*3.7. Substrate Specificity*

The substrate specificity of CamPhoD was determined by adding 2 mM CMP, CDP, CTP, dCMP, dCTP, UMP, UDP, UTP, TMP, TTP, GMP, GDP, GTP, dGMP, dGTP, AMP, ATP, dAMP, bis-pNPP, 5 -pNP-TMP, c-di-GMP, and pNPP to a standard incubation mixture, containing 25 mM Tris-HCl buffer, pH 9.0, 2 mM CoCl2, 2 mM FeCl3. The mixture was incubated at 37 ◦C for 60 min. The volume of the reaction mixture was 1 mL. Then, a molybdate reagent with ascorbic acid (4 mL) was added to the incubation mixture and again incubated at 37 ◦C for 60 min. The mixture was cooled to room temperature, and absorbance of the formed product of dephosphorylation by CamPhoD was measured at an optical density of 820 nm. The amount of inorganic phosphate Pi (mkM) released during dephosphorylation of the studied substrates with the enzyme was determined using a calibration curve with KH2PO4, according to Chen's method [54].

#### *3.8. Determination of Thermostability and Temperature Optimum*

The effect of temperature on CamPhoD and the optimum temperature were determined by incubating the standard incubation mixture at temperatures ranging from 15 to 65 ◦C, at 10 ◦C intervals. Alkaline phosphatase activity was determined using the standard method for determining for CamPhoD, as described above.
