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
2020-03-29
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 Biotechnol12:379–386[CrossRef]
    [Google Scholar]
  2. Bayer E. A., Shimon L. J. W., Shoham Y., Lamed R.. 1998; Cellulosomes – structure and ultrastructure. J Struct Biol124:221–234[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 J299:381–387
    [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. Biochemistry35:16195–16204[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 Lett83:305–310[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 Lett113:211–218[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 J269:261–264
    [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 J307:151–158
    [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 Bacteriol179:6596–6601
    [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 Genet210:551–556[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 Microbiol4:759–767[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 Bacteriol170:4582–4588
    [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 Bacteriol179:4246–4253
    [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 Biol61:211–241
    [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 Bacteriol180:1709–1714
    [Google Scholar]
  16. Kemp P., Lander D. J., Orpin C. G.. 1984; The lipids of the anaerobic rumen fungus Piromyces communis. . J Gen Microbiol130:27–37
    [Google Scholar]
  17. Lamed R., Bayer E. A.. 1988; The cellulosome of Clostridium thermocellum. . Adv Appl Microbiol33:1–46
    [Google Scholar]
  18. McGavin M., Forsberg C. W.. 1989; Catalytic and substrate-binding domains of endoglucanase-2 from Bacteroides succinogenes. . J Bacteriol171:3310–3315
    [Google Scholar]
  19. Miller G. L.. 1959; Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem31:426–428[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 Technol14:289–292[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 Microbiol61:1980–1986
    [Google Scholar]
  22. Perlman D., Halvorson H. O.. 1983; A putative signal peptidase site and sequence on eukaryotic and prokaryoptic signal peptides. J Mol Biol167:391–409[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 Microbiol133:1297–1307
    [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 Biol4:810–818[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 Chem266:11335–11340
    [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 Chem268:20756–20761
    [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 Lett291:185–188[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 Biochem170:575–581[CrossRef]
    [Google Scholar]
  30. Tomme P., Warren R. A. J., Gilkes N. R.. 1995; Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol37:1–77
    [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 J15:5739–5751
    [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 Microbiol49:656–659
    [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...

Most cited this month

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