Applied Microbiology and Biotechnology

, Volume 63, Issue 1, pp 42–50 | Cite as

Cloning of a gene encoding thermostable cellobiohydrolase from Thermoascus aurantiacus and its expression in yeast

Original Paper

Abstract.

A gene encoding a cellobiohydrolase (CBH) was isolated from Thermoascus aurantiacus IFO 9748 and designated as cbh1. The deduced amino acid sequence encoded by cbh1 showed high homology with the sequence of glycoside hydrolase family 7. To confirm the sequence of the gene encoding the CBH, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The recombinant enzyme was confirmed as a CBH by analysis of the reaction product and designated as CBHI. Recombinant CBHI retained more than 80% of its initial activity after 1 h of incubation at 65 °C and was stable in the pH range 3.0–9.0. The optimal temperature for enzyme activity was about 65 °C and the optimal pH was about 6.0. The recombinant enzyme was found to be highly glycosylated and this glycosylation was shown to contribute to the thermostability of the enzyme. CBHI expression was shown to be induced at higher temperature in T. aurantiacus.

References

  1. 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 25:3389–3402PubMedGoogle Scholar
  2. Aro N, Saloheimo A, Ilmen M, Penttila M (2001) ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei. J Biol Chem 276:24309–24314PubMedGoogle Scholar
  3. Bradford MM (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein–dye binding. Anal Biochem 72:248–252CrossRefPubMedGoogle Scholar
  4. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal Biochem 162:156–159PubMedGoogle Scholar
  5. Deshpande MV, Pettersson LG, Eriksson KE (1988) Selective assay for exo-1,4-beta-glucanase. In: Wood WA, Kellogg ST (eds) Methods in enzymology, vol 160. Academic Press, San Diego, pp 126–130Google Scholar
  6. Feldman KA, Lovett JS, Tsao GT (1988) Isolation of the cellulase enzymes from the thermophilic fungus Thermoascus aurantiacus and regulation of enzyme production. Enzyme Microb Technol 10:262–271CrossRefGoogle Scholar
  7. Gielkens MM, Dekkers E, Visser J, Graaff LH de (1999) Two cellobiohydrolase-encoding genes from Aspergillus niger Require d-xylose and the xylanolytic transcriptional activator XlnR for their expression. Appl Environ Microbiol 65:4340–4345PubMedGoogle Scholar
  8. Gomes I, Gomes J, Gomes DJ, Steiner W (2000) Simultaneous production of high activities of thermostable endoglucanase and β-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus. Appl Microbiol Biotechnol 53:461–468CrossRefPubMedGoogle Scholar
  9. Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263–269PubMedGoogle Scholar
  10. Hong J, Tamaki H, Akiba S, Yamamoto K, Kumagai H (2001) Cloning of a gene encoding a highly stable endo-β-1,4-glucanase from Aspergillus niger and its expression in yeast. J Biosci Bioeng 92:434–441CrossRefGoogle Scholar
  11. Hong J, Tamaki H, Yamamoto K, Kumagai H (2003) Cloning of a gene encoding thermo-stable endo-b-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast. Biotechnol Lett 25:657–661CrossRefGoogle Scholar
  12. Johnson PE, McKnight SL (1989) Eukaryotic transcriptional regulatory proteins. Annu Rev Biochem 58:799–839CrossRefPubMedGoogle Scholar
  13. Kawamori M, Takayama K, Takasawa S (1987) Production of cellulase by a thermophilic fungus, Thermoascus aurantiacus A-131. Agric Biol Chem 51:647–654Google Scholar
  14. Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol Rev 64:461–488PubMedGoogle Scholar
  15. Miller GL, Blum R, Glennon WE, Burton AL (1960) Measurement of carboxymethyl cellulose activity. Anal Biochem 1:127–132Google Scholar
  16. Ortner J, Albert M, Terler K, Steiner W, Dax K (2000) Transglycosylation reactions with a crude culture filtrate from Thermoascus aurantiacus. Carbohydr Res 327:483–487CrossRefPubMedGoogle Scholar
  17. Parry NJ, Beever DE, Owen E, Vandenberghe I, Van Beeumen J, Bhat MK (2001) Biochemical characterization and mechanism of action of a thermostable beta-glucosidase purified from Thermoascus aurantiacus. Biochem J 353:117–127CrossRefPubMedGoogle Scholar
  18. Parry NJ, Beever DE, Owen E, Nerinckx W, Claeyssens M, Van Beeumen J (2002) Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus. Arch Biochem Biophys 404:243–253CrossRefPubMedGoogle Scholar
  19. Peij NN van, Gielkens MM, Vries RP de, Visser J, Graaff LH de (1998) The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger. Appl Environ Microbiol 64:3615–3619PubMedGoogle Scholar
  20. Saloheimo A, Aro N, Ilmen M, Penttila M (2000) Isolation of the ace1 gene encoding a Cys(2)-His(2) transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei. J Biol Chem 275:5817–5825PubMedGoogle Scholar
  21. Sherman F (1991) Getting started with yeast. In: Guthrie C, Fink GR (eds) Methods in enzymology, vol 194. Academic Press, San Diego, pp 3–21Google Scholar
  22. Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:19–23Google Scholar
  23. Suto M, Tomita F (2001) Induction and catabolite repression mechanisms of cellulase in fungi. J Biosci Bioeng 92:305–311CrossRefGoogle Scholar
  24. Tong C, Anthony LC, Maxwell GS (1980) Purification and properties of the cellulases from the thermophilic fungus Thermoascus aurantaicus. Biochem J 191:83–94PubMedGoogle Scholar
  25. Wood TM, Bhat KM(1988) Methods for measuring cellulase activity. In: Wood WA, Kellogg ST (eds) Methods in enzymology, vol 160. Academic Press, San Diego, pp 87–116Google Scholar
  26. Xu J, Nogawa M, Okada H, Morikawa Y (2000) Regulation of xyn3 gene expression in Trichoderma reesei PC-3-7. Appl Microbiol Biotechnol 54:370–375CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Division of Integrated Life Sciences, Graduate School of BiostudiesKyoto UniversityKyotoJapan
  2. 2.Division of Applied Life Sciences, Graduate School of AgricultureKyoto UniversityKyotoJapan
  3. 3.College of BioscienceBeijing Normal UniversityBeijingChina

Personalised recommendations