Produção, purificação e caracterização bioquímica de dextranase de Paecilomyces marquandii

• Autor(a) principal: Machado, Fabiano de Paula Pereira
• Data de Publicação: 2009
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/296
• topic: Dextranase; Paecilomyces marquandii; Dextranase; Paecilomyces marquandii; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA

Dextranases (EC 3.2.1.-) catalyses the hydrolysis of α-1,6-glycosidic linkages on dextran polysaccharides. They are produced by fungi, bacteria and some yeasts. Its main application is in the elimination of dextrans produced by bacteria which can cause serious problems in sugar processing. Other potential applications for dextranases are in the prevention and treatment of dental plaques, production of isomaltooligosaccharides and oligodextrans with prebiotic functions, elaboration of cleaning products and synthesis of dextrans for clinical applications. The objective of this study was to produce, purify and characterize dextranase produced by the fungi Paecilomyces marquandii. The effects of various components of culture medium and incubation temperature were studied on the production of dextranase using a Plackett-Burman design. Dextran, ammonium nitrate and yeast extract showed a significant positive effect on dextranase production, while triptone presented a negative effect. According to the full factorial design, dextranase maximum production conditions are within the experimental region utilized in this study. It appeared to be adequate for modeling of the surface response and determination of the value ranges of these variables for dextranase maximum production. Dextranase was purified using chromatographies in DEAE-Sepharose and CM-Sepharose resins, yielding of 24% and specific activity of 1036 U.mg-1. Molecular mass was estimated to be 73 kDa by SDS-PAGE. Dextranase presented greatest activity in pH 6.0 and stability in the pH range of 5 to 7 at 40 °C for 30 min. The optimal temperature was 55 °C and during 4 h at 37 °C the enzyme was stable. Half-life of dextranase was 55 hs at 37 °C and 14 min at 55 °C. Reduction of enzymatic activity was 40%, 80%, 51% and 53% in the presence of Ag+, SDS, Cu+ and Pb2+, respectively. Increase in dextranase activity in the presence of Fe2+ and Mn2+ was 20% and 15%, respectively, all at the concentration of 10 mM. The determined values of KM and Vmax were 0.59 g.L-1 and 0.89 mmol glucose.min-1, respectively, at 40 °C for dextran (molecular mass between 60,000 and 90,000).