title: Identification and characterization of endo-β-N-acetylglucosaminidase from methylotrophic yeast Ogataea minuta.
abstract: In four yeast strains, Ogataea minuta, Candida parapolymorpha, Pichia anomala and Zygosaccharomyces rouxii, we identified endo-β-N-acetylglucosaminidase (ENGase) homologous sequences by database searches; in each of the four species, a corresponding enzyme activity was also confirmed in crude cell extract obtained from each strain. The O. minuta ENGase (Endo-Om)-encoding gene was directly amplified from O. minuta genomic DNA and sequenced. The Endo-Om-encoding gene contained a 2319-bp open-reading frame; the deduced amino acid sequence indicated that the putative protein belonged to glycoside hydrolase family 85. The gene was introduced into O. minuta, and the recombinant Endo-Om was overexpressed and purified. When the enzyme assay was performed using an agalacto-biantennary oligosaccharide as a substrate, Endo-Om exhibited both hydrolysis and transglycosylation activities. Endo-Om exhibited hydrolytic activity for high-mannose, hybrid, biantennary and (2,6)-branched triantennary N-linked oligosaccharides, but not for tetraantennary, (2,4)-branched triantennary, bisecting N-acetylglucosamine structure and core-fucosylated biantennary N-linked oligosaccharides. Endo-Om also was able to hydrolyze N-glycans attached to RNase B and human transferrin under both denaturing and nondenaturing conditions. Thus, the present study reports the detection and characterization of a novel yeast ENGase.
authors: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba 305-8566, Japan.
authors: Satoshi
authors: S
authors: Murakami
authors: Yuki
authors: Y
authors: Takaoka
authors: Hisashi
authors: H
authors: Ashida
authors: Kenji
authors: K
authors: Yamamoto
authors: Hisashi
authors: H
authors: Narimatsu
authors: Yasunori
authors: Y
authors: Chiba
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010802:Phylogeny; D011499:Protein Processing, Post-Translational; D012260:Ribonucleases / Q000737:chemistry; D004718:Saccharomycetales / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014168:Transferrin / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D009844:Oligosaccharides; D014168:Transferrin; D012260:Ribonucleases; C029600:ribonuclease B; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
keywords: Endo-Om; Endo-β-N-acetylglucosaminidase; Ogataea minuta; transglycosylation; yeast