Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase
Autor(a) principal: | |
---|---|
Data de Publicação: | 2020 |
Tipo de documento: | Tese |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/12468 |
Resumo: | Industrial processes are increasingly requiring environmentally sustainable technologies and the use of enzymes is relevant due to the selectivity and specificity of these biocatalysts. However, the application of soluble enzymes in catalytic processes is often impracticable due to the high cost of these biocatalysts and the catalytic instability under severe physical-chemical process conditions. In this sense, the immobilization of enzymes guarantees the reuse of these biomolecules and often improves their stability. Among the supports used as immobilizing agents, nanoparticles have been increasingly studied due to the possibility of creating structures with high surface area to adsorb proteins and also to demonstrate low resistance to mass transfer, thus ensuring good accessibility of the catalyst to the substrate. Hydroxyapatite (HA) is a solid inorganic potential for immobilizing enzymes, since it is non-toxic, has good chemical and physical resistance, good ability to interact with proteins and can be synthesized in the form of nanoparticles; however, it has been little explored as a support for enzymatic immobilization. Given this context, the objective of this research was to evaluate the immobilization of the enzymes β-glucosidase, xylanase and phytase on hydroxyapatite nanoparticles (HA), as a strategy to improve the catalytic efficiency and stability of these enzymes. These enzymes have wide application in different sectors of the agribusiness, such as biofuels, food, animal feed and drugs. For this, HA nanoparticles were first characterized in order to understand their composition, size, morphology and surface area. Then, a systematic study of the immobilization of these enzymes in HA was carried out. The biochemical aspects of enzymatic immobilization were investigated, such as the physicochemical conditions of adsorption and desorption, that helps to understand the type of chemical interaction between support and enzyme. Changes in enzymatic activity profile, thermo-stability, conversion capacity during enzymatic hydrolysis and reuse of these biomolecules were also evaluated. The results obtained showed that the β-glucosidase, phytase and xylanase enzymes were efficiently immobilized on the HA nanoparticles using a simple and fast adsorption protocol, which occur mainly through coordination interactions between the Ca2+ sites of the HA with the carboxylic acids (COO-) of the enzyme amino acids. The biochemical behavior of the enzymes in the presence of HA was evaluated under different physicochemical conditions of adsorption and desorption (pH and ionic strength), indicating a strong and highly stable interaction between enzymes and support, with immobilization yields close to 100% and recovered activities in the range of 70-100%. For β-glucosidase it was possible to recycle the immobilized enzyme and retain 70% of the initial activity during at least 10 hydrolysis cycles. For phytase, the immobilized enzyme showed broader activity profile as to pH and temperature, and higher stability at high temperatures than free enzyme, whose improvement in its properties suggested the potential of applying the immobilized form of phytase in animal feed. For xylanase, the immobilized enzyme demonstrated greater affinity for the HA support that was modified with Cu2+ ions, promoting chelation interaction with enzyme amino acids, generating derivatives with maintenance of their catalytic activity and with activity profile similar to that of the free enzyme. Finally, the three enzymes were immobilized on HA magnetic nanoparticles (synthesized with cobalt ferrite, CoFe2O4), demonstrating excellent recovery capacity from the reaction medium through the application of a magnetic field. The results obtained showed the potential of HA to act as a support for enzyme immobilization, creating derivatives with promising industrial applications, which can improve processes and make them more sustainable. |
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Coutinho, Thamara CarvalhoFarinas, Cristiane Sanchezhttp://lattes.cnpq.br/9933650905615452http://lattes.cnpq.br/515353537762575575311f44-055d-4e1d-9a21-05086ef4e2002020-04-24T14:01:12Z2020-04-24T14:01:12Z2020-03-10COUTINHO, Thamara Carvalho. Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase. 2020. Tese (Doutorado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2020. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12468.https://repositorio.ufscar.br/handle/ufscar/12468Industrial processes are increasingly requiring environmentally sustainable technologies and the use of enzymes is relevant due to the selectivity and specificity of these biocatalysts. However, the application of soluble enzymes in catalytic processes is often impracticable due to the high cost of these biocatalysts and the catalytic instability under severe physical-chemical process conditions. In this sense, the immobilization of enzymes guarantees the reuse of these biomolecules and often improves their stability. Among the supports used as immobilizing agents, nanoparticles have been increasingly studied due to the possibility of creating structures with high surface area to adsorb proteins and also to demonstrate low resistance to mass transfer, thus ensuring good accessibility of the catalyst to the substrate. Hydroxyapatite (HA) is a solid inorganic potential for immobilizing enzymes, since it is non-toxic, has good chemical and physical resistance, good ability to interact with proteins and can be synthesized in the form of nanoparticles; however, it has been little explored as a support for enzymatic immobilization. Given this context, the objective of this research was to evaluate the immobilization of the enzymes β-glucosidase, xylanase and phytase on hydroxyapatite nanoparticles (HA), as a strategy to improve the catalytic efficiency and stability of these enzymes. These enzymes have wide application in different sectors of the agribusiness, such as biofuels, food, animal feed and drugs. For this, HA nanoparticles were first characterized in order to understand their composition, size, morphology and surface area. Then, a systematic study of the immobilization of these enzymes in HA was carried out. The biochemical aspects of enzymatic immobilization were investigated, such as the physicochemical conditions of adsorption and desorption, that helps to understand the type of chemical interaction between support and enzyme. Changes in enzymatic activity profile, thermo-stability, conversion capacity during enzymatic hydrolysis and reuse of these biomolecules were also evaluated. The results obtained showed that the β-glucosidase, phytase and xylanase enzymes were efficiently immobilized on the HA nanoparticles using a simple and fast adsorption protocol, which occur mainly through coordination interactions between the Ca2+ sites of the HA with the carboxylic acids (COO-) of the enzyme amino acids. The biochemical behavior of the enzymes in the presence of HA was evaluated under different physicochemical conditions of adsorption and desorption (pH and ionic strength), indicating a strong and highly stable interaction between enzymes and support, with immobilization yields close to 100% and recovered activities in the range of 70-100%. For β-glucosidase it was possible to recycle the immobilized enzyme and retain 70% of the initial activity during at least 10 hydrolysis cycles. For phytase, the immobilized enzyme showed broader activity profile as to pH and temperature, and higher stability at high temperatures than free enzyme, whose improvement in its properties suggested the potential of applying the immobilized form of phytase in animal feed. For xylanase, the immobilized enzyme demonstrated greater affinity for the HA support that was modified with Cu2+ ions, promoting chelation interaction with enzyme amino acids, generating derivatives with maintenance of their catalytic activity and with activity profile similar to that of the free enzyme. Finally, the three enzymes were immobilized on HA magnetic nanoparticles (synthesized with cobalt ferrite, CoFe2O4), demonstrating excellent recovery capacity from the reaction medium through the application of a magnetic field. The results obtained showed the potential of HA to act as a support for enzyme immobilization, creating derivatives with promising industrial applications, which can improve processes and make them more sustainable.Processos industriais requerem cada vez mais a aplicação de tecnologias ambientalmente sustentáveis e o uso de enzimas se destaca devido à seletividade e especificidade desses biocatalisadores. Contudo, a aplicação de enzimas solúveis em processos catalíticos torna-se muitas vezes inviável devido ao alto custo desses biocatalisadores e a instabilidade catalítica em condições físico-químicas severas de processo. Nesse sentido, a imobilização de enzimas garante o reaproveitamento dessas biomoléculas e muitas vezes, o ganho de estabilidade. Dentre os suportes utilizados para imobilização de enzimas, as nanopartículas têm-se mostrado bastante atraentes devido à elevada área superficial específica e baixa resistência à transferência de massa, garantindo alta capacidade de imobilização enzimas e boa acessibilidade do substrato ao catalisador. A hidroxiapatita (HA) é um potencial sólido inorgânico para imobilização de enzimas, uma vez que não é tóxico, apresenta boa resistência química e física, boa capacidade de interação com proteínas e ainda pode ser sintetizada na forma de nanopartículas; contudo tem sido pouco explorada como suporte para imobilização enzimática. O objetivo deste projeto de doutorado foi avaliar a imobilização das enzimas β-glicosidase, xilanase e fitase em nanopartículas de HA, a fim de obter derivados mais interessantes para serem aplicados na indústria. Essas enzimas têm vasta aplicação em diferentes setores da agroindústria, como de biocombustíveis, de alimentos, de ração animal e de fármacos. Para isso, as nanopartículas de HA foram primeiramente caracterizadas com o objetivo de compreender sua composição, tamanho, morfologia e área de superfície. Em seguida, realizou-se um estudo sistemático da imobilização dessas enzimas em HA, avaliando os aspectos bioquímicos da imobilização, como as condições físico-químicas de adsorção e dessorção, que ajudaram a compreender o tipo de interação química entre suporte e enzima; alterações no perfil de atividade enzimática; melhora na estabilidade térmica; capacidade de hidrólise enzimática e reutilização dessas biomoléculas. Os resultados obtidos mostraram que as enzimas β-glicosidase, fitase e xilanase foram eficientemente imobilizadas nas nanopartículas de HA através de um protocolo de adsorção simples e rápido, principalmente através de interações de coordenação entre os sítios de Ca2+ da HA com os ácidos carboxílicos (COO-) dos aminoácidos das enzimas. Foi avaliado o comportamento bioquímico das enzimas com a HA em diferentes condições físico-químicas de adsorção e dessorção (pH e força iônica), indicando uma interação forte e altamente estável entre enzimas e suporte, com rendimentos de imobilização próximos a 100% e atividades recuperadas na faixa de 70-100%. A β-glicosidase imobilizada em HA reteve até 70% de sua atividade catalítica após 10 ciclos de hidrólise, demonstrando alta capacidade de reuso. Já a fitase imobilizada apresentou maior atuação em ampla faixa de pH e temperatura, e maior termoestabilidade a 80 e 90 °C do que a enzima livre, demonstrando propriedades de interesse para aplicação em ração animal. A xilanase demonstrou maior afinidade pelo suporte de HA modificado com íons de Cu2+, os quais promoveram quelação com aminoácidos da enzima, gerando derivados com manutenção de sua atividade catalítica e perfil de atividade semelhante ao da enzima livre. Por fim, as três enzimas foram imobilizadas em nanopartículas magnéticas de HA (sintetizadas com ferrita de cobalto, CoFe2O4), demonstrando excelente capacidade de recuperação desses biocatalisadores do meio reacional através da aplicação de um campo magnético. Os resultados obtidos mostraram o potencial da HA para atuar como suporte para imobilização de enzimas, gerando derivados enzimáticos com aplicações promissoras na indústria, que podem melhorar os processos e torná-los mais sustentáveis.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES: código de financiamento - 001engUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Engenharia Química - PPGEQUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessimobilizaçãoenzimasnanopartículashidroxiapatitaImmobilizationEnzymesNanoparticlesHydroxyapatiteENGENHARIAS::ENGENHARIA QUIMICAImmobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytaseImobilização de enzimas de interesse agroindustrial em nanopartículas de hidroxiapatita: β-glicosidase, xilanase e fitaseinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis600600dd9ceb6c-d509-421a-a31e-bcb55bb21e02reponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALTese de Doutorado correções pós defesa.pdfTese de Doutorado correções pós defesa.pdfTese Finalapplication/pdf4935409https://repositorio.ufscar.br/bitstream/ufscar/12468/1/Tese%20de%20Doutorado%20corre%c3%a7%c3%b5es%20p%c3%b3s%20defesa.pdf798d3903fee7f3700301348404e4aca5MD51Carta Cristiane.pdfCarta Cristiane.pdfCarta comprovante orientadorapplication/pdf248610https://repositorio.ufscar.br/bitstream/ufscar/12468/2/Carta%20Cristiane.pdfa763fc80d85502eea6748bdbef0a3fc9MD52CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufscar.br/bitstream/ufscar/12468/4/license_rdfe39d27027a6cc9cb039ad269a5db8e34MD54TEXTTese de Doutorado correções pós defesa.pdf.txtTese de Doutorado correções pós defesa.pdf.txtExtracted texttext/plain348090https://repositorio.ufscar.br/bitstream/ufscar/12468/5/Tese%20de%20Doutorado%20corre%c3%a7%c3%b5es%20p%c3%b3s%20defesa.pdf.txt2e76323ec201f520d5ad9cd3ace5c54cMD55Carta Cristiane.pdf.txtCarta Cristiane.pdf.txtExtracted texttext/plain1https://repositorio.ufscar.br/bitstream/ufscar/12468/7/Carta%20Cristiane.pdf.txt68b329da9893e34099c7d8ad5cb9c940MD57THUMBNAILTese de Doutorado correções pós defesa.pdf.jpgTese de Doutorado correções pós defesa.pdf.jpgIM Thumbnailimage/jpeg10619https://repositorio.ufscar.br/bitstream/ufscar/12468/6/Tese%20de%20Doutorado%20corre%c3%a7%c3%b5es%20p%c3%b3s%20defesa.pdf.jpg8dde2acb7b0e2508e11a711b4979baeaMD56Carta Cristiane.pdf.jpgCarta Cristiane.pdf.jpgIM Thumbnailimage/jpeg10886https://repositorio.ufscar.br/bitstream/ufscar/12468/8/Carta%20Cristiane.pdf.jpg35e3f2b5040bff7982895cf4898d1c74MD58ufscar/124682023-09-18 18:31:53.562oai:repositorio.ufscar.br:ufscar/12468Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:53Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.eng.fl_str_mv |
Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase |
dc.title.alternative.por.fl_str_mv |
Imobilização de enzimas de interesse agroindustrial em nanopartículas de hidroxiapatita: β-glicosidase, xilanase e fitase |
title |
Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase |
spellingShingle |
Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase Coutinho, Thamara Carvalho imobilização enzimas nanopartículas hidroxiapatita Immobilization Enzymes Nanoparticles Hydroxyapatite ENGENHARIAS::ENGENHARIA QUIMICA |
title_short |
Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase |
title_full |
Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase |
title_fullStr |
Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase |
title_full_unstemmed |
Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase |
title_sort |
Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase |
author |
Coutinho, Thamara Carvalho |
author_facet |
Coutinho, Thamara Carvalho |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/5153535377625755 |
dc.contributor.author.fl_str_mv |
Coutinho, Thamara Carvalho |
dc.contributor.advisor1.fl_str_mv |
Farinas, Cristiane Sanchez |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/9933650905615452 |
dc.contributor.authorID.fl_str_mv |
75311f44-055d-4e1d-9a21-05086ef4e200 |
contributor_str_mv |
Farinas, Cristiane Sanchez |
dc.subject.por.fl_str_mv |
imobilização enzimas nanopartículas hidroxiapatita |
topic |
imobilização enzimas nanopartículas hidroxiapatita Immobilization Enzymes Nanoparticles Hydroxyapatite ENGENHARIAS::ENGENHARIA QUIMICA |
dc.subject.eng.fl_str_mv |
Immobilization Enzymes Nanoparticles Hydroxyapatite |
dc.subject.cnpq.fl_str_mv |
ENGENHARIAS::ENGENHARIA QUIMICA |
description |
Industrial processes are increasingly requiring environmentally sustainable technologies and the use of enzymes is relevant due to the selectivity and specificity of these biocatalysts. However, the application of soluble enzymes in catalytic processes is often impracticable due to the high cost of these biocatalysts and the catalytic instability under severe physical-chemical process conditions. In this sense, the immobilization of enzymes guarantees the reuse of these biomolecules and often improves their stability. Among the supports used as immobilizing agents, nanoparticles have been increasingly studied due to the possibility of creating structures with high surface area to adsorb proteins and also to demonstrate low resistance to mass transfer, thus ensuring good accessibility of the catalyst to the substrate. Hydroxyapatite (HA) is a solid inorganic potential for immobilizing enzymes, since it is non-toxic, has good chemical and physical resistance, good ability to interact with proteins and can be synthesized in the form of nanoparticles; however, it has been little explored as a support for enzymatic immobilization. Given this context, the objective of this research was to evaluate the immobilization of the enzymes β-glucosidase, xylanase and phytase on hydroxyapatite nanoparticles (HA), as a strategy to improve the catalytic efficiency and stability of these enzymes. These enzymes have wide application in different sectors of the agribusiness, such as biofuels, food, animal feed and drugs. For this, HA nanoparticles were first characterized in order to understand their composition, size, morphology and surface area. Then, a systematic study of the immobilization of these enzymes in HA was carried out. The biochemical aspects of enzymatic immobilization were investigated, such as the physicochemical conditions of adsorption and desorption, that helps to understand the type of chemical interaction between support and enzyme. Changes in enzymatic activity profile, thermo-stability, conversion capacity during enzymatic hydrolysis and reuse of these biomolecules were also evaluated. The results obtained showed that the β-glucosidase, phytase and xylanase enzymes were efficiently immobilized on the HA nanoparticles using a simple and fast adsorption protocol, which occur mainly through coordination interactions between the Ca2+ sites of the HA with the carboxylic acids (COO-) of the enzyme amino acids. The biochemical behavior of the enzymes in the presence of HA was evaluated under different physicochemical conditions of adsorption and desorption (pH and ionic strength), indicating a strong and highly stable interaction between enzymes and support, with immobilization yields close to 100% and recovered activities in the range of 70-100%. For β-glucosidase it was possible to recycle the immobilized enzyme and retain 70% of the initial activity during at least 10 hydrolysis cycles. For phytase, the immobilized enzyme showed broader activity profile as to pH and temperature, and higher stability at high temperatures than free enzyme, whose improvement in its properties suggested the potential of applying the immobilized form of phytase in animal feed. For xylanase, the immobilized enzyme demonstrated greater affinity for the HA support that was modified with Cu2+ ions, promoting chelation interaction with enzyme amino acids, generating derivatives with maintenance of their catalytic activity and with activity profile similar to that of the free enzyme. Finally, the three enzymes were immobilized on HA magnetic nanoparticles (synthesized with cobalt ferrite, CoFe2O4), demonstrating excellent recovery capacity from the reaction medium through the application of a magnetic field. The results obtained showed the potential of HA to act as a support for enzyme immobilization, creating derivatives with promising industrial applications, which can improve processes and make them more sustainable. |
publishDate |
2020 |
dc.date.accessioned.fl_str_mv |
2020-04-24T14:01:12Z |
dc.date.available.fl_str_mv |
2020-04-24T14:01:12Z |
dc.date.issued.fl_str_mv |
2020-03-10 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
COUTINHO, Thamara Carvalho. Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase. 2020. Tese (Doutorado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2020. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12468. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/12468 |
identifier_str_mv |
COUTINHO, Thamara Carvalho. Immobilization of enzymes of agroindustrial interest on hydroxyapatite nanoparticles: β-glucosidase, xylase and phytase. 2020. Tese (Doutorado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2020. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12468. |
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https://repositorio.ufscar.br/handle/ufscar/12468 |
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eng |
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eng |
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600 600 |
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dd9ceb6c-d509-421a-a31e-bcb55bb21e02 |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ info:eu-repo/semantics/openAccess |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ |
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openAccess |
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Universidade Federal de São Carlos Câmpus São Carlos |
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Programa de Pós-Graduação em Engenharia Química - PPGEQ |
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UFSCar |
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Universidade Federal de São Carlos Câmpus São Carlos |
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