Imobilização de ficina em pó de sabugo de milho e obtenção de hidrolisados proteicos
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Data
2021-12-17
Autores
Davanso, Maísa [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Ficina (EC 3.4.22.3) é um grupo de endoproteases extraída a partir do látex de figueira (Ficus carica). Para a execução e conclusão desse trabalho, a ficina foi extraída, caracterizada, imobilizada e aplicada na hidrólise de extratos proteicos. Foram testados três diferentes métodos de concentração proteica, precipitação com acetona, liofilização e precipitação com sulfato de amônio. A atividade enzimática do extrato foi avaliada em diferentes valores de pH e temperaturas, usando dois substratos a caseína (macromolecular) e BAPNA (sintético), apresentando picos de atividade em 55°C com o pH 9,0 (84,69 ± 1,21 U mg-1) e 6,0 (0,202 ± 0,01 U mg-1), respectivamente. A enzima foi imobilizada em dois suportes, agarose e pó de sabugo de milho, com três ativações químicas distintas, imobilizando a enzima de forma covalente e iônica. Foi avaliada a manutenção de atividade dos derivados produzidos em diferentes condições de temperatura (45, 55 e 65ºC) e pH (5, 7 e 9). De todos os derivados obtidos por imobilização em pó de sabugo de milho (SM), SM-Glioxil-F e SM-Gluta-F foram selecionados considerando as estabilidades e atividades recuperadas, para serem utilizados na produção de hidrolisados proteicos a partir de extratos de grão-de-bico, ora-pro-nóbis e clara de ovo que também foram submetidos a hidrólise com a ficina na forma solúvel (livre) em um sistema de mistura operado em batelada. Os hidrolisados produzidos foram avaliados através da dosagem de proteínas, SDS-PAGE, atividade antioxidante pelo radical ABTS e atividade quelante de ferro. A partir da análise desses resultados, o derivado enzimático SM-Glioxil-F foi utilizado para hidrólise de extrato de grão de bico em reator de leito fixo operado de forma contínua por até 10h, com tempo espacial de 71 min. Foi possível realizar a imobilização da ficina em suporte alternativo e os derivados utilizados foram capazes de promover a hidrólise de proteínas presentes nos extratos avaliados, em ambas as formas de operação, indicando que a ficina imobilizada apresenta potencial de hidrólise de proteínas e produção de peptídeos bioativos.
Ficin (EC 3.4.22.3) is a group of endoproteases extracted from the latex of the fig tree (Ficus carica). For the execution and conclusion of this work, ficin was extracted, characterized, immobilized and applied in the hydrolysis of protein extracts. Three different methods of protein concentration were tested, acetone precipitation, lyophilization and ammonium sulfate precipitation. The enzymatic activity of the extract was evaluated at different pH and temperatures, using two substrates, casein (macromolecular) and BAPNA (synthetic), showing activity peaks at 55°C at pH 9.0 (84.69 ± 1.21 U mg-1) and 6.0 (0.202 ± 0.01 U mg-1), respectively. The enzyme was immobilized on two supports, agarose and corncob powder, with three different chemical activations, immobilizing the enzyme in a covalent and ionic way. The maintenance of activity of the derivatives produced was evaluated under different conditions of temperature (45, 55 and 65ºC) and pH (5, 7 and 9). From all the derivatives obtained by immobilization in corncob powder (SM), SM-Glyoxyl-F and SM-Gluta-F were selected, considering the stability and activities recovered, to be used in the production of protein hydrolysates from extracts of chickpea, ora-pro-nóbis and egg white that were also subjected to hydrolysis with ficin in soluble form (free) in a mixed system batch-operated. The hydrolysates produced were evaluated through protein dosage, SDS-PAGE, antioxidant activity by ABTS radical and iron chelating activity. From the analysis of these results, the enzymatic derivative SM-Glyoxyl-F was used for hydrolysis of chickpea extract in a fixed-bed reactor operated continuously for up to 10 hours, with residence time of 71 min. It was possible to execute the immobilization of ficin in an alternative support and the derivatives used were able to promote the hydrolysis of proteins present in the extracts evaluated, in both forms of operation, indicating that the immobilized ficin has the potential for protein hydrolysis and production of bioactive peptides.
Ficin (EC 3.4.22.3) is a group of endoproteases extracted from the latex of the fig tree (Ficus carica). For the execution and conclusion of this work, ficin was extracted, characterized, immobilized and applied in the hydrolysis of protein extracts. Three different methods of protein concentration were tested, acetone precipitation, lyophilization and ammonium sulfate precipitation. The enzymatic activity of the extract was evaluated at different pH and temperatures, using two substrates, casein (macromolecular) and BAPNA (synthetic), showing activity peaks at 55°C at pH 9.0 (84.69 ± 1.21 U mg-1) and 6.0 (0.202 ± 0.01 U mg-1), respectively. The enzyme was immobilized on two supports, agarose and corncob powder, with three different chemical activations, immobilizing the enzyme in a covalent and ionic way. The maintenance of activity of the derivatives produced was evaluated under different conditions of temperature (45, 55 and 65ºC) and pH (5, 7 and 9). From all the derivatives obtained by immobilization in corncob powder (SM), SM-Glyoxyl-F and SM-Gluta-F were selected, considering the stability and activities recovered, to be used in the production of protein hydrolysates from extracts of chickpea, ora-pro-nóbis and egg white that were also subjected to hydrolysis with ficin in soluble form (free) in a mixed system batch-operated. The hydrolysates produced were evaluated through protein dosage, SDS-PAGE, antioxidant activity by ABTS radical and iron chelating activity. From the analysis of these results, the enzymatic derivative SM-Glyoxyl-F was used for hydrolysis of chickpea extract in a fixed-bed reactor operated continuously for up to 10 hours, with residence time of 71 min. It was possible to execute the immobilization of ficin in an alternative support and the derivatives used were able to promote the hydrolysis of proteins present in the extracts evaluated, in both forms of operation, indicating that the immobilized ficin has the potential for protein hydrolysis and production of bioactive peptides.
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Palavras-chave
Enzimas proteolíticas, Enzimas imobilizadas, Peptídeos, Biorreatores, Hidrolisados de proteína