**5. Conclusions**

The recombinant and mutated α-PsGal were shown to be expressed as soluble proteins with the use of pET-40 b (+)-based constructions, and were purified successfully with the His-tag approach. The wild α-PsGal from the cold-adapted marine bacterium *Pseudoalteromonas* sp. KMM 701 has the traits of a cold-active enzyme that catalyzes the hydrolysis and weak transglycosylation reactions. The combination of TLC, MALDI-MS, and NMR spectroscopy methods to analyze the reaction mixture sugars allowed us to define the regioselectivity of the transglycosylation reaction, to qualitatively and quantitatively identify the reaction products obtained under the action of the recombinant analogues of α-PsGal, and to evaluate the yield of these products. The yield of transglycosylation products ranged from 6 to 12%. α-PsGal has a narrow acceptor specificity but rather wide regioselectivity. D-glucose, D-fructose, L-fucose, and D-xylose are not acceptors in the transglycosylation reaction. Together with the major α(1→6)-links under experimental conditions, the enzyme produced minor (traces) α(1→4) or -α(1→3)-links in bigalactosides at the saturating concentrations of melibiose and pNP-α-Gal, respectively. The point mutation D451A resulted in the completely loss of α-PsGal activity, indicating crucial significance of the residue A451 in the performance of the α-PsGal-mediated hydrolysis as well as transglycosylation. The C494N mutation slightly changed the structure, properties, and substrate specificity of the enzyme. Thus, *Pseudoalteromonas* KMM 701 α-D-galactosidase of the GH 36 family, which is important in biomedical technology, demonstrates weak glycosynthase properties in vitro.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/16/10/ 349/s1, Figure S1: MALDI-MS of mixtures of reaction products with: (**a**) melibiose and pNP-α-Gal as donor and acceptor, respectively; (**b**) wild α-PsGal with pNP-α-Gal as substrate; (**c**) D451A mutant with pNP-α-Gal; (**d**) C494N mutant with pNP-α-Gal; and (**e**) MALDI-MS of cellobiose and melibiose as standards, Figure S2: Electrospray ionization mass (ESIMS-MS) spectra of the transglycosylation products catalyzed by wild α-PsGal with melibiose as substrate: (**a**) ESIMS-MS spectra; (**b**) fragmentation of *m*/*z* 527 ion; (**c**) fragmentation of *m*/*z* 365 ion, Figure S3: ESIMS-MS spectra of the transglycosylation products from pNP-α-Gal catalyzed by wild α-PsGal: (**a**) fragmentation of *m*/*z* 365 ion; (**b**) fragmentation of *m*/*z* 486 ion, Figure S4: ESIMS-MS spectra of the transglycosylation products catalyzed by mutant C494N: (**a**) melibiose as substrate; (**b**) fragmentation of *m*/*z* 365 ion; (**c**) pNP-α-Gal as substrate; (**d**) fragmentation of *m*/*z* 486 ion; (**e**) and fragmentation of *m*/*z* 347 ion. Figure S5: Schemes for presumable mechanism of hydrolysis and transglycosylation of substrates melibiose (**a**) and pNP-α-Gal (**b**) with recombinant α-PaGal, Table S1: Ligand Interactions Report.

**Author Contributions:** Conceptualization, enzyme investigation, writing-original draft preparation: I.B.; construction, expression, and purification of recombinant and mutant enzymes: L.S. and L.B.; mass-spectra registration and interpretation: S.A.; NMR experiments: V.I.; CD spectra registration and analysis: N.K.; bioinformatics analysis and computer modeling of protein structure: G.L; formal analysis: O.S.; resources: L.T.

**Funding:** Financial support was provided by Ministry of Education and Science of Russia (Agreement 02.G25.31.0172, 01.12.2015); Mass spectrometric experiments using heavy-oxygen water were funded by Russian Science Foundation (Grand No. 16-13-10185).

**Conflicts of Interest:** The authors declare no conflicts of interest.
