A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro

Autores
Colombatto, Dario; Beauchemin, K. A.
Año de publicación
2009
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In vitro experiments were conducted to examine the characteristics and mode of action of a protease that increased the ruminai fiber digestibility of alfalfa hay. A commercial source of protease (Protex 6L, Genencor Int., Rochester, NY), already characterized for its main activities, was further analyzed to determine protease activity in response to pH, molecular size by SDS-PAGE, specificity to degrade model or feed substrates, response to autoclaving, and action of specific protease inhibitors in the absence or presence of ruminai fluid. In addition, batch culture in vitro incubations in buffered ruminal fluid were conducted to compare the enzyme product with purified protease sources, and dose-response studies (0 to 10 μL/g of forage DM) were carried out using alfalfa hay as a substrate. The enzyme product was shown to be an alkaline protease (optimum pH >8.5) of approximately 30 kDa. Specificity in the absence of ruminai fluid showed that the enzyme was active against gelatin and casein to the same extent, whereas it had limited (21% of the total) activity on BSA. In the presence of ruminai fluid and with the use of feed substrates, the protease increased (P < 0.05) 22-h IVDMD (%) of alfalfa hay, fresh corn silage, dry-rolled corn, and a total mixed ration composed of the 3 ingredients (39.5 vs. 44.7; 50.3 vs. 54.5; 63.8 vs. 68.4; and 55.4 vs. 56.4 for control vs. protease for each feed, respectively). Inhibitor studies in the absence of ruminai fluid indicated that the enzyme was inhibited most by a serine protease inhibitor but not by cysteineor metalloprotease inhibitors (10 vs. 1.9 and 0.1%, respectively). In the presence of ruminal fluid, the serine protease inhibitor reversed (P < 0.05) the increase in alfalfa IVDMD achieved by the enzyme product, such that IVDMD was similar to that of the control treatment. Comparisons among different proteases revealed that only pure subtilisin achieved increases in IVDMD that were similar to those with protease, suggesting the serine protease was subtilisin-like (EC 3.4.1.62). Dose-response studies using alfalfa hay as substraje showed quadratic responses in IVDMD, NDF digestion, and hemicellulose and protein disappearance. It is postulated that this enzyme acts by removing structural proteins in the cell wall, allowing ruminal microbes to gain faster access to digestible substrates.
Fil: Colombatto, Dario. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina
Fil: Beauchemin, K. A.. Agriculture Et Agroalimentaire Canada; Canadá
Materia
EXOGENOUS PROTEASE
FIBER
IN VITRO DISAPPEARANCE
RUMEN DIGESTIBILITY
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:11336/112666

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oai_identifier_str oai:ri.conicet.gov.ar:11336/112666
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitroColombatto, DarioBeauchemin, K. A.EXOGENOUS PROTEASEFIBERIN VITRO DISAPPEARANCERUMEN DIGESTIBILITYhttps://purl.org/becyt/ford/4.3https://purl.org/becyt/ford/4In vitro experiments were conducted to examine the characteristics and mode of action of a protease that increased the ruminai fiber digestibility of alfalfa hay. A commercial source of protease (Protex 6L, Genencor Int., Rochester, NY), already characterized for its main activities, was further analyzed to determine protease activity in response to pH, molecular size by SDS-PAGE, specificity to degrade model or feed substrates, response to autoclaving, and action of specific protease inhibitors in the absence or presence of ruminai fluid. In addition, batch culture in vitro incubations in buffered ruminal fluid were conducted to compare the enzyme product with purified protease sources, and dose-response studies (0 to 10 μL/g of forage DM) were carried out using alfalfa hay as a substrate. The enzyme product was shown to be an alkaline protease (optimum pH >8.5) of approximately 30 kDa. Specificity in the absence of ruminai fluid showed that the enzyme was active against gelatin and casein to the same extent, whereas it had limited (21% of the total) activity on BSA. In the presence of ruminai fluid and with the use of feed substrates, the protease increased (P < 0.05) 22-h IVDMD (%) of alfalfa hay, fresh corn silage, dry-rolled corn, and a total mixed ration composed of the 3 ingredients (39.5 vs. 44.7; 50.3 vs. 54.5; 63.8 vs. 68.4; and 55.4 vs. 56.4 for control vs. protease for each feed, respectively). Inhibitor studies in the absence of ruminai fluid indicated that the enzyme was inhibited most by a serine protease inhibitor but not by cysteineor metalloprotease inhibitors (10 vs. 1.9 and 0.1%, respectively). In the presence of ruminal fluid, the serine protease inhibitor reversed (P < 0.05) the increase in alfalfa IVDMD achieved by the enzyme product, such that IVDMD was similar to that of the control treatment. Comparisons among different proteases revealed that only pure subtilisin achieved increases in IVDMD that were similar to those with protease, suggesting the serine protease was subtilisin-like (EC 3.4.1.62). Dose-response studies using alfalfa hay as substraje showed quadratic responses in IVDMD, NDF digestion, and hemicellulose and protein disappearance. It is postulated that this enzyme acts by removing structural proteins in the cell wall, allowing ruminal microbes to gain faster access to digestible substrates.Fil: Colombatto, Dario. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Beauchemin, K. A.. Agriculture Et Agroalimentaire Canada; CanadáAmerican Society of Animal Science2009-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/112666Colombatto, Dario; Beauchemin, K. A.; A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro; American Society of Animal Science; Journal of Animal Science; 87; 3; 3-2009; 1097-11050021-8812CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.2527/jas.2008-1262info:eu-repo/semantics/altIdentifier/url/https://academic.oup.com/jas/article-abstract/87/3/1097/4731175info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:41:51Zoai:ri.conicet.gov.ar:11336/112666instacron: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 10:41:52.001CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro
title A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro
spellingShingle A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro
Colombatto, Dario
EXOGENOUS PROTEASE
FIBER
IN VITRO DISAPPEARANCE
RUMEN DIGESTIBILITY
title_short A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro
title_full A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro
title_fullStr A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro
title_full_unstemmed A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro
title_sort A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro
dc.creator.none.fl_str_mv Colombatto, Dario
Beauchemin, K. A.
author Colombatto, Dario
author_facet Colombatto, Dario
Beauchemin, K. A.
author_role author
author2 Beauchemin, K. A.
author2_role author
dc.subject.none.fl_str_mv EXOGENOUS PROTEASE
FIBER
IN VITRO DISAPPEARANCE
RUMEN DIGESTIBILITY
topic EXOGENOUS PROTEASE
FIBER
IN VITRO DISAPPEARANCE
RUMEN DIGESTIBILITY
purl_subject.fl_str_mv https://purl.org/becyt/ford/4.3
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv In vitro experiments were conducted to examine the characteristics and mode of action of a protease that increased the ruminai fiber digestibility of alfalfa hay. A commercial source of protease (Protex 6L, Genencor Int., Rochester, NY), already characterized for its main activities, was further analyzed to determine protease activity in response to pH, molecular size by SDS-PAGE, specificity to degrade model or feed substrates, response to autoclaving, and action of specific protease inhibitors in the absence or presence of ruminai fluid. In addition, batch culture in vitro incubations in buffered ruminal fluid were conducted to compare the enzyme product with purified protease sources, and dose-response studies (0 to 10 μL/g of forage DM) were carried out using alfalfa hay as a substrate. The enzyme product was shown to be an alkaline protease (optimum pH >8.5) of approximately 30 kDa. Specificity in the absence of ruminai fluid showed that the enzyme was active against gelatin and casein to the same extent, whereas it had limited (21% of the total) activity on BSA. In the presence of ruminai fluid and with the use of feed substrates, the protease increased (P < 0.05) 22-h IVDMD (%) of alfalfa hay, fresh corn silage, dry-rolled corn, and a total mixed ration composed of the 3 ingredients (39.5 vs. 44.7; 50.3 vs. 54.5; 63.8 vs. 68.4; and 55.4 vs. 56.4 for control vs. protease for each feed, respectively). Inhibitor studies in the absence of ruminai fluid indicated that the enzyme was inhibited most by a serine protease inhibitor but not by cysteineor metalloprotease inhibitors (10 vs. 1.9 and 0.1%, respectively). In the presence of ruminal fluid, the serine protease inhibitor reversed (P < 0.05) the increase in alfalfa IVDMD achieved by the enzyme product, such that IVDMD was similar to that of the control treatment. Comparisons among different proteases revealed that only pure subtilisin achieved increases in IVDMD that were similar to those with protease, suggesting the serine protease was subtilisin-like (EC 3.4.1.62). Dose-response studies using alfalfa hay as substraje showed quadratic responses in IVDMD, NDF digestion, and hemicellulose and protein disappearance. It is postulated that this enzyme acts by removing structural proteins in the cell wall, allowing ruminal microbes to gain faster access to digestible substrates.
Fil: Colombatto, Dario. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina
Fil: Beauchemin, K. A.. Agriculture Et Agroalimentaire Canada; Canadá
description In vitro experiments were conducted to examine the characteristics and mode of action of a protease that increased the ruminai fiber digestibility of alfalfa hay. A commercial source of protease (Protex 6L, Genencor Int., Rochester, NY), already characterized for its main activities, was further analyzed to determine protease activity in response to pH, molecular size by SDS-PAGE, specificity to degrade model or feed substrates, response to autoclaving, and action of specific protease inhibitors in the absence or presence of ruminai fluid. In addition, batch culture in vitro incubations in buffered ruminal fluid were conducted to compare the enzyme product with purified protease sources, and dose-response studies (0 to 10 μL/g of forage DM) were carried out using alfalfa hay as a substrate. The enzyme product was shown to be an alkaline protease (optimum pH >8.5) of approximately 30 kDa. Specificity in the absence of ruminai fluid showed that the enzyme was active against gelatin and casein to the same extent, whereas it had limited (21% of the total) activity on BSA. In the presence of ruminai fluid and with the use of feed substrates, the protease increased (P < 0.05) 22-h IVDMD (%) of alfalfa hay, fresh corn silage, dry-rolled corn, and a total mixed ration composed of the 3 ingredients (39.5 vs. 44.7; 50.3 vs. 54.5; 63.8 vs. 68.4; and 55.4 vs. 56.4 for control vs. protease for each feed, respectively). Inhibitor studies in the absence of ruminai fluid indicated that the enzyme was inhibited most by a serine protease inhibitor but not by cysteineor metalloprotease inhibitors (10 vs. 1.9 and 0.1%, respectively). In the presence of ruminal fluid, the serine protease inhibitor reversed (P < 0.05) the increase in alfalfa IVDMD achieved by the enzyme product, such that IVDMD was similar to that of the control treatment. Comparisons among different proteases revealed that only pure subtilisin achieved increases in IVDMD that were similar to those with protease, suggesting the serine protease was subtilisin-like (EC 3.4.1.62). Dose-response studies using alfalfa hay as substraje showed quadratic responses in IVDMD, NDF digestion, and hemicellulose and protein disappearance. It is postulated that this enzyme acts by removing structural proteins in the cell wall, allowing ruminal microbes to gain faster access to digestible substrates.
publishDate 2009
dc.date.none.fl_str_mv 2009-03
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/11336/112666
Colombatto, Dario; Beauchemin, K. A.; A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro; American Society of Animal Science; Journal of Animal Science; 87; 3; 3-2009; 1097-1105
0021-8812
CONICET Digital
CONICET
url http://hdl.handle.net/11336/112666
identifier_str_mv Colombatto, Dario; Beauchemin, K. A.; A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro; American Society of Animal Science; Journal of Animal Science; 87; 3; 3-2009; 1097-1105
0021-8812
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.2527/jas.2008-1262
info:eu-repo/semantics/altIdentifier/url/https://academic.oup.com/jas/article-abstract/87/3/1097/4731175
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 American Society of Animal Science
publisher.none.fl_str_mv American Society of Animal Science
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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