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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...

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Autores principales: Dayi, Defne I., Eschenhagen, Ursula, Seidinger, Henrike, Schneider, Holger, Schmidt, Magnus S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
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.
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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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