Enzymatic Assay of CmFUM Activity

The 100 μl assay solution containing 100 mM Tris-HCl (pH 7.5), 50 pmol CmFUM, and 1 M disodium fumarate was incubated at 52°C for 24 h. Thereafter, HCl was added to the assay solution to be 100 mM. The sample is analyzed by HPLC using an LC-2000Plus System (JASCO, Tokyo, Japan). Organic acids were quantified using 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer and peaks were detected at 445 nm, as described previously (Osanai et al., 2015 (link)).

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Ito S., Iwazumi K., Sukigara H, & Osanai T. (2020). Fumarase From Cyanidioschyzon merolae Stably Shows High Catalytic Activity for Fumarate Hydration Under High Temperature Conditions. Frontiers in Microbiology, 11, 2190.

Publication 2020

LC-2000Plus System

Acids Assay Bromothymol blue Buffer Fumarate Hplc Sodium phosphate Tris

Corresponding Organization : Meiji University

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Variable analysis

independent variables

Incubation temperature (52°C)
Incubation time (24 h)

dependent variables

Concentration of organic acids (quantified using 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer and detected at 445 nm)

control variables

Assay solution composition (100 mM Tris-HCl (pH 7.5), 50 pmol CmFUM, and 1 M disodium fumarate)
HCl concentration in the assay solution (100 mM)

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