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

  1. Czarnievicz, Nicolette 1
  2. Rubanu, Maria Grazia 1
  3. Iturralde, Marialen 1
  4. Albarran-Velo, Jesús 2
  5. Diamanti, Eleftheria 1
  6. Gotor-Fernandez, Vicente 2
  7. Skolimowski, Maciej 3
  8. López-Gallego, Fernando 1
  1. 1 Center for cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA)
  2. 2 Organic and Inorganic Chemistry Department, University of Oviedo
  3. 3 Micronit BV
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Editor: Zenodo

Any de publicació: 2022

Tipus: Dataset

CC BY 4.0

DOI: 10.5281/ZENODO.7947610 lock_openAccés obert editor

Resum

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.