(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-228REACH compliant product

GLUCOSE DEHYDROGENASE [FAD-GDHⅡ]

from Microorganism
（FAD dependent glucose dehydrogenase, EC 1.1.5.9）

D‒Glucose + acceptor → D‒glucono-1,5-lactone + reduced acceptor

Preparation and Specification

Appearance: : Light yellow to yellow lyophilizate

Specific activity: : More than 400 U/mg solid

Properties

Molecular weight: : 66 kDa (SDS-PAGE, as deglycosylated form)

Michaelis constant: : 1.3 × 10^-2M (glucose)

Optimum pH: : See Figure 1

pH stability: : See Figure 2

Optimum temperature: : See Figure 3

Thermal stability: : See Figure 4

Stability (solid form): : See Figure 5

Substrate specificity: : See Table 1

Light stability: : See Table 2

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of glucose.

Table 1. Substrate specificity

Substrate	FAD-GDHⅡ	FAD-GDH from Aspergillus sp.
Glucose	100	100
Galactose	0.6	0.3
Xylose	2.4	13.5
Maltose	0.4	0
Mannose	1.0	1.3
2-deoxy-glucose	31.8	29.2

Fig.1 Optimum pH

Table 2. Light stability

Form of FAD-GDH	Light source	Residual activity (%)
Powder	LED	93.2
Powder	Fluorescent lamp	93.7
Solution (1mg/ml, water)	LED	95.7
Solution (1mg/ml, water)	Fluorescent lamp	96.4

Radiation for 24 hours

Fig.2 pH Stability

37℃ 60min

Fig.3 Optimum Temperature

Fig.4 Thermal stability

Fig.5 Stability (solid form)

Assay

Principle

The assay is based on the decrease in absorbance at
600 nm as the reduction of 2,6-dichloroindophenol in the
following reactions:

FAD-GDH Ⅱ
D-Glucose + PMS	→	D-Glucono-1,5-lactone + PMS (red)
PMS (red) + DCIP	→	PMS + DCIP (red)

PMS : Phenazine methosulfate
DCIP : 2,6-Dichloroindophenol

Unit definition

One unit is defined as the amount of enzyme which
converts 1 μmole of D-Glucose to D-Glucono-1,5-lactone
per minute at 37 ℃ under the conditions specified in the
assay procedure.

Reagents

Reaction mixture
0.1 M NaH₂PO₄‒Na₂HPO₄ buffer pH 6.5 containing 0.222 M D-Glucose and 0.0833% Triton X-100
20 mM PMS solution
0.2 N HCl solution
2.0 mM DCIP solution
Enzyme dilution buffer
50 mM NaH2PO4‒Na2HPO4 buffer pH 6.5 containing 0.1 % BSA and 0.1 % Triton X-100
Reagents
Triton X-100 : The Dow Chemical Company
BSA : Millipore Fraction V pH5.2 #81053
D-Glucose : FUJIFILM Wako Pure Chemical Corporation #049-31165
PMS (Phenazine methosulfate) : Tokyo Chemical Industry #P0083
DCIP (2,6-Dichloroindophenol) : Sigma-Aldrich #119814

Enzyme solution

Accurately weigh about 10 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.

Procedure

Pipette accurately 1.8 ml of reaction mixture and 20 μl of enzyme solution into a small test tube. Add 100 μl of 20 mM PMS solution, mix and preincubate at 37 ℃.

※ In the case of a blank test, add 20 μl of enzyme dilution buffer at this time.
After 3 min, add 100 μl of 2.0 mM DCIP solution and mix to start the reaction at 37 ℃.
After starting the reaction, measure the rate of increase per minute in absorbance at 600 nm. The rate must be measured within the linear portion of the absorbance curve.

Absorbance sample : As/min

blank : Ab/min

△ A/min =（As/min−Ab/min）≦ 0.1Abs/min

Calculation

Activity（U/mg of powder）= {(△A/min)/(16.3)} × 2.02/0.02 × 1/x

16.3 :	millimolar extinction coefficient of DCIP at 600 nm (cm²/μmole)
2.02 :	final volume (ml)
0.02 :	volume of enzyme solution (ml)
X :	concentration of the sample in enzyme solution (mg/ml)

Storage

Storage at -20 ℃ in the presence of a desiccant is recommended.

References

Matsushita, K. et al. (1980) Agric. Biol. Chem. 44, 1505-1512.
Shinagawa, E. et al. (1990) J. Biochem. 107, 863-867.

FAD-GDH Ⅱ活性測定法(Japanese)

試薬液

反応試薬混合液
0.222 M グルコース及び0.0833%トリトンX-100含む0.1 M リン酸緩衝液 pH 6.5
20 mM PMS 溶液
2.0 mM DCIP 溶液
酵素溶解希釈用液
0.1 % BSA 及び0.1 % トリトンX-100 を含む50mM リン酸緩衝液 pH 6.5
試薬
トリトンX-100：Dow Chemical製
BSA (Bovine serum albumin) Fra. V, pH5.2：Millipore製　#81053
グルコース：富士フイルム和光純薬製　特級　#049-31165
PMS (フェナジンメチルスルファート)：東京化成製　#P0083
DCIP (2,6-ジクロロインドフェノールナトリウム塩)：シグマ製　#119814

酵素試料液

検品約10 mg を精密に量り、酵素溶解希釈用液に溶解して全容20 ml とする。その液を酵素溶解希釈用液で適宜希釈する。

測定操作法

小試験管に反応試薬混合液1.8 ml 及び酵素試料液20μl を正確に分注し、20 mM PMS 溶液100 μl を加えて混和し、37 ℃で予備加温する。

※ 盲検は酵素試料液の代わりに酵素溶解希釈用液20μl を加える。
3 分経過後、2.0 mM DCIP 溶液100 μl を加えて混和し、37 ℃で反応を開始する。
反応開始後、600 nm における吸光度を測定して直線的に反応している1 分間当たりの吸光度変化を求める。
求められた吸光度変化を試料液についてはAs/min、盲検液についてはAb/min とする。
ΔA/min =（As/min − Ab/min）≦ 0.1 Abs/min

計算

活性（U/mg）= {(ΔA/min)/(16.3)} × 2.02/0.02 × 1/x

16.3 :	DCIP の600nm におけるミリモル分子吸光係数（cm²/μmole）
2.02 :	反応総液量（ml）
0.02 :	反応に供した酵素試料液量（ml）
X :	酵素試料液中の検品濃度（mg/ml）

Absorbance sample :	As/min
blank :	Ab/min

GLUCOSE DEHYDROGENASE [FAD-GDHⅡ] (T-228)