Abstract
The recombinant α-galactosidase of the marine bacterium (α-PsGal) 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 Pseudoalteromonas sp. KMM 701, transformed into the Escherichia coli 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-PsGal was measured by 1H 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.
Highlights
INTRODUCTION αGalactosidase (α-D-galactoside galactohydrolase; EC 3.2.1.22) catalyzes the hydrolysis of terminal α-linked galactose residues in oligosaccharides and polymeric galactomannans
Α-Galactosidases are wide distributed in marine bacteria, especially among γ-proteobacteria (Ivanova et al, 1998) and Bacteroidetes (Bakunina et al, 2012), an α-galactosidase isolated from the marine bacterium Pseudoalteromonas sp
The recombinant plasmid 40Gal of 8306 base pairs for the synthesis of the recombinant protein α-PsGal consisted of the NcoI/SalI-fragment of plasmid pET-40b (+) (Novagen) and the gene of 2130 bp corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp
Summary
Galactosidase (α-D-galactoside galactohydrolase; EC 3.2.1.22) catalyzes the hydrolysis of terminal α-linked galactose residues in oligosaccharides and polymeric galactomannans. They have found a number of useful potential biotechnological and medical applications. KMM 701 was the first biochemicaly characterized marine αgalactosidase It catalyzes the hydrolysis of α-galactose residues from non-reducing end of B-trisaccharides and is capable for reducing a serological activity of B-red blood cells at neutral pH. The α-galactosidase is able to interrupt the adhesion of pathogens to human buccal epithelium (Balabanova et al, 2010) These properties showed the great therapeutic potential and opened up broad prospects for application of the enzyme in medicine. KMM 701 α-galactosidase has been found to belong to GH36 family in CAZy classification, according to its amino acids sequence (Balabanova et al, 2010)
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