Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits

Autores
Rocha, Gabriela F.; Obregon, Walter David; Muñoz, Fernando Felipe; Guevara, Maria Gabriela; Fernandez, Graciela del Valle; Rosso, Adriana M.; Parisi, Mónica G.
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
This report describes the purification of an aspartic protease (salpichroin) from ripe fruits of Salpichroa origanifolia (Solanaceae) starting with precipitation using organic solvents and anionexchange chromatography with 32.1% recovery and 13.4-fold purification. SDS-PAGE and zymograms of this enzyme showed a single band corresponding to an apparent molecular mass of approximately 32 kDa. The biochemical and kinetic characterization of the pure enzyme showed an acidic behavior with an optimal pH value around 3.0–4.5 with hemoglobin and 5.5–6.0 with casein. Salpichroin activity was inhibited by pepstatin but not by phenylmethylsulfonyl fluoride, E-64, EDTA or 1,10-phenanthroline, thus suggesting an aspartic protease behavior. Salpichroin hydrolyzed natural substrates, such as casein and hemoglobin, with high specific activity. Kinetic studies conducted with the synthetic peptide H-Pro- Thr-Glu-Phe-p-(NO2)-Phe-Arg-Leu-OH showed lower affinity (Km 494 µM) than other representative aspartic proteases. By investigating the cleavage of oxidized insulin β-chain to establish the hydrolytic specificity of salpichroin, we found six cleavage sites on the substrate of peptide bonds similar to those of chymosin. MALDI-TOF/TOF-MS of the tryptic ingel digest of salpichroin showed that the isolated protease shared homologous sequences with other plant proteases of the A1 aspartic protease family. This is the first report concerning the isolation and biochemical characterization of an aspartic protease isolated from Salpichroa origanifolia fruits.
Fil: Rocha, Gabriela F. . Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; Argentina
Fil: Obregon, Walter David. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biologicas. Laboratorio de Investigacion de Proteinas Vegetales; Argentina
Fil: Muñoz, Fernando Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina
Fil: Guevara, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina
Fil: Fernandez, Graciela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; Argentina
Fil: Rosso, Adriana M. . Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; Argentina
Fil: Parisi, Mónica G. . Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; Argentina
Materia
Caseinolytic Activity
Peptide Mass Fingerprinting
Plant Aspartic Protease
Purification
Salpichroa Origanifolia
Salpichroin
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/13206
Acceder
id CONICETDig_678ca4d2c6337de058012cead7970836
oai_identifier_str oai:ri.conicet.gov.ar:11336/13206
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia FruitsRocha, Gabriela F. Obregon, Walter DavidMuñoz, Fernando FelipeGuevara, Maria GabrielaFernandez, Graciela del ValleRosso, Adriana M. Parisi, Mónica G. Caseinolytic ActivityPeptide Mass FingerprintingPlant Aspartic ProteasePurificationSalpichroa OriganifoliaSalpichroinhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1This report describes the purification of an aspartic protease (salpichroin) from ripe fruits of Salpichroa origanifolia (Solanaceae) starting with precipitation using organic solvents and anionexchange chromatography with 32.1% recovery and 13.4-fold purification. SDS-PAGE and zymograms of this enzyme showed a single band corresponding to an apparent molecular mass of approximately 32 kDa. The biochemical and kinetic characterization of the pure enzyme showed an acidic behavior with an optimal pH value around 3.0–4.5 with hemoglobin and 5.5–6.0 with casein. Salpichroin activity was inhibited by pepstatin but not by phenylmethylsulfonyl fluoride, E-64, EDTA or 1,10-phenanthroline, thus suggesting an aspartic protease behavior. Salpichroin hydrolyzed natural substrates, such as casein and hemoglobin, with high specific activity. Kinetic studies conducted with the synthetic peptide H-Pro- Thr-Glu-Phe-p-(NO2)-Phe-Arg-Leu-OH showed lower affinity (Km 494 µM) than other representative aspartic proteases. By investigating the cleavage of oxidized insulin β-chain to establish the hydrolytic specificity of salpichroin, we found six cleavage sites on the substrate of peptide bonds similar to those of chymosin. MALDI-TOF/TOF-MS of the tryptic ingel digest of salpichroin showed that the isolated protease shared homologous sequences with other plant proteases of the A1 aspartic protease family. This is the first report concerning the isolation and biochemical characterization of an aspartic protease isolated from Salpichroa origanifolia fruits.Fil: Rocha, Gabriela F. . Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; ArgentinaFil: Obregon, Walter David. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biologicas. Laboratorio de Investigacion de Proteinas Vegetales; ArgentinaFil: Muñoz, Fernando Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Guevara, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Fernandez, Graciela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; ArgentinaFil: Rosso, Adriana M. . Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; ArgentinaFil: Parisi, Mónica G. . Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; ArgentinaBentham Science Publishers2015-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/13206Rocha, Gabriela F. ; Obregon, Walter David; Muñoz, Fernando Felipe; Guevara, Maria Gabriela; Fernandez, Graciela del Valle; et al.; Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits; Bentham Science Publishers; Protein And Peptide Letters; 22; 4; 3-2015; 379-3900929-86651875-5305enginfo:eu-repo/semantics/altIdentifier/url/http://www.eurekaselect.com/128937/articleinfo:eu-repo/semantics/altIdentifier/doi/10.2174/0929866522666150302111059info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:46:50Zoai:ri.conicet.gov.ar:11336/13206instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:46:50.721CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits
title Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits
spellingShingle Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits
Rocha, Gabriela F.
Caseinolytic Activity
Peptide Mass Fingerprinting
Plant Aspartic Protease
Purification
Salpichroa Origanifolia
Salpichroin
title_short Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits
title_full Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits
title_fullStr Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits
title_full_unstemmed Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits
title_sort Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits
dc.creator.none.fl_str_mv Rocha, Gabriela F.
Obregon, Walter David
Muñoz, Fernando Felipe
Guevara, Maria Gabriela
Fernandez, Graciela del Valle
Rosso, Adriana M.
Parisi, Mónica G.
author Rocha, Gabriela F.
author_facet Rocha, Gabriela F.
Obregon, Walter David
Muñoz, Fernando Felipe
Guevara, Maria Gabriela
Fernandez, Graciela del Valle
Rosso, Adriana M.
Parisi, Mónica G.
author_role author
author2 Obregon, Walter David
Muñoz, Fernando Felipe
Guevara, Maria Gabriela
Fernandez, Graciela del Valle
Rosso, Adriana M.
Parisi, Mónica G.
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Caseinolytic Activity
Peptide Mass Fingerprinting
Plant Aspartic Protease
Purification
Salpichroa Origanifolia
Salpichroin
topic Caseinolytic Activity
Peptide Mass Fingerprinting
Plant Aspartic Protease
Purification
Salpichroa Origanifolia
Salpichroin
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv This report describes the purification of an aspartic protease (salpichroin) from ripe fruits of Salpichroa origanifolia (Solanaceae) starting with precipitation using organic solvents and anionexchange chromatography with 32.1% recovery and 13.4-fold purification. SDS-PAGE and zymograms of this enzyme showed a single band corresponding to an apparent molecular mass of approximately 32 kDa. The biochemical and kinetic characterization of the pure enzyme showed an acidic behavior with an optimal pH value around 3.0–4.5 with hemoglobin and 5.5–6.0 with casein. Salpichroin activity was inhibited by pepstatin but not by phenylmethylsulfonyl fluoride, E-64, EDTA or 1,10-phenanthroline, thus suggesting an aspartic protease behavior. Salpichroin hydrolyzed natural substrates, such as casein and hemoglobin, with high specific activity. Kinetic studies conducted with the synthetic peptide H-Pro- Thr-Glu-Phe-p-(NO2)-Phe-Arg-Leu-OH showed lower affinity (Km 494 µM) than other representative aspartic proteases. By investigating the cleavage of oxidized insulin β-chain to establish the hydrolytic specificity of salpichroin, we found six cleavage sites on the substrate of peptide bonds similar to those of chymosin. MALDI-TOF/TOF-MS of the tryptic ingel digest of salpichroin showed that the isolated protease shared homologous sequences with other plant proteases of the A1 aspartic protease family. This is the first report concerning the isolation and biochemical characterization of an aspartic protease isolated from Salpichroa origanifolia fruits.
Fil: Rocha, Gabriela F. . Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; Argentina
Fil: Obregon, Walter David. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biologicas. Laboratorio de Investigacion de Proteinas Vegetales; Argentina
Fil: Muñoz, Fernando Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina
Fil: Guevara, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina
Fil: Fernandez, Graciela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; Argentina
Fil: Rosso, Adriana M. . Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; Argentina
Fil: Parisi, Mónica G. . Universidad Nacional de Luján. Departamento de Ciencias Básicas. Area de Química Biológica; Argentina
description This report describes the purification of an aspartic protease (salpichroin) from ripe fruits of Salpichroa origanifolia (Solanaceae) starting with precipitation using organic solvents and anionexchange chromatography with 32.1% recovery and 13.4-fold purification. SDS-PAGE and zymograms of this enzyme showed a single band corresponding to an apparent molecular mass of approximately 32 kDa. The biochemical and kinetic characterization of the pure enzyme showed an acidic behavior with an optimal pH value around 3.0–4.5 with hemoglobin and 5.5–6.0 with casein. Salpichroin activity was inhibited by pepstatin but not by phenylmethylsulfonyl fluoride, E-64, EDTA or 1,10-phenanthroline, thus suggesting an aspartic protease behavior. Salpichroin hydrolyzed natural substrates, such as casein and hemoglobin, with high specific activity. Kinetic studies conducted with the synthetic peptide H-Pro- Thr-Glu-Phe-p-(NO2)-Phe-Arg-Leu-OH showed lower affinity (Km 494 µM) than other representative aspartic proteases. By investigating the cleavage of oxidized insulin β-chain to establish the hydrolytic specificity of salpichroin, we found six cleavage sites on the substrate of peptide bonds similar to those of chymosin. MALDI-TOF/TOF-MS of the tryptic ingel digest of salpichroin showed that the isolated protease shared homologous sequences with other plant proteases of the A1 aspartic protease family. This is the first report concerning the isolation and biochemical characterization of an aspartic protease isolated from Salpichroa origanifolia fruits.
publishDate 2015
dc.date.none.fl_str_mv 2015-03
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/13206
Rocha, Gabriela F. ; Obregon, Walter David; Muñoz, Fernando Felipe; Guevara, Maria Gabriela; Fernandez, Graciela del Valle; et al.; Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits; Bentham Science Publishers; Protein And Peptide Letters; 22; 4; 3-2015; 379-390
0929-8665
1875-5305
url http://hdl.handle.net/11336/13206
identifier_str_mv Rocha, Gabriela F. ; Obregon, Walter David; Muñoz, Fernando Felipe; Guevara, Maria Gabriela; Fernandez, Graciela del Valle; et al.; Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits; Bentham Science Publishers; Protein And Peptide Letters; 22; 4; 3-2015; 379-390
0929-8665
1875-5305
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.eurekaselect.com/128937/article
info:eu-repo/semantics/altIdentifier/doi/10.2174/0929866522666150302111059
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Bentham Science Publishers
publisher.none.fl_str_mv Bentham Science Publishers
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.070432

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