Orientadores: Francisco Maugeri Filho, Renato de Azevedo Moreira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-21T19:53:48Z (GMT). No. of bitstreams: 1 Oliveira_Gilcenara_M.pdf: 1974223 bytes, checksum: 647b7e3918cb207ca7de16931074a977 (MD5) Previous issue date: 1996 / 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 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. / Mestrado / Mestre em Engenharia de Alimentos

Orientador: Hélia Harumi Sato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T13:30:04Z (GMT). No. of bitstreams: 1 Bedani_CarolinaCasagrande_M.pdf: 909079 bytes, checksum: 17274331f891e68042559fd5f2939310 (MD5) Previous issue date: 2010 / Resumo: As ß-glicosidases (ß-D-glucosídeo glucohidrolase, EC 3.2.1.21) catalisam a hidrólise de dissacarídeos e glicosídeos conjugados a partir da extremidade não redutora. A enzima ß-glicosidase apresenta inúmeras aplicações na indústria de alimentos e farmacêutica, atuando na hidrólise da celobiose em glicose, no processo de conversão da celulose em glicose em combinação com outras enzimas celulolíticas; na liberação de compostos de aroma em sucos de frutas e vinho; na hidrólise de compostos cianogênicos, presentes em plantas e resíduos agroindustriais e na transformação de isoflavonas glicosiladas de soja em isoflavonas agliconas, que podem ser utilizadas para reposição hormonal. Entre as linhagens de Aspergillus sp., Aspergillus oryzae, Mucor miehei, Penicillium sp. E Trichoderma viride a primeira apresentou a maior produção de ß-glicosidase. Na fermentação da linhagem Aspergillus sp. em meio semi sólido composto de farelo de trigo (Natur¿s): casca de maracujá: água destilada, na proporção 1:1:0,4; foram obtidos, respectivamente, 25,75 U/g, 45,88 U/g e 9,29 U/g de ß-glicosidase utilizando-se os substratos p-NPG, celobiose e amigdalina, após 30 minutos a 50°C. A ß-glicosidase bruta de Aspergillus oryzae apresentou atividade ótima na faixa de pH 5,0 - 5,5 e uma fração com atividade em pH 7,0. A enzima apresentou temperatura ótima de atividade na faixa de 45 - 50°C e mostrou-se estável a 45°C após 30 minutos de incubação em pH 5,0 e na faixa de pH 5,0 - 7,0 após 2 h a 47°C. A ß-glicosidase bruta de Penicillium sp. apresentou atividade ótima em pH 4,0 a 50°C e a 60ºC, evidenciando a presença de iso enzimas. Mostrou-se estável a 55°C após 30 minutos de incubação em pH 5,0 e apr esentou maior estabilidade na faixa de pH 3,0 - 7,0 após 2 h a 60°C. A ß-glicosidase bruta de Trichoderma viride mostrou atividade ótima em pH 5,0 - 5,5 e 45ºC e mostrou-se estável a 45°C após 30 minutos de incubação em pH 5,0 e na faixa de pH 5,0 - 7,0 após 2 h a 45°C . A ß-glicosidase bruta de Aspergillus sp. apresentou atividade ótima em pH 4,5 e 60°C, estabilidade a 60°C após 30 minutos de incubação em pH 4,5 e na faixa de pH 3,0 - 8,5 após 2 h de incubação a 60°C. A ß-glicosidase de Aspergillus sp. apresentou valores de Km de 3,41 mM e Vmax de 72,46 µmol p nitrofenol/mL.min para o substrato p-NPG. Após fracionamento com sulfato de amônio 80% e cromatografia em coluna DEAE-celulose, a ß-glicosidase de Aspergillus sp. foi parcialmente purificada cerca de 38 vezes com recuperação de 2,5%. Utilizando-se planejamento experimental a ß-glicosidase parcialmente purificada apresentou atividade ótima em pH 4,5 e 60°C e estabilidade na faixa de pH de 3,6 - 5,0 e temperatura de 30 - 50°C após 2 h de incubação. A ß-glicosidase parcialmente purificada de Aspergillus sp. apresentou atividade de hidrólise da celobiose, p-NPG e amigdalina / Abstract: The ß-glucosidases (ß-D-glucoside glucohydrolase, EC 3.2.1.21) catalyze the hydrolysis of disaccharides and conjugate glycosides from the non-reducing end. The ß-glucosidases have several applications in the food and pharmaceutical industries such as the hydrolysis of cellobiose into glucose, the conversion of cellulose into glucose together with other cellulolytic enzymes; the release of flavour compounds into fruit juices and wine; the hydrolysis of cyanogenic compounds present in plants and agro-industrial residues and the conversion of isoflavone glycosides into the soy isoflavone aglycone, which can be used for hormone replacement. Of the strains Aspergillus sp., Aspergillus oryzae, Mucor miehei, Penicillium sp. and Trichoderma viride, the first showed the greater production of ß-glucosidase. In the fermentation by the Aspergillus sp. strain in a semi-solid medium containing wheat bran: passion fruit peel: distilled water, in a ratio of 1:1:0.4; 25.75 U/g, 45.88 U/g and 9.29 U/g of ß-glucosidase were obtained, respectively, using p-NPG, amygdalin and cellobiose as the substrate, after 30 minutes at 50°C. The crude ß-glucosidase obtained from Aspergillus oryzae showed a fraction with optimal activity at pH 5.0 ¿ 5.5, and another fraction with activity at pH 7.0. The enzymes showed optimum activity in the temperature range from 45 to 50°C and were stable after 30 minutes of incubation at 45°C and pH 5.0, and also after 2 hours at 47°C in the pH range from 5.0 to 7.0. The crude ß-glucosidase from Penicillium sp. showed optimal activity at pH 4.0 and isoenzymes with optimal activity at 50°C and 60ºC. The enzymes showed stability after 30 minutes of incubation at 55°C and pH 5.0, and were stable in the pH range from pH 3.0 to 7.0 after 2 hours at 60°C. The crude ß-glucosidase from Trichoderma viride showed optimal activity in the pH range from 5.0 to 5.5 at 45°C; stability after 30 minutes of incubation at 45°C and pH 5.0 and stabil ity in the pH range from 5.0 to 7.0 after 2 hours at 45°C. The crude ß-glucosidase from Aspergillus sp. Showed optimal activity at pH 4.5 and 60°C; stability afte r 30 minutes of incubation at 60°C and pH 4.5, and stability in the pH range from 3.0 to 8.5 after 2 hours of incubation at 60°C. The ß-glucosidase from Aspergillus sp. showed a Km value of 3.41 mM and Vmax of 72.46 mmol p-nitrophenyl/mL.min with p-NPG as the substrate. After fractionation with 80% ammonium sulphate and chromatography on a DEAEcellulose column, the ß-glucosidase from Aspergillus sp. was purified 38 fold with a recovery of 2.5%. Using an experimental design the partially purified ß-glucosidase showed optimal activity at pH 4.5 and 60°C, and sta bility in the pH range from 3.6 to 5.0 and temperature range from 30°- 50°C, after 2 hours of incubation. The partially purified ß-glucosidase from Aspergillus sp. showed hydrolytic activity with respect to cellobiose, p-NPG and amygdalin / Mestrado / Mestre em Ciência de Alimentos