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A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes

BACKGROUND: Glycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzy...

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Autores principales: Herlet, J., Kornberger, P., Roessler, B., Glanz, J., Schwarz, W. H., Liebl, W., Zverlov, V. V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637330/
https://www.ncbi.nlm.nih.gov/pubmed/29046720
http://dx.doi.org/10.1186/s13068-017-0923-9
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author Herlet, J.
Kornberger, P.
Roessler, B.
Glanz, J.
Schwarz, W. H.
Liebl, W.
Zverlov, V. V.
author_facet Herlet, J.
Kornberger, P.
Roessler, B.
Glanz, J.
Schwarz, W. H.
Liebl, W.
Zverlov, V. V.
author_sort Herlet, J.
collection PubMed
description BACKGROUND: Glycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzymes are generally determined by two separate measurements. However, these lead to a two-dimensional assessment of the pH range at one temperature (and vice versa) and do not allow prediction of the relative enzymatic performance at any pH/temperature combination of interest. In this work, we demonstrate a new method that is based on experimental data and visualizes the relationship among pH, temperature, and activity at a glance in a three-dimensional contour plot. RESULTS: In this study, we present a method to determine the relative activity of an enzyme at 96 different combinations of pH and temperature in parallel. For this purpose, we used a gradient PCR cycler and a citrate–phosphate-based buffer system in microtiter plates. The approach was successfully tested with various substrates and diverse assays for glycoside hydrolases. Furthermore, its applicability was demonstrated for single enzymes using the endoglucanase Cel8A from Clostridium thermocellum as well as the commercially available complex enzyme mixture Celluclast(®). Thereby, we developed a fast and adaptable method to determine simultaneously both pH and temperature ranges of enzymes over a wide range of conditions, an easy transformation of the experimental data into a contour plot for visualization, and the necessary controls. With our method, the suitability of an enzyme or enzyme mixture for any chosen combination of temperature and pH can easily be assessed at a glance. CONCLUSIONS: We propose a method that offers significant advantages over commonly used methods to determine the pH and temperature ranges of enzymes. The overall relationship among pH, temperature, and activity is visualized. Our method could be applied to evaluate exactly what conditions have to be met for optimal utilization of an enzyme or enzyme mixture for both lab-scale and industrial processes. Adaptation to other enzymes, including proteases, should be possible and the method may also lead to a platform for additional applications, such as inactivation kinetics analysis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-017-0923-9) contains supplementary material, which is available to authorized users.
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spelling pubmed-56373302017-10-18 A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes Herlet, J. Kornberger, P. Roessler, B. Glanz, J. Schwarz, W. H. Liebl, W. Zverlov, V. V. Biotechnol Biofuels Methodology BACKGROUND: Glycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzymes are generally determined by two separate measurements. However, these lead to a two-dimensional assessment of the pH range at one temperature (and vice versa) and do not allow prediction of the relative enzymatic performance at any pH/temperature combination of interest. In this work, we demonstrate a new method that is based on experimental data and visualizes the relationship among pH, temperature, and activity at a glance in a three-dimensional contour plot. RESULTS: In this study, we present a method to determine the relative activity of an enzyme at 96 different combinations of pH and temperature in parallel. For this purpose, we used a gradient PCR cycler and a citrate–phosphate-based buffer system in microtiter plates. The approach was successfully tested with various substrates and diverse assays for glycoside hydrolases. Furthermore, its applicability was demonstrated for single enzymes using the endoglucanase Cel8A from Clostridium thermocellum as well as the commercially available complex enzyme mixture Celluclast(®). Thereby, we developed a fast and adaptable method to determine simultaneously both pH and temperature ranges of enzymes over a wide range of conditions, an easy transformation of the experimental data into a contour plot for visualization, and the necessary controls. With our method, the suitability of an enzyme or enzyme mixture for any chosen combination of temperature and pH can easily be assessed at a glance. CONCLUSIONS: We propose a method that offers significant advantages over commonly used methods to determine the pH and temperature ranges of enzymes. The overall relationship among pH, temperature, and activity is visualized. Our method could be applied to evaluate exactly what conditions have to be met for optimal utilization of an enzyme or enzyme mixture for both lab-scale and industrial processes. Adaptation to other enzymes, including proteases, should be possible and the method may also lead to a platform for additional applications, such as inactivation kinetics analysis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-017-0923-9) contains supplementary material, which is available to authorized users. BioMed Central 2017-10-11 /pmc/articles/PMC5637330/ /pubmed/29046720 http://dx.doi.org/10.1186/s13068-017-0923-9 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology
Herlet, J.
Kornberger, P.
Roessler, B.
Glanz, J.
Schwarz, W. H.
Liebl, W.
Zverlov, V. V.
A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes
title A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes
title_full A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes
title_fullStr A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes
title_full_unstemmed A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes
title_short A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes
title_sort new method to evaluate temperature vs. ph activity profiles for biotechnological relevant enzymes
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637330/
https://www.ncbi.nlm.nih.gov/pubmed/29046720
http://dx.doi.org/10.1186/s13068-017-0923-9
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