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Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity
Crude enzyme from Geotrichum candidum S12 exhibited high activity towards hexanol at pH 4.0, distinguishing it from currently known enzymes. To identify the dominant enzyme contributing to this activity, the crude enzyme extract was separated into different fractions by ammonium sulfate precipitatio...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209314/ https://www.ncbi.nlm.nih.gov/pubmed/28050850 http://dx.doi.org/10.1186/s13568-016-0307-8 |
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author | Zhu, Jing Lu, Kuan Xu, Xiaoguang Wang, Xinglong Shi, Junling |
author_facet | Zhu, Jing Lu, Kuan Xu, Xiaoguang Wang, Xinglong Shi, Junling |
author_sort | Zhu, Jing |
collection | PubMed |
description | Crude enzyme from Geotrichum candidum S12 exhibited high activity towards hexanol at pH 4.0, distinguishing it from currently known enzymes. To identify the dominant enzyme contributing to this activity, the crude enzyme extract was separated into different fractions by ammonium sulfate precipitation, MonoQ anion-exchange chromatography, and Sephacryl S-200 gel filtration chromatography. Afraction with high activity towards hexanol at pH 4.0 was obtained, exhibiting 38-fold improved purity and a specific activity of 3802.7 U/mg. After electrophoretic analysis, the fraction showed a molecular weight of 223 kDa by Native-PAGE and 51.4 kDa by SDS-PAGE. The purified fraction was identified as a glutamate dehydrogenase (GDH) by peptide mass fingerprinting data. This fraction showed high activity towards glutamate, α-ketoglutarate, hexanol, and isoamyl alcohol with a Km value of 41.74, 4.01, 20.37, and 19.37 mM, respectively, but with no activity towards methanol, ethanol, 1-propanol, and isobutanol. As a comparison, the GDH from yeast had no activity towards hexanol and other alcohols. Kinetic analysis revealed that glutamate and hexanol served as competitive inhibitors to each other for the purified GDH. The GDH showed the highest activity towards hexanol at pH 4.0 and 30 °C, and was the most stable at pH 2.2–7.0 and ≤40 °C. The presence of ADP, Fe(2+), K(+), and Zn(2+) increased the enzymatic activity towards hexanol and EDTA, Pb(2+), Mn(2+), ATP, and DTT decreased the activity. These novel characteristics expand the reported properties of GDH and suggest the newly characterized GDH has unique potential for practical application. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13568-016-0307-8) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5209314 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-52093142017-01-18 Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity Zhu, Jing Lu, Kuan Xu, Xiaoguang Wang, Xinglong Shi, Junling AMB Express Original Article Crude enzyme from Geotrichum candidum S12 exhibited high activity towards hexanol at pH 4.0, distinguishing it from currently known enzymes. To identify the dominant enzyme contributing to this activity, the crude enzyme extract was separated into different fractions by ammonium sulfate precipitation, MonoQ anion-exchange chromatography, and Sephacryl S-200 gel filtration chromatography. Afraction with high activity towards hexanol at pH 4.0 was obtained, exhibiting 38-fold improved purity and a specific activity of 3802.7 U/mg. After electrophoretic analysis, the fraction showed a molecular weight of 223 kDa by Native-PAGE and 51.4 kDa by SDS-PAGE. The purified fraction was identified as a glutamate dehydrogenase (GDH) by peptide mass fingerprinting data. This fraction showed high activity towards glutamate, α-ketoglutarate, hexanol, and isoamyl alcohol with a Km value of 41.74, 4.01, 20.37, and 19.37 mM, respectively, but with no activity towards methanol, ethanol, 1-propanol, and isobutanol. As a comparison, the GDH from yeast had no activity towards hexanol and other alcohols. Kinetic analysis revealed that glutamate and hexanol served as competitive inhibitors to each other for the purified GDH. The GDH showed the highest activity towards hexanol at pH 4.0 and 30 °C, and was the most stable at pH 2.2–7.0 and ≤40 °C. The presence of ADP, Fe(2+), K(+), and Zn(2+) increased the enzymatic activity towards hexanol and EDTA, Pb(2+), Mn(2+), ATP, and DTT decreased the activity. These novel characteristics expand the reported properties of GDH and suggest the newly characterized GDH has unique potential for practical application. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13568-016-0307-8) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2017-01-03 /pmc/articles/PMC5209314/ /pubmed/28050850 http://dx.doi.org/10.1186/s13568-016-0307-8 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. |
spellingShingle | Original Article Zhu, Jing Lu, Kuan Xu, Xiaoguang Wang, Xinglong Shi, Junling Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity |
title | Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity |
title_full | Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity |
title_fullStr | Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity |
title_full_unstemmed | Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity |
title_short | Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity |
title_sort | purification and characterization of a novel glutamate dehydrogenase from geotrichum candidum with higher alcohol and amino acid activity |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209314/ https://www.ncbi.nlm.nih.gov/pubmed/28050850 http://dx.doi.org/10.1186/s13568-016-0307-8 |
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