1887

Microbiology Society logo

Volume 136, Issue 10

Purification and characterization of alkaline endo-1,4--glucanases from sp. KSM-635 Free

Abstract

Two carboxymethylcellulases (CMCase, 1,4-1,4---glucan glucanohydrolase, EC 3.2.1.4), designated E-H and E-L, were purified to homogeneity from a culture filtrate of the alkalophilic sp. KSM-635, by chromatography on DEAE-Toyopearl 650S and gel filtration on Bio-Gel A-0·5m. The purified CMCases both contained approximately 2–3% (w/w) glucosamine. Molecular masses deduced from SDS-PAGE were 130 kDa for E-H and 103 kDa for E-L. The pH optima of the enzymes were both about 9·5, and their optimum temperatures were around 40 °C. Activities of both enzymes were inhibited by Hg, Cu, Fe and Fe, but sulphydryl inhibitors, such as -ethylmaleimide, monoiodoacetate and 4-chloromercuribenzoate, had either no effect or a slightly inhibitory effect. -Bromosuccinimide was strongly inhibitory, suggesting that a tryptophan residue is essential for the activity of the CMCases from . In addition, the activities of both E-H and E-L were stimulated by Co, and they required Mg, Ca, Mn or Co for stabilization. Both enzymes efficiently hydrolysed carboxymethylcellulose (-1,4-linkage) and lichenan (-1,3; 1,4-linkage), but crystalline cellulosic substrates, curdlan (-1,3-linkage), laminarin (-1,3; 1,6-linkage) and 4-nitrophenyl---glucopyranoside were hydrolysed very little, if at all. 4-Nitrophenyl---cellobioside was hydrolysed by both enzymes to liberate 4-nitrophenol, and their hydrolysis rates were higher at neutral pH than at alkaline pH.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology 1990
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-136-10-1973
1990-10-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/136/10/mic-136-10-1973.html?itemId=/content/journal/micro/10.1099/00221287-136-10-1973&mimeType=html&fmt=ahah

References

  1. Au K.-S., Chan K.-Y. 1987; Purification and properties of the endo-l,4-β-glucanase from Bacillus subtilis. . Journal of General Microbiology 133:2155–2162
     [Google Scholar]
  2. Béguin P. 1983; Detection of cellulase activity in polyacrylamide gels using Congo red-stained agar replicas. Analytical Biochemistry 131:333–336
     [Google Scholar]
  3. Beldman G., Serle-Vanleeuwen F.M., Rombout F.M., Woragen G.J. 1985; The cellulase of Trichoderma viride.Purification, characterization and comparison of all detectable endoglucanases, exoglucanases and β-glucosidases. European Journal of Biochemistry 146:301–308
     [Google Scholar]
  4. Berghem L.E.R., Pettersson L.G., Axio-Fredriksson U.B. 1976; The mechanism of enzymatic cellulose degradation. Purification and some properties of two different 1,4-β-glucan glucanohydro- lases from Trichoderma viride. . European Journal of Biochemistry 61:620–630
     [Google Scholar]
  5. Bernard B.A., Newton S.A., Olden K. 1983; Effect of size and location of oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease. Journal of Biological Chemistry 258:12198–12202
     [Google Scholar]
  6. Beaufay H., Amar-Costesec A. 1976; Cell fractionation techniques. Methods in Membrane Biology 6:1–100
     [Google Scholar]
  7. Bradbury S., Steward V.W. 1964; A note on the electron staining of diformazan deposits in tissue sections. Journal of the Royal Microscopical Society 83:467–470
     [Google Scholar]
  8. Bradford M.M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72248–254
     [Google Scholar]
  9. Coleman G.S. 1978; Rumen entodiniomorphid protozoa. In Methods of Cultivating Parasites In Vitro, pp 39–54 Taylor A. E. R., Baker J. R. Edited by London & New York: Academic Press;
     [Google Scholar]
  10. Contantinescu E., Dragos N. 1984; Unele aspecte de structura electronmicroscopica a infuzorilor ruminali de la ovine. Buletinul Institutului Agronomic Cluj-Napoca, Seria Zootehnie si Medicina Veterinara 38:57–60
     [Google Scholar]
  11. Delfosse-Debuscher J. 1977; Ultrastructure de l’endoplasme des ophryoscolecides. Cellule 72:77–90
     [Google Scholar]
  12. Ellis J.E., Lloyd D., Williams A.G. 1989a; Protozoal contribution to ruminal oxygen utilization. In Biochemistry and Molecular Biology of “Anaerobic” Protozoa, pp 32–41 Lloyd D., Coombs G. H., Paget T. Edited by Chur, Switzerland: Harwood Academic Publishers;
     [Google Scholar]
  13. Ellis J.E., Williams A.G., Lloyd D. 1989b; Oxygen consumption by ruminal microorganisms: protozoal and bacterial contributions. Applied and Environmental Microbiology 55:2583–2587
     [Google Scholar]
  14. DiFanchiotti L.A.C., Jones G.R.W., Bradbury S. 1971; The intramitochondrial localisation of succinate-yellow tetrazolium reductase with the electron microscope. Histochemie 27:28–35
     [Google Scholar]
  15. Hanker J.S. 1975; Oxidoreductases. In Electron Microscopy of Enzymes, 4 pp 1–139 Hayat M. A. Edited by New York: Van Nostrand Reinhold.;
     [Google Scholar]
  16. Hillman K., Lloyd D., Scott R.I., Williams A.G. 1985; The effects of oxygen on hydrogen production by rumen holotrich protozoa, as determined by membrane-inlet mass spectrometry. In Microbial Gas Metabolism: Mechanistic, Metabolic and Biotechnological Aspects, pp 271–277 Poole R. K., Dow C. S. Edited by London & New York: Academic Press;
     [Google Scholar]
  17. Lindmark D.G., Müller M. 1973; Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate Tritrichomonas foetus and its role in pyruvate metabolism. Journal of Biological Chemistry 248:7724–7728
     [Google Scholar]
  18. Lindmark D.G., Müller M. 1974; Biochemical cytology of trichomonad flagellates. II. Subcellular distribution of oxidoreductases and hydrolases in Monoceromonas sp. Journal of Protozoology 21:374–378
     [Google Scholar]
  19. Lloyd D., Hillman K., Yarlett N., Williams A.G. 1989; Hydrogen production by rumen holotrich protozoa: effects of oxygen and implications for metabolic control by in situ conditions. Journal of Protozoology 36:205–213
     [Google Scholar]
  20. Lockwood B.C., Coombs G.H., Williams A.G. 1988; Proteinase activity in rumen ciliate protozoa. Journal of General Microbiology 134:2605–2614
     [Google Scholar]
  21. Müller M. 1980; The hydrogenosome. In The Eukaryotic Microbial Cell, pp 127–142 Gooday G. W., Lloyd D., Trinci A. P. J. Edited by Cambridge: Cambridge University Press;
     [Google Scholar]
  22. Müller M. 1988; Energy metabolism of protozoa without mitochondria. Annual Review of Microbiology 42:465–488
     [Google Scholar]
  23. Snyers L., Hellings P., Bovy-Kesler C., Thines-Sempoux D. 1982; Occurrence of hydrogenosomes in the rumen ciliates Ophryoscolecidae. . FEBS Letters 137:35–39
     [Google Scholar]
  24. Steinbüchel A., Müller M. 1986; Anaerobic pyruvate metabolism of Tritrichomonas foetus and Trichomonas vaginalis hydrogenosomes. Molecular and Biochemical Parasitology 20:57–65
     [Google Scholar]
  25. Thorneley R.N.F. 1974; A convenient electrochemical preparation of reduced methylviologen and kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus. Biochimica et Biophysica Acta 333:487–496
     [Google Scholar]
  26. Williams A.G. 1986; Rumen holotrich ciliate protozoa. Microbiological Reviews 50:25–49
     [Google Scholar]
  27. Williams A.G., Coleman G.S. 1988; The rumen protozoa. In The Rumen Microbial Ecosystem, pp 77–128 Hobson P. N. Edited by London & New York: Elsevier Applied Science;
     [Google Scholar]
  28. Williams A.G., Ellis A.B. 1985; Subcellular distribution of glycoside hydrolase and polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliate Polyplastron multivesiculatum. . Current Microbiology 12:175–182
     [Google Scholar]
  29. Yarlett N., Lloyd D., Williams A.G. 1982; Respiration of the rumen ciliate Dasytricha ruminantium Schuberg. Biochemical Journal 206:259–266
     [Google Scholar]
  30. Yarlett N., Coleman G.S., Williams A.G., Lloyd D. 1984; Hydrogenosomes in known species of rumen entodiniomorphid protozoa. FEMS Microbiology Letters 21:15–19
     [Google Scholar]
  31. Yarlett N., Hann A.C., Lloyd D., Williams A.G. 1981; Hydrogenosomes in the rumen protozoon Dasytricha ruminantiumSchuberg. Biochemical Journal 200:365–372
     [Google Scholar]
  32. Yarlett N., Hann A.C., Lloyd D., Williams A.G. 1983a; Hydrogenosomes in a mixed isolate of Isotricha prostoma and Isotricha intestinalis from ovine rumen contents. Comparative Biochemistry and Physiology 74B:357–364
     [Google Scholar]
  33. Yarlett N., Orpin C.G., Munn E.A., Yarlett N., Greenwood C.A. 1986; Hydrogenosomes in the rumen fungus Neocallimastix patriciarum. . Biochemical Journal 236:729–739
     [Google Scholar]
  34. Yarlett N., Scott R.I., Williams A.G., Lloyd D. 1983b; A note on the effects of oxygen on hydrogen production by the rumen protozoon Dasytricha ruminantium Schuberg. Journal of Applied Bacteriology 55:359–361
     [Google Scholar]
  35. Zwart K.B., Goosen N.K., VanSchijndel M.W., Broers C.A.M., Stumm C.K., Vogels G.D. 1988; Cytochemical localization of hydrogenase activity in the anaerobic protozoa Trichomonas vaginalis, Plagiopyla nasuta and Trimyema compressum. . Journal of General Microbiology 134:2165–2170
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-136-10-1973
Loading
Purification and characterization of alkaline endo-1,4-β-glucanases from alkalophilic Bacillus sp. KSM-635
Microbiology 136, 1973 (1990); https://doi.org/10.1099/00221287-136-10-1973
/content/journal/micro/10.1099/00221287-136-10-1973
/content/journal/micro/10.1099/00221287-136-10-1973
Loading

Data & Media loading...

Most cited 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