Hydroxylamine → 5'-Adp

Participants

Reaction Steps

The crude enzyme solution was pretreated at 30 to 80° C. for 1, 3, 5 or 10 minutes and reacted with a reaction mixture at 30° C. The reaction mixture was prepared by adding the crude enzyme solution to an aqueous solution containing 100 mM Tris-HCl (pH 7.5), 10 mM ATP (pH 7.5), 600 mM hydroxylamine, 600 mM ammonium sulfate, 10 mM magnesium sulfate, 50 mM L-aspartic acid (pH 7.5), and adjusted to the total volume of 500 μl with sterilized water. The reaction was allowed to proceed for a predetermined period of time, and the reaction was terminated by addition of 750 μl of a reaction terminating solution. The reaction terminating solution had a composition of 4% trichloroacetic acid, 10% FeCl2 and 1.4 N hydrochloric acid. The reaction mixture added with the reaction terminating solution was centrifuged at 15,000 rpm for 5 minutes, and the absorbance of the supernatant was measured at a wavelength of 540 nm. This measurement was performed for quantifying color development of hydroxymate produced by the reaction in which L-aspartic acid phosphoric acid salt, ADP and hydroxymate were produced from L-aspartic acid, ATP and hydroxylamine. The reaction mixture not containing L-aspartic acid was used as a blank.

Provenance & Evidence