*2.5. Catalytic Properties of CamPhoD*

The Michaelis constant (Km) of the alkaline phosphatase/phosphodiesterase CamPhoD at pH 9.0 in the presence of Co2<sup>+</sup> and Fe<sup>3</sup> <sup>+</sup> with the use of chromogenic pNPP as a substrate was 4.2 mM, the maximum velocity (Vmax) was 0.203 mM/min, and the efficiency (kcat/Km) was 7988.6 S−1/mM. Using the chromogenic phosphate bis-pNPP, Km was determined to have values of 6.71 mM, Vmax = 0.046 mM/min, and efficiency (kcat/Km) = 1133.0 S−1/mM. The kinetic parameters obtained for CamPhoD are similar to those previously obtained for other phosphatases and phosphodiesterases (Table 3). For example, Km of the alkaline phosphatase from *Termatoga maritima* had a value of 175 mM [44], while Km of the alkaline phosphodiesterases from *Sphingobium* sp. TCM1 and *A. halophytica* for pNPP were 1.5 mM and 3.38 mM, respectively [10,18].


**Table 3.** Biochemical characteristics of the reported bacterial phosphatases and phosphodiesterases.

The CamPhoD catalytic efficiency (*k*cat/*K*m) in relation to pNPP was seven-fold higher than that with the use of bis-pNPP as the substrate, indicating that the enzyme mainly has a phosphomonoesterase structure with phosphodiesterase capability, similar to other PhoD-like enzymes [15]. However, the value of its catalytic efficiency for diester bonds is much higher when compared with many monospecific phosphodiesterases, excluding the enzyme from an unknown protein family with recently established structure and properties, which was isolated from the metagenome [18,19,45].
