Enzymatic synthesis of a neoglycoconjugate by transglycosylation with Arthrobacter endo-beta-N-acetylglucosaminidase: a substrate for colorimetric detection of endo-beta-N-acetylglucosaminidase activity

The transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-p-nitrophenyl-alpha-D-glucose (Man6GlcNAc-Glc-pNP). The reaction was efficiently induced in aqueous solution containing dim...

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Bibliographic Details
Published in:Analytical biochemistry 1998-03, Vol.257 (2), p.218-223
Main Authors: Takegawa, K, Fujita, K, Fan, J Q, Tabuchi, M, Tanaka, N, Kondo, A, Iwamoto, H, Kato, I, Lee, Y C, Iwahara, S
Format: Article
Language:English
Subjects:
Acetylglucosaminidase - chemistry
Acetylglucosaminidase - metabolism
Arthrobacter - enzymology
Carbohydrate Sequence
Colorimetry
Dimethyl Sulfoxide
Flavobacterium - enzymology
Glycosylation
Hydrolysis
Magnetic Resonance Spectroscopy
Molecular Sequence Data
Oligosaccharides - chemical synthesis
Oligosaccharides - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Streptomyces - enzymology
Yeasts - enzymology
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Summary:The transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-p-nitrophenyl-alpha-D-glucose (Man6GlcNAc-Glc-pNP). The reaction was efficiently induced in aqueous solution containing dimethyl sulfoxide. In the medium containing 20% (v/v) dimethyl sulfoxide with 0.1 M Glc-pNP as an acceptor, the transglycosylation attained yields of 75% by high-performance anion-exchange chromatography. The structure of Man6GlcNAc-Glc-pNP was confirmed by ion mass spectrometry and 400 MHz 1H NMR spectrometry. Various endo-beta-N-acetylglucosaminidases hydrolyzed this oligosaccharide and Man6GlcNAc and Glc-pNP were released from the oligosaccharide by endo-beta-N-acetylglucosaminidase digestion. We have established a new procedure for the colorimetric detection of endo-beta-N-acetylglucosaminidase activity using Man6Glc-NAc-Glc-pNP, which is simple as that for other exoglycosidase assays with pNP-glycosides as substrates.
ISSN:0003-2697
DOI:10.1006/abio.1997.2543