Partial Purification and Thermal Stability of Two Peroxidases from Pithecellobium dulce (Roxb.) Benth. Aril

Abstract

Resumo: Duas formas de peroxidase (POX), denominadas PdPI e PdPII, foram parcialmente purificadas de arilos de Pithecellobium dulce, utilizando precipitação com sulfato de amônio e cromatografia de troca iônica. O zimograma das proteínas obtidas após precipitação com sulfato de amônio na faixa de 60 a 90% de saturação (fração F6090), mostrou duas bandas com atividade peroxidásica. Na coluna de celulose DE-52, a atividade peroxidásica foi detectada nos picos não adsorvido (PdPI) e adsorvido (PdPII) eluído com NaCl 0,2 mol L -1 , sugerindo que a PdPI (~114 KDa) e a PdPII (~77 KDa) são proteínas básicas e ácidas, respectivamente, sendo a PdPI estável a 80 ºC. Este é o primeiro relato de purificação parcial de POX obtida de arilos de P. dulce, indicando que esta espécie pode ser uma nova fonte de enzimas para utilização em biossensores e outras aplicações biotecnológicas. Palavras-chave:Enzimas; Atividade peroxidásica; P. dulce; Zimograma. AbstractTwo peroxidase (POX) forms, named PdPI and PdPII, were partially purified from the Pithecellobium dulce aril, using ammonium sulfate precipitation and anion exchange chromatography. Zymography of the proteins obtained after precipitation in the range of 60-90% ammonium sulfate (F6090 fraction) showed two bands with peroxidase activity. In DE-52 cellulose column, the peroxidase activity was detected in unadsorbed peak (PdPI) and adsorbed peak eluted with 0.2 mol L -1 NaCl (PdPII) suggesting that PdPI (~114 KDa) and PdPII (~77 KDa) are basic and acidic proteins, respectively, being PdPI stable at 80 ºC. This is the first report of partial purification of POX from P. dulce aril, indicating that this species may be a new source of enzymes for use in biosensors and other biotechnological applications.

Extraction, Purification and Characterization of Peroxidase from Okra (Abelmoschus esculentus)

“…The optimum pH for the activity of the enzyme was determined using sodium acetate buffer solution of (0.2 M) with pH values ranging from 3.5-5.5, sodium phosphate buffer solution with pH value ranging between 6.0-7.5, and Tris-HCL buffer solution with a pH value (8)(9) to prepare the substrate solution. The reaction carried out by incubating the substrate with enzyme for each pH (3.5-9.0) in test tubes for 15 minutes in a water bath at 37 ° C, then it was cooled directly in an ice bath to stop the enzymatic reaction and the enzymatic activity determined as explained previously and then the relationship between the enzyme activity and the pH values is plotted.…”

“…In addition, peroxidase can modify the gluten network by cross-linking the gluten proteins or by introducing arabinoxylans to gluten proteins [6,7]. Research on peroxidase enzymes from a wide variety of plants suggested that the enzyme's physical and kinetic properties, as well as its substrate choice, may vary greatly, even within a single source [8,9]. Furthermore, it is well believed that peroxidase activity and related enzyme patterns fluctuate with changes in plant development.…”