Glucose Dehydrogenase (FADGDH-AB) | 龟甲万百欧凯米发株式会社

Glucose Dehydrogenase (FADGDH-AB) 60101 葡萄糖脱氢酶

临床检测用酶

The enzyme is useful for the determination of d-glucose in clinical analysis and self-monitoring blood glucose meters.

系统名称	D-Glucose : acceptor 1-oxidoreductase
EC编号	1.1.5.9
反应式	D-Glucose + acceptor →→→ D-Glucono-1,5-lactone + reduced acceptor

规格

Appearance	Yellow lyophilizate
Activity	≧700 U/mg lyophilizate
Contaminant	NAD Glucose Dehydrogenase　 ≺1.0×10^-2％
	Hexokinase　≺1.0×10^-2％
	α-Glucosidase　≺1.0×10^-2％
	β-Glucosidase　≺1.0×10^-2％
Storage	below -20℃

特性

Molecular weight	ca. 85 kDa (SDS-PAGE)
Structure	monomer, one mole of FAD per mole of enzyme glycoprotein
Michaelis constant	2.2×10^-2M (D-Glucose)
pH Optimum	7.0–7.5
pH Stability	4.5–8.0
Optimum temperature	40–50℃
Thermal stability	below 45℃
Specificity	D-glucose (100), Maltose (≺0.3),
	D-xylose (≺10), D-galactose (≺1.2)
	sucrose (≺0.0)

适用类型

The enzyme is useful for the determination of D-glucose in clinical analysis and self-monitoring blood glucose meters.

ASSAY PROCEDURE

Principle

The disappearance of the blue color of DCIP by the reduction is measured spectrophotometrically at 600 nm.

Definition of unit

One unit (U) causes the reduction of one micromole of DCIP per minute under the conditions described below.

Reagents

D-Glucose solution, 2 M: 72 g of d-glucose/200 ml of distilled water.
Potassium phosphate buffer, 0.1 M; pH 7.0: mix 0.1 M KH₂PO₄ solution and 0.1 m K₂HPO₄ solution to make a pH 7.0 solution.
2,6-Dichloroindophenol (DCIP) solution, 1.8 mM: 58.7 mg of DCIP/100 ml of distilled water.
5-Methylphenazinium methyl sulfate (PMS) solution, 30 mM: 91.9 mg of PMS/10 ml of distilled water.
Enzyme dilution buffer: 10 mM potassium phosphate buffer, pH 6.0, containing 0.1% bovine serum albumin(BSA).

Sample: dissolve the lyophilized enzyme to final concentration about 0.4 μg/ml with enzyme dilution buffer (ReagentE) immediately before measurement.

Procedure

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

600 μL D-Glucose solution (Reagent A)

2050 μL Potassium phosphate buffer pH 7.0 (Reagent B)

150 μL DCIP solution (Reagent C)
Equilibrate at 37℃ for about 3 min.
Add 0.1 ml of PMS solution (Reagent D) and mix.
Add 0.1 ml of sample and mix.
Record the decrease of absorbance at 600 nm against water for 1 min. (30–90 sec) in a spectrophotometer thermostated at 37℃, 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 E) instead of sample (ΔA₀).

600 μL	D-Glucose solution	(Reagent A)
2050 μL	Potassium phosphate buffer pH 7.0	(Reagent B)
150 μL	DCIP solution	(Reagent C)

Calculation

Activity can be calculated by using the following formula:

20.4 : Millimolar extinction coefficient of DCIP under the assay condition (cm²/μmol)
1.0 : Light pass length (cm)
df : Dilution factor

EXPERIMENTAL DATA

Line-up

FAQ

What precautions should be taken when handling the product after opening?: To avoid moisture absorption of the enzyme powder, please allow it to return to room temperature in a sealed state before use. (Recommended conditions: around 25°C, humidity below 55%)

How should FADGDH-AB be stored?: Please store at or below -20°C.

How is the activity of FADGDH-AB measured?: An example of activity measurement: The dehydrogenase activity is assayed in a reaction mixture containing glucose as a substrate, phenazine methosulfate as a mediator and dichlorophenolindophenol (DCIP). The activity is calculated by monitoring the decrease in the absorbance of DCIP at 600 nm.

What is the substrate specificity of FADGDH-AB?: It shows less than 1% relative activity toward maltose compared to 100% for glucose.

What stabilizers are used in FADGDH-AB?: No stabilizers are used.

How thermostable is FADGDH-AB?: It is stable in liquid form below 45°C (Fig.4). In powder form, it remains stable at 30°C for at least 20 days (Fig.5).

What are the optimal pH and temperature for FADGDH-AB?: The optimal pH is 7.0–7.5, and the optimal temperature is 40–50°C.

What is the molecular weight of FADGDH-AB?: It exhibits a monomeric structure with a molecular weight of approximately 85 kDa (SDS-PAGE).

What are the main applications of FADGDH-AB?: It is useful for the determination of blood glucose levels in diabetes diagnosis, with minimal interference from substances such as maltose.

参考文献

Hatada M, Loew N, Inose-Takahashi Y, Okuda-Shimazaki J, Tsugawa W, Mulchandani A, Sode K (2018)
Development of a glucose sensor employing quick and easy modification method with mediator for altering electron acceptor preference.
Bioelectrochemistry, 121, 185-190

Okuda-Shimazaki, J, Yoshida H, Sode K (2020)
FAD dependent glucose dehydrogenases – Discovery and engineering of representative glucose sensing enzymes –
Bioelectrochemistry, 132, 107414

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