Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da UFRPE |
Texto Completo: | http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/7217 |
Resumo: | The fibrinolytic proteases, due to its potential use in the treatment of thrombosis, have drawn attention from researchers because such diseases are a leading cause of death worldwide. The objective of this research was to produce and extract simultaneously the fibrinolytic protease by Mucor subtilissimus UCP 1262 through extractive fermentation with PEG/sodium sulfate. Homogeneous fermentations were carried out by changing the nitrogen source (soybean flour or wheat -1%), carbon source with 1% glucose, agitation 120 rpm and 30°C. Extractive fermentation assays were made using a 23 factorial design, changing the PEG molar mass (4,000, 6,000 and 8,000 g/mol), the PEG concentration (18, 24 and 30%) and salt concentration (10; 11,5; 13%). After 72 hours fermentation, the fibrinolytic protease had its best output in soy flour obtaining 13.43 U/mL and specific activity 749.27 U/mg in homogeneous cultures. However in the extractive fermentation, the sodium sulfate rich phase showed 15.40 U/mL fibrinolytic activity and 34.17 U/mg for specific activity in the assay comprises of sodium sulphate concentration (10%) and PEG (18%) and PEG molar mass (8000g/mol) and yields (Y) of 80% of the fibrinolytic protease. The protease has a pH optimum between 7.0 and stability pH 6.0 to 8.5 and optimum temperature 50°C, stable between 10°C to 50°C. It has similar specificity to chymotrypsin enzyme it has been classified as a serine protease having molecular weight of 52-kDa. Thus it was possible to pre-purify the fibrinolytic protease having a low cost process and considerably faster when compared to other isolated production and purification techniques by replacing the initial stages of conventional separation processes. |
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PORTO, Tatiana SouzaGOMES, Bruno SeveroPONTUAL, Emmanuel VianaCUNHA, Marcia Nieves Carneiro daPORTO, Camila Souzahttp://lattes.cnpq.br/4160699401113199CLEMENTINO, Ellen Leal2018-05-03T14:46:00Z2016-02-26CLEMENTINO, Ellen Leal. Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262. 2016. 45 f. Dissertação (Programa de Pós-Graduação em Biociência Animal) - Universidade Federal Rural de Pernambuco, Recife.http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/7217The fibrinolytic proteases, due to its potential use in the treatment of thrombosis, have drawn attention from researchers because such diseases are a leading cause of death worldwide. The objective of this research was to produce and extract simultaneously the fibrinolytic protease by Mucor subtilissimus UCP 1262 through extractive fermentation with PEG/sodium sulfate. Homogeneous fermentations were carried out by changing the nitrogen source (soybean flour or wheat -1%), carbon source with 1% glucose, agitation 120 rpm and 30°C. Extractive fermentation assays were made using a 23 factorial design, changing the PEG molar mass (4,000, 6,000 and 8,000 g/mol), the PEG concentration (18, 24 and 30%) and salt concentration (10; 11,5; 13%). After 72 hours fermentation, the fibrinolytic protease had its best output in soy flour obtaining 13.43 U/mL and specific activity 749.27 U/mg in homogeneous cultures. However in the extractive fermentation, the sodium sulfate rich phase showed 15.40 U/mL fibrinolytic activity and 34.17 U/mg for specific activity in the assay comprises of sodium sulphate concentration (10%) and PEG (18%) and PEG molar mass (8000g/mol) and yields (Y) of 80% of the fibrinolytic protease. The protease has a pH optimum between 7.0 and stability pH 6.0 to 8.5 and optimum temperature 50°C, stable between 10°C to 50°C. It has similar specificity to chymotrypsin enzyme it has been classified as a serine protease having molecular weight of 52-kDa. Thus it was possible to pre-purify the fibrinolytic protease having a low cost process and considerably faster when compared to other isolated production and purification techniques by replacing the initial stages of conventional separation processes.As proteases fibrinolíticas, em virtude do seu potencial para uso no tratamento de trombose, têm chamado a atenção de pesquisadores, pois tal doença é uma das principais causas de morte no mundo. O objetivo desta pesquisa foi produzir e extrair de forma integrada a protease fibrinolítica por Mucor subtilissimus UCP 1262 através de fermentação extrativa utlilizandoSistema de Duas Fases Aquosas (SDFA) PEG/Sulfato de sódio. Foram realizadas fermentações homogêneas alterando a fonte de nitrogênio (farinha de soja ou trigo - 1%), fonte de carbono com 1% de glicose, agitação de 120 rpm e temperatura de 30°C. Os ensaios das fermentações extrativas foram constituídos através de um planejamento fatorial 23, modificando a massa molar do PEG (4000, 6000 e 8000 g/mol), concentração do PEG (18, 24 e 30%) e concentração do sal (10; 11,5 e 13%). Após 72 horas de fermentação, a protease fibrinolítica teve sua melhor produção na farinha de soja obtendo 13,43 U/mL e com atividade específica de 749,27 U/mg em cultivos homogêneos. No entanto na fermentação extrativa, a fase rica em sulfato de sódio apresentou 15,40 U/mL de atividade fibrinolítica e 34,17 U/mg de atividade específica no ensaio composto por concentração de sulfato de sódio (10%) e de PEG (18%) e massa molar do PEG (8000 g/mol), tendo recuperação (Y) de 80% da protease fibrinolítica. A protease tem pH ótimo 7,0 e estabilidade entre os valores de pH 6,0 e 8,5, e temperatura ótima aos 50°C, sendo estável entre 10°C a 50°C. Tem especificidade semelhante à enzima quimiotripsina e foi classificada como uma serino protease, apresentando massa molar de 52kDa. Deste modo foi possível pré-purificar a protease fibrinolítica com um processo de baixo custo e de considerável rapidez quando comparado a outras técnicas de produção e purificação isoladas substituindo etapas iniciais de processos de separação convencionais.Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-05-03T14:46:00Z No. of bitstreams: 1 Ellen Leal Clementino.pdf: 1364888 bytes, checksum: 1a3b7cf184f74c3f95b093c74e973c3a (MD5)Made available in DSpace on 2018-05-03T14:46:00Z (GMT). No. of bitstreams: 1 Ellen Leal Clementino.pdf: 1364888 bytes, checksum: 1a3b7cf184f74c3f95b093c74e973c3a (MD5) Previous issue date: 2016-02-26application/pdfporUniversidade Federal Rural de PernambucoPrograma de Pós-Graduação em Biociência AnimalUFRPEBrasilDepartamento de Morfologia e Fisiologia AnimalProtease fibrinolíticaMucor subtilissimusFermentação extrativaCIENCIAS AGRARIAS::MEDICINA VETERINARIAProdução e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis-1510757014399315592600600600-8922364187987396204453670264235017319info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UFRPEinstname:Universidade Federal Rural de Pernambuco (UFRPE)instacron:UFRPEORIGINALEllen Leal Clementino.pdfEllen Leal Clementino.pdfapplication/pdf1364888http://www.tede2.ufrpe.br:8080/tede2/bitstream/tede2/7217/2/Ellen+Leal+Clementino.pdf1a3b7cf184f74c3f95b093c74e973c3aMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82165http://www.tede2.ufrpe.br:8080/tede2/bitstream/tede2/7217/1/license.txtbd3efa91386c1718a7f26a329fdcb468MD51tede2/72172018-05-03 11:46:00.109oai:tede2: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Biblioteca Digital de Teses e Dissertaçõeshttp://www.tede2.ufrpe.br:8080/tede/PUBhttp://www.tede2.ufrpe.br:8080/oai/requestbdtd@ufrpe.br ||bdtd@ufrpe.bropendoar:2018-05-03T14:46Biblioteca Digital de Teses e Dissertações da UFRPE - Universidade Federal Rural de Pernambuco (UFRPE)false |
dc.title.por.fl_str_mv |
Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262 |
title |
Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262 |
spellingShingle |
Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262 CLEMENTINO, Ellen Leal Protease fibrinolítica Mucor subtilissimus Fermentação extrativa CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
title_short |
Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262 |
title_full |
Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262 |
title_fullStr |
Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262 |
title_full_unstemmed |
Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262 |
title_sort |
Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262 |
author |
CLEMENTINO, Ellen Leal |
author_facet |
CLEMENTINO, Ellen Leal |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
PORTO, Tatiana Souza |
dc.contributor.referee1.fl_str_mv |
GOMES, Bruno Severo |
dc.contributor.referee2.fl_str_mv |
PONTUAL, Emmanuel Viana |
dc.contributor.referee3.fl_str_mv |
CUNHA, Marcia Nieves Carneiro da |
dc.contributor.referee4.fl_str_mv |
PORTO, Camila Souza |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/4160699401113199 |
dc.contributor.author.fl_str_mv |
CLEMENTINO, Ellen Leal |
contributor_str_mv |
PORTO, Tatiana Souza GOMES, Bruno Severo PONTUAL, Emmanuel Viana CUNHA, Marcia Nieves Carneiro da PORTO, Camila Souza |
dc.subject.por.fl_str_mv |
Protease fibrinolítica Mucor subtilissimus Fermentação extrativa |
topic |
Protease fibrinolítica Mucor subtilissimus Fermentação extrativa CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
dc.subject.cnpq.fl_str_mv |
CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
description |
The fibrinolytic proteases, due to its potential use in the treatment of thrombosis, have drawn attention from researchers because such diseases are a leading cause of death worldwide. The objective of this research was to produce and extract simultaneously the fibrinolytic protease by Mucor subtilissimus UCP 1262 through extractive fermentation with PEG/sodium sulfate. Homogeneous fermentations were carried out by changing the nitrogen source (soybean flour or wheat -1%), carbon source with 1% glucose, agitation 120 rpm and 30°C. Extractive fermentation assays were made using a 23 factorial design, changing the PEG molar mass (4,000, 6,000 and 8,000 g/mol), the PEG concentration (18, 24 and 30%) and salt concentration (10; 11,5; 13%). After 72 hours fermentation, the fibrinolytic protease had its best output in soy flour obtaining 13.43 U/mL and specific activity 749.27 U/mg in homogeneous cultures. However in the extractive fermentation, the sodium sulfate rich phase showed 15.40 U/mL fibrinolytic activity and 34.17 U/mg for specific activity in the assay comprises of sodium sulphate concentration (10%) and PEG (18%) and PEG molar mass (8000g/mol) and yields (Y) of 80% of the fibrinolytic protease. The protease has a pH optimum between 7.0 and stability pH 6.0 to 8.5 and optimum temperature 50°C, stable between 10°C to 50°C. It has similar specificity to chymotrypsin enzyme it has been classified as a serine protease having molecular weight of 52-kDa. Thus it was possible to pre-purify the fibrinolytic protease having a low cost process and considerably faster when compared to other isolated production and purification techniques by replacing the initial stages of conventional separation processes. |
publishDate |
2016 |
dc.date.issued.fl_str_mv |
2016-02-26 |
dc.date.accessioned.fl_str_mv |
2018-05-03T14:46:00Z |
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 |
CLEMENTINO, Ellen Leal. Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262. 2016. 45 f. Dissertação (Programa de Pós-Graduação em Biociência Animal) - Universidade Federal Rural de Pernambuco, Recife. |
dc.identifier.uri.fl_str_mv |
http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/7217 |
identifier_str_mv |
CLEMENTINO, Ellen Leal. Produção e purificação integrada de protease fibrinolítica por Mucor subtilissimus UCP 1262. 2016. 45 f. Dissertação (Programa de Pós-Graduação em Biociência Animal) - Universidade Federal Rural de Pernambuco, Recife. |
url |
http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/7217 |
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por |
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por |
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600 600 600 |
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453670264235017319 |
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info:eu-repo/semantics/openAccess |
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openAccess |
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Universidade Federal Rural de Pernambuco |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Biociência Animal |
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UFRPE |
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Brasil |
dc.publisher.department.fl_str_mv |
Departamento de Morfologia e Fisiologia Animal |
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Universidade Federal Rural de Pernambuco |
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