Glucose oxidase is used in the food and beverage industry as a preservative and stabilizer and is commonly derived from the fungus Aspergillus niger. Glucose oxidase can react with intracellular glucose and oxygen (O₂) to produce hydrogen peroxide (H₂O₂) and gluconic acid, which can cut off the nutrition source of cancer cells and consequently inhibit their proliferation.
CATALYSIS : β-D-Glucose + O₂ → D-Glucono-δ-lactone + H₂O₂
SPECIFICATIONS:
Activity: ≥180 U/mg enzyme powder
The activity may increase up to 160%, if the reaction mixture is saturated with oxygen.
Appearance: Yellow powder, lyophilized
Contaminants: Catalase ≤1.0 U/mg
Additives: Not added
CHARACTERISTICS:
Isoelectric point: 4.3
Km value: 1.9 × 10⁻² M (Glucose)
Inhibitors: Ag⁺, Hg²⁺, Cu²⁺
Opt. pH: 5.0 – 6.5
Opt. temperature: 37℃
Stable pH range: 3.0 – 8.0 (40℃, 6 hrs.)
Stable temp. range: Below 50℃ (pH7.0, 15 min.)
APPLICATIONS:
This enzyme is used for the enzymatic determination of glucose in blood or urine by glucose sensor etc.
Assay Method of Glucose Oxidase
Principle
The appearance of quinoneimine dye formed by coupling with 4-aminoantipyrine and phenol is measured at 500 nm by spectrophotometry.
Unit Definition
One unit is defined as the enzyme quantity which oxidizes one μ mole of β-D-glucose per minute under the conditions described below.
Reagents
A. Aminoantipyrine Solution (4 mg/ml)
Weigh 0.20 g of 4-aminoantipyrine and dissolve in 20 ml of deionized water. Fill up to 50 ml with deionized water.
B. Triton X-100 Solution (50 mg/ml)
Weigh 5.0 g of Triton X-100 and dissolve in 30 ml of deionized water with heating. After cooling, fill up to 100 ml with deionized water.
C. Phenol Solution (50 mg/ml)
Weigh 5.0 g of phenol and dissolve in 30 ml of deionized water. Fill up to 100 ml with deionized water.
D. Peroxidase Solution
Dissolve 250 purpurogallin units of Peroxidase in 10 ml of deionized water. Keep in an ice bath. (Expires after 11 days at 2–8℃)
E. 0.1 M Phosphate Buffer (KH₂PO₄-NaOH, pH7.0)
F. Phenol-Buffer Solution
Weigh 1.36 g of KH₂PO₄ and dissolve in 80 ml of deionized water. Add 3 ml of Phenol Solution (C) and 3 ml of Triton X-100 Solution (B), then adjust the pH to 7.0 with 1N NaOH. Fill up to 100 ml with deionized water. This reagent should be used after incubation at 25℃ for 24 hours. (Expires after 2 weeks at 25℃)
G. Substrate Solution
Weigh 2.5 g of D-glucose and dissolve in deionized water. Fill up to 25ml with deionized water. (Expires after 11 days at room temperature)
H. Enzyme Solution
Weigh approx. 50 mg of Glucose Oxidase and dissolve in chilled 0.1 M Phosphate Buffer (E).
Enzyme Solution should be prepared so that the value of ΔOD/minute becomes in the range of 0.030 ± 0.005.
Procedure
Pipette 2.0 ml of Phenol-Buffer Solution (F), 0.5 ml of Substrate Solution (G), 0.5 ml of Peroxidase Solution (D) and 0.1 ml of Aminoantipyrine Solution (A) respectively into a cuvette (d=10 mm), and keep at 37 ± 0.1℃ for 10 minutes. Then, pipette 0.1 ml of Enzyme Solution (H) into the cuvette and mix well immediately. Keep the reaction mixture at 37 ± 0.1℃.
Exactly at 2 minutes and 5 minutes after the addition of Enzyme Solution (H), measure the absorbances of the reaction mixture at 500 nm (A2 and A5).
As a blank, pipette 0.1 M Phosphate Buffer (E) into another cuvette (d=10 mm) instead of the Enzyme Solution (H) and take the same procedure described above (Ab2 and Ab5).
Calculation
3 : Reaction time
12.88 : Millimolar extinction coefficient of quinoneimine dye
1/2 : Factor based on the fact that two moles of H₂O₂ form one mole of quinoneimine dye
3.2 : Final volume of the reaction mixture
0.1 : Volume of Enzyme Solution
Dm : Dilution multiple of Enzyme Solution
1.339 : Exchange coefficient to the original method using O-dianisidine which is found to be a carcinogenic substances
G2624193
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