GLUCOSE DEHYDROGENASE (PQQ-dependent) from Microorganism


Appearance Purple amorphous powder, lyophilized
Activity GradeⅢ 500U/mg-solid or more
Contaminants Glucose dehydrogenase ≤1.0×10⁻³%
Hexokinase ≤1.0×10⁻³%
Stabilizers Ca⁺⁺, BSA
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight approx. 100,000 (by gel filtration)
Michaelis constant 4.8mM (D-Glucose)
Inhibitors Cu⁺⁺, Pb⁺⁺, Ag⁺
Optimum pH 7.0(Fig.2)
Optimum temperature 37℃(Fig.3)
pH Stability pH 3.5-8.5 (25℃, 16hr)(Fig.4)
Thermal stability below 50°C (pH 7.5, 30min)(Fig.5)
Substrate specificty  (Table 1)
Effect of various chemicals (Table 2)


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



PQQ-glucose dehydrogenase

D-glucose+PMS                                  D-glucono-1, 5-lactone+PMS (red)

2PMS (red)+NTB                                  ►2PMS+Diformazan

The appearance of diformazan formed by the reduction of nitrotetrazorium blue (NTB) with phenazine methosulfate (PMS)(red) is measured at 570nm by spectrophotometry.

Unit definition:

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


A. D-Glucose solution 1M [1.8g D-Glucose (MW=180.16)/10ml HO]keep this solution at room temperature at least 3 hours before use
B. PIPES-NaOH buffer, pH 6.5 50mM [Weight 1.51g of PIPES (MW=302.36), suspended in 60ml of HO,
dissolve with 5N NaOH and add 2.2ml of 10% Triton X-100. After adjusting pH to 6.5±0.05 at 25°C with 5N NaOH, fill up to 100ml with HO]
C. PMS solution 3.0mM [9.19mg Phenazine methosulfate (MW=306.34)/10ml HO]
D. NTB solution 6.6mM [53.96mg nitrotetrazorium blue (MW=817.65)/10ml HO]
E. Enzyme diluent 50mM PIPES-NaOH buffer, pH 6.5 containing 1mM CaCl, 0.1% Triton X-100, 0.1% BSA


Concentration in assay mixture
PIPES-buffer 42 mM
D-Glucose 30 mM
PMS 0.20mM
NTB 0.22mM

1. Prepare the following reaction mixture in a brownish bottle and store on ice. (Prepare freshly)

0.9 ml D-Glucose solution (A)
25.5ml PIPES-NaOH buffer, pH 6.5 (B)
2.0ml PMS solution (C)
1.0ml NTB solution (D)

2. Pipet 3.0ml of working solution into a test tube (plastic tube) and equilibrate at 37°C for about 5 minutes.

3. Add 0.1ml of enzyme solution* and mix by gentle inversion.

4. Record the increase of optical density at 570nm against water for 4 to 5 minutes in a spectrophotometer
thermostated at 37°C, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by the same method as test except that the enzyme diluent (E) is added instead of the enzyme solution.

* Dissolve the enzyme preparation on ice cold enzyme diluent (E) and dilute to 0.1-0.8U/ml with the same buffer, immediately before assay. (The use of plastic tube is recommended because of sticky nature.)


Activity can be calculated by using the following formula :

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

Volume activity (U/ml) =                                                               =ΔOD×1.54×df


Weight activity (U/mg)=(U/ml)×1/C

: Total volume (3.1ml)
: Sample volume (0.1ml)
: Half a millimolar extinction coefficient of diformazan (㎠/0.5micromole)
: Light path length (cm)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  1. K.Matsushita et al.; FEMS Microbiology Letters, 55, 53 (1988).
Table 1. Substrate Specificity of PQQ-Glucose dehydrogenase
Substrate (50mM) Relative activity(%) Substrate (50mM) Relative activity(%)
D-Glucose 100 Galactose 16.0
L-Glucose 0.3 D-Lactose 68.9
D-Xylose 15.0 D-Sorbitole 0.2
2-Deoxy-glucose 4.9 D-Mannitol 0.0
L-Sorbose 0.5 Sucrose 0.2
D-Mannose 10.8 Inositol 0.0
D-Fructose 0.3 Maltose 107.0

Table 2. Effect of Various Chemicals on PQQ-Glucose dehydrogenase

[The enzyme dissolved in 50mM PIPES-NaOH buffer, pH 6.5 contg. 1mM CaCl₂, 0.1% Triton X-100 (5U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 MIA 2.0 87
Metal salt 2.0   NEM 2.0 100
  108 IAA 2.0 98
CaCl₂   108 Hydroxylamine 2.0 19
Ba(OAc)₂ 105
EDTA 5.0 79
FeCl₃   79
o-Phenanthroline 2.0 7
CoCl₂   42 α,α′-Dipyridy 1.0 103
MnCl₂   105 Borate 5.0 110
ZnCl₂   45 NAF 2.0 111
Cd(OAc)₂   107 NaN 2.0 115
NiCl₂   101 Triton X-100 0.10% 101
CuSO₄   0 Brij 35 0.10% 22
Pb(OAc)₂   0 Tween 20 0.10% 104
AgNO₃   0 Span 20 0.10% 60
HgCl₂   77 Na-Cholate 0.10% 67
2-Mercaptoethanol 2.0 99 SDS
0.05% 33
PCMB 1.0 97 DAC 0.05% 113

Ac, CHCO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, Iodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzyl-alkyl-ammonium chloride.


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