1887

Microbiology Society logo

Volume 148, Issue 1

A newly described cellulosomal cellobiohydrolase, CelO, from : investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose

The GenBank accession number for the sequence determined in this work is AJ275975.

Free

Abstract

The sequence of the gene from F7 was determined. The gene product, cellulase CelO (Ct-Cel5F), had a modular structure consisting of a carbohydrate-binding module of the CBM3 family and a catalytic domain of the glycosyl hydrolase family 5. The presence of the dockerin module indicated that the enzyme was a component of the cellulosome complex. The thermostable recombinant gene product was active on cellodextrins, barley β-glucan, carboxymethylcellulose and insoluble cellulose. Cellobiose was the only product released from amorphic and crystalline cellulose, cellotetraose and higher cello-oligosaccharides, identifying CelO as a cellobiohydrolase. The cleavage pattern of nitrophenyl β-D-cellotetraoside, blockage of the hydrolysis of NaBH-reduced cellopentaose and the reduction in substrate viscosity suggested activity from the reducing end in a processive mode after making random cuts. Binding to insoluble, i.e. amorphous, and crystalline cellulose was mediated by the carbohydrate-binding module CBM3b, with a preference for the crystalline substrate.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): carbohydrate-binding module CBM3 , CBM, carbohydrate-binding module , cellulase CelO , cellulosome , CMC, carboxymethylcellulose , GHF, glycosyl hydrolase family , PASC, phosphoric acid swollen cellulose , pNP, p-nitrophenyl and reducing end
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-1-247
2002-01-01
2023-04-01
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/1/1480247a.html?itemId=/content/journal/micro/10.1099/00221287-148-1-247&mimeType=html&fmt=ahah

References

  1. Barr B., Hsieh Y.-L., Ganem B., Wilson D. B. 1996; Identification of two functionally different classes of exocellulases. Biochemistry 35:586–592 [CrossRef]
     [Google Scholar]
  2. Bayer E. A., Chanzy H., Lamed R., Shoham Y. 1998a; Cellulose, cellulases and cellulosomes. Curr Opin Struct Biol 8:548–557 [CrossRef]
     [Google Scholar]
  3. Bayer E. A., Morag E., Lamed R., Yaron S., Shoham Y. 1998b; Cellulosome structure: four-pronged attack using biochemistry, molecular biology, crystallography and bioinformatics. In Carbohydrases from Trichoderma reesei and Other Microorganisms pp 39–65 Edited by Claeyssens M. Nerinckx W., Piens K. London: Royal Society of Chemistry;
     [Google Scholar]
  4. Béguin P., Chauvaux S., Chaveroche M.-K., Guglielmi G., Kataeva I., Leibovitz E., Miras I. 1998; The cellulosome: a versatile system for coupling cellulolytic enzymes and attaching them to the cell surface. In Carbohydrases from Trichoderma reesei and Other Microorganisms pp 66–72 Edited by Claeyssens M. Nerinckx W., Piens K. London: Royal Society of Chemistry;
     [Google Scholar]
  5. Boisset C., Armand S., Drouillard S., Chanzy H., Driguez H., Henrissat B. 1998; Structure–function relationships in cellulases: the enzymatic degradation of insoluble cellulose. In Carbohydrases from Trichoderma reesei and Other Microorganisms pp 124–132 Edited by Claeyssens M. Nerinckx W., Piens K. London: Royal Society of Chemistry;
     [Google Scholar]
  6. Bolam D. N., Ciruela A., McQueen-Mason S., Simpson P., Williamson M. P., Rixon J. E., Boraston A., Hazlewood G. P., Gilbert H. J. 1998; Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity. Biochem J 331:775–781
     [Google Scholar]
  7. Bumazkin B. K., Velikodvorskaya G. A., Tuka K., Mogutov M. A., Strongin A. Y. 1990; Cloning of Clostridium thermocellum endoglucanase genes in Escherichia coli . Biochem Biophys Res Commun 167:1057–1064 [CrossRef]
     [Google Scholar]
  8. Divne C., Stahlberg J., Reinikainen T., Ruohonen L., Pettersson G., Knowles J. K. C., Teeri T. T., Jones T. A. 1994; The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei . Science 265:524–528 [CrossRef]
     [Google Scholar]
  9. Ferreira L. M., Hazlewood G. P., Barker P. J., Gilbert H. J. 1991; The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains. Biochem J 279:793–799
     [Google Scholar]
  10. Hazlewood G. P., Davidson K., Laurie J. I., Huskisson N. S., Gilbert H. J. 1993; Gene sequence and properties of CelI, a family E endoglucanase from Clostridium thermocellum . J Gen Microbiol 139:307–316 [CrossRef]
     [Google Scholar]
  11. Henikoff S., Henikoff J. G. 1992; Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA 89:10915–10919 [CrossRef]
     [Google Scholar]
  12. Henrissat B., Teeri T. T., Warren R. A. J. 1998; A scheme for designating enzymes that hydrolyse the polysaccharides in the cell wall of plants. FEBS Lett 425:352–354 [CrossRef]
     [Google Scholar]
  13. Jauris S., Schwarz W. H., Kratzsch P., Bronnenmeier K., Staudenbauer W. L., Rücknagel K. P. 1990; Sequence analysis of the Clostridium stercorarium celZ gene encoding a thermoactive cellulase (Avicelase I): identification of catalytic and cellulose-binding domains. Mol Gen Genet 223:258–267
     [Google Scholar]
  14. Joliff G., Juy M., Millet J., Ryter A., Poljak R., Aubert J. P., Béguin P. 1986; Isolation, crystallization and properties of a new cellulase of Clostridium thermocellum overproduced in Escherichia coli . Bio/Technology 4:896–900 [CrossRef]
     [Google Scholar]
  15. Kataeva I., Li X. L., Chen H., Choi S. K., Ljungdahl L. G. 1999; Cloning and sequence analysis of a new cellulase gene encoding CelK, a major cellulosome component of Clostridium thermocellum : evidence for gene duplication and recombination. J Bacteriol 181:5288–5295
     [Google Scholar]
  16. Kruus K., Wang W. K., Ching J., Wu J. H. D. 1995; Exoglucanase activities of the recombinant Clostridium thermocellum CelS, a major cellulosome component. J Bacteriol 177:1641–1644
     [Google Scholar]
  17. Lamed R., Setter E., Kenig R., Bayer E. A. 1983; The cellulosome – a discrete cell surface organelle of Clostridium thermocellum which exhibits separate antigenic, cellulose-binding and various cellulolytic activities. Biotechnol Bioeng 13:163–181
     [Google Scholar]
  18. Lemaire M., Béguin P. 1993; Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG. J Bacteriol 175:3353–3360
     [Google Scholar]
  19. Mechaly A., Yaron S., Lamed R., Fierobe H.-P., Belaich A., Belaich J.-P., Shoham Y., Bayer E. A. 2000; Cohesin-dockerin recognition in cellulosome assembly: experiment versus hypothesis. Proteins 39:170–177 [CrossRef]
     [Google Scholar]
  20. Meinke A., Gilkes N. R., Kilburn D. G., Miller R. C., Warren R. A. J. 1993; Cellulose-binding polypeptides from Cellulomonas fimi : endoglucanase D (CenD), a family A β-1,4-glucanase. J Bacteriol 175:1910–1918
     [Google Scholar]
  21. Nutt A., Sild V., Pettersson G., Johansson G. 1998; Progress curves. A means for functional classification of cellulases. Eur J Biochem 258:200–206 [CrossRef]
     [Google Scholar]
  22. Pagès S., Gal L., Gaudin C., Tardif C., Bélaich A., Bélaich J.-P. 1997; Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation. J Bacteriol 179:2810–2816
     [Google Scholar]
  23. Parsiegla G., Reverbel-Leroy C., Tardif C., Driguez H., Haser R., Bélaich J. P. 2000; Crystal structures of the cellulase Cel48F in complex with inhibitors and substrates give insights into its processive action. Biochemistry 39:11238–11246 [CrossRef]
     [Google Scholar]
  24. Reverbel-Leroy C., Pages S., Tardif C., Bélaich A., Bélaich J.-P. 1997; The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form. J Bacteriol 179:46–52
     [Google Scholar]
  25. Riedel K., Ritter J., Bauer S., Bronnenmeier K. 1998; The modular cellulase CelZ of the thermophilic bacterium Clostridium stercorarium contains a thermostabilizing domain. FEMS Microbiol Lett 164:261–267 [CrossRef]
     [Google Scholar]
  26. Sakon J., Adney W. S., Himmel M. E., Thomas S. R., Karplus P. A. 1996; Crystal structure of thermostable family 5 endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose. Biochemistry 35:10648–10660 [CrossRef]
     [Google Scholar]
  27. Salamitou S., Lemaire M., Fujino T., Ohayon H., Gounon P., Aubert J.-P., Béguin P. 1994; Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a reporter for the docking sequence borne by the catalytic components of the cellulosome. J Bacteriol 176:2828–2834
     [Google Scholar]
  28. 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]
  29. Schwarz W. H. 2001; The cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol 56:634–649 [CrossRef]
     [Google Scholar]
  30. Sedmak J. J., Grossberg S. E. 1977; A rapid, sensitive assay for protein using Coomassie brilliant blue G250. Anal Biochem 79:544–552 [CrossRef]
     [Google Scholar]
  31. Teeri T. T. 1997; Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends Biotechnol 15:160–167 [CrossRef]
     [Google Scholar]
  32. Thompson J. D., Higgins D. G., Gibson T. J. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 [CrossRef]
     [Google Scholar]
  33. Tokatlidis K., Salamitou S., Aubert J.-P., Béguin P., Dhurjati P. 1991; Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components. FEBS Lett 291:185–188 [CrossRef]
     [Google Scholar]
  34. Tomme P., Warren R. A. J., Gilkes N. R. 1995; Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol 37:1–81
     [Google Scholar]
  35. Tomme P., Boraston A., McLean B. 7 other authors 1998; Characterization and affinity applications of cellulose-binding domains. J Chromatogr B Biomed Appl 715:283–296 [CrossRef]
     [Google Scholar]
  36. Tormo J., Lamed R., Chirino A. J., Morag E., Bayer E. A., Shoham Y., Steitz T. A. 1996; Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose. EMBO J 15:5739–5751
     [Google Scholar]
  37. Tuka K., Zverlov V. V., Bumazkin B. K., Velikodvorskaya G. A., Strongin A. Ya. 1990; Cloning and expression of Clostridium thermocellum genes coding for thermostable exoglucanases (cellobiohydrolases) in Escherichia coli . Biochem Biophys Res Commun 169:1055–1060 [CrossRef]
     [Google Scholar]
  38. Wood T. M. 1988; Preparation of crystalline, amorphous and dyed cellulase substrates. Methods Enzymol 160:19–25
     [Google Scholar]
  39. Wood T. M., Bhat K. M. 1988; Methods for measuring cellulase activities. Methods Enzymol 160:87–112
     [Google Scholar]
  40. Young M., Minton N. P., Staudenbauer W. L. 1989; Recent advances in the genetics of the clostridia. FEMS Microbiol Rev 63:301–326
     [Google Scholar]
  41. Zverlov V. V., Velikodvorskaya G. A., Schwarz W. H., Bronnenmeier K., Kellermann J., Staudenbauer W. L. 1998; Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA. J Bacteriol 180:3091–3099
     [Google Scholar]
  42. Zverlov V. V., Velikodvorskaya G. A., Schwarz W. H., Kellermann J., Staudenbauer W. L. 1999; Duplicated Clostridium thermocellum cellobiohydrolase gene encoding cellulosomal subunits S3 and S5. Appl Microbiol Biotechnol 51:852–859 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-1-247
Loading
A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline celluloseThe GenBank accession number for the sequence determined in this work is AJ275975.
Microbiology 148, 247 (2002); https://doi.org/10.1099/00221287-148-1-247
/content/journal/micro/10.1099/00221287-148-1-247
/content/journal/micro/10.1099/00221287-148-1-247
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