Glutamate dehydrogenase gdh enzyme

The whole-cell glutaminase activity was measured as described previously^{52 (link)} with some modifications as follows. E. coli B834 cells containing empty vector, gatA or tse8 in pET41a were grown to OD₆₀₀ ~ 0.6 when expression was induced by addition of IPTG (0.5 mM) and grown at 18 °C for 16 h. Cells pellets equivalent to 45 OD₆₀₀ units were washed in sodium acetate solution (sodium acetate (100 mM, pH 6), L-glutamine (20 mM)) and resuspended in a final volume of 600 μL sodium acetate solution, and incubated at 37 °C for 30 min. 20 μL of cells were retained and serially diluted to quantify the CFUs present. The remaining cell volume was then lysed by heating at 99 °C for 3 min. Once cooled to room temperature 100 μL of cell lysate was added to 2 mL of glutamate dehydrogenase solution (sodium acetate (10 mm), NAD⁺ (4 mM), hydroxylamine HCl (400 mM), 30 U of glutamate dehydrogenase (GDH) enzyme (Sigma) in potassium phosphate buffer (100 mM, pH 7.2)) and incubated at 60 °C for 60 min. 150 μL of the reaction was added to a 96 well clear plate and the relative accumulation of NADPH was calculated using the measured absorbance at 340 nm.