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Applied Microbiology and Biotechnology

, Volume 49, Issue 3, pp 301–308 | Cite as

Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products

  • H. Okada
  • T. Sekiya
  • K. Yokoyama
  • H. Tohda
  • H. Kumagai
  • Y. Morikawa
ORIGINAL PAPER

Abstract

A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin, was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin. The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and Vmax values toward phosphoric-acid-swollen cellulose as substrate, except that their Km values were about fourfold higher than that of the native enzyme.

Keywords

Enzyme Cellulose Gentamicin Trichoderma Molecular Species 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • H. Okada
    • 1
  • T. Sekiya
    • 1
  • K. Yokoyama
    • 1
  • H. Tohda
    • 2
  • H. Kumagai
    • 2
  • Y. Morikawa
    • 1
  1. 1.Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-21, Japan Tel.: +81 258 479407 Fax: +81 258 479407JP
  2. 2.Research Center, Asahi Glass Co. Ltd., Hazawa, Kanagawa, Yokohama, Kanagawa 221, JapanJP

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