TY - JOUR
T1 - Enzymatic synthesis of a neoglycoconjugate by transglycosylation with Arthrobacter endo-β-N-acetylglucosaminidase
T2 - A substrate for colorimetric detection of endo-β-N-acetylglucosaminidase activity
AU - Takegawa, Kaoru
AU - Fujita, Kiyotaka
AU - Fan, Jian Qiang
AU - Tabuchi, Mitsuaki
AU - Tanaka, Naotaka
AU - Kondo, Akihiro
AU - Iwamoto, Hiroyuki
AU - Kato, Ikunoshin
AU - Lee, Yuan C.
AU - Iwahara, Shojiro
N1 - Funding Information:
We thank Dr. Kenji Yamamoto (Kyoto University) for helpful advice and Satomi Suda for her excellent technical assistance. This work was partly supported by Kagawa Techno Foundation and a Grant-in-Aid for Scientific Research (09358013) from the Ministry of Education, Science, Sports, and Culture of Japan (to K.T.).
PY - 1998/3/15
Y1 - 1998/3/15
N2 - The transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-p-nitrophenylα-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 specrometry. Various endo-β-N-acetylglucosaminidases hydrolyzed this oligosaccharide and Man6GlcNAc and Glc-pNP were released from the oligosaccharide by endo-β-N-acetylglucosaminidase digestion. We have established a new procedure for the colorimetric detection of endo-βN- acetylglucosaminidase activity using Man6GlcNAc-Glc-pNP, which is simple as that for other exoglycosidase assays with pNP-glycosides as substrates.
AB - The transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-p-nitrophenylα-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 specrometry. Various endo-β-N-acetylglucosaminidases hydrolyzed this oligosaccharide and Man6GlcNAc and Glc-pNP were released from the oligosaccharide by endo-β-N-acetylglucosaminidase digestion. We have established a new procedure for the colorimetric detection of endo-βN- acetylglucosaminidase activity using Man6GlcNAc-Glc-pNP, which is simple as that for other exoglycosidase assays with pNP-glycosides as substrates.
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U2 - 10.1006/abio.1997.2543
DO - 10.1006/abio.1997.2543
M3 - Article
C2 - 9514782
AN - SCOPUS:0032521067
SN - 0003-2697
VL - 257
SP - 218
EP - 223
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -