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

Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and... 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 V max values toward phosphoric-acid-swollen cellulose as substrate, except that their K m values were about fourfold higher than that of the native enzyme. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Microbiology and Biotechnology Springer Journals

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

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Publisher
Springer Journals
Copyright
Copyright © 1998 by Springer-Verlag Berlin Heidelberg
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
ISSN
0175-7598
eISSN
1432-0614
DOI
10.1007/s002530051173
Publisher site
See Article on Publisher Site

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 V max values toward phosphoric-acid-swollen cellulose as substrate, except that their K m values were about fourfold higher than that of the native enzyme.

Journal

Applied Microbiology and BiotechnologySpringer Journals

Published: Mar 27, 1998

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