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
Glutamate Oxidaseグルタミン酸オキシダーゼ

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

  1. 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.
  2. TOOS solution, 15 mM: 0.50 g of TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine) / 100 mL of distilled water.
  3. Peroxidase (POD) solution, 300 U/mL; 15 mg of POD (200 guaiacol U/mg)/10 mL of distilled water.
  4. 4-Aminoantipyrine (4-AA) solution, 30 mM: 610 mg of 4-AA/100 mL of distilled water.
  5. Sodium Hydrogen L-Glutamate Monohydrate solution, 300 mM: 2.36 g of Sodium Hydrogen L-Glutamate Monohydrate/50 mL of distilled water.
  6. 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

  1. 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)
  2. Equilibrate at 30℃ for about 3 min.
  3. Add 0.10 mL of sample and incubate for 1 min at 30℃.
  4. Add 0.10 mL of Sodium Hydrogen L-Glutamate Monohydrate solution (Reagent E) and mix.
  5. 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 (ΔAS).
    The blank solution is prepared by adding enzyme dilution buffer (Reagent F) instead of sample (A0).

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)

実験データ

FAQ

GLOD-Eの主な用途は何ですか？
GLOD-Eの分子量を教えてください。
GLOD-Eの至適pHと至適温度について教えてください。
GLOD-Eの熱安定性について教えてください。
GLOD-Eの安定化剤は何を使用していますか？
GLOD-Eの保存方法を教えてください。
開封後の取扱い方法に注意事項はありますか？

参考文献

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