Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba
Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a...
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ftdoajarticles:oai:doaj.org/article:6d8889f675764f75837121388814fb49 2023-05-15T16:08:26+02:00 Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba Jikun Xia Wanmeng Xin Fang Wang Wancui Xie Yi Liu Jiakun Xu 2022-09-01T00:00:00Z https://doi.org/10.3390/ijms231810478 https://doaj.org/article/6d8889f675764f75837121388814fb49 EN eng MDPI AG https://www.mdpi.com/1422-0067/23/18/10478 https://doaj.org/toc/1661-6596 https://doaj.org/toc/1422-0067 doi:10.3390/ijms231810478 1422-0067 1661-6596 https://doaj.org/article/6d8889f675764f75837121388814fb49 International Journal of Molecular Sciences, Vol 23, Iss 10478, p 10478 (2022) fructose-1,6-bisphosphate aldolase Euphausia superba heterologous expression molecular simulation point mutation Biology (General) QH301-705.5 Chemistry QD1-999 article 2022 ftdoajarticles https://doi.org/10.3390/ijms231810478 2022-12-31T00:34:17Z Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a 365-amino-acid protein. The fructose-1,6-bisphosphate aldolase gene was expressed in Escherichia coli ( E. coli ). A highly purified protein was obtained using HisTrap HP affinity chromatography and size-exclusion chromatography. The predicted three-dimensional structure of EsFBA showed a 65.66% homology with human aldolase, whereas it had the highest homology (84.38%) with the FBA of Penaeus vannamei . Recombinant EsFBA had the highest activity at 45 °C and pH 7.0 in phosphate buffer. By examining the activity of metal ions and EDTA, we found that the effect of metal ions and EDTA on EsFBA’s enzyme activity was not significant, while the presence of borohydride severely reduced the enzymatic activity; thus, EsFBA was confirmed to be a class I aldolase. Furthermore, targeted mutations at positions 34, 147, 188, and 230 confirmed that they are key amino acid residues for EsFBA. Article in Journal/Newspaper Euphausia superba Directory of Open Access Journals: DOAJ Articles International Journal of Molecular Sciences 23 18 10478 |
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topic |
fructose-1,6-bisphosphate aldolase Euphausia superba heterologous expression molecular simulation point mutation Biology (General) QH301-705.5 Chemistry QD1-999 |
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fructose-1,6-bisphosphate aldolase Euphausia superba heterologous expression molecular simulation point mutation Biology (General) QH301-705.5 Chemistry QD1-999 Jikun Xia Wanmeng Xin Fang Wang Wancui Xie Yi Liu Jiakun Xu Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba |
topic_facet |
fructose-1,6-bisphosphate aldolase Euphausia superba heterologous expression molecular simulation point mutation Biology (General) QH301-705.5 Chemistry QD1-999 |
description |
Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a 365-amino-acid protein. The fructose-1,6-bisphosphate aldolase gene was expressed in Escherichia coli ( E. coli ). A highly purified protein was obtained using HisTrap HP affinity chromatography and size-exclusion chromatography. The predicted three-dimensional structure of EsFBA showed a 65.66% homology with human aldolase, whereas it had the highest homology (84.38%) with the FBA of Penaeus vannamei . Recombinant EsFBA had the highest activity at 45 °C and pH 7.0 in phosphate buffer. By examining the activity of metal ions and EDTA, we found that the effect of metal ions and EDTA on EsFBA’s enzyme activity was not significant, while the presence of borohydride severely reduced the enzymatic activity; thus, EsFBA was confirmed to be a class I aldolase. Furthermore, targeted mutations at positions 34, 147, 188, and 230 confirmed that they are key amino acid residues for EsFBA. |
format |
Article in Journal/Newspaper |
author |
Jikun Xia Wanmeng Xin Fang Wang Wancui Xie Yi Liu Jiakun Xu |
author_facet |
Jikun Xia Wanmeng Xin Fang Wang Wancui Xie Yi Liu Jiakun Xu |
author_sort |
Jikun Xia |
title |
Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba |
title_short |
Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba |
title_full |
Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba |
title_fullStr |
Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba |
title_full_unstemmed |
Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba |
title_sort |
cloning and characterization of fructose-1,6-bisphosphate aldolase from euphausia superba |
publisher |
MDPI AG |
publishDate |
2022 |
url |
https://doi.org/10.3390/ijms231810478 https://doaj.org/article/6d8889f675764f75837121388814fb49 |
genre |
Euphausia superba |
genre_facet |
Euphausia superba |
op_source |
International Journal of Molecular Sciences, Vol 23, Iss 10478, p 10478 (2022) |
op_relation |
https://www.mdpi.com/1422-0067/23/18/10478 https://doaj.org/toc/1661-6596 https://doaj.org/toc/1422-0067 doi:10.3390/ijms231810478 1422-0067 1661-6596 https://doaj.org/article/6d8889f675764f75837121388814fb49 |
op_doi |
https://doi.org/10.3390/ijms231810478 |
container_title |
International Journal of Molecular Sciences |
container_volume |
23 |
container_issue |
18 |
container_start_page |
10478 |
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1766404499093585920 |