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. 1990 Dec;56(12):3798–3804. doi: 10.1128/aem.56.12.3798-3804.1990

Subunit Composition and Glycosidic Activities of the Cellulase Complex from Clostridium thermocellum JW20

S Kohring 1, J Wiegel 1, F Mayer 1,*
PMCID: PMC185070  PMID: 16348380

Abstract

The subunit composition of the extracellular complex from Clostridium thermocellum was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Twenty-six bands, representing proteins with apparent molecular sizes ranging from 37,500 to 185,000 Da, could be detected by silver staining. Cultivation of the bacteria with the substrate Avicel, Sigma cellulose, Solka floc, or cellobiose as the carbon source had no influence on the number of detectable protein bands. By activity staining with the substrate carboxymethyl cellulose or xylan added to the SDS-polyacrylamide gels, 15 of the 26 bands exhibited endoglucanase activity and 13 showed xylanase activity. In 8 of the 26 bands, both activities could be found. As minor activities, β-glucosidase, β-xylosidase, β-galactosidase, and β-mannosidase activities could be demonstrated in the cellulase complex. Upon measuring the release of para-nitrophenol (PNP) from PNP-cellobioside and determining the amount of glucose formed, the presence of exoglucanase activity was indicated. Upon glycoprotein staining of SDS-polyacrylamide gels, 14 of the 26 bands reacted positive, indicating the glycoprotein nature of the respective proteins. Four proteins (apparent molecular sizes, 58,000, 72,500, 94,000, and 110,000 Da) could be enriched from the originally bound cellulase complex by preparative SDS-PAGE. The two smaller proteins exhibited xylanase activity, whereas the 94,000-Da protein had endo- and exoglucanase activity, and the 110,000-Da protein degraded PNP-pyranosides.

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Selected References

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  1. Almin K. E., Eriksson K. E. Enzymic degradation of polymers. I. Viscometric method for the determination of enzymic activity. Biochim Biophys Acta. 1967 Jul 11;139(2):238–247. doi: 10.1016/0005-2744(67)90028-9. [DOI] [PubMed] [Google Scholar]
  2. Aït N., Creuzet N., Cattanéo J. Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum. Biochem Biophys Res Commun. 1979 Sep 27;90(2):537–546. doi: 10.1016/0006-291x(79)91269-5. [DOI] [PubMed] [Google Scholar]
  3. Bayer E. A., Kenig R., Lamed R. Adherence of Clostridium thermocellum to cellulose. J Bacteriol. 1983 Nov;156(2):818–827. doi: 10.1128/jb.156.2.818-827.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bayer E. A., Lamed R. Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose. J Bacteriol. 1986 Sep;167(3):828–836. doi: 10.1128/jb.167.3.828-836.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bayer E. A., Setter E., Lamed R. Organization and distribution of the cellulosome in Clostridium thermocellum. J Bacteriol. 1985 Aug;163(2):552–559. doi: 10.1128/jb.163.2.552-559.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coughlan M. P., Hon-Nami K., Hon-Nami H., Ljungdahl L. G., Paulin J. J., Rigsby W. E. The cellulolytic enzyme complex of Clostridium thermocellum is very large. Biochem Biophys Res Commun. 1985 Jul 31;130(2):904–909. doi: 10.1016/0006-291x(85)90502-9. [DOI] [PubMed] [Google Scholar]
  7. Davidson T., Cooke J., Parsons C., Westbury G. Pre-operative assessment of soft tissue sarcomas by computed tomography. Br J Surg. 1987 Jun;74(6):474–478. doi: 10.1002/bjs.1800740615. [DOI] [PubMed] [Google Scholar]
  8. Deshpande M. V., Eriksson K. E., Pettersson L. G. An assay for selective determination of exo-1,4,-beta-glucanases in a mixture of cellulolytic enzymes. Anal Biochem. 1984 May 1;138(2):481–487. doi: 10.1016/0003-2697(84)90843-1. [DOI] [PubMed] [Google Scholar]
  9. Forsberg C. W., Groleau D. Stability of the endo-beta-1,4-glucanase and beta-1,4-glucosidase from Bacteroides succinogenes. Can J Microbiol. 1982 Jan;28(1):144–148. doi: 10.1139/m82-017. [DOI] [PubMed] [Google Scholar]
  10. Gilkes N. R., Warren R. A., Miller R. C., Jr, Kilburn D. G. Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J Biol Chem. 1988 Jul 25;263(21):10401–10407. [PubMed] [Google Scholar]
  11. Grépinet O., Chebrou M. C., Béguin P. Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum. J Bacteriol. 1988 Oct;170(10):4582–4588. doi: 10.1128/jb.170.10.4582-4588.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobs E., Clad A. Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap. Anal Biochem. 1986 May 1;154(2):583–589. doi: 10.1016/0003-2697(86)90033-3. [DOI] [PubMed] [Google Scholar]
  13. John M., Schmidt B., Schmidt J. Purification and some properties of five endo-1,4-beta-D-xylanases and a beta-D-xylosidase produced by a strain of Aspergillus niger. Can J Biochem. 1979 Feb;57(2):125–134. doi: 10.1139/o79-016. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lamed R., Setter E., Bayer E. A. Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum. J Bacteriol. 1983 Nov;156(2):828–836. doi: 10.1128/jb.156.2.828-836.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mayer F. Cellulolysis: ultrastructural aspects of bacterial systems. Electron Microsc Rev. 1988;1(1):69–85. doi: 10.1016/s0892-0354(98)90006-5. [DOI] [PubMed] [Google Scholar]
  18. Mayer F., Coughlan M. P., Mori Y., Ljungdahl L. G. Macromolecular Organization of the Cellulolytic Enzyme Complex of Clostridium thermocellum as Revealed by Electron Microscopy. Appl Environ Microbiol. 1987 Dec;53(12):2785–2792. doi: 10.1128/aem.53.12.2785-2792.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ng T. K., Zeikus J. G. Purification and characterization of an endoglucanase (1,4-beta-D-glucan glucanohydrolase) from Clostridium thermocellum. Biochem J. 1981 Nov 1;199(2):341–350. doi: 10.1042/bj1990341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Petre J., Longin R., Millet J. Purification and properties of an endo-beta-1,4-glucanase from Clostridium thermocellum. Biochimie. 1981 Jul;63(7):629–639. doi: 10.1016/s0300-9084(81)80061-2. [DOI] [PubMed] [Google Scholar]
  21. Romaniec M. P., Clarke N. G., Hazlewood G. P. Molecular cloning of Clostridium thermocellum DNA and the expression of further novel endo-beta-1,4-glucanase genes in Escherichia coli. J Gen Microbiol. 1987 May;133(5):1297–1307. doi: 10.1099/00221287-133-5-1297. [DOI] [PubMed] [Google Scholar]
  22. Schwarz W. H., Bronnenmeier K., Gräbnitz F., Staudenbauer W. L. Activity staining of cellulases in polyacrylamide gels containing mixed linkage beta-glucans. Anal Biochem. 1987 Jul;164(1):72–77. doi: 10.1016/0003-2697(87)90369-1. [DOI] [PubMed] [Google Scholar]
  23. Schwarz W. H., Gräbnitz F., Staudenbauer W. L. Properties of a Clostridium thermocellum Endoglucanase Produced in Escherichia coli. Appl Environ Microbiol. 1986 Jun;51(6):1293–1299. doi: 10.1128/aem.51.6.1293-1299.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schwarz W. H., Schimming S., Rücknagel K. P., Burgschwaiger S., Kreil G., Staudenbauer W. L. Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum. Gene. 1988;63(1):23–30. doi: 10.1016/0378-1119(88)90542-2. [DOI] [PubMed] [Google Scholar]
  25. Teeri T. T., Lehtovaara P., Kauppinen S., Salovuori I., Knowles J. Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II. Gene. 1987;51(1):43–52. doi: 10.1016/0378-1119(87)90472-0. [DOI] [PubMed] [Google Scholar]
  26. Wardi A. H., Michos G. A. Alcian blue staining of glycoproteins in acrylamide disc electrophoresis. Anal Biochem. 1972 Oct;49(2):607–609. doi: 10.1016/0003-2697(72)90472-1. [DOI] [PubMed] [Google Scholar]
  27. Wiegel J., Mothershed C. P., Puls J. Differences in Xylan Degradation by Various Noncellulolytic Thermophilic Anaerobes and Clostridium thermocellum. Appl Environ Microbiol. 1985 Mar;49(3):656–659. doi: 10.1128/aem.49.3.656-659.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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