Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Feb 1;377(Pt 3):749–755. doi: 10.1042/BJ20031145

Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1-->6)-galactanase gene.

Toshihisa Kotake 1, Satoshi Kaneko 1, Aya Kubomoto 1, Md Ashraful Haque 1, Hideyuki Kobayashi 1, Yoichi Tsumuraya 1
PMCID: PMC1223900  PMID: 14565843

Abstract

The nucleotide sequence depicted in Figure 1 has been submitted to the DDBJ nucleotide sequence database under the accession no. AB104898. A gene encoding endo-beta-(1-->6)-galactanase from Trichoderma viride was cloned by reverse transcriptase-PCR and expressed in Escherichia coli. The gene contained an open reading frame consisting of 1437 bp (479 amino acids). The deduced amino acid sequence of the protein showed little similarity with other known glycoside hydrolases. A signal sequence (20 amino acids) was found at the N-terminal region of the protein and the molecular mass of the mature form was calculated to be 50.488 kDa. The gene product expressed in E. coli as a recombinant protein fused with thioredoxin and His(6) tags had almost the same substrate specificity and mode of action as native enzyme purified from a commercial cellulase preparation of T. viride, i.e. recombinant enzyme endo-hydrolysed beta-(1-->6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse alpha-L-arabinofuranosidase-treated arabinogalactan protein from radish. It produced beta-(1-->6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and beta-(1-->6)-galactobiose as the major products at the final reaction stage. These results indicate that the cloned gene encodes an endo-beta-(1-->6)-galactanase. As far as we know, this is the first time an endo-beta-(1-->6)-galactanase has been cloned.

Full Text

The Full Text of this article is available as a PDF (189.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cutfield S. M., Davies G. J., Murshudov G., Anderson B. F., Moody P. C., Sullivan P. A., Cutfield J. F. The structure of the exo-beta-(1,3)-glucanase from Candida albicans in native and bound forms: relationship between a pocket and groove in family 5 glycosyl hydrolases. J Mol Biol. 1999 Dec 3;294(3):771–783. doi: 10.1006/jmbi.1999.3287. [DOI] [PubMed] [Google Scholar]
  2. Gaspar Y., Johnson K. L., McKenna J. A., Bacic A., Schultz C. J. The complex structures of arabinogalactan-proteins and the journey towards understanding function. Plant Mol Biol. 2001 Sep;47(1-2):161–176. [PubMed] [Google Scholar]
  3. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1991 Dec 1;280(Pt 2):309–316. doi: 10.1042/bj2800309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993 Aug 1;293(Pt 3):781–788. doi: 10.1042/bj2930781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hilge M., Gloor S. M., Rypniewski W., Sauer O., Heightman T. D., Zimmermann W., Winterhalter K., Piontek K. High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca - substrate specificity in glycosyl hydrolase family 5. Structure. 1998 Nov 15;6(11):1433–1444. doi: 10.1016/s0969-2126(98)00142-7. [DOI] [PubMed] [Google Scholar]
  6. Kelley L. A., MacCallum R. M., Sternberg M. J. Enhanced genome annotation using structural profiles in the program 3D-PSSM. J Mol Biol. 2000 Jun 2;299(2):499–520. doi: 10.1006/jmbi.2000.3741. [DOI] [PubMed] [Google Scholar]
  7. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  8. Majewska-Sawka A., Nothnagel E. A. The multiple roles of arabinogalactan proteins in plant development. Plant Physiol. 2000 Jan;122(1):3–10. doi: 10.1104/pp.122.1.3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Nothnagel E. A. Proteoglycans and related components in plant cells. Int Rev Cytol. 1997;174:195–291. doi: 10.1016/s0074-7696(08)62118-x. [DOI] [PubMed] [Google Scholar]
  10. Okemoto Kazuo, Uekita Takamasa, Tsumuraya Yoichi, Hashimoto Yohichi, Kasama Takeshi. Purification and characterization of an endo-beta-(1-->6)-galactanase from Trichoderma viride. Carbohydr Res. 2003 Jan 31;338(3):219–230. doi: 10.1016/s0008-6215(02)00405-6. [DOI] [PubMed] [Google Scholar]
  11. SMOGYI M. Notes on sugar determination. J Biol Chem. 1952 Mar;195(1):19–23. [PubMed] [Google Scholar]
  12. Sabini E., Schubert H., Murshudov G., Wilson K. S., Siika-Aho M., Penttilä M. The three-dimensional structure of a Trichoderma reesei beta-mannanase from glycoside hydrolase family 5. Acta Crystallogr D Biol Crystallogr. 2000 Jan;56(Pt 1):3–13. doi: 10.1107/s0907444999013943. [DOI] [PubMed] [Google Scholar]
  13. Schultz C. J., Johnson K. L., Currie G., Bacic A. The classical arabinogalactan protein gene family of arabidopsis. Plant Cell. 2000 Sep;12(9):1751–1768. doi: 10.1105/tpc.12.9.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Schultz Carolyn J., Rumsewicz Michael P., Johnson Kim L., Jones Brian J., Gaspar Yolanda M., Bacic Antony. Using genomic resources to guide research directions. The arabinogalactan protein gene family as a test case. Plant Physiol. 2002 Aug;129(4):1448–1463. doi: 10.1104/pp.003459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Shi Huazhong, Kim YongSig, Guo Yan, Stevenson Becky, Zhu Jian-Kang. The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion. Plant Cell. 2003 Jan;15(1):19–32. doi: 10.1105/tpc.007872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tsumuraya Y., Mochizuki N., Hashimoto Y., Kovác P. Purification of an exo-beta-(1----3)-D-galactanase of Irpex lacteus (Polyporus tulipiferae) and its action on arabinogalactan-proteins. J Biol Chem. 1990 May 5;265(13):7207–7215. [PubMed] [Google Scholar]
  17. Tsumuraya Y., Ogura K., Hashimoto Y., Mukoyama H., Yamamoto S. Arabinogalactan-Proteins from Primary and Mature Roots of Radish (Raphanus sativus L.). Plant Physiol. 1988 Jan;86(1):155–160. doi: 10.1104/pp.86.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wu H. M., Wang H., Cheung A. Y. A pollen tube growth stimulatory glycoprotein is deglycosylated by pollen tubes and displays a glycosylation gradient in the flower. Cell. 1995 Aug 11;82(3):395–403. doi: 10.1016/0092-8674(95)90428-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES