1887

Abstract

Summary: A specific isocitrate lyase (ICL) activity of 0.17 U (mg protein) was detected in cultures of the riboflavin-producing fungus during growth on soybean oil. Enzyme activity was not detectable during growth on glucose [<0.005 U (mg protein)], indicating a regulation. The enzyme was purified 108-fold by means of ammonium sulphate fractionation, gel filtration and cation-exchange chromatography. SDS-PAGE of the purified protein showed a homogeneous band with an of 66000. The of 254000 determined by gel-filtration chromatography indicated a tetrameric structure of the native protein. The enzyme was found to have a pH optimum for the isocitrate cleavage of 7.0, and the for -DL-isocitrate was determined as 550 μ. Enzyme activity was Mg dependent. In regulation studies ICL was weakly inhibited by central metabolites. A concentration of 10 mM phosphoenolpyruvate or 6-phosphogluconate revealed a residual activity of more than 40%. On the other hand, oxalate ( : 4 μM) and itaconate ( : 170 μM) showed a strong inhibition and may therefore be interesting as antimetabolites.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-142-2-411
1996-02-01
2021-10-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/142/2/mic-142-2-411.html?itemId=/content/journal/micro/10.1099/13500872-142-2-411&mimeType=html&fmt=ahah

References

  1. Aebi H. E. 1974; Catalase. Methods of Enzymatic Analysis673–686 Edited by Bergmeyer H. U. New York: Academic Press;
    [Google Scholar]
  2. Ashworth J. M., Kornberg H. L. 1963; Fine control of the glyoxylate cycle by allosteric inhibition of isocitrate lyase. Biochim Biophys Acta 73:519–522
    [Google Scholar]
  3. Bacher A. 1990; Biosynthesis of flavins. Chemistry and Biochemistry of Flavoenzymes 1215–249 Edited by Müller F. Boca Raton: CRC Press;
    [Google Scholar]
  4. Bigelis R. 1989; Industrial products of biotechnology: application of gene technology. Biotechnology 7243 Edited by Rehm H. J., Reed G. Weinheim: VCH;
    [Google Scholar]
  5. Bowyer P., De Lucas J. R., Turner G. 1994; Regulation of the expression of isocitrate lyase gene (acuD) of Aspergillus nidulans. Mol & Gen Genet 242:484–489
    [Google Scholar]
  6. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
    [Google Scholar]
  7. Cioni M., Pinzauti G., Vanni P. 1981; Comparative biochemistry of the glyoxylate cycle. Comp Biochem Physiol B 70B:1–26
    [Google Scholar]
  8. Demain A. L. 1972; Riboflavin oversynthesis. Annu Rev Microbiol 26:369–388
    [Google Scholar]
  9. Dixon G. H., Kornberg H. L. 1959; Assay methods for key enzymes of the glyoxylate cycle. Biochem J 72:3
    [Google Scholar]
  10. Gainey L. D. S., Kolble K., Connerton I. F. 1991; Isolation and expression of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa: evidence for a second constitutive enzyme. Mol & Gen Genet 229:253–260
    [Google Scholar]
  11. Giachetti E., Pinzauti G., Bonaccorsi R., Vanni P. 1988; Isocitrate lyase from Pinus pinea. Eur J Biochem 172:85–91
    [Google Scholar]
  12. Guilliermond P. 1928; Recherches sur quelques Ascomycetes inferieurs isoles de la stigmatomycose des graines de cotonnier. Essai sur la phylogenie des Ascomycetes. Rev Gen Bol 40:328–342 397-14, 474-85, 555-574, 606-624, 690-704
    [Google Scholar]
  13. Hanson A. M. 1967; Microbial production of pigments and vitamins. Microbial Technology222–250 Edited by Peppier H. J. New York: Reinhold;
    [Google Scholar]
  14. Hones I., Simon M., Weber H. 1991; Characterization of isocitrate lyase from the yeast Yarrowia lipolytica. J Basic Microbiol 31:251–258
    [Google Scholar]
  15. Johanson R. A., Hill J. M., McFadden B. A. 1974; Isocitrate lyase from Neurospora crassa. I. Purification, kinetic mechanism, and interaction with inhibitors. Biochim Biophys Acta 364:327–340
    [Google Scholar]
  16. Khan A. S., van Driessche E., Kanarek L., Beeckmans S. 1992; The purification and physicochemical characterization of maize (Zea mais L.) isocitrate lyase. Arch Biochem Biophys 297:9–18
    [Google Scholar]
  17. Kornberg H. L. 1965a; Anaplerotische Sequenzen im mikro-biologischen Stoffwechsel. Angew Chem 11:601–609
    [Google Scholar]
  18. Kornberg H. L. 1965b; The co-ordination of metabolic routes. Symp Soc Gen Microbiol 15:8–31
    [Google Scholar]
  19. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 221:680–685
    [Google Scholar]
  20. Lago B. D., Kaplan L. 1981; Vitamin fermentations: B2 and B12. Adv Biotechnol 3:241–246
    [Google Scholar]
  21. Leatherbarrow R. J. 1987 Emitter Manual Amsterdam: Elsevier Science Publishers;
    [Google Scholar]
  22. Lewis K. F., Weinhouse S. 1957; Synthesis and degradation of isotopically labeled glycolic, glyoxylic, and oxalic acids. Methods Enzymol 4:609–614
    [Google Scholar]
  23. Mehta S. M., Mattoo A. K., Modi V. V. 1972; Ribitol and flavinogenesis in Eremothecium ashbyii. Biochem J 130:159–166
    [Google Scholar]
  24. Nakamura K., Amano Y., Nakadate M., Kagami M. 1989; Purification and properties of isocitrate lyase from Candida brassicae E-17. J Ferment Bioeng 67:153–157
    [Google Scholar]
  25. Ozbas T., Kutsal T. 1986; Comparative study of riboflavin production from two microorganisms: Eremothecium ashbyii and Ashbya gossypii. Enzyme Microb Technol 8:593–596
    [Google Scholar]
  26. Pinzauti G., Giachetti E., Camici G., Manao G., Cappugi G., Vanni P. 1986; An isocitrate lyase of higher plants: analysis and comparison of some molecular properties. Arch Biochem Biophys 244:85–93
    [Google Scholar]
  27. Rogers J. E., McFadden B. A. 1977; Isocitrate lyase from Neurospora crassa: pH dependence of catalysis and interaction with substrates and inhibitors. Arch Biochem Biophys 180:348–353
    [Google Scholar]
  28. Rua J., De Arriaga D., Busto F., Soler J. 1989; Effect of glucose on isocitrate lyase in Phycomyces blakesleeanus. J Bacteriol 171:6391–6393
    [Google Scholar]
  29. Rua J., De Arriaga D., Busto F., Soler J. 1990; Isocitrate lyase from Phycomyces blakesleeanus. Biochem J 272:359–367
    [Google Scholar]
  30. Segel I. H. 1975 Enzyme Kinetics New York: Wiley Interscience;
    [Google Scholar]
  31. Stahmann K.-P., Kupp C., Feldmann S. D., Sahm H. 1994; Formation and degradation of lipid bodies found in the riboflavin-producing fungus Ashbya gossypii. Appl Microbiol Bio technol 42:121–127
    [Google Scholar]
  32. Tanaka A., Nabeshima S., Tokuda M., Fukui S. 1977; Partial purification of isocitrate lyase from Candida tropicalis and some kinetic properties of the enzyme. Agric Biol Chem 41:795–801
    [Google Scholar]
  33. Vanni P., Giachetti E., Pinzauti G., McFadden B. A. 1990; Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol 95B:431–458
    [Google Scholar]
  34. Wickerham L. J., Flickinger M. H., Johnsten R. M. 1946; The production of riboflavin by Ashbya gossypii. Arch Biochem 9:95–98
    [Google Scholar]
  35. Zacharius M. R., Zell T. M., Morrison J. H., Woodlock J. J. 1969; Glycoprotein staining following electrophoresis on acryl-amide gels. Anal Biochem 30:148–152
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-142-2-411
Loading
/content/journal/micro/10.1099/13500872-142-2-411
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error