Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products.
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 micrograms cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 micrograms/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 alpha-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 four-fold higher than that of the native enzyme.
Okada
H
H
Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
Sekiya
T
T
Yokoyama
K
K
Tohda
H
H
Kumagai
H
H
Morikawa
Y
Y
D002480:Cellulase / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000378:metabolism*; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification
D005656:Fungal Proteins; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase