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Recombinant expression and characterization of XynD from Bacillus subtilis subsp. subtilis ATCC 6051: a GH 43 arabinoxylan arabinofuranohydrolase

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Abstract

The complete genome sequence of Bacillus subtilis reveals that sequences encoding several hemicellulases are co-localised with a gene (xynD) encoding a putative family 43 glycoside hydrolase that has not yet been characterised. In this work, xynD has been isolated from genomic DNA of B. subtilis subsp. subtilis ATCC 6051 and cloned for cytoplasmatic expression in Escherichia coli. Recombinant XynD (rXynD) was purified using ion-exchange chromatography and gel permeation chromatography. The enzyme had a molecular mass of approximately 52 kDa, a pI above 9.0 and releases α-l-arabinose from arabinoxylo-oligosaccharides as well as arabinoxylan polymers with varying degree of substitution. Using para-nitrophenyl-α-l-arabinofuranoside as substrate, maximum activity was observed at pH 5.6 and 45°C. The enzyme retained its activity over a large pH range, while activity was lost after pre-incubation above 50°C. Gas–liquid chromatography and proton nuclear magnetic resonance spectrometry analysis indicated that rXynD specifically releases arabinofuranosyl groups from mono-substituted C-(O)-2 and C-(O)-3 xylopyranosyl residues on the xylan backbone. As rXynD did not display endoxylanase, xylosidase or arabinanase activity and was inactive on arabinan, we conclude that this enzyme is best described as an arabinoxylan arabinofuranohydrolase.

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Acknowledgements

We acknowledge the financial support from the Flemish IWT (Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen SBO project funding) and from the Bijzonder Onderzoeksfonds K.U.Leuven (post-doctoral fellowship to Dr. S. Van Campenhout).

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Authors and Affiliations

  1. Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, 3001, Leuven, Belgium

    Tine M. Bourgois, Steven Van Campenhout & Guido Volckaert

  2. Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001, Leuven, Belgium

    Valerie Van Craeyveld, Steven Van Campenhout, Christophe M. Courtin & Jan A. Delcour

  3. Biomedical Research Institute, Limburgs Universitair Centrum and School of Life Sciences, University Hasselt, 3590, Diepenbeek, Belgium

    Johan Robben

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  1. Tine M. Bourgois
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  2. Valerie Van Craeyveld
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Correspondence to Tine M. Bourgois.

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Bourgois, T.M., Van Craeyveld, V., Van Campenhout, S. et al. Recombinant expression and characterization of XynD from Bacillus subtilis subsp. subtilis ATCC 6051: a GH 43 arabinoxylan arabinofuranohydrolase. Appl Microbiol Biotechnol 75, 1309–1317 (2007). https://doi.org/10.1007/s00253-007-0956-2

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