The hydrolysis of indoxyl acetate: A versatile reaction to assay carbonic anhydrase activity by high-throughput screening

The interest in CO2 capture and conversion by biological methodologies into various beneficial products is increased. In nature, there are enzymes, with hydration activity, which catalyze the reversible hydration of the CO2 molecule and, thus, are of high interest for biotechnological applications. Such enzymes are carbonic anhydrases (CAs). Structural, functional and mutational studies have shown, that besides hydratase activity, CAs have exposed hydrolytic, particularly esterase activity, and importantly, both activities follow similar catalytic mechanisms in the same catalytic pocket. CAs activity measurement methods based on electrometric assays for hydration activity and nitrophenyl based esters for hydrolytic activity assays do not fulfill the requirements amenable for enzyme activity screening methods. By this study, we were aiming to develop an indigogenic assay method based on the esterase activity of CAs. The first time use of indoxyl acetate as a substrate for CA has shown promising results to gain simplicity, repeatability, and applicability to implement high-throughput screening methods.