CO2 hydration activity
CO2 hydration activity was determined, using an electrometric method, in 5ml of 20 mM Tris (pH 8.3) at 0ºC. This reaction was started by the addition of 1.5 ml of ice-cold CO2 saturated water to the enzyme solution. The activity was calculated as µmol CO2hydrated min-1 (mg Chl)-1 mg Chl-1 using the following equation:
CA activity = [2×(T0−T)]/[T× amount of Chl in reaction mixture (mg)],
where, T0 and T are the times for pH drop from 8.3 to 6.8 for the non-enzymatic and the enzymatic reactions, respectively (Wilbur and Anderson, 1948).
To eliminate the non-enzymatic CA reaction, the enzyme extract was inactivated at 95ºC and the CA activity was determined. The heat-denatured CA had negligible activity.