A Selective Phenyl Azo Acetate Substrate for Enhanced Carbonic Anhydrase Functional Analysis

Carbon dioxide (CO₂) sequestration is a key strategy for mitigating climate change, and biocatalytic approaches offer sustainable, cost-effective alternatives to chemical and physical methods. Carbonic anhydrases (CAs) efficiently catalyse the reversible hydration of CO₂ to bicarbonate, yet their large-scale application is constrained by limited stability and the lack of robust analytical tools for activity and/or inhibition profiling. In this work, we synthesised and characterised a novel phenyl azo dye acetate to address these limitations. The substrate was optimised for spectrophotometric assays, enabling reliable kinetic and inhibition analyses of CAs. Compared with conventional substrates such as p-nitrophenyl acetate and indoxyl acetate, the new assay demonstrated more than 4.5-fold higher sensitivity, substantial thermal stability and wider applicability across tested hydrolases. These findings provide a more robust platform for evaluating CA activity, supporting the development of more efficient biocatalytic systems for CO₂ sequestration.