CODE No.:Z1A111

ALCOHOL DEHYDROGENASE (ZM-ADH)

[EC 1.1.1.1]
from Zymomonas mobilis

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 400 U/mg protein
Contaminants	:	(as ZM-ADH activity = 100%)
		Glucose-6-phosphate dehydrogenase	<0.10%
		Glucokinase	<0.02%
		Pyruvate kinase	<0.02%
		NADH oxidase	<0.01%
		Lactate dehydrogenase	<0.01%

PROPERTIES

Molecular weight	:	ca. 148,000
Subunit molecular weight	:	ca. 37,000
Optimum pH	:	9.5 - 10.0	(Fig.1)
pH stability	:	7.0 - 9.0	(Fig.2)
Thermal stability	:	No detectable decrease in activity up to 40°C.	(Fig.3, 4)
Michaelis constants	:	(100mM Glycine-KOH buffer, pH 9.0, at 30°C)
		Ethanol	110mM
		Methanol	350mM
		NAD+	0.12mM
		Acetaldehyde	1.66mM
		NADH	0.03mM
Substrate specificity	:	Ethanol	100%
		Methanol	0.05%
		n - Propanol	42.3%
		n - Butanol	0.28%

STORAGE

stable at -20 °C for at least six months

APPLICATION

The enzyme is useful for determination of alcohols or aldehydes.

ASSAY

Principle

The change in absorbance is measured at 340 nm according to the following reaction.

Unit Definition

One unit of activity is defined as the amount of ZM-ADH that forms 1 μmol of NADH per minute at 30°C.

Solutions

1. Buffer solution ; 80mM Glycine-KOH, pH 9.5
2. NAD⁺ solution ; 10mM (0.0663 g NAD⁺ free acid/10mL distilled water)
3. Ethanol solution ; Ethanol (96 %)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50mM Tris succinate buffer containing 1mg/mL BSA and 0.2 mM CoCl₂, pH 7.0

Procedure

1. Prepare the following reaction mixture and pipette 3.00mL of reaction mixture into a cuvette.

   Solution I	22.90mL
   Solution II	6.00mL
   Solution III	1.10mL

2. Incubate at 30°C for about 3 minutes.
3. Add 0.01 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute (ΔAbs₃₄₀) in the linear portion of curve.

Calculation

• d.f. ; dilution factor
• 6.22 ; millimolar extinction coefficient of NADH (cm²/μmol)
• *Protein concentration ; determined by Bradford's method

REFERENCE

1. Neale, A.D., Scopes. R.K., Kelly, J.M., and Wettenhall, R.E.H.; Eur. J. Biochem., 154, 119 (1986)

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