1887

Microbiology Society logo

Volume 145, Issue 11

A family 26 mannanase produced by as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungi

The GenBank/EMBL accession number for the sequence reported in this paper is AJ242666.

Free

Abstract

Cellulosomes prepared by the cellulose affinity digestion method from culture supernatant hydrolysed carob galactomannan during incubation at 60 °C and pH 65. A recombinant phage expressing mannanase activity was isolated from a library of genomic DNA constructed in λZAPII. The cloned fragment of DNA containing a putative mannanase gene () was sequenced, revealing an ORF of 1767 nt, encoding a protein (mannanase A; Man26A) of 589 aa with a molecular mass of 66816 Da. The putative catalytic domain (CD) of Man26A, identified by gene sectioning and sequence comparisons, displayed up to 32% identity with other mannanases belonging to family 26. Immediately downstream of the CD and separated from it by a short proline/threonine linker was a duplicated 24-residue dockerin motif, which is conserved in all cellulosomal enzymes described thus far and mediates their attachment to the cellulosome-integrating protein (CipA). Man26A consisting of the CD alone (Man26A′) was hyperexpressed in BL21(DE3) and purified. The truncated enzyme hydrolysed soluble and insoluble mannan, displaying a temperature optimum of 65 °C and a pH optimum of 65, but exhibited no activity against other plant cell wall polysaccharides. Antiserum raised against Man26A′ cross-reacted with a polypeptide with a molecular mass of 70000 Da that is part of the cellulosome. A second variant of Man26A containing the N-terminal segment of 130 residues and the CD (Man26A′′) bound to ivory-nut mannan and weakly to soluble Carob galactomannan and insoluble cellulose. Man26A′ consisting of the CD alone did not bind to these polysaccharides. These results indicate that the N-terminal 130 residues of mature Man26A may constitute a weak mannan-binding domain. Sequence comparisons revealed a lack of identity between this region of Man26A and other polysaccharide-binding domains, but significant identity with a region conserved in the three family 26 mannanases from the anaerobic fungus .

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): CBD, cellulose-binding domain , CD, catalytic domain , cellulosome , Clostridium thermocellum , family 26 and mannanase
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-11-3101
1999-11-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/145/11/1453101a.html?itemId=/content/journal/micro/10.1099/00221287-145-11-3101&mimeType=html&fmt=ahah

References

  1. Bayer E. A., Morag E., Lamed R. 1994; The cellulosome – a treasure trove for biotechnology. Trends Biotechnol 12:379–386 [CrossRef]
     [Google Scholar]
  2. Bayer E. A., Shimon L. J. W., Shoham Y., Lamed R. 1998; Cellulosomes – structure and ultrastructure. J Struct Biol 124:221–234 [CrossRef]
     [Google Scholar]
  3. Black G. W., Hazlewood G. P., Xue G. P., Orpin C. R., Gilbert H. J. 1994; Xylanase B from Neocallimastix patriciarum contains a non-catalytic 465-residue linker sequence comprised of 57 repeats of an octapeptide. Biochem J 299:381–387
     [Google Scholar]
  4. Bolam D. N., Hughes N., Virden R., Lakey J. H., Hazlewood G. P., Henrissat B., Braithwaite K. L., Gilbert H. J. 1996; Mannanase A from Pseudomonas fluorescens subsp. cellulosa is a retaining glycosyl hydrolase in which E212 and E320 are the putative catalytic residues. Biochemistry 35:16195–16204 [CrossRef]
     [Google Scholar]
  5. Clarke J. H., Laurie J. I., Gilbert H. J., Hazlewood G. P. 1991; Multiple xylanases of Cellulomonas fimi are encoded by distinct genes. FEMS Microbiol Lett 83:305–310 [CrossRef]
     [Google Scholar]
  6. Coutinho J. B., Gilkes N. R., Kilburn D. G., Warren R. A. J., Miller R. C. Jr 1993; The nature of the cellulose-binding domain affects the activities of a bacterial endoglucanase on different forms of cellulose. FEMS Microbiol Lett 113:211–218 [CrossRef]
     [Google Scholar]
  7. Ferreira L. M.A., Durrant A. J., Hall J., Hazlewood G. P., Gilbert H. J. 1990; Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase. Biochem J 269:261–264
     [Google Scholar]
  8. Fontes C. M. G. A., Hazlewood G. P., Morag E., Hall J., Hirst B. H., Gilbert H. J. 1995; Evidence for a general role for non-catalytic thermostabilizing domains in zylanases from thermophilic bacteria. Biochem J 307:151–158
     [Google Scholar]
  9. Gal L., Gaudin C., Belaich A., Pagès S., Tardif C., Belaich J.-P. 1997; CelG from Clostridium cellulolyticum: a multidomain endoglucanase acting efficiently on crystalline cellulose. J Bacteriol 179:6596–6601
     [Google Scholar]
  10. Gilbert H. J., Jenkins G., Sullivan D. A., Hall J. 1987; Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa. . Mol Gen Genet 210:551–556 [CrossRef]
     [Google Scholar]
  11. Gilbert H. J., Hall J., Hazlewood G. P., Ferreira L. M. A. 1990; The N-terminal region of an endoglucanase from Pseudomonas fluorescens subsp. cellulosa constitutes a cellulose binding domain that is distinct from the catalytic centre. Mol Microbiol 4:759–767 [CrossRef]
     [Google Scholar]
  12. Grépinet, O., Chebrou M.-C., Béguin, P. 1988; Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum. . J Bacteriol 170:4582–4588
     [Google Scholar]
  13. Hayashi H., Takagi K.-I., Fukumura M., Kimura T., Karita S., Sakka K., Ohmiya K. 1997; Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome. J Bacteriol 179:4246–4253
     [Google Scholar]
  14. Hazlewood G. P., Gilbert H. J. 1997; Structure and function analysis of Pseudomonas fluorescens subsp. cellulosa plant cell wall hydrolases. Prog Nucleic Acid Res Mol Biol 61:211–241
     [Google Scholar]
  15. Irwin D., Shin D.-H., Zhang S., Barr B. K., Sakon J., Karplus P. A., Wilson D. D. 1998; Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis. J Bacteriol 180:1709–1714
     [Google Scholar]
  16. Kemp P., Lander D. J., Orpin C. G. 1984; The lipids of the anaerobic rumen fungus Piromyces communis. . J Gen Microbiol 130:27–37
     [Google Scholar]
  17. Lamed R., Bayer E. A. 1988; The cellulosome of Clostridium thermocellum. . Adv Appl Microbiol 33:1–46
     [Google Scholar]
  18. McGavin M., Forsberg C. W. 1989; Catalytic and substrate-binding domains of endoglucanase-2 from Bacteroides succinogenes. . J Bacteriol 171:3310–3315
     [Google Scholar]
  19. Miller G. L. 1959; Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428 [CrossRef]
     [Google Scholar]
  20. Morag E., Bayer E. A., Lamed R. 1992; Affinity digestion for the near total recovery of purified cellulosome from Clostridium thermocellum. . Enzyme Microb Technol 14:289–292 [CrossRef]
     [Google Scholar]
  21. Morag E., Lapidot A., Govorko D., Lamed R., Wilchek M., Bayer E. A., Shoham Y. 1995; Expression, purification and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum. . Appl Environ Microbiol 61:1980–1986
     [Google Scholar]
  22. Perlman D., Halvorson H. O. 1983; A putative signal peptidase site and sequence on eukaryotic and prokaryoptic signal peptides. J Mol Biol 167:391–409 [CrossRef]
     [Google Scholar]
  23. Puls J., Schuseil J. 1993; Chemistry of hemicelluloses: relationship between hemicelluose structure and enzymes required for hydrolysis. In Hemicellulase and Hemicellulases pp. 1–27Edited by Coughlan M. P., Hazlewood G. P. London: Portland Press;
     [Google Scholar]
  24. Romaniec M. P. M., Clarke N., Hazlewood G. P. 1987; Molecular cloning of Clostridium thermocellum DNA and the expression of further novel endo-β-1,4-glucanase genes in Escherichia coli. . J Gen Microbiol 133:1297–1307
     [Google Scholar]
  25. Sakon J., Irwin D., Wilson D. B., Karplus P. A. 1997; Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca. . Nature Struct Biol 4:810–818 [CrossRef]
     [Google Scholar]
  26. Shen H., Schmuck M., Pilz I., Gilkes N. R., Kilburn D. G., Miller R. C. Jr 1991; Deletion of the linker connecting the catalytic and cellulose binding domains of endoglucanase A (CENA) of Cellulomonas fimi alter its conformation and catalytic activity. J Biol Chem 266:11335–11340
     [Google Scholar]
  27. Srisodsuk M., Reinikainen T., Penttila M., Teeri T. 1993; Role of the interdomain linker peptide of Trichoderma reesei cellobiohydrolase-I in its interaction with crystalline cellulose. J Biol Chem 268:20756–20761
     [Google Scholar]
  28. Tokatlidis K., Salamitou S., Béguin, P., Dhurjati P., Aubert J. P. 1991; Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components. FEBS Lett 291:185–188 [CrossRef]
     [Google Scholar]
  29. Tomme P., Vantilbeurgh H., Pettersson G., Vandamme J., Vandekerckhove J., Knowles J., Teeri T., Claeyssens M. 1988; Studies of the cellulolytic system of Trichoderma reesei QM-9414 – analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J Biochem 170:575–581 [CrossRef]
     [Google Scholar]
  30. Tomme P., Warren R. A. J., Gilkes N. R. 1995; Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol 37:1–77
     [Google Scholar]
  31. 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]
  32. Wiegel J., Mothershed C. P., Puls J. 1985; Differences in xylan degradation by various noncellulolytic thermophilic anaerobes and Clostridium thermocellum. . Appl Environ Microbiol 49:656–659
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-145-11-3101
Loading
A family 26 mannanase produced by Clostridium thermocellum as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungiThe GenBank/EMBL accession number for the sequence reported in this paper is AJ242666.
Microbiology 145, 3101 (1999); https://doi.org/10.1099/00221287-145-11-3101
/content/journal/micro/10.1099/00221287-145-11-3101
/content/journal/micro/10.1099/00221287-145-11-3101
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