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A β-glucosidase from Sclerotinia sclerotiorum

Biochemical characterization and use in oligosaccharide synthesis

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Abstract

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one β-glucosidase (β-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that β-glu x may be a homodimer. For p-nitrophenyl β-d-glucopyranoside hydrolysis, apparent K m and V max values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55–60°C and pH 5.0, respectively. β-Glu x was strongly inhibited by Fe2+ and activated about 35% by Ca2+. β-Glu x possesses strong transglucosylation activity in comparison with commercially available β-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50°C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 13C nuclear magnetic resonance spectroscopy.

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Authors and Affiliations

  1. Unité de Génie Biologique, Institut National de Sciences Appliquées et de Technologie (INSAT), BP 676, 1080, Tunis Cedex, Tunisia

    Smaali M. Issam, Gargouri Mohamed & Marzouki Nejib

  2. Laboratoire de Génie Protéique et Cellulaire (LGPC), Université de La Rochelle, Avenue Michel Crépeau, 17042, La Rochelle, France

    Legoy Marie Dominique & Maugard Thierry

  3. Laboratoire de Biochimie, Institut National de Recherche Scientifique et Technique (INRST), BP 95, 2050, Hammam Lif, Tunisia

    Limam Farid

Authors
  1. Smaali M. Issam
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  2. Gargouri Mohamed
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  3. Legoy Marie Dominique
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  4. Maugard Thierry
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  5. Limam Farid
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  6. Marzouki Nejib
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Correspondence to Marzouki Nejib.

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Issam, S.M., Mohamed, G., Dominique, L.M. et al. A β-glucosidase from Sclerotinia sclerotiorum . Appl Biochem Biotechnol 112, 63–77 (2004). https://doi.org/10.1385/ABAB:112:2:63

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  • DOI: https://doi.org/10.1385/ABAB:112:2:63

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