Name: GLUCOSE OXIDASE from Aspergillus sp.
SKU: A8GD-1005
Availability: InStock

PREPARATION and SPECIFICATION

Appearance	Yellowish amorphous powder, lyophilized
Activity	GradeⅠ	180 U/mg-solid or more
	GradeⅡ	100 U/mg-solid or more
		(containing approx. 50 % of stabilizers)
Contaminant	Catalase	GradeⅠ	≤ 5.0×10^-3 %
Contaminant		GradeⅡ	≤ 3.0 %
Stabilizers	Potassium gluconate, sodium glutamate

PROPERTIES

Stability	Stable at −20 ℃ for at least one year(Fig.1)
Molecular weight	approx. 153,000
Michaelis constants	3.3×10^-2 M (β-D-Glucose),³⁾
Michaelis constants	6.1×10^-2 M (2-Deoxyglucose)
Structure	Glycoprotein with 2 moles of FAD
Inhibitors	p-Chloromercuribenzoate, heavy metal ions (Cu^2＋, Hg^2＋, Ag^＋)
Optimum pH	4.5(Fig.3)
Optimum temperature	40 − 50 ℃(Fig.4)
pH Stability	pH 4.5−6.0 (30 ℃, 20 hr)(Fig.5)
Thermal stability	below 50 ℃ (pH 5.7, 1 hr)(Fig.6)
Substrate specificty	(Table 1)
Effect of various chemicals	(Table 2)

APPLICATIONS

This enzyme is useful for enzymatic determination of glucose, and for an amylase-activity assay in combination with α-glucosidase (AGH-211, if maltooligosaccharide or modified starch is used as a substrate), in clinical analysis.

ASSAY

Principle

4-AA : 4-Aminoantipyrine

EHSPT : N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine

The formation of quinoneimine dye is measured at 555 nm by spectrophotometry.

Unit definition

One unit catalyzes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per minute under the conditions detailed below.

Method

Reagents

A. MES-Na buffer pH 5.7	0.1 M: Dissolve 2.13 g of 2-(N-morpholino) ethansulfonic acid (MW ＝ 213.25) in approx. 60 mL of H₂O and, after adjusting the pH to 5.7 with 1 N NaOH at 25℃, make up to 100 mL with H2O (stable at 5℃ for 1 month).
B. Glucose solution	15 %: Dissolve 1.5 g of β-D-glucose in H₂O, and make up to 10 mL, at least 2 hours before, but on the same day as, the assay.
C. 4-AA solution	0.5 %: 50 mg of 4-aminoantipyrine (MW ＝ 203.25) / 10 mL of H₂O (stable at 5℃ in a brownish bottle for at least 1 week).
D. EHSPT solution	40 mM: 118 mg of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (MW ＝ 295.3) / 10 mL of H₂O (stable at 5℃ in a brownish bottle for at least 1 week).
E. Peroxidase solution	500 purpurogalin units (U) /mL H₂O
F. Enzyme diluent	10 mM MES-Na buffer, pH 5.7, containing 0.1 % Triton X-100

Procedure

1.Prepare the following working solution in a brownish bottle and store on ice.

(Should be prepared fresh)
30 mL	Buffer solution	(A)
6.0 mL	Substrate solution	(B)
0.3 mL	4-AA solution	(C)
0.3 mL	EHSPT solution	(D)
0.3 mL	POD solution	(E)

Concentration in assay mixture
MES buffer	79 mM
D-glucose	131 mM
4-AA	0.2 mM
EHSPT	0.3 mM
POD	ca.4 U/ml

2.Pipette 3.0 mL of working solution into a cuvette (d ＝ 1.0cm) and equilibrate at 37 ℃ for approximately 5 minutes.

3.Add 0.1 mL of the enzyme solution^* and mix by gentle inversion.

4.Record the increase in optical density at 555 nm against water for 2 to 3 minutes with a spectrophotometer thermostated at 37 ℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).

At the same time, measure the blank rate (ΔOD blank) using the same method as the test, except that enzyme diluent (F) is added instead of the enzyme solution.

^*Dissolve the enzyme preparation in ice cold enzyme diluent (F) and dilute to 0.05−0.2 U/mL with the same buffer, immediately before the assay.

Calculation

Activity can be calculated by using the following formula :

Volume activity (U/mL) =
ΔOD/min (ΔOD test−ΔOD blank)×Vt×df
32.8×1/2×1.0×Vs
＝ ΔOD/min×1.89×df

Weight activity (U/mg) ＝ (U/mL)×1/C

Vt	: Total volume (3.1 mL)
Vs	: Sample volume (0.1 mL)
32.8	: Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm²/micromole)
1/2	: Factor based on the fact that one mole of H₂O₂ produces a half of quinoneimine dye.
1.0	: Light path length (cm)
df	: Dilution factor
C	: Enzyme concentration in dissolution (c mg/mL)

REFERENCES

1)The Enzymes, Vol.ⅫB, P.421 (P.D.Boyer, ed.), Academic Press (1975).

2)Method in Enzymology, Vol.Ⅸ, p.82 (S.P.Colowick and N.O.Kaplan, ed.),Academic Press (1966).

3)B.E.P.Swoboda and V.Massay; J.Biol.Chem., 240, 2209 (1965).

4)P.J.Auses, S.L.Cook and J.T.Maloy; Anal.Chem., 47, 244 (1975).

5)D.C.Williams, G.F.Huff and W.R.Gaitz; Clin.Chem., 22, 372 (1976).

Table 1. Substrate Specificity of Glucose oxidase

[0.1M of Substrate, 79mM MES buffer, pH 5.7, at 30 ℃ ]

Substrate (0.1M) Relative activity(%)
D-Glucose 100
2-Dexy-D-glucose 16.2
Glucono-1,5-lactone 0.06
L-Glucose 0.00
Galactose 3.10
Mannose 2.10
Substrate (0.1M) Relative activity(%)
Fructose 0.24
Xylose 0.93
Ribose 0.00
Maltose 0.69
Lactose 0.00

Table 2. Effect of Various Chemicals on Glucose oxidase

［The enzyme dissolved in 0.1M MES buffer, pH 5.7 (10 U/mL) was incubated with each chemical for 2 hr at 25 ℃.］

Chemical Concn.(mM) Residual
activity(%)
None – 100
Metal salt 2.0
MgCl₂ 92.6
CaCl₂ 93.6
BaCl₂ 94.4
CoCl₂ 98.1
MnCl₂ 95.1
ZnSO₂ 94.3
FeCl₂ 96.8
NiCl₂ 91.7
CuSO₄ 71.6
AgNO₃ 58.6
HgCl₂ 0.7
PCMB 2.0 31.6
MIA 2.0 96.8
NaF 2.0 97.1

Chemical	Concn.(mM)	Residual activity(%)
NaN₃	20	96.3
EDTA	5.0	97.3
o-Phenanthroline	2.0	95.3
α,α′-Dipyridyl	2.0	99.5
Borate	50.0	96.1
IAA	2.0	96.1
MIA	2.0	101.1
Hydroxylamine	10.0	98.3
Sodium bisulfite	10.0	100.0
hydrazine	10.0	103.1
Triton X-100	0.1 %	111.2
Brij 35	0.1 %	108.0
Tween 20	0.1 %	110.7
Span 20	0.1 %	106.7
Na-Cholate	0.1 %	106.1
SDS	0.1 %	113.1

PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediamimetetraacetate; IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate.

Fig.1. Stability (Powder form)
(kept under dry conditions)
Fig.2. Stability (Powder form)
(kept under dry conditions)
Fig.3. pH-Activity
37 ℃, 5min-reaction in 79 mM buffer solution : ○̶̶○ . acetate;●̶̶● MES;△̶̶△, BES;×̶̶×, BICINE
Fig.4. Temperature activity
(5 min-reaction in 79 mM MES buffer, pH5.7)
Fig.5. pH-Stability
30 ℃, 20hr-treatment with 0.1 M buffer solution : ○̶̶○ . acetate : ●̶̶● MES : △̶̶△, BES : ×̶̶×, BICINE
Fig.6. Thermal stability
(1hr-treatment in 79 mM MES buffer , pH5.7)

2. MSDS

3. Tech Data Sheets/Manuals

Size	1 MG, 10 MG, 5 MG

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GLUCOSE OXIDASE from Aspergillus sp.

PREPARATION and SPECIFICATION

PROPERTIES

APPLICATIONS

ASSAY

Principle

Unit definition

Method

Reagents

Procedure

Calculation

REFERENCES

Table 1. Substrate Specificity of Glucose oxidase

Table 2. Effect of Various Chemicals on Glucose oxidase

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Appearance :	Yellowish amorphous powder, lyophilized
Activity :	GradeⅠ	180 U/mg-solid or more
	GradeⅡ	100 U/mg-solid or more
		(containing approx. 50 % of stabilizers)
Contaminant :	Catalase	GradeⅠ	≤ 5.0×10^-3 %
Contaminant :		Grade Ⅱ	≤ 3.0 %
Stabilizers :	Potassium gluconate, sodium glutamate

Substrate (0.1M)	Relative activity(%)
D-Glucose	100
2-Dexy-D-glucose	16.2
Glucono-1,5-lactone	0.06
L-Glucose	0.00
Galactose	3.10
Mannose	2.10

Chemical	Concn.(mM)	Residual activity(%)
None	–	100
Metal salt	2.0
MgCl₂		92.6
CaCl₂		93.6
BaCl₂		94.4
CoCl₂		98.1
MnCl₂		95.1
ZnSO₂		94.3
FeCl₂		96.8
NiCl₂		91.7
CuSO₄		71.6
AgNO₃		58.6
HgCl₂		0.7
PCMB	2.0	31.6
MIA	2.0	96.8
NaF	2.0	97.1

Substrate (0.1M)	Relative activity(%)
Fructose	0.24
Xylose	0.93
Ribose	0.00
Maltose	0.69
Lactose	0.00

GLUCOSE OXIDASE from Aspergillus sp.

PREPARATION and SPECIFICATION

PROPERTIES

APPLICATIONS

ASSAY

Principle

Unit definition

Method

Reagents

Procedure

Calculation

REFERENCES

Table 1. Substrate Specificity of Glucose oxidase

Table 2. Effect of Various Chemicals on Glucose oxidase

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Reviews

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