Characterization of Properties and Transglycosylation Abilities of Recombinant -Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants

A novel wild-type recombinant cold-active -d-galactosidase (-PsGal) from the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701, and its mutants D451A and C494N, were studied in terms of their structural, physicochemical, and catalytic properties. Homology models of the three-dimensional -PsGal structure, its active center, and complexes with D-galactose were constructed for identification of functionally important amino acid residues in the active site of the enzyme, using the crystal structure of the -galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild -PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl--galactopiranoside (pNP--Gal). Thin-layer chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and nuclear magnetic resonance spectroscopy methods were used to identify transglycosylation products in reaction mixtures. -PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. -PsGal synthesized -(16)- and -(14)-linked galactobiosides from melibiose as well as -(16)- and -(13)-linked p-nitrophenyl-digalactosides (Gal(2)-pNP) from pNP--Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-(13)-Gal-pNP synthesis and increased the Gal-(14)-Gal yield compared to Gal-(16)-Gal-pNP.