Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1990 Jun;56(6):1844–1850. doi: 10.1128/aem.56.6.1844-1850.1990

Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1.

K C Doerner 1, B A White 1
PMCID: PMC184520  PMID: 2383014

Abstract

The extracellular endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1 were analyzed by high-performance liquid chromatography (HPLC) by using DEAE ion-exchange, hydroxylapatite, and gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). Two endo-1,4-beta-glucanase peaks were resolved by DEAE-HPLC and termed endoglucanases A and B. Carboxymethyl cellulose (CMC) zymograms were achieved by enzyme separation using nondenaturing PAGE followed by incubation of the gel on top of a CMC-agarose gel. This revealed no less than 13 and 5 endo-1,4-beta-glucanase components present in endoglucanases A and B, respectively. Hydroxylapatite chromatography of endoglucanases A and B revealed one activity peak for each preparation, which contained 4 and 5 endo-1,4-beta-glucanase components, respectively. Gel filtration chromatography of endoglucanase A following hydroxylapatite chromatography resolved the most active carboxymethylcellulase (CMCase) component from other endo-1,4-beta-glucanase activities. Gel filtration of endoglucanase B following hydroxylapatite chromatography showed one CMCase activity peak. Protein stains of sodium dodecyl sulfate-PAGE and nondenaturing PAGE gels of endoglucanases A and B from hydroxylapatite and gel filtration chromatography revealed multiple protein components. When xylan was substituted for CMC in zymograms, identical separation patterns for CMCase and xylanase activities were observed for both endoglucanases A and B. These data suggest that both 1,4-beta linkage-hydrolyzing activities reside on the same polypeptide or protein complex. The highest endo-1,4-beta-glucanase-specific activities were observed following DEAE-HPLC chromatography, with 16.2 and 7.5 mumol of glucose equivalents per min per mg of protein for endoglucanases A and B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Full text

PDF
1844

Images in this article

1847Image
on p.1847
1847Image
on p.1847
1848Image
on p.1848
1848Image
on p.1848

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barros M. E., Thomson J. A. Cloning and expression in Escherichia coli of a cellulase gene from Ruminococcus flavefaciens. J Bacteriol. 1987 Apr;169(4):1760–1762. doi: 10.1128/jb.169.4.1760-1762.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Biely P., Markovic O., Mislovicová D. Sensitive detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in gels. Anal Biochem. 1985 Jan;144(1):147–151. doi: 10.1016/0003-2697(85)90096-x. [DOI] [PubMed] [Google Scholar]
  3. Béguin P. Detection of cellulase activity in polyacrylamide gels using Congo red-stained agar replicas. Anal Biochem. 1983 Jun;131(2):333–336. doi: 10.1016/0003-2697(83)90178-1. [DOI] [PubMed] [Google Scholar]
  4. Béguin P., Eisen H. Purification and partial characterization of three extracellular cellulases from Cellulomonas sp. Eur J Biochem. 1978 Jul 3;87(3):525–531. doi: 10.1111/j.1432-1033.1978.tb12403.x. [DOI] [PubMed] [Google Scholar]
  5. Gardner R. M., Doerner K. C., White B. A. Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1. J Bacteriol. 1987 Oct;169(10):4581–4588. doi: 10.1128/jb.169.10.4581-4588.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gerwig G. J., de Waard P., Kamerling J. P., Vliegenthart J. F., Morgenstern E., Lamed R., Bayer E. A. Novel O-linked carbohydrate chains in the cellulase complex (cellulosome) of Clostridium thermocellum. 3-O-Methyl-N-acetylglucosamine as a constituent of a glycoprotein. J Biol Chem. 1989 Jan 15;264(2):1027–1035. [PubMed] [Google Scholar]
  7. Gilbert H. J., Jenkins G., Sullivan D. A., Hall J. Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa. Mol Gen Genet. 1987 Dec;210(3):551–556. doi: 10.1007/BF00327211. [DOI] [PubMed] [Google Scholar]
  8. Howard G. T., White B. A. Molecular Cloning and Expression of Cellulase Genes from Ruminococcus albus 8 in Escherichia coli Bacteriophage lambda. Appl Environ Microbiol. 1988 Jul;54(7):1752–1755. doi: 10.1128/aem.54.7.1752-1755.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hu Y. J., Wilson D. B. Cloning of Thermomonospora fusca genes coding for beta 1-4 endoglucanases E1, E2 and E5. Gene. 1988 Nov 30;71(2):331–337. doi: 10.1016/0378-1119(88)90050-9. [DOI] [PubMed] [Google Scholar]
  10. Huang L., Forsberg C. W., Thomas D. Y. Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85. J Bacteriol. 1988 Jul;170(7):2923–2932. doi: 10.1128/jb.170.7.2923-2932.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kalb V. F., Jr, Bernlohr R. W. A new spectrophotometric assay for protein in cell extracts. Anal Biochem. 1977 Oct;82(2):362–371. doi: 10.1016/0003-2697(77)90173-7. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Lamed R., Naimark J., Morgenstern E., Bayer E. A. Specialized cell surface structures in cellulolytic bacteria. J Bacteriol. 1987 Aug;169(8):3792–3800. doi: 10.1128/jb.169.8.3792-3800.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Langsford M. L., Gilkes N. R., Singh B., Moser B., Miller R. C., Jr, Warren R. A., Kilburn D. G. Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains. FEBS Lett. 1987 Dec 10;225(1-2):163–167. doi: 10.1016/0014-5793(87)81150-x. [DOI] [PubMed] [Google Scholar]
  15. MacKenzie C. R., Bilous D., Johnson K. G. Purification and characterization of an exoglucanase from Streptomyces flavogriseus. Can J Microbiol. 1984 Sep;30(9):1171–1178. doi: 10.1139/m84-183. [DOI] [PubMed] [Google Scholar]
  16. McGavin M., Forsberg C. W. Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85. J Bacteriol. 1988 Jul;170(7):2914–2922. doi: 10.1128/jb.170.7.2914-2922.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  18. Nebreda A. R., Vazquez C. R., Villa T. G., Villanueva J. R., del Rey F. Heterogeneous glycosylation of the EXG1 gene product accounts for the two extracellular exo-beta-glucanases of Saccharomyces cerevisiae. FEBS Lett. 1987 Aug 10;220(1):27–30. doi: 10.1016/0014-5793(87)80869-4. [DOI] [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. PARK J. T., JOHNSON M. J. A submicrodetermination of glucose. J Biol Chem. 1949 Nov;181(1):149–151. [PubMed] [Google Scholar]
  21. Rasmussen M. A., Hespell R. B., White B. A., Bothast R. J. Inhibitory Effects of Methylcellulose on Cellulose Degradation by Ruminococcus flavefaciens. Appl Environ Microbiol. 1988 Apr;54(4):890–897. doi: 10.1128/aem.54.4.890-897.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shikata S., Nsizawa K. Purification and properties of an exo-cellulase component of novel type from Trichoderma miride. J Biochem. 1975 Sep;78(3):499–512. doi: 10.1093/oxfordjournals.jbchem.a130934. [DOI] [PubMed] [Google Scholar]
  23. Shoemaker S. P., Brown R. D., Jr Characterization of endo-1,4-beta-D-glucanases purified from Trichoderma viride. Biochim Biophys Acta. 1978 Mar 14;523(1):147–161. doi: 10.1016/0005-2744(78)90017-7. [DOI] [PubMed] [Google Scholar]
  24. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  25. Teather R. M., Wood P. J. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol. 1982 Apr;43(4):777–780. doi: 10.1128/aem.43.4.777-780.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wood T. M., Wilson C. A., Stewart C. S. Preparation of the cellulase from the cellulolytic anaerobic rumen bacterium Ruminococcus albus and its release from the bacterial cell wall. Biochem J. 1982 Jul 1;205(1):129–137. doi: 10.1042/bj2050129. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES