Abstract
The conventional method of transketolase (TKT) activity assay uses ribose 5-phosphate and xylulose 5-phosphate as substrates. However, a new method of TKT assay is currently required since xylulose 5-phosphate is no longer commercially available and is difficult to synthesize chemically. Although there are effective assays for TKT using non-natural substrates, these are inadequate for evaluating changes in enzyme activity and affinity toward real substrates. As a solution to such problems, we describe a novel assay system using xylulokinase (XK) from Saccharomyces cerevisiae. As for this purpose, the XK was overexpressed in E. coli, separated and purified in a single step, added to induce a reaction that generated xylulose 5-phosphate, which was integrated into the conventional TKT assay. The new coupling assay gave reproducible results with E. coli TKT and had a detection limit up to 5 × 10−4 unit/mg protein. A reliable result was also achieved for the incorporation of XK and TKT into a single reaction.
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Lee, JY., Cheong, DE. & Kim, GJ. A novel assay system for the measurement of transketolase activity using xylulokinase from Saccharomyces cerevisiae . Biotechnol Lett 30, 899–904 (2008). https://doi.org/10.1007/s10529-007-9616-y
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DOI: https://doi.org/10.1007/s10529-007-9616-y