In enzymology, a quinoprotein glucose dehydrogenase (EC 1.1.5.2) is an enzyme that catalyzes the chemical reaction: D-glucose + ubiquinone ↔D-glucono-1,5-lactone + ubiquinol. Thus, the two substrates of this enzyme are D-glucose and ubiquinone, whereas its two products are D-glucono-1,5-lactone and ubiquinol. This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with a quinone or similar compound as acceptor. This enzyme participates in pentose phosphate pathway. It employs one cofactor, PQQ.
D-Glucose + Acceptor → D-glucono-1,5-lactone + reduced Acceptor
PREPARATION and SPECIFICATION
| Appearance | : Purple amorphous powder, lyophilized | |
|---|
| Activity | : ≥500U/mg powder | |
| Contaminants | : Glucose dehydrogenase (NAD-dependent) ≤1.0×10⁻³% | |
| Hexokinase ≤1.0×10⁻³% | |
| Stabilizers | : Ca²⁺, BSA |
PROPERTIES
| Stability | Stable at -20℃ for at least one year | Fig.1 | | Molecular weight | approx. 100,000 (by gel filtration) |
| | Michaelis constant | 4.8mM (D-Glucose) |
| | Inhibitors | Cu²⁺, Pb²⁺, 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℃ (pH 7.5, 30min) | Fig.5 | | Substrate specificity |
| (Table 1)
| | Effect of various chemicals |
| (Table 2) |
|
APPLICATIONS
This enzyme is useful for enzymatic determination of D-Glucose.
ASSAY
Principle:
Unit definition:
One unit causes the formation of one half micromole of diformazan per minute under the conditions described below.
Method:
Reagents
A. D-Glucose solution: 1M [1.8g D-Glucose (MW=180.16)/10ml H₂O], 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 H₂O, dissolve with 5N NaOH and add 2.2ml of 10% Triton X-100. After adjusting pH to 6.5±0.05 at 25℃ with 5N NaOH, fill up to 100ml with H₂O]
C. PMS solution: 3.0mM [9.19mg Phenazine methosulfate (MW=306.34)/10ml H₂O]
D. NTB solution: 6.6mM [53.96mg nitrotetrazorium blue (MW=817.65)/10ml H₂O]
E. Enzyme diluent: 50mM PIPES-NaOH buffer, pH 6.5 containing 1mM CaCl₂, 0.1% Triton X-100, 0.1% BSA
Procedure
1. Prepare the following reaction mixture in a brownish bottle and store on ice. (Prepare freshly)
| 0.9ml | D-Glucose solution | (A) | | 25.5ml | PIPES-NaOH buffer, pH 6.5 | (B) | | 2.0ml | PMS solution | (C) | | 1.0ml | NTB solution | (D) |
|
Concentration in assay mixture
| PIPES-buffer | 42mM | | D-Glucose | 30mM | | NTB | 0.20mM | | PMS | 0.22mM |
|
2. Pipet 3.0ml of working solution into a test tube (plastic tube) and equilibrate at 37℃ 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℃, 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.)
Calculation
Activity can be calculated by using the following formula:
| Vt | :Total volume (3.1ml) |
| Vs | :Sample volume (0.1ml) |
| 20.1 | :Half a millimolar extinction coefficient of diformazan (cm²/0.5 micromole) |
| 1.0 | :Light path length (cm) |
| df | :Dilution factor |
| C | :Enzyme concentration in dissolution (c mg/ml) |
Table 1. Substrate Specificity of PQQ-Glucose dehydrogenase
| Substrate (50mM) | Relative activity(%) | Substrate (50mM) | Relative activity(%) |
|---|
| D-Glucose | 100.0 | 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℃ for 1hr.]
| Chemical | Concn.(mM) | Activity(%) | Chemical | Concn.(mM) | Activity(%) |
|---|
| None | - | 100 | MIA | 2.0 | 87 | | Metal salt | 2.0 |
| NEM | 2.0 | 100 | | MgSO₄ |
| 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′-Dipyridyl | 1.0 | 103 | | MnCl₂ |
| 105 | Borate | 5.0 | 110 | | ZnCl₂ |
| 45 | NAF | 2.0 | 111 | | Cd(OAc)₂ |
| 107 | NaN₃ | 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, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, Iodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.
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