Skip to main content

Advertisement

SpringerLink
Log in

Molecular cloning and characterization of two intracellular β-glucosidases belonging to glycoside hydrolase family 1 from the basidiomycete Phanerochaete chrysosporium

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

cDNAs encoding two glycoside hydrolase family 1 β-glucosidases (BGL1A and BGL1B) were cloned from the basidiomycete Phanerochaete chrysosporium, and the substrate specificities of the recombinant enzymes and the expression patterns of the two genes were investigated in relation to cellobiose metabolism by the fungus. The cDNA sequences contained open reading frames of 1,389 base pairs (bp) (bgl1A) and 1,623 bp (bgl1B), encoding 462 and 530 amino acids, respectively. Although high sequence identity (65%) was observed between the deduced amino acid sequences of the two enzymes, an apparent difference was observed at the C-terminal region: BGL1B has a 63-amino acid extension, which has no similarity with any known protein. Both recombinant enzymes expressed in Escherichia coli showed hydrolytic activity towards several β-glycosidic compounds. However, the substrate recognition patterns of the two enzymes were quite different from each other. In particular, cellobiose was hydrolyzed more effectively by BGL1B than by BGL1A. The expression of the two genes in the fungus was monitored by reverse transcription-PCR, which showed that bgl1A was expressed constitutively in both glucose- and cellobiose-containing culture, whereas bgl1B was expressed in cellobiose culture but was repressed in glucose culture, possibly because of carbon catabolite repression. We conclude that BGL1B contributes to cellobiose metabolism during cellulose degradation by P. chrysosporium.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ayers AR, Ayers SB, Eriksson KE (1978) Cellobiose oxidase, purification and partial characterization of a hemoprotein from Sporotrichum pulverulentum. Eur J Biochem 90:171–181

    Article  CAS  PubMed  Google Scholar 

  • Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: signalP 3.0. J Mol Biol 340:783–795

    Article  PubMed  Google Scholar 

  • Broda P, Birch PR, Brooks PR, Sims PF (1995) PCR-mediated analysis of lignocellulolytic gene transcription by Phanerochaete chrysosporium: substrate-dependent differential expression within gene families. Appl Environ Microbiol 61:2358–2364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eriksson KE (1978) Enzyme mechanisms involved in cellulose hydrolysis by rot fungus Sporotrichum pulverulentum. Biotechnol Bioeng 20:317–332

    Article  CAS  Google Scholar 

  • Eriksson KE, Pettersson B, Westermark U (1974) Oxidation: an important enzyme reaction in fungal degradation of cellulose. FEBS Lett 49:282–285

    Article  CAS  PubMed  Google Scholar 

  • Habu N, Igarashi K, Samejima M, Pettersson B, Eriksson KE (1997) Enhanced production of cellobiose dehydrogenase in cultures of Phanerochaete chrysosporium supplemented with bovine calf serum. Biotechnol Appl Biochem 26:97–102

    CAS  PubMed  Google Scholar 

  • Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293:781–788

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Henrissat B, Bairoch A (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316:695–696

    Article  PubMed  PubMed Central  Google Scholar 

  • Igarashi K, Samejima M, Saburi Y, Habu N, Eriksson KE (1997) Localization of cellobiose dehydrogenase in cellulose-grown cultures of Phanerochaete chrysosporium. Fungal Genet Biol 21:214–222

    Article  CAS  PubMed  Google Scholar 

  • Igarashi K, Tani T, Kawai R, Samejima M (2003) Family 3 β-glucosidase from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium is a glucan 1,3-β-glucosidase. J Biosci Bioeng 95:572–576

    Article  CAS  PubMed  Google Scholar 

  • Johnsrud SC, Eriksson KE (1985) Cross-breeding of selected and mutated homokaryotic strains of Phanerochaete chrysosporium K-3—new cellulase deficient strains with increased ability to degrade lignin. Appl Microbiol Biotechnol 21:320–327

    Article  CAS  Google Scholar 

  • Kawai R, Yoshida M, Tani T, Igarashi K, Ohira T, Nagasawa H, Samejima M (2003) Production and characterization of recombinant Phanerochaete chrysosporium β-glucosidase in the methylotrophic yeast Pichia pastoris. Biosci Biotechnol Biochem 67:1–7

    Article  CAS  PubMed  Google Scholar 

  • Kawai R, Igarashi K, Kitaoka M, Ishii T, Samejima M (2004) Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1->3)-β-glucosidase from the white-rot fungus Phanerochaete chrysosporium. Carbohydr Res 339:2851–2857

    Article  CAS  PubMed  Google Scholar 

  • Kawai R, Igarashi K, Yoshida M, Kitaoka M, Samejima M (2005) Hydrolysis of β-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-β-glucanase from the basidiomycete Phanerochaete chrysosporium. Appl Microbiol Biotechnol (in press) https://doi.org/10.1007/s00253-005-0214-4

    Article  PubMed  Google Scholar 

  • Kremer SM, Wood PM (1992) Evidence that cellobiose oxidase from Phanerochaete chrysosporium is primarily an Fe(III) reductase—kinetic comparison with neutrophil NADPH oxidase and yeast flavocytochrome b 2. Eur J Biochem 205:133–138

    Article  CAS  PubMed  Google Scholar 

  • Li B, Renganathan V (1998) Gene cloning and characterization of a novel cellulose-binding β-glucosidase from Phanerochaete chrysosporium. Appl Environ Microbiol 64:2748–2754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lymar ES, Li B, Renganathan V (1995) Purification and characterization of a cellulose-binding β-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium. Appl Environ Microbiol 61:2976–2980

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Martinez D, Larrondo LF, Putnam N, Gelpke MD, Huang K, Chapman J, Helfenbein KG, Ramaiya P, Detter JC, Larimer F, Coutinho PM, Henrissat B, Berka R, Cullen D, Rokhsar D (2004) Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nat Biotechnol 22:695–700

    Article  CAS  PubMed  Google Scholar 

  • Mchale A, Coughlan MP (1981a) The cellulolytic system of Talaromyces emersonii—identification of the various components produced during growth on cellulosic media. Biochim Biophys Acta 662:145–151

    Article  CAS  Google Scholar 

  • Mchale A, Coughlan MP (1981b) The cellulolytic system of Talaromyces emersonii—purification and characterization of the extracellular and intracellular β-glucosidases. Biochim Biophys Acta 662:152–159

    Article  CAS  Google Scholar 

  • Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

    Article  CAS  PubMed  Google Scholar 

  • Peralta RM, Terenzi HF, Jorge JA (1990) β-D-Glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme. Biochim Biophys Acta 1033:243–249

    Article  CAS  PubMed  Google Scholar 

  • Saloheimo M, Kuja-Panula J, Ylosmaki E, Ward M, Penttila M (2002) Enzymatic properties and intracellular localization of the novel Trichoderma reesei β-glucosidase BGLII (cel1A). Appl Environ Microbiol 68:4546–4553

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Smith MH, Gold MH (1979) Phanerochaete chrysosporium β-glucosidases: induction, cellular localization, and physical characterization. Appl Environ Microbiol 37:938–942

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Takashima S, Nakamura A, Masaki H, Uozumi T (1996) Purification and characterization of cellulases from Humicola grisea. Biosci Biotechnol Biochem 60:77–82

    Article  CAS  Google Scholar 

  • Takashima S, Nakamura A, Hidaka M, Masaki H, Uozumi T (1999) Molecular cloning and expression of the novel fungal β-glucosidase genes from Humicola grisea and Trichoderma reesei. J Biochem (Tokyo) 125:728–736

    Article  CAS  Google Scholar 

  • Tempelaars CA, Birch PR, Sims PF, Broda P (1994) Isolation, characterization, and analysis of the expression of the cbhII gene of Phanerochaete chrysosporium. Appl Environ Microbiol 60:4387–4393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Westermark U, Eriksson KE (1974) Cellobiose: quinone oxidoreductase, a new wood-degrading enzyme from white-rot fungi. Acta Chem Scand B 28:209–214

    Article  CAS  Google Scholar 

  • Yoshida M, Ohira T, Igarashi K, Nagasawa H, Aida K, Hallberg BM, Divne C, Nishino T, Samejima M (2001) Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris. Biosci Biotechnol Biochem 65:2050–2057

    Article  CAS  PubMed  Google Scholar 

  • Yoshida M, Igarashi K, Kawai R, Aida K, Samejima M (2004) Differential transcription of β-glucosidase and cellobiose dehydrogenase genes in cellulose degradation by the basidiomycete Phanerochaete chrysosporium. FEMS Microbiol Lett 235:177–182

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are grateful to Dr. Shinya Fushinobu (Department of Biotechnology, University of Tokyo) for valuable discussions about the kinetics of GH family 1 BGL. This research was supported by a Grant-in-Aid for Scientific Research to M. Samejima (number 17380102) from the Japanese Ministry of Education, Culture, Sports and Technology.

Author information

Author notes

  1. Makoto Yoshida

    Present address: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan

Authors and Affiliations

  1. Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Takeshi Tsukada, Kiyohiko Igarashi, Makoto Yoshida & Masahiro Samejima

Authors
  1. Takeshi Tsukada
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kiyohiko Igarashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Makoto Yoshida
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Masahiro Samejima
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masahiro Samejima.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tsukada, T., Igarashi, K., Yoshida, M. et al. Molecular cloning and characterization of two intracellular β-glucosidases belonging to glycoside hydrolase family 1 from the basidiomycete Phanerochaete chrysosporium . Appl Microbiol Biotechnol 73, 807–814 (2006). https://doi.org/10.1007/s00253-006-0526-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-006-0526-z

Keywords

Search

Navigation