Purification and Characterization of an α-Glucosidase from Rhizobium sp. ( Robinia pseudoacacia L.) Strain USDA 4280

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.