Affiliation:
1. Glycobiologie, Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique UPR 4301, Université d’Orléans, 45071 Orléans cedex 2, France
Abstract
ABSTRACT
A novel α-glucosidase with an apparent subunit mass of 59 ± 0.5 kDa was purified from protein extracts of
Rhizobium
sp. strain USDA 4280, a nodulating strain of black locust (
Robinia pseudoacacia
L), and characterized. After purification to homogeneity (475-fold; yield, 18%) by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic chromatography, dye chromatography, and gel filtration, this enzyme had a pI of 4.75 ± 0.05. The enzyme activity was optimal at pH 6.0 to 6.5 and 35°C. The activity increased in the presence of NH
4
+
and K
+
ions but was inhibited by Cu
2+
, Ag
+
, Hg
+
, and Fe
2+
ions and by various phenyl, phenol, and flavonoid derivatives. Native enzyme activity was revealed by native gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis with fluorescence detection in which 4-methylumbelliferyl α-glucoside was the fluorogenic substrate. The enzyme was more active with α-glucosides substituted with aromatic aglycones than with oligosaccharides. This α-glucosidase exhibited Michaelis-Menten kinetics with 4-methylumbelliferyl α-
d
-glucopyranoside (
K
m
, 0.141 μM;
V
max
, 6.79 μmol min
−1
mg
−1
) and with
p
-nitrophenyl α-
d
-glucopyranoside (
K
m
, 0.037 μM;
V
max
, 2.92 μmol min
−1
mg
−1
). Maltose, trehalose, and sucrose were also hydrolyzed by this enzyme.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
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