An investigation of the transferase activity of cellulase from Trichoderma reesei
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.