Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita |
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Authors: | Zhongwei Gan Jinkui Yang Nan Tao Lianming Liang Qili Mi Juan Li Ke-Qin Zhang |
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Affiliation: | (1) Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, 3001 Leuven, Belgium;(2) Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001 Leuven, Belgium;(3) Biomedical Research Institute, Limburgs Universitair Centrum and School of Life Sciences, University Hasselt, 3590 Diepenbeek, Belgium |
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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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Keywords: | Bacillus subtilis Arabinoxylan arabinofuranohydrolase Glycoside hydrolase family 43 Arabinoxylan-derived oligosaccharides α - font-variant:small-caps" >l-arabinose |
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