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Glycobiology. 2013 Jun;23(6):736-44. doi: 10.1093/glycob/cwt012. Epub 2013 Feb 22.

Identification and characterization of endo-β-N-acetylglucosaminidase from methylotrophic yeast Ogataea minuta.

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  • 1Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba 305-8566, Japan.

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.

KEYWORDS:

Endo-Om; Endo-β-N-acetylglucosaminidase; Ogataea minuta; transglycosylation; yeast

PMID:
23436287
[PubMed - indexed for MEDLINE]
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