Purificação e caracterização de uma β-xilosidase do fungo fitopatogênico Ceratocystis fimbriata RM 35

• Autor(a) principal: Coura, Roberta Ribeiro
• Data de Publicação: 2012
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2448
• topic: β-xilosidase; Ceratocystis fimbriata RM 35; β-xilosidase; Ceratocystis fimbriata RM 35; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA

The objectives were to cultivate the plant pathogenic fungus Ceratocystis fimbriata RM 35 for the production of β-xilosidases, purify and characterize this enzyme, for their application in industrial bleaching of cellulose pulp, biofuel production and production of xylitol, used in the food industry and indental and medical. For production of extracellular β-xylosidase, the fungus was grown for 168 h in mineral medium containing wheat bran as carbon source. After this period, the medium was filtered, centrifuged and used as a source of β-xilosidases enzymes. The enzyme was purified by ion exchange chromatography, gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. At the end of the purification process, the β-xylosidase showed a purification factor of 18.29 fold with a yield of 22.5 %. The molecular weight of the enzyme was approximately 161.6 kDa, as estimated by SDS-PAGE, and about 204.2 kDa, as estimated by gel filtration. The enzyme showed maximum activity at pH 3.9 and temperature 65 ° C and was stable at 60 ° C for 24h. The KM and Vmax values, using the substrate ρ-NP-βXil were 0.326 mM to 0.91×10-3 mM/min, respectively. The enzyme showed very low activity with the synthetic substrate ρ-nitrophenyl-β-glucopyranoside. The enzymatic activity was partially inhibited by copper II sulfate, aluminum chloride III SDS and xylose, all final concentrations of 1 and 10 mM. C. fimbriata is a plant pathogenic fungus causing disease and found in various cultures of high economic value. Thus, the biochemical and kinetic characterization of β-xylosidase, and assessment of potential for application of these enzymes may be a promising and cost to improve the quality of industrial processes for the production of xylitol of bleaching of cellulose pulp and the production of biofuels, and an important step in the elucidation of plant-pathogen interaction in the development of disease caused by this fungus.