1887

Abstract

The production of pectinase was studied in , using the hyperproducer mutant , which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36·6–37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. and for polypectate hydrolysis were 5·0 mg ml and 357 μmol min (mg protein), respectively. Temperature and pH optima were 45°C and 6·0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50%, with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified enzyme was classified as an endopolygalacturonase [poly(1,4---galacturonide) glycanohydrolase; EC 3.2.1.15].

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-137-8-1815
1991-08-01
2021-10-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/137/8/mic-137-8-1815.html?itemId=/content/journal/micro/10.1099/00221287-137-8-1815&mimeType=html&fmt=ahah

References

  1. Aguilar G., Huitron C. 1987; Stimulation of the production of extracellular pectinolytic activities of Aspergillus sp. by galacturonic acid and glucose addition. Enzyme and Microbial Technology 9:690–696
    [Google Scholar]
  2. Ayers W. A., Papavizas G. C., Diem A. F. 1966; Polygalacturonate trans-eliminase and polygalacturonase production by Rhizoctonia solanix. Phytopathology 56:1006–1011
    [Google Scholar]
  3. Barash I., Klein L. 1969; The surface localization of polygalacturonase in spores of Geotrichum candidum. Phytopathology 59:319–324
    [Google Scholar]
  4. Barash I., Zilberman E., Marcus L. 1984; Purification of Geotrichum candidum endopolygalacturonase from culture and from host tissue by affinity chromatography on cross-linked polypectate. Physiological Plant Pathology 25:161–169
    [Google Scholar]
  5. Burton E. G., Metzenberg R. L. 1974; Properties of represssible alkaline phosphatase from wild type and wall-less mutant of Neurospora crassa. Journal of Biological Chemistry 249:4679–4688
    [Google Scholar]
  6. Chamier A.-C., Dixon P. A. 1982; Pectinases in leaf degradation by aquatic hyphomycetes: the enzyme and leaf maceration. Journal of General Microbiology 128:2469–2483
    [Google Scholar]
  7. Cohen B. L., Drucker H. 1977; Regulation of exocellular protease in Neurospora crassa: Induction and repression under conditions of nitrogen starvation. Archives of Biochemistry and Biophysics 182:601–613
    [Google Scholar]
  8. Collmer A., Ried J. L., Mount M. S. 1988; Assay methods for pectic enzymes. Methods in Enzymology 161:326–335
    [Google Scholar]
  9. Cooke R. D., Ferber C. E., Kanagasabapathy L. 1976; Purification and characterization of polygalacturonase from a commercial Aspergillus niger preparation. Biochimica et Biophysica Acta 452:440–451
    [Google Scholar]
  10. Cooper R. M., Wood R. K. S. 1973; Induction of synthesis of extracellular cell-wall degrading enzymes in vascular wilt fungi. Nature, London 246:309–311
    [Google Scholar]
  11. Davis B. J. 1964; Disc electrophoresis. II. Method and application to human serum proteins. Annals of the New York Academy of Sciences 121:404–427
    [Google Scholar]
  12. De Lorenzo G., Salvi G., Degra L., D’Ovidio R., Cervone F. 1987; Induction of extracellular polygalacturonase and its mRNA in the phytopathogenic fungus Fusarium moniliforme. Journal of General Microbiology 133:3365–3373
    [Google Scholar]
  13. Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A., Smith F. 1956; Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28:350–356
    [Google Scholar]
  14. Eberhart B. M., Beck R. S. 1973; Induction of β-glucosidase in Neurospora crassa. Journal of Bacteriology 116:295–303
    [Google Scholar]
  15. Eberhart B. M., Beck R. S., Goolsby K. M. 1977; Cellulase of Neurospora crassa. Journal of Bacteriology 130:181–186
    [Google Scholar]
  16. Fanellii C., Cacace M. G., Cervone F. 1978; Purification and properties of two polygalacturonases from Trichoderma koningii. Journal of General Microbiology 104:305–309
    [Google Scholar]
  17. Fontana J. D., Gebara M., Blumel M., Schneider H., Mackenzie C. R., Johnson K. G. 1988; α-4-O-Methyl-d-glucuronidase component of xylanolytic complexes. Methods in Enzymology 160:560–571
    [Google Scholar]
  18. Gratzner H. G., Sheehan D. N. 1969; Neurospora mutant exhibiting hyperproduction of amylase and invertase. Journal of Bacteriology 97:544–549
    [Google Scholar]
  19. Gysler C., Harmsen J. A. M., Kester H. C. M., Visser J., Heim J. 1990; Isolation and structure of the pectin lyase D-encoding gene from Aspergillus niger. Gene 89:101–108
    [Google Scholar]
  20. Khanh N. Q., Albrecht H., Ruttkowski E., Läffler F., Gottschalk M., Jany K.-D. 1990; Nucleotide and derived amino acid sequence of a pectinesterase cDNA isolated from Aspergillus niger strain R.H. 5344. Nucleic Acids Research 18:4262
    [Google Scholar]
  21. Leone G., Van Den Heuvel J. 1987; Regulation by carbohydrates of the sequential ‘in vitro’ production of pectic enzymes by Botrytis cinerea. Canadian Journal of Botany 65:2133–2141
    [Google Scholar]
  22. Lindberg R. A., Drucker H. 1984; Characterization and comparison of a Neurospora crassa RNAse purified from cultures undergoing each of three different states of derepression. Journal of Bacteriology 157:375–379
    [Google Scholar]
  23. Lisker N., Katan J., Henis Y. 1975; Sequential production of polygalacturonase, cellulase, and pectin lyase by Rhizoctonia solani. Canadian Journal of Microbiology 21:1298–1304
    [Google Scholar]
  24. Manachini M. G., Fortina C., Parini C. 1987; Purification and properties of an endopolygalacturonase produced by Rhizopus stolonifer. Biotechnology tetters 9:219–224
    [Google Scholar]
  25. Marcus L., Barash I., Sneh B., Koltin Y., Finkler A. 1986; Purification and characterization of pectolytic enzymes produced by virulent and hipovirulent isolates of Rhizoctonia solani Kuhn. Physiological and Molecular Plant Pathology 29:325–336
    [Google Scholar]
  26. Martinez M. J., Martinez R., Reyes F. 1988; Effect of pectin on pectinases in autolysis of Botrytis cinerea. Mycopathologia 102:37–43
    [Google Scholar]
  27. Miller G. L. 1959; Use of dinitrosalicylic acid reagent for determination of reducing sugars. Analytical Biochemistry 31:426–428
    [Google Scholar]
  28. Mishra C., Keskar S., Rao M. 1984; Production and properties of extracellular endoxylanase from Neurospora crassa. Applied and Environmental Microbiology 48:224–228
    [Google Scholar]
  29. Nagel C. W., Anderson M. M. 1965; Action of a bacterial transeliminase on normal and unsaturated oligogalacturonic acids. Archives of Biochemistry and Biophysics 112:322–330
    [Google Scholar]
  30. Pietro R. C. L. R., Jorge J. A., Terenzi H. F. 1990; Effects of cell wall deficiency in ‘slime’ strains of Neurospora crassa: a study on mycelial and wall-less phenotypic derivatives of a single fz; sg; os-1 (’slime’-like) segregant. Journal of General Microbiology 136:121–129
    [Google Scholar]
  31. Pitt D. 1988; Pectin lyase from Phoma medicaginis var. pinodella. Methods in Enzymology 161:350–354
    [Google Scholar]
  32. Polizeli M. L. T. M., Pietro R. C. L. R., Jorge J. A., Terenzi H. F. 1990; Effects of cell wall deficiency on the synthesis of polysaccharide-degrading exoenzymes. A study on mycelial and wall-less phenotypes of the fz, sg, os-1 (’slime’) triple mutant of Neurospora crassa. Journal of General Microbiology 136:1463–1468
    [Google Scholar]
  33. Roboz E., Barratt R. W., Tatum E. L. 1952; Breakdown of pectic substances by a new enzyme from Neurospora. Journal of Biological Chemistry 195:459–471
    [Google Scholar]
  34. Rombouts F. M., Pilnik W. 1980; Pectic enzymes. Economic Microbiology 5227–282 Rose A. H. Academic Press; London:
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-137-8-1815
Loading
/content/journal/micro/10.1099/00221287-137-8-1815
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