Joncour, Karine (1998) An investigation of the transferase activity of cellulase from Trichoderma reesei. Doctoral thesis, University of Huddersfield.

A study of the transglycosylationr eactionsc atalysedb y a multi-enzymec omplex,
cellulase from Trichoderma reesei, was undertaken. An activated substrate donor,
p-nitrophenyl P-D-cellobioside (PNPQ, and various mono- and disaccharide
acceptors were tested in the studies which were performed under kinetically
controlled conditions.

Surprisingly, three main transfer products were obtained as opposed to the single
product cited in the literature for the cellulase catalysed reaction. Two were
identified as the N-(p-nitrophenyl)-p-D-ceUobioside (a P-(14) linked
disaccharide)a nd the N-(p-nitrophenyl)-p-D-gentiobiosylamine(a P-(1-6) linked
disaccharide)A. number of experimentalp arametersw ere varied and their effects
on the yield of the transglycosylation reaction were determined. The variables
investigated included: increasing of substrate concentration, increasing the
acceptor concentration, varying the pH and the temperature of the reaction. The
effect of the addition of a co-solvent (ACN, t-butanol, dioxane or acetone) was
also studied.T he reactionsw ere found to be stereospecificb ut not regioselective.
The latter was found to vary with the substrate concentration: at low
concentrations (< 1.5 mM), the P-(1-6) linked disaccharide was the preferred
transfer product whereasa t higher concentrationst,h e P-(14) linked disaccharide
was favoured. Increasing the acceptor concentration was found to increase the
transglycosylationy ield (6% to 19%) whereast he addition of co-solvent resulted
in a decrease. Ilese results are discussed in relation to the components of the
complexw hich are responsiblef or the production of the various transferp roducts.
Interestingly,t he use of p-nitrophenyl 1-thio-p-D-glucopyranosidea s an acceptor
proved to give a higher yield of the transfer products (-40 %) showing the
importanceo f the acceptors tructure in the transglycosylationr eaction

Moreover, the transglycosylation studies were also undertaken in the presence of
the P-glucosidasein hibitor, 1,5-glucono-S-lactoneT. his resulted in the formation
of a single product, the P-(14) linked disaccharide and therefore P-glucosidase
was the only cellulase component responsible for producing the P-(1-6) transfer
product. A difference in the degree of orientation of the acceptor between the
different enzyme components of the cellulase complex was then suggested.

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