Monoclonal antibodies against different domains of cellobiohydrolase I and II from Trichoderma reesei.
Monoclonal antibodies have been produced against two functionally different domains present in two cellobiohydrolases from Trichoderma reesei (CBH I and CBH II). Four groups of antibodies were obtained, which specifically recognized (Western blotting, ELISA) (a) the core protein within CBH I, (b) the core protein within CBH II, (c) the BA region of CBH I, and (d) the ABB' region of CBH II. No cross-reactivities within these four groups were observed. The antibodies reacted also specifically with proteins of similar size to CBH I and CBH II (SDS-PAGE) from other Trichoderma strains (Western blotting), whereas no reaction was observed with cellulases from other fungal sources. Analysis of culture filtrates of T. reesei QM 9414 harvested at various times of growth on cellulose under buffered conditions (pH 5-6) indicated the presence of only single bands of CBH I and CBH II, even after prolonged cultivation (160 h). Cultivation on cellulose in unbuffered media, however, showed the appearance (Western blotting) of additional lower molecular weight proteins, which reacted with the monoclonal antibodies directed against the cores of CBH I and II, but not with those recognizing the respective BA and ABB' regions. The appearance of these lower molecular weight bands was most pronounced in unbuffered media, supplemented with a 3-fold (w/w) amount of organic nitrogen (peptone). Analysis of some commercial cellulase preparations from T. harzianum revealed the same pattern of lower molecular weight proteins, in contrast to samples from other fungal cellulases. Those samples or preparations, showing a multiple pattern of CBH I and CBH II, exhibited higher activities of an acid proteinase. These results imply that the use of unbuffered, high nitrogen-supplemented culture conditions for production of cellulases may lead to considerable proteolytic modification of the secreted cellobiohydrolases.
Mischak
H
H
Institut für Biochemie, Universität Wien, Austria.
Hofer
F
F
Messner
R
R
Weissinger
E
E
Hayn
M
M
Tomme
P
P
Esterbauer
H
H
Küchler
E
E
Claeyssens
M
M
Kubicek
C P
CP
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D015153:Blotting, Western; D043366:Cellulose 1,4-beta-Cellobiosidase; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000276:immunology*; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D010206:Papain; D010446:Peptide Fragments; D014242:Trichoderma / Q000201:enzymology* / Q000254:growth & development
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D006026:Glycoside Hydrolases; D043366:Cellulose 1,4-beta-Cellobiosidase; D010206:Papain