1887

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 .

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-11-3101
1999-11-01
2019-10-22
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. 1-27. Edited by M. P. Coughlan & G. P. Hazlewood. London: Portland Press.
  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
/content/journal/micro/10.1099/00221287-145-11-3101
Loading

Data & Media loading...

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