Thermostable Enzymes forClinical Chemistry

NIPRO Enzymes

From Zymomonas mobilis

ALCOHOL DEHYDROGENASE (ZM-ADH)
GLUCOKINASE (ZM-GlcK)GLUCOSE-6-PHOSPHATE DEHYDROGENASE (ZM-G6PDH)

From Bacillus stearothermophilus

ADENYLATE KINASE (AdK)
ALANINE DEHYDROGENASE (AlaDH)
ALANINE RACEMASE (AlaR)LEUCINE DEHYDROGENASE (LeuDH)
PHOSPHOGLUCOSE ISOMERASE (PGI)
SUPEROXIDE DISMUTASE (SOD)

From Others

ACETATE KINASE (AK2)
BILIRUBIN OXIDASE (BOD3)
DIAPHORASE3 (DI-3)
DIAPHORASE22 (Di-22)
GALACTOSE DEHYDROGENASE (GalDH)
GLUCOKINASE2 (GlcK2)
GLUCOSE DEHYDROGENASE (GlcDH2)
GLYCEROKINASE (GlyK)GLYCEROL-3-PHOSPHATE DEHYDROGENASE (G3PDH)
D-LACTATE DEHYDROGENASE (D-LDH)
MALATE DEHYDROGENASE (MDH)
MUTAROTASE (MRO)PHENYLALANINE DEHYDROGENASE (PheDH)
PHOSPHOTRANSACETYLASE (PTA2)POLYNUCLEOTIDE PHOSPHORYLASE (PNPase3)
PYRUVATE KINASE (PK2)

ご照会は下記へお願い申し上げます。
ニプロ株式会社
本社/〒566-8510 大阪府摂津市千里丘新町3番26号
Tel 06-6310-6596

For more information, please contact
NIPRO CORPORATION
3-26, Senriokashinmachi, Settsu,
Osaka, 566-8510, Japan
Phone +81 6 6310 6596
Fax
+81 50 3730 4942

e-mail : nipro-web@nipro.co.jp
http://www.nipro.co.jp/
Bacillus stearothermophilus is used as a synonym of Geobacillus stearothermophilus .

2024.06

Ｑｕａｌｉｔｙ
The Quality Management System of Enzyme Center, NIPRO Corp. has been certified
as to meet the requirements of ISO9001 in the scope of design, development andmanufacture of enzymes for analytical reagents and industrial use by JAPANCHEMICAL QUALITY ASSURANCE LTD.

NIPRO ENZYMESALCOHOL DEHYDROGENASE (ZM-ADH)[EC 1 .1 .1 .1]
from Zymomonas mobilis
Alcohol + NAD+ ↔ Aldehyde + NADH + H+
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants

Substrate specificity: Lyophilized: more than 400 U/mg protein: (as ZM-ADH activity = 100 %)Glucose-6-phosphate dehydrogenase
Glucokinase
Pyruvate kinase
NADH oxidase
Lactate dehydrogenase: ca. 148,000: ca. 37,000: 9.5 - 10.0
: 7.0 - 9.0: No detectable decrease in activity up to 40 °C.
: (100 mM Glycine-KOH buffer, pH 9.0, at 30 °C)
Ethanol
MethanolNAD+
Acetaldehyde
NADH: Ethanol
Methanoln-Propanol
n-Butanol

STORAGE
Stable at -20 °C for at least six months
APPLICATIONThe enzyme is useful for determination of alcohols or aldehydes.

< 0.10 %< 0.02 %< 0.02 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
110 mM
350 mM
0.12 mM
1.66 mM
0.03 mM
100 %0.05 %
42.3 %
0.28 %NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
Ethanol + NAD+ZM-ADH

Acetaldehyde + NADH + H+

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
Ⅰ Buffer solution ; 80 mM Glycine-KOH, pH 9.5
Ⅱ NAD+ solution ; 10 mM (0.0663 g NAD+ free acid/10 mL distilled water)
Ⅲ Ethanol solution ; Ethanol (96 %)Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50 mM Tris
succinate buffer containing 1mg/mL BSA and 0.2 mM CoCl2, pH 7.0
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 22.90 mL
SolutionⅡ 6.00 mL
SolutionⅢ 1.10 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 （∆Abs340） 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 (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)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

10

11

12

50

0

4

5

7

8

9

10

pH

pH

Fig. 1 pH profile

Fig. 2 pH stability○ phosphate, ● Tris-HCl,■ Gly-KOH

11

12

Remaining activity (%)

treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M),
containing 0.5 mM CoCl2;△ acetate,○ phosphate,● Tris-HCl,■ Gly-KOH

100

Remaining activity (%)

100

50

0

6

20

30

40
50
60
70
Temperature (℃)
Fig. 3 Thermal stability

treated for 15 min in 0.1 M
phosphate buffer containing
0.2 mM CoCl2, pH 6.5

80

50

0

0

15

30

45

60

Time (min)Fig. 4 Thermal stability
treated in 0.1 M phosphate buffer
containing 0.2 mM CoCl2, pH 6.5○ 50 ℃, □ 55 ℃, ● 60 ℃

NIPRO ENZYMESGLUCOKINASE (ZM-GlcK)[EC 2. 7. 1. 2]from Zymomonas mobilis
ATP + D-Glucose ↔ ADP + D-Glucose-6-phosphate
SPECIFICATION
State
Specific 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 NNaOH))
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 (cm2/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 (%)

100

50

100

0
4

5

6

7

8

9

50

0

10 11 12

4

5

6

pH

8

9

10 11 12pH

Fig. 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 (%)

7

50

100

50

0

0
20

30

40

50

60

70

Temperature (°C)

80

0

15

30
45
Time (min)60

Fig. 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

NIPRO ENZYMESGLUCOSE-6-PHOSPHATE
DEHYDROGENASE (ZM-G6PDH)[EC 1. 1. 1. 49]
from Zymomonas mobilis
D-Glucose-6-phosphate + NAD(P)+ ↔ D-Gluconolactone-6-phosphate + NAD(P)H + H+
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants

Substrate specificity: Lyophilized: more than 250 U/mg protein: (as ZM-G6PDH activity = 100 %)Glucokinase
Phosphoglucomutase
6-Phosphogluconate dehydrogenase
Hexose-6-phosphate isomerase
Glutathione reductase: ca. 208,000: ca. 52,000: 8.0: 5.0 - 10.0
: No detectable decrease in activity up to 50 °C.
: (30 mM Tris-HCI buffer, pH 8.0, at 30 °C)
Glucose-6-phosphate
NADP+
NAD+
: NADP+NAD+< 0.02 %< 0.01 %< 0.02 %< 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.

0.14 mM
0.02 mM
0.14 mM
70 %100 %NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
Glucose-6-phosphate + NAD+ZM-G6PDH

Gluconolactone-6-phosphate + NADH + H+

Unit Definition
One unit of activity is defined as the amount of ZM-G6PDH that forms 1 μmol of NADH per minuteat 30 °C.
Solutions
Ⅰ Buffer solution ; 50 mM Tris-HCI, pH 8.0
Ⅱ NAD+ solution ; 100 mM (0.663 g NAD+ free acid/10 mL distilled water)
Ⅲ Glucose-6-phosphate (G6P) solution ; 33 mM (0.112 g G6P disodium salt 2H2O/10 mL distilledwater)
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 26.40 mL
SolutionⅡ 0.90 mL
SolutionⅢ 2.70 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 (∆Abs340) 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 (cm2/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)

100

Remaining activity (%)

Relative activity (%)

NIPRO ENZYMES

50

100

0
4

5

6

7

9

0

10 11 12

4

5

6

7

8

9

10 11 12

pH

pH

Fig. 1 pH profile

Fig. 2 pH stability△ acetate,● Tris-HCl,treated for 24 hr at 4 °C in thefollowing buffer solution (0.1 M);○ phosphate,△ acetate,● Tris-HCl,■ Gly-KOH○ phosphate,■ Gly-KOH

100

Remaining activity (%)

Remaining activity (%)

8

50

50

100

50

0

0
20

30

40

50

60

70

Temperature (°C)
Fig. 3 Thermal stability
treated for 15 min in 0.1 M
phosphate buffer, pH 7.0

80

0

15

30
45
Time (min)60

Fig. 4 Thermal stability
treated in 0.1 M phosphatebuffer, pH 7.0○ 50 °C, □ 55 °C, ● 60 °C

NIPRO ENZYMESADENYLATE KINASE (AdK)[EC 2. 7. 4. 3]
from Bacillus stearothermophilus
ATP + AMP ↔ 2 ADP
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Optimum pH
pH stability
Isoelectric point
Thermal stability
Michaelis constants: Lyophilized: more than 200 U/mg protein: (as AdK activity = 100 %)ATPasePhosphoglycerate kinase: ca. 20,000: 6.5: 8.0 - 10.5
: 5.0
: No detectable decrease in activity up to 65 °C.
: (89 mM Imidazole-HCI buffer, pH 6.5, at 30 °C)
ATP
ADP
AMP

< 0.01 %< 0.10 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
0.04 mM
0.05 mM
0.02 mM

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for determination of AMP or for system involving ATP regeneration.

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

AdK

2 ADP + 2PEP

2 ADP

PK

2 ATP + 2 Pyruvate

2 Pyruvate + 2 NADH + 2 H+

LDH

2 Lactate + 2 NAD+

Unit Definition
One unit of activity is defined as the amount of AdK that forms 2 μmol of ADP per minute at 30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Imidazole-HCl, pH 6.5
Ⅱ AMP solution ; 50 mM (0.250 g AMP disodium salt∙6H2O/10 mL distilled water)
Ⅲ ATP solution ; 100 mM (0.605 g ATP disodium salt∙3H2O/(8.2 mL distilled water + 1.8 mL 1 NNaOH))
Ⅳ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O /10 mL distilled water)
Ⅴ Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10 mL distilled water)
Ⅵ MgCl2 solution ; 1 M (20.33 g MgCl2∙6H2O/100 mL distilled water)
Ⅶ KCI solution ; 2.5 M (18.64 g KCl/100mL distilled water)
Ⅷ Pyruvate kinase (PK) ; (from rabbit muscle, Roche Diagnostics K.K., No. 128 155) crystalline
suspension in 3.2 M (NH4)2SO4 solution (10 mg/mL) approx. 200 U/mg at 25 °C
Ⅸ Lactate dehydrogenase (LDH) ; (from pig heart, Oriental Yeast Co. Ltd., Product Code: LDH-02)
ammonium sulfate suspension, approx. 5,000 U/mL at 25 °C
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 2.5 to 5 U/mL with 50 mM Tris-HCI
buffer, pH 8.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 26.70 mL
SolutionⅥ 0.60 mL
SolutionⅡ 0.24 mL
SolutionⅦ 1.20 mL
SolutionⅢ 0.30 mL
SolutionⅧ 0.09 mL
SolutionⅣ 0.60 mL
SolutionⅨ 0.09 mL
SolutionⅤ 0.18 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 (∆Abs340) in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (3.00 + 0.01)
2 X 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
2; according to the reaction that forms 2 μmol of ADP, one unit of activity of Adk is defined toform 2 μmol of ADP.

NIPRO ENZYMES
6.22 ; millimolar extinction coefficient of NADH (cm2/μmol)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Imahori, K., Nakajima, H., Nagata, K., and lwasaki, T.; Seikagaku, 53, 829 (1981)

NIPRO ENZYMES

100
Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

10

11

50

0

12

pH

4

5

6

○ phosphate,▲ carbonate

10

11

treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);△ acetate,○ phosphate,● Tris-HCl, ▲ carbonate

100
Remaining activity (%)

Remainig activity (%)

9Fig. 2 pH stability

100

50

0

8
pH

Fig. 1 pH profile△ acetate,● Tris-HCl,

7

20

30

40
50
60
70
Temperature (℃)

80

50

0

0

15

30
45
Time (min)60

Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1 M
Tris-HCl buffer, pH 9.0

treated in 0.1 M Tris-HCl buffer,
pH 9.0○ 60 ℃, □ 65 ℃, ● 70 ℃

12

NIPRO ENZYMESALANINE DEHYDROGENASE (AIaDH)[EC 1. 4. 1. 1]
from Bacillus stearothermophilus
L-Alanine + NAD+ + H2O ↔ Pyruvate + NH4+ + NADH
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants
Substrate specificity: Lyophilized: more than 10 U/mg protein: (as AlaDH activity = 100 %)NADH oxidase
Lactate dehydrogenase< 0.01 %< 0.10 %: ca. 230,000: ca. 38,000: 10.4(Fig. 1)
: 7.0 - 11.5
(Fig. 2)
: No detectable decrease in activity up to 70 °C. (Fig. 3, 4): (125 mM Glycine-NaOH buffer, pH 10.5, at 30 °C)
L-Alanine
10.0 mM
0.26 mM
NAD+: L-Alanine
100 %L-Leucine0%
L-Isoleucine
0%

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for determination of L-alanine.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
AlaDH

L-Alanine + NAD+ + H2O

Pyruvate + NH4+ + NADH

Unit Definition
One unit of activity is defined as the amount of AlaDH that forms 1 μmol of NADH per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 250 mM Glycine-NaOH, pH 10.5
Ⅱ L-Alanine solution ; 150 mM (1.336 g L-alanine/80 mL distilled water, adjusted to pH 10.5 with 1 NNaOH and filled up to 100 mL with distilled water)
Ⅲ NAD+ solution ; 100 mM (0.663 g NAD+/ 10 mL with distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 100 mM glycine NaOH buffer, pH 9.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 15.00 mL
SolutionⅢ 1.50 mL
3.50 mL
SolutionⅡ 10.00 mL
H2O
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 (∆Abs340) 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 (cm2/μmol)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Sakamoto, Y., Nagata, S., Esakl, N., Tanaka, H. and Soda, K.; J. Ferment. Bioeng., 69, 154 (1990)

100

Remaining activity (%)

Relative activity (%)

NIPRO ENZYMES

50

0
8

9

11

10

100

50

0

12

4

5

6

pH

8

9

10 11 12pH

Fig. 1 pH profile

Fig. 2 pH stability● Tris-HCl, ■ Gly-KOH,○ phosphate

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 (%)

7

50

100

50

0

0
20

30

40

50

60

70

80

Temperature (°C)

90

0

15

30
45
Time (min)60

Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1 M
Gly-KOH buffer, pH 9.0

treated in 0.1 M Gly-KOH
buffer, pH 9.0○ 70 °C, □ 80 °C, ● 90 °C

NIPRO ENZYMESALANINE RACEMASE (AlaR)[EC 5. 1. 1. 1]
from Bacillus stearothermophilus
D-Alanine ↔ L-Alanine
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants
Substrate specificity
STORAGE
Stable at least one year at -25 °C.

: Liquid: more than 950 U/mg protein: (as AlaR activity = 100 %)Lactate dehydrogenase
NADH oxidase
Alanine dehydrogenase: ca. 78,000: ca. 39,000: 10.5 - 12.0
: 5.5 - 11.0
: No detectable decrease in activity up to 70 °C.
: (100 mM Carbonate buffer, pH 10.5, at 30 °C)
D-Alanine:< 0.01 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
31 mM

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
D-Alanine

AlaR

L-Alanine

L-Alanine + NAD+ + H2O

AlaDH

Pyruvate + NH4+ + NADH

Unit Definition
One unit of activity is defined as the amount of AlaR that forms 1 μmol of L-alanine per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 200 mM Sodium hydrogencarbonate, pH 10.5
Ⅱ D-Alanine solution ;1 M (0.891 g D-alanine/10 mL distilled water)
Ⅲ NAD+ solution ; 100 mM (0.663 g NAD+/10 mL distilled water)
Ⅳ L-Alanine dehydrogenase (AlaDH) ; 1000 U/mL (from Bacillus stearothermophilus, Nipro Corp.,
Dissolve with distilled water)
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, pH 7.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 16.50 mL
SolutionⅣ 1.50 mL
8.25 mL
SolutionⅡ 3.00 mL
H2O
SolutionⅢ 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 minute (∆Abs340) 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 (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)

100

Remaining activity (%)

Relative activity (%)

NIPRO ENZYMES

50

0
4

5

6

7

8

9

100

50

0

10 11 12

4

5

6

8

9

10 11 12pH

Fig. 1 pH profile

Fig. 2 pH stability○ phosphate,● Tris-HCl,■ Gly-KOH, □ NaHCO3-NaOH

treated for 24 hr at 4 °C in thefollowing buffer solution (0.2 M);△ acetate,○ phosphate,● Tris-HCl, ■ Gly-KOH

100

Remaining activity (%)

Remaining activity (%)

pH

7

50

0

100

50

0
20

30

40

50

60

70

80

Temperature (°C)

90

0

15

30
45
Time (min)60

Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 50 mM
Tris-HCl buffer, pH 9.0

treated in 50 mM Tris-HCl
buffer, pH 9.0○ 70 °C, □ 80 °C, ● 85 °C

NIPRO ENZYMESRemaining activity (%)

140
120
100
80
60
40
20
0

0

10

20

30

Period (months)
Fig. 5 Stability (Liquid form) at -25 °C

NIPRO ENZYMESLEUCINE DEHYDROGENASE (LeuDH)[EC 1. 4. 1. 9]from Bacillus stearothermophilus
L-Leucine + NAD+ + H2O ↔ α-Ketoisocaproate+ NH4+ + NADH
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants
Substrate specificity: Lyophilized: more than 40 U/mg protein: (as LeuDH activity = 100 %)NADH oxidase
Lactate dehydrogenase< 0.01 %< 0.01 %: ca. 300,000: ca. 49,000: 10.6(Fig. 1)
: 6.0 - 11.5
(Fig. 2)
: No detectable decrease in activity up to 60 °C. (Fig. 3, 4): (125 mM Sodium phosphate buffer, pH 10.5, at 30 °C)
L-Leucine
3.4 mM
0.3 mMNAD+: L-Leucine
100 %L-Valine
86 %L-Isoleucine
73 %STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for determination of L-leucine, L-valine or L-isoleucine.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
L-Leucine + NAD+ + H2O

LeuDH

α-Ketoisocaproate + NH4+ + NADH

Unit Definition
One unit of activity is defined as the amount of LeuDH that forms 1 μmol of NADH per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 250 mM Sodium phosphate, pH 10.5
Ⅱ L-Leucine solution ; 60 mM (0.787 g L-leucine/80 mL distilled water, adjusted to pH 10.5 with 1 NNaOH and filled up to 100 mL with distilled water)
Ⅲ NAD+ solution ; 100mM (0.663 g NAD+/ 10mL with distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 100 mM sodium
phosphate buffer, pH 9.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette,
SolutionⅠ 15.00 mL
SolutionⅢ 0.93 mL
4.07 mL
SolutionⅡ 10.00 mL
H2O
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 (cm2/μmol)*Protein concentration ; determined by Bradford's method
REFERENCE
1. Ohshima, T., Nagata, S., and Soda, K.; Arch. Microbiol., 141, 407 (1985)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

10

11

50

0

12

4

5

6

pH
Fig. 1 pH profile■ Gly-KOH,

9

10

11

12

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

100

Remaining activity (%)

Remaining activity (%)

8pHFig. 2 pH stability

100

50

0

7

50

20

30

40

50
60
70
Temperature (℃)

80

0

0

15

30
45
Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1 M GlyKOH buffer, pH 9.0

treated in 0.1M Gly-KOH
buffer, pH 9.0○ 60 ℃, □ 70 ℃, ● 80 ℃

60

NIPRO ENZYMESPHOSPHOGLUCOSE ISOMERASE (PGI)[EC 5. 3. 1. 9]from Bacillus stearothermophilus
D-Glucose-6-phosphate ↔ D-Fructose-6-phosphate
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Isoelectric point
Thermal stability
Michaelis constants: Lyophilized: more than 400 U/mg protein: (as PGI activity = 100 %)Phosphofructokinase
6-Phosphogluconate dehydrogenase
Phosphoglucomutase
NADPH oxidase
Glutathione reductase: ca. 200,000: ca. 54,000: 9.0 - 10.0
: 6.0 - 10.5
: 4.2
: No detectable decrease in activity up to 60 °C.
: (95 mM Tris-HCI buffer, pH 9.0, at 30 °C)
Fructose-6-phospate

STORAGE
Stable at -20 °C for at least one year

< 0.01 %< 0.01 %< 0.01 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
0.27 mM

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
Fructose-6-phosphate

PGI

Glucose-6-phosphate + NADP+

Glucose-6-phosphate
G6PDH

Gluconolactone-6-phosphate + NADPH + H+

Unit Definition
One unit of activity is defined as the amount of PGI that forms 1 μmol of glucose-6-phosphate perminute at 30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Tris-HCl, pH 9.0
Ⅱ Fructose-6-phosphate (F6P) solution ; 100 mM (0.310 g F6P disodium salt/10 mL distilled water)
Ⅲ NADP+ solution ; 22.5 mM (0.188 g NADP+ sodium salt∙4H2O/10 mL distilled water)
Ⅳ Glucose-6-phosphate dehydrogenase (G6PDH) ; (from yeast, Roche Diagnostics K.K., No. 127
671) suspension in 3.2 M (NH4)2SO4 solution (10 mg/2 mL) approx. 140 U/mg at 25 °C
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50 mM Tris-HCI
buffer, pH 8.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 28.44 mL
SolutionⅢ 0.60 mL
SolutionⅡ 0.90 mL
SolutionⅣ 0.06 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 the 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 NADPH (cm2/μmol)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Muramatsu, N., and Nosoh, T.; Arch. Biochem. Biophys., 144, 245 (1971)

100

Remaining activity (%)

Relative activity (%)

NIPRO ENZYMES

50

100

0

50

0
4

5

6

7

8

9

10 11 12

4

6

7

8

9

10 11 12

pH

pH

Fig. 1 pH profile

Fig. 2 pH stability△ acetate, ○ phosphate,● Tris-HCl, ■ Gly-KOH

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

100

Remaining activity (%)

Remaining activity (%)

5

50

0

100

50

0
20

30

40

50

60

70

80

0

15

30

45

Temperature (°C)

Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 50 mM
Tris-HCl buffer, pH 8.5

60

treated in 50 mM Tris-HCl
buffer, pH 8.5○ 60 °C, □ 65 °C, ● 70 °C

NIPRO ENZYMESSUPEROXIDE DISMUTASE (SOD)[EC 1.15.1.1]
from Bacillus stearothermophilus
O2- + O2- + 2H+ ↔ O2 + H2O2
SPECIFICATION
State
Specific activity
Contaminants
PROPERTIES
Molecular weight
Subunit molecular weight
Metal content
Optimum pH
pH stability
Isoelectric point
Thermal stability: Lyophilized: more than 9,000 U/mg protein: (as SOD activity = 100 %)Catalase: ca. 50,000: ca. 25,000: 1.5 g atoms of Mn per mole of enzyme: 9.5: 6.0 - 9.0: 4.5: No detectable decrease in activity up to 60 °C.

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for medicine, cosmetic material and nutrition or antioxidant.

< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)

NIPRO ENZYMES
ASSAY
Principle
To determine the enzyme activity of cytochrome c reduction is measured by the following reactions.
Xanthine + O2
O2-

Xanthine oxidase

Urate + O2- + H2O2

cytochrome cSODcytochrome c (red.)O2 + H2O2

Unit Definition
One unit of activity is defined as the amount of SOD required to inhibit the rate of reduction ofcytochrome C by 50 % at 30 °C.
Solutions
Ⅰ Buffer solution ; 75 mM Potassium phosphate buffer, pH 7.8
Ⅱ Xanthine solution ; 0.75 mM (0.010 g xanthine/50 mL N/250 NaOH)
Ⅲ Cytochrome c solution ; 0.15 mM (0.019 g cytochrome c/10 mL distilled water, Sigma-Aldrich Co.,
No. C-2506, from horse heart)Ⅳ EDTA solution ; 1.5 mM (0.028 g EDTA disodium salt∙2H2O/50 mL distilled water)
Ⅴ Xanthine oxidase (XOD) ; (from buttermilk, Sigma-Aldrich Co., No. X-1875) suspension in 2.3 M(NH4)2SO4 solution is diluted to 0.04 U/mL with distilled water. (prepare freshly)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to approx. 600 U/mL with 50 mM
potassium phosphate buffer, pH 7.5.
Procedure
1. Prepare the following reaction mixture and pipette 2.80 mL of reaction mixture and 0.005 mL of
enzyme solution into a cuvette.
SolutionⅠ 22.00 mL
SolutionⅢ 2.00 mL
SolutionⅡ 2.00 mL
SolutionⅣ 2.00 mL
2. Incubate at 30 °C for about 3 minutes.
3. Add 0.20 mL of SolutionⅤ into the cuvette and mix.
4. Read absorbance change at 550 nm per minute for the linear portion of curve (ΔAbs•test)*.
5. Add 0.005 mL of SolutionⅠ in place of enzyme solution and measure the same above 4(ΔAbs•blank).
*Dilute enzyme solution with 50 mM potassium phosphate buffer, pH 7.5, because the decrease in theinitial rate should not fall outside the range of 40 to 60 % for the results to be valid.
Calculation
Volume activity (U/mL) =(ΔAbs•blank)(ΔAbs•test)

Specific activity (U/mg protein) =

-1

X

601
1

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

d.f. ; dilution factor*Protein concentration ; determined by Bradford's method
REFERENCE

X d.f.

NIPRO ENZYMES
1. Bridgen, J., Harris, J.I., and Kolb, E.; J. Mol. Biol., 105, 333 (1976)
2. Brock, C.J., Harris, J.I., and Sato, S.; ibid., 107, 175 (1976)
3. Brock, C.J., and Walker, J.E.; Biochemistry, 19, 2873 (1980)
4. Auffret, A.D., Blake, T,J., and Williams, D.H.; Eur. J. Biochem., 113, 333 (1981)
5. Atkinson, T., Banks, G.T., Bruton, C.J., Comer, M.J., Jakes, R., Kamalagharan, T., Whitak, A.R.,
and Winter, G.P.; J. Appl. Biochem., 1, 247 (1979)

Remaining activity (%)

Relative activity (%)

NIPRO ENZYMES

100

50

0

100

50

0
4

6

5

7

8

9

10 11 128

9

10 11 12Fig. 2 pH stability
treated for 24 hr at 4 ℃ in the
Britton-Robinson buffer

Remaining activity (%)

Remaining activity (%)

7

Fig. 1 pH profile

50

30

6

pH

100

20

5

pH

△ acetate, ○ phosphate,● Tris-HCl, ▲ carbonate

0

4

40

50

60

70

80

Temperature (℃)
Fig. 3 Thermal stability
treated for 15 min in 0.1 M potassium
phosphate buffer, pH 7.5

100

50

0

0

15

30

45

Time (min)Fig. 4 Thermal stability
treated in 0.1 M potassium
phosphate buffer, pH 7.5○ 60 ℃, □ 70 ℃, ● 80 ℃

60

NIPRO ENZYMESACETATE KINASE (AK2)[EC 2. 7. 2. 1]
from recombinant E. coli
ATP + Acetate ↔ ADP + AcetyIphosphate
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants

Substrate specificity

Activator: Lyophilized: more than 1,100 U/mg protein: (as AK2 activity = 100 %)Lactate dehydrogenase
Adenylate kinase
NADH oxidase
GOT
GPT: ca. 172,000: ca. 39,400: 6.0: 6.0 - 11.0
: No detectable decrease in activity up to 65 °C.
: (57 mM Imidazole-HCI buffer, pH 7.2, at 30 °C)
AcetateAcetyIphosphate
ATP
ADP: Acetate
FormatePropionate
ButyrateOxalateMalateGlycine: Fructose-1,6-bisphosphate

STORAGE
Stable at -20 °C for at least one year

< 0.01 %< 0.01 %< 0.01 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
130 mM
3.3 mM
5.4 mM
0.6 mM
100 %
0%
4%
1%
0%
0%
0%

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

AK2
PK

Pyruvate + NADH + H+

ADP + AcetyIphosphate
Pyruvate + ATP
LDH
Lactate + NAD+

Unit Definition
One unit of activity is defined as the amount of AK that forms 1 μmol of ADP per minute at 30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Imidazole-HCl, pH 7.2
Ⅱ ATP solution ; 100 mM (0.605 g ATP disodium salt∙3H2O/(8.2 mL distilled water + 1.8 mL 1 NNaOH))
Ⅲ Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10 mL distilled water)
Ⅳ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Ⅴ MgCl2 solution ; 1 M (20.33 g MgCl2∙6H2O /100 mL distilled water)
Ⅵ KCl solution ; 2.5 M (18.64 g KCI/100 mL distilled water)
Ⅶ Pyruvate kinase (PK) ; (from rabbit muscle, Roche Diagnostics K.K., No. 128 155) crystalline
suspension in 3.2 M (NH4)2SO4 solution (10 mg/mL) approx. 200 U/mg at 25 °C
Ⅷ Lactate dehydrogenase (LDH) ; (from pig heart, Oriental Yeast Co. Ltd., Product Code: LDH-02)
ammonium sulfate suspension, approx. 5,000 U/mL at 25 °C
Ⅸ Sodium acetate solution ; 2 M (27.22 g sodium acetate∙3H2O/100 mL distilled water)
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, pH 7.5.
Procedure
1. Prepare the following reaction mixture and pipette 2.4 mL of reaction mixture into a cuvette.
SolutionⅠ 16.92 mL
SolutionⅤ 0.60 mL
SolutionⅡ 3.00 mL
SolutionⅥ 0.90 mL
SolutionⅢ 1.80 mL
SolutionⅦ 0.12 mL
SolutionⅣ 0.60 mL
SolutionⅧ 0.06 mL
2. Incubate at 30 °C for about 3 minutes.
3. Add 0.60 mL of Solution Ⅸ and 0.01 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute (∆Abs340) 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 (cm2/μmol)*Protein concentration ; determined by Bradford's method

NIPRO ENZYMESREFERENCE
1. Nakajima, H., Suzuki, K., and Imahori, K. ; J. Biochem., 84, 193 (1978)
2. Nakajima, H., Suzuki, K., and Imahori, K. ; ibid., 84, 1139 (1978)
3. Nakajima, H., Suzuki, K., and Imahori, K. ; ibid., 86, 1169 (1979)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

10

11

50

0

12

4

7

8

9

10

Fig. 1 pH profile

Fig. 2 pH stability

11

12

treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);△ acetate, ○ phosphate,● Tris-HCl, ▲ Carbonate

100

100

Remaining activity (%)

Remaining activity (%)

6pH△ acetate, ○ phosphate,● Tris-HCl, ■ Gly-KOH

50

0

5

pH

30

40

50

60

70

Temperature (℃)

Fig. 3 Thermal stability
treated for 15 min in 0.1 M

phosphate buffer, pH 7.5

80

50

0

0

15

30

45

Time (min)Fig. 4 Thermal stability
treated in 0.1 M phosphatebuffer, pH 7.5○ 60 ℃, □ 65 ℃, ● 70 ℃

60

NIPRO ENZYMESBILIRUBIN OXIDASE (BOD3)[EC 1.3.3.5]
from Trachyderma tsunodae
2 Bilirubin + O2 → 2 Biliverdin + 2 H2O
SPECIFICATION
State
Specific activity
PROPERTIES
Molecular weight
Optimum pH
pH stability
Isoelectric point (calculation)
Optimum temperature
Thermal stability
Michaelis constants
Substrate specificity: Lyophilized: more than 100 U/mg protein: ca. 60,000(SDS-electrophoresis): ca. 80,000(Gel filtration): 5.0(Fig. 1)
: 4.0 – 11.0
(4 °C, 24 hr)(Fig. 2)
: 3.8
: 65 – 80 °C(Fig. 3)
: No detectable decrease in activity up to 50 °C. (pH 7.0)
(Fig. 4, 5): See table 1: See table 1

STORAGE
Stable at -20 °C for one year
APPLICATIONThe enzyme is useful for enzymatic determination of bilirubin.
It could be used as a cathode catalyst in biofuel cells.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 500 nm according to the following reaction.
Phenol +1O2 + H2O
2

2 H2O2 + 4-Aminoantipyrine + Phenol

BOD3
POD

Quinone and/or Phenoxy radical + H2O2
Quinoneimine + 4 H2OUnit Definition
One unit of activity is defined according to the calculation formula below.
Solutions
Ⅰ Buffer solution ; 300 mM Potassium phosphate buffer, pH7.0
Ⅱ 4-Aminoantipyrine (4-AA) solution ; 24.6 mM (0.25 g 4-AA / 50 mL distilled water)
Ⅲ Phenol solution ; 420 mM (1.98 g phenol/50mL distilled water)
Ⅳ Peroxidase*1 (POD) solution ; 240 U/mL (2,400 U/10mL distilled water)*1POD: TOYOBO Co., LTD. Grade Ⅲ #PEO-302
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 15 to 60 U/mL with 10 mM
potassium phosphate buffer, pH 7.0 containing 0.1 % BSA.
Procedure
1. Prepare the following reaction mixture and pipette 0.90 mL of reaction mixture into a cuvette.
SolutionⅠ 4.00 mL
SolutionⅡ 0.40 mL
SolutionⅢ 0.40 mL
SolutionⅣ 0.40 mL
6.40 mL
H2O
2. Incubate at 37 °C for about 3 minutes.
3. Add 0.005 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 500 nm per minute (∆Abs (test)) in linear portion of curve. Repeat theprocedure 3 using distilled water in place of enzyme solution, and ∆Abs (blank) is obtained.
Calculation
Volume activity (U/mL) =(∆Abs (test) - ∆Abs (blank)) X (0.90 + 0.005)

Specific activity (U/mg protein) =11.11 X 0.005 X 1/20

X d.f.

Volume activity (U/mL)
Protein concentration (mg/mL)*2

d.f. ; dilution factor
11.11 ; millimolar extinction coefficient of quinoneimine dye at 500 nm (cm2/μmol)
1/20 ; coefficient of transformation for internal unit definition*2Protein concentration ; determined by Bradford’s method

NIPRO ENZYMESNIPRO ENZYMESNIPRO ENZYMESNIPRO ENZYMESDIAPHORASE 3 (DI-3)[EC 1. 6. 99. - ]from recombinant E. coli
NAD(P)H + Acceptor(ox.) + H+ ↔ NAD(P) + + Acceptor(red.)
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Isoelectric point
Thermal stability
Michaelis constants: Lyophilized: more than 1,000 U/mg protein: (as Diaphorase activity = 100 %)Adenylate kinase
NADH oxidase: ca. 20,000(SDS-electrophoresis): 8.0: 7.5 - 9.5: 4.7: No detectable decrease in activity up to 60 °C.
: See Table 1< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)

STORAGE
Stable at -20 to 5 °C for one year
APPLICATIONThe enzyme is useful for the measurement of various dehydrogenase reactions in visible spectral
range.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 600 nm according to the following reaction.
NAD(P)H + DCIP(ox.) + H+

DI-3

NAD(P)+ + DCIP(red.)

Unit Definition
One unit of activity is defined as the amount of DI-3 that reduces 1 μmol of DCIP per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 500 mM Tris-HCI, pH 8.5
Ⅱ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Ⅲ 2,6-Dichlorophenolindophenol (DCIP) solution ; 1.2 mM (2.0 mg DCIP sodium salt∙2H2O/5mL
distilled water) (prepare freshly)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 1.0 to 2.0 U/mL with 50 mM
potassium phosphate buffer, pH 7.5.
Procedure
1. Prepare the following reaction mixture and pipette 2.28 mL of reaction mixture and 0.12 mL of
Solution Ⅲ into a cuvette.
SolutionⅠ 3.00 mL
SolutionⅡ 2.28 mL
23.22 mL
H2O
2. Incubate at 30 °C for about 3 minutes.
3. Add 0.008 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 600 nm per minute (∆Abs(test)) in linear portion of curve. Repeat theProcedure 3 using distilled water in place of enzyme solution, and ∆Abs(blank) is obtained.
Calculation
Volume activity (U/mL) =(∆Abs (test) - ∆Abs (blank)) X (2.40 + 0.008)

Specific activity (U/mg protein) =19 X 0.008

X d.f.

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

d.f. ; dilution factor
19 ; millimolar extinction coefficient of DCIP (cm2/μmol)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Mains, I., Power, D.M., Thomas, E.W. and Buswell J. A.; Biochem. J., 191, 457 (1980)

NIPRO ENZYMESRemaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

100

50

0

10 11 124

5

Fig. 1 pH profile● Tris-HCl,

9

10 11 12treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);○ phosphate,△ acetate,● Tris-HCl

100

Remaining activity (%)

Remaining activity (%)

8

Fig. 2 pH stability

100

50

0

7

pH

pH

○ phosphate,■ Gly-KCl-KOH

6

50

0
20 30 40 50 60 70 80 90 100
Temperature (℃)
Fig. 3 Thermal stability
treated for 15 min in 0.1 M
potassium phosphate buffer,
pH 7.50

10 20 30 40 50 60 70
Time (min)Fig. 4 Thermal stability
treated in 0.1 M potassium
phosphate buffer, pH 7.5○ 50 ℃, □ 60 ℃, ● 70 ℃

NIPRO ENZYMESTable 1. SUBSTRATE SPECIFICITY OF DIAPHORASE
Acceptor

DCIP*1

NTB*2

MTT*3

Km Acceptor

(mM)

0.02

0.15

0.9

Km

NADH

(mM)

0.37

0.01

0.05

Km NADPH

(mM)

32.7

0.31

2.0

8.0

10

8.0

Optimum pH
Assay Mixture

Tris-HCI(pH 8.5)
NAD(P)HDCIP

Wavelength for
Measurement(nm)

Extinction Coefficient(cm2/μmol)

50 mM
1 mM
0.06 mM

TEA(pH 7.0)
50 mM
NAD(P)H
1 mM
NBT
0.5 mM
Triton X-100 0.1 %TEA(pH 7.0)
NAD(P)H
MTT
Triton X-100

600

550

565

19

12.4

20

50 mM
1 mM
0.5 mM
0.5 %*1 2,6-Dichlorophenolindophenol*2 Nitrotetrazolium Blue
*3 Thiazolyl Blue Tetrazolium Bromide

pH profiles of DI-3 (Acceptor; NTB or MTT)

100

Relative activity (%)

Relative activity (%)

100

50

50

0

6

7

8

9

10

11

12

pH
Fig. 5 pH profile (NTB)□ triethanolamine, ○ phosphate,● Tris-HCl,■ Gly-KCl-KOH

0

6

7

8

9

10

11

pH
Fig. 6 pH profile (MTT)□ triethanolamine, ○ phosphate,● Tris-HCl,■ Gly-KCl-KOH

12

NIPRO ENZYMESDIAPHORASE 22 (Di-22)[EC 1. 8. 1. 4]from recombinant E.coli
NADH + Acceptor(ox.) + H+ ↔ NAD+ + Acceptor(red.)
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants

Substrate specificity: Lyophilized: more than 150 U/mg protein: (as Diaphorase activity = 100 %)Adenylate kinase
NADH oxidase< 0.01 %< 0.20 %: ca. 110,000: ca. 50,000: 8.0(Fig,1): 6.0 – 9.0(Fig.2): No detectable decrease inactivity up to 70 °C.
(Fig. 3, 4): (50 mM HEPES buffer, pH 7.0, at 30 °C)
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT)
0.345 mM
NADH
0.033 mM
(Table 1): NADH
100 %
NADPH
1%
MTT
100 %
Lipoate103 %(Table 1)

STORAGEStore at -20℃
APPLICATIONThe enzyme is useful for measurement of various dehydrogenase reactions in the visible spectral range.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 565 nm according to the following reaction.
NADH + MTT(ox.) + H+

Di-22

NAD+ + MTT(red.)

Unit Definition
One unit of activity is defined as the amount of Diaphorase that forms 1 μmol of NAD+ per minute at
30 °C
Solutions
Ⅰ Buffer solution ; 100 mM HEPES, pH 7.0
Ⅱ 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution; 10 mM (20 mg MTT disodium salt･2H2O/5 mL distilled water)
Ⅲ NADH solution ; 13.1 mM (0.100 g NADH disodium salt・3H2O /10 mL distilled water)
Ⅳ Triton solution ; 10 % (1 mL TritonX-100 dilute with distilled water up to10 mL)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 1 to 5 U/mL with 50 mM potassium
phosphate buffer, pH 7.5, 1 mg/mL BSA.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 15.00 mL
SolutionⅣ
1.50 mL
10.80 mL
SolutionⅡ 1.50 mL
H2O
SolutionⅢ 1.20 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 565 nm per minute (∆Abs565 ) in the linear portion of curve.
Calculation(∆Abs565) X (3.00 + 0.01)
Volume activity (U/mL) =20.0 X 0.01

Specific activity (U/mg protein) =X d.f.

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

d.f. ; dilution factor
20.0 ; millimolar extinction coefficient of MTT (cm2/μmol)*Protein concentration ; determined by Bradford's method
REFERENCE
1. Packman, L.C., and Perham. R.N.; FEBS Lett., 139, 155 (1982)

NIPRO ENZYMESTable 1. SUBSTRATE SPECIFICITY OF DIAPHORASE 22
Acceptor

MTT

LipoateKm Acceptor

(mM)

0.345

2.0

Km NADH

(mM)

0.033

0.01

100

103

Relative Activity
Assay Mixture

Wavelength for
Measuremen
Extinction
coefficient

HEPESI (pH 7.0)
NADH
MTT
Triton X-100(nm)(cm2/μmol)

50 mM
0.5 mM
0.5 mM
0.5 %Potassium Phosphate(pH 6.5)
70.5 mM
NADH
0.2 mM
NAD
0.3 mM
Lipoate10.2 mM
EDTA
0.81 mM
BSA
0.7 mg/mL

565

340

20

6.22

NIPRO ENZYMES
200

Remaining activity (%)

Relative activity (%)

100.0

150

100

50

0

4

5

6

7

8

9

10

11

0.0

12

5

6

7

8

9

pH

Fig. 1 pH profile

Fig. 2 pH stability○ Bicine,▲ HEPES10

treated for 24 hr at 4 ℃ in the following
buffer solution (0.1 M), :△ Gly-KOH,○ Bicine,● phosphate, ▲ HEPES,□ MES,■ Citrate

100

100

Remaining activity (%)

Remaining activity (%)

4pH△ Gly-KOH,● phosphate,

50

0

50.0

20

30

40

50

60

70

80

Temperature (℃)

90

50

0

0

15

30

45

60

Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1M
potassium phosphate buffer,
pH 7.5treated for in 0.1M potassium
phosphate buffer, pH 7.5○ 60 ℃, □ 70 ℃, ● 80 ℃

11

NIPRO ENZYMESGALACTOSE DEHYDROGENASE (GalDH)[EC 1. 1. 1. 48]
from recombinant E. coli
D-Galactose + NAD(P)+ ↔ D-Galactono-δ-lactone + NAD(P)H + H+
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants
Substrate specificity (100mM): Ammonium sulphate suspension: more than 80 U/mg protein: (as GalDH activity = 100 %)NADH oxidase
LDH
ADH< 0.10 %< 0.10 %< 0.01 %: ca. 33,800: 10.5(Fig. 1)
: 5.0 - 10.0
(Fig. 2)
: No significant decrease in activity up to 50 °C with Ammonium
sulphate and 40 °C without Ammonium sulphate . (Fig. 3, 4): D-Galactose
0.25 mM
0.15 mM
NAD+: D-Galactose
100 %D-Glucose
0.2 %D-Xylose
8.7 %D-Maltose
0.1 %D-Sucrose
0.1 %STORAGEStore at 2 to 10 °C (Do not freeze)
Stable at 4 °C for at least one year
APPLICATIONThis enzyme is useful for determination of galactose.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
D-Galactose + NAD+

GalDH

D-Galactono-δ-lactone + NADH + H+

Unit Definition
One unit of activity is defined as the amount of GalDH that forms 1 μmol of NADH per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Tris-HCl, pH9.1 (at 30 °C)
Ⅱ NAD+ solution ; 100 mM
Ⅲ D-Galactose solution ; 1 M
Ⅳ Enzyme diluent ; 20 mM potassium phosphate, 0.1 % bovine serum albumin, pH 7.5
Preparation of Enzyme Solution
Dilute the enzyme suspension to approx. 5 U/mL with the enzyme diluent.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 27.60 mL
SolutionⅡ 0.90 mL
SolutionⅢ 1.50 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 （∆Abs340） 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 (cm2/μmol)*Protein concentration ; determined by the Bradford's method

100

100

Relative activity (%)

Remaining activity (%)

NIPRO ENZYMES

50

4

5

6

7

8
pH

9

10

11

4

5

6

7

8
pH

9

10

11

Fig. 1 pH profile

Fig. 2 pH stability△ acetate, ○ phosphate,● Tris-HCl, ▲ Glycine-KOH

treated for 24 hr at 4 ℃ in thefolowing buffer solution (0.1M); △ acetate, ○ phosphate,● Tris-HCl, ▲ Glycine-KOH○ with AmS● without AmS

Remaining activity (%)

100

50

0

0

100

Remaining activity (%)

0

50

50

30

40

50
60
70
Temperature (℃)

80Fig. 3 Thermal stability
treated for 15 min in 25 mM
potassium phosphate buffer
pH 7.5, with or without 3.2 M
ammonium sulphate (AmS).

0

0

15

30
45
Time (min)Fig. 4 Thermal stability
treated in 25 mM potassium
phosphate buffer pH 7.5 at ○40 ℃, □ 50 ℃, ● 60 ℃
without ammonium sulphate.

60

NIPRO ENZYMESRemaining activity (%)

120
100
80
60
40
20
0

0

200

400

600

Period (days)
Fig. 5 Storage Stability
ammonium suiphate suspension(ca. 1300 U/mL) store at 4 ℃(○)

NIPRO ENZYMES
1GLUCOKINASE 2 (GlcK2)[EC 2. 7. 1. 2]from Recombinant E.coli
ATP + D-Glucose ↔ ADP + D-Glucose-6-phosphate
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Optimum temperature
Thermal stability
Michaelis constants
Substrate specificity: Lyophilized: more than 350 U/mg protein: (as GlcK2 activity = 100 %)Glucose-6-phosphate dehydrogenase
Phosphoglucomutase
6-Phosphogluconate dehydrogenase
Hexose-6-phosphate isomerase
Glutathione reductase: ca. 32,000: 9.0: 7.0 - 10.0
: 70 °C: No detectable decrease in activity up to 60 °C.
: (60 mM Tris-HCI buffer, pH 8.5, at 30 °C)
GlucoseATP: D-Glucose
D-Mannose
D-Fructose< 0.01 %< 0.01 %< 0.01 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 5)
(Fig. 3, 4)
0.1 mM
0.05 mM
100 %
20 %
0%

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 forthe specific determination of glucose.

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

GlcK2

ADP + Glucose-6-phosphate

Glucose-6-phosphate + NADP+

G6PD

Gluconolactone-6-phosphate + NADPH + H+

Unit Definition
One unit of activity is defined as the amount of GlcK2 that forms 1 μmol of glucose-6-phosphate perminute at 30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Tris-HCl, pH 9.0
Ⅱ ATP solution ; 100 mM (0.605 g ATP disodium salt∙3H2O/(8.2 mL distilled water + 1.8 mL 1 NNaOH))
Ⅲ MgCl2 solution ; 1 M (20.33 g MgCl2∙6H2O/100 mL distilled water)
Ⅳ NADP+ solution ; 22.5 mM [(0.172 g NADP+ monosodium salt or 0.177 g NADP+ disodium salt)/10
mL distilled water]
Ⅴ Glucose solution ; 40 mM (0.072 g glucose (anhyd.)/10 mL distilled water)
Ⅵ Glucose-6-phosphate dehydrogenase (G6PDH) ; (from yeast. Roche Diagnostics K.K., No. 127
671) suspension in 3.2 M (NH4)2SO4 solution (10 mg/2 mL) approx. 140 U/mg at 25 °C
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50 mM Tris-HCI
buffer, pH 8.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 17.97 mL
SolutionⅣ 1.20 mL
SolutionⅡ 1.20 mL
SolutionⅤ 9.00 mL
SolutionⅢ 0.60 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 NADPH (cm2/μmol)*Protein concentration; determined by Bradford's method
REFERENCE
1. Hengartner, H., and Zuber, H.; FEBS Lett., 37, 212 (1973)
2. Kamei, S., Tomita, K., Nagata, K., Okuno, H., Shiraishi, T., Motoyama, A., Ohkubo, A., andYamanaka, H.; J. Clin. Biochem. Nutr., 3,1 (1987)

NIPRO ENZYMES
3. Tomita, K., Kamei, S., Nagata, K., Okuno, H., Shiraishi, T., Motoyama, A., Ohkubo, A., andYamanaka, M.; ibid., 3, 11 (1987)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

10

11

0

12

5

6

7

8

9

10

11

12

pH

Fig. 1 pH profile

Fig. 2 pH stability△ acetate, ○ phosphate,● Tris-HCl, ▲ carbonate

treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M), :△ acetate,○ phosphate,● Tris-HCl, ▲ carbonate

100

Remaining activity (%)

Remaining activity (%)

4

pH

100

50

0

50

20

30

40

50

60

70

80

50

0

0

15

30

45

Temperature (℃)

Time (min.)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min. in 0.1 M
Tris-HCl buffer, pH 9.0

treated in 0.1 M Tris-HCl
buffer , pH 9.0○ 60 ℃, □ 70 ℃, ● 80 ℃

60

NIPRO ENZYMES

Relative activity (%)

100

50

0

20

40

60

80

Temperature (℃)
Fig. 5 Thermal activity
defined as 100 % at 70 ℃

100

NIPRO ENZYMESGLUCOSE DEHYDROGENASE (GlcDH2)[EC 1. 1. 1. 47]
from recombinant E. coli
D-Glucose + NAD(P)+ ↔ D-Glucono-δ-lactone + NAD(P)H + H+
SPECIFICATION
State
Specific activity
Contaminants
PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants
Substrate specificity (100mM): Lyophilized: more than 900 U/mg protein: (as GlcDH2 activity = 100 %)NADH oxidase: ca. 126,000: ca. 31,500: 8.5: 5.0 - 10.0 (with 3M NaCl): No significant decrease in activity up to 70 °C.
(with 3M NaCl and 0.1% BSA)
: D-Glucose
NAD+
NADP+
: D-Glucose
D-Maltose
D-Galactose
D-Xylose
D-Fructose
D-Mannose
D-Arabinose
Trehalose
D-Lactose
D-Sucrose
2-Deoxy-D-Glucose
D-Glucose-1-Phosphate
D-Glucose-6-Phosphate
D-Sorbitol

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

< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
3.7 mM
0.06 mM
0.02 mM
100 %
1.1 %
0.1 %
3.0 %
0.3 %
4.8 %
0%
0%
1.3 %
0%
100 %
0%
0%
0%

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
D-Glucose + NAD+GlcDH2

D-Glucono-δ-lactone + NADH + H+

Unit Definition
One unit of activity is defined as the amount of GlcDH2 that forms 1 μmol of NADH per minute at
37 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Tris-HCl, pH 8.5 (at 25 °C)
Ⅱ NAD+ solution ; 100 mM (0.663 g NAD+ free acid/10 mL distilled water)
Ⅲ D-Glucose solution ; 1 M ( 1.802 g glucose (anhyd.)/10 mL distilled water)
Ⅳ NaCl solution ; 5 M ( 2.92 g NaCl/10 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 15 U/mL with 20 mM potassium
phosphate buffer containing 1 mg/mL BSA and 2 M NaCl, pH 6.5.
Procedure
1. Prepare the following reaction mixture and pipette 2.70 mL of reaction mixture into a cuvette.
SolutionⅠ 17.22 mL
SolutionⅡ 0.50 mL
SolutionⅢ 2.00 mL
SolutionⅣ 0.28 mL
2. Incubate at 37 °C for about 3 minutes.
3. Add 0.015 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute （∆Abs340） in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (2.70 + 0.015)
6.22 X 0.015

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 (cm2/μmol)*Protein concentration ; determined by the absorbance at 280nm (Abs280),
where 1 Abs280 = 1 mg/mL
REFERENCE
1. Ramaley, R.F. and Vasantha, N.; J. Biol. Chem. 258, 12558-12565 (1983)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0
6

8

7

9

0

10

pH

3

7
8
pH

Fig. 1 pH profile

Fig. 2 pH stability○ phosphate, ● Tris-HCl, ▲ glycine

treated for 24 hr at 4 ℃ in the following
buffer solution (0.1 M) containg 3 M
NaCl : △ acetate, ○ phosphate,● Tris-HCl, ▲ glycine

100

4

5

6

9

10

11

100

Remaining activity (%)

Remaining activity (%)

50

50

0
20

30

40

50

60

70

80

50

0

Temperature (℃)

3045Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1M phosphatebuffer, pH 6.5, containing 3 M NaCland 0.1 % BSA

0

15

60

treated for in 0.1 M phosphate buffer,
pH 6.5, containing 3M NaCl and 0.1 %BSA, ○ 60℃, □ 70℃, ● 80℃

NIPRO ENZYMESGLYCEROKINASE (GlyK)[EC 2.7.1.30]
from recombinant E. coli
Glycerol + ATP ↔ Glycerol-3-phosphate + ADP
SPECIFICATION
State
Specific activity
PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Optimum temperature
Michaelis constants: Lyophilized: more than 80 U/mg protein: ca. 54,700: 9.8(Fig. 1)
: 5.5 - 9.0(Fig. 2)
: No detectable decrease in activity up to 40 °C.
(Fig. 3): above 50 °C(Fig. 4): (186 mM Glycine-Hydrazine-KOH buffer pH 9.8, at 30 °C)
Glycerol
0.026 mM
ATP
0.025 mM

STORAGE
Stable at -20 °C for at least six months
APPLICATIONThe enzyme is useful for enzymatic determination of glycerol and triglyceride when coupled withglycerol-3-phosphate dehydrogenase

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

GlyK

Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + NAD+

G3PDH

Dihydroxyacetone phosphate + NADH + H+

Unit Definition
One unit of activity is defined as the amount of GlyK that forms 1 μmol of NADH per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 200 mM Glycine-Hydrazine-KOH, pH 9.8 (Dissolve 1.5 g glycine and 5 mL
hydrazine hydrate in 80 mlmL distilled water. After adjusting pH to 9.8 with 1 M KOH, fill up to 100
mL with distilled water.)
Ⅱ MgCl2 solution ; 100 mM (2.03 g MgCl2∙6H2O/100 mL distilled water)
Ⅲ ATP solution ; 100 mM (0.605 g ATP disodium salt∙3H2O/(8.2 mL distilled water + 1.8 mL 1 M
NaOH))Ⅳ NAD+ solution; 100 mM (0.663 g NAD+ free acid/10 mL distilled water)
Ⅴ Glycerol-3-phosphate dehydrogenase ; 1700 U/mL (from rabbit muscle, Roche Diagnostics)
Ⅵ Glycerol solution ; 330 mM (3.04 g Glycerol/100 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute the enzyme solution to 0.1 to 1.0
U/mL with 50 mM Tris-HCl buffer pH 9.0 containing 0.1 % bovine serum albumin.
Procedure
1. Prepare the following reaction mixture and pipette 2.70 mL of reaction mixture into a cuvette.
SolutionⅠ
27.90 mL
SolutionⅣ 0.15 mL
SolutionⅡ0.57 mL
SolutionⅤ 0.30 mL
SolutionⅢ
0.39 mL
SolutionⅥ 0.30 mL
Distilled water
0.39 mL
2. Incubate at 30 °C for about 3 minutes.
3. Add 0.015 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute (∆Abs340) in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (2.70 + 0.015)
6.22 X 0.015

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 (cm2/μmol)*Protein concentration ; determined by the absorbance at 280 nm (Abs280),
where 1 Abs280 = 1 mg/mL
REFERENCE
1. Mike J. Comer, Chris J. Bruton, and Tony Atkinson ; J. App. Biochem. 1, 259-270 (1979)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

10

11

50

0

12

4

5

6

7

8

9

10

pH

pH

Fig. 1 pH profile

Fig. 2 pH stability

11

12

treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);△ MES, ○ phosphate,● Tris-HCl, ■ GlycineHydrazine, ◇ Bicine△ MES, ○ phosphate,● Tris-HCl, ◇ Bicine,▲ Glycine-Hydrazine

120
100

Relative activity (%)

Remaining activity (%)

100

50

80
60
40
20

0

30

40

50

60

70

80

Temperature (℃)

Fig. 3 Thermal stability
treated for 15 min in 100 mM
Bicine buffer, pH 8.5

90

0

10

20

30

40

50

Temperature (℃)

Fig. 4 Thermal activity

60

NIPRO ENZYMESGLYCEROL-3-PHOSPHATE
DEHYDROGENASE (G3PDH)[EC 1.1.1.8]
from recombinant E. coli
Glycerol-3-phosphate + NAD+ ↔ Dihydroxyacetone phosphate + NADH + H+
SPECIFICATION
State
Specific activity
PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Optimum temperature
Michaelis constants: Lyophilized: more than 7 U/mg protein: ca. 36,800: 9.0: 6.5 - 10.0
: No detectable decrease in activity up to 80 °C.
: above 60 °C: (90 mM Bicine buffer pH 9.0, at 37 °C)
Glycerol-3-phosphate
NAD+(Fig. 1)
(Fig. 2)
(Fig. 3)(Fig. 4)0.074 mM
0.022 mM

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for enzymatic determination of glycerol and triglyceride when coupled withglycerokinase

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
Glycerol-3-phosphate + NAD+

G3PDH

Dihydroxyacetone phosphate + NADH + H+

Unit Definition
One unit of activity is defined as the amount of G3PDH that forms 1 μmol of NADH per minute at
37 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Bicine-NaOH, pH 9.0
Ⅱ Glycerol-3-phosphate solution ; 50 mM (0.172 g Glycerol-3-phosphate bis(cyclohexylammonium)
salt/20 mL distilled water)
Ⅲ NAD+ solution ; 100 mM (0.663 g NAD+ free acid/10 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute the enzyme solution to 0.3 to 1.2
U/mL with 50 mM BICINE-NaOH buffer pH 9.0 containing 0.1 % bovine serum albumin.
Procedure
1. Prepare the following reaction mixture and pipette 2.70 mL of reaction mixture into a cuvette.
SolutionⅠ
27.00 mL
SolutionⅡ1.02 mL
SolutionⅢ
0.51 mLDistilled water
1.47 mL
2. Incubate at 37 °C for about 3 minutes.
3. Add 0.015 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute (∆Abs340) in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (2.70 + 0.015)
6.22 X 0.015

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 (cm2/μmol)*Protein concentration ; determined by the absorbance at 280 nm (Abs280),
where 1 Abs280 = 1 mg/mL

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

10

11

50

0

12

4

5

6

7

8

9

10

pH

pH

Fig. 1 pH profile

Fig. 2 pH stability

11

12

60

70

treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);△ MES, ○ phosphate,● Tris-HCl, ■ GlycineHydrazine, ◇ Bicine△ MES, ○ phosphate,● Tris-HCl, ◇ Bicine,▲ CHES

120
100

Relative activity (%)

Remaining activity (%)

100

50

80
60
40
20

0

30

40

50

60

70

80

90

Temperature (℃)

Fig. 3 Thermal stability
treated for 15 min in 100 mM
Bicine buffer, pH 9.0

100

0

10

20

30

40

50

Temperature (℃)

Fig. 4 Thermal activity

NIPRO ENZYMESD-LACTATE DEHYDROGENASE (D-LDH)[EC 1. 1. 1. 28]
from Microorganism
D-Lactate + NAD+ ↔ Pyruvate + NADH + H+
FOR PYRUVATE → LACTATE REACTION
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Isoelectric point
Thermal stability
Michaelis constants
Stabilizers
Inhibitors
STORAGE
Stable at -20 °C at least one year

: Lyophilized: more than 2,500 U/mg protein: (as D-LDH activity = 100 %)NADH oxidase
GOT
GPT< 0.01 %< 0.01 %< 0.01 %: ca. 80,000: ca. 40,000: 7.5(Fig. 1)
: 5.5 - 10.0
(Fig. 2)
: 4.1
: No detectable decrease in activity up to 40 °C. (Fig. 3, 4): (94 mM Potassium phosphate buffer, pH 7.5, at 30 °C)
Pyruvate
0.80 mM
NADH
0.18 mM: (NH4)2 SO4, BSA: Zn2+, Cu2+

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
D-LDH
D-Lactate + NAD+
Pyruvate + NADH + H+
Unit Definition
One unit is defined as the amount of D-LDH that forms 1 μmol of NAD+ per minute at 30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Potassium phosphate buffer, pH 7.5
Ⅱ Sodium pyruvate solution ; 100 mM (100 mg sodium pyruvate/10 mL distilled water)
Ⅲ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 3 to 5 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 28.00 mL
SolutionⅡ 1.20 mL
SolutionⅢ 0.80 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 (∆Abs340) 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 (cm2/μmol)*Protein concentration ; determined by Bradford’s method

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8
pH

9

10

11

50

0

12

4

5

6

Fig. 1 pH profile△ acetate,○ phosphate,

9

10

11

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

100

Remaining activity (%)

Remaining activity (%)

8pHFig. 2 pH stability

100

50

0

7

20

30

40
50
60
Temperature (℃)

70

50

0

0

15

30
45
Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1 M
potassium phosphate buffer,
pH 7.0treated in 0.1 M potassium
phosphate buffer, pH 7.0○ 40 ℃, □ 45 ℃, ● 50 ℃

60

12

NIPRO ENZYMESMALATE DEHYDROGENASE (MDH)[EC 1. 1. 1. 37]
from Microorganism
L-Malate+ NAD+ ↔ Oxaloacetate + NADH + H+
FOR OXALATE → MALATE REACTION
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants: Lyophilized: more than 1,200 U/mg protein: (as MDH activity = 100 %)GOT
GPTNADHoxidase
Glutamate dehydrogenase
Fumarase: ca. 72,000: ca. 36,000: 9.0: 5.5 - 11.0
: No detectable decrease in activity up to 50 °C.
: (90 mM Tris-HCI buffer, pH 9.0, at 30 °C)
Oxaloacetate
NADH< 0.01 %< 0.01 %< 0.01 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
0.027 mM
0.014 mM

STORAGE
Stable at -20 °C for at least six months
APPLICATIONThis enzyme is useful for enzymatic determination of L- malate and of glutamate oxaloacetatetransaminase in clinical analysis.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
Oxaloacetate + NADH + H+

MDH

L-Malate + NAD+

Unit Definition
One unit of activity is defined as the amount of MDH that forms 1 μmol of NAD+ per minute at 30 °C.
Solutions
Ⅰ Buffer solution ; 200 mM Tris-HCl, pH 9.0
Ⅱ Oxaloacetate solution ; 15 mM (0.020 g oxaloacetate/10 mL distilled water)
Ⅲ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 3 to 5 U/mL with 100 mM Tris-HCl
buffer, pH 9.0.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 13.50 mL
SolutionⅡ 1.00 mL
SolutionⅢ 0.57 mL
14.93 mL
H2O
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 (cm2/μmol)*Protein concentration ; determined by Bradford’s method

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

7

6

8
pH

9

10

11

50

0

12

4

5

6

Fig. 1 pH profile△ acetate,● Tris-HCl,○ phosphate,■ Gly-KOH

11

100

Remaining activity (%)

Remaining activity (%)

8
9
10
pH
Fig. 2 pH stability

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

100

50

0

7

20

30

40

50
60
70
Temperature (°C)

Fig. 3 Thermal stability
treated for 15 min in 0.1 M
Tris-HCl buffer, pH 9.0

80

50

0

0

15

30
45
Time (min)60

Fig. 4 Thermal stability
treated in 0.1 M Tris-HCl
buffer, pH 9.0○ 50 ℃, □ 55 ℃, ● 60 ℃

12

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

α-D-Glucose ↔ β-D-Glucose
SPECIFICATION
State
Specific 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 ENZYMES
ASSAY
Principle
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-δ-lactone + NADH + H+

Unit Definition
One unit of activity is defined as the amount of Mutarotase that forms 10 μmol of NADH per minuteat 25 °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 (cm2/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 (%)

100

50

0

3

4

5

6

7

8

9

10

50

0

11

3

6

7

8

9

Fig. 1 pH profile

Fig. 2 pH stability

10

11

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

100

100

Remaining activity (%)

Remaining activity (%)

5pH△ acetate, □ MES, ◆ PIPES,◇ HEPES, ● Tris-HCl,■Bicine, ▲Glycine-KOH

50

0

4

pH

20

30

40

50

60

70

Temperature (℃)

Fig. 3 Thermal stability
treated for 15 min in 0.1 M potassium
phosphate buffer, pH 6.5, 0.1 % BSA.

80

50

0

0

15

30

45

60

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

NIPRO ENZYMESPHENYLALANINE DEHYDROGENASE (PheDH)[EC 1.4.1.20]
from Thermoactinomyces intermedius
L-Phenylalanine + NAD+ + H2O ↔ Phenylpyruvate + NH4+ + NADH
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants
Substrate specificity: Ammonium sulphate suspension: more than 30 U/mg protein: (as PheDH activity = 100 %)NADH oxidase
Lactate dehydrogenase< 0.01 %< 0.01 %: ca. 380,000: ca. 40,000: 11.5(Fig. 1)
: 5.0 - 10.0
(Fig. 2)
: No detectable decrease in activity up to 50 °C. (Fig. 3, 4): (200 mM Gly-KCl-KOH buffer, pH 11.0, at 30 °C)
L-Phenylalanine
0.66 mM
0.05 mM
NAD+: L-Phenylalanine
100 %L-Tyrosine
7.6 %L-Methionine
1.5 %STORAGE
Stable at 4 °C for at least six months (Do not freeze)NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
L-Phenylalanine + NAD+ + H2O

PheDH

Phenylpyruvate + NH4+ + NADH

Unit Definition
One unit of activity is defined as the amount of PheDH that forms 1 μmol of NADH per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 400 mM Gly-KCI-KOH, pH 11.0
Ⅱ L-Phenylalanine solution ; 100 mM (0.165 g L-phenylalanine/10 mL distilled water)
Ⅲ NAD+ solution ; 100 mM (0.663 g NAD+ free acid/10 mL distilled water)
Preparation of Enzyme Solution
Dilute the ammonium sulphate suspension of enzyme to 2 to 6 U/mL with 10 mM Tris-HCl buffer, pH
8.0, containing 50 mM NaCl.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 15.00 mL
SolutionⅡ 3.00 mL
SolutionⅢ 0.15 mL
11.85 mL
H2O
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 340nm 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 (cm2/μmol)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Ohshima, T., Takada, H., Yoshimura, T., Esaki, N., and Soda, K.; J. Bacteriol., 173, 3943 (1991)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

5

6

7

8

9

10

11

12

50

0

13

pH

6

7

8

9

10

11

12

Fig. 1 pH profile

Fig. 2 pH stability■ Gly-KCl-KOH

treated for 24 hr at 4 ℃ in thefollowing buffer solution (50 mM);
△ acetate, ○ phosphate,■ Gly-KCl-KOH

100

Remaining activity (%)

Remaining activity (%)

5

pH

100

50

0

4

20

30

40

50

60

70

Temperature (℃)

80

50

0

0

15

30
45
Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 10
mM potassium phosphatebuffer, pH 7.2

treated in 10 mM potassium
phosphate buffer, pH 7.2○ 50 ℃, □ 60 ℃, ● 70 ℃

60

NIPRO ENZYMESPHOSPHOTRANSACETYLASE (PTA2)[EC 2. 3. 1. 8]
from recombinant E. coli
Acetyl-CoA + Pi ↔ AcetyIphosphate + CoA
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants: Lyophilized: more than 5,000 U/mg protein: (as PTA2 activity = 100 %)Acetate kinase
Adenylate kinase
Lactate dehydrogenase: ca. 69,700: ca. 33,500: 7.4: 4.0 - 11.0
: No detectable decrease in activity up to 60 °C.
: (87mM Tris-HCl buffer, pH 7.5, at 30 °C)
Coenzyme A
Acetyl Phosphate

STORAGE
Stable at -20 °C for at least one year

< 0.01 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
0.1 mM
0.5 mMNIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 233 nm according to the following reaction.
Acetylphosphate + CoA

PTA2Acetyl-CoA + Pi

Unit Definition
One unit of activity is defined as the amount of PTA that forms 1 μmol of acetyl-CoA per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Tris-HCl, pH 7.5
Ⅱ CoA solution ; 6.4 mM (50 mg CoA trilithium salt/10 mL distilled water)
Ⅲ Acetylphosphate solution ; 217 mM (0.400 g acetylphosphate potassium lithium salt/10 mL distilledwater)
Ⅳ Ammonium sulfate (AmS) solution ; 1 M (13.2 g AmS/100 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 20 U/mL with 50 mM Tris-HCI
buffer, pH 8.0.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 26.0 mL
SolutionⅢ 1.0 mL
SolutionⅡ 2.0 mL
SolutionⅣ 1.0 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 233 nm per minute (ΔAbs233) in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs233) X (3.00 + 0.01)
4.44 X 0.01

Specific activity (U/mg protein) =X d.f.

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

d.f. ; dilution factor
4.44 ; differential millimolar extinction coefficient between acety-CoA and CoA (cm2/μmol)*Protein concentration ; determined by Bradford's method

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

10

11

50

0

12

4

7

8

9

10

Fig. 1 pH profile

Fig. 2 pH stability

11

12

treated for 24 hr at 4 ℃ in thefollowing buffer solution (50 mM);
△ acetate, ○ phosphate,● Tris-HCl, ■ Glycine-KOH

100

100

Remaining activity (%)

Remaining activity (%)

6pH△ acetate, ○ phosphate,● Tris-HCl, ■ Gly-KOH

50

0

5

pH

30

40

50

60

70

Temperature (℃)

80

50

0

0

15

30

45

Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 50 mM
Tris-HCl buffer, pH 8.5, 0.1 %BSA

treated in 50 mM Tris-HCl
buffer, pH 8.5, 0.1 % BSA○ 55 ℃, □ 60 ℃, ● 65 ℃

60

NIPRO ENZYMESPOLYNUCLEOTIDE PHOSPHORYLASE (PNPase3)[EC 2. 7. 7. 8]
from recombinant E. coli
RNAn+1 + Pi ↔ RNAn + Nucleoside diphosphate

FOR DEPOLYMERIZATION REACTION
SPECIFICATION
State
Specific activity: Liquid: more than 2,000 U/mg protein

PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Effectors: ca. 79,000: 9.0 - 9.5(Fig. 1)
: 8.0 - 11.0
(Fig. 2)
: No detectable decrease in activity up to 55 °C. (Fig. 3, 4): cations and anions(Fig. 5, 6)

STORAGE
at -20 °C
APPLICATIONThe enzyme is useful for the preparation of polyribonucleotide.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
Poly An + Pi
ADP + PEPPNPase3PK

Pyruvate + NADH + H+

Poly An-1 + ADP(I)

ATP + Pyruvate
LDH

Lactate + NAD+(II)

Unit Definition
One unit of activity is defined as the amount of PNPase that forms 1 μmol of ADP per hour aｔ 60 °C
by depolymerizing of Poly A.
Solutions(Reaction I)
Ⅰ Buffer solution ; 100 mM Tris-HCl, pH 9.5 ((1.212 g Tris + 0.074 g EDTA + 0.014 mL 2mercaptoethanol + 0.610 g MgCl2∙6H2O + 0.746 g KCl)/80 mL distilled water, adjusted to pH 9.5
with 1 N-HCI and filled up to 100 mL with distilled water)
Ⅱ KH2PO4 solution ; 65 mM (0.088 g KH2PO4/10 mL distilled water)
Ⅲ polyadenylate (Poly A) solution ; (25 mg PoIy A potassium salt/1 mL distilled water; ca. 35 mM
based on AMP concentration)(Reaction II)
Ⅳ Buffer solution ; 100 mM Triethanolamine buffer, pH 7.6 ((9.300 g triethanolamine-HCI + 0.407 g
MgCl2∙6H2O + 0.373 g KCl)/400 mL distilled water, adjusted to pH 7.6 with 1 N-NaOH and filled up
to 500 mL with distilled water)
Ⅴ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Ⅵ Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10 mL distilled water)
Ⅶ Pyruvate kinase (PK) ; (from rabbit muscle, Roche Diagnostics K.K., No. 128 155) crystalline
suspension in 3.2 M (NH4)2SO4 solution (10 mg/mL) approx. 200 U/mg at 25 °C
Ⅷ Lactate dehydrogenase (LDH) ; (from pig heart, Oriental Yeast Co. Ltd., Product Code: LDH-02)
ammonium sulfate suspension, approx. 5,000 U/mL at 25 °C
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 2 to 150 U/mL with 50 mM Tris-HCl
buffer, pH 8.5.
Procedure(Reaction I)
1. Prepare the following reaction mixture and pipette 0.55 mL of reaction mixture into a test tube.
SolutionⅠ 2.50 mL
SolutionⅢ 1.00 mL
1.00 mL
SolutionⅡ 1.00 mL
H2O
2. Add 0.10 mL of enzyme solution and mix.
3. Incubate at 60 °C for exactly 10 minutes.
4. After incubation, add 0.01 mL conc. HCI and mix.
5. Centrifuge at 10,000 rpm for 30 seconds.
At the same time, repeat the Procedure 1 to 5 using distilled water in place of enzyme solution in
Procedure 2 (as blank).
(Reaction II)
6. Prepare the following reaction mixture and pipette 2.50 mL of the reaction mixture into a cuvette.
SolutionⅣ 24.18 mL
SolutionⅦ 0.12 mLNIPRO ENZYMESSolutionⅤ 0.40 mL
SolutionⅧ
SolutionⅥ 0.25 mL
7. Incubate at 30 °C for about 3 minutes.
8. Add 0.10 mL of supernatant of Procedure 5 and mix.
9. Read absorbance at 340 nm (Abs•test).
Repeat the Procedure using blank (Abs•blank).
Calculation
Volume activity (U/mL) = ((Abs•blank) - (Abs•test)) X

Specific activity (U/mg protein) =0.05 mL

2.60 х 0.65
6.22 X 0.10 х 0.10

X

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

d.f. ; dilution factor
6.22 ; millimolar extinction coefficient of NADH (cm2/μmol)*Protein concentration ; determined by the absorbance at 280 nm (Abs280),
where 1 Abs280 = 1 mg/mL
REFERENCES
1. Smith, J.C., and Eaton, M.A.W.; Nucleic Acids Research, 1, 1763 (1974)
2. Wood, J.N., and Hutchinson, D.W.; ibid., 3, 219 (1976)

60
10

X d.f.

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

50

0

4

5

6

7

8

9

10

11

50

0

12

6

7

8

9

10

Fig. 1 pH profile

Fig. 2 pH stability

11

12

treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);● Tris-HCl, ■ Glycine-KOH

100

100

Remaining activity (%)

Remaining activity (%)

5pH● Tris-HCl

50

0

4

pH

30

40

50

60

70

Temperature (℃)

Fig. 3 Thermal stability
treated for 15 min in 0.1 M
Tris-HCl buffer, pH 8.5

80

50

0

0

15

30

45

Time (min)Fig. 4 Thermal stability
treated in 50 mM Tris-HCl
buffer, pH 8.5○ 55 ℃, □ 60 ℃, ● 65 ℃

60

NIPRO ENZYMES
150

Relative activity (%)

Relative activity (%)

150

100

50

0

0

50

100

150

200

100

50

0

0

50

100

150

200

Concentration (mM)Concentration (mM)

Fig. 5 Effect of various cations on theactivity of PNPase3 in the following Assay

Fig. 6 Effect of various anions on theactivity of PNPase3 in the following Assay

Measurement: 0.015 mL of each cation
solution, 0.010 mL of enzyme solution and0.055 mL of reaction mixture were mixed,
and reacted at 60 ℃. After 10 minutes, thequantity of ADP was determined.
○ NaCl, △ KCl, □ MgCl2

Measurement: 0.015 mL of each anion
solution, 0.010 mL of enzyme solution and0.055 mL of reaction mixture were mixed,
and reacted at 60 ℃. After 10 minutes, thequantity of ADP was determined.
○ NaCl, △ CH3COONa, □ Na2SO4, ●NaH2PO4NIPRO ENZYMESPYRUVATE KINASE (PK2)[EC 2.7.1.40]
from Recombinant E. coli
ATP + Pyruvate ↔ ADP + Phosphoenolpyruvate
SPECIFICATION
State
Specific activity
Contaminants

PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants: Lyophilized: more than 230 U/mg protein: (as PK activity = 100 %)Adenylate kinase
Lactate dehydrogenase: ca. 60,000: 6.0 - 6.5: 6.0 - 11.0
: No detectable decrease in activity up to 60 °C.
: (76 mM Imidazole-HCl buffer, pH 7.2, at 30 °C)
Phosphoenolpyruvate
ADP

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for diagnostic reagent, for example, ADP determination.

< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
1.0 mM
1.5 mMNIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reaction.
ADP + PEP

PK2

ATP + Pyruvate

Pyruvate + NADH + H+

LDH

Lactate + NAD+

Unit Definition
One unit of activity is defined as the amount of PK2 that forms 1 μmol of pyruvate per minute at
30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Imidazole-HCl, pH 7.2
Ⅱ ADP solution ; 100 mM (0.45 g ADP sodium salt, Sigma-Aldrich A2754/(9.0 mL distilled water + 1.0
mL 1 N NaOH))
Ⅲ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Ⅳ Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10 mL distilled water)
Ⅴ MgCl2 solution ; 1 M (20.33 g MgCl2∙6H2O/100 mL distilled water)
Ⅵ KCI solution ; 2.5 M (18.64 g KCI/100 mL distilled water)
Ⅶ Lactate dehydrogenase (LDH) ; (from pig heart, Oriental Yeast Co. Ltd., LDH (P.H.)) ammonium
sulfate suspension, approx. 5,000 U/mL at 25 °C
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50 mM Tris-HCl
buffer, pH 8.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 22.71 mL
SolutionⅤ
0.48 mL
SolutionⅡ 2.40 mL
SolutionⅥ
0.90 mL
SolutionⅢ 0.45 mL
SolutionⅦ
0.06 mL
SolutionⅣ 3.00 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 (cm2/μmol)*Protein concentration ; determined by Bradford’s method
REFERENCE
1. Sakai, H., Suzuki, K., and Imahori, K.; J. Biochem., 99, 1157 (1986)

NIPRO ENZYMES

100
Remaining activity (%)

Relative activity (%)

100

50

0

3

4

5

6

7
pH

8

9

10

50

0

11

3

4

5

Fig. 1 pH profile

8
pH

9

10 11 12treated for 24 hr at 4 ℃ in thefollowing buffer solution (50 mM);
△ Acetate, □ Imidazole-HCl,● Tris-HCl, ■ Glycine-KOH

100
Remaining activity (%)

100
Remaining activity (%)

7

Fig. 2 pH stability△ Acetate, □ Imidazole-HCl,● Tris-HCl, ■ Glycine-KOH

50

0

6

20

30

40
50
60
70
Temperature (℃)

80

50

0

0

15

30
45
Time (min)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 50 mM
Tris-HCl buffer, pH 8.5

treated in 50 mM Tris-HCl
buffer, pH 8.5○ 55 ℃, □ 60 ℃, ● 65 ℃

60