In vivo assay to identify bacteria with β-glucosidase activity
[Background]: β-Glucosidase assay is performed with purified or semipurified enzymes extracted from cell lysis. However, in screening studies, to find bacteria with β-glucosidase activity among many tested bacteria, a fast method without cell lysis is desirable. In that objective, we report an in vi...
| Autores: | , , , , , |
|---|---|
| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2017 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/171408 |
| Acceso en línea: | http://hdl.handle.net/10261/171408 |
| Access Level: | acceso abierto |
| Palabra clave: | β-Glucosidase assay Screening β-Glucosidase producer strain Antimicrobial p-Nitrophenyl-β-glucopyranoside Bifidobacterium Cell lysis Enzymatic test Lactobacillus p-Nitrophenol |
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In vivo assay to identify bacteria with β-glucosidase activityStrahsburger, ErwinLópez de Lacey, Ana M.Marotti, IlariaDi Gioia, DianaBiavati, BrunoDinelli, Giovanniβ-Glucosidase assayScreeningβ-Glucosidase producer strainAntimicrobialp-Nitrophenyl-β-glucopyranosideBifidobacteriumCell lysisEnzymatic testLactobacillusp-Nitrophenol[Background]: β-Glucosidase assay is performed with purified or semipurified enzymes extracted from cell lysis. However, in screening studies, to find bacteria with β-glucosidase activity among many tested bacteria, a fast method without cell lysis is desirable. In that objective, we report an in vivo β-glucosidase assay as a fast method to find a β-glucosidase producer strain. [Results]: The method consists in growing the strains for testing in a medium supplemented with the artificial substrate p-nitrophenyl-β-glucopyranoside (pNPG). The presence of β-glucosidases converts the substrate to p-nitrophenol (pNP), a molecule that can be easily measured in the supernatant spectrophotometrically at 405 nm. The assay was evaluated using two Bifidobacterium strains: Bifidobacterium longum B7254 strain that lacks β-glucosidase activity and Bifidobacterium pseudocatenulatum B7003 strain that shows β-glucosidase activity. The addition of sodium carbonate during pNP measurement increases the sensitivity of pNP detection and avoids the masking of absorbance by the culture medium. Furthermore, we show that pNP is a stable enzymatic product, not metabolized by bacteria, but with an inhibitory effect on cell growth. The β-glucosidase activity was measured as units of enzyme per gram per minute per dry cell weight. This method also allowed the identification of Lactobacillus strains with higher β-glucosidase activity among several lactobacillus species. [Conclusion]: This in vivo β-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with β-glucosidase activity but also with high β-glucosidase activity.The study was funded by the University of Bologna, RFO Program number J61J10000790001. Erwin Strahsburger received a scholarship within the Erasmus Mundus Program. Ana M. Lopez de Lacey received a fellowship within the JAE-CSIC predoctoral Programme. The study was also financially funded by the Universidad Arturo Prat, internal projects number VRIIP0218-15 and VRIIP01-14.Peer ReviewedElsevierUniversità di BolognaEuropean CommissionConsejo Superior de Investigaciones Científicas (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2018201820172018info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/171408reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttps://doi.org/10.1016/j.ejbt.2017.08.010Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1714082026-05-22T06:33:51Z |
| dc.title.none.fl_str_mv |
In vivo assay to identify bacteria with β-glucosidase activity |
| title |
In vivo assay to identify bacteria with β-glucosidase activity |
| spellingShingle |
In vivo assay to identify bacteria with β-glucosidase activity Strahsburger, Erwin β-Glucosidase assay Screening β-Glucosidase producer strain Antimicrobial p-Nitrophenyl-β-glucopyranoside Bifidobacterium Cell lysis Enzymatic test Lactobacillus p-Nitrophenol |
| title_short |
In vivo assay to identify bacteria with β-glucosidase activity |
| title_full |
In vivo assay to identify bacteria with β-glucosidase activity |
| title_fullStr |
In vivo assay to identify bacteria with β-glucosidase activity |
| title_full_unstemmed |
In vivo assay to identify bacteria with β-glucosidase activity |
| title_sort |
In vivo assay to identify bacteria with β-glucosidase activity |
| dc.creator.none.fl_str_mv |
Strahsburger, Erwin López de Lacey, Ana M. Marotti, Ilaria Di Gioia, Diana Biavati, Bruno Dinelli, Giovanni |
| author |
Strahsburger, Erwin |
| author_facet |
Strahsburger, Erwin López de Lacey, Ana M. Marotti, Ilaria Di Gioia, Diana Biavati, Bruno Dinelli, Giovanni |
| author_role |
author |
| author2 |
López de Lacey, Ana M. Marotti, Ilaria Di Gioia, Diana Biavati, Bruno Dinelli, Giovanni |
| author2_role |
author author author author author |
| dc.contributor.none.fl_str_mv |
Università di Bologna European Commission Consejo Superior de Investigaciones Científicas (España) Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72] |
| dc.subject.none.fl_str_mv |
β-Glucosidase assay Screening β-Glucosidase producer strain Antimicrobial p-Nitrophenyl-β-glucopyranoside Bifidobacterium Cell lysis Enzymatic test Lactobacillus p-Nitrophenol |
| topic |
β-Glucosidase assay Screening β-Glucosidase producer strain Antimicrobial p-Nitrophenyl-β-glucopyranoside Bifidobacterium Cell lysis Enzymatic test Lactobacillus p-Nitrophenol |
| description |
[Background]: β-Glucosidase assay is performed with purified or semipurified enzymes extracted from cell lysis. However, in screening studies, to find bacteria with β-glucosidase activity among many tested bacteria, a fast method without cell lysis is desirable. In that objective, we report an in vivo β-glucosidase assay as a fast method to find a β-glucosidase producer strain. [Results]: The method consists in growing the strains for testing in a medium supplemented with the artificial substrate p-nitrophenyl-β-glucopyranoside (pNPG). The presence of β-glucosidases converts the substrate to p-nitrophenol (pNP), a molecule that can be easily measured in the supernatant spectrophotometrically at 405 nm. The assay was evaluated using two Bifidobacterium strains: Bifidobacterium longum B7254 strain that lacks β-glucosidase activity and Bifidobacterium pseudocatenulatum B7003 strain that shows β-glucosidase activity. The addition of sodium carbonate during pNP measurement increases the sensitivity of pNP detection and avoids the masking of absorbance by the culture medium. Furthermore, we show that pNP is a stable enzymatic product, not metabolized by bacteria, but with an inhibitory effect on cell growth. The β-glucosidase activity was measured as units of enzyme per gram per minute per dry cell weight. This method also allowed the identification of Lactobacillus strains with higher β-glucosidase activity among several lactobacillus species. [Conclusion]: This in vivo β-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with β-glucosidase activity but also with high β-glucosidase activity. |
| publishDate |
2017 |
| dc.date.none.fl_str_mv |
2017 2018 2018 2018 |
| dc.type.none.fl_str_mv |
info:eu-repo/semantics/article http://purl.org/coar/resource_type/c_6501 Publisher's version info:eu-repo/semantics/publishedVersion |
| format |
article |
| status_str |
publishedVersion |
| dc.identifier.none.fl_str_mv |
http://hdl.handle.net/10261/171408 |
| url |
http://hdl.handle.net/10261/171408 |
| dc.language.none.fl_str_mv |
Inglés |
| language_invalid_str_mv |
Inglés |
| dc.relation.none.fl_str_mv |
https://doi.org/10.1016/j.ejbt.2017.08.010 Sí |
| dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess |
| eu_rights_str_mv |
openAccess |
| dc.publisher.none.fl_str_mv |
Elsevier |
| publisher.none.fl_str_mv |
Elsevier |
| dc.source.none.fl_str_mv |
reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC instname:Consejo Superior de Investigaciones Científicas (CSIC) |
| instname_str |
Consejo Superior de Investigaciones Científicas (CSIC) |
| reponame_str |
DIGITAL.CSIC. Repositorio Institucional del CSIC |
| collection |
DIGITAL.CSIC. Repositorio Institucional del CSIC |
| repository.name.fl_str_mv |
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| repository.mail.fl_str_mv |
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1869405027770040320 |
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15,812429 |