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

Bakunina IY, Balabanova LA, Golotin VA, Slepchenko LV, Isakov VV, Rasskazov VA - Front Chem (2014)

Bottom Line: 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 (1)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.

View Article: PubMed Central - PubMed

Affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences Vladivostok, Russia.

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 (1)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.

No MeSH data available.


Related in: MedlinePlus

Results of 12.5% SDS-PAGE electrophoresis. Line 1: Cell extract of E. coli Rosetta(DE3)/40Gal; Line 2: α-PsGal after final purification step; Line 3: MW-standards. Filled arrow indicates the band corresponding to the chimeric protein with DsbC overhang (112.5 kDa); empty arrow indicates the band corresponding to the mature protein after enterokinase treatment (80 kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4195319&req=5

Figure 2: Results of 12.5% SDS-PAGE electrophoresis. Line 1: Cell extract of E. coli Rosetta(DE3)/40Gal; Line 2: α-PsGal after final purification step; Line 3: MW-standards. Filled arrow indicates the band corresponding to the chimeric protein with DsbC overhang (112.5 kDa); empty arrow indicates the band corresponding to the mature protein after enterokinase treatment (80 kDa).

Mentions: PAGE-SDS showed a single band of the purified recombinant protein with a molecular mass of approximately 80 kDa corresponding to the one subunit of α-PsGal (Figure 2).


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

Bakunina IY, Balabanova LA, Golotin VA, Slepchenko LV, Isakov VV, Rasskazov VA - Front Chem (2014)

Results of 12.5% SDS-PAGE electrophoresis. Line 1: Cell extract of E. coli Rosetta(DE3)/40Gal; Line 2: α-PsGal after final purification step; Line 3: MW-standards. Filled arrow indicates the band corresponding to the chimeric protein with DsbC overhang (112.5 kDa); empty arrow indicates the band corresponding to the mature protein after enterokinase treatment (80 kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4195319&req=5

Figure 2: Results of 12.5% SDS-PAGE electrophoresis. Line 1: Cell extract of E. coli Rosetta(DE3)/40Gal; Line 2: α-PsGal after final purification step; Line 3: MW-standards. Filled arrow indicates the band corresponding to the chimeric protein with DsbC overhang (112.5 kDa); empty arrow indicates the band corresponding to the mature protein after enterokinase treatment (80 kDa).
Mentions: PAGE-SDS showed a single band of the purified recombinant protein with a molecular mass of approximately 80 kDa corresponding to the one subunit of α-PsGal (Figure 2).

Bottom Line: 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 (1)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.

View Article: PubMed Central - PubMed

Affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences Vladivostok, Russia.

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 (1)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.

No MeSH data available.


Related in: MedlinePlus