Heterologous expression and characterization of processing α-glucosidase I from Aspergillus brasiliensis ATCC 9642.
A gene for processing α-glucosidase I from a filamentous fungus, Aspergillus brasiliensis (formerly called Aspergillus niger) ATCC 9642 was cloned and fused to a glutathione S-transferase tag. The active construct with the highest production level was a truncation mutant deleting the first 16 residues of the hydrophobic N-terminal domain. This fusion enzyme hydrolyzed pyridylaminated (PA-) oligosaccharides Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA and the products were identified as Glc(2)Man(9)GlcNAc(2)-PA and Glc(2)Man(4)-PA, respectively. Saturation curves were obtained for both Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA, and the K (m) values for both substrates were estimated in the micromolar range. When 1 μM Glc(3)Man(4)-PA was used as a substrate, the inhibitors kojibiose and 1-deoxynojirimycin had similar effects on the enzyme; at 20 μM concentration, both inhibitors reduced activity by 50%.
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
Takatsugu
T
Miyazaki
Yuji
Y
Matsumoto
Kana
K
Matsuda
Yuma
Y
Kurakata
Ichiro
I
Matsuo
Yukishige
Y
Ito
Atsushi
A
Nishikawa
Takashi
T
Tonozuka
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000187:drug effects / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D065089:Glycoside Hydrolase Inhibitors; D006868:Hydrolysis / Q000187:drug effects; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000520:alpha-Glucosidases / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D065089:Glycoside Hydrolase Inhibitors; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; C042059:glucosidase I; D000520:alpha-Glucosidases; D000117:Acetylglucosamine