A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-β-d-cellobioside

A simple and reliable continuous assay procedure for measurement of cellulase activity from several species using the new substrate resorufin-β-d-cellobioside (Res-CB) has been developed. The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pKa 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 reesei has been shown to give the kinetic parameters Km of 112 μM and Vmax of 0.000673 μmol/mL/min. Methods for performing the assay using cellulases isolated from both live Arabidopsis thaliana plant and Aspergillus niger fungal species are presented.