D-FRUCTOSE DEHYDROGENASE from Gluconobacter sp.

D-Fructose:(acceptor) 5-oxidoreductase(EC 1.1.99.11)¹ ˜ ²⁾
D-Fructose + Acceptor                        5-Keto-D-fructose + Acceptor(red)

PREPARATION and SPECIFICATION
Appearance Red-yellowish amorphous powder, lyophilized
Activity GradeⅢ 20U/mg-solid or more
(containing approx. 80% of stabilizers)
Stabilizers Sugars, amino acids, BSA
PROPERTIES
Stability Stable at -20℃for at least one year (Fig.1)
Molecular weight approx. 140,000 (by gel filtration)
Isoelectric point 5.0±0.1
Michaelis constant 5×10⁻³M (D-Fructose)
Inhibitors Ag⁺, Hg⁺⁺, SDS
Optimum pH 4.0(Fig.2)
Optimum temperature: 37℃(Fig.3)
pH Stability pH 4.0-6.0 (25℃, 16hr)(Fig.4)
Thermal stability  below 40℃ (pH 4.5, 15min)(Fig.5)
Substrate specificty (Table 1)
Effect of various chemicals (Table 2)

APPLICATIONS ³⁾

This enzyme is useful for enzymatic determination of D-fructose in clinical analysis.

FCD-302

ASSAY

Principle:

D-fructose dehydrogenase

D-Fructose+2Potassium ferricyanide                                  ►5-Keto-D-fructose+2Potassium ferrocyanide

2Potassium ferrocyanide+ Ferric sulfate                        ►2Prussian blue+3KSO

Prussian blue : KFeIII [ FeII (CN)₆ ]

The appearance of prussian blue formed by chelate reaction is measured at 660nm by spectrophotometry.

Unit definition:

One unit causes the oxidation of one micromole of D-fructose (the formation of two micromoles of prussian blue) per
minute under the conditions described below.

Method:

Reagents
A. McⅠ lvaine buffer, pH 4.5 Prepare by mixing of 0.1M citric acid and 0.2M disodium phosphate, at 25℃
B. D-Fructose solution 1.0M (1.80g D-fructose (MW=180.16)/10ml McIlvaine buffer (A) contg. 0.1% Triton X-100)
C. Potassium ferricyanide solution 0.1M (0.33g potassium ferricyanide (MW=329.25)/10ml McIlvaine buffer (A) contg. 0.1% Triton X-100)
D. Ferric sulfate-SDS solution 5.0g Fe(SO₄)₃・HO, 3.0g SDS (sodium dodecyl sulfate), 95ml 85% phosphoric acid/1,000ml of HO
E. Enzyme diluent McⅠ lvaine buffer (A) contg. 0.1% Triton X-100 and 0.05% BSA

Procedure

Concentration in assay mixture
McⅠ lvaine buffer ×1
Triton X-100 0.1%
D-Fructose 0.1M
Potassium ferricyanide 10mM

1. Pipette 0.7ml of Reagent E, 0.1ml of Reagent B and 0.1ml of the enzyme solution* into a test tube and equilibrate at 37℃ for about 5 minutes.

2. Add 0.1ml of Raegent C and mix.

3. After exactly 5 minutes at 37°C, add 0.5ml of Reagent D to stop
the reaction, and then incubate at 37°C for further 20 minutes.

4. Add 3.5ml of distilled water and measure the optical density at 660nm against water (OD test).
At the same time, prepare the blank by using the same method as the test except that Reagent E (0.1ml) is used instead of the Reagent B (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent and dilute to 1.0-3.0U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula :

ΔOD (OD test−OD blank ) ×Vt × df

Volume activity (U/ml) =                                                               =ΔOD×2.5×df

2.0×2× t ×1.0×Vs

 

Weight activity (U/mg) = (U/ml) × 1/C

Vt
: Total volume (5.0ml)
Vs
: Sample volume (0.1ml)
2.0
: Millimolar extinction coefficient of prussian blue under the assay conditions (㎠/micromole)
2
: Factor based on the fact that oxidation of one mole of D-fructose produces
two moles of prussian blue
t
: Reaction time (5 minutes)
1.0
: Light path length (cm)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

  1. M.Ameyama, E.Shinagawa, K.Matsushita and O.Adachi; J.Bacteriol., 145, 814 (1981).
  2. M.Ameyama; Methods in Enzymology, vol.89, p.20 (1982).
  3. K.Nakashima, H.Takei, O.Adachi, E.Shinagawa and M.Ameyama; Clinica Chimica Acta, 151, 307 (1985).

Table 1. Substrate Specificity of D-Fructose dehydrogenase
Substrate Concn.(mM) Relative activity(%) Substrate Concn.(mM) Relative activity(%)
D-Fructose 100 100 D-Mannitol 100 0
D-Galactose 100 0.1 D-Xylitol 100 0
D-Glucose 100 0.1 Glucose-1-phosphate 20 0
D-Mannose 100 0.4 Fructose-6-phosphate 12.5 0
L-Sorbose 100 0 Fructose-1.6-diphosphate 12.5 0
D-Arabinose 100 0.3 Glycerol
100 0
D-Xylose 100 0.2 D-Glyceraldehyde 20 0
D-Ribose 100 0.2 D-Dihydroxyacetone 100 0.1
D-Rhamnose 100 0 Ethanol 100 0
Sucrose 100 0 Malic acid 100 0
Lactose 20 0 3α-Hydroxy-n-butyric acid 100 0
Maltose 100 0 Choline chloride 100 0.1
Raffinose 10 0 Potassium gluconate 100 19
D-Sorbitol 100 0      
Table 2. Effect of Various Chemicals on D-Fructose dehydrogenase
[The enzyme dissolved in McIlvaine buffer, pH 4.5(3U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical Concn.(mM) Residual
activity(%)
Chemical Concn.(mM) Residual
activity(%)
None 100 NaF 2.0 96
Metal salt 2.0   NaN₃ 2.0 88
MgCl₂
  96 EDTA 4.0 81
CaCl₂   98 o-Phenanthroline 2.0 88
Ba(OAc)₂ 98 α,α′-Dipyridyl 1.5 83
FeCl₃   88 Borate
40 89
CoCl₂   95 IAA 2.0 95
MnCl₂   80 NEM 2.0 92
ZnSO₄   91 Hydroxylamine 2.0 88
Cd(OAc)₂   82 PCMB 1.5 87
NiCl₂   93 MIA 2.0 91
CuSO₄   92 Triton X-100 0.10% 89
Pb(OAc)₂   82
Brij 35
0.10% 98
AgNO₃   0.20 Na-cholate 0.10% 101
HgCl₂   0.07 SDS 0.05% 6.5
      DAC 0.05% 69
           

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

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