一种使用荧光底物6,8-二氟-4-甲基伞形酮基-β-D-木二糖苷的木聚糖酶超灵敏连续检测法。
An ultrasensitive, continuous assay for xylanase using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside.
作者信息
Ge Yue, Antoulinakis Evan G, Gee Kyle R, Johnson Iain
机构信息
Invitrogen Corp., 29851 Willow Creek Road, Eugene, OR 97402, USA.
出版信息
Anal Biochem. 2007 Mar 1;362(1):63-8. doi: 10.1016/j.ab.2006.11.034. Epub 2006 Dec 20.
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.
我们描述了一种基于荧光的分析方法，用于使用新型荧光底物6,8-二氟-4-甲基伞形酮基β-D-木二糖苷（DiFMUX(2)）分析木聚糖酶活性。从底物生成荧光性的6,8-二氟-4-甲基伞形酮（DiFMU）直接对应于木聚糖酶活性。对消化产物的高效液相色谱分析表明，木聚糖酶将DiFMUX(2)直接水解为DiFMU和木二糖。该分析方法提供了以高通量形式测量木聚糖酶活性或筛选木聚糖酶抑制剂所需的速度、灵敏度和便利性，适用于对来自各种来源的木聚糖酶进行动力学分析。
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