TOYOBO Ideas & Chemistry バイオ事業総括部

D-FRUCTOSE DEHYDROGENASE

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 specificity (Table 1)
Effect of various chemicals (Table 2)

APPLICATIONS 3)

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

ASSAY

Principle

Principle

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. McⅠlvaine 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 Fe2(SO4)3・H2O, 3.0g SDS (sodium dodecyl sulfate), 95ml 85% phosphoric acid/1,000ml of H2O
E. Enzyme diluent McⅠlvaine buffer (A) contg. 0.1% Triton X-100 and 0.05% BSA

Procedure

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.

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

2.Add 0.1ml of Raegent C and mix.

3.After exactly 5 minutes at 37℃, add 0.5ml of Reagent D to stop the reaction, and then incubate at 37℃ 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 :

  • Volume activity (U/ml) =

  • ΔOD (OD test−OD blank)×Vt×df

    2.0×2×t×1.0×Vs

  • ＝ ΔOD×2.5×df

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 (cm2/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) Residual
    activity(%)
    D-Fructose 100 100
    D-Galactose 100 0.1
    D-Glucose 100 0.1
    D-Mannose 100 0.4
    L-Sorbose 100 0
    D-Arabinose 100 0.3
    D-Xylose 100 0.2
    D-Ribose 100 0.2
    D-Rhamnose 100 0
    Sucrose 100 0
    Lactose 20 0
    Maltose 100 0
    Raffinose 10 0
    D-Sorbito 100 0
  • Substrate Concn.(mM) Residual
    activity(%)
    D-Mannitol 100 0
    D-Xylitol 100 0
    Glucose-1-phosphate 20 0
    Fructose-6-phosphate 12.5 0
    Fructose-1.6-diphosphate 12.5 0
    Glycerol 100 0
    D-Glyceraldehyde 20 0
    D-Dihydroxyacetone 100 0.1
    Ethanol 100 0
    Malic acid 100 0
    3α-Hydroxy-n-butyric acid 100 0
    Choline chloride 100 0.1
    Potassium gluconate 100 19

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℃ for 1hr.]

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt 2.0
    MgCl2 96
    CaCl2 98
    Ba(OAc)2 98
    FeCl3 88
    CoCl2 95
    MnCl2 80
    ZnSO4 91
    Cb(OAc)2 82
    NiCl2 93
    CuSO4 92
    Pb(OAc)2 82
    AgNO3 0.20
    HgCl2 0.07
  • Chemical Concn.(mM) Residual
    activity(%)
    NaF 2.0 96
    NaN3 2.0 88
    EDTA 4.0 81
    o-Phenanthroline 2.0 88
    α,α′-Dipyridyl 1.5 83
    Borate 40 89
    IAA 2.0 95
    NEM 2.0 92
    Hydroxylamine 2.0 88
    PCMB 1.5 87
    MIA 2.0 91
    Triton X-100 0.10% 89
    Brij 35 0.10% 98
    Na-cholate 0.10% 101
    SDS 0.05% 6.5
    DAC 0.05% 69

Ac, CH3CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride

  • Fig.1. Stability (Powder form)

    Fig.1. Stability (Powder form)

    (kept under dry conditions)

  • Fig.2. pH-Activity

    Fig.2. pH-Activity

    37℃,5min-reaction in McⅠIvaine buffer solution

  • Fig.3. Temperature activity

    Fig.3. Temperature activity

    (in McⅠIvaine buffer,pH4.5)

  • Fig.4. pH-Stability

    Fig.4. pH-Stability

    25℃,16hr-treatment with McⅠIvaine buffer solution

  • Fig.5. Thermal stability

    Fig.5. Thermal stability

    15min-treatment with McⅠIvaine buffer pH4.5. enzyme concn. :3U/ml

活性測定法（Japanese）

1. 原理

原理

Prussian blue : KFeⅢ〔FeⅡ(CN)6〕
プルシャンブルーの生成量を660nmの吸光度で測定する。

2.定義

下記条件下で1分間に1マイクロモルのD-フラクトースが 酸化される(2マイクロモルのプルシャンブルーが生成され る)酵素量を1単位 (U)とする。

3.試薬

  • McⅠlvaine緩衝液,pH4.5:0.1Mのクエン酸と 0.2Mリン酸ナトリウムを混合して,pHを4.5に調整 する。
  • 1.0M D-フラクトース溶液:1.80gのD-フラクトース (MW＝180.16)を0.1%トリトンX-100を含むMcⅠ lvaine緩衝液 (A)に溶解し,10mlとする。
  • 0.1Mフェリシアン化カリ溶液:0.33gのフェリシア ン化カリ(MW＝329.25)を0.1%トリトンX-100を 含むMcⅠlvaine緩衝液 (A)に溶解し,10mlとする。
  • 硫酸第二鉄-SDS溶液:5.0gの硫酸第二鉄・xH2O, 3.0gのSDS(sodium dodecyl sulfate)及び,95ml の85%リン酸を蒸留水に溶解し1,000mlとする。
  • 酵素希釈液:0.1%トリトンX-100と0.05%のBSA を含むMcⅠlvaine緩衝液 (A)。

酵素溶液：酵素標品を予め氷冷した酵素希釈液 (E)で 溶解し,分析直前に同希釈液で1.0〜3.0 U/mlに希釈する。

4.手順

1.試験管に試薬E 0.7ml,試薬B 0.1ml,酵素溶液0.1ml を採り,37℃で約５分間予備加温する。

2.試薬Cを0.1mlを加えて，反応を開始する。

3.37℃で正確に５分間反応させた後,試薬Dを0.5ml加え て反応を停止させ,37℃で更に20分間静置する。

4.蒸留水3.5mlを加え,水と対照にして660nmの吸光度 を測定する(ODtest)。

5.盲検は試薬Bの代わりに試薬E (0.1ml)を加え,上記同 様に操作を行って吸光度を測定する(ODblank)。

5.計算式

  • U/ml ＝

  • ΔOD (OD test−OD blank)×5.0(ml)×希釈倍率

    2.0×2×5(分)×1.0×0.1(ml)

＝ ΔOD×2.5×希釈倍率
U/mg ＝ U/ml×1/C
2.0 : プルシャンブルーの上記測定条件でのミ リモル分子吸光係数(cm2/micromole)
2 : 1モルのD-フラクトースの酸化から生成するプ ルシャンブルーは2分子である事による係数
1.0 : 光路長(cm)
C : 溶解時の酵素濃度(c mg/ml)