*3.2. Construction of Plasmid pET40CamPhoD*

The recombinant plasmid pET40CamPhoD was constructed by insertion into the NcoI/SacI fragment of plasmid pET-40b (+) (Thermo Fisher Scientific-RU, Moscow, Khimki, Russia), the gene encoding for the PhoD-like full-length phosphatase/phosphodiesterase, which was synthesized by polymerase chain reaction (PCR) using the genomic DNA of *Cobetia amphilecti* KMM 296 (Collection of Marine Microorganisms PIBOC FEB RAS) as a template and the gene-specific primers CamPhoD-NcoI-dir: 5 -TATACCATGGAAGGACGGCGCCCGCGCATGCCCTC-3 and CamPhoD-SacI-rev: 5 -TATAGAGCTCTTAGACACTGGCGGCGGCGGGGGTC-3 .

The reaction conditions were: 1 μL 10 × Encyclo buffer, 0.2 μl 50 × Encyclo polymerase mixture (Encyclo PCR kit; Evrogen, Moscow, Russia), 0.2 μl 50 × dNTP mixture (10 mM of each), a mixture of primers (1 μl 5 μM of each), and 1 μl 20 ng DNA. The volume of the reaction mixture was 10 μL. The amplification process consisted of 40 cycles of PCR (15 s, 95 ◦C, 1 min 40 s, 72 ◦C). After amplification, the PCR product was purified by electrophoresis in 1% agarose gel. The PCR fragment (1 μg) was treated with the restriction enzymes NcoI and SacI in an optimal buffer (Thermo Fisher Scientific RU) for 3 h at 37 ◦C, and then the enzymes were removed from the reaction mixture using phenol (1: 1). Here, 1/10 volume of 0.3 M sodium acetate, pH 5.2, and 1/2 volume of isopropyl alcohol were added to the aqueous fraction containing the PCR fragment, then incubated at −20 ◦C for 30 min. Then, this was centrifuged at 14,000 rpm for 20 min, the precipitate was washed with 75% ethanol, then dried at room temperature. The precipitate was dissolved in 20 μl of deionized water.

In total, 2 μg of the pET-40b (+) plasmid DNA (Thermo Fisher Scientific-RU) was treated with the NcoI and SacI restriction endonucleases in accordance with the procedure described above.

The obtained fragment of the CamPhoD gene and the NcoI /SacI part of the plasmid pET-40b (+) were ligated using a ligase reaction in 50 μl of ligation buffer, according to the instructions (Thermo Fisher Scientific RU). Then, 10 μL of the reaction mixture was used to transform the competent *E. coli* Rosetta cells (DE3). Transformants were grown on the Luria–Bertani (LB) agar containing 25 μg/mL kanamycin. After incubation for 16 h at 37 ◦C, the clones were screened, and then the targeted plasmid DNA was isolated and screened for mutations.

#### *3.3. Optimization of Conditions for CamPhoD Expression*

To determine the optimal IPTG concentration, the *E. coli* Rosetta (DE3) cells transformed with the pET40 plasmid carrying the CamPhoD gene were grown on LB agar containing 25 mg/mL kanamycin overnight at 37 ◦C. Single colonies were selected and grown in 5 mL of the liquid LB medium containing 25 mg/mL kanamycin at 200 rpm for 16 h at 37 ◦C. Then, the inoculum was placed into the flasks with 20 mL of fresh LB medium containing kanamycin at a concentration of 25 mg/mL and incubated at 37 ◦C in a shaker at 200 rpm, up to an optical density 0.6–0.8 (λ 600 nm). Next, 0.1 mM, 0.2 mM, 0.3 mM, and 0.5 mM IPTG were added to induce the expression of CamPhoD, then incubation was continued at 37 ◦C. To determine the phosphatase activity, 5 mL of each sample was taken at 0, 3, 6, and 24 h after the start of expression and ultra-sonication for the bacterial cells and determination of the CamPhoD alkaline phosphatase activity.

To determine the dependence of CamPhoD activity on the presence of phosphate in the growth medium, the *E. coli* Rosetta (DE3) cells transformed with the pET40CamPhoD plasmid were grown in 25 mL of the liquid medium containing: bacto-trypton 10 g/L, yeast extract 7.5 g/L, sorbitol 70 g/L, MgCl2 5 mM, KH2PO4 80 mM, and 25 mg/mL kanamycin at 200 rpm for 16 h at 37 ◦C [46]. Then, the cells were placed in 1 L of the fresh medium of the abovementioned composition and incubated at 37 ◦C on a rocking chair at 200 rpm, up to an optical density 0.6–0.8 (λ 600 nm). Next, 0.1 mM IPTG was added to induce the expression of CamPhoD and incubation was continued at 37 ◦C for 6 h at 200 rpm. After this, CamPhoD was isolated, purified, and its activity was determined as described below.

#### *3.4. The Recombinant CamPhoD Production*

The recombinant *E. coli* Rosetta (DE3) strain was grown in 25 mL of the LB liquid medium containing 25 mg/mL kanamycin at 200 rpm for 16 h at 37 ◦C. Then, the cells were placed in the fresh LB medium (1 L) containing kanamycin at a concentration of 25 mg/mL and incubated at 37 ◦C in a shaker at 200 rpm, up to an optical density at 600 nm of 0.6–0.8. After that, 0.1 mM IPTG was added to induce expression of the enzyme and incubation was continued at 37 ◦C for 6 h at 200 rpm.

The cells were precipitated by centrifugation at 4000 rpm for 15 min at 8 ◦C, suspended in 35 mL of 25 mM Tris-HCl buffer (pH 9.0) with phenylmethylsulfonyl fluoride (PMSF) added to a final concentration of 0.15 mM, and subjected to ultrasonication at 22 kHz and 0–4 ◦C with intervals of 30 s, up to clarification of the suspension. The suspension was centrifuged at 11,000 rpm for 30 min at 8 ◦C, the precipitate was discarded, and the activity and properties of CamPhoD were determined in the resulting extract.
