A comparative study of two retaining enzymes of Trichoderma reesei: transglycosylation of oligosaccharides catalysed by the cellobiohydrolase I, Cel7A, and the beta-mannanase, Man5A

HPLC, MALDI-TOF MS and NMR spectroscopy were used to investigate the hydrol ysis of cello- and manno-oligosaccharides by Cel7A and Man5A from Trichoder ma reesei. The experimental progress curves were analysed by fitting the nu merically integrated kinetic equations, which provided cleavage patterns fo r oligosaccharides, This data evaluation procedure accounts for product inh ibition and avoids the initial slope approximation. In addition, a transgly cosylation step had to be included in the model to reproduce the experiment al progress curves. For the hydrolysis of manno-oligosaccharides, Man(4-6), by Man5A no mannose was detected at the beginning of the reaction showing that only the internal linkages are hydrolysed. For cellotriose and cellote traose hydrolysis by Cel7A, the main product is cellobiose and glucose is r eleased from the non-reducing end of the substrate, Intermediary products l onger than the substrates were detected by MALDI-TOF RIS when oligosacchari des (Glc(4-6) or Man(4-6)) were hydrolysed by either Cel7A or Man5A. Intere stingly, two distinct transglycosylation pathways could be observed. Cel7A produced intermediates that are one unit longer than the substrate, whereas Man5A produced intermediates that are two units longer than the substrate, (C) 1999 Federation of European Biochemical Societies.