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Previous issue date: 2011-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior, CAPES / The production of enzymes for application at different areas of food agroindustry presents promising future perspectives, due to several intrinsic properties regarded to the performance of the enzymes as natural and biodegradable compounds, responsible for achieving specific reactions with better quality. Cellulases have been the most employed enzymes on food industry, acting sinergically on the hydrolysis of the glucosidic links ?-1,4 from the molecules of cellulose, and being used on several applications in this sector as in vegetal oils extraction, fruit maceration and juice clarification. Based on this context, the present study aimed to produce, concentrate and partially characterize an enzymatic extract by a mutant fungus strain of Aspergillus niger. Production was performed by solid-state fermentation (SSF) in aerated columns, using humidified wheat bran with (NH4)2SO4 solution on 0,1N HCl as substrate and cellobiose as inducer. Cellulolytic extract was a blend of extracts from three different assays selected on previous studies as the best conditions for the enzymes caboxymethilcellulase, ?-glucosidase and filter paper cellulose (FPase). During the characterization of the enzymatic extract, besides cellulases activity, the presence of protease and other enzymes with similar action to cellulases as xylanase and poligalacturonase was evaluated. For enzymatic extract concentration, three different strategies were performed: ultrafiltration, using a stainless steel plates system through a 20 kDa molecular weight cut-off polyethersulphone membrane and 0,014 m2 area; precipitation with ammonium sulphate under 20%, 40 %, 60% and 80% saturation level and lyophiilization. The best results were achieved by the ultrafiltration process, partially purified sample and providing enzymatic activities recovery between 75% and 99%, except for FPase. SDS-PAGE analysis presented 15 visible protein bands on cellulolytic extract with molecular weights ranging from 13.3 to 104.6 kDa. Zymography test was applied for cellulases and correlate enzymes as well as to protease, however, just for the last one the conditions were considered appropriate, identifying bands on 88, 103 and 145 kDa. The effective performance of ?-glucosidase and xylanase over xylan and cellobiose hydrolysis was confirmed by thin layer chromatography. A central rotational statistical design 22 with 4 central points was used for evaluating optimal temperature and pH for carboxymethylcellulase and ?-glucosidase. The analysis of the results obtained for both enzymes demonstrated that all variables were significative at a 95% confidence level. Based on the conditions studied it can be concluded that optimal pH and temperature ranges for efficient and combined action of carboxymethylcellulase and ?-glucosidase are 3.7 to 5.5 and 60 to 65?C, respectively. / A produ??o de enzimas para uso em diferentes ?reas da agroind?stria de alimentos mostra perspectivas futuras promissoras, devido ?s v?rias caracter?sticas inerentes ? a??o das enzimas que s?o compostos naturais, biodegrad?veis e capazes de desempenhar rea??es espec?ficas com melhor qualidade. Entre as enzimas mais utilizadas pelo setor de alimentos est?o as celulases, um complexo de enzimas que atuam de forma sin?rgica sobre a hidr?lise das liga??es glicos?dicas ?-1,4 das mol?culas de celulose, e possuem v?rias aplica??es industriais neste setor, como na extra??o de ?leos vegetais, na macera??o de frutas e na clarifica??o de sucos. Dentro deste contexto, este trabalho teve como objetivo produzir, concentrar e caracterizar parcialmente um extrato celulol?tico obtido por linhagem f?ngica mutante de Aspergillus niger. A produ??o foi realizada por fermenta??o no estado s?lido (FES) em colunas aeradas, utilizando como substrato farelo de trigo triturado umidificado com solu??o de (NH4)2SO4 em HCl 0,1N e celobiose, como indutor. O extrato celulol?tico consistiu de uma mistura de extratos obtidos em 3 ensaios fermentativos diferentes, selecionados em trabalhos anteriores como as melhores condi??es para produ??o de cada uma das enzimascarboximetilcelulase (CMCase), ?-glicosidase e celulase em papel de filtro (FPase). Durante a caracteriza??o do extrato enzim?tico, al?m da atividade das celulases, tamb?m era avaliado o teor de prote?na, a presen?a de protease e de enzimas correlatas ? a??o de celulases como xilanase e poligalacturonase. Para concentra??o do extrato enzim?tico foram realizadas tr?s diferentes estrat?gias: ultrafiltra??o em um sistema de quadro e placas em a?o inox, utilizando uma membrana de polietersulfona com massa molar de corte de 20 kDa e ?rea de 0,014m2; precipita??o com sulfato de am?nio utilizando satura??es de 20%, 40%, 50%, 60%, 80% e liofiliza??o. O processo de ultrafiltra??o foi o que obteve o melhor resultado, purificando parcialmente a amostra e proporcionando uma recupera??o das atividades enzim?ticas entre 75% e 99% para todas as atividades avaliadas, exceto FPase. A an?lise eletrofor?tica em SDS-PAGE demonstrou a presen?a de 15 bandas vis?veis de prote?nas no extrato celulol?tico com pesos moleculares que compreendem uma faixa entre 13,3 e 104,6 kD. O teste de zimografia foi realizado para as celulases e enzimas correlatas, bem como para protease, no entanto somente para esta ?ltima, as condi??es testadas foram adequadas tornando-se poss?vel identificar bandas em 88, 103 e 145 kDa. A efetiva a??o das enzimas ?-glicosidase e xilanase na hidr?lise de celobiose e xilana, respectivamente,foi comprovada em cromatografia de camada fina. Al?m disso, a temperatura e pH ?timos de atua??o de carboximetilcelulase e ?-glicosidase foram determinados utilizando o delineamento composto central rotacional 22, com 4 pontos centrais. A an?lise dos resultados de ambas as enzimas demonstrou que as vari?veis eram significativas, a um n?vel de confian?a de 95%. Com base nas condi??es estudadas, concluiu-se que as faixas de pH e temperatura ?timos para a atua??o eficiente e conjunta de CMCase e ?-glicosidase est?o entre 3,7 a 5,5 e 60 a 65 ?C, respectivamente.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:jspui/1152 |
| Date | 28 February 2011 |
| Creators | Santos, Alex da Silva |
| Contributors | Damaso, Monica Caramez Triches, Couri, Sonia |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Ci?ncia e Tecnologia de Alimentos, UFRRJ, Brasil, Instituto de Tecnologia |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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