NIPRO ENZYMESGLUCOSE DEHYDROGENASE (GlcDH2)[EC 1. 1. 1. 47]
from recombinant E. coli
D-Glucose + NAD(P)+ ↔ D-Glucono--lactone + NAD(P)H + H+SPECIFICATIONStateSpecific activity
Contaminants
PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants
Substrate specificity (100mM): Lyophilized: more than 900 U/mg protein: (as GlcDH2 activity = 100 %)NADH oxidase: ca. 126,000: ca. 31,500: 8.5: 5.0 - 10.0 (with 3M NaCl): No significant decrease in activity up to 70 °C.
(with 3M NaCl and 0.1% BSA)
: D-Glucose
NAD+NADP+: D-Glucose
D-Maltose
D-Galactose
D-Xylose
D-Fructose
D-Mannose
D-Arabinose
Trehalose
D-Lactose
D-Sucrose
2-Deoxy-D-Glucose
D-Glucose-1-Phosphate
D-Glucose-6-Phosphate
D-Sorbitol

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThis enzyme is useful for determination of glucose.

< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
3.7 mM
0.06 mM
0.02 mM
100 %
1.1 %
0.1 %
3.0 %
0.3 %
4.8 %0%0%
1.3 %0%
100 %0%0%0%

NIPRO ENZYMESASSAYPrinciple
The change in absorbance is measured at 340 nm according to the following reaction.
D-Glucose + NAD+GlcDH2

D-Glucono--lactone + NADH + H+

Unit Definition
One unit of activity is defined as the amount of GlcDH2 that forms 1 μmol of NADH per minute at37 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Tris-HCl, pH 8.5 (at 25 °C)
Ⅱ NAD+ solution ; 100 mM (0.663 g NAD+ free acid/10 mL distilled water)
Ⅲ D-Glucose solution ; 1 M ( 1.802 g glucose (anhyd.)/10 mL distilled water)
Ⅳ NaCl solution ; 5 M ( 2.92 g NaCl/10 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 15 U/mL with 20 mM potassium
phosphate buffer containing 1mg/mL BSA and 2 M NaCl, pH 6.5.
Procedure
1. Prepare the following reaction mixture and pipette 2.70 mL of reaction mixture into a cuvette.
SolutionⅠ 17.22 mL
SolutionⅡ 0.50 mL
SolutionⅢ 2.00 mL
SolutionⅣ 0.28 mL
2. Incubate at 37 °C for about 3 minutes.
3. Add 0.015 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute （∆Abs340） in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (2.70 + 0.015)
6.22 X 0.015

Specific activity (U/mg protein) =X d.f.

Volume activity (U/mL)
Protein concentration (mg/mL)*

d.f. ; dilution factor
6.22 ; millimolar extinction coefficient of NADH (cm 2/μmol)*Protein concentration ; determined by the absorbance at 280nm (Abs280),
where 1 Abs280 = 1 mg/mL
REFERENCE
1. Ramaley, R.F. and Vasantha, N.; J. Biol. Chem. 258, 12558-12565 (1983)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

1005006789010pH

378pHFig. 1 pH profile

Fig. 2 pH stability○ phosphate, ● Tris-HCl, ▲ glycine

treated for 24 hr at 4 ℃ in the following
buffer solution (0.1 M) containg 3 MNaCl : △ acetate, ○ phosphate,● Tris-HCl, ▲ glycine

10045691011100

Remaining activity (%)

Remaining activity (%)

5050020304050607080500Temperature (℃)

3045Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1M phosphatebuffer, pH 6.5, containing 3 M NaCland 0.1 % BSA

01560

treated for in 0.1 M phosphate buffer,
pH 6.5, containing 3M NaCl and 0.1 %BSA, ○ 60℃, □ 70℃, ● 80℃