Imobilização da enzima β-Galactosidase de origem microbiana em diferentes suportes
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2018-08-07
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Souza, Claucia Fernanda Volken De
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A β-galactosidase é empregada pelas indústrias de laticínios no desenvolvimento de produtos lácteos com baixos teores de lactose. Entretanto, estes biocatalisadores possuem algumas limitações que dificultam sua aplicação industrial. Estas podem ser superadas através da imobilização enzimática, pois permite a reutilização da enzima e um maior controle reacional. O Immobead 150 é um suporte comercial formado de polímeros de metacrilato que contém grupamentos epóxi na sua superfície permitindo a ligação com grupos funcionais. Entre os suportes de baixo custo que podem ser utilizados na imobilização enzimática está o colágeno, uma proteína pouco solúvel na sua forma natural, organizada em fibras resistentes. A celulose
microcristalina é outro material promissor para este processo, pois além de ser abundante e barata, possui excelentes propriedades físicas. O objetivo geral desse trabalho foi estudar o processo de imobilização das β-galactosidases de Aspergillus oryzae e Kluyveromyces lactis utilizando diferentes suportes. As matrizes utilizadas no processo foram o colágeno em pó, a celulose microcristalina e o Immobead. Estudos de cargas foram realizados a fim de determinar a melhor proporção enzima-suporte, além de diferentes tratamentos para funcionalizar essas matrizes. Os derivados obtidos que apresentaram melhores rendimentos e eficiências foram avaliados quanto a sua estabilidade térmica e ao armazenamento, condições operacionais de pH
e temperatura e ciclos de reuso. Além disso, seus parâmetros cinéticos foram comparados com os da enzima livre. As cargas da enzima de A. oryzae na imobilização em Immobead, colágeno e celulose foram de 100, 1000 e 100 mg de proteína/g de suporte, respectivamente. Já para a β-galactosidase de K. lactis a carga de 100 foi a melhor nos três suportes. Em relação aos parâmetros cinéticos e as condições ótimas de catálise, diferentes comportamentos foram observados entre as diferentes enzimas e suportes. A aplicação em batelada da enzima imobilizada de A. oryzae nas soluções de substrato com pHs ácidos (solução de lactose e
permeado) pode ser realizada por aproximadamente 35, 60 e 30 vezes quando imobilizada em Immobead, colágeno e celulose, respectivamente. A β-galactosidase de K. lactis por sua vez, resistiu por 35 ciclos de reuso, em soro de queijo e/ou leite, quando imobilizada em Immobead e 30 em colágeno e celulose. Com base nos resultados obtidos nesse trabalho, verificou-se que o suporte comercial Immobead, e outros dois materiais, colágeno e celulose, demonstram potencial como materiais para imobilização de β-galactosidases microbianas.
β-galactosidase is used by the dairy industries in the development of dairy products with low lactose content. However, these biocatalysts have some limitations that hinder their industrial application. These can be overcome through enzymatic immobilization, as it allows the reuse of the enzyme and a greater reaction control. Immobead 150 is a commercial support formed of methacrylate polymers containing epoxy groups on its surface allowing attachment to functional groups. Among the low-cost supports that can be used in enzymatic immobilization is collagen, a poorly soluble protein in its natural form, organized into resistant fibers. Microcrystalline cellulose is another promising material for this process, as it is abundant and inexpensive and has excellent physical properties. The general objective of this project was to study the immobilization process of the β-galactosidases of Aspergillus oryzae and Kluyveromyces lactis using different supports. The matrices used in the process were collagen powder, microcrystalline cellulose and Immobead. Load studies were performed in order to determine the best enzyme-support ratio, as well as different treatments to functionalize these matrices. The obtained derivatives that presented better yields and efficiencies were evaluated for their thermal and storage stability, pH and temperature operating conditions and reuse cycles. In addition, their kinetic parameters were compared with those of the free enzyme. The protein loads of the immobilization of A. oryzae on Immobead, collagen and cellulose were 100, 1000 and 100 mg protein/g support, respectively. For K. lactis β-galactosidase, the loading of 100 was the best in all three supports. Regarding the kinetic parameters and the optimal conditions of catalysis, different behaviors were observed between the different enzymes and supports. Batch application of the A. oryzae immobilized enzyme in substrate solutions with acid pHs (lactose solution and permeate) could be carried out for approximately 35, 60 and 30 times when immobilized on Immobead, collagen and cellulose, respectively. The β-galactosidase of K. lactis, in turn, resisted for 35 cycles of reuse in cheese and/or milk, when immobilized on Immobead and 30 on collagen and cellulose. Based on the results obtained in this work, the commercial support Immobead, and other two materials, collagen and cellulose, have shown potential as materials for the immobilization of microbial β-galactosidases.
β-galactosidase is used by the dairy industries in the development of dairy products with low lactose content. However, these biocatalysts have some limitations that hinder their industrial application. These can be overcome through enzymatic immobilization, as it allows the reuse of the enzyme and a greater reaction control. Immobead 150 is a commercial support formed of methacrylate polymers containing epoxy groups on its surface allowing attachment to functional groups. Among the low-cost supports that can be used in enzymatic immobilization is collagen, a poorly soluble protein in its natural form, organized into resistant fibers. Microcrystalline cellulose is another promising material for this process, as it is abundant and inexpensive and has excellent physical properties. The general objective of this project was to study the immobilization process of the β-galactosidases of Aspergillus oryzae and Kluyveromyces lactis using different supports. The matrices used in the process were collagen powder, microcrystalline cellulose and Immobead. Load studies were performed in order to determine the best enzyme-support ratio, as well as different treatments to functionalize these matrices. The obtained derivatives that presented better yields and efficiencies were evaluated for their thermal and storage stability, pH and temperature operating conditions and reuse cycles. In addition, their kinetic parameters were compared with those of the free enzyme. The protein loads of the immobilization of A. oryzae on Immobead, collagen and cellulose were 100, 1000 and 100 mg protein/g support, respectively. For K. lactis β-galactosidase, the loading of 100 was the best in all three supports. Regarding the kinetic parameters and the optimal conditions of catalysis, different behaviors were observed between the different enzymes and supports. Batch application of the A. oryzae immobilized enzyme in substrate solutions with acid pHs (lactose solution and permeate) could be carried out for approximately 35, 60 and 30 times when immobilized on Immobead, collagen and cellulose, respectively. The β-galactosidase of K. lactis, in turn, resisted for 35 cycles of reuse in cheese and/or milk, when immobilized on Immobead and 30 on collagen and cellulose. Based on the results obtained in this work, the commercial support Immobead, and other two materials, collagen and cellulose, have shown potential as materials for the immobilization of microbial β-galactosidases.
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Palavras-chave
imobilização; β-galactosidase; colágeno; celulose; immobead
Citação
GENNARI, Adriano. Imobilização da enzima β-Galactosidase de origem microbiana em diferentes suportes. 2018. Dissertação (Mestrado) – Curso de Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, 26 fev. 2018. Disponível em: http://hdl.handle.net/10737/2162.