A re-appraisal of multiplicity of endoglucanase I from Trichoderma reesei using monoclonal antibodies and plasma desorption mass spectrometry.
An endo beta-1,4-glucanase (EC 3.2.1.4, 1.4-(1,3;1,4)-beta-D-glucan 4 glucanhydrolase) was purified to apparent homogeneity from culture filtrates of Trichoderma reesei QM 9414. Identity of the protein with endoglucanase I (EG I) was examined by subjecting CNBr fragments of the protein to analysis by plasma desorption mass spectrometry. Seven non-glycosylated fragments, mapped on the eg1 gene sequence, could be identified, hence proving at least 39.4% identity of the amino acid sequence. No sign for microheterogeneity was observed. Purified EG I was used to prepare monoclonal antibodies. 17 stable clones were obtained, of which one--Mab EG 3--was used to analyze several commercial T. reesei cellulase preparations as well as culture filtrates from T. pseudokoningii and T. longibrachiatum for the presence of EG I. Most of them contained immunoreactive material migrating as a prominent 50-55 kDa band on SDS-PAGE, resembling EG I, but in some instances additional lower molecular weight bands were also observed. Cultivation of T. reesei at low pH led to an increase of these lower molecular weight bands. EG I was rather stable against proteolysis by papain in vitro, but after prolonged treatment, immunopositive products of 50 and 45 kDa were produced at the expense of the 55 kDa band. Our monoclonal antibodies failed to react with a low-molecular-weight endoglucanase, which was previously shown to be detectable with polyclonal antiserum against EG I. However, all monoclonals reacted with a 118 kDa protein which is most probably a dimer of EG I. These results are discussed with respect to the occurrence of multiple forms of EG I in T. reesei cellulase preparations.
Luderer
M E
ME
Abteilung für Mikrobielle Biochemie, Institut für Biochemische Technologie und Mikrobiologie, Wien, Austria.
Hofer
F
F
Hagspiel
K
K
Allmaier
G
G
Blaas
D
D
Kubicek
C P
CP
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D043366:Cellulose 1,4-beta-Cellobiosidase; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide; D006026:Glycoside Hydrolases / Q000276:immunology / Q000302:isolation & purification*; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification*; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D010449:Peptide Mapping; D014242:Trichoderma / Q000201:enzymology*; D014462:Ultrafiltration
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D006026:Glycoside Hydrolases; D043366:Cellulose 1,4-beta-Cellobiosidase; D003488:Cyanogen Bromide