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

Author:
Czarnievicz, Nicolette; Rubanu, Maria Grazia; Iturralde, Maialen; Albarrán Velo, JesúsUniovi authority; Diamanti, Eleftheria; Gotor Fernández, VicenteUniovi authority; Skolimowski, Maciej; López Gallego, Fernando
Publication date:
2023
Publisher version:
https://doi.org/10.1002/cbic.202200614
Citación:
ChemBioChem, 24(2) (2023); doi:10.1002/cbic.202200614
Abstract:

The development of methods to engineer and immobilize Amine transaminases (ATAs) improving their functionality and operational stability is gaining momentum. Nowadays, 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 allows 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 adapt 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

The development of methods to engineer and immobilize Amine transaminases (ATAs) improving their functionality and operational stability is gaining momentum. Nowadays, 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 allows 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 adapt 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

URI:
http://hdl.handle.net/10651/65871
ISSN:
1439-4227
DOI:
10.1002/cbic.202200614
Patrocinado por:

The research was mainly carried out at the facility of CIC biomaGUNE and partially supported by the Spanish Ministry of Science and Innovation under the Maria de Maeztu Units of Excellence Programme (MDM‐ 2017 ‐ 0720). The authors acknowledge the funding of Horizon 2020 MSCA-ITN-EID (INTERFACES, 860414). FLG acknowledges the funding of IKERBASQUE. VGF and FLG thank to the Spanish Biocatalysis Network funded by the Spanish State Research Agency (AIE) (RED2018-102403-T) for its support.

Id. Proyecto:

info:eu‐repo/grantAgreement/EC/H2020/860414

RED2018-102403-T

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