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Expression and Purification of Cellulase Xf818 from Xylella fastidiosa in Escherichia coli

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Abstract

Xylella fastidiosa was the first plant pathogen whose complete genome sequence was available. X. fastidiosa causes citrus variegated chlorosis, but the physiological basis of the disease in unknown. Through comparative sequence analysis, several putative plant cell wall–degrading enzymes were identified on the X. fastidiosa genome. We have cloned Xf818, a putative endoglucanase ORF, into expression vectors pET20b and pET28b, and purified a recombinant form of Xf818 containing a His6 tag. Through biochemical assays, we have characterized the endoglucanase activity of this protein. The best conditions for hydrolysis over carboxymethyl cellulose (CMC) were on pH 5.2 at 65°C. Xf818 hydrolyzed CMC, acid swollen cellulose, Avicel, birch wood, oat spels xylans, and the oligosaccharides cellotetraose and cellopentaose. Xf818 carried out transglycosylation and had a functional cellulose-binding domain.

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Acknowledgments

This work was supported by FAPESP grant-in-aid for Xylella fastidiosa Functional Genomics Project. N.A. Wulff received a PhD scholarship from FAPESP and S.F. Pascholati is a Fellow at CNPq.

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  1. Escola Superior de Agricultura “Luiz de Queiroz,” USP, Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, Av. Pádua Dias, 11, CEP 13418-900, Piracicaba, SP, Brasil, CP 9

    Nelson Arno Wulff, Helaine Carrer & Sérgio Florentino Pascholati

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  1. Nelson Arno Wulff
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  2. Helaine Carrer
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  3. Sérgio Florentino Pascholati
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Correspondence to Nelson Arno Wulff.

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Wulff, N.A., Carrer, H. & Pascholati, S.F. Expression and Purification of Cellulase Xf818 from Xylella fastidiosa in Escherichia coli . Curr Microbiol 53, 198–203 (2006). https://doi.org/10.1007/s00284-005-0475-2

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  • DOI: https://doi.org/10.1007/s00284-005-0475-2

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