An ultrasensitive, continuous assay for xylanase using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside.
We describe a fluorescence-based assay for the analysis of xylanase activity using a novel fluorogenic substrate, 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside (DiFMUX(2)). Generation of fluorescent 6,8-difluoro-4-methylumbelliferone (DiFMU) from the substrate corresponded directly to xylanase activity. High-performance liquid chromatography analysis of the digestion products showed that xylanase hydrolyzed DiFMUX(2) directly to DiFMU and xylobiose. The assay provides the speed, sensitivity, and convenience required for measuring xylanase activity or for screening xylanase inhibitors in a high-throughput format and is suitable for the kinetic assay of xylanases from a variety of sources.
Ge
Yue
Y
Invitrogen Corp., 29851 Willow Creek Road, Eugene, OR 97402, USA.
Antoulinakis
Evan G
EG
Gee
Kyle R
KR
Johnson
Iain
I
D001234:Aspergillus niger / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D015203:Reproducibility of Results; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology
C504401:6,8-difluoro-4-methylumbelliferyl sulfate; D005456:Fluorescent Dyes; D006027:Glycosides; C010807:xylosides; D006923:Hymecromone; D043364:Endo-1,4-beta Xylanases