CA Activity Measurements

The activity of CA constructs was assayed using a proprietary commercial ester substrate (BioVision) that upon conversion by CA releases p-nitrophenol, which was monitored via UV/vis spectrometry. Briefly, 2 μL of the substrate was mixed with 10 μL of CA (4.8 μM) and 88 μL of 100 mM HEPES buffer containing 150 mM NaCl and 50 μM ZnSO₄ (pH 7.5). The activity was measured by recording the absorption of p-nitrophenol at 405 nm using an Infinite M Plex plate reader (Tecan). Background hydrolysis was subtracted by measuring the release of p-nitrophenol in identical conditions in the absence of enzyme. Relative activity was determined by normalizing the activity of the immobilized form of each CA variant to the activity of the same variant in solution.