CODE No.:M1G221

GLUCOSE DEHYDROGENASE (GlcDH2)

[EC 1.1.1.47]
from recombinant E. coli

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 900 U/mg protein
Contaminants	:	(as GlcDH2 activity = 100 %)
		NADH oxidase	<0.01%

PROPERTIES

Molecular weight	:	ca. 126,000
Subunit molecular weight	:	ca. 31,500
Optimum pH	:	8.5	(Fig.1)
pH stability	:	5.0 - 10.0 (with 3M NaCl)	(Fig.2)
Thermal stability	:	No significant decrease in activity up to 70 °C. (with 3M NaCl and 0.1% BSA)	(Fig.3, 4)
Michaelis constants	:	D-Glucose	3.7mM
		NAD+	0.06mM
		NADP+	0.02mM
Substrate specificity (100mM)	:	D-Glucose	100%
	:	D-Maltose	1.1%
	:	D-Galactose	0.1%
	:	D-Xylose	3.0%
	:	D-Fructose	0.3%
	:	D-Mannose	4.8%
	:	D-Arabinose	0%
	:	Trehalose	0%
	:	D-Lactose	1.3%
	:	D-Sucrose	0%
	:	2-Deoxy-D-Glucose	100%
	:	D-Glucose-1-Phosphate	0%
	:	D-Glucose-6-Phosphate	0%
	:	D-Sorbitol	0%

STORAGE

Stable at -20 °C for at least one year.

APPLICATION

The enzyme is useful for determination of glucose.

ASSAY

Principle

The change in absorbance is measured at 340 nm according to the following reaction.

Unit Definition

One unit of activity is defined as the amount of GDH that forms 1μmol of NADH per minute at 37°C.

Solutions

1. Buffer solution ; 100 mM Tris-HCl, pH8.5 (at 25°C)
2. NAD⁺ solution ; 100 mM (0.663g NAD⁺ free acid/10 mL distilled water)
3. D-Glucose solution ; 1 M ( 1.802g glucose (anhyd.)/10mL distilled water)
4. NaCl solution ; 5 M ( 2.92g NaCl/10mL distilled water)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 15 U/mL with 20mM potassium phosphate buffer containing 1mg/mL BSA and 2 M NaCl, pH 6.5.

Procedure

1. Prepare the following reaction mixture and pipette 2.70mL of reaction mixture into a cuvette.

   Solution I	17.26mL
   Solution II	0.50mL
   Solution III	2.00mL
   Solution IV	0.24mL

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 340nm per minute (ΔAbs₃₄₀) in the linear portion of curve.

Calculation

• d.f. ; dilution factor
• 6.22 ; millimolar extinction coefficient of NADH (cm²/mol)
• *Protein concentration; determined by the absorbance at 280nm (Abs280),
   where 1 Abs280 = 1mg/ml

REFERENCE

2. Ramaley, R.F. and Vasantha, N.; J. Biol. Chem. 258, 12558-12565 (1983)

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