Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFMS |
Texto Completo: | https://repositorio.ufms.br/handle/123456789/3798 |
Resumo: | β-glycosidases are enzymes that are part of the cellulolytic complex with the ability to act on lignocellulosic materials, promoting their hydrolysis. These enzymes are produced by microorganisms, mainly filamentous fungi. They have varied applications such as in the pulp and paper industry, food, animal feed and in the production of second generation ethanol. The present work involves the purification, biochemical characteristics and immobilization of a β-glucosidase resistant to the thermophilic fungus Rasamsonia composticola. The enzyme was purified using two chromatographic steps, including DEAE-Fractogel and hydrophobic Phenyl-Sepharose ion exchange. It was purified 19 times with a final yield of 14%. A purified β-glucosidase having an optimum temperature of 70 °C, remaining stable at a temperature of 65 to 70 °C for up to 8 hours, pH 5.0, and a molecular mass of approximately 45 kDa. The enzyme was not stimulated by most of the chemical compounds tested, however it was slightly stimulated by the detergents saponin and triton X-100, in addition to the chelators EDTA and EGTA and the reducing agent Temed. The enzyme has activity on the synthetic substrates pNP-Glc and pNP-Xyl, and is shown to be glucose tolerant and stimulated by xylose (1-100 mM). The estimated kinetic parameters for a β-glucosidase were Km and Vmax of 2.3 mM and 0.0221 µmol / min / mg for pNP-Glc. The catalytic efficiency (Kcat / Km) of the enzyme was 7.7x10-11. For pNP-Glc + glucose and pNP-Glc + xylose, the enzyme presented Km and Vmax of 2.33 mM and 0.0271 µmol / min / mg and 2.02 mM and 0.0255 µmol / min / mg, respectively . The enzyme was immobilized in glyoxyl agarose about 40% and 56% in the presence of glucose for 60 minutes. It demonstrated optimal activity at pH 5.0 and 75 °C. The immobilized enzyme is stable at temperatures from 75 to 80 °C during 60 minutes of reaction. The results demonstrate that the fungus Rasamsonia composticola can be a candidate to contribute to the knowledge of the properties of a β-glucosidase with great potential for biotechnological application. |
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2021-06-28T12:34:06Z2021-09-30T19:57:39Z2021https://repositorio.ufms.br/handle/123456789/3798β-glycosidases are enzymes that are part of the cellulolytic complex with the ability to act on lignocellulosic materials, promoting their hydrolysis. These enzymes are produced by microorganisms, mainly filamentous fungi. They have varied applications such as in the pulp and paper industry, food, animal feed and in the production of second generation ethanol. The present work involves the purification, biochemical characteristics and immobilization of a β-glucosidase resistant to the thermophilic fungus Rasamsonia composticola. The enzyme was purified using two chromatographic steps, including DEAE-Fractogel and hydrophobic Phenyl-Sepharose ion exchange. It was purified 19 times with a final yield of 14%. A purified β-glucosidase having an optimum temperature of 70 °C, remaining stable at a temperature of 65 to 70 °C for up to 8 hours, pH 5.0, and a molecular mass of approximately 45 kDa. The enzyme was not stimulated by most of the chemical compounds tested, however it was slightly stimulated by the detergents saponin and triton X-100, in addition to the chelators EDTA and EGTA and the reducing agent Temed. The enzyme has activity on the synthetic substrates pNP-Glc and pNP-Xyl, and is shown to be glucose tolerant and stimulated by xylose (1-100 mM). The estimated kinetic parameters for a β-glucosidase were Km and Vmax of 2.3 mM and 0.0221 µmol / min / mg for pNP-Glc. The catalytic efficiency (Kcat / Km) of the enzyme was 7.7x10-11. For pNP-Glc + glucose and pNP-Glc + xylose, the enzyme presented Km and Vmax of 2.33 mM and 0.0271 µmol / min / mg and 2.02 mM and 0.0255 µmol / min / mg, respectively . The enzyme was immobilized in glyoxyl agarose about 40% and 56% in the presence of glucose for 60 minutes. It demonstrated optimal activity at pH 5.0 and 75 °C. The immobilized enzyme is stable at temperatures from 75 to 80 °C during 60 minutes of reaction. The results demonstrate that the fungus Rasamsonia composticola can be a candidate to contribute to the knowledge of the properties of a β-glucosidase with great potential for biotechnological application.As β-glucosidases são enzimas que fazem parte do complexo celulolítico com a capacidade de atuar sobre materiais lignocelulósicos promovendo sua hidrólise. Essas enzimas são produzidas por microrganismos, principalmente os fungos filamentosos. Possuem variadas aplicações como na indústria de papel e celulose, alimentícia, ração animal e na produção de etanol de segunda geração. O presente trabalho descreve a purificação, características bioquímicas e imobilização de uma β-glucosidase produzida pelo fungo termofílico Rasamsonia composticola. A enzima foi purificada utilizando duas etapas cromatográficas, incluindo a troca iônica DEAE-Fractogel e hidrofóbica Phenyl-Sepharose. Foi purificada 19 vezes com rendimento final de 14%. A β-glucosidase purificada apresentou temperatura ótima de 70°C, permanecendo estável em temperaturas de 65 a 70°C durante até 8 horas, pH 5,0, e uma massa molecular de aproximadamente 45 kDa. A enzima não foi estimulada pela maioria dos compostos químicos testados, entretanto foi levemente estimulada pelos detergentes saponina e triton X-100, além dos quelantes EDTA e EGTA e o agente redutor Temed. A enzima apresentou atividade sobre os substratos sintéticos pNP-Glc e pNP-Xyl, e demonstrou ser tolerante a glicose e estimulada por xilose (1-100 mM). Os parâmetros cinéticos estimados para a β-glucosidase foram de Km e Vmax de 2,3 mM e 0,0221 µmol/min/mg para pNP-Glc. A eficiência catalítica (Kcat/Km) da enzima foi de 7,7x10-11. Para pNP-Glc + glicose e pNP-Glc + xilose a enzima apresentou Km e Vmax de 2,33 mM e 0,0271 µmol/min/mg e 2,02 mM e 0,0255 µmol/min/mg, respectivamente. A enzima foi imobilizada em glioxil agarose cerca de 40% e 56% na presença de glicose durante 60 minutos. Demonstrou atividade ótima em pH 5,0 e 75°C. A enzima imobilizada apresentou estabilidade nas temperaturas de 75 a 80°C durante 60 minutos de reação. Os resultados demonstram que o fungo Rasamsonia composticola pode ser um candidato a contribuir para o conhecimento das propriedades de uma β-glucosidase com grande potencial de aplicação biotecnológica.Fundação Universidade Federal de Mato Grosso do SulUFMSBrasilRasamsonia composticola, fungo termofílico, β-glucosidase, caracterização bioquímica.Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticolainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisFabiana Fonseca ZanoeloISABELA PAVAO VARGASinfo:eu-repo/semantics/openAccessporreponame:Repositório Institucional da UFMSinstname:Universidade Federal de Mato Grosso do Sul (UFMS)instacron:UFMSTHUMBNAILDissertação_Final_Revisado_Isabela_Pavao_2021.pdf.jpgDissertação_Final_Revisado_Isabela_Pavao_2021.pdf.jpgGenerated Thumbnailimage/jpeg1165https://repositorio.ufms.br/bitstream/123456789/3798/3/Disserta%c3%a7%c3%a3o_Final_Revisado_Isabela_Pavao_2021.pdf.jpg610e37a0dbe3fa83d4a2630bd553053dMD53TEXTDissertação_Final_Revisado_Isabela_Pavao_2021.pdf.txtDissertação_Final_Revisado_Isabela_Pavao_2021.pdf.txtExtracted texttext/plain113614https://repositorio.ufms.br/bitstream/123456789/3798/2/Disserta%c3%a7%c3%a3o_Final_Revisado_Isabela_Pavao_2021.pdf.txt49d0e0b4cc9079304bf95aed544c29f9MD52ORIGINALDissertação_Final_Revisado_Isabela_Pavao_2021.pdfDissertação_Final_Revisado_Isabela_Pavao_2021.pdfapplication/pdf1722578https://repositorio.ufms.br/bitstream/123456789/3798/1/Disserta%c3%a7%c3%a3o_Final_Revisado_Isabela_Pavao_2021.pdf38272dadc3f944eb5a2fd3efada1d208MD51123456789/37982021-09-30 15:57:39.352oai:repositorio.ufms.br:123456789/3798Repositório InstitucionalPUBhttps://repositorio.ufms.br/oai/requestri.prograd@ufms.bropendoar:21242021-09-30T19:57:39Repositório Institucional da UFMS - Universidade Federal de Mato Grosso do Sul (UFMS)false |
dc.title.pt_BR.fl_str_mv |
Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola |
title |
Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola |
spellingShingle |
Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola ISABELA PAVAO VARGAS Rasamsonia composticola, fungo termofílico, β-glucosidase, caracterização bioquímica. |
title_short |
Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola |
title_full |
Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola |
title_fullStr |
Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola |
title_full_unstemmed |
Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola |
title_sort |
Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola |
author |
ISABELA PAVAO VARGAS |
author_facet |
ISABELA PAVAO VARGAS |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Fabiana Fonseca Zanoelo |
dc.contributor.author.fl_str_mv |
ISABELA PAVAO VARGAS |
contributor_str_mv |
Fabiana Fonseca Zanoelo |
dc.subject.por.fl_str_mv |
Rasamsonia composticola, fungo termofílico, β-glucosidase, caracterização bioquímica. |
topic |
Rasamsonia composticola, fungo termofílico, β-glucosidase, caracterização bioquímica. |
description |
β-glycosidases are enzymes that are part of the cellulolytic complex with the ability to act on lignocellulosic materials, promoting their hydrolysis. These enzymes are produced by microorganisms, mainly filamentous fungi. They have varied applications such as in the pulp and paper industry, food, animal feed and in the production of second generation ethanol. The present work involves the purification, biochemical characteristics and immobilization of a β-glucosidase resistant to the thermophilic fungus Rasamsonia composticola. The enzyme was purified using two chromatographic steps, including DEAE-Fractogel and hydrophobic Phenyl-Sepharose ion exchange. It was purified 19 times with a final yield of 14%. A purified β-glucosidase having an optimum temperature of 70 °C, remaining stable at a temperature of 65 to 70 °C for up to 8 hours, pH 5.0, and a molecular mass of approximately 45 kDa. The enzyme was not stimulated by most of the chemical compounds tested, however it was slightly stimulated by the detergents saponin and triton X-100, in addition to the chelators EDTA and EGTA and the reducing agent Temed. The enzyme has activity on the synthetic substrates pNP-Glc and pNP-Xyl, and is shown to be glucose tolerant and stimulated by xylose (1-100 mM). The estimated kinetic parameters for a β-glucosidase were Km and Vmax of 2.3 mM and 0.0221 µmol / min / mg for pNP-Glc. The catalytic efficiency (Kcat / Km) of the enzyme was 7.7x10-11. For pNP-Glc + glucose and pNP-Glc + xylose, the enzyme presented Km and Vmax of 2.33 mM and 0.0271 µmol / min / mg and 2.02 mM and 0.0255 µmol / min / mg, respectively . The enzyme was immobilized in glyoxyl agarose about 40% and 56% in the presence of glucose for 60 minutes. It demonstrated optimal activity at pH 5.0 and 75 °C. The immobilized enzyme is stable at temperatures from 75 to 80 °C during 60 minutes of reaction. The results demonstrate that the fungus Rasamsonia composticola can be a candidate to contribute to the knowledge of the properties of a β-glucosidase with great potential for biotechnological application. |
publishDate |
2021 |
dc.date.accessioned.fl_str_mv |
2021-06-28T12:34:06Z |
dc.date.available.fl_str_mv |
2021-09-30T19:57:39Z |
dc.date.issued.fl_str_mv |
2021 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
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masterThesis |
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https://repositorio.ufms.br/handle/123456789/3798 |
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openAccess |
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Fundação Universidade Federal de Mato Grosso do Sul |
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UFMS |
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Brasil |
publisher.none.fl_str_mv |
Fundação Universidade Federal de Mato Grosso do Sul |
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