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

ABSTRACT: 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 -?(1?6)- and -?(1?4)-linked galactobiosides from melibiose as well as -?(1?6)- and -?(1?3)-linked p-nitrophenyl-digalactosides (Gal?-pNP) from pNP-?-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-?(1?3)-Gal-pNP synthesis and increased the Gal-?(1?4)-Gal yield compared to Gal-?(1?6)-Gal-pNP.

SUBMITTER: Bakunina I 

PROVIDER: S-EPMC6213131 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature