Cloning and overexpression of beta-N-acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide.
We isolated a beta-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture. The beta-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21% and 0.15% yield, respectively.
Matsuo
Ichiro
I
Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Kim
Sunhwa
S
Yamamoto
Yuichi
Y
Ajisaka
Katsumi
K
Maruyama
Jun-ich
JI
Nakajima
Harushi
H
Kitamoto
Katsuhiko
K
D000117:Acetylglucosamine / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics*; D002240:Carbohydrate Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D005800:Genes, Fungal / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D007536:Isomerism; D007785:Lactose / Q000378:metabolism; D008895:Milk, Human / Q000378:metabolism*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014312:Trisaccharides / Q000096:biosynthesis* / Q000737:chemistry
D014312:Trisaccharides; C098555:lacto-N-triose II; D000118:Acetylglucosaminidase; D007785:Lactose; D000117:Acetylglucosamine