基于酶触发的点击化学反应测定β-葡萄糖苷酶活性

Determination of β-Glucosidase Activity Based on Enzyme-Triggered Click Chemistry

建立了一种新的β-葡萄糖苷酶活性定量检测的方法,其原理是通过β-葡萄糖苷酶对2-O-β-D-葡萄糖基-L-抗坏血酸的特异性水解,释放出抗坏血酸来还原Cu(II),原位生成的Cu(I)催化荧光较弱的香豆素和苄基叠氮进行环加成点击反应,产生高荧光强度的三氮唑,从而利用荧光光谱检测体系荧光强度的变化,反映出β-葡萄糖苷酶的活性.实验结果显示,在1～40U/L范围内,荧光强度增强程度与β-葡萄糖苷酶活性呈线性关系,可实现定量检测,其最低检测限为0.456 U/L.

A novel method to quantify β-glucosidase activity was developed by coupling the cleavage of 2-O-（β-glucopyranosyl）ascorbic acid with the reduction of Cu（II）. The in situ generated Cu（I） catalyzed the cycloaddition between weakly fluorescent coumarin and benzyl azide to yield a highly fluorescent triazole product. The fluorescence intensity was dependently enhanced on the increase of the activity of β-glucosidase and a linear relationship was found between 1～40 U/L. This cascade allows detection of β-glucosidase with a limit of detection of 0.456 U/L.