3.4. CO2 Hydration Assay

Enzymatic activity of Saz_CA variants was performed using CO₂ hydration assay and presented as Wilbur–Anderson units (WAU) [43] using methods originally devised by Roughton and Booth [28]. Since CO₂ hydration assay results in formation of bicarbonate ions and protons, it results in a pH shift that can be monitored using bromothymol blue as an indicator. The indicator is yellow at pH lower than 6.0 and blue when pH is higher than 7.6. The protocol for the estimation of enzymatic activity was adapted from Capasso et al. [44] and executed as follows: 1 mL of ice-cold 25 mM Tris-SO₄ pH 8.3 containing 0.2 g L⁻¹ bromothymol blue was kept on ice, and 10 μL of enzymatic solution and 1 mL of ice-cold CO₂-saturated water were subsequently added to the buffer, and the stopwatch was started. The CO₂ hydration activity was calculated as one Wilbur–Anderson unit (WAU), i.e., time required in seconds for a saturated CO₂ solution to lower the pH of 0.012 M Tris-SO4 buffer from 8.3 to 6.3 at 0 °C, one WAU = (To − T)/T where To and T are the time needed for colour change from blue (pH 8.3) to yellow (pH 6.0) in the absence and presence of catalyst, respectively. The CO₂-saturated solution was prepared by bubbling CO₂ into ultrapure water on an ice bath for at least 1 h. All activity measurements were performed in triplicates.