1887

Abstract

produced an inducible extracellular chitosanase when grown on chitosan. Large-scale production of the enzyme was obtained using hyphae as substrate. Chitosanase was purified 38-fold to homogeneity by ammonium sulphate fractionation and sequential chromatography on DEAE-Biogel A, Biogel P and Crude enzyme was unstable at 37 °C, but was stabilized by 1·0 m-Ca. The pH optimum for activity was broad and dependent on the solubility of the chitosan substrate. Various physical and chemical properties of the purified enzyme were determined.

chitosanase cleaved chitosan in an endo-splitting manner with maximal activity on polymers of 30 to 60% acetylation. No activity was found on chitin (100% acetylated chitosan) or trimers and tetramers of The latter two oligomers and all small oligomers of glucosamine inhibited the activity of chitosanase on 30% acetylated chitosan. The pentamer of -acetylglucosamine and glucosamine oligomers were slowly cleaved by the enzyme. Analysis of the reaction products from 30% acetylated chitosan indicated that the major oligomeric product was a trimer; with 60% acetylated chitosan as substrate a dimer was also found. The new terminal reducing groups produced by chitosanase hydrolysis of 30% acetylated chitosan were reduced by sodium boro[H]hydride. The new end residues were found to be -acetylglucosamine. The analyses strongly indicated that chitosanase cleaved chitosan between -acetylglucosamine and glucosamine. Both residues were needed for cleavage, and polymers containing equal proportions of acetylated and non-acetylated sugars were optimal for chitosanase activity. The products of reaction depended on the degree of acetylation of the polymer.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-126-1-151
1981-09-01
2021-10-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/126/1/mic-126-1-151.html?itemId=/content/journal/micro/10.1099/00221287-126-1-151&mimeType=html&fmt=ahah

References

  1. Andrews P. 1964; Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochemical Journal 91:222–233
    [Google Scholar]
  2. Araki Y., Ito E. 1974; A pathway of chitosan formation in Mucor rouxii: enzymatic deacetylation of chitin. Biochemical and Biophysical Research Communications 56:669–675
    [Google Scholar]
  3. Bartnicki-Garcia S. 1968; Cell wall composition and other biochemical markers in fungal phylogeny. Annual Review of Microbiology 22:87–108
    [Google Scholar]
  4. Bartnicki-Garcia S., Nickerson W. J. 1962; Isolation, composition and structure of cell walls of filamentous and yeast-like forms of Mucor rouxii. . Biochimica et biophysica acta 58:102–119
    [Google Scholar]
  5. Bartnicki-Garcia S., Reyes E. 1968; Polyuronides in the cell walls of Mucor rouxii. . Biochimica et biophysica acta 170:54–62
    [Google Scholar]
  6. Bruck H. M., Nash G., Foley F. D., Pruitt B. A. Jr 1971; Opportunistic fungal infections of the burn wound with Phycomyces and Aspergillus. . Archives of Surgery 102:476–482
    [Google Scholar]
  7. Datema R., Wessels J. G. H., Van Den Ende H. 1977; The hyphal wall of Mucor mucedo: hexosamine-containing polymers. European Journal of Biochemistry 80:621–626
    [Google Scholar]
  8. Davis B. J. 1964; Disc gel electrophoresis. II. Method and application to human serum proteins. Annals of the New York Academy of Sciences 121:404–409
    [Google Scholar]
  9. Davis L., Bartnicki-Garcia S. 1980; The structure and biosynthesis of chitosan from Mucor rouxii. . Abstracts, Mycological Society of America no. C27.
    [Google Scholar]
  10. Fenton D. M., Davis B., Rotgers C., Eveleigh D. E. 1978; Enzymatic hydrolysis of chitosan. In First International Symposium on Chitin and Chitosan pp. 525–541 Muzzarrelli R. A. A., Pariser E. R. Edited by Boston:: MIT Press.;
    [Google Scholar]
  11. French D., Wild G. M. 1953; Correlation of carbohydrate structure with papergram mobility. Journal of the American Chemical Society 75:2612–2616
    [Google Scholar]
  12. Hayes R., Davies D. H. 1978; Characterization of chitosan. II. The determination of the degree of acetylation of chitosan and chitin. In First International Symposium on Chitin and Chitosan pp. 406–420 Muzzarrelli R. A. A., Pariser E. R. Edited by Boston:: MIT Press.;
    [Google Scholar]
  13. Hedges A., Wolfe R. S. 1974; Extracellular enzyme from Myxobacter A1-1 that exhibits both β-l,4-glucanase and chitosanase activities. Journal of Bacteriology 120:844–853
    [Google Scholar]
  14. Hirano S., Yagi Y. 1980; The effects of N-substitution on chitosan and the physical form of the products on the rate of hydrolysis by chitinase from Streptomyces griseus. . Carbohydrate Research 83:103–108
    [Google Scholar]
  15. Hirano S., Ohe Y., Ono H. 1976; Selective N-acetylation of chitosan. Carbohydrate Research 47:315–320
    [Google Scholar]
  16. Hirs C. H. W. 1967; Determination of cystine as cysteic acid. Methods in Enzymology 2:59–62
    [Google Scholar]
  17. Horowitz S. T., Roseman S., Blumenthal H. 1957; The preparation of glucosamine oligosaccharides. I. Separation. Journal of the American Chemical Society 79:5046–5049
    [Google Scholar]
  18. Horton D. 1969; Monosaccharide amino sugars. In The Amino Sugars 1A p. 128 Jeanloz R. W. Edited by New York:: Academic Press.;
    [Google Scholar]
  19. Horton D., Lineback D. R. 1965; N-Deacetylation: chitosan from chitin. Methods in Carbohydrate Chemistry403–406
    [Google Scholar]
  20. Hough L., Jones J. K. N., Wadman W. H. 1950; Quantitative analysis of mixtures of sugars by the method of partition chromatography. V. Improved methods for the separation and detection of the sugars and their methylated derivatives on the paper chromatogram. Journal of the Chemical Society1702–1706
    [Google Scholar]
  21. Jones D., Bacon J. S. D., Farmer V. C., Webley D. M. 1968; Lysis of the cell walls of Mucor ramannianus Moller by a Streptomycete sp. Antonie van Leeuwenhoek 35:173–182
    [Google Scholar]
  22. Kreger D. R. 1954; Observations of cell walls of yeast and some other fungi by X-ray diffraction and solubility tests. Biochimica et biophysica acta 13:1–9
    [Google Scholar]
  23. Krystal G., Graham A. F. 1976; A sensitive method for estimating the carbohydrate content of glycoproteins. Analytical Biochemistry 57:336–345
    [Google Scholar]
  24. Letourneau D. R., Deven J. M., Manocha M. S. 1976; Structure and composition of the cell wall of Choanephora curcurbitarum. . Canadian Journal of Microbiology 22:486–494
    [Google Scholar]
  25. Monaghan R. L., Eveleigh D. E., Tewari R., Reese E. T. 1972; Chitosanase, a novel enzyme. Nature New Biology 245:79–81
    [Google Scholar]
  26. Monreal J., Reese E. T. 1969; The chitinase of Serratia marcescens. . Canadian Journal of Microbiology 15:689–696
    [Google Scholar]
  27. Muzzarrelli R. A. A. 1973; Chitosan. In Natural Chelating Polymers pp. 144–176 New York:: Pergamon Press.;
    [Google Scholar]
  28. Muzzarrelli R. A. A. 1977 Chitin pp. 155–181 New York:: Pergamon Press.;
    [Google Scholar]
  29. Nelson W. 1944; A photometric adaptation of the Somogyi method for determination of glucose. Journal of Biological Chemistry 153:375–380
    [Google Scholar]
  30. Price J. S., Storck R. 1975; Production, purification and characterization of an extracellular chitosanase from Streptomyces. . Journal of Bacteriology 124:1574–1585
    [Google Scholar]
  31. Ruiz-Herrera J. 1978; The distribution and quantitative importance of chitin in fungi. In First International Symposium on Chitin and Chitosan pp. 11–12 Muzzarrelli R. A. A., Pariser E. R. Edited by Boston:: MIT Press.;
    [Google Scholar]
  32. Ruiz-Herrera J., Ramirez-Leon I. F. 1972; Purification of chitosanases active against the cell walls of Mucorales. Abstract, Third International Symposium on Yeast Protoplasts.
    [Google Scholar]
  33. Rupley J. A. 1964; The hydrolysis of chitin by concentrated hydrochloric acid and the preparation of low molecular weight substrates for lysozyme. Biochimica et biophysica acta 83:245–255
    [Google Scholar]
  34. Schindler M., Mirelman D., Sharon S. 1977; Substrate-induced evolution of lysozymes. Biochimica et biophysica acta 482:386–392
    [Google Scholar]
  35. Shiveley J. E., Conrad H. E. 1970; Stoichiometry of the nitrous acid deaminative cleavage of model amino sugar glucosides and glucosamino- glycuronans. Biochemistry 9:33–43
    [Google Scholar]
  36. Storck R., Price J. S. 1977; Sensitivity of germinating spores of Mucor rouxii to chitosanase. Experimental Mycology 1:323–338
    [Google Scholar]
  37. Tabata S., Terui G. 1962; Studies on microbial enzymes active in hydrolyzing yeast walls. I. Isolation of a strain and culture conditions for enzyme formation. Journal of Fermentation Technology 40:366–373
    [Google Scholar]
  38. Tominaga Y., Tsujisaka Y. 1975; Purification and some enzymatic properties of the chitosanase from Bacillus R-4 which lyses Rhizopus cell walls. Biochimica et biophysica acta 410:145–155
    [Google Scholar]
  39. Tracey M. V. 1955; Chitin. In Modern Methods of Plant Analysis II pp. 264–274 Paech K., Tracey M. V. Edited by Berlin:: Springer-Verlag.;
    [Google Scholar]
  40. Trevelyan W. E., Proctor D. P., Harrison J. S. 1950; Detection of sugar on paper chromatograms. Nature London: 166:444–445
    [Google Scholar]
  41. Udenfriend J., Stien J., Bohlen P., Dairman W. 1972; Fluorescamine as a reagent for assay of amino acids, peptides, proteins and primary amines in picomole range. Science 178:871
    [Google Scholar]
  42. Vessey J. C., Pegg G. P. 1973; Autolysis and chitinase production in cultures of Verticillium alboatrum. . Transactions of the British Mycological Society 60:133–143
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-126-1-151
Loading
/content/journal/micro/10.1099/00221287-126-1-151
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