LACTATE OXIDASE from Microorganism
Appearance | Yellowish amorphous powder, lyophilized | |
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Activity | GradeⅢ 80 U/mg-solid or more | |
Contaminants | Pyruvate oxidase | ≤1.0×10-3 % |
Cholesterol oxidase | ≤1.0×10-3 % | |
Uricase | ≤1.0×10-3 % | |
Glucose oxidase | ≤1.0×10-3 % |
Stability | Stable at −20 ℃ for at least one year(Fig.1) |
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Molecular weight | approx. 160,000 (by gel filtration) |
Isoelectric point | 4.3±0.2 |
Michaelis constant | 1.0×10-3 M (L-Lactate) |
Inhibitors | Fe3+, SDS |
Optimum pH | 7.5(Fig.2) |
Optimum temperature | 35−40 ℃(Fig.3) |
pH Stability | 4.0−9.8 (25 ℃, 16 hr)(Fig.4) |
Thermal stability | below 50 ℃ (pH 7.0, 10 min)(Fig.5) |
Effect of various chemicals | (Table 1) |
This enzyme is useful for enzymatic determination of L-lactate
The formation of quinoneimine dye is measured at 555 nm by spectrophotometry.
One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per minute under the conditions detailed below.
A. DL-Lactate solution | 0.125 M [240 mg of DL-lithium lactate (MW=96.01)/20 mL of 50 mM K-PB pH 7.5] (Should be prepared fresh) | |
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B. 4-AA solution | 0.5 % (500 mg of 4-aminoantipyrine/100 mL of H2O) (Store at 4 ℃ in a brownish bottle) | |
C. EHSPT(TOOS) solution | 20 mM [591 mg N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (MW=295.3)/100 mL of H2O] (Store at 4 ℃ in a brownish bottle) | |
D. Peroxidase solution | 25 U/mL [ca. 23 mg of horseradish peroxidase (Toyobo GradeⅢ, 110 purpurogallin units/mg)/100 mL of H2O] | |
E. SDS solution | 0.25 % (500 mg sodium dodecyl sulfate/200 mL of H2O) | |
F. Enzyme diluent | 20 mM K-PB, pH 7.0 containing 0.1 %(w/v) sodium cholate |
1. Prepare the following working solution for 20 tests in a brownish bottle, and store on ice.
8.0 mL | DL-lactate solution | (A) |
1.2 mL | 4-AA solution | (B) |
0.8 mL | EHSPT solution | (C) |
2.0 mL | Peroxidase solution | (D) |
8.0 mL | Distilled water |
Concentration in assay mixture | |
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Potassium-phosphate buffer | 20 mM |
DL-lactate | 48 mM |
4-Aminoantipyrine | 1.2 mM |
EHSPT | 0.76 mM |
Peroxidase | 2.4 U/mL |
2. Pipette 1.0 mL of working solution into a test tube and equilibrate at 37 ℃ for approximately 5 minutes.
3. Add 0.05 mL of the enzyme solution* and mix.
4. After exactly 15minutes at 37 ℃, add 2.0 mL of SDS solution (E) to stop the reaction, and measure the optical density at 555 nm against water (OD test).
At the same time, prepare the blank using the same method as the test except that the enzyme diluent (F) is used instead of the enzyme solution (OD blank).
*Dissolve the enzyme preparation in ice-cold 20 mM ACES-NaOH, pH 7.0, containing 1 mM EDTA and 0.5 % (w/v) sodium cholate, and dilute to 0.04−0.1 U/mL with the enzyme diluent (F) immediately before the assay.
Activity can be calculated by using the following formula :
Volume activity (U/mL) =
ΔOD (OD test−OD blank)×Vt×df
34.3×1/2×t×1.0×Vs
= ΔOD×0.237×df
Weight activity (U/mg) = (U/mL)×1/C
Vt | : Total volume (3.05 mL) |
Vs | : Sample volume (0.05 mL) |
34.3 | : Millimolar extinction coefficient of quinoneimine dye under the assay condition (㎠/micromole) |
1/2 | : Factor based on the fact that one mole of H2O2 produced half a mole of quinoneimine dye |
t | : Reaction time (15 minutes) |
1.0 | : Light path length (cm) |
C | : Enzyme concentration in dissolution (C mg/mL) |
1) A. Toda, and Y. Nishiya; J. Ferment. Technol., 85, 507 (1998)
[The enzyme solution dissolved in 20 mM ACES-NaOH, pH 7.0 (50 U/mL) was incubated with each chemical at 25 ℃ for 1 hr.]
Chemical | Concn.(mM) | Residual activity(%) |
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None | - | 100 |
Metal salt | 2.0 | |
MgCl2 | 100 | |
CaCl2 | 101 | |
Ba(OAc)2 | 101 | |
FeCl3 | 5 | |
CoCl2 | 100 | |
MnCl2 | 100 | |
ZnCl2 | 94 | |
Cd(OAc)2 | 91 | |
NiCl2 | 99 | |
CuSO4 | 94 | |
AgNO3 | 54 | |
MIA | 2.0 | 94 |
NEM | 2.0 | 99 |
Chemical | Concn.(mM) | Residual activity(%) |
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IAA | 2.0 | 90 |
Hydroxylamine | 2.0 | 99 |
EDTA | 5.0 | 94 |
o-Phenanthroline | 2.0 | 100 |
α,α′-Dipyridyl | 2.0 | 94 |
Borate | 50.0 | 97 |
NaF | 2.0 | 99 |
NaN3 | 2.0 | 100 |
Triton X-100 | 0.10 % | 98 |
Brij 35 | 0.10 % | 86 |
Tween 20 | 0.10 % | 81 |
Span 20 | 0.10 % | 96 |
Na-cholate | 0.10 % | 101 |
DAC | 0.05 % | 76 |
SDS | 0.05 % | 0 |
Ac, CH3CO; MIA, Monoiodoacetate; NEM, N-ethylmaleimide; IAA, Iodoacetate; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.
Fig.1. Stability (Powder form)
(kept under dry conditions)
Fig.2. pH-Activity
(37 ℃, in 20 mM buffer solution
●, pH 5.7-7.9 K-phosphate;
○, pH 8.2-9.0 borate;
■, pH 8.6-9.6 glycine-NaOH)
Fig.3. Temperature activity
(in 20 mM K-phosphate , pH 7.5)
Fig.4. pH-Stability
(25 ℃, 16 hr-treatment with 50 mM buffer solution:
●, pH 3.6-5.0 acetate;
○, pH 5.6-7.8 K-phosphate;
■, pH 7.5-8.5 Tris-HCl; □, pH 8.9-10.2 glycine-NaOH)
Fig.5. Thermal stability
(10 min-treatment with 20 mM ACES-NaOH, pH 7.0. Enzyme concentration: 100 U/mL)
1. 原理
4-AminoantipyrineとEHSPTの酸化縮合物であるQuinoneimine色素を555nmで測定し, 上記反応で生成したH2O2量を定量する。
2.定義
下記条件下で1分間に1マイクロモルのH2O2を生成する酵素量を1単位(U)とする。
3.試薬
酵素溶液:酵素標品を予め氷冷した1.0mM EDTA,0.5%(w/v)コール酸ナトリウムを含む20mMACES-NaOH,pH7.0で溶解し,分析直前に0.1%(w/v)コール酸ナトリウムを含む20mMK-リン酸緩衝液, pH7.0で希釈する。
4.手順
1.下記反応混液(20テスト分)を調製する(褐色瓶にて氷冷保存)
8.0ml | DL-乳酸溶液 | (A) |
1.2ml | 4-AA水溶液 | (B) |
0.8ml | EHSPT水溶液 | (C) |
2.0ml | POD水溶液 | (D) |
8.0ml | 蒸留水 |
2.反応混液1.0mlを試験管に採り, 37℃で約5分間予備加温する。
3.酵素溶液0.05mlを加え, 反応を開始する。
4.37℃で正確に15分反応させた後, SDS水溶液(E)2.0mlを加えて反応を停止させる。この液につき水を対照に555nmにおける吸光度を測定する(ODtest)。
5.盲検は酵素溶液の代わりに酵素希釈液〔0.1%(w/v)コール酸ナトリウムを含む20mM K-リン酸緩衝液,pH7.0〕を用い, 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)×3.05(ml)×希釈倍率
34.3×1/2×15(分)×1.0×0.05(ml)
= ΔOD×0.237×希釈倍率 | |
U/mg | = U/ml×1/C |
34.3 | : Quinoneimine色素の上記測定条件下でのミリモル分子吸光係数(cm2/micromole) |
1/2 | : 酵素反応で生成したH2O2の1分子から形成するQuinoneimine色素は1/2分子であることによる係数 |
1.0 | : 光路長(cm) |
C | : 溶解時の酵素濃度(c mg/ml) |
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