Purification and characterization of a novel alpha-glucuronidase from Aspergillus niger specific for O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid.
A new alpha-glucuronidase that specifically hydrolyzed O-alpha-D-glucosyluronic acid alpha-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-alpha-D-glucosyluronic acid alpha-D-glucoside, O-alpha-D-glucosyluronic acid beta-D-glucosiduronic acid, O-alpha-D-glucosyluronic acid-(1-->2)-beta-D-fructosiduronic acid, p-nitrophenyl-O-alpha-D-glucosiduronic acid, methyl-O-alpha-D-glucosiduronic acid, or 6-O-alpha-(4-O-alpha-D-glucosyluronic acid)-D-glucosyl-beta-cyclodextrine. Furthermore, it showed no activity on alpha-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-alpha-(1-->2)-xylooligosaccharides, derived from xylan, a supposed substrate of alpha-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 degrees C. It was stable in pH 2.0-4.5 and below 30 degrees C. It hydrolyzed O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid to produce alpha- and beta-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.
Kiryu
Takaaki
T
Department of Biochemistry and Materials for Human Life, Osaka Municipal Technical Research Institute, Japan. kiryu@omtri.city.osaka.jp
Nakano
Hirofumi
H
Kiso
Taro
T
Murakami
Hiromi
H
D001234:Aspergillus niger / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D013379:Substrate Specificity
D006026:Glycoside Hydrolases; C096734:alpha-glucuronidase