Studies of the cellulolytic system of the filamentous fungus Trichoderma reesei QM 9414. Substrate specificity and transfer activity of endoglucanase I.
Endoglucanase I from the filamentous fungus Trichoderma reesei catalyses hydrolysis and glycosyl-transfer reactions of cello-oligosaccharides. Initial bond-cleaving frequencies determined with 1-3H-labelled cello-oligosaccharides proved to be substrate-concentration-dependent. Using chromophoric glycosides and analysing the reaction products by h.p.l.c., kinetic data are obtained and, as typical for an endo-type depolymerase, apparent hydrolytic parameters (kcat., kcat./Km) increase steadily as a function of the number of glucose residues. At high substrate concentrations, and for both free cellodextrins and their aromatic glycosides, complex patterns (transfer reactions) are, however, evident. In contrast with the corresponding lactosides and 1-thiocellobiosides, and in conflict with the expected specificity, aromatic 1-O-beta-cellobiosides are apparently hydrolysed at both scissile bonds, yielding the glucoside as one of the main reaction products. Its formation rate is clearly non-hyperbolically related to the substrate concentration and, since the rate of D-glucose formation is substantially lower, strong indications for dismutation reactions (self-transfer) are again obtained. Evidence for transfer reactions catalysed by endoglucanase I further results from experiments using different acceptor and donor substrates. A main transfer product accumulating in a digest containing a chromophoric 1-thioxyloside was isolated and its structure elucidated by proton n.m.r. spectrometry (500 MHz). The beta 1-4 configuration of the newly formed bond was proved.
Laboratorium voor Biochemie, Rijksuniversiteit Gent, Belgium.
M
M
Claeyssens
H
H
van Tilbeurgh
J P
JP
Kamerling
J
J
Berg
M
M
Vrsanska
P
P
Biely
D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006027:Glycosides / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology* / Q000378:metabolism
D006027:Glycosides; D009844:Oligosaccharides; D002482:Cellulose; D014166:Transferases; D002480:Cellulase