*2.3. Physicochemical and Enzymatic Properties of CamPhoD*

The optimum pH for the CamPhoD maximum activity was determined to be 9.2, which lies in the range of pH 8.0–11.0 inherent for most alkaline phosphatases and phosphodiesterases (Figure 3). For comparison, the phosphodiesterases from *Sphingobium* sp. TCM1 and *E. coli* exhibited the maximal activity at pH 9.5 and 8.5–9.8, respectively [18,19].

**Figure 3.** Effect of the pH value on the CamPhoD phosphatase activity for conducting the enzymatic reaction in various buffer solutions: (1) glycine buffer, (2) acetate buffer, (3) Tris-HCl buffer, and (4) bicarbonate buffer.

The significant effect of KCl on the activity and the absence of activity without any salt in the incubation medium indicated that CamPhoD was a highly salt-tolerant enzyme, which is a common trait of many enzymes of marine origin [6,9,48,49]. Moreover, the PhoD alkaline phosphatase/phosphodiesterase from a halotolerant cyanobacterium, *Aphanothece halophytica*, has been shown to be induced and secreted out of cells by salt stress [9]. In the presence of KCl in a concentration up to 1 M, CamPhoD was 2–3 times more active than in the presence of NaCl at the same concentration (Figure 4).

**Figure 4.** Effect of NaCl and KCl on the CamPhoD alkaline phosphatase activity.

Concentration of both KCl and NaCl of more than 1 M decreased the CamPhoD activity. A similar effect was noted for the salt-tolerant alkaline phosphatase from eggs of the sea urchin *Strongylocentrotus intermedius* [48,50] and the enzyme from the halophilic bacterium *Halomonas* sp. 593 [49].

It was found that the addition of Co2<sup>+</sup> and Mg2<sup>+</sup> to the incubation mixture containing the lysate with the recombinant CamPhoD after ultrasonic homogenization of the *E. coli* cells increased its activity by 100% compared to the control. The addition of CaCl2 and FeCl3 activated CamPhoD from the cell lysate by 70% and 60%, respectively. For the CamPhoD purified up to level of homogenous protein, the salts of divalent metals Zn <sup>2</sup>+, Mn <sup>2</sup>+, and Cu <sup>2</sup><sup>+</sup> at a concentration of 2 mM had almost no effect on its ability to cleave pNPP, while the salts of Co <sup>2</sup>+, Mg <sup>2</sup>+, Ca <sup>2</sup>+, Fe <sup>3</sup>+, and Ni <sup>2</sup><sup>+</sup> drastically activated the enzyme, indicating its metal dependence (Table 1).

**Table 1.** Effect of metal cations on the alkaline phosphatase CamPhoD from *Cobetia amphilecti* KMM 296.


\* The various metal ions were added to the reaction mixture at the 2 mM salt concentration. The phosphatase activity was measured using the standard method described in Experimental Procedures. The retained activity is expressed as the specific phosphatase activity of CamPhoD relative to the activity of the enzyme incubated in the control reaction mixture in the absence of any metal ion.

However, the maximal CamPhoD activity was achieved by the simultaneous addition of Co2<sup>+</sup> and Fe3<sup>+</sup> to the incubation mixture, whereas only an 80% or 50% increase of the activity was observed after the addition of Co2<sup>+</sup> and Fe3<sup>+</sup> separately. It is evident that CamPhoD is the first bimetal Co2<sup>+</sup>–Fe3<sup>+</sup>-dependent phosphatase/phosphodiesterase characterized to date. Previously, the PhoD-like phosphatase from *B. subtilis* was described as being closely related to purple acid phosphatases (PAPs) with tyrosinate-ligated Fe3<sup>+</sup> ions, but differed from them by having two Ca2<sup>+</sup> ions instead of a single extra Fe2<sup>+</sup>, Mn2<sup>+</sup>, or Zn2<sup>+</sup> ion [15]. The Ca2<sup>+</sup> dependence of the phosphatase/phosphodiesterase from *Aphanothece halophytica* [9] as well as the activation of the alkaline phosphatase from *Pyrococcus abyssi* in the presence of Mg2<sup>+</sup>, Zn2<sup>+</sup>, and Co2<sup>+</sup> ions have already been shown [43]. In addition, the alkaline phosphatase of the hyperthermophilic bacterium *Termatoga maritima* was shown to contain Co2<sup>+</sup> and Mg2<sup>+</sup> in the active center [44], while the active center of an *E. coli* alkaline phosphatase possessing the phosphodiesterase activity contained two Zn2<sup>+</sup> and one Mg2<sup>+</sup> [51], similar to most of the PhoA alkaline phosphatases, for example CmAP from *C. amphilecti* KMM 296 [6,24,52].

In spite of the obvious CamPhoD metal-dependence, the addition of EDTA and EGTA to the incubation medium at a concentration of 2 mM led to almost no effect on its activity, probably due to the deeply hidden metal-binding site in the core domain, a common trait of extracellular marine enzymes [6]. The narrow enzymatic cavity directed towards the catalytic aa residues, which is packed with metal ions, protects the extracellular *C. amphilecti* KMM 296 alkaline phosphatase CmAP from the damaging effects of chelating agents [6].

The treatment of CamPhoD with dithiothreitol (DTT) at a concentration of 10 mM completely inhibited enzyme activity (Figure 5). The sensitivity to sulfhydryl reagent has been shown previously for the highly active alkaline phosphatase CmAP, which was previously isolated from the same bacterium *C. amphilecti* KMM 296 [6]. This indicates that the presence of SH groups in the protein structure is necessary for enzyme activity, although CmAP does not have any intermolecular disulfide bond [6,12].

**Figure 5.** Effect of dithiothreitol (DTT) on the CamPhoD phosphatase activity.

Adding the non-ionic detergent Triton X-100 to the CamPhoD incubation mixture at concentrations of 1%, 0.1%, and 0.01% also reduced its activity by 80%, 70%, and 46%, respectively. This could influence the hydrophobic interactions in the protein, the importance of which were shown for the overall thermal stability of psychrophilic and mesophilic enzymes [53].

The alkaline phosphatase CamPhoD retained its activity during incubation for 60 min at temperatures ranging from 15 to 45 ◦C, while incubation at 65 ◦C completely inhibited its activity after 20 min (Figure 6). The CamPhoD thermostability and the optimal temperature of 45 ◦C (Figure 7) are similar to the CmAP properties [6]. As for phosphatases and phosphodiesterases, their temperature optimums cover a wide range, allowing them to belong to both thermolabile and thermostable enzymes. Alkaline phosphodiesterases from *Sphingobium* sp. and *Delftia acidovorans* exhibit maximum activity at 55 ◦C and 65 ◦C, respectively [17,18].

**Figure 6.** Effect of temperature on the CamPhoD stability.

**Figure 7.** Effect of temperature on the CamPhoD phosphatase activity.
