Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    The activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH
    Perugino, G. ; Trincone, A. ; Giordano, A. ; Oost, J. van der; Kaper, T. ; Rossi, M. ; Moracci, M. - \ 2003
    Biochemistry 42 (2003). - ISSN 0006-2960 - p. 8484 - 8493.
    agrobacterium beta-glucosidase - oligosaccharide synthesis - sulfolobus-solfataricus - glycosyl transfer - glycosidase - intermediate - pyrococcus - mechanism - evolution - protein
    We have previously shown that the hyperthermophilic glycosynthase from Sulfolobus so fataricus (Ssbeta-glyE387G) can promote the synthesis of branched oligosaccharides from activated beta-glycosides, at pH 6.5, in the presence of 2 M sodium formate as an external nucleophile. In an effort to increase the synthetic potential of hyperthermophilic glycosynthases, we report a new method to reactivate the Ssbeta-glyE387G glycosynthase and two novel mutants in the nucleophile of the P-glycosidases from the hyperthermophilic Archaea Thermosphaera aggregans (Tabeta-gly) and Pyrococcus furiosus (CelB). We describe here that, at pH 3.0 and low concentrations of sodium formate buffer, the three hyperthermophilic glycosynthases show k(cat) values similar to those of the wild-type enzymes and 17-fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5. Moreover, at acidic pH the three reactivated mutants have wide substrate specificity and improved efficiency in the synthetic reaction. The data reported suggest that the reactivation conditions modify the ionization state of the residue acting as an acid/base catalyst. This new reactivation method can be of general applicability on hyperthermophilic glycosynthases whose intrinsic stability allows their exploitation as synthetic tools at low pH.
    Activity and stability of hyperthermophilic enzymes : a comparative study on two archaeal b-glycosidases
    Pouwels, J. ; Moracci, M. ; Cobucci-Ponzano, B. ; Perugino, G. ; Oost, J. van der; Kaper, T. ; Lebbink, J.H.G. ; Vos, W.M. de; Ciaramella, M. ; Rossi, M. - \ 2000
    Extremophiles 4 (2000). - ISSN 1431-0651 - p. 157 - 164.
    Identification and molecular characterization of the first a-xylosidase from an Archaeon
    Moracci, M. ; Cobucci-Ponzano, B. ; Trincone, A. ; Fusco, S. ; Rosa, M. de; Oost, J. van der; Sensen, C.W. ; Charlebois, R.L. ; Rossi, M. - \ 2000
    Journal of Biological Chemistry 275 (2000). - ISSN 0021-9258 - p. 22082 - 22089.
    We here report the first molecular characterization of an -xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 °C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to -glucosidases, -xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a -glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The -xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 °C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the -xylosidase and the -glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.
    Molecular biology of hyperthermophilic Archaea.
    Oost, J. van der; Ciaramella, M. ; Moracci, M. ; Pisani, F.M. ; Rossi, M. ; Vos, W.M. de - \ 1998
    Advances in biochemical engineering 61 (1998). - ISSN 0065-2210 - p. 87 - 115.
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