bacteriol 72:227–232
    [Google Scholar]
  34. Warmke R., Belitz H.D. 1993; Influence of glutamic acid on the bitter taste of various compounds. Z.. Lebensm Untersuch Forsch 197:132–133
    [Google Scholar]
  35. Yamada R., Mizutani S., Kurauchi O., Okano K., Imaizumi H., Narita O., Tomoda Y. 1988; Purification and characterization of human placental aminopeptidase A.. Enzyme 40:223–230
    [Google Scholar]
  36. Yoshpe-Besançon I., Auriol D., Paul F., Monsan P., Gripon J.-C., Ribadeau-Dumas B. 1993; Purification and characterization of an aminopeptidase A from Staphylococcus chromogenes and its use for the synthesis of amino-acid derivatives and dipeptides.. Eur J Biochem 211:105–110
    [Google Scholar]
  37. Zevaco C., Monnet V., Gripon J.C. 1990; Intracellular X-prolyl-dipeptidyl peptidase from Lactococcus lactis ssp.lactis: purification and properties.. J Appl Bacteriol 68:357–366
    [Google Scholar]
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