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A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese
BACKGROUND: Lipolysis is an important process of cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is perform...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2008
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2442053/ https://www.ncbi.nlm.nih.gov/pubmed/18498642 http://dx.doi.org/10.1186/1475-2859-7-16 |
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author | Dherbécourt, Julien Falentin, Hélène Canaan, Stéphane Thierry, Anne |
author_facet | Dherbécourt, Julien Falentin, Hélène Canaan, Stéphane Thierry, Anne |
author_sort | Dherbécourt, Julien |
collection | PubMed |
description | BACKGROUND: Lipolysis is an important process of cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027(T )was recently obtained in our laboratory. The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study of their activity and specificity of hydrolysed substrates. RESULTS: The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among P. freudenreichii CIP103027(T )predicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in E. coli and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method. CONCLUSION: We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a P. freudenreichii strain, more exhaustively than classical approaches. This study highlights the interest in using the automatic search of motifs, with the manual search of homology to previously characterised enzymes as a complementary method. Only further characterisations would permit the identification of the esterases of P. freudenreichii involved in the lipolysis in Emmental cheese. |
format | Text |
id | pubmed-2442053 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-24420532008-07-01 A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese Dherbécourt, Julien Falentin, Hélène Canaan, Stéphane Thierry, Anne Microb Cell Fact Research BACKGROUND: Lipolysis is an important process of cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027(T )was recently obtained in our laboratory. The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study of their activity and specificity of hydrolysed substrates. RESULTS: The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among P. freudenreichii CIP103027(T )predicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in E. coli and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method. CONCLUSION: We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a P. freudenreichii strain, more exhaustively than classical approaches. This study highlights the interest in using the automatic search of motifs, with the manual search of homology to previously characterised enzymes as a complementary method. Only further characterisations would permit the identification of the esterases of P. freudenreichii involved in the lipolysis in Emmental cheese. BioMed Central 2008-05-22 /pmc/articles/PMC2442053/ /pubmed/18498642 http://dx.doi.org/10.1186/1475-2859-7-16 Text en Copyright © 2008 Dherbécourt et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Dherbécourt, Julien Falentin, Hélène Canaan, Stéphane Thierry, Anne A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese |
title | A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese |
title_full | A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese |
title_fullStr | A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese |
title_full_unstemmed | A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese |
title_short | A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese |
title_sort | genomic search approach to identify esterases in propionibacterium freudenreichii involved in the formation of flavour in emmental cheese |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2442053/ https://www.ncbi.nlm.nih.gov/pubmed/18498642 http://dx.doi.org/10.1186/1475-2859-7-16 |
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