Purification and characterization of a novel α-glucuronidase from Aspergillus niger specific for O-α-D-glucosyluronic acid α-D-glucosiduronic acid

Abstract

A new α-glucuronidase that specifically hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was purified from a commercial enzyme preparation from Aspergillus niger, and its properties were examined. The enzyme did not degrade O-α-D-glucosyluronic acid α-D-glucoside, O-α-D-glucosyluronic acid β-D-glucosiduronic acid, O-α-D-glucosyluronic acid-(1 → 2)-β-D-fructosiduronic acid, p-nitrophenyl-O-α-D-glucosiduronic acid, methyl-O-α-D-glucosiduronic acid, or 6-O-α-(4-O-α-D- glucosyluronic acid)-D-glucosyl-β-cyclodextrine. Furthermore, it showed no activity on α-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-α-(1 → 2)-xylooligosaccharides, derived from xylan, a supposed substrate of α-glucuronidases. The molecular mass of the enzyme was estimated to be 120 kDa by gel filtration and 58 kDa by SDS-PAGE suggesting, the enzyme is composed of two identical subunits. It was most active at pH 3.0-3.5 and at 40°C. It was stable in pH 2.0-4.5 and below 30°C. It hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid to produce α- and β-anomers of D-glucuronic acid in an equimolar ratio. This result suggests that inversion of the anomeric configuration of the substrate is involved in the hydrolysis mechanism.