Document Type : Original Article
Author
Abstract
A glycoside hydrolase enzyme known as α-galactosidase (EC 3.2.1.22) is
responsible for hydrolyzing the terminal alpha-galactosyl moieties found in
glycolipids and glycoproteins. Bacteria that produce this enzyme are isolated from
Erysipelothrix rhusiopathia. Following the release of α-galactosidase from the cells,
the purification process involves precipitating the protein with 40% ammoniumsulfate, resuspending it, and dialyzing it using a dialyze tube cut-off number (10–14
KD) overnight against phosphate buffer (pH=7). Finally, the protein is further purified
using DEAE-cellulose column chromatography with (33.4) folds. At 45 °C, the
enzyme had maximal activity, while 6.5 °C was its optimum pH. Through the use of
SDS-PAGE technique, the molecular weight of the enzyme was estimated to be (55
KD). The enzyme's Km and Vmax were found to be (6.6 mM) and (833.3μmol/min)
of protein, respectively, using p-nitrophenyl-α-D-galactopyranoside as the substrate.
Following treatments of the purified enzyme with chlorogenic acid and caffeine,
studies involving caffeine indicate that the inhibitor has the same affinity for the
enzyme as the substrate pNPG. Measurements of the reaction rates at different
concentrations of substrate and inhibitor observed a non-competitive inhibition and
showed that the Km value was ineffective (6.66) mM and a decrease in Vmax
values (555.5) μmol/min. While when we treated chlorogenic acid with α -
galactosidase that is purified from Erysipelothrix rhusiopathiae this substrate acted
as uncompetitive inhibition with substrate pNPG as the catalytic site that reflected
the enzyme has single active that results from the change of Km and Vmax (3.22 mM)
and (400 μmol/min) respectively
Keywords
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