For the α-PsGal gene amplification, Encyclo Taq Polymerase (Eurogene), genomic DNA of the marine bacterium Pseudoalteromonas sp. KMM 701 and the gene-specific the upstream primer, G2-NcoI-for40: 5′-ATTACCATGGATGACGACGACAAGGCCGACACTAAATCATTTTATCGATTAGACA-3′, and the downstream primer, G3-SalI-rev40: 5′-ACACGTCGACTTACGCTTTGTTGAGCTCAAATATAAGC-3′ were used. The resultant PCR products were purified with agarose gel (Helicon). PCR was carried out in automatic amplifier “Eppendorf.” Restriction endonucleases and T4 DNA ligase were purchased from “Thermo Scientific.” The restricted PCR product and plasmid pET-40b (+) were purified from agarose gel with the use of a “Qiagen” column. The recombinant insert of the resultant plasmid 40Gal was sequenced using the automated PE/ABI 310 DNA sequencer and the PE/ABI-ABI PRISM BigDye Terminator cycle sequencing Ready Reaction Kit (PE Applied Biosystems). Preparation of Escherichia coli competent cells and heat shock transformation were carried out according to the standard methods (Sambrook et al., 1989).

For producing α-PsGal of marine bacterium Pseudoalteromonas sp. KMM 701 the recombinant plasmid 40Gal was transformed in to the E. coli Rosetta(DE3) cells. The obtained recombinant cells were grown on LB plate containing 25 μg/mL of kanamycin overnight at 37°C. A single colony was picked and grown at 200 rpt in 20 mL of LB, with 25 μg/mL of kanamycin at 37°C for 12 h. Overnight culture was transferred to 1 L of fresh LB with 25 μg/mL of kanamycin. When the cell density reached an OD₆₀₀ of 0.6–0.8, 0.2 mM IPTG was added to induce the expression and the incubation was continued at 16°C up to 12 h at 200 rpt. The E. coli Rosetta(DE3) cells were transformed with the pET-40b (+) plasmid as a control.

All purification steps were carried out at +6°C. After harvesting, the transgenic E. coli Rosetta (DE3)/40Gal cells were resuspended in 200 mL of buffer A (0.01 M NaH₂PO₄, pH 7.7, 0.01% NaN₃) and sonificated by ultrasonic treatment, then centrifuged at 10,000 g for 30 min. The supernatant was applied to a column (2.5 × 30 cm) of Macro-Prep (BioRad). Elution of the protein was performed with the use a linear gradient of NaCl concentration (0.05–0.3 M) in the buffer A. The enzymatic active fractions were collected and applied to a column (1 × 2.5 cm) of Ni-NTA agarose (Qiagen). Elution of the protein was carried out in buffer B (0.01 M NaH₂PO₄, pH 7.7, 0.01% NaN₃, 0.04 M EDTA). The active fractions were collected and desalted with the use of Bio-Scale™ Mini Macro-Prep® High Q 1 mL cartridge, then incubated with enterokinase (Invitrogen) at 21°C for 15 h to cleave N-terminal plasmid overhang from the chimeric protein (32.5 kDa). Then, the protein solution is applied to a gel filtration column (1.5 × 170 cm) of Superdex 200 (Sigma) equilibrated with the buffer C (0.01 M NaH₂PO₄, pH 7.7, 0.01% NaN₃, 0.1 M NaCl). All purification steps were examined by SDS-PAGE. The concentration of the protein was determined according to Bredford (1976). The obtained recombinant polypeptide is determined by the first 10 amino acids on an automatic sequencer Procise model 492 (Applied Biosystems, USA). All biochemicals and reagents were from “Thermo scientific” and “Sigma-Aldrich.”

Enzyme activity of the α-PsGal was determined with 3.3 mM of 4-NPGP in 50 mM sodium phosphate buffer pH 7.5, at 20°C. One unit of the α-galactosidase activity was defined as amount of the enzyme that hydrolyzed 1 μmol of 4-NPGP per min. Total quantity of p-nitrophenol was determined spectrophotometrically at 400 nm (ε₄₀₀ = 18300 M⁻¹ cm⁻¹).

The molecular size of the active α-PsGal after treatment of enterokinase was determined by gel filtration on the column of Superdex 200 (Sigma) (1.5 × 170 cm) in the buffer A at a flow of 0.16 mL/min at 6°C and calibrated using Bio-Rad standard molecular weight markers: Thyroglobulin bovine (670 kDa), γ-globulin bovine (158 kDa), Ovalbumin chicken (44 kDa), Mioglobin horse (17 kDa), Vitamin B₁₂ (1.35 kDa). The molecular mass of the enzyme was determined by 12.5 % polyacrylamide gel electrophoresis (PAGE). The protein preparation was mixed with Laemmli sample buffer with heat treatment at 95°C, and then applied to PAGE. The SDS-PAGE gels were stained according to Laemmli (1970).

Determination of the anomeric center configuration of the product of the hydrolysis reaction was monitored by ¹H NMR spectroscopy. The experiment was performed at 20°C with the use of NMR DRX-500 spectrometer (Bruker, Germany). ¹H NMR spectra were acquired over 32,000 data points using a spectral width 5000 Hz.

Prior to analysis by NMR, 0.6 mL 50 mM sodium phosphate buffer, pH 7.5, and substrate were dried on rotary evaporator and dissolved in 0.6 mL D₂O. The deuterium-exchanged α-PsGal was obtained on vivaspin turbo 10 K MWCO (Sartorius, Germany). Chemical shifts were measured relative to an external standard—acetone (trace) in D₂O. CH₃—signal acetone was set to δ = 2.22 ppm. After recording of the original spectrum at τ = 0 min 6.0 mmol of the deuterium-exchanged p-nitrophenyl galactopyranoside in 0.6 mL of D₂O, 0.1 mL of the deuterium-exchanged α-PsGal containing 1.14 U was added for initiation of the reaction. ¹H NMR spectra were recorded automatically at intervals of 3 min for 60 min from the start of the reaction.

¹H NMR anomer signals (αH-1) of α-4-NPGP, α-galactopyranose, and (β H-1) of β-galactopyranose, as well as proton signals of free 4-nitrophenol ring were analyzed and integrated by the standard software “TopSpin 3.2.” Integral intensities of signals of each anomer were calculated as a percentage of the total integral intensities of all signals of anomers and were plotted depend on time. The degree of substrate hydrolysis (4-NPGP) was defined as a percent ratio of the ¹H NMR integral intensities of proton signals of free 4-nitrophenol ring at 8.109 ppm to proton signals of 4-NPGP at 8.275 ppm and also was plotted depend on time.

The recombinant plasmid 40Gal of 8306 base pairs (bp) for the synthesis of the recombinant protein α-PsGal consisted of the NcoI/SalI-fragment of plasmid pET-40b (+) (Novagen) and the gene of 2130 bp corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp. KMM 701 (Figure 1).

The IPTG concentration of 0.2 mM, strain cultivation temperature of 16°C and duration of the cultivation of 12 h at 200 rpt were optimal conditions for the α-PsGal expression. The induced α-PsGal expression in 1 L culture of E.coli Rosetta(DE3)/40Gal cells yielded up to 10 mg of the functionally active protein with the specific activity 160 U/mg at the final purification step. α-PsGal has been found to provide the complete inactivation of serological activity of B-red blood cells and its conversion into the O-red blood cells in the same condition as for the native enzyme (data not shown).

PAGE-SDS showed a single band of the purified recombinant protein with a molecular mass of approximately 80 kDa corresponding to the one subunit of α-PsGal (Figure 2).

The molecular mass of the mature protein determined by gel filtration was to be 160 kDa, suggesting the two-subunit molecular organization of the active α-PsGal.

¹H NMR spectra of deuterium-exchanged 4-NPGP and products of it's hydrolysis under the action of the recombinant α-PsGal are shown on Figure 3.

In ¹H NMR spectrum 4-NPGP (Figure 3, spectrum 1, formula of the compound is shown on the right) one signal was a doublet at 5.84 ppm [J_{(1, 2)} = 3.43 Hz] and two groups signals at 8.28 and 7.31 ppm corresponded to αH-1 anomer and 4-nitrophenol ring protons in 4-NPGP, respectively. Moreover, signals in region of resonance from 4.13 to 3.96 ppm are observed. These signals corresponded to H-2, H-3, H-4, H-5, and H-6 protons of α-galactopyranose in 4-NPGP (Figure 3, spectrum 1).

After addition of the enzyme, the doublet signal was appeared at 5.26 ppm [J_{(1, 2)} = 3.8 Hz] during the first 3 min (Figure 3, spectrum 2, reaction equation is shown on the right). The doublet was identical in chemical shift to the αH-1 resonance of free α-D-galactopyranose (Angyal and Pickles, 1972). Signals arising at 8.11 and 6.68 ppm related to protons of free 4-nitrophenol formed through the reaction. Decrease in the amplitude of signals at 8.28 and 7.31 ppm indicates disappearance the substrate. A doublet signal at 4.57 ppm [J_{(1, 2)} = 7.8 Hz], which was identical in chemical shift to the H-1 resonance of free β-D-galactopyranose (β H-1) (Angyal and Pickles, 1972), appeared after 10 min of the reaction in accordance with the time of spontaneous mutarotation (Figure 3, spectrum 4). Signals of protons H-2, H-3, H-4, H-5, and H-6 of free α- and β-D-galactopyranose occurred at 3.40–4.20 ppm, minor signals at 5.21 ppm [J_{(1, 2)} = 3.30] rising at late time of reaction corresponded to β-D-galactofuranose (Angyal and Pickles, 1972) (Figure 3, spectra 4 and 5, equation of equilibrium of D-galactose is shown on the right Zhu et al., 2001). Signals in the anomeric region, which could indicate the emergence of new products of the reaction or formation of O-glycosidic bond in the spectra were not found.

NMR spectroscopic monitoring of the hydrolysis of 4-NPGP by α-PsGal is shown on Figure 4.

Signal at 5.26 ppm the α-D-galactopyranose αH-1 appeared at the first minutes after the addition of α-PsGal (Figure 4, curve 1). Disappearance of doublet at 5.84 ppm of αH-1 of 4-NPGP (Figure 4, curve 2) simultaneously with appearance of the signal at 5.26 ppm of αH-1 of α-D-galactopyranose increasing in intensity (Figure 4, curve 1) were observed within next 30 min. On curve of the time-dependent changes in signal amplitude of β H-1 at 4.57 ppm lag-period were observed during 9 min (Figure 4, curve 3), when degree of the substrate hydrolysis reached up to ~30% (Figure 4, curve 4). After 33 min, the degree of substrate hydrolysis reached up to 98–100% (Figure 4, curve 4). After 60 min, the ratio of integral intensities of αH-1 and β H-1 signal was 40:60. Typical equilibrium ratio 31:69 between αH-1 and β H-1 D-galactopyranose anomers (Angyal and Pickles, 1972; Zhu et al., 2001) was formed for 180 min from the start of the reaction in experimental conditions pH 7.5 and 20°C.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.