The aim of this work is to obtain, purify and characterize biochemically a peroxidase from Copaifera langsdorffii leaves (COP). COP was obtained by acetone precipitation followed by ion-exchange chromatography. Purification yielded 3.5% of peroxidase with the purification factor of 46.86. The COP optimum pH is 6.0 and the temperature is 35 ºC. COP was stable in the pH range of 4.5 to 9.3 and at temperatures below 50.0 ºC. The apparent Michaelis-Menten constants (Km) for guaiacol and H2O2 were 0.04 mM and 0.39 mM respectively. Enzyme turnover was 0.075 s-1 for guaiacol and 0.28 s-1 for hydrogen peroxide. Copaifera langsdorffii leaves showed to be a rich source of active peroxidase (COP) during the whole year. COP could replace HRP, the most used peroxidase, in analytical determinations and treatment of industrial effluents at low cost
RESUMOObjetivou-se neste trabalho extrair peroxidase de folha de Copaifera langsdorffii (COP), medir sua atividade, compará-la com a peroxidase de raiz forte (Horseradish peroxidase -HRP) e determinar o pH ótimo, a melhor solução extratora e o efeito de aditivos sobre a atividade da COP. Os resultados mostraram que a COP atingiu 81,6% da atividade de HRP e a faixa de pH ótimo foi de 5,5 a 6,0. A melhor solução extratora da enzima foi o tampão fosfato de sódio 50 mM, pH 6,0 e o melhor aditivo foi o PVPP. Concluindo, a COP apresenta atividade mais alta que outras peroxidases de diferentes fontes citadas na literatura. Palavras-chave: Copaifera langsdorffii, peroxidase, atividade enzimática, horseradish peroxidase, extrato vegetal. SUMMARYOBTENTION OF A NEW SOURCE OF PEROXIDASE FROM Copaifera langsdorffii LEAF, Desf. WITH HIGH ACTIVITY. The purpose of this work was to extract peroxidase from Copaifera langsdorffii leaves (COP), measure its activity, compare it to that of Horseradish peroxidase and determine the optimum pH, the best extraction solution and the effect of additives on the COP activity. The results showed that COP has 81.6% of the activity of HRP and an optimum pH range between 5.5-6.0. The best extraction solution was a sodium phosphate buffer 50 mM, pH 6.0 and the best additive was PVPP. In conclusion, COP presents higher activity than peroxidases from different sources reported in the literature.
ResumoEm recentes publicações têm sido descritos vários processos para obtenção de peroxidases. O propósito deste trabalho foi extrair peroxidase de folhas de Copaifera langsdorffii e caracterizar parcialmente a enzima usando planejamento experimental e teste univariado, para confirmação dos resultados obtidos por planejamento experimental. A atividade da peroxidase foi medida usando sistema guaiacol: peróxido de hidrogênio. A peroxidase isolada apresentou 81,6% da atividade da horseradish peroxidase e é de fácil obtenção, a partir de folhas de uma árvore abundante em todo o país. A peroxidase semi-purificada (COP) foi obtida pela precipitação do extrato bruto com acetona 65% (v.v -1 ), produzindo o pó cetônico. A COP apresentou atividade ótima na faixa de pH 5,0 a 7,0 e temperatura de 5 a 45 °C, com atividade máxima em pH 6,0 e 35 °C. A enzima mostrou-se estável em temperaturas inferiores a 50 °C e pH entre 4,5 e 9,0, por até 24 horas. A peroxidase foi inativada após 4 horas a 80 °C e após 3 minutos a 96 °C. Esta enzima demonstra possibilidade para ser usada como reagente para diagnósticos, construção de biossensores e outros métodos analíticos em vários campos da ciência. Palavras-chave: copaíba; enzima; extrato vegetal. AbstractIn the literature, several processes have been described to obtain peroxidases. The purpose of this work was to obtain peroxidase from Copaifera langsdorffii leaves and characterize it partially using a factorial design of experiments and univaried tests, to confirm the results obtained by the factorial design of experiments. Peroxidase activity was measured using the guaiacol: hydrogen peroxide system. The isolated peroxidase presented 81.6% of horseradish peroxidase activity and was easy to obtain from leaves of an abundant tree distributed all over the country. Semi-purified peroxidase (COP) was precipitated with acetone 65% (v.v -1 ) of the crude extract, obtaining the acetone powder. The COP optimum reaction pH values were between 5.0-7.0 and the temperatures between 5 and 45 °C, with a maximum activity at pH 6.0 and 35 °C. The enzyme was stable in temperatures below 50 °C and pH from 4.5 to 9.0 for up to 24 hours. The peroxidase was inactivated after 4 hours at 80 °C and after 3 minutes at 96 °C. This enzyme can possibly be used as a diagnostic reagent, biosensor and for other analytical methods in several fields of Sciences. Keywords: copaiba; enzyme; vegetal extract.Extração e caracterização parcial de peroxidase de folhas de Copaifera langsdorffii Desf. Extraction and partial characterization of peroxidase from Copaifera langsdorffii Desf. LeavesHermelinda Penha Freire MACIEL 1 , Cibele Marli Cação Paiva GOUVÊA 2 *, Gláucia Maria PASTORE 1 IntroduçãoAs peroxidases (E.C. 1.11.1.7, doador H 2 O 2 oxidorredutase) catalisam a redução do peróxido de hidrogênio ou outro peróxido orgânico, enquanto um doador de elétrons é oxidado. São abundantes em vários organismos incluindo as plantas superiores, com múltiplas isoformas sintetizadas e reguladas por estímulos diversos 8,22 . Nas últ...
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