NIPRO ENZYMESMUTAROTASE (MRO)[EC 5. 1. 3. 3]from Microorganism

α-D-Glucose ↔ β-D-Glucose
SPECIFICATIONStateSpecific activity
Contaminants
PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Thermal stability: Lyophilized: more than 120 U/mg protein: (as MRO activity = 100 %)NADHoxidase< 0.01 %: ca. 39,500: 7.0 - 9.0: 3.5 - 10.0
: No detectable decrease in activity up to 50 °C.

(Fig. 1)
(Fig. 2)
(Fig. 3, 4)

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThis enzyme is useful for enzymatic determination of glucose.

NIPRO ENZYMESASSAYPrinciple
Acceleration of the glucose dehydrogenase reaction by Mutarotase is measured according to thefollowing reactions.
β-Fructosidase
Sucros
α-D-Glucose + Fructose
α-D-Glucose

MRO

β-D-Glucose + NAD+

β-D-Glucose

GlucoseDH

D-Glucono--lactone + NADH + H+

Unit Definition
One unit of activity is defined as the amount of Mutarotase that forms 10 μmol of NADH per minute at25 °C.
SolutionsⅠ HEPES buffer ; 50 mM (1.19 g HEPES / 100 mL distilled water, adjust pH to 7.5 with NaOH)
Ⅱ Sucrose solution ; 16.7 mM (57 mg Sucrose / 10 mL distilled water)
Ⅲ NAD+ solution ; 100 mM (0.663 g NAD+ free acid / 10 mL distilled water)
Ⅳ Glucose dehydrogenase solution ; 3 kU/mL (GlcDH2, Nipro Corp. / 20 mM potassium phosphate
containing 2 M NaCl, pH6.5)
Ⅴ β-Fructosidase solution ; ≧30 kU/mL (100 mg Invertase from baker’s yeast, Sigma-Aldrich I4504 /1 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 0.7 to 1.4 U/mL with 20 mM
potassium phosphate pH 7.3 containing 1 mg/mL BSA.
Procedure
1. Prepare the following reaction mixture and pipette 2.70 mL of reaction mixture into a cuvette.
SolutionⅠ 19.90 mL
SolutionⅣ 0.166 mL
SolutionⅡ 1.00 mL
SolutionⅢ 0.60 mL
2. Add 0.015 mL of the enzyme solution into the cuvette and mix.
3. Incubate at 25 °C for about 3 minutes.
4. Add 0.06 mL of the Solution Ⅴ into the cuvette and mix.
5. Read absorbance change at 340 nm per minute (ΔAbs1) in the linear portion of curve.
6. Run the procedure 1 to 5 with the enzyme diluent instead of the enzyme solution (ΔAbs2).
Calculation
Volume activity (U/mL) =(∆Abs1 - ΔAbs2) X (2.70 + 0.015 + 0.060)

Specific activity (U/mg protein) =6.22 X 0.015 X 10

X d.f.

Volume activity (U/mL)
Protein concentration (mg/mL)*

d.f. ; dilution factor
6.22 ; millimolar extinction coefficient of NADH (cm 2/μmol)
10 ; conversion factor*Protein concentration ; determined by the absorbance at 280 nm (Abs280),
where 1 Abs280 = 1 mg/mL

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

10050034567891050011356789pH

Fig. 1 pH profile

Fig. 2 pH stability△ acetate, □ MES, ◆ PIPES,◇ HEPES, ● Tris-HCl,■Bicine, ▲Glycine-KOH

1011treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M)
containing 0.1 % BSA;
△ acetate, ○ phosphate,● Tris-HCl, ▲ Glycine-KOH

100100

Remaining activity (%)

Remaining activity (%)

4pH505000203040506070Temperature (℃)

Fig. 3 Thermal stability

treated for 15 min in 0.1 M potassium
phosphate buffer, pH 6.5, 0.1 % BSA.

80015304560Time (min)Fig. 4 Thermal stability
treated in 0.1 M potassium
phosphate buffer, pH 6.5, 0.1 % BSA
at ○ 40 ℃, □ 50 ℃, ● 60 ℃