title: Expression and purification of cellulase Xf818 from Xylella fastidiosa in Escherichia coli.
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 His(6) 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 degrees 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.
authors.affiliation: 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. neawulff@yahoo.com.br
authors.forename: Nelson Arno
authors.initials: NA
authors.lastname: Wulff
authors.forename: Helaine
authors.initials: H
authors.lastname: Carrer
authors.forename: Sérgio Florentino
authors.initials: SF
authors.lastname: Pascholati
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013780:Tetroses / Q000378:metabolism; D014169:Transformation, Bacterial / Q000235:genetics; D014990:Xylans / Q000378:metabolism; D044167:Xylella / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013780:Tetroses; D014990:Xylans; C019193:maltopentaose; C048468:cellotetraose; D002482:Cellulose; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium