(Diagnostic Reagent Grade) INQUIRY NEEDED
ASAHI KASEI ENZYMES T-38REACH適合品

ALCOHOL OXIDASE [ALOD]

from Candida sp.
(Alcohol: oxygen oxidoreductase, EC 1.1.3.13)

R–CH₂OH + O₂ → R–CHO + H₂O₂

Preparation and Specification

Appearance: : Light yellowish amorphous powder, lyophilized

Specific activity: : More than 7 U/mg solid

Properties

Substrate specificity: : See Table 1

Molecular weight: : 520 kDa (gel filtration)
75 kDa (SDS–PAGE)

Isoelectric point: : pH 4.1

Michaelis constants: : Methanol 2.9 × 10^-3M
　Ethanol 8.2 × 10^-3M

Optimum pH: : 7.5–9.0Figure 1

pH stability: : 6.0–9.5 (37℃, 60 min) Figure 2

Thermal stability: : Stable at 40℃ and below (pH 7.5, 10 min) Figure 3

Effect of chemicals: : See Table 2 and Table 3

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of blood alcohol.

	ALOD
R－CH₂OＨ＋ O₂	→	R－CHO ＋ H₂O₂
	POD
2 H₂O₂ ＋ 4－AA ＋ Phenol	→	Quinoneimine dye ＋ 4 H₂O

Table 1.Substrate specificity

Substrate	Relative activity (%)
Methanol	100
Ethanol	79.3
n–Propanol	46.5
iso–Propanol	25.8
n–Butanol	39.6
Form aldehyde	48.2
Acetoaldehyde	0

Table 2. Effect of metal ions on ALOD activity

Metal ion	Concentration (mM)	Relative activity (％)
None	–	100.0
KCl	10	103.4
NaCl	10	99.3
LiCl	10	99.3
NH₄Cl	10	95.2
MgCl₂	1	100.0
CaCl₂	1	93.1
CoCl₂	1	68.4
BaCl₂	1	69.8
NiCl₂	1	70.5
MnCl₂	1	100.6
EDTA	1	108.2

Table 3. Effect of detergents on ALOD activity

Detergent	Concentration (％)	Relative activity (％)
Nonidet P–40	0.1	96.3
Triton X–100	0.1	98.7
Adekatol PC–8	0.1	91.5
Adekatol SO–120	0.1	96.3
Tween 80	0.1	98.7
Briji 35	0.1	95.1
Deoxycholate	0.1	69.8

Fig.1 pH Optimum

△: Phosphate buffer
●: Tris-HCl buffer

Fig.2 pH Stability

(＋0.2M NaCl) 37℃, 60 min.
〇: 3,3-Dimethylglutarate-NaOH
buffer
●: Tris-HCl buffer

Fig.3 Thermal Stability

pH 7.5, 10 min.
10 mM Tris-HCl buffer

Assay

Principle

The assay is based on the increase in absorbance at 480 nm as the formation of quinoneimine dye proceeds in the following reactions:

	ALOD
CH₃OH+O₂	→	HCHO+H₂O₂
	POD
2 H₂O₂+4–AA+Phenol	→	Quinoneimine dye+4 H₂O

Unit definition

One unit is defined as the amount of enzyme which generates 1 μmole of H₂O₂ and formaldehyde from methanol per minute at 37℃ under the conditions specified in the assay procedure.

Reagents

Reaction mixture

0.2 M Tris–HCl buffer　pH8.0 0.20ml

15 mM 4–AA solution 0.10ml

0.2% (W/V) Phenol solution 0.10ml

2.0 M Methanol solution 0.25ml

50 U/ml POD solution ¹⁾ 0.10 ml

Distilled water 0.25 ml

1) : 50 U/ml POD solution
Dissolve 500 U (PPU) of POD with 10 ml of distilled water.
Reaction stopper
Ethanol
Enzyme dilution buffer
10 mM Tris–HCl buffer　pH 8.0
Reagents
4–AA: NACALAI TESQUE, INC. Special grade #01907–52
POD: Sigma Chemical Co.　Type Ⅱ #P–8250
Ethanol: FUJIFILM Wako Pure Chemical Corporation
Japanese Pharmacopoeia Grade #324–00015

Enzyme solution

Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.

Procedure

Pipette accurately 1.0 ml of reaction mixture into a small test tube and preincubate at 37℃.
After 5 min, add exactly 50 μl of enzyme solution and mix to start the reaction at 37℃.

※ In the case of a test blank, add 50 μl of enzyme dilution buffer in place of enzyme solution.
At 5 min after starting the reaction, add 2.0 ml of the reaction stopper to stop the reaction.
Measure the absorbance at 480 nm.

Absorbance sample : As

blank : Ab/min

△A = (As－Ab) ≦ 0.20 Abs

Calculation

Activity (U/mg of powder) = {(△ A/5)/(17.17 × 1/2)} × 3.05/0.05 × 1/x

17.17 :	millimolar extinction coefficient of quinoneimine dye at 480 nm (cm² / μmole)
1/2 :	a multiplier derived from the fact that 2 mole of H₂O₂ produces 1 mole of quinoneimine dye
5 :	reaction time (min)
3.05 :	final volume (ml)
0.05 :	volume of enzyme solution (ml)
X :	concentration of the sample in enzyme solution (mg/ml)

Storage

Storage at －20℃ in the presence of a desiccant is recommended.

References

Fujii, T. and Tonomura. K. (1972) Agric. Biol. Chem., 36, 2297–2306.
Sahm, H. and Wagner, F. (1973) Eur. J. Biochem., 36, 250–256.
Kato, N., Omori, Y., Tani. Y. and Ogata. K. (1976) Eur. J. Biochem., 64, 341–350.
Tani, Y., Miya, T., Nishikawa. H. and Ogata. K (. 1972) Agric. Biol. Chem., 36, 68–75.

ALOD 活性測定法 (Japanese)

試薬液

反応試薬混合液

0.2Mトリス–HCl 緩衝液 pH8.0	0.20 ml
15mM 4–AA 溶液	0.10 ml
0.2% (W/V) フェノール溶液	0.10 ml
2M メタノール液	0.25 ml
50U/ml POD 溶液 ¹⁾	0.10 ml
精製水 l	0.25 m

1) :	50U/ml POD 溶液 POD 500 単位 (PPU) を精製水10ml で溶解する。

反応停止液
エタノール原液をそのまま使用する。
酵素溶解希釈用液
10mM トリス–HCl 緩衝液 pH8.0
試薬
4–AA：ナカライテスク製　特級　#01907–52
POD：シグマ製　Type Ⅱ　#P–8250
エタノール：富士フイルム和光純薬製
日本薬局方　#324–00015

酵素試料液

検品約20mg を精密に量り、酵素溶解希釈用液に溶解して全容20ml とする。
その液を酵素溶解希釈用液で適宜希釈する。

測定操作法

小試験管に反応試薬混合液1.0ml を正確に分注して37℃で予備加温する。
5 分経過後、酵素試料液50 μl を正確に加えて混和後、37℃で反応を開始する。

※ 盲検は酵素試料液の代わりに酵素溶解希釈用液50μl を加える。
5 分経過後、反応停止液2.0ml を正確に加え反応を停止する。
480nm における吸光度を測定する。
求められた吸光度を試料液はAs、盲検液はAb とする。
ΔA = (As－Ab) ≦ 0.20 Abs

計算

活性 (U/mg) = {(△ A/5)/(17.17 × 1/2)} × 3.05/0.05 × 1/x

17.17 :	キノンイミン色素の480nm におけるミリモル分子吸光係数 (cm²/ μmole)
1/2 :	H₂O₂ 2 モルからキノンイミン色素1 モルが生成することによる係数
5 :	反応時間 (min)
3.05 :	反応総液量 (ml)
0.05 :	反応に供した酵素試料液量 (ml)
X :	酵素試料液中の検品濃度 (mg/ml)

0.2 M Tris–HCl buffer　pH8.0	0.20ml
15 mM 4–AA solution	0.10ml
0.2% (W/V) Phenol solution	0.10ml
2.0 M Methanol solution	0.25ml

ALCOHOL OXIDASE [ALOD] (T-38)