Advertisement

Current Microbiology

, Volume 31, Issue 1, pp 49–54 | Cite as

Characterization of fructose 6 phosphate phosphoketolases purified from Bifidobacterium species

  • Jean-Pierre Grill
  • Joel Crociani
  • Jean Ballongue
Article

Abstract

Fructose 6 phosphate phosphoketolases (F6PPKs) were purified from Bifidobacterium longum BB536, B. dentium ATCC 27534, B. globosum ATCC 25864, and Bifidobacterium animalis ATCC 25527. Concerning ions (Cu++, Zn++, Ca++, Mg++, Fe++, Co++, Mn++) and common enzyme inhibitors (fructose, ammonium sulfate, iodoacetate, and parachloromercuribenzoic acid), no difference appeared between the enzymes. Cu++, parachloromercuribenzoic acid (pCMB), and mercuric acetate induced high enzymatic inhibition. The study of pCMB demonstrated a noncompetitive inhibition. Additional results showed that the sulfhydryl group was not involved in catalytic reaction. Photooxidation experiments and determination of ionizable group pKas (5.16–7.17) suggested the presence of one or more histidines necessary for the catalytic reaction and explained the inhibition observed with pCMB. In light of the noncompetitive inhibition, this group was not directly involved in substrate binding. Determination of Kmdemonstrated that the affinities for fructose 6 phosphate in the case of animal and human origin strains were close. In addition, the same enzymatic efficiency (Kcat/Km)was obtained for each strain. The F6PPK activity was regulated by sodium pyrophosphate, ATP, and especially by ADP.

Keywords

Fructose Pyrophosphate Catalytic Reaction Ammonium Sulfate Sulfhydryl 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature Cited

  1. 1.
    Benno Y, Sawada K, Mitsuoka T (1984) The intestinal microflora of infants: composition of faecal flora in breast-fed and bottle-fed infants. Microbiol Immunol 28:975–986Google Scholar
  2. 2.
    Bourget N, Simonet JM, Decaris B (1993) Analysis of the genome of the five Bifidobacterium breve strains: plasmid content, pulsed-field gel electrophoresis genome size estimation and rm loci number. FEMS Microbiol Lett. 110:11–20Google Scholar
  3. 3.
    Bradford MM (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principles of protein dye binding. Anal Biochem 72:248–254Google Scholar
  4. 4.
    Goldberg M, Fessenden JM, Racker E (1966) Phosphoketolase. Methods Enzymol IX:515–520Google Scholar
  5. 5.
    Kenyon GL, Bruice TW (1977) Novel sulfhydryl reagents. Methods Enzymol 47:407–430Google Scholar
  6. 6.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  7. 7.
    Lipman F, Tuttle LC (1945) A specific micromethod for determination of acyl phosphates. J Biol Chem 159:21–28Google Scholar
  8. 8.
    Mangin I, Bourget N, Bouhnick Y, Bisetti N, Simonet JM, Decaris B (1994) Identification of Bifidobacterium strains by rRNA gene restriction patterns. Appl Env Microbiol 60:1451–1458Google Scholar
  9. 9.
    Racker E (1962) Fructose 6 phosphate phosphoketolase from Acetobacter xylinum. Methods Enzymol V:276–280Google Scholar
  10. 10.
    Scardovi V (1986) Bifidobacterium. In: Sneath HA, Mair NS, Sharpe ME, Holt JG (eds) The bergey's manual of systematic bacteriology, Vol 2, 9th ed. Baltimore: Williams and Wilkins, pp 1418–1434Google Scholar
  11. 11.
    Scardovi V, Trovatelli LD (1965) The fructose 6 phosphate shunt as peculiar pattern of hexose degradation in the genus Bifidobacterium. Ann Microbiol 15:19–29Google Scholar
  12. 12.
    Scardovi V, Trovatelli LD, Zani G, Crociani F, Matteuzzi D (1971a) Deoxyribonucleic acid homology relationships among species of the genus Bifidobacterium. Int J Syst Bacteriol 21:276–294Google Scholar
  13. 13.
    Scardovi V, Sgorbati B, Zani G (1971b) Starch gel electrophoresis of fructose 6 phosphate phosphoketolase in the genus Bifidobacterium. J Bacteriol 106:1036–1039Google Scholar
  14. 14.
    Schramm M, Klybas V, Racker E (1958) Phosphorolytic cleavage of fructose 6 phosphate phosphoketolase from Acetobacter xylinum. J Biol Chem 233:1283–1288Google Scholar
  15. 15.
    Sebald M, Gasser F, Werner H (1965) DNA base composition and classification. Application to group of bifidobacteria and to related genera. Ann Inst Pasteur 109:251–269Google Scholar
  16. 16.
    Sgorbati G, Lenaz Z, Casalicchio F (1976) Purification and properties of two fructose 6 phosphate phosphoketolases in Bifidobacterium. Antonie van Leeuwenhoek 45:557–564Google Scholar
  17. 17.
    Tissier H (1900) Recherche sur la flore intestinale des nourissons (Etat normal et pathologique). Ph D thesis, University of Medecine, ParisGoogle Scholar
  18. 18.
    Weil L, Seibles TS (1955) Photooxidation of crystalline ribonuclease in the presence of methylene blue. Arch Biochem Biophys 54:368–377Google Scholar
  19. 19.
    Yamamoto T, Marotomi M, Tanaka R (1993) Species specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora. Appl Env Microbiol 58:4076–4079Google Scholar

Copyright information

© Springer-Verlag New York Inc 1995

Authors and Affiliations

  • Jean-Pierre Grill
    • 1
  • Joel Crociani
    • 1
  • Jean Ballongue
    • 1
  1. 1.Institut Henry Tissier, Laboratoire de Chimie Biologique IUniversité de Nancy IVandoeuvre les NancyCédex France

Personalised recommendations