Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1
Autor(a) principal: | |
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Data de Publicação: | 2011 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | http://locus.ufv.br/handle/123456789/2429 |
Resumo: | The objectives of this work were to establish the best conditions for Debaryomyces hansenii UFV-1 yeast cultivation for β-glucosidase production, purification and characterization. In addition, another objective was to immobilize the yeast cells containing the intracellular β-glucosidase in calcium alginate and characterize the immobilized enzyme. The yeast produced an intracellular β-glucosidase when grown for 12 h in YP medium containing cellobiose as carbon source. The enzyme was purified by ion exchange, gel filtration and hydrophobic interaction chromatografies and at the end of the process, the β-glucosidase showed a purification factor of 99.3 times, with a yield of 8 %. The molecular mass was approximately 198 kDa when estimated by SDS-PAGE, and approximately 178 kDa when estimated by gel filtration. The maximum activity occurred at pH 6.0 and temperature of 45 °C. The intracellular β-glucosidase half-life values were 356, 312, 73 and 5 minutes, at temperatures of 40, 45, 50 and 55 °C, respectively. The KM and Vmax values using the substrate ρNPβGlc were 0.434 mM and 0.021 mM/min, respectively. The enzyme showed absolute specificity for glucose in β position and the enzymatic activity was completely inhibited by ferric chloride, silver nitrate and SDS, all of them at final concentrations of 0.2 and 2 mM. The yeast cells were immobilized in calcium alginate and the enzyme showed pH and temperature optima of 5.5 and 50 °C, respectively. The immobilization provided higher enzyme thermostability at temperatures of 45 and 50 °C. The KM app and Vmax app. values, with the substrate ρNPβGlc, were 4.35 mM and 3.63 mmol of ρNP/min/g cell, respectively. D. hansenii is a nonpathogenic yeast and it is found in various types of food. So, the use of immobilized cells of this yeast in calcium alginate in food industry would not cause problems for human health. In this case, the enzyme is more stable and the process is more economical because there is no spending on enzyme purification and also the possibility of reuse of alginate beads containing the enzyme β-glucosidase. |
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Maitan, Gabriela Piccolohttp://lattes.cnpq.br/7554486723696822Viana, Pollyanna Amaralhttp://lattes.cnpq.br/5442738991535153Rezende, Sebastião Tavares dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787599A3Guimarães, Valéria Montezehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3Oliveira, Eduardo Basílio dehttp://lattes.cnpq.br/4091528830821027Fietto, Luciano Gomeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H82015-03-26T13:07:31Z2011-11-032015-03-26T13:07:31Z2011-02-22MAITAN, Gabriela Piccolo. Production, purification and characterization of free and immobilized β-glucosidases from Debaryomyces hansenii UFV-1. 2011. 105 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2011.http://locus.ufv.br/handle/123456789/2429The objectives of this work were to establish the best conditions for Debaryomyces hansenii UFV-1 yeast cultivation for β-glucosidase production, purification and characterization. In addition, another objective was to immobilize the yeast cells containing the intracellular β-glucosidase in calcium alginate and characterize the immobilized enzyme. The yeast produced an intracellular β-glucosidase when grown for 12 h in YP medium containing cellobiose as carbon source. The enzyme was purified by ion exchange, gel filtration and hydrophobic interaction chromatografies and at the end of the process, the β-glucosidase showed a purification factor of 99.3 times, with a yield of 8 %. The molecular mass was approximately 198 kDa when estimated by SDS-PAGE, and approximately 178 kDa when estimated by gel filtration. The maximum activity occurred at pH 6.0 and temperature of 45 °C. The intracellular β-glucosidase half-life values were 356, 312, 73 and 5 minutes, at temperatures of 40, 45, 50 and 55 °C, respectively. The KM and Vmax values using the substrate ρNPβGlc were 0.434 mM and 0.021 mM/min, respectively. The enzyme showed absolute specificity for glucose in β position and the enzymatic activity was completely inhibited by ferric chloride, silver nitrate and SDS, all of them at final concentrations of 0.2 and 2 mM. The yeast cells were immobilized in calcium alginate and the enzyme showed pH and temperature optima of 5.5 and 50 °C, respectively. The immobilization provided higher enzyme thermostability at temperatures of 45 and 50 °C. The KM app and Vmax app. values, with the substrate ρNPβGlc, were 4.35 mM and 3.63 mmol of ρNP/min/g cell, respectively. D. hansenii is a nonpathogenic yeast and it is found in various types of food. So, the use of immobilized cells of this yeast in calcium alginate in food industry would not cause problems for human health. In this case, the enzyme is more stable and the process is more economical because there is no spending on enzyme purification and also the possibility of reuse of alginate beads containing the enzyme β-glucosidase.Os objetivos deste trabalho foram estabelecer melhores condições de cultivo da levedura Debaryomyces hansenii UFV-1 para produção de β-glicosidases, purificar e caracterizar esta enzima, imobilizar as células da levedura contendo a β-glicosidase intracelular em alginato de cálcio e caracterizar a enzima imobilizada. Para produção da β-glicosidase intracelular, a levedura foi cultivada por 12 h em meio YP contendo celobiose como fonte de carbono. A enzima foi purificada por cromatografias de troca iônica, filtração em gel e interação hidrofóbica e, ao final do processo, a β-glicosidase apresentou um fator de purificação de 99,3 vezes, com um rendimento de 8 %. A massa molecular da enzima foi de aproximadamente 198 kDa, quando estimada por SDS-PAGE, e de aproximadamente 178 kDa, quando estimada por filtração em gel. A enzima apresentou atividade máxima em pH 6,0 e na temperatura de 45 °C. Os valores de meia-vida para a β-glicosidase foram de 356, 312, 73 e 5 minutos, nas temperaturas de 40, 45, 50 e 55 °C, respectivamente. Os valores de KM e Vmax, utilizando o substrato ρNPβGlc, foram 0,434 mM e 0,021 mM/min, respectivamente. A enzima apresentou especificidade absoluta para glicose em posição β e a atividade enzimática foi completamente inibida por cloreto de ferro, nitrato de prata e SDS, todos nas concentrações finais de 0,2 e 2 mM. Após imobilização das células de D. hansenii em alginato de cálcio, a enzima apresentou máxima atividade em pH 5,5 e na temperatura de 50 °C. A imobilização proporcionou à enzima maior termoestabilidade nas temperaturas de 45 e 50 °C. Os valores de KM app. e de Vmax app., com o substrato ρNPβGlc, foram, respectivamente, 4,35 mM e 3,63 μmol de ρNP/min/g célula. D. hansenii é uma levedura não patogênica e encontrada em diversos tipos de alimentos. Assim, a utilização das células desta levedura imobilizadas em alginato de cálcio, na indústria de alimentos, não acarretaria problemas para a saúde humana. Neste caso, a enzima é mais estável e o processo é mais econômico por não haver gastos com purificação enzimática e ainda pela possibilidade de reutilização das esferas de alginato contendo a enzima β-glicosidase.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaMestrado em Bioquímica AgrícolaUFVBRBioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animalβ-glicosidases livreDebaryomyces hanseniiβ-glucosidasesDebaryomyces hanseniiCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIAProdução, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1Production, purification and characterization of free and immobilized β-glucosidases from Debaryomyces hansenii UFV-1info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf877904https://locus.ufv.br//bitstream/123456789/2429/1/texto%20completo.pdf3aade8903bb34fdf9472fd1eba21c72bMD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain184069https://locus.ufv.br//bitstream/123456789/2429/2/texto%20completo.pdf.txtbc2f601af8999bcf8b51d415f344b359MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3598https://locus.ufv.br//bitstream/123456789/2429/3/texto%20completo.pdf.jpg6a895b8d17de4ad92f2d64205e47619cMD53123456789/24292017-10-06 15:27:56.481oai:locus.ufv.br:123456789/2429Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452017-10-06T18:27:56LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1 |
dc.title.alternative.eng.fl_str_mv |
Production, purification and characterization of free and immobilized β-glucosidases from Debaryomyces hansenii UFV-1 |
title |
Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1 |
spellingShingle |
Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1 Maitan, Gabriela Piccolo β-glicosidases livre Debaryomyces hansenii β-glucosidases Debaryomyces hansenii CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA |
title_short |
Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1 |
title_full |
Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1 |
title_fullStr |
Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1 |
title_full_unstemmed |
Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1 |
title_sort |
Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1 |
author |
Maitan, Gabriela Piccolo |
author_facet |
Maitan, Gabriela Piccolo |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/7554486723696822 |
dc.contributor.author.fl_str_mv |
Maitan, Gabriela Piccolo |
dc.contributor.advisor-co1.fl_str_mv |
Viana, Pollyanna Amaral |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/5442738991535153 |
dc.contributor.advisor-co2.fl_str_mv |
Rezende, Sebastião Tavares de |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787599A3 |
dc.contributor.advisor1.fl_str_mv |
Guimarães, Valéria Monteze |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3 |
dc.contributor.referee1.fl_str_mv |
Oliveira, Eduardo Basílio de |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/4091528830821027 |
dc.contributor.referee2.fl_str_mv |
Fietto, Luciano Gomes |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8 |
contributor_str_mv |
Viana, Pollyanna Amaral Rezende, Sebastião Tavares de Guimarães, Valéria Monteze Oliveira, Eduardo Basílio de Fietto, Luciano Gomes |
dc.subject.por.fl_str_mv |
β-glicosidases livre Debaryomyces hansenii |
topic |
β-glicosidases livre Debaryomyces hansenii β-glucosidases Debaryomyces hansenii CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA |
dc.subject.eng.fl_str_mv |
β-glucosidases Debaryomyces hansenii |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA |
description |
The objectives of this work were to establish the best conditions for Debaryomyces hansenii UFV-1 yeast cultivation for β-glucosidase production, purification and characterization. In addition, another objective was to immobilize the yeast cells containing the intracellular β-glucosidase in calcium alginate and characterize the immobilized enzyme. The yeast produced an intracellular β-glucosidase when grown for 12 h in YP medium containing cellobiose as carbon source. The enzyme was purified by ion exchange, gel filtration and hydrophobic interaction chromatografies and at the end of the process, the β-glucosidase showed a purification factor of 99.3 times, with a yield of 8 %. The molecular mass was approximately 198 kDa when estimated by SDS-PAGE, and approximately 178 kDa when estimated by gel filtration. The maximum activity occurred at pH 6.0 and temperature of 45 °C. The intracellular β-glucosidase half-life values were 356, 312, 73 and 5 minutes, at temperatures of 40, 45, 50 and 55 °C, respectively. The KM and Vmax values using the substrate ρNPβGlc were 0.434 mM and 0.021 mM/min, respectively. The enzyme showed absolute specificity for glucose in β position and the enzymatic activity was completely inhibited by ferric chloride, silver nitrate and SDS, all of them at final concentrations of 0.2 and 2 mM. The yeast cells were immobilized in calcium alginate and the enzyme showed pH and temperature optima of 5.5 and 50 °C, respectively. The immobilization provided higher enzyme thermostability at temperatures of 45 and 50 °C. The KM app and Vmax app. values, with the substrate ρNPβGlc, were 4.35 mM and 3.63 mmol of ρNP/min/g cell, respectively. D. hansenii is a nonpathogenic yeast and it is found in various types of food. So, the use of immobilized cells of this yeast in calcium alginate in food industry would not cause problems for human health. In this case, the enzyme is more stable and the process is more economical because there is no spending on enzyme purification and also the possibility of reuse of alginate beads containing the enzyme β-glucosidase. |
publishDate |
2011 |
dc.date.available.fl_str_mv |
2011-11-03 2015-03-26T13:07:31Z |
dc.date.issued.fl_str_mv |
2011-02-22 |
dc.date.accessioned.fl_str_mv |
2015-03-26T13:07:31Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
MAITAN, Gabriela Piccolo. Production, purification and characterization of free and immobilized β-glucosidases from Debaryomyces hansenii UFV-1. 2011. 105 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2011. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/2429 |
identifier_str_mv |
MAITAN, Gabriela Piccolo. Production, purification and characterization of free and immobilized β-glucosidases from Debaryomyces hansenii UFV-1. 2011. 105 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2011. |
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http://locus.ufv.br/handle/123456789/2429 |
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por |
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por |
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Universidade Federal de Viçosa |
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Mestrado em Bioquímica Agrícola |
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UFV |
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BR |
dc.publisher.department.fl_str_mv |
Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal |
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Universidade Federal de Viçosa |
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LOCUS Repositório Institucional da UFV |
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