GLUCOSE DEHYDROGENASE (NAD(P)-dependent) from Microorganism


Appearance White amorphous powder, lyophilized
Activity GradeⅢ 250U/mg-solid or more
Contaminants NADH oxidase ≤1.0×10⁻³%
α-Glucosidase ≤1.0×10⁻³%
Glucose-6-phosphate dehydrogenase ≤1.0×10⁻³%
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight approx. 101,000 (Gel filtration)
Isoelectric point 4.5
Michaelis constants NAD⁺linked : 1.38×10⁻²M (D-Glucose) 3.09×10⁻⁴M (NAD⁺)
NADP⁺linked : 1.25×10⁻²M (D-Glucose) 4.07×10⁻⁵M (NADP⁺)
Inhibitors Ag⁺, Hg²⁺, Monoiodoacetate
Optimum pH 9.0(Fig.4)
Optimum temperature 55℃(Fig.5)
pH Stability pH 6.0-7.5 (20℃, 16hr)(Fig.6)
Thermal stability  45℃ (15min-treatment with 50mM K-phosphate buffer, pH 7.0)(Fig.7)
Substrate specificty Specific for ß-D,-Glucose or 2-Deoxy-glucose (Table.1)
(Either NAD⁺ or NADP⁺ serves as coenzyme.)


This enzyme is useful for enzymatic determination of D-Glucose.



glucose dehydrogenase

ß-D-Glucose+NAD⁺                                  D-Glucono-δ-lactone+NADH+H⁺

The appearance of NADH is measured at 340nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of NADH per minute under the conditions described below.


A. Tris-HCl buffer, pH 8.0 0.1M
B. D-Glucose solution 1.5M
C. ß-NAD⁺ solution 80mg/ml
D. Enzyme diluent 50mM K-phosphate buffer, pH 7.0 contg. 0.1% BSA


Concentration in assay mixture
Tris-HCl buffer 85.25mM
D-Glucose 147.54mM
NAD⁺ 3.66mM

1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.

2.6 ml Tris-HCl buffer, pH 8.0 (A)
0.3ml Substrate solution (B)
0.1ml ß-NAD⁺ solution(C)

2. Add 0.05ml of the enzyme solution* and mix by gentle inversion

3. Record the increase in optical density at 340nm against water for 2 to 5 minutes in a spectrophotometer
thermostated at 37℃, and calculate theΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent (D) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (D), dilute to 0.8-1.2U/ml with the same buffer and store on ice.


Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test−ΔOD blank ) ×Vt × df

Volume activity (U/ml) =                                                               =ΔOD/min×9.807×df


Weight activity (U/mg)=(U/ml)×1/C

: Total volume (3.05ml)
: Sample volume (0.05ml)
: Millimolar extinction coefficient of NADH under the assay conditions (㎠/micromole)
: Light path length (cm)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)
Table 1. Substrate Specificity of Glucose dehydrogenase
Substrate (150mM) Relative activity(%) Substrate (150mM) Relative activity(%)
D-Glucose 100 Galactose 1.7
L-Glucose 0.0 D-Lactose 1.5
D-Xylose 16.2 D-Sorbitole 0.0
2-Deoxy-glucose 127.0 D-Mannitol 0.0
L-Sorbose 0.0 Sucrose 0.0
D-Mannose 5.1 Inositol 0.0
D-Fructose 0.0 Maltose 1.4

Table 2. Effect of Various Chemicals on Glucose dehydrogenase

[The enzyme dissolved in 50mM K-phosphate buffer, pH 7.0 (2.8U/ml) was incubated with each chemical for 1hr at 30℃.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 KF 2.0 98.7
Metal salt 2.0   NaF 10.0 100.6
  7.1 NaN 20.0 101.6
Ba(OAc)₂   98.2 NEM 2.0 97.6
CaCl₂ 98.9 MIA 2.0 0.4
Cd(OAc)₂   96.6 IAA
2.0 92.2
CoCl₂   96.4 EDTA 5.0 107.2
CuSO₄   99.5 (NH)SO 20.0 96.0
FeCl₃   98.1 Borate 20.0 101.4
FeSO₄   96.6 o-Phenanthroline 2.0 97.7
HgCl₂   5.9 α,α′-Dipyridy 1.0 100.3
MgCl₂   101.5 Urea 2.0 122.5
MnCl₂   100.9 Guanidine 2.0 99.2
NiCl₂   93.4 Hydroxylamine 2.0 107.2
Pb(OAc)₂   99.8      
ZnSO₄   102.1      

Ac, CHCO; PNEM, N-Ethylmaleimide; MIA, Monoiodoacetate; IAA, lodoacetamide; EDTA, Ethylenediaminetetraacetate.


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