1887

Abstract

A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium ‘’. Full-length CelA and a truncated enzyme species designated CelA' were purified to homogeneity from culture supernatants. CelA has an apparent molecular mass of 230 kDa. The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and β-glucan. Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 ° (CM-cellulase) and 85 ° (Avicelase). Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation. The CelA-encoding gene was isolated from genomic DNA of ‘’ by PCR and the nucleotide sequence was determined. The gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the N-terminus of CelA purified from ‘’. Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family III cellulose-binding domain linked by Pro-Thr-Ser-rich regions. The enzyme is most closely related to CelA of (sequence identities of 96 and 97% were found for the N- and C-terminal catalytic domains, respectively). Endoglucanase CelZ of shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain. Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound to microcrystalline cellulose. Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-144-2-457
1998-02-01
2021-05-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/144/2/mic-144-2-457.html?itemId=/content/journal/micro/10.1099/00221287-144-2-457&mimeType=html&fmt=ahah

References

  1. Ahsan M. M., Kimura T., Karita S., Sakka K., Ohmiya K. 1996; Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome.. J Bacterial 178:5732–5740
    [Google Scholar]
  2. Béguin P., Aubert J. P. 1994; The biological degradation of cellulose.. FEMS Microbiol Rev 13:25–58
    [Google Scholar]
  3. Bronnenmeier K., Staudenbauer W. L. 1990; Cellulose hydrolysis by a highly thermostable endo-l,4-β-glucanase (Avicelase I) from Clostridium stercorarium . Enzyme Microb Technol 12:431–436
    [Google Scholar]
  4. Bronnenmeier K., RUcknagel K. P., Staudenbauer W. L. 1991; Purification and properties of a novel type of exo-l,4-β-glucanase (Avicelase II) from the cellulolytic thermophile Clostridium stercorarium . Eur J Biochem 200:379–385
    [Google Scholar]
  5. Bronnenmeier K., Kundt K., Riedel K., Schwarz W. H., Staudenbauer W. L. 1997; Structure of the Clostridium stercorarium gene celY encoding the exo-l,4- β -glucanase Avicelase II.. Microbiology 143:891–898
    [Google Scholar]
  6. Flint H. J., Martin J., McPherson C. A., Daniel A. S., Zhang J. -X. 1993; A bifunctional enzyme, with separate xylanase and β (l,3-l,4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens . J Bacteriol 175:2943–2951
    [Google Scholar]
  7. Gibbs M. D., Saul D. J., Luthi E., Bergquist P. L. 1992; The β -mannanase from Caldocellum saccharolyticum is part of a multidomain enzyme.. Appl Environ Microbiol 58:3864–3867
    [Google Scholar]
  8. Hall J., Black G. W., Ferreira L. M., Millward-Sadler S. J., Ali B. R., Hazlewood G. P., Gilbert H. J. 1995; The non-catalytic cellulose-binding domain of a novel cellulase from Pseudomonas fluorescens subsp. cellulosa is important for the efficient hydrolysis of Avicel.. Biochem J 309:749–756
    [Google Scholar]
  9. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids.. J Mol Biol 16:557–580
    [Google Scholar]
  10. Hazlewood G. P., Devidson K., Laurie J. I., Huskisson N. S., Gilbert H. J. 1993; Gene sequence and properties of Cell, a family E endoglucanase from Clostridium thermocellum . J Gen Microbiol 139:307–316
    [Google Scholar]
  11. Herrmann J. L., O'Gaora P., Gallagher A., Thole J. E. R., Young D. B. 1996; Bacterial glycoproteins: a link between glycosylation and proteolytic cleavage of a 19 kDa antigen from Mycobacterium tuberculosis . EMBO J 15:3547–3554
    [Google Scholar]
  12. Jauris S., Rücknagel K. P., Schwarz W. H., Kratzsch P., Bronnenmeier K., Staudenbauer W. L. 1990; Sequence analysis of the Clostridium stercorarium celZ gene encoding a thermostable cellulase (Avicelase I): identification of catalytic and cellulose-binding domains.. Mol Gen Genet 223:258–267
    [Google Scholar]
  13. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature 277:680–685
    [Google Scholar]
  14. Langsford M. L., Gilkes N. R., Singh B., Moser B., Miller R. C., Warren R. A. J., Kilburn D. G. 1987; Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains.. FEBS Lett 225:163–167
    [Google Scholar]
  15. Kruus K., Wang W. K., Ching J., Wu J. H. D. 1995; Exoglucanase activities of the recombinant Clostridium tbermocellum CelS, a major cellulosome component.. J Bacteriol 177:1641–1644
    [Google Scholar]
  16. Montgomery L., Fu Y. -K. 1988; Detection of cellulose-binding proteins in electrophoresis gels by filter paper affinity blotting.. Anal Biochem 174:204–208
    [Google Scholar]
  17. Morris D. D., Reeves R. A., Gibbs M. D., Saul D. J., Bergquist P. L. 1995; Correction of the β -mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp.. Appl Environ Microbiol 61:2262–2269
    [Google Scholar]
  18. Ong E., Kilburn D. G., Miller R. C., Warren R. A. J. 1994; Streptomyces lividans glycosylates the linker region of a β -l,4- glycanase from Cellulomonas fimi.. J Bacteriol 176:999–1008
    [Google Scholar]
  19. Rainey F. A., Ward N. L., Morgan H. W., Toalster R., Stackebrandt E. 1993; Phylogenetic analysis of anaerobic thermophilic bacteria: aid for their reclassification.. J Bacteriol 175:4772–4779
    [Google Scholar]
  20. Rainey F. A., Donnison A. M., Janssen P. H., Saul D., Rodrigo A., Bergquist P. L., Daniel R. M., Stackebrandt E., Morgan H. W. 1994; Description of Caldicellulosiruptor saccharolyticus gen. nov., spec. nov.: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium.. FEMS Microbiol Lett 120:263–266
    [Google Scholar]
  21. Riedel K., Ritter J., Bronnenmeier K. 1997; Synergistic interaction of the Clostridium stercorarium cellulases Avicelase I (CelZ) and Avicelase II (CelY) in the degradation of microcrystalline cellulose.. FEMS Microbiol Lett 147:239–243
    [Google Scholar]
  22. Sakamoto R., Arai M., Murao S. 1984; Enzymatic properties of hydrocellulase from Aspergillus aculeatus . J Ferment Technol 62:561–567
    [Google Scholar]
  23. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual 2nd edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  24. Sandercock L. E., MacLeod A. M., Ong E., Warren R. A. J. 1994; Non-S-layer glycoproteins in eubacteria.. FEMS Microbiol Lett 118:1–8
    [Google Scholar]
  25. Saul D. J., Williams L. C., Grayling R. A., Chamley L. W., Love D. R., Bergquist P. L. 1990; celB, a gene coding for a bifunctional cellulase from the extreme thermophile ‘Caldocellum saccharolyticum’ . Appl Environ Microbiol 56:3117–3124
    [Google Scholar]
  26. Schwarz W. H., Bronnenmeier K., Gräbnitz F., Staudenbauer W. L. 1987; Activity staining of cellulases in polyacrylamide gels containing mixed linkage β-glucans.. Anal Biochem 164:72–77
    [Google Scholar]
  27. Sedmak J. J., Grossberg S. E. 1977; A rapid, sensitive assay for protein using Coomassie brilliant blue G250.. Anal Biochem 79:544–552
    [Google Scholar]
  28. Svetlichnyi V. A., Svetlichnaya T. P., Chernykh N. A., Zavarzin G. A. 1990; Anaerocellum thermophilum gen. nov. sp. nov.: an extremely thermophilic cellulolytic eubacterium isolated from hot springs in the valley of geysers.. Microbiology (English translation of Mikrobiologiya) 59:598–603
    [Google Scholar]
  29. Te'o V. S. J., Saul D. J., Bergquist P. L. 1995; celA, another gene coding for a multidomain cellulase from the extreme thermophile Caldocellum saccharolyticum . Appl Microbiol Biotechnol 43:291–296
    [Google Scholar]
  30. Tomme P., Van Tilbeurgh H., Pettersson G., Van Damme J., Vandekerckhove J., Knowles J., Teeri T., Claeyssens M. 1988; Studies of the cellulolytic system of Trichoderma reesei QM 9414.. Eur J Biochem 170:575–581
    [Google Scholar]
  31. Warren R. A. J. 1996; Microbial hydrolysis of polysaccharides.. Annu Rev Microbiol 50:183–212
    [Google Scholar]
  32. Weimer P. J., Wagner W., Knowlton S., Ng T. K. 1984; Thermophilic anaerobic bacteria which ferment hemicellulose: characterization of organisms and identification of plasmids.. Arch Microbiol 138:31–36
    [Google Scholar]
  33. Wood T. M., Bhat K. M. 1988; Methods for measuring cellulase activities.. Methods Enzymol 160:87–112
    [Google Scholar]
  34. Yanisch-Perron C., Vieira J., & Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors.. Gene 33:103–119
    [Google Scholar]
  35. Zhang J., -X. & Flint H. J. 1992; A bifunctional xylanase encoded by the xynA gene of the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 comprises two dissimilar domains linked by an asparagine/glutamine-rich sequence.. Mol Microbiol 6:1013–1023
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-144-2-457
Loading
/content/journal/micro/10.1099/00221287-144-2-457
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