A Multiplex Assay to Assess the Transaminase Activity toward Chemically Diverse Amine Donors

The development of methods to engineer and immobilize amine transaminases (ATAs) to improve their functionality and operational stability is gaining momentum. The quest for robust, fast, and easy-to-use methods to screen the activity of large collections of transaminases, is essential. This work pre...

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Hauptverfasser:	Czarnievicz, Nicolette, Rubanu, Maria Grazia, Iturralde, Marialen, Albarran-Velo, Jesús, Diamanti, Eleftheria, Gotor-Fernandez, Vicente, Skolimowski, Maciej, López-Gallego, Fernando
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Sprache:	eng
Schlagworte:	amine donor screening biocatalysis protein immobilization single-particle assays transaminases kinetics
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creator	Czarnievicz, Nicolette Rubanu, Maria Grazia Iturralde, Marialen Albarran-Velo, Jesús Diamanti, Eleftheria Gotor-Fernandez, Vicente Skolimowski, Maciej López-Gallego, Fernando
description	The development of methods to engineer and immobilize amine transaminases (ATAs) to improve their functionality and operational stability is gaining momentum. The quest for robust, fast, and easy-to-use methods to screen the activity of large collections of transaminases, is essential. This work presents a novel and multiplex fluorescence-based kinetic assay to assess ATA activity using 4-dimethylamino-1-naphthaldehyde as an amine acceptor. The developed assay allowed us to screen a battery of amine donors using free and immobilized ATAs from different microbial sources as biocatalysts. As a result, using chromatographic methods, 4-hydroxybenzylamine was identified as the best amine donor for the amination of hydroxy methyl furfural. Finally, we adapted this method to determine the apparent Michaelis-Menten parameters of a model immobilized ATA at the microscopic (single-particle) level. Our studies promote the use of this multiplex, multidimensional assay to screen ATAs for further improvement.
doi_str_mv	10.5281/zenodo.7947609
format	Dataset
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identifier	DOI: 10.5281/zenodo.7947609
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subjects	amine donor screening biocatalysis protein immobilization single-particle assays transaminases kinetics
title	A Multiplex Assay to Assess the Transaminase Activity toward Chemically Diverse Amine Donors
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