Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.
Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.
Aho
S
S
Research Laboratories, Alko Ltd., Helsinki, Finland.
Olkkonen
V
V
Jalava
T
T
Paloheimo
M
M
Bühler
R
R
Niku-Paavola
M L
ML
Bamford
D H
DH
Korhola
M
M
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D015153:Blotting, Western; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D000939:Epitopes / Q000276:immunology; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics / Q000276:immunology*; D010206:Papain; D010446:Peptide Fragments / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014242:Trichoderma / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D002482:Cellulose; D006026:Glycoside Hydrolases; D043366:Cellulose 1,4-beta-Cellobiosidase; D010206:Papain