Apo D-Amino Acid Oxidase
(D-Amino acid: oxygen oxidoreductase (deaminating); EC 126.96.36.199)
D-Amino acid oxidase catalyzes the oxidation of D-amino acids
as shown below:
RCHNH2COOH + O2 + H2O ------> RCOCOOH + NH3 + H2O2
The D isomers of alanine, methionine, valine, isoleucine,
phenylalanine and proline serve as good substrates while the L
isomers do not react at all. The enzyme is a
flavoprotein. D-amino acid oxidase from porcine kidney has
been extensively studied. It has a monomeric molecular weight
of 38,000-39,000 (Tu, S.C., Edelstein, S.J. and McCormick,
D.B., Arch. Biochem. Biophys., 159, 889, 1973)
D-Amino acid oxidase has several possible applications such as
the determination of D-amino acids, the separation of natural
L- amino acid isomers from a racemic mixture and in the
preparation of keto acids. The usefulness and application of
D-amino acid oxidase can be significantly increased if it is
available in an immobilized form (Parkin, K. and Hultin, H.O.,
Biotech. and Bioeng., Vol XXI, 939, 1979).
||Distilled water or dilute buffer
||Store at -20° C (-4° F)
||25-30 U/mg protein
||Free of FAD and Alkaline Phosphatase
The amount of enzyme that will deaminate by oxidation one micromole of D-alanine to pyruvate per minute at pH 8.3, at 37°C in the presence of catalase.
The assay is based on the method described by Bergmeyer (Methods of Enzymatic Analysis, Bergmeyer, H.U. ed. Vol 1, 431, 1974, Academic Press, New York). The decrease in the absorbance at 340 nm, due to the oxidation of NADH, is a measure of D-amino acid oxidase activity.
- 0.2 M Tris-HCl buffer, pH 8.3.
- 0.02 M D-Alanine (17.8 mg/ml) in buffer.
- 0.008 M NADH disodium salt (5 mg/ml) in buffer.
- Catalase (200 U/ml) in buffer. Prepare fresh.
- Lactate dehydrogenase (LDH) (200 U/ml) in buffer. Prepare fresh.
- FAD (Prepare1 mg/ml solution)
- D-Amino acid oxidase solution. Dilute in buffer to give a concentration of 0.1-0.5 U/ml. Must be prepared fresh prior to assay.
- Set spectrophotometer (equipped with a strip chart recorder and temperature
control) at 340 nm and 37°C.
- Bubble oxygen through the buffer for 5-10 min. to saturate it with oxygen.
- In a cuvette, pipette the following reagents in the amounts
Tris buffer (oxygenated) 2.00 ml
D-Alanine 0.50 ml
Catalase 0.10 ml
LDH 0.10 ml
FAD 0.10 ml
Incubate in spectrophotometer at 37°C for 5 min. to attain temperature
equilibration. Record absorbance at 340 nm (blank).
- Initiate the reaction by adding 0.1 ml D-amino acid oxidase (enzyme) to the
cuvette. Follow the reaction by recording the decrease in the absorbance at 340
nm for 5-8 min.
- Calculate E340nm/min
|Activity (U/mg) =
||(ΔE340nm/min)(Total Vol.)(Enz. Diln.)
|(6.22)(Enz. Vol.)(mg Enz./ml)