Orientadores: Francisco Maugeri Filho, Renato de Azevedo Moreira

Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos

Resumo: A celulose é um material amplamente encontrado na natureza. A hidrólise deste material é resultado de um complexo enzimático que age em sinergismo, denominado celulase. Neste complexo a enzima ß-glicosidase (EC 3.2.1.21) é responsável pelo passo final da hidrólise para formação do produto...

Resumo: A celulose é um material amplamente encontrado na natureza. A hidrólise deste material é resultado de um complexo enzimático que age em sinergismo, denominado celulase. Neste complexo a enzima ß-glicosidase (EC 3.2.1.21) é responsável pelo passo final da hidrólise para formação do produto glicose. Esta enzima tem várias aplicações nos diversos setores industriais, atuando principalmente de forma suplementar, ou seja, é acrescida ao sistema enzimático assim aumentando a cinética da hidrólise. Na indústria alimentícia pode-se destacar a extração e clarificação de sucos, a hidrólise de vegetais para preparação de pures, para alimentação infantil ou geriátrica. Este trabalho se propõe a determinar os parâmetros envolvidos no processo de separação e purificação da ß-glicosidase, a partir de um complexo enzimático, Celulase Tr (Cellumax), para então aplicá-los ao sistema CARE (Continuous Affinity Recycle Extraction). No processo de purificação foram utilizadas técnicas cromatográficas. Iniciou-se com cromatografia de filtração em gel, seguida de cromatografia de troca iônica. Obtiveram-se melhores resultados com o uso de CM-Sephadex C-50. As frações correspondentes aos picos obtidos nesta cromatografia foram concentradas e aplicadas em coluna de Superose 12 R acoplada ao sistema de FPLC (Faster Performance Liquid Chromatography) a fim de confirmar a pureza da enzima ß-glicosidase. O material obtido foi submetido a análise de eletroforese em gel de poliacrilamida/dodecil sulfeto de Sódio (PAGE/SDS), garantindo desta forma que a enzima estava realmente pura. Os resultados obtidos mostram que é possível a aplicação do processo CARE para separar e purificar a ß-glicosidase. Como continuidade a este trabalho, sugere-se a determinação das constantes cinéticas envolvidas na adsorção-dessorção da resina usada no sistema CARE

Abstract: Cellulose is a very common natural substance. The hydrolysis of this material is caused by an enzyme complex, acting synergistically, known as cellulase. In such a complex, the enzyme ß-glucosidase (EC 3.2.1.21) is responsible for the final step of hydrolysis to form glucose. This enzyme...

Abstract: Cellulose is a very common natural substance. The hydrolysis of this material is caused by an enzyme complex, acting synergistically, known as cellulase. In such a complex, the enzyme ß-glucosidase (EC 3.2.1.21) is responsible for the final step of hydrolysis to form glucose. This enzyme has numerous industrial applications. Particularly important among applications in the food industry we may include the lightening of juices and the hydrolysis of vegetables for production of purees, for infants and elderly. This work was carried out to try to find the parameters involved in the separation and purification of , ß-glucosidase, from an enzymatic complex called Cellulase Tr (Cellumax), and then, to apply them in the CARE (Continuous Affinity Recycle Extraction) system. The purification process began with gel filtration chromatography and followed by ion-exchange chromatography. The best results were obtained using CM-Sephadex C-50. The peak fractions obtained with ion-exchange cromatography were concentrated and applied to a Superose 12 R column in a FPLC system to confirm the purity of the , Abstract: Cellulose is a very common natural substance. The hydrolysis of this material is caused by an enzyme complex, acting synergistically, known as cellulase. In such a complex, the enzyme Abstract: Cellulose is a very common natural substance. The hydrolysis of this material is caused by an enzyme complex, acting synergistically, known as cellulase. In such a complex, the enzyme ß-glucosidase (EC 3.2.1.21) is responsible for the final step of hydrolysis to form glucose. This enzyme has numerous industrial applications. Particularly important among applications in the food industry we may include the lightening of juices and the hydrolysis of vegetables for production of purees, for infants and elderly. This work was carried out to try to find the parameters involved in the separation and purification of , ß-glucosidase, from an enzymatic complex called Cellulase Tr (Cellumax), and then, to apply them in the CARE (Continuous Affinity Recycle Extraction) system. The purification process began with gel filtration chromatography and followed by ion-exchange chromatography. The best results were obtained using CM-Sephadex C-50. The peak fractions obtained with ion-exchange cromatography were concentrated and applied to a Superose 12 R column in a FPLC system to confirm the purity of the , ß-glucosidase. For the material obtained with Superose, a PAGE/SDS analysis was undertaken to garantee the purity of the enzime. The results obtained make possible the application of CARE process to the separation and purification of ß-glucosidase. In addition, from the results it is possible to calculate the kinetic constants involved in the resin adsorption-desorption used in the CARE system. ß-glucosidase (EC 3.2.1.21) is responsible for the final step of hydrolysis to form glucose. This enzyme has numerous industrial applications. Particularly important among applications in the food industry we may include the lightening of juices and the hydrolysis of vegetables for production of purees, for infants and elderly. This work was carried out to try to find the parameters involved in the separation and purification of , ß-glucosidase, from an enzymatic complex called Cellulase Tr (Cellumax), and then, to apply them in the CARE (Continuous Affinity Recycle Extraction) system. The purification process began with gel filtration chromatography and followed by ion-exchange chromatography. The best results were obtained using CM-Sephadex C-50. The peak fractions obtained with ion-exchange cromatography were concentrated and applied to a Superose 12 R column in a FPLC system to confirm the purity of the , ß-glucosidase. For the material obtained with Superose, a PAGE/SDS analysis was undertaken to garantee the purity of the enzime. The results obtained make possible the application of CARE process to the separation and purification of ?-glucosidase. In addition, from the results it is possible to calculate the kinetic constants involved in the resin adsorption-desorption used in the CARE system.-glucosidase. For the material obtained with Superose, a PAGE/SDS analysis was undertaken to garantee the purity of the enzime. The results obtained make possible the application of CARE process to the separation and purification of ß-glucosidase. In addition, from the results it is possible to calculate the kinetic constants involved in the resin adsorption-desorption used in the CARE system.