A novel fluorescence-based assay for the transglycosylation activity of endo-beta-N-acetylglucosaminidases.
A fluorescence-based assay for the transglycosylation activity of endo-beta-N-acetylglucosaminidases (ENGases) was developed. The assay was based on the findings that a coupled chitinase can specifically capture and hydrolyze the fluorogenic intermediate that is formed by the ENGase-catalyzed transglycosylation to release a fluorophore, but does not hydrolyze the donor asparagine-linked N-glycan and the acceptor 4-methylumbelliferyl N-acetylglucosaminide. The assay method was verified by detecting the transglycosylation activities of the known ENGases. Its application for assessing the effects of organic solvents on transglycosylation activity was demonstrated. The novel coupled assay provides a highly sensitive, easy, and quantitative method for screening endo-beta-N-acetylglucosaminidases with transglycosylation activities useful for glycoconjugate synthesis.
Hauser
Steven
S
Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA.
Song
Haijing
H
Li
Hengguang
H
Wang
Lai-Xi
LX
D002688:Chitinases / Q000032:analysis* / Q000737:chemistry*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004789:Enzyme Activation; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000032:analysis* / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D002688:Chitinases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D004121:Dimethyl Sulfoxide