Development of a colourimetric assay for glycosynthases
The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput meth...
Published in: | Journal of Biotechnology |
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Elsevier Science
2017
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ftfzjuelichnvdb:oai:juser.fz-juelich.de:836199 2023-05-15T14:00:31+02:00 Development of a colourimetric assay for glycosynthases Hayes, Marc R. Bochinsky, Kevin Seibt, Lisa S. Elling, Lothar Pietruszka, Jörg DE 2017 https://juser.fz-juelich.de/record/836199 https://juser.fz-juelich.de/search?p=id:%22FZJ-2017-05320%22 eng eng Elsevier Science info:eu-repo/semantics/altIdentifier/issn/0168-1656 info:eu-repo/semantics/altIdentifier/pmid/pmid:28193496 info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jbiotec.2017.02.005 info:eu-repo/semantics/altIdentifier/issn/1873-4863 info:eu-repo/semantics/altIdentifier/wos/WOS:000408021800020 https://juser.fz-juelich.de/record/836199 https://juser.fz-juelich.de/search?p=id:%22FZJ-2017-05320%22 info:eu-repo/semantics/closedAccess Journal of biotechnology 257, 162-170 (2017). doi:10.1016/j.jbiotec.2017.02.005 info:eu-repo/classification/ddc/540 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 2017 ftfzjuelichnvdb https://doi.org/10.1016/j.jbiotec.2017.02.005 2022-07-14T11:13:18Z The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput methods for reaction analysis and screening of potential glycosynthase variants. Fluoride detection, via silyl ether chemosensors, has recently shown high potential for the identification of glycosynthase mutants in a high-throughput manner, though limited by the low maximal detection concentration. In the present paper, we describe a new version of a glycosynthase activity assay using a silyl ether of p-nitrophenol, allowing fast reliable detection of fluoride even at concentrations of 4 mM and higher. This improvement of detection allows not only screening and identification but also kinetic characterisation of glycosynthases and synthetic reactions in a fast microtiter plate format. The applicability of the assay was successfully demonstrated by the biochemical characterisation of the mesophilic β-glucosynthase of Abg-E358S (Rhizobium radiobacter) and psychrotolerant β-glucosynthase BglU-E377A (Micrococcus antarcticus). The limitation of hyperthermophilic glycosidases as potential glycosynthases, when using glycosyl fluoride donors, was also illustrated by the example of the putative β-galactosidase GalPf from Pyrococcus furiosus. Article in Journal/Newspaper Antarc* antarcticus Forschungszentrum Jülich: JuSER (Juelich Shared Electronic Resources) Journal of Biotechnology 257 162 170 |
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Forschungszentrum Jülich: JuSER (Juelich Shared Electronic Resources) |
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ftfzjuelichnvdb |
language |
English |
topic |
info:eu-repo/classification/ddc/540 |
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info:eu-repo/classification/ddc/540 Hayes, Marc R. Bochinsky, Kevin Seibt, Lisa S. Elling, Lothar Pietruszka, Jörg Development of a colourimetric assay for glycosynthases |
topic_facet |
info:eu-repo/classification/ddc/540 |
description |
The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput methods for reaction analysis and screening of potential glycosynthase variants. Fluoride detection, via silyl ether chemosensors, has recently shown high potential for the identification of glycosynthase mutants in a high-throughput manner, though limited by the low maximal detection concentration. In the present paper, we describe a new version of a glycosynthase activity assay using a silyl ether of p-nitrophenol, allowing fast reliable detection of fluoride even at concentrations of 4 mM and higher. This improvement of detection allows not only screening and identification but also kinetic characterisation of glycosynthases and synthetic reactions in a fast microtiter plate format. The applicability of the assay was successfully demonstrated by the biochemical characterisation of the mesophilic β-glucosynthase of Abg-E358S (Rhizobium radiobacter) and psychrotolerant β-glucosynthase BglU-E377A (Micrococcus antarcticus). The limitation of hyperthermophilic glycosidases as potential glycosynthases, when using glycosyl fluoride donors, was also illustrated by the example of the putative β-galactosidase GalPf from Pyrococcus furiosus. |
format |
Article in Journal/Newspaper |
author |
Hayes, Marc R. Bochinsky, Kevin Seibt, Lisa S. Elling, Lothar Pietruszka, Jörg |
author_facet |
Hayes, Marc R. Bochinsky, Kevin Seibt, Lisa S. Elling, Lothar Pietruszka, Jörg |
author_sort |
Hayes, Marc R. |
title |
Development of a colourimetric assay for glycosynthases |
title_short |
Development of a colourimetric assay for glycosynthases |
title_full |
Development of a colourimetric assay for glycosynthases |
title_fullStr |
Development of a colourimetric assay for glycosynthases |
title_full_unstemmed |
Development of a colourimetric assay for glycosynthases |
title_sort |
development of a colourimetric assay for glycosynthases |
publisher |
Elsevier Science |
publishDate |
2017 |
url |
https://juser.fz-juelich.de/record/836199 https://juser.fz-juelich.de/search?p=id:%22FZJ-2017-05320%22 |
op_coverage |
DE |
genre |
Antarc* antarcticus |
genre_facet |
Antarc* antarcticus |
op_source |
Journal of biotechnology 257, 162-170 (2017). doi:10.1016/j.jbiotec.2017.02.005 |
op_relation |
info:eu-repo/semantics/altIdentifier/issn/0168-1656 info:eu-repo/semantics/altIdentifier/pmid/pmid:28193496 info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jbiotec.2017.02.005 info:eu-repo/semantics/altIdentifier/issn/1873-4863 info:eu-repo/semantics/altIdentifier/wos/WOS:000408021800020 https://juser.fz-juelich.de/record/836199 https://juser.fz-juelich.de/search?p=id:%22FZJ-2017-05320%22 |
op_rights |
info:eu-repo/semantics/closedAccess |
op_doi |
https://doi.org/10.1016/j.jbiotec.2017.02.005 |
container_title |
Journal of Biotechnology |
container_volume |
257 |
container_start_page |
162 |
op_container_end_page |
170 |
_version_ |
1766269702094454784 |