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

Daniel J. Coleman; Missy J. Studler; John J. Naleway

doi:10.1016/j.ab.2007.08.027

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

Daniel J. Coleman, Missy J. Studler, John J. Naleway

Research output: Contribution to journal › Article › peer-review

38 Scopus citations

Abstract

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 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 reesei has been shown to give the kinetic parameters K_m of 112 μM and V_max 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.

Original language	English (US)
Pages (from-to)	146-153
Number of pages	8
Journal	Analytical Biochemistry
Volume	371
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2007.08.027
State	Published - Dec 15 2007
Externally published	Yes

Keywords

Cellulase
Continuous assay
Fluorescence
Resorufin

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.ab.2007.08.027

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

abstract = "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.",

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doi = "10.1016/j.ab.2007.08.027",

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AU - Studler, Missy J.

AU - Naleway, John J.

PY - 2007/12/15

Y1 - 2007/12/15

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AB - 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.

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KW - Continuous assay

KW - Fluorescence

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

Abstract

Keywords

ASJC Scopus subject areas

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