Croatica Chemica Acta, Vol. 99 No. 1, 2026.
Izvorni znanstveni članak
https://doi.org/10.5562/cca4256
An Enantioselective High-Throughput Colorimetric Assay for Rapid Screening of Oxidoreductases Producing Chiral Amines or Alcohols
Vasilis Tseliou
orcid.org/0000-0001-5923-9735
; Van ’t Hoff Institute for Molecular Sciences, HIMS-Biocat, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands
Kyriakos Tsatsis
orcid.org/0009-0002-9452-6734
; Van ’t Hoff Institute for Molecular Sciences, HIMS-Biocat, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands
Francesco G. Mutti
orcid.org/0000-0002-6771-5102
; Van ’t Hoff Institute for Molecular Sciences, HIMS-Biocat, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands
*
* Dopisni autor.
Sažetak
High-throughput screening of oxidoreductases for enantioselectivity remains a key challenge in enzyme engineering, as traditional screening formats commonly provide activity data but not stereochemical discrimination. Herein, we describe a stereoselective colorimetric platform in which a monoamine oxidase variant selectively oxidizes (S)-configured amines to imines, releasing stoichiometric hydrogen peroxide that is detected through a horseradish peroxidase–mediated color reaction. This enables rapid screening in aqueous microplate formats, with color development that can be monitored in real time by standard plate readers. Control experiments confirmed the absence of background signal from (R)-configured amines or lysate components, demonstrating the assay’s high selectivity. The concept was further extended to other oxidoreductases by coupling enzymatic product oxidation to oxidase-mediated hydrogen peroxide formation and subsequent horseradish peroxidase–driven color development. Validation with engineered amine dehydrogenase (AmDH) and alcohol dehydrogenase (ADH) confirmed both sensitivity and enantioselectivity of the colorimetric assay, with visual results also validated by gas chromatography. This approach provides a scalable and versatile tool for accelerating the discovery and optimization of stereoselective biocatalysts.
Ključne riječi
biocatalysis; high-throughput screening; colorimetric assay; enantioselectivity; amine dehydrogenase; monoamine oxidase; alcohol dehydrogenases; enzyme engineering
Hrčak ID:
348352
URI
Datum izdavanja:
17.1.2026.
Posjeta: 0 *