Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural

Detalhes bibliográficos
Ano de defesa: 2015
Autor(a) principal: SALES, Amanda Emmanuelle lattes
Orientador(a): PORTO, Tatiana Souza
Banca de defesa: TAKAKI, Galba Maria de Campos, CORREIA, Maria Tereza dos Santos, STAMFORD, Tânia Lúcia Montenegro, PONTUAL, Emmanuel Viana
Tipo de documento: Tese
Tipo de acesso: Acesso aberto
Idioma: por
Instituição de defesa: Universidade Federal Rural de Pernambuco
Programa de Pós-Graduação: Programa de Pós-Graduação em Biociência Animal
Departamento: Departamento de Morfologia e Fisiologia Animal
País: Brasil
Palavras-chave em Português:
Palavras-chave em Inglês:
Área do conhecimento CNPq:
Link de acesso: http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4455
Resumo: Fibrinolytic proteases are enzymes that degrade fibrin, the main component of blood clots. The accumulation of this protein leads to thrombosis responsible for cardiovascular disease including myocardial infarction. A promising alternative to thrombolytic therapy has been the production of these enzymes by microorganisms which promotes low cost, high efficiency and capacity for large scale production. This study aimed to select species of filamentous fungi isolated from Caatinga soil samples - Pernambuco - Brazil and assess their potential for production of proteases with fibrinolytic activity. Among the 36 isolates studied, 58% showed fibrinolytic activity above 100 U/mL. The microorganism with the higher activity in terms of enzyme production was Mucor subtilissimus UCP 1262 with 415 U/mL. Further optimization of the fermentation process resulted in the production of 1075 U/mL of enzymatic activity. The fibrinolytic enzyme had a capacity of enzymatic degradation of the blood clot of 16.7 % in vitro. Extraction of fibrinolytic protease produced at submerged fermentation was carried out using a PEG/ammonium sulphate aqueous two-phase system (ATPS). PEG 8000 15% and 25% ammonium sulphate were selected as the most appropriate components for extraction with Fibrinolytic Activity in salt phase: 345 U/mL; K: 0.65; Y: 253.1 % and FP: 8.8. The fibrinolytic enzyme from Mucor subitilissimus UCP 1262 was pre-purified using extractive fermentation in PEG and ammonium sulphate ATPS, in which the fungal strain was able to grown even in high salt concentration, produced and extracted simultaneously to the PEG phase. A novel protease with fibrinolytic activity was purified also by chromatographic methods using a two-step purification protocol. Compared to the crude enzyme extract, the specific activity of the enzyme increased 5.30 fold with a recovery of 36.31%. The initial crude extract with the enzyme was pre-purified using acetone precipitation and adsorbed by ion exchange chromatography on DEAE-sephadex G50. The two-dimensional electrophoresis system (2DE) coupled with SDS-PAGE showed a single protein band of approximately 15.3 kDa and isoelectric focusing point of 3.9, exhibiting a nature as an acidic enzyme. Additionally, the activity was slightly inhibited by EDTA, but significantly inhibited by PMSF and also had a higher affinity for the N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAApNA) and azocasein substrates, suggesting to be a chymotrypsin-like protease. Protein unfolding induced by pH and temperature were applied to study the protein conformational changes and showed from the thermal denaturation curve, change in ellipticity at 222 nm, indicated Tm (Melting temperature) of the protein to be 58.14°C. The far UV circular dichroism (CD) of the fibrinolytic protease showed the secondary structure with most content percentage of α-helix. These results demonstrate an economical, viable enzyme purification protocol. And studying the purified fibrinolytic enzyme have established basis for elucidating mechanisms responsible for the changes in conformation of the new fibrinolytic enzyme under varying conditions of temperature and pH. This novel fibrinolytic enzyme may represent a new source of therapeutic agents to treat thrombosis diseases.
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spelling PORTO, Tatiana SouzaPORTO, Ana Lúcia FigueiredoTAKAKI, Galba Maria de CamposCORREIA, Maria Tereza dos SantosSTAMFORD, Tânia Lúcia MontenegroPONTUAL, Emmanuel Vianahttp://lattes.cnpq.br/6244031450915488SALES, Amanda Emmanuelle2016-05-19T12:14:27Z2015-12-21SALES, Amanda Emmanuelle. Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural. 2015. 157 f. Tese (Programa de Pós-Graduação em Biociência Animal) - Universidade Federal Rural de Pernambuco, Recife.http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4455Fibrinolytic proteases are enzymes that degrade fibrin, the main component of blood clots. The accumulation of this protein leads to thrombosis responsible for cardiovascular disease including myocardial infarction. A promising alternative to thrombolytic therapy has been the production of these enzymes by microorganisms which promotes low cost, high efficiency and capacity for large scale production. This study aimed to select species of filamentous fungi isolated from Caatinga soil samples - Pernambuco - Brazil and assess their potential for production of proteases with fibrinolytic activity. Among the 36 isolates studied, 58% showed fibrinolytic activity above 100 U/mL. The microorganism with the higher activity in terms of enzyme production was Mucor subtilissimus UCP 1262 with 415 U/mL. Further optimization of the fermentation process resulted in the production of 1075 U/mL of enzymatic activity. The fibrinolytic enzyme had a capacity of enzymatic degradation of the blood clot of 16.7 % in vitro. Extraction of fibrinolytic protease produced at submerged fermentation was carried out using a PEG/ammonium sulphate aqueous two-phase system (ATPS). PEG 8000 15% and 25% ammonium sulphate were selected as the most appropriate components for extraction with Fibrinolytic Activity in salt phase: 345 U/mL; K: 0.65; Y: 253.1 % and FP: 8.8. The fibrinolytic enzyme from Mucor subitilissimus UCP 1262 was pre-purified using extractive fermentation in PEG and ammonium sulphate ATPS, in which the fungal strain was able to grown even in high salt concentration, produced and extracted simultaneously to the PEG phase. A novel protease with fibrinolytic activity was purified also by chromatographic methods using a two-step purification protocol. Compared to the crude enzyme extract, the specific activity of the enzyme increased 5.30 fold with a recovery of 36.31%. The initial crude extract with the enzyme was pre-purified using acetone precipitation and adsorbed by ion exchange chromatography on DEAE-sephadex G50. The two-dimensional electrophoresis system (2DE) coupled with SDS-PAGE showed a single protein band of approximately 15.3 kDa and isoelectric focusing point of 3.9, exhibiting a nature as an acidic enzyme. Additionally, the activity was slightly inhibited by EDTA, but significantly inhibited by PMSF and also had a higher affinity for the N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAApNA) and azocasein substrates, suggesting to be a chymotrypsin-like protease. Protein unfolding induced by pH and temperature were applied to study the protein conformational changes and showed from the thermal denaturation curve, change in ellipticity at 222 nm, indicated Tm (Melting temperature) of the protein to be 58.14°C. The far UV circular dichroism (CD) of the fibrinolytic protease showed the secondary structure with most content percentage of α-helix. These results demonstrate an economical, viable enzyme purification protocol. And studying the purified fibrinolytic enzyme have established basis for elucidating mechanisms responsible for the changes in conformation of the new fibrinolytic enzyme under varying conditions of temperature and pH. This novel fibrinolytic enzyme may represent a new source of therapeutic agents to treat thrombosis diseases.Proteases fibrinolíticas são enzimas que degradam a fibrina, o principal componente dos coágulos sanguíneos. O acúmulo da fibrina nos vasos sanguíneos leva a trombose, fenômeno responsável por doenças cardiovasculares. Uma alternativa promissora para a terapia trombolítica tem sido a produção dessas enzimas por micro-organismos que promovem baixo custo, alta eficiência e capacidade de produção em larga escala. Produzir proteases fibrinolíticas por linhagens de fungos filamentosos por fermentação submersa e desenvolver o processo de purificação utilizando Sistemas de Duas Fases Aquosas (SDFA) e cromatografia líquida, além de caracterizar bioquímico e estruturalmente a enzima. Dentre as 36 espécies estudadas, 58% apresentaram atividade fibrinolítica acima de 100 U/mL A espécie com maior atividade foi Mucor subtilissimus UCP 1262 com 415 U/mL. Foram realizados processos fermentativos que resultaram na produção de 1075 U/mL de atividade fibrinolítica, com capacidade de degradação do coágulo sanguíneo de 16,7% in vitro. A extração da protease fibrinolítica produzida por fermentação submersa foi realizada utilizando o sistema de duas fases aquosas (SDFA) com Polietileno glicol (PEG) e sulfato de amônio. O PEG 8000 (g/mol) a 15% e sulfato de amônio a 25% foi selecionado como a condição mais eficiente para a extração da enzima na fase do sal, apresentando 345 U/mL de atividade, coeficiente de partição K=0,65; Recuperação Y=253,1% e Fator de purificação FP=8,8. A protease fibrinolítica produzida por Mucor subitilissimus UCP 1262 foi também pré-purificada utilizando fermentação extrativa com SDFA (PEG e sulfato de amônio), onde a espécie fúngica foi capaz de crescer mesmo em altas concentrações de sal, produzir e extrair simultaneamente para a fase do PEG do sistema. A protease fibrinolítica foi purificada também através de métodos cromatográficos utilizando um protocolo de purificação com dois passos. O extrato bruto inicial com a enzima foi pré-clarificado utilizando precipitação com acetona e adsorção em cromatografia de troca-iônica em DEAE-sephadex G50, o qual foi capaz de aumentar a pureza em 5,30 vezes com a recuperação de 36, 31%. O sistema de eletroforese bidimensional 2DE acoplado ao SDS-PAGE mostrou uma banda única de aproximadamente 15,3 kDa e a focalização isoelétrica apresentou o ponto isoelétrico no pH 3,9, exibindo uma natureza de enzima ácida. Adicionalmente a enzima foi significativamente inibida por PMSF e alta afinidade catalítica para o substrato sintético amidolítico N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAApNA) e azocaseína. Sugerindo ser uma serino-protease semelhante à quimotripsina. Desdobramento proteico induzido por pH e temperatura foram aplicados para estudar as mudanças conformacionais da enzima e mostraram através da curva de desnaturação térmica, mudança da elipticidade a 222 nm, indicando um Tm (Temperatura de desnaturação) da proteína de 58,14°C. O dicroísmo circular no UV distante (far UV CD) da protease fibrinolítica mostrou a estrutura secundária da proteína com maior teor de α-hélix. Estes resultados demonstram um protocolo de purificação de enzimas eficiente. E o estudo da enzima purificada estabeleceu bases para elucidar mecanismos responsáveis pelas mudanças de conformação de uma nova enzima fibrinolítica sob a variação de condições variadas de temperatura e pH. Esta enzima fibrinolítica pode representar uma nova fonte de agente terapêutico no tratamento de doenças trombolíticas.Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-05-19T12:14:27Z No. of bitstreams: 1 Amanda Emmanuelle Sales.pdf: 4982712 bytes, checksum: 90011cbdafe7a0363213b2fff5735b44 (MD5)Made available in DSpace on 2016-05-19T12:14:27Z (GMT). No. of bitstreams: 1 Amanda Emmanuelle Sales.pdf: 4982712 bytes, checksum: 90011cbdafe7a0363213b2fff5735b44 (MD5) Previous issue date: 2015-12-21Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESapplication/pdfporUniversidade Federal Rural de PernambucoPrograma de Pós-Graduação em Biociência AnimalUFRPEBrasilDepartamento de Morfologia e Fisiologia AnimalProtease fibrinolíticaPurificação de enzimaDicroísmo circularFibrinolytic proteaseCircular dichroismPurified enzymeCIENCIAS AGRARIAS::MEDICINA VETERINARIAProtease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estruturalinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis-1510757014399315592600600600600-89223641879873962044536702642350173193590462550136975366info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UFRPEinstname:Universidade Federal Rural de Pernambuco (UFRPE)instacron:UFRPELICENSElicense.txtlicense.txttext/plain; charset=utf-82089http://www.tede2.ufrpe.br:8080/tede2/bitstream/tede2/4455/1/license.txt7b5ba3d2445355f386edab96125d42b7MD51ORIGINALAmanda Emmanuelle Sales.pdfAmanda Emmanuelle Sales.pdfapplication/pdf4982712http://www.tede2.ufrpe.br:8080/tede2/bitstream/tede2/4455/2/Amanda+Emmanuelle+Sales.pdf90011cbdafe7a0363213b2fff5735b44MD52tede2/44552024-02-23 16:49:03.911oai: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:2024-05-28T12:31:47.770697Biblioteca Digital de Teses e Dissertações da UFRPE - Universidade Federal Rural de Pernambuco (UFRPE)false
dc.title.por.fl_str_mv Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural
title Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural
spellingShingle Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural
SALES, Amanda Emmanuelle
Protease fibrinolítica
Purificação de enzima
Dicroísmo circular
Fibrinolytic protease
Circular dichroism
Purified enzyme
CIENCIAS AGRARIAS::MEDICINA VETERINARIA
title_short Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural
title_full Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural
title_fullStr Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural
title_full_unstemmed Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural
title_sort Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural
author SALES, Amanda Emmanuelle
author_facet SALES, Amanda Emmanuelle
author_role author
dc.contributor.advisor1.fl_str_mv PORTO, Tatiana Souza
dc.contributor.advisor-co1.fl_str_mv PORTO, Ana Lúcia Figueiredo
dc.contributor.referee1.fl_str_mv TAKAKI, Galba Maria de Campos
dc.contributor.referee2.fl_str_mv CORREIA, Maria Tereza dos Santos
dc.contributor.referee3.fl_str_mv STAMFORD, Tânia Lúcia Montenegro
dc.contributor.referee4.fl_str_mv PONTUAL, Emmanuel Viana
dc.contributor.authorLattes.fl_str_mv http://lattes.cnpq.br/6244031450915488
dc.contributor.author.fl_str_mv SALES, Amanda Emmanuelle
contributor_str_mv PORTO, Tatiana Souza
PORTO, Ana Lúcia Figueiredo
TAKAKI, Galba Maria de Campos
CORREIA, Maria Tereza dos Santos
STAMFORD, Tânia Lúcia Montenegro
PONTUAL, Emmanuel Viana
dc.subject.por.fl_str_mv Protease fibrinolítica
Purificação de enzima
Dicroísmo circular
topic Protease fibrinolítica
Purificação de enzima
Dicroísmo circular
Fibrinolytic protease
Circular dichroism
Purified enzyme
CIENCIAS AGRARIAS::MEDICINA VETERINARIA
dc.subject.eng.fl_str_mv Fibrinolytic protease
Circular dichroism
Purified enzyme
dc.subject.cnpq.fl_str_mv CIENCIAS AGRARIAS::MEDICINA VETERINARIA
description Fibrinolytic proteases are enzymes that degrade fibrin, the main component of blood clots. The accumulation of this protein leads to thrombosis responsible for cardiovascular disease including myocardial infarction. A promising alternative to thrombolytic therapy has been the production of these enzymes by microorganisms which promotes low cost, high efficiency and capacity for large scale production. This study aimed to select species of filamentous fungi isolated from Caatinga soil samples - Pernambuco - Brazil and assess their potential for production of proteases with fibrinolytic activity. Among the 36 isolates studied, 58% showed fibrinolytic activity above 100 U/mL. The microorganism with the higher activity in terms of enzyme production was Mucor subtilissimus UCP 1262 with 415 U/mL. Further optimization of the fermentation process resulted in the production of 1075 U/mL of enzymatic activity. The fibrinolytic enzyme had a capacity of enzymatic degradation of the blood clot of 16.7 % in vitro. Extraction of fibrinolytic protease produced at submerged fermentation was carried out using a PEG/ammonium sulphate aqueous two-phase system (ATPS). PEG 8000 15% and 25% ammonium sulphate were selected as the most appropriate components for extraction with Fibrinolytic Activity in salt phase: 345 U/mL; K: 0.65; Y: 253.1 % and FP: 8.8. The fibrinolytic enzyme from Mucor subitilissimus UCP 1262 was pre-purified using extractive fermentation in PEG and ammonium sulphate ATPS, in which the fungal strain was able to grown even in high salt concentration, produced and extracted simultaneously to the PEG phase. A novel protease with fibrinolytic activity was purified also by chromatographic methods using a two-step purification protocol. Compared to the crude enzyme extract, the specific activity of the enzyme increased 5.30 fold with a recovery of 36.31%. The initial crude extract with the enzyme was pre-purified using acetone precipitation and adsorbed by ion exchange chromatography on DEAE-sephadex G50. The two-dimensional electrophoresis system (2DE) coupled with SDS-PAGE showed a single protein band of approximately 15.3 kDa and isoelectric focusing point of 3.9, exhibiting a nature as an acidic enzyme. Additionally, the activity was slightly inhibited by EDTA, but significantly inhibited by PMSF and also had a higher affinity for the N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAApNA) and azocasein substrates, suggesting to be a chymotrypsin-like protease. Protein unfolding induced by pH and temperature were applied to study the protein conformational changes and showed from the thermal denaturation curve, change in ellipticity at 222 nm, indicated Tm (Melting temperature) of the protein to be 58.14°C. The far UV circular dichroism (CD) of the fibrinolytic protease showed the secondary structure with most content percentage of α-helix. These results demonstrate an economical, viable enzyme purification protocol. And studying the purified fibrinolytic enzyme have established basis for elucidating mechanisms responsible for the changes in conformation of the new fibrinolytic enzyme under varying conditions of temperature and pH. This novel fibrinolytic enzyme may represent a new source of therapeutic agents to treat thrombosis diseases.
publishDate 2015
dc.date.issued.fl_str_mv 2015-12-21
dc.date.accessioned.fl_str_mv 2016-05-19T12:14:27Z
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 SALES, Amanda Emmanuelle. Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural. 2015. 157 f. Tese (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/tede2/4455
identifier_str_mv SALES, Amanda Emmanuelle. Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural. 2015. 157 f. Tese (Programa de Pós-Graduação em Biociência Animal) - Universidade Federal Rural de Pernambuco, Recife.
url http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4455
dc.language.iso.fl_str_mv por
language por
dc.relation.program.fl_str_mv -1510757014399315592
dc.relation.confidence.fl_str_mv 600
600
600
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dc.relation.department.fl_str_mv -8922364187987396204
dc.relation.cnpq.fl_str_mv 453670264235017319
dc.relation.sponsorship.fl_str_mv 3590462550136975366
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universidade Federal Rural de Pernambuco
dc.publisher.program.fl_str_mv Programa de Pós-Graduação em Biociência Animal
dc.publisher.initials.fl_str_mv UFRPE
dc.publisher.country.fl_str_mv Brasil
dc.publisher.department.fl_str_mv Departamento de Morfologia e Fisiologia Animal
publisher.none.fl_str_mv Universidade Federal Rural de Pernambuco
dc.source.none.fl_str_mv reponame:Biblioteca Digital de Teses e Dissertações da UFRPE
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