AUTHOR=Bakunina Irina Y. , Balabanova Larissa A. , Golotin Vasiliy A. , Slepchenko Lyubov V. , Isakov Vladimir V. , Rasskazov Valeriy A. 

TITLE=Stereochemical course of hydrolytic reaction catalyzed by alpha-galactosidase from cold adaptable marine bacterium of genus Pseudoalteromonas

JOURNAL=Frontiers in Chemistry

VOLUME=2

YEAR=2014

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2014.00089

DOI=10.3389/fchem.2014.00089

ISSN=2296-2646

ABSTRACT=<p>The recombinant α-galactosidase of the marine bacterium (α-<italic>Ps</italic>Gal) was synthesized with the use of the plasmid 40Gal, consisting of plasmid pET-40b (+) (Novagen) and the gene corresponding to the open reading frame of the mature α-galactosidase of marine bacterium <italic>Pseudoalteromonas</italic> sp. KMM 701, transformed into the <italic>Escherichia coli</italic> Rosetta(DE3) cells. In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-<italic>Ps</italic>Gal was measured by <sup>1</sup>H NMR spectroscopy. The kinetics of formation of α- and β-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of β-anomer appeared as a result of mutarotation. The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction.</p>