Desenvolvimento e caracterização de suportes para imobilização orientada da β-Galactosidade recombinante com cauda de histidina
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2024-10
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Souza, Claucia Fernanda Volken de
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Lehn , Daniel Neutzling
Contini , Veronica
Klein, Manuela Poletto
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Abstract
A β-galactosidase (β-Gal) é amplamente usada na produção de alimentos com baixo teor de lactose, mas seu uso em larga escala é limitado pelos altos custos de produção. Uma alternativa para reduzir os custos é o uso de enzimas recombinantes, que aumentam a atividade e a adaptabilidade enzimática. A imobilização orientada com marcadores de afinidade facilita a separação e reutilização das enzimas em vários ciclos. Suportes magnéticos, como nanopartículas, são utilizados para essa imobilização, mas as partículas de ferro oxidam facilmente, tornando o cobalto e o níquel melhores opções. A tecnologia core-shell pode aumentar a resistência desses suportes. Este trabalho visa desenvolver um sistema biotecnológico para imobilização de β-galactosidase recombinante (HisβGal) de Kluyveromyces sp. Foram sintetizados três tipos de suportes: nanopartículas de cobalto, nanopartículas de níquel e core-shell de cobalto, que foram empregados para imobilizar a HisβGal de forma eficiente. Para os suportes de nanopartículas magnéticas (NMP) de cobalto e de níquel os maiores valores de eficiência de imobilização foram obtidos para uma carga de 30 U/g suporte. Os resultados experimentais mostraram que as nanopartículas de cobalto foram mais eficazes para a imobilização orientada da HisβGal, com base em análises de difração de raios X, termogravimetria, espectroscopia de energia dispersiva e infravermelho. A imobilização em nanopartículas de níquel e cobalto alterou a temperatura ótima da β galactosidase para 40 °C e 50 °C, respectivamente, e ampliou a faixa de pH para atividade enzimática. Os biocatalisadores puderam ser reutilizados por 25 ciclos. A imobilização em nanopartículas de cobalto reduziu o efeito inibitório da galactose e aumentou a afinidade da enzima pela lactose no leite. Para as cápsulas core-shell de cobalto, a imobilização alcançou 85,5% de rendimento e 83,17% de eficiência com uma carga de 2 U/cápsula. Após a imobilização, a temperatura ótima da β-galactosidase caiu de 55 °C para 50 °C, mantendo o pH estável em 7,0. A enzima imobilizada mostrou maior afinidade pelos substratos comparada à forma livre. Em reatores de coluna de leito fixo, foi possível hidrolisar 50% da lactose em leite desnatado em cerca de 10 horas de operação. Este estudo destaca um suporte inovador de imobilização orientada para β-galactosidase recombinante com His-tag, usando um sistema magnético sem ferro. A estratégia permite a reutilização da enzima, facilita a separação e melhora as propriedades catalíticas.
β-Galactosidase (β-Gal) is widely used in the production of low-lactose foods, but its large scale use is limited by high production costs. An alternative to reduce costs is the use of recombinant enzymes, which increase enzymatic activity and adaptability. Oriented immobilization with affinity markers facilitates the separation and reuse of enzymes in multiple cycles. Magnetic supports, such as nanoparticles, are used for this immobilization, but iron particles easily oxidize, making cobalt and nickel better options. Core-shell technology can enhance the resistance of these supports. This study aims to develop a biotechnological system for immobilizing recombinant β-galactosidase (HisβGal) from Kluyveromyces sp. Three types of supports were synthesized: cobalt nanoparticles, nickel nanoparticles, and cobalt core-shell, which were used to efficiently immobilize HisβGal. For the cobalt and nickel magnetic nanoparticle (MNP) supports, the highest immobilization efficiency values were obtained for a load of 30 U/g support. Experimental results showed that cobalt nanoparticles were more effective for oriented immobilization of HisβGal, based on X-ray diffraction, thermogravimetry, energy-dispersive spectroscopy, and infrared analyses. Immobilization on nickel and cobalt nanoparticles altered the optimal temperature of β-galactosidase to 40 °C and 50 °C, respectively, and expanded the pH range for enzymatic activity. The biocatalysts could be reused for 25 cycles. Immobilization on cobalt nanoparticles reduced the inhibitory effect of galactose and increased the enzyme's affinity for lactose in milk. For cobalt core-shell capsules, immobilization achieved 85.5% yield and 83.17% efficiency with a load of 2 U/capsule. After immobilization, the optimal temperature of β-galactosidase decreased from 55 °C to 50 °C, maintaining the pH stable at 7.0. The immobilized enzyme showed greater substrate affinity compared to the free form. In fixed-bed column reactors, it was possible to hydrolyze 50% of the lactose in skim milk in about 10 hours of operation. This study highlights an innovative oriented immobilization support for recombinant β-galactosidase with His-tag, using a non-iron magnetic system. The strategy enables enzyme reuse, facilitates separation, and improves catalytic properties.
β-Galactosidase (β-Gal) is widely used in the production of low-lactose foods, but its large scale use is limited by high production costs. An alternative to reduce costs is the use of recombinant enzymes, which increase enzymatic activity and adaptability. Oriented immobilization with affinity markers facilitates the separation and reuse of enzymes in multiple cycles. Magnetic supports, such as nanoparticles, are used for this immobilization, but iron particles easily oxidize, making cobalt and nickel better options. Core-shell technology can enhance the resistance of these supports. This study aims to develop a biotechnological system for immobilizing recombinant β-galactosidase (HisβGal) from Kluyveromyces sp. Three types of supports were synthesized: cobalt nanoparticles, nickel nanoparticles, and cobalt core-shell, which were used to efficiently immobilize HisβGal. For the cobalt and nickel magnetic nanoparticle (MNP) supports, the highest immobilization efficiency values were obtained for a load of 30 U/g support. Experimental results showed that cobalt nanoparticles were more effective for oriented immobilization of HisβGal, based on X-ray diffraction, thermogravimetry, energy-dispersive spectroscopy, and infrared analyses. Immobilization on nickel and cobalt nanoparticles altered the optimal temperature of β-galactosidase to 40 °C and 50 °C, respectively, and expanded the pH range for enzymatic activity. The biocatalysts could be reused for 25 cycles. Immobilization on cobalt nanoparticles reduced the inhibitory effect of galactose and increased the enzyme's affinity for lactose in milk. For cobalt core-shell capsules, immobilization achieved 85.5% yield and 83.17% efficiency with a load of 2 U/capsule. After immobilization, the optimal temperature of β-galactosidase decreased from 55 °C to 50 °C, maintaining the pH stable at 7.0. The immobilized enzyme showed greater substrate affinity compared to the free form. In fixed-bed column reactors, it was possible to hydrolyze 50% of the lactose in skim milk in about 10 hours of operation. This study highlights an innovative oriented immobilization support for recombinant β-galactosidase with His-tag, using a non-iron magnetic system. The strategy enables enzyme reuse, facilitates separation, and improves catalytic properties.
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Keywords
Enzima recombinante;; His-tag; β-galactosidase; Partículas magnéticas sem ferro; Core-shell; Reator de coluna; Recombinant enzyme; Non-iron magnetic particles; column reactor
Citation
PALUDO, Graziela. Desenvolvimento e caracterização de suportes para imobilização orientada da β-Galactosidade recombinante com cauda de histidina. 2024. Tese (Doutorado) – Curso de Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, 29 out. 2024. Disponível em: http://hdl.handle.net/10737/4906.