Glutamate Oxidase(GLOD-E) | キッコーマンバイオケミファ

Glutamate Oxidase(GLOD-E) 61227 グルタミン酸オキシダーゼ(GLOD-E)

臨床診断用酵素

グルタミン酸に対し高い特異性を示すFAD依存性グルタミン酸酸化酵素です。遺伝子組換え技術を用いて生産しており、品質の安定した酵素です。開発中のため、詳細はお問合せください。

由来	recombinant E. coli
系統名	L-glutamate:oxygen oxidoreductase (deaminating)
EC 番号	1.4.3.11
反応式	L-glutamate + O₂ + H₂O →→→ 2-oxoglutarate + NH₃ + H₂O₂

仕様

Appearance	Light yellow to yellow lyophilizate
Activity	≧15 U/mg lyophilizate
Stabilizer	raffinose
Storage	below -20℃

特性

Molecular weight	ca. 115 kDa (gel filtration)
Structure	2 subunits of 70 kDa (SDS-PAGE) 1 mole of FAD per 1 mole of enzyme subunit
Michaelis constant	3.2 × 10-⁴ M (L-glutamate)
pH Optimum	6.0 – 8.0 (Fig. 1)
pH Stability	6.0 – 8.5 (Fig. 2)
Optimum temperature	40 – 50℃ (Fig. 3)
Thermal stability	below 55℃ (Fig. 4)
Thermal stability (liquid form)	stable at 37℃ for at least two weeks (Fig. 5)
Stability (powder form)	stable at 30℃ for at least one month (Fig. 6)

アプリケーション

GLOD-Eとトリンダー試薬を用いた比色法によりグルタミン酸を定量可能です。また、GLOD-Eを電極に固定化し、グルタミン酸を電極法により連続測定できます。

分析手順

Principle

The appearance of quinoneimine dye is measured spectrophotometrically at 555 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of hydrogen peroxide per min at 30℃ and pH 7.4 under the conditions described below.

Reagents

Potassium phosphate buffer, 0.25 M; pH 7.4: mix 0.25 M KH₂PO₄ solution and 0.25 M K₂HPO₄ solution to make a pH 7.4 solution.
TOOS solution, 15 mM: 0.50 g of TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine) / 100 mL of distilled water.
Peroxidase (POD) solution, 300 U/mL; 15 mg of POD (200 guaiacol U/mg)/10 mL of distilled water.
4-Aminoantipyrine (4-AA) solution, 30 mM: 610 mg of 4-AA/100 mL of distilled water.
Sodium Hydrogen L-Glutamate Monohydrate solution, 300 mM: 2.36 g of Sodium Hydrogen L-Glutamate Monohydrate/50 mL of distilled water.
Enzyme dilution buffer: 10 mM Potassium phosphate buffer, pH 7.4, containing 0.1% bovine serum albumin (BSA).

Sample: dissolve the lyophilized enzyme to a volume activity of 0.3-0.6 U/mL in ice-cold enzyme dilution buffer (Reagent F) immediately before

Procedure

Pipette the following reagents into a cuvette (light path: 1 cm).

2.40 mL Potassium phosphate buffer (Reagent A)

0.20 mL TOOS solution (Reagent B)

0.10 mL POD solution (Reagent C)

0.10 mL 4-AA solution (Reagent D)
Equilibrate at 30℃ for about 3 min.
Add 0.10 mL of sample and incubate for 1 min at 30℃.
Add 0.10 mL of Sodium Hydrogen L-Glutamate Monohydrate solution (Reagent E) and mix.
Record the increase of absorbance at 555 nm in a spectrophotometer thermostated at 30℃ and calculate the ΔA per min using the linear portion of the curve (ΔA_S).
The blank solution is prepared by adding enzyme dilution buffer (Reagent F) instead of sample (A₀).

2.40 mL	Potassium phosphate buffer	(Reagent A)
0.20 mL	TOOS solution	(Reagent B)
0.10 mL	POD solution	(Reagent C)
0.10 mL	4-AA solution	(Reagent D)

Calculation

Activity can be calculated by using the following formula:

39.2: Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm²/μmol)
1/2 : Factor based on the fact that 1 mol of hydrogen peroxide produces 1/2 mol of quinoneimine dye
df : Dilution factor
C : Content of sarcosine oxidase preparation in sample (mg/mL)