CODE No.:B5A111

ALANINE RACEMASE (AlaR)

[EC 5.1.1.1]
from Bacillus stearothermophilus

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 950 U/mg protein
Contaminants	:	(as AlaR activity = 100%)
		Lactate dehydrogenase	<0.01%
		NADH oxidase	<0.01%
		Alanine dehydrogenase	<0.01%

PROPERTIES

Molecular weight	:	ca. 78,000
Subunit molecular weight	:	ca. 39,000
Optimum pH	:	10.5 - 12.0	(Fig.1)
pH stability	:	5.5 - 11.0	(Fig.2)
Thermal stability	:	No detectable decrease in activity up to 70°C.	(Fig.3, 4)
Michaelis constants	:	(100mM Carbonate buffer, pH 10.5, at 30°C)
		D-Alanine	31mM
Substrate specificity	:

STORAGE

stable at -20°C at least one year

ASSAY

Principle

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

Unit Definition

One unit of activity is defined as the amount of AlaR that forms 1μmol of L-alanine per minute at 30°C.

Solutions

1. Buffer solution ; 200mM Sodium hydrogencarbonate, pH 10.5
2. D-Alanine solution ;1 M (0.891 g D-alanine/10mL distilled water)
3. NAD⁺ solution ; 100mM (0.663 g NAD⁺/10mL distilled water)
4. L-Alanine dehydrogenase (AlaDH) ; 1000 U/mL (from Bacillus stearothermophilus, Unitika Ltd., Dissolve with distilled water)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50mM potassium phosphate buffer, pH 7.5.

Procedure

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

   Solution I	16.50mL	Solution IV	1.50mL
   Solution II	3.00mL	H2O	8.25 mL
   Solution III	0.75 mL

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 minutes (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. Inagaki, K., Tanizawa, K., Badet, B., Walsh, C.T., Tanaka, H., and Soda, K.; Biochemistry, 25, 3268 (1986)

• 
• 
• 
•