A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity:: resorufin-β-D-cellobioside
A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity:: resorufin-β-D-cellobioside
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DOI：
10.1016/j.ab.2007.08.027
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发表时间：
2007-12-15
影响因子：
2.9
通讯作者：
Naleway, John J.
中科院分区：
文献类型：
--
作者：
Coleman, Daniel J.;Studler, Missy J.;Naleway, John J.
以试卤灵-β-D-纤维二糖苷（Res-CB）为底物，建立了一种简便、可靠的连续测定纤维素酶活力的方法。酶反应的产物试卤灵在571 nm激发下在585 nm处显示荧光发射，pK（a）为5.8，这允许在生理pH值或接近生理pH值处连续测量荧光转换。使用纯化的纤维素酶从微观真菌Trichoderma rcesei进行的测定已被证明给动力学参数K为112 μ M和V-最大值为0.000673 μ mol/mL/min。使用纤维素酶从两个活拟南芥植物和尼日尔曲霉真菌物种分离进行测定的方法。(C)2007爱思唯尔公司All rights reserved.
A simple and reliable continuous assay procedure for measurement of cellulase activity from several species using the new substrate resorufin-beta-D-cellobioside (Res-CB) has been developed. The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 urn and has a pK(a) of 5.8, which allows continuous measurement of fluorescence turnover at or near physiological pH values. The assay performed using purified cellulase from the microscopic fungus Trichoderma rcesei has been shown to give the kinetic parameters K of 112 mu M and V-max of 0.000673 mu mol/mL/min. Methods for performing the assay using cellulases isolated from both live Arabidopsis thaliana plant and Aspergillus niger fungal species are presented. (C) 2007 Elsevier Inc. All rights reserved.