title: Biophysical and biochemical studies of a major endoglucanase secreted by Xanthomonas campestris pv. campestris.
abstract: Endoglucanases are the main cellulolytic enzymes secreted by the bacterium Xanthomonas campestris pv. campestris (Xcc). The major endoglucanase exported by this bacterium into an external milieu is an enzyme XccCel5A, which belongs to GH5 family subfamily 1 and is encoded by the gene engXCA. We purified XccCel5A using ammonium sulfate precipitation followed by size exclusion chromatography and identified it by zymogram analysis. Circular dichroism and fluorescence spectroscopy studies showed that XccCel5A is stable in a wide pH range and up to about 55°C and denatures at the higher temperatures. The optimal conditions for enzyme activity were identified as T=45°C and pH=7.0. Under the optimum conditions the catalytic efficiency (kcat/KM) of the enzyme was determined as 5.16×10(4)s(-1)M(-1) using carboxymethylcellulose (CMC) as a substrate. Our SAXS studies revealed extended tadpole-shape molecular assembly, typical for cellulases, and allowed to determine an overall shape of the enzyme and a relative position of the catalytic and cellulose binding domains.
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São Carlense 400-Centro, São Carlos-SP 13560-970, Brazil.
authors.forename: Flávio Rodolfo
authors.initials: FR
authors.lastname: Rosseto
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São Carlense 400-Centro, São Carlos-SP 13560-970, Brazil.
authors.forename: Livia Regina
authors.initials: LR
authors.lastname: Manzine
authors.affiliation: Instituto de Biociências, Universidade Estadual Paulista, Distrito de Rubião Jr. s/n, Botucatu-SP 18618-970, Brazil.
authors.forename: Mario
authors.initials: M
authors.lastname: de Oliveira Neto
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São Carlense 400-Centro, São Carlos-SP 13560-970, Brazil. Electronic address: ipolikarpov@ifsc.usp.br.
authors.forename: Igor
authors.initials: I
authors.lastname: Polikarpov
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055592:Biophysical Phenomena; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D053838:Scattering, Small Angle; D013050:Spectrometry, Fluorescence; D014961:X-Ray Diffraction; D016959:Xanthomonas campestris / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C407024:endoglucanase Cel5A; D002480:Cellulase
keywords: Endoglucanases; Hydrolysis; SAXS; Xanthomonas campestris