| Autor: |
Irina Bakunina, Lubov Slepchenko, Stanislav Anastyuk, Vladimir Isakov, Galina Likhatskaya, Natalya Kim, Liudmila Tekutyeva, Oksana Son, Larissa Balabanova |
| Jazyk: |
angličtina |
| Rok vydání: |
2018 |
| Předmět: |
|
| Zdroj: |
Marine Drugs, Vol 16, Iss 10, p 349 (2018) |
| Druh dokumentu: |
article |
| ISSN: |
1660-3397 |
| DOI: |
10.3390/md16100349 |
| Popis: |
A novel wild-type recombinant cold-active α-d-galactosidase (α-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 α-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 α-galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild α-PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl-α-galactopiranoside (pNP-α-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. α-PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. α-PsGal synthesized -α(1→6)- and -α(1→4)-linked galactobiosides from melibiose as well as -α(1→6)- and -α(1→3)-linked p-nitrophenyl-digalactosides (Gal2-pNP) from pNP-α-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-α(1→3)-Gal-pNP synthesis and increased the Gal-α(1→4)-Gal yield compared to Gal-α(1→6)-Gal-pNP. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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