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p-Phenylene Diisothiocyanate-Based Covalent Immobilization of β-d-Galactosidase and Determination of Enzyme Activity by Cleavage of X-Gal and ONPG on Solid Support
[Image: see text] Herein, we present the immobilization of a technical grade β-d-galactosidase on amino-functionalized microtiter plates. Afterward, we transferred the results to a resin-based approach. For the covalent binding of the enzyme, an amino-functionalized microtiter plate was prefunctiona...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10399185/ https://www.ncbi.nlm.nih.gov/pubmed/37546597 http://dx.doi.org/10.1021/acsomega.3c03279 |
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author | Dayi, Defne I. Eschenhagen, Ursula Seidinger, Henrike Schneider, Holger Schmidt, Magnus S. |
author_facet | Dayi, Defne I. Eschenhagen, Ursula Seidinger, Henrike Schneider, Holger Schmidt, Magnus S. |
author_sort | Dayi, Defne I. |
collection | PubMed |
description | [Image: see text] Herein, we present the immobilization of a technical grade β-d-galactosidase on amino-functionalized microtiter plates. Afterward, we transferred the results to a resin-based approach. For the covalent binding of the enzyme, an amino-functionalized microtiter plate was prefunctionalized with 1,4-phenylendiisothiocyanate. The cleavage of the substrate 5-bromo-4-chloro-3-indoxyl-β-d-galactopyranoside (X-Gal) produces a deep blue dye, which was quantified in a microtiter plate reader at 595 nm. The maximum reaction rates and the Michaelis–Menten constant were calculated. In addition, the unwanted blue precipitate formed during the experiments could be minimized by optimizing the experiments. When transferring the immobilization method to Rink amide resin, o-nitrophenyl-β-d-galactopyranoside was used as the substrate and the measurement was carried out in a photometer at 420 nm. |
format | Online Article Text |
id | pubmed-10399185 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-103991852023-08-04 p-Phenylene Diisothiocyanate-Based Covalent Immobilization of β-d-Galactosidase and Determination of Enzyme Activity by Cleavage of X-Gal and ONPG on Solid Support Dayi, Defne I. Eschenhagen, Ursula Seidinger, Henrike Schneider, Holger Schmidt, Magnus S. ACS Omega [Image: see text] Herein, we present the immobilization of a technical grade β-d-galactosidase on amino-functionalized microtiter plates. Afterward, we transferred the results to a resin-based approach. For the covalent binding of the enzyme, an amino-functionalized microtiter plate was prefunctionalized with 1,4-phenylendiisothiocyanate. The cleavage of the substrate 5-bromo-4-chloro-3-indoxyl-β-d-galactopyranoside (X-Gal) produces a deep blue dye, which was quantified in a microtiter plate reader at 595 nm. The maximum reaction rates and the Michaelis–Menten constant were calculated. In addition, the unwanted blue precipitate formed during the experiments could be minimized by optimizing the experiments. When transferring the immobilization method to Rink amide resin, o-nitrophenyl-β-d-galactopyranoside was used as the substrate and the measurement was carried out in a photometer at 420 nm. American Chemical Society 2023-07-21 /pmc/articles/PMC10399185/ /pubmed/37546597 http://dx.doi.org/10.1021/acsomega.3c03279 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Dayi, Defne I. Eschenhagen, Ursula Seidinger, Henrike Schneider, Holger Schmidt, Magnus S. p-Phenylene Diisothiocyanate-Based Covalent Immobilization of β-d-Galactosidase and Determination of Enzyme Activity by Cleavage of X-Gal and ONPG on Solid Support |
title | p-Phenylene Diisothiocyanate-Based
Covalent Immobilization of β-d-Galactosidase
and Determination of Enzyme Activity by Cleavage of X-Gal and
ONPG on Solid Support |
title_full | p-Phenylene Diisothiocyanate-Based
Covalent Immobilization of β-d-Galactosidase
and Determination of Enzyme Activity by Cleavage of X-Gal and
ONPG on Solid Support |
title_fullStr | p-Phenylene Diisothiocyanate-Based
Covalent Immobilization of β-d-Galactosidase
and Determination of Enzyme Activity by Cleavage of X-Gal and
ONPG on Solid Support |
title_full_unstemmed | p-Phenylene Diisothiocyanate-Based
Covalent Immobilization of β-d-Galactosidase
and Determination of Enzyme Activity by Cleavage of X-Gal and
ONPG on Solid Support |
title_short | p-Phenylene Diisothiocyanate-Based
Covalent Immobilization of β-d-Galactosidase
and Determination of Enzyme Activity by Cleavage of X-Gal and
ONPG on Solid Support |
title_sort | p-phenylene diisothiocyanate-based
covalent immobilization of β-d-galactosidase
and determination of enzyme activity by cleavage of x-gal and
onpg on solid support |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10399185/ https://www.ncbi.nlm.nih.gov/pubmed/37546597 http://dx.doi.org/10.1021/acsomega.3c03279 |
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