A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.)
Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process h...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2015-12-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fpls.2015.01154/full |
| _version_ | 1811268232746631168 |
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| author | Anna eGasperl Annette eMorvan-Bertrand Annette eMorvan-Bertrand Annette eMorvan-Bertrand Marie-Pascale ePrud'Homme Marie-Pascale ePrud'Homme Marie-Pascale ePrud'Homme Eric eVan Der Graaff Eric eVan Der Graaff Thomas eRoitsch Thomas eRoitsch Thomas eRoitsch |
| author_facet | Anna eGasperl Annette eMorvan-Bertrand Annette eMorvan-Bertrand Annette eMorvan-Bertrand Marie-Pascale ePrud'Homme Marie-Pascale ePrud'Homme Marie-Pascale ePrud'Homme Eric eVan Der Graaff Eric eVan Der Graaff Thomas eRoitsch Thomas eRoitsch Thomas eRoitsch |
| author_sort | Anna eGasperl |
| collection | DOAJ |
| description | Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding. |
| first_indexed | 2024-04-12T21:18:08Z |
| format | Article |
| id | doaj.art-63a39b664a2d4468a928271a6aeacb06 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-12T21:18:08Z |
| publishDate | 2015-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-63a39b664a2d4468a928271a6aeacb062022-12-22T03:16:22ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2015-12-01610.3389/fpls.2015.01154150409A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.)Anna eGasperl0Annette eMorvan-Bertrand1Annette eMorvan-Bertrand2Annette eMorvan-Bertrand3Marie-Pascale ePrud'Homme4Marie-Pascale ePrud'Homme5Marie-Pascale ePrud'Homme6Eric eVan Der Graaff7Eric eVan Der Graaff8Thomas eRoitsch9Thomas eRoitsch10Thomas eRoitsch11Karl-Franzens-Universität GrazNormandie UniversitéUCBNINRANormandie UniversitéUCBNINRAKarl-Franzens-Universität GrazCopenhagen UniversityCopenhagen UniversityKarl-Franzens-Universität GrazCzech Globe AS CRDespite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding.http://journal.frontiersin.org/Journal/10.3389/fpls.2015.01154/fullEnzymatic activitykinetic assayPerennial ryegrassFructan degradationFructan exohydrolase1-FEH |
| spellingShingle | Anna eGasperl Annette eMorvan-Bertrand Annette eMorvan-Bertrand Annette eMorvan-Bertrand Marie-Pascale ePrud'Homme Marie-Pascale ePrud'Homme Marie-Pascale ePrud'Homme Eric eVan Der Graaff Eric eVan Der Graaff Thomas eRoitsch Thomas eRoitsch Thomas eRoitsch A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.) Frontiers in Plant Science Enzymatic activity kinetic assay Perennial ryegrass Fructan degradation Fructan exohydrolase 1-FEH |
| title | A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.) |
| title_full | A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.) |
| title_fullStr | A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.) |
| title_full_unstemmed | A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.) |
| title_short | A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.) |
| title_sort | simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass lolium perenne l |
| topic | Enzymatic activity kinetic assay Perennial ryegrass Fructan degradation Fructan exohydrolase 1-FEH |
| url | http://journal.frontiersin.org/Journal/10.3389/fpls.2015.01154/full |
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