Glutamate oxidase(GLOD-E)

  • Enzymes for Clinical Chemistry
Glutamate Oxidase

CD : 61227

The enzyme is useful for the determination of L-glutamate. GLOD-E is a FAD-dependent glutamate oxidase has high specificity for glutamate. It is produced using genetic recombination technology, so it is an enzyme with stable quality.

Origin Streptomyces sp. 　
Systematic name

L-glutamate:oxygen oxidoreductase (deaminating)
EC Number 1.4.3.11
Reaction formula

L-glutamate + O2 + H2O →→→ 2-oxoglutarate + NH3 + H2O2

SPECIFICATION

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

PROPERTIES

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)

APPLICATIONS

Glutamate can be quantified by colorimetry using GLOD-E and Trinder's reagent. GLOD-E can also be immobilized on an electrode to continuously measure glutamate using the electrode method.

ASSAY PROCEDURE

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)

EXPERIMENTAL DATA

FAQ

What precautions should be taken when handling the product after opening?
How should GLOD-E be stored?
What stabilizers are used in GLOD-E?
How thermostable is GLOD-E?
What are the optimal pH and temperature for GLOD-E?
What is the molecular weight of GLOD-E?
What are the main applications of GLOD-E?

REFERENCES

CONTACT / SAMPLE

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