β-galactosidase activity againts different substrates and in the presence of lipid interfaces
- Autores
- Flores Mamani, Sandra Soledad; Perillo, Maria Angelica; Sanchez, Julieta Maria
- Año de publicación
- 2013
- Idioma
- inglés
- Tipo de recurso
- documento de conferencia
- Estado
- versión publicada
- Descripción
- Previously we demonstrated that the activity of a soluble wild-type E. coli β-galactosidase (β-Galwt) against both lactose (the natural substrate) as ortho-nitrofenilgalactopiranósido (ONPG, artificial substrate) increases in the presence of multilamellar vesicles (MLVs) composed of neutral and charged phospholipids. The aim of this study was to compare the activity of a recombinant β-Gal (β-GalHis6) against two different substrates in the presence of MLVs of different lipid composition. β-Gal-His6 was overexpressed in E.coli, and the six histidine residues (His-tag) fused to the carboxyl terminus facilitated purification by ion metal affinity chromatography (IMAC). The enzyme activity was measured by visible spectrophotometry, in the absence or presence of MLVs of pure egg phosphatidylcholine (EPC interface) or at 80:20 molar ratio with dioleoylphosphatidyl glycerol (EPC80/DOPG20) negative zwitterionic interface). Kinetic parameters were determined by fitting the michaelian model to the experimental data using nonlinear regression. Our results showed that the enzyme activity was more efficient against ONPG compared to lactose (kcat/KMONPG >kcat/KMLactosa) as previously described for other beta galactosidase. An activation of the enzymatic activity but a decrease in the substrate affinity were observed in the presence of lipid interfaces against both substrates. Those effects were enhanced by the presence of charged interfaces favored by electrostatic interactions mediated by the presence His residues of the enzyme. The nature of the substrate not qualitatively affected the kinetics of the reaction catalyzed by β-Gal-His6 in the presence of lipid interfaces.
Fil: Flores Mamani, Sandra Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina
Fil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina
Fil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina
XLII Reunión Anual de la Sociedad Argentina de Biofísica
Villa Carlos Paz
Argentina
Sociedad Argentina de Biofísica - Materia
-
Recombinant beta-galactosidase
Surface charge
Catalytic activity
Surface adsorption - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
.jpg)
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/234821
Ver los metadatos del registro completo
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β-galactosidase activity againts different substrates and in the presence of lipid interfacesFlores Mamani, Sandra SoledadPerillo, Maria AngelicaSanchez, Julieta MariaRecombinant beta-galactosidaseSurface chargeCatalytic activitySurface adsorptionhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Previously we demonstrated that the activity of a soluble wild-type E. coli β-galactosidase (β-Galwt) against both lactose (the natural substrate) as ortho-nitrofenilgalactopiranósido (ONPG, artificial substrate) increases in the presence of multilamellar vesicles (MLVs) composed of neutral and charged phospholipids. The aim of this study was to compare the activity of a recombinant β-Gal (β-GalHis6) against two different substrates in the presence of MLVs of different lipid composition. β-Gal-His6 was overexpressed in E.coli, and the six histidine residues (His-tag) fused to the carboxyl terminus facilitated purification by ion metal affinity chromatography (IMAC). The enzyme activity was measured by visible spectrophotometry, in the absence or presence of MLVs of pure egg phosphatidylcholine (EPC interface) or at 80:20 molar ratio with dioleoylphosphatidyl glycerol (EPC80/DOPG20) negative zwitterionic interface). Kinetic parameters were determined by fitting the michaelian model to the experimental data using nonlinear regression. Our results showed that the enzyme activity was more efficient against ONPG compared to lactose (kcat/KMONPG >kcat/KMLactosa) as previously described for other beta galactosidase. An activation of the enzymatic activity but a decrease in the substrate affinity were observed in the presence of lipid interfaces against both substrates. Those effects were enhanced by the presence of charged interfaces favored by electrostatic interactions mediated by the presence His residues of the enzyme. The nature of the substrate not qualitatively affected the kinetics of the reaction catalyzed by β-Gal-His6 in the presence of lipid interfaces.Fil: Flores Mamani, Sandra Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaXLII Reunión Anual de la Sociedad Argentina de BiofísicaVilla Carlos PazArgentinaSociedad Argentina de BiofísicaSociedad Argentina de BiofísicaFanani, Maria LauraWilke, NataliaFidelio, Gerardo Daniel2013info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónBookhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/234821β-galactosidase activity againts different substrates and in the presence of lipid interfaces; XLII Reunión Anual de la Sociedad Argentina de Biofísica; Villa Carlos Paz; Argentina; 2013; 75-75978-987-27591-2-4CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://biofisica.org.ar/reuniones-cientificas/reunionsab-previas/Nacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:44:57Zoai:ri.conicet.gov.ar:11336/234821instacron: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 09:44:57.998CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
| dc.title.none.fl_str_mv |
β-galactosidase activity againts different substrates and in the presence of lipid interfaces |
| title |
β-galactosidase activity againts different substrates and in the presence of lipid interfaces |
| spellingShingle |
β-galactosidase activity againts different substrates and in the presence of lipid interfaces Flores Mamani, Sandra Soledad Recombinant beta-galactosidase Surface charge Catalytic activity Surface adsorption |
| title_short |
β-galactosidase activity againts different substrates and in the presence of lipid interfaces |
| title_full |
β-galactosidase activity againts different substrates and in the presence of lipid interfaces |
| title_fullStr |
β-galactosidase activity againts different substrates and in the presence of lipid interfaces |
| title_full_unstemmed |
β-galactosidase activity againts different substrates and in the presence of lipid interfaces |
| title_sort |
β-galactosidase activity againts different substrates and in the presence of lipid interfaces |
| dc.creator.none.fl_str_mv |
Flores Mamani, Sandra Soledad Perillo, Maria Angelica Sanchez, Julieta Maria |
| author |
Flores Mamani, Sandra Soledad |
| author_facet |
Flores Mamani, Sandra Soledad Perillo, Maria Angelica Sanchez, Julieta Maria |
| author_role |
author |
| author2 |
Perillo, Maria Angelica Sanchez, Julieta Maria |
| author2_role |
author author |
| dc.contributor.none.fl_str_mv |
Fanani, Maria Laura Wilke, Natalia Fidelio, Gerardo Daniel |
| dc.subject.none.fl_str_mv |
Recombinant beta-galactosidase Surface charge Catalytic activity Surface adsorption |
| topic |
Recombinant beta-galactosidase Surface charge Catalytic activity Surface adsorption |
| purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| dc.description.none.fl_txt_mv |
Previously we demonstrated that the activity of a soluble wild-type E. coli β-galactosidase (β-Galwt) against both lactose (the natural substrate) as ortho-nitrofenilgalactopiranósido (ONPG, artificial substrate) increases in the presence of multilamellar vesicles (MLVs) composed of neutral and charged phospholipids. The aim of this study was to compare the activity of a recombinant β-Gal (β-GalHis6) against two different substrates in the presence of MLVs of different lipid composition. β-Gal-His6 was overexpressed in E.coli, and the six histidine residues (His-tag) fused to the carboxyl terminus facilitated purification by ion metal affinity chromatography (IMAC). The enzyme activity was measured by visible spectrophotometry, in the absence or presence of MLVs of pure egg phosphatidylcholine (EPC interface) or at 80:20 molar ratio with dioleoylphosphatidyl glycerol (EPC80/DOPG20) negative zwitterionic interface). Kinetic parameters were determined by fitting the michaelian model to the experimental data using nonlinear regression. Our results showed that the enzyme activity was more efficient against ONPG compared to lactose (kcat/KMONPG >kcat/KMLactosa) as previously described for other beta galactosidase. An activation of the enzymatic activity but a decrease in the substrate affinity were observed in the presence of lipid interfaces against both substrates. Those effects were enhanced by the presence of charged interfaces favored by electrostatic interactions mediated by the presence His residues of the enzyme. The nature of the substrate not qualitatively affected the kinetics of the reaction catalyzed by β-Gal-His6 in the presence of lipid interfaces. Fil: Flores Mamani, Sandra Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina Fil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina Fil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina XLII Reunión Anual de la Sociedad Argentina de Biofísica Villa Carlos Paz Argentina Sociedad Argentina de Biofísica |
| description |
Previously we demonstrated that the activity of a soluble wild-type E. coli β-galactosidase (β-Galwt) against both lactose (the natural substrate) as ortho-nitrofenilgalactopiranósido (ONPG, artificial substrate) increases in the presence of multilamellar vesicles (MLVs) composed of neutral and charged phospholipids. The aim of this study was to compare the activity of a recombinant β-Gal (β-GalHis6) against two different substrates in the presence of MLVs of different lipid composition. β-Gal-His6 was overexpressed in E.coli, and the six histidine residues (His-tag) fused to the carboxyl terminus facilitated purification by ion metal affinity chromatography (IMAC). The enzyme activity was measured by visible spectrophotometry, in the absence or presence of MLVs of pure egg phosphatidylcholine (EPC interface) or at 80:20 molar ratio with dioleoylphosphatidyl glycerol (EPC80/DOPG20) negative zwitterionic interface). Kinetic parameters were determined by fitting the michaelian model to the experimental data using nonlinear regression. Our results showed that the enzyme activity was more efficient against ONPG compared to lactose (kcat/KMONPG >kcat/KMLactosa) as previously described for other beta galactosidase. An activation of the enzymatic activity but a decrease in the substrate affinity were observed in the presence of lipid interfaces against both substrates. Those effects were enhanced by the presence of charged interfaces favored by electrostatic interactions mediated by the presence His residues of the enzyme. The nature of the substrate not qualitatively affected the kinetics of the reaction catalyzed by β-Gal-His6 in the presence of lipid interfaces. |
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2013 |
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2013 |
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info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/conferenceObject Reunión Book http://purl.org/coar/resource_type/c_5794 info:ar-repo/semantics/documentoDeConferencia |
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http://hdl.handle.net/11336/234821 β-galactosidase activity againts different substrates and in the presence of lipid interfaces; XLII Reunión Anual de la Sociedad Argentina de Biofísica; Villa Carlos Paz; Argentina; 2013; 75-75 978-987-27591-2-4 CONICET Digital CONICET |
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β-galactosidase activity againts different substrates and in the presence of lipid interfaces; XLII Reunión Anual de la Sociedad Argentina de Biofísica; Villa Carlos Paz; Argentina; 2013; 75-75 978-987-27591-2-4 CONICET Digital CONICET |
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eng |
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Sociedad Argentina de Biofísica |
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