(3) Measurement of activity and concentration of enzyme
The enzyme activity of native and refolded CA was determined via esterase activity assessment (Ikai et al., 1978). A mixture of 1.5 mL of pure water and 1 mL of p -nitrophenyl acetate solution (3 mL in acetone) was placed in a quartz cell. After 60 seconds 0.4 mL of 0.05 M Tris-HCl buffer was added, and after another 60 seconds 0.1 mL of each of the CA solution samples prepared as described in Section (2) was added. The increase in absorbance at 400 nm due to hydrolysis ofp -nitrophenyl acetate was monitored.
The concentration of CA was measured using Coomassie Plus (Bradford) Assay kits (Thermo Fischer Scientific. co., Waltham, USA) and was normalized using 0.25 mg/mL native CA.