NIPRO ENZYMESGLUCOKINASE (ZM-GlcK)[EC 2. 7. 1. 2]from Zymomonas mobilis
ATP + D-Glucose ↔ ADP + D-Glucose-6-phosphate
SPECIFICATIONStateSpecific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants
Activator: Lyophilized: more than 150 U/mg protein: (as ZM-GlcK activity = 100 %)Glucose-6-phosphate dehydrogenase
Phosphoglucomutase
6-Phosphogluconate dehydrogenase
Hexose-6-phosphate isomerase
Glutathione reductase: ca. 66,000: ca. 33,000: 7.0 - 8.0: 6.0 - 8.0: No detectable decrease in activity up to 40 °C.
: (60 mM Phosphate buffer, pH 7.0, at 30 °C)
GlucoseATP: Pi< 0.02 %< 0.01 %< 0.01 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for diagnostic reagent, for example, glucose determination or CK
determination, and for the specific determination of glucose.
Tris-HCI buffer is not suitable for the practical use of ZM-GlcK.

0.10 mM
0.65 mM

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
ATP + Glucose

ZM-GlcK

ADP + Glucose-6-phosphate

Glucose-6-phosphate + NAD+G6PDH

Gluconolactone-6-phosphate + NADH + H+

Unit Definition
One unit of activity is defined as the amount of ZM-GlcK that forms 1 μmol of glucose-6-phosphate
per minute at 30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Triethanolamine - NaOH and 3 mM K2HPO4, pH 7.5
Ⅱ ATP solution ; 100 mM (0.605 g ATP disodium salt∙3H2O/(8.2 mL distilled water + 1.8 mL 1 N-NaOH))
Ⅲ MgCl2 solution ; 1 M (20.33 g MgCl2∙6H2O/100 mL distilled water)
Ⅳ NAD+ solution ; 100 mM (0.663 g NAD+ free acid/10 mL distilled water)
Ⅴ Glucose solution ; 40mM (0.072 g glucose (anhyd.)/10 mL distilled water)
Ⅵ Glucose-6-phosphate dehydrogenase (G6PDH) ; 2000 U/mL (from Zymomonas mobilis, Nipro Corp.,
Dissolve with Buffer solutionⅠ)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50 mM potassium
phosphate buffer containing 1 mg/mL BSA, pH 7.0.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 20.07 mL
SolutionⅣ 0.60 mL
SolutionⅡ 1.50 mL
SolutionⅤ 7.50 mL
SolutionⅢ 0.30 mL
SolutionⅥ 0.03 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 minute （ΔAbs340） in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (3.00 + 0.01)
6.22 X 0.01

Specific activity (U/mg protein) =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)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Scopes. R.K., Testolin, V., Stoter, A., Griffiths-Smith, K., and Algar, E.M.; Biochem. J., 228, 627(1985)NIPRO ENZYMESRemaining activity (%)

Relative activity (%)

10050

100050045678910 11 12456pH8910 11 12pHFig. 1 pH profile

Fig. 2 pH stability○ MES-KOH, □ TEA-NaOH,■ Gly-KOH

treated for 24 hr at 4 °C in thefollowing buffer solution (0.1 M);△ acetate,○ phosphate,● Tris-HCl,■ Gly-KOH

100

Remaining activity (%)

Remaining activity (%)

750

100500020304050

6070

Temperature (°C)

800153045Time (min)60Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1 M
phosphate buffer, pH 7.0

treated in 0.1 M phosphatebuffer , pH 7.0○ 40 °C, □ 50 °C, ● 60 °C