GLUCOSE OXIDASE from Aspergillus sp.

GLO-101, 201

Appearance Yellowish amorphous powder, lyophilized
Activity Grade I: 180U/mg-solid or more
Grade Ⅱ: 100U/mg-solid or more
(containing approx. 50% of stabilizers)
Contaminant Catalase
Grade I ≤5.0×10⁻³%
GradeⅡ ≤3.0%
Stabilizers Potassium gluconate, sodium glutamate
Stability GLO-101: Stable at -20°C for at least 3 years(Fig.1)
GLO-201: Stable at -20°C for at least One year
Molecular weight approx. 153,000
Michaelis constants 3.3×10⁻²M (ß-D-Glucose),³ ⁾
6.1×10⁻²M (2-Deoxyglucose)
Structure Glycoprotein with 2 moles of FAD
Inhibitors p-Chloromercuribenzoate,
heavy metal ions (Cu⁺⁺, Hg⁺⁺, Ag⁺)
Optimum pH 4.5(Fig.3)
Optimum temperature 40-50℃(Fig.4)
pH Stability pH 4.5-6.0 (30℃, 20hr)(Fig.5)
Thermal stability below 50℃ (pH 5.7, 1hr)(Fig.6)
Substrate specificty  (Table 1)
Effect of various chemicals (Table 2)


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



glucose oxidase

β-D-Glucose+O+HO                                  D-Glucono-δ-lactone+HO


2HO+4-AA+EHSPT                                  ►Quinoneimine dye+4HO

4-AA : 4-Aminoantipyrine
EHSPT : N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine

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

Unit definition:

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


A. MES-Na buffer pH 5.7 0.1M [Dissolve 2.13g of 2-(N-morpholino) ethansulfonic acid (MW=213.25) in ca. 60ml of HO and, after adjusting the pH to 5.7 with 1N NaOH at 25°C, fill up to 100ml with HO](Stable at 5°C for one month)
B. Glucose solution 15% [Dissolve 1.5g of β-D-glucose and fill up to 10ml with HO]for at least 2hrs before assay.) (Should prepare fresh)
C. 4-AA solution 0.5% [50mg fo 4-aminoantipyrine (MW=203.25)/10ml of HO](Stable at 5°C in a brownish bottle for at least one week)
D. EHSPT solution 40mM [118mg of N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (MW= 295.3)/10ml of HO] (Stable at 5°C in a brownish bottle for at least one week)
E. Peroxidase solution 500U (purpurogalin unit)/ml of HO
F. Enzyme diluent 10mM MES-Na buffer, pH 5.7, containing 0.1% Triton X-100


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

1. Prepare the following working solution in a brownish bottle and store on ice.
(Should be prepared fresh)

30 ml Buffer solution (A)
6 ml Substrate solution (B)
0.3 ml 4-AA solution (C)
0.3 ml EHSPT solution (D)
0.3 ml POD solution (E)

2. Pipette 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37°C for about 5 minutes.

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

4. Record the increase in optical density at 555nm against water for 2 to 3 minutes in a spectrophotometer
thermostated at 37°C, 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) by 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 0.05-0.2U/ml with the same buffer, immediately before the assay.


Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test−ΔOD blank ) ×Vt × df

Volume activity (U/ml) =                                                               =ΔOD×1.89×df


Weight activity (U/mg)=(U/ml)×1/C

: Total volume (3.1ml)
: Sample volume (0.1ml)
: Millimolar extinction coefficient of quinoneimine dye under the assay conditions (㎠/micromole)
: Factor based on the fact that one mole of HO produces a half of quinoneimine dye.
: Light path length (cm)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  1. The Enzymes, Vol.XII B, 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 oxydase
[0.1M of Substrate, 79mM MES buffer, pH 5.7, at 30°C ]
Substrate (0.1M) Relative activity(%) Substrate (0.1M) Relative activity(%)
D-Glucose 100 Fructose 0.24
2-Dexy-D-glucose 16.2 Xylose 0.93
Glucono-1, 5-lactone 0.06 Ribose 0.00
L-Glucose 0.00 Maltose 0.69
Galactose 3.10 Lactose 0.00
Mannose 2.10    

Table 2. Effect of Various Chemicals on Glucose oxydase

[The enzyme dissolved in 0.1M MES buffer, pH 5.7 (10U/ml) was incubated with each chemical for 2hr at 25°C.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 NaN 20 96.3
Metal salt 2.0   EDTA 5.0 97.3
  92.6 o-Phenanthroline
2.0 95.3
CaCl₂   93.6 α,α′-Dipyridy 2.0 99.5
BaCl₂ 94.4 Borate 50 96.1
CoCl₂   98.1
IAA 2.0 96.1
MnCl₂   95.1
NEM 2.0 101.1
ZnSO₂   94.3 Hydroxylamine 10 98.3
FeCl₂   96.8 Sodium bisulfite 10 100.0
NiCl₂   91.7 hydrazine 10 103.1
CuSO₄   71.6 Triton X-100 0.1% 111.2
AgNO₃   58.6
Brij 35 0.1% 108.0
  0.7 Tween 20 0.1% 110.7
PCMB 2.0 31.6 Span 20 0.1% 106.7
MIA 2.0 96.8 Na-Cholate 0.1% 106.1
NaF 2.0 97.1 SDS
0.1% 113.1

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


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