*2.1. CamPhoD Isolation and Characterization by Enzymatic Activity and Primary Structure*

The heterologous expression of the *C. amphilecti* KMM 296 gene (GenBank ID: WP\_043333989) corresponding to the open reading frame (ORF) of the PhoD-like phosphatase (CamPhoD) resulted in obtaining an enzymatically active recombinant protein with a specific phosphatase activity of 18.2 U/mg (with p-NPP as a substrate) after purification using the modified scheme described earlier [6]. The CamPhoD phosphodiesterase activity at the cleavage of bis-pNPP was 0.3 U/mg. The isolation of this enzyme confirmed the ability of *C. amphilecti* to produce the functionally active alkaline bifunctional phosphatase/phosphodiesterase CamPhoD with a calculated molecular weight of 54839.8 kDa for the mature protein, without the 33-letter leader peptide, according to the Simple Modular Architecture Research Tool (SMART) database [37]. The obtained data were in agreement with the polyacrylamide gel electrophoresis (PAGE) estimation of its molecular weight (Figure 1).

Apart from the leader peptide indicating an extracellular intent of the enzyme, the CamPhoD 492 amino acid (aa) sequence includes the region of the fibronectin type III repeat (FN3) from 43 to 118 aa residues, containing a cell recognition region of Arg-Gly-Asp (RGD) in a flexible loop between 2 strands, according to the new functional classification of proteins via subfamily domain architectures [38]. RGD is the cell attachment site of a large number of adhesive extracellular matrix and cell surface proteins, which are recognized by transmembrane receptors activating signal transduction. FN3-like domains were also found in bacterial glycosyl hydrolases [38]. It has been shown that the bacterial extracellular proteins with the RGD motif may be located on the surface of the bacterial type IV secretion pili. They mimic fibronectin in triggering cell spreading, focal adhesion formation, and activation of several tyrosine kinases during interaction with various mammalian cell lines [39,40]. Thus, the RGD motif of CamPhoD may be a player in pathogenesis or the symbiotic relationships between the bacterium and host mollusk during its shell mineralization [12,36]. The part of the CamPhoD molecule from 149 to 505 aa residues is the PhoD-like phosphatase (pfam09423), with characteristic active and ion-binding sites [38].

**Figure 1.** SDS-PAGE image of CamPhoD. Line 1-the crude extract from the recombinant *E. coli* cells; line 2-the purified CamPhoD before the enterokinase treatment; line 3-the purified CamPhoD after the treatment with enterokinase to remove the 34.2 kDa His-tagged N-end of the plasmid pET 40b(+) sequence (shaperon DsbC); line 4-the marker of the protein molecular weights (BioRad).

The values of CamPhoD-specific activities corresponded to the activities of the PhoD phosphatase/phosphodiesterase from *Aphanothece halophytica* and alkaline phosphatase from *Vibrio* sp., whose molecular weights were also similar to CamPhoD and other alkaline phosphatases [10,41–45]. Thus, the molecular weights of the alkaline phosphatases' monomers from *Streptomyces griseus* IMRU 3570, *Pyrococcus abyssi*, and *Thermotoga maritima* were 62 kDa, 54 kDa, and 45 kDa, respectively [42–44]. The enzymes with exclusively phosphodiesterase-related activity possess subunits with lower molecular weight, such as in the enzyme ZiPD from *E. coli* (36 kDa) [45]. Alkaline phosphatases generally have a dimeric structure, but the literature also describes a trimer for the phosphodiesterase from *Delftia acidovorans* with a molecular weight of 85 kDa [17]. According to the gel chromatography data, the native CamPhoD tended to form an active dimer with a molecular weight of approximately 100–120 kDa in the conditions used (see Experimental Procedure section).

#### *2.2. Expression Conditions for CamPhoD Production*

The study of optimal expression conditions for the recombinant CamPhoD showed that its highest yield was achieved when cultivating the recombinant *E. coli* strain over 6 h at 37 ◦C, with an addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) in the Luria–Bertani (LB) medium (Figure 2). It was also found that the alkaline phosphatase activity of the recombinant CamPhoD increased when the cells were cultured in a medium depleted by phosphorus. The growth of the recombinant *E. coli* cells in a MX medium containing 80 mM KH2PO4 described earlier [46] significantly reduced the alkaline phosphatase activity of CamPhoD down to 0.035 U/mg compared with the activity of the enzyme, which was produced in the standard phosphate-free LB medium, similarly to the phoD phosphatase isolated from *Streptomyces coelicolor* [47].

**Figure 2.** Determination of the expression conditions for the recombinant CamPhoD production: (**A**) the dependence of the CamPhoD concentration (mg/mL) on the cultivation time at 37 ◦C in the presence of (1) 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), (2) 0.2 mM IPTG, (3) 0.3 mM IPTG, and (4) 0.5 mM IPTG; (**B**) the dependence of the CamPhoD phosphatase activity (U/mg) on the cultivation time at 37 ◦C in the presence of (1) 0.1 mM IPTG, (2) 0.2 mM IPTG, (3) 0.3 mM IPTG, and (4) 0.5 mM IPTG.
