تفاعل #1355851
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Glucose isomerase assays: TNXI 1F1 and Gensweet™ were assayed routinely with glucose as the substrate. The enzyme (1–1.5 mg/mL) was incubated in 100 mM MOPS buffer (pH 7.0) [or 100 mM sodium acetate buffer (pH 5.5)] containing 1 mM CoCl2 and 0.4 mM glucose at 80° C. for 10 min. The reaction was stopped by transferring the tube to an ice bath. The amount of fructose produced was determined by the modified resorcinol-ferric ammonium sulfate-hydrochloric acid method (Schenk and Bisswanger, 1998). To determine the effect of temperature on the activity of TNXI 1F1 and Gensweet™, the holo-enzyme was incubated in the reaction mixture at the temperatures of interest in a heated water bath (45–95° C.) or a heated oil bath (95–110° C.) for 10 min. The effect of pH on activity was determined using the routine assay described above except that the MOPS buffer was substituted with 100 mM sodium acetate buffer (pH 4.3–5.8), 100 mM PIPES buffer (pH 6.1–7.0), or 100 mM EPPS buffer (pH 7.2–8.1). All pHs were adjusted at room temperature, and the ΔpKa/Δt's for acetate, PIPES, and EPPS (0, −0.0085, −0.011, respectively) (USB, Cleveland, Ohio) were taken into account for the results. To determine the kinetic parameters, assays were performed in the presence of 10–1,500 mM glucose, 50 mM MOPS (pH 7.0) and 1 mM CoCl2. One unit of glucose isomerase activity is defined as the amount of enzyme that produces 1 μmol of fructose per minute under the assay conditions.