IGEM:IMPERIAL/2009/Assays Protocols/Trehalose/OtsB

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As this enzyme catalyses the reaction:

alpha,alpha-trehalose 6-phosphate + H2O -> alpha,alpha-trehalose + phosphate

we could use a standard trehalose assay as already defined

Also, there are assays for the T6PP enzyme:
OtsB Information and Basic Assay

  • Gives a quantitative output for the T6PP activity

"Initially, the phosphatase activity of T6PP was assayed against para-Nitrophenyl phosphate (pNPP) as substrate by continuously following production of p-nitrophenolate at 410 nM using Beckman DU530 spectrophotometer (Chen et al. 1956; Parsons et al. 2002). A substrate concentration series was prepared by dissolving pNPP in a solution of 50 mM Tris-HCl (pH 8.0), 5 mM DTT, and 20 mM MgCl2. The reaction was carried out in a total volume of 100 μL using different substrate concentrations (5–50 mM) for 20 min at 50°C in the presence of T6PP and was stopped by diluting with 100 mM NaOH (final concentration). Reactions performed without T6PP served as control. The assay was also carried out keeping the substrate concentration fixed and varying the duration of the reaction time. The amount of reaction product released was calculated by measuring the difference in absorbance between the controls and the corresponding enzyme-containing reactions with a molar absorptive coefficient of 18,400 M−1cm−1 for pNPP (Parsons et al. 2002). Kinetic constants were determined from the rate of hydrolysis of the substrate over a range of concentrations using the Michaelis-Menten equation. The trehalose-6-phosphate phosphatase activity was determined using trehalose-6-phosphate (T6P; molar absorptive coefficient 25,000 M−1cm−1) as substrate (1–10 mM). Release of inorganic phosphate was estimated by a published method (Chen et al. 1956). The experiment was also repeated with the enzyme treated with EDTA to remove any metal ion."

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