Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal

• Autor(a) principal: Monteiro, Paulo Sérgio
• Data de Publicação: 2011
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/305
• topic: Fitases; Fungos; Ração animal; Phytases; Fungi; Feed; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA

In this work, two fungi strains isolated from macadamia nut and sugar cane bagasse were evaluated for potential productions of phytases, focusing on biotechnological applications, mainly in animal feed. The fungi Rhizopus stolonifer isolated from macadamia nut was cultivated in submerged fermentation containing potato infusion, sucrose, peptone, Tween-20 and CaCl2, using response surface methodology (RSM) to optimize the phytase production. The use of MSR resulted in an enzyme activity of 0.65 U/mL, a value 9-times the initial phytase activity. The enzyme showed optimum pH and temperature in the range of 5.0-5.5 and of 65 oC, respectivamente, with t1/2 of 50 minutes at 50 oC. The KM app and Vmax app value were 0.692 mM and 0.780 μmol/min, respectively. The enzyme was efficient in phytate hydrolysis in animal feed, releasing 5.14 μmol PO4 3-/mL after 5 h of treatment. This value is equivalent to the initial phosphorus concentration. The fungi Aspergillus niger UFV-1 isolated from sugar cane bagasse was also cultivated in liquid medium containing potato infusion, sucrose, peptone, Tween-20 and CaCl2. After eight days of fermentation, the crude extract produced was collected and analyzed for enzymatic activity of phytases. The enzyme was purified by ultrafiltration, acid precipitation, and ion exchange and gel filtration chromatography. The phytase showed a molecular mass of 161 kDa determined by gel filtration and 81 kDa by SDS-PAGE electrophoresis, suggesting that the enzyme is composed of two subunits. The optimal values of pH and temperature were 2.0 and 60 oC, respectively. The kinetic parameters, KM and Vmax, determined by the Lineweaver-Burk plot, were 30.9 mM and 7.48 Tmol/min, respectively. The kcat estimated was 1.46 x 105 s-1 and kcat/KM was 4724919 s-1.M-1. The phytase showed high thermostability with t½ of 6.2 h at 80 oC and was resistant to pepsin and trypsin. Additionally, it efficiently in release of phosphorus, when evaluated in commercial animal feed, releasing 15.3 Tmol PO4 3-/mL, equivalent to 4-times the initial phosphorus concentration. The obtained results suggest that the two fungi strains evaluated have great potential as phytase producers for industrial application, mainly the strain Aspergillus niger UFV-1 that showed acidic optimum pH and high protease resistance.