*4.2. Homology Model of α-PsGal 3D Structure*

The target-template alignment customization of the modeling process and 3D model building of α-PsGalA (GenBank: ABF72189.2) were carried out using the Molecular Operating Environment version 2018.01 [14] package (Chemical Computing Group ULC: 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2018) using the forcefield Amber12: EHT. The α-D-galactosidase from *Lactobacillus acidophilus* NCFM (PDB code: 2XN2) with a high-resolution crystal structure was used as a template. The evaluation of structural parameters, contact structure analysis, physicochemical properties, molecular docking, and visualization of the results were carried out with the Ligand interaction and Dock modules in the MOE 2018.01 program (Chemical Computing Group ULC: 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2018). The results were obtained using the equipment of Shared Resource Center Far Eastern Computing Resource of Institute of Automation and Control Processes Far Eastern Branch of the Russian Academy of Sciences (IACP FEB RAS) [56]

#### *4.3. Production of Recombinant Enzymes*

The recombinant wild α-D-galactosidase α-PsGal was produced as described earlier [13]. The D451A and C494N mutants were produced by polymerase chain reaction (PCR)-mediated

site-directed mutagenesis using the full-length wild gene of α-PsGal. The mutations were inserted in the sequences of synthetic oligonucleotides for each DNA chain of the wild gene:


The plasmid DNA pET40 containing an insertion of the α-D-galactosidase gene of the marine bacterium *Pseudoalteromonas* sp. KMM 701 (α-PsGal) or its D451A and C494N mutants were transformed in the *E. coli* strain Rosetta (DE3). Heterological expression was carried out at optimal conditions into *E. coli*, as described previously [57]. Purification of the recombinant α-PaGal and its D451A and C494N mutant forms were performed according to the procedures described previously [13].
