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An investigation of the transferase activity of cellulase from Trichoderma reesei

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

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    Abstract

    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.

    Item Type: Thesis (Doctoral)
    Additional Information: EThOS Persistent ID uk.bl.ethos.285630
    Uncontrolled Keywords: Transglycosylation, Cellulase, Kinetic control, Biochemistry Chemistry, Organic chemistry
    Subjects: Q Science > QD Chemistry
    Schools: School of Applied Sciences
    Depositing User: Graham Stone
    Date Deposited: 29 Jun 2009 16:34
    Last Modified: 28 Jul 2010 19:38
    URI: http://eprints.hud.ac.uk/id/eprint/4837

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