Glucose dehydrogenase (FADGDH-AD)

Enzymes for Clinical Chemistry

CD : 60102

The enzyme is useful for the determination of D-glucose in clinical analysis. FADGDH-AD is an FAD-dependent glucose dehydrogenase with low reactivity toward maltose and xylose. It is one of the most stable glucose dehydrogenases for blood glucose measurement and is suitable for continuous glucose monitoring sensor.

Origin	recombinant Aspergillus sojae
Systematic name	D-Glucose : acceptor 1-oxidoreductase
EC Number	1.1.5.9
Reaction formula	D-Glucose + acceptor →→→ D-Glucono-1,5-lactone + reduced acceptor

SPECIFICATION

Appearance	yellow to brown lyophilizate
Activity	≧700 U/mg
Contaminants	NAD glucose dehydrogenase　 <0.01 U/U%
	Hexokinase　 <0.01 U/U%
	α-glucosidase　 <0.01 U/U%
	β-glucosidase　 <0.01 U/U%
Storage condition	below -20℃

PROPERTIES

Molecular weight	ca. 90 kDa (SDS-PAGE)
Structure	monomer, one mole of FAD per mole of enzyme glycoprotein
Michaelis constant	6.4×10^-2M (D-glucose)
pH Optimum	7.0–7.5 (Fig.1)
pH Stability	2.5–9.5 (Fig.2)
Optimum temperature	45℃ (Fig.3)
Thermal stability (liquid form)	below 60℃ (Fig.4)
Thermal stability (powder form)	stable at 30℃ for at least one month (Fig.5)
Inhibitors	Mn²⁺, Ag⁺
Specificity (Table.1)	D-glucose (100%), maltose (0.2%), D-xylose (0.9%), D-galactose (0.8%) sucrose (<0.1%), D-mannose (0.4%) 2-deoxy-D-glucose (23.5%)

APPLICATIONS

By using FADGDH-AD and an electron mediator-modified polymer, glucose can be continuously measured by an electrode method.

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 (Reagent E) 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).

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-AD be stored?: Please store at or below -20°C.

How is the activity of FADGDH-AD 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-AD?: It shows less than 1% relative activity toward maltose, xylose, and galactose compared to 100% for glucose.

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

How thermostable is FADGDH-AD?: It is stable in liquid form below 60°C (Fig.4). In powder form, it remains stable at 30°C for at least one month (Fig.5).

What are the optimal pH and temperature for FADGDH-AD?: The optimal pH is 7.0–7.5, and the optimal temperature is 45°C.

What is the molecular weight of FADGDH-AD?: It exhibits a monomeric structure with a molecular weight of approximately 90 kDa (SDS-PAGE).

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

REFERENCES

Satake R, Ichiyanagi A, Ichikawa K, Hirokawa K, Araki Y, Yoshimura T, Gomi K (2015)
Novel glucose dehydrogenase from Mucor prainii: Purification,
characterization, molecular cloning and gene expression in Aspergillus sojae J. Biosci Bioeng., 120, 498-503

Masakari Y, Hara C, Araki Y, Gomi K, Ito K (2020)
Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization Enzyme Microb Technol., 132, 109387

CONTACT / SAMPLE