Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate

Autores
Rosales, Joel Andrés; Perillo, Maria Angelica; Nolan, María Verónica
Año de publicación
2021
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
The yeast β-galactosidase or lactase [EC 3.2.1.23] (β-Gal) is a soluble enzyme capable of catalyzing lactose hydrolysis into its constitutive monosaccharides: glucose and galactose. In addition, and depending on the conditions of the environment, fundamentally high lactose concentration, β-Gal catalyzes the transglycosylation reaction whose products will be the Galacto-oligosaccharides (GOS). These molecules are considered prebiotics because they are not degraded in the digestive tract, reaching the intestine where they are a substrate for the growth of beneficial bacteria. GOS production is favored by: high lactose concentration, high reaction temperature and low water availability. These experimental conditions can be achieved if macromolecular crowded media (MCM) are used as the reaction medium. In this work we investigate the effect that molecular crowding induces on the activity and thermal stability of β-galactosidase from Kluyveromices lactis. PEG6000, a non-charged highly water-soluble polymer with wellknown effects on water dynamics was used to produce the crowded environment. The effect of PEG6000 on β-Gal kinetic parameters was studied using lactose as substrate. Results obtained showed that enzymatic activity is improved in MCM: the affinity increased while the Vmax remained unchanged. Temperature-dependent β-Gal activity profile was studied both in the absence or in the presence of molecular crowded agent in a range from 37 to 50 °C. Results obtained showed that β-Gal thermal activity profile was enhanced in molecular crowded environment. The enzyme maintained its activity when it was incubated at temperatures 5 degrees higher in the presence than in the absence of molecular crowding agent. Thermal inactivation kinetic was also studied: in this type of experiments, the enzyme was pre-incubated at 37 and 50 °C during different periods of time and after that, the enzymatic activity was measured in optimal conditions. Results obtained show again that molecular crowding conditions protect the enzyme from heat denaturation. In this case, it was observed that the enzyme maintains its activity even when it is subjected for a considerable period of time at high temperature when it is in the presence of the molecular crowding agent.
Fil: Rosales, Joel Andrés. 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. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; 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. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; Argentina
Fil: Nolan, María Verónica. 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. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; Argentina
XLIX Reunión Anual SAB
Buenos Aires
Argentina
Sociedad Argentina de Biofísica
Materia
LACTASE
GALACTO-OLIGOSACHARIDES
MOLECULAR CROWDING
THERMAL STABILITY
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:11336/188184
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/188184
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrateRosales, Joel AndrésPerillo, Maria AngelicaNolan, María VerónicaLACTASEGALACTO-OLIGOSACHARIDESMOLECULAR CROWDINGTHERMAL STABILITYhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The yeast β-galactosidase or lactase [EC 3.2.1.23] (β-Gal) is a soluble enzyme capable of catalyzing lactose hydrolysis into its constitutive monosaccharides: glucose and galactose. In addition, and depending on the conditions of the environment, fundamentally high lactose concentration, β-Gal catalyzes the transglycosylation reaction whose products will be the Galacto-oligosaccharides (GOS). These molecules are considered prebiotics because they are not degraded in the digestive tract, reaching the intestine where they are a substrate for the growth of beneficial bacteria. GOS production is favored by: high lactose concentration, high reaction temperature and low water availability. These experimental conditions can be achieved if macromolecular crowded media (MCM) are used as the reaction medium. In this work we investigate the effect that molecular crowding induces on the activity and thermal stability of β-galactosidase from Kluyveromices lactis. PEG6000, a non-charged highly water-soluble polymer with wellknown effects on water dynamics was used to produce the crowded environment. The effect of PEG6000 on β-Gal kinetic parameters was studied using lactose as substrate. Results obtained showed that enzymatic activity is improved in MCM: the affinity increased while the Vmax remained unchanged. Temperature-dependent β-Gal activity profile was studied both in the absence or in the presence of molecular crowded agent in a range from 37 to 50 °C. Results obtained showed that β-Gal thermal activity profile was enhanced in molecular crowded environment. The enzyme maintained its activity when it was incubated at temperatures 5 degrees higher in the presence than in the absence of molecular crowding agent. Thermal inactivation kinetic was also studied: in this type of experiments, the enzyme was pre-incubated at 37 and 50 °C during different periods of time and after that, the enzymatic activity was measured in optimal conditions. Results obtained show again that molecular crowding conditions protect the enzyme from heat denaturation. In this case, it was observed that the enzyme maintains its activity even when it is subjected for a considerable period of time at high temperature when it is in the presence of the molecular crowding agent.Fil: Rosales, Joel Andrés. 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. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; 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; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; ArgentinaFil: Nolan, María Verónica. 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. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; ArgentinaXLIX Reunión Anual SABBuenos AiresArgentinaSociedad Argentina de BiofísicaSociedad Argentina de BiofísicaDelfino, José MaríaCelej, Maria SoledadPietrasanta, LiaAmbroggio, Ernesto EstebanMangialavori, Irene CeciliaAcierno, Juan Pablo2021info: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/11336/188184Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate; XLIX Reunión Anual SAB; Buenos Aires; Argentina; 2021; 128-128978-987-27591-9-3CONICET 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 Técnicas2025-10-22T11:06:43Zoai:ri.conicet.gov.ar:11336/188184instacron: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:34982025-10-22 11:06:43.707CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate
title Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate
spellingShingle Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate
Rosales, Joel Andrés
LACTASE
GALACTO-OLIGOSACHARIDES
MOLECULAR CROWDING
THERMAL STABILITY
title_short Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate
title_full Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate
title_fullStr Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate
title_full_unstemmed Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate
title_sort Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate
dc.creator.none.fl_str_mv Rosales, Joel Andrés
Perillo, Maria Angelica
Nolan, María Verónica
author Rosales, Joel Andrés
author_facet Rosales, Joel Andrés
Perillo, Maria Angelica
Nolan, María Verónica
author_role author
author2 Perillo, Maria Angelica
Nolan, María Verónica
author2_role author
author
dc.contributor.none.fl_str_mv Delfino, José María
Celej, Maria Soledad
Pietrasanta, Lia
Ambroggio, Ernesto Esteban
Mangialavori, Irene Cecilia
Acierno, Juan Pablo
dc.subject.none.fl_str_mv LACTASE
GALACTO-OLIGOSACHARIDES
MOLECULAR CROWDING
THERMAL STABILITY
topic LACTASE
GALACTO-OLIGOSACHARIDES
MOLECULAR CROWDING
THERMAL STABILITY
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The yeast β-galactosidase or lactase [EC 3.2.1.23] (β-Gal) is a soluble enzyme capable of catalyzing lactose hydrolysis into its constitutive monosaccharides: glucose and galactose. In addition, and depending on the conditions of the environment, fundamentally high lactose concentration, β-Gal catalyzes the transglycosylation reaction whose products will be the Galacto-oligosaccharides (GOS). These molecules are considered prebiotics because they are not degraded in the digestive tract, reaching the intestine where they are a substrate for the growth of beneficial bacteria. GOS production is favored by: high lactose concentration, high reaction temperature and low water availability. These experimental conditions can be achieved if macromolecular crowded media (MCM) are used as the reaction medium. In this work we investigate the effect that molecular crowding induces on the activity and thermal stability of β-galactosidase from Kluyveromices lactis. PEG6000, a non-charged highly water-soluble polymer with wellknown effects on water dynamics was used to produce the crowded environment. The effect of PEG6000 on β-Gal kinetic parameters was studied using lactose as substrate. Results obtained showed that enzymatic activity is improved in MCM: the affinity increased while the Vmax remained unchanged. Temperature-dependent β-Gal activity profile was studied both in the absence or in the presence of molecular crowded agent in a range from 37 to 50 °C. Results obtained showed that β-Gal thermal activity profile was enhanced in molecular crowded environment. The enzyme maintained its activity when it was incubated at temperatures 5 degrees higher in the presence than in the absence of molecular crowding agent. Thermal inactivation kinetic was also studied: in this type of experiments, the enzyme was pre-incubated at 37 and 50 °C during different periods of time and after that, the enzymatic activity was measured in optimal conditions. Results obtained show again that molecular crowding conditions protect the enzyme from heat denaturation. In this case, it was observed that the enzyme maintains its activity even when it is subjected for a considerable period of time at high temperature when it is in the presence of the molecular crowding agent.
Fil: Rosales, Joel Andrés. 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. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; 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. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; Argentina
Fil: Nolan, María Verónica. 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. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; Argentina
XLIX Reunión Anual SAB
Buenos Aires
Argentina
Sociedad Argentina de Biofísica
description The yeast β-galactosidase or lactase [EC 3.2.1.23] (β-Gal) is a soluble enzyme capable of catalyzing lactose hydrolysis into its constitutive monosaccharides: glucose and galactose. In addition, and depending on the conditions of the environment, fundamentally high lactose concentration, β-Gal catalyzes the transglycosylation reaction whose products will be the Galacto-oligosaccharides (GOS). These molecules are considered prebiotics because they are not degraded in the digestive tract, reaching the intestine where they are a substrate for the growth of beneficial bacteria. GOS production is favored by: high lactose concentration, high reaction temperature and low water availability. These experimental conditions can be achieved if macromolecular crowded media (MCM) are used as the reaction medium. In this work we investigate the effect that molecular crowding induces on the activity and thermal stability of β-galactosidase from Kluyveromices lactis. PEG6000, a non-charged highly water-soluble polymer with wellknown effects on water dynamics was used to produce the crowded environment. The effect of PEG6000 on β-Gal kinetic parameters was studied using lactose as substrate. Results obtained showed that enzymatic activity is improved in MCM: the affinity increased while the Vmax remained unchanged. Temperature-dependent β-Gal activity profile was studied both in the absence or in the presence of molecular crowded agent in a range from 37 to 50 °C. Results obtained showed that β-Gal thermal activity profile was enhanced in molecular crowded environment. The enzyme maintained its activity when it was incubated at temperatures 5 degrees higher in the presence than in the absence of molecular crowding agent. Thermal inactivation kinetic was also studied: in this type of experiments, the enzyme was pre-incubated at 37 and 50 °C during different periods of time and after that, the enzymatic activity was measured in optimal conditions. Results obtained show again that molecular crowding conditions protect the enzyme from heat denaturation. In this case, it was observed that the enzyme maintains its activity even when it is subjected for a considerable period of time at high temperature when it is in the presence of the molecular crowding agent.
publishDate 2021
dc.date.none.fl_str_mv 2021
dc.type.none.fl_str_mv 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
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/188184
Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate; XLIX Reunión Anual SAB; Buenos Aires; Argentina; 2021; 128-128
978-987-27591-9-3
CONICET Digital
CONICET
url http://hdl.handle.net/11336/188184
identifier_str_mv Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate; XLIX Reunión Anual SAB; Buenos Aires; Argentina; 2021; 128-128
978-987-27591-9-3
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://biofisica.org.ar/reuniones-cientificas/reunionsab-previas/
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
application/pdf
dc.coverage.none.fl_str_mv Nacional
dc.publisher.none.fl_str_mv Sociedad Argentina de Biofísica
publisher.none.fl_str_mv Sociedad Argentina de Biofísica
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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