Glucose Oxidase

A. J. Vroemen

DSM Food Specialties, Delft, The Netherlands

INTRODUCTION

Glucose oxidase (GOX) is produced predominantly by
fungi as part of an enzyme complex also containing
catalase. The best-studied microorganisms are species
of Aspergillus (1–3) and Penicillium (4, 5). In
Aspergillus

the enzymatic complex is located in cell

compartments called peroxisomes (6), which results
in the need to open the fungal cell walls before the
enzyme can be released and puriﬁed. The enzyme com-
plex of Penicillium is secreted into the extracellular
environment (7). Almost all GOX preparations avail-
able on the market are produced by Aspergillus niger.
These preparations are either highly or somewhat or
completely exempt of catalase (see Sec. II.A.1). This
chapter will focus mainly on GOX of Aspergillus niger.

The question can be raised why fungi like

Aspergillus niger

invest a high amount of nutrients

and energy in the synthesis of the enzymatic complex.
As we will see later, the catalyzed reaction of glucose to
gluconic acid does not yield energy for the microorgan-
ism. Most probably the enzymatic complex gives the
microorganiism an ecological advantage in the soil. In
nature, GOX is produced by the fungus only when
glucose is present: It is an inducible enzyme. When
glucose is liberated from, for instance, starch or cellu-
lose, the synthesis of the enzymatic complex starts and
in the presence of air, glucose is oxidized to gluconic

acid with simultaneous production of hydrogen perox-
ide. The decrease of the pH caused by the gluconic acid
as well as the antibacterial effect of the hydrogen per-
oxide produced, are favorable for the fungus. In addi-
tion, A. niger is able to consume the gluconic acid
produced, which is not the case for most of the com-
peting microorganisms. Bacteria like Gluconobacter are
also capable of converting glucose into gluconic acid,
but

these

bacteria

use

different

mechanism.

Gluconobacter oxidans

contains two types of glucose

dehydrogenases, one PQQ and one NADP dependent,
which convert glucose to gluconic acid without forma-
tion of hydrogen peroxide. The PQQ-dependent
enzyme seems to be the most important (8). This oxi-
dation process yields energy for the bacteria con-
cerned. It is notable that these bacteria are rather
acid tolerant.

II.

REACTION CATALYZED

As substrates GOX uses b(D)-glucose and oxygen.
Two hydrogen atoms are ﬁrst transferred from glucose
to the coenzyme FAD during formation of d-glucono-
lactone (9). Subsequently the enzyme transfers the two
hydrogen atoms directly to molecular oxygen during
formation of hydrogen peroxide. The d-gluconolactone
is hydrolyzed to gluconic acid spontaneously or by the
enzyme gluconolactonase.

+ E-FAD

Ð C

+ E-FADH

(1)

glucose

gluconolactone

+ H

! C

(2)

gluconolactone

gluconic acid

E-FADH

+ O

! E-FAD

(3)

+ H

O + O

Ð C

glucose

gluconic acid

hydrogen
peroxide

The fact that GOX uses only b-glucose as substrate
does not mean that glucose can be only partially oxi-
dized; during the process a-glucose spontaneously
mutarotates to a-,b-glucose, making all the glucose
available as substrate. GOX is rather speciﬁc for glu-
cose. There is < 1% activity on related sugars like
xylose and galactose. Also, the activity on other nat-
ural mono- and disaccharides is very low. In a systema-
tic study Whitaker showed that in the deoxyglucoses,
replacement of the hydroxyl group by a hydrogen
atom at successively the 6, 4, 2, 3 and 5 position
resulted in respectively 10%, 4%, 3%, 1% and 0%
remaining activity (10).

The hydrogen peroxide formed causes the reaction

to stop since it inactivates the GOX rapidly and irre-
versibly. In principle, hydrogen peroxide can be hydro-
lyzed by the enzyme catalase, or the active oxygen
can be transferred spontaneously or enzymatically to
another molecule.

As mentioned before, the fungi concerned also pro-

duce catalase, which oxidizes/reduces the hydrogen
peroxide produced into water and oxygen. The overall
reaction of the enzyme complex becomes:

+ 2H

O + 2O

Ð 2C

+ 2H

(4)

O + O

(5)

Overall reaction:
2C

+ O

! C

(6)

III.

ENZYME CHARACTERISTICS

Michaelis-Menten Constants

The enzyme has two substrates: glucose and molecular
oxygen. Simple Michaelis-Menten kinetics results in

¼ k

cat

½E

glu

þ c

glu

þ c

The K

values as determined by Gibson et al. (11)

glu

¼ 110 mM;

¼ 0:48 mM

(both determined at 278C.)

Here the molarity of glucose is deﬁned as total glucose,
not as b-glucose.

The value of 110 mM mentioned by Gibson seems

rather high. Other values mentioned in the literature
are 37 mM (12), which is more in agreement with GOX
from other microorganisms. The Penicillium amagasa-
kiense

enzyme has a higher afﬁnity for glucose: a K

value of 5.2 mM is reported (13). The problem in the
determination of the Michaelis-Menten constant for
glucose is that precautions must be taken to ensure
that the concentration of dissolved oxygen, which
is consumed during the reaction, remains constant.
Technically it seems possible to maintain the dissolved
oxygen concentration constant during the reaction in
for instance a fermenter, enabling the determination of
correct velocity values at different glucose concentra-
tions. However, to the author’s best knowledge such an
approach has never been published so far. Initial velo-
city, v

, can also be used to obtain correct data.

These K

values mentioned above show that the

afﬁnities of GOX for the two substrates are rather
poor. This is especially true for the substrate oxygen
when we compare the K

for oxygen with the solubility

of oxygen in water. At 1 bar, 258C, and in the presence
of air, the maximum solubility of oxygen is

6 mg/L or

0.2 mM, equaling < 50% of the K

. Even with pure

oxygen the maximum solubility of

30 mg/L is only

twice the K

. With glucose concentrations far below

the K

value, which is between 7 to 20 g/L for the

Aspergillus

enzyme, the velocity of the reaction will

be low. As a consequence it can be concluded that
for the elimination of low quantities of glucose or oxy-
gen from certain foods, high quantities of enzyme and/
or long incubation times are necessary even under opti-
mal conditions. The afﬁnity of the enzyme catalase for
its substrate hydrogen peroxide is extremely low: K

= 1.1 M or 37.4 g/L. This aspect is very important in
certain applications.

Determination of GOX Activity

From a theoretical point of view, the analysis of GOX
is rather complicated. The enzyme has two substrates:
glucose and oxygen. In principle high glucose concen-
trations can be chosen, far above the K

, so that the

velocity of the reaction is no longer dependent on the
glucose concentration. This is more difﬁcult for the
second substrate oxygen. To obtain oxygen concentra-
tions in solution far above the K

, very high oxygen

pressures have to be chosen, which is not practical. The
concentration of oxygen is below the K

if the reaction

is performed under normal conditions, and as a result
the velocity of the reaction is below the maximum
velocity.

An extra complication is that the reaction consumes

oxygen. When there is insufﬁcient supply to maintain
the oxygen concentration at a saturation level, there
will not be a linear production of gluconic acid in
time at a ﬁxed enzyme concentration or a linear rela-
tionship between the amount of gluconic acid pro-
duced within a ﬁxed time and amount of enzyme.
Such linear relationships are desirable for an analytical
method. Therefore, an analytical method for GOX is
preferably based on a system in which only very small
quantities of oxygen are consumed. A sensitive method
is not realized when it is based on the consumption of
glucose or the formation of gluconic acid. If the ana-
lysis is based on the consumption of oxygen, the result
is inﬂuenced by the presence of catalase. In this type of
method high quantities of GOX-free catalase have to
be added. This is the principle underlying a method
described in 1953 by Scott (14) and in 1957 by
Underkoﬂer (15). In a Warburg equipment the amount
of oxygen consumed under optimal conditions was
determined. The unit most frequently used is the
Sarrett unit, which is deﬁned as the amount of enzyme
which catalyzes the uptake of 10

L oxygen/ min at

308C, pH 5.9, and a glucose concentration of 3%.
Working with a Warburg is rather laborious, and
therefore Underkoﬂer also describes a method using
a sodium hydroxide solution neutralizing the gluconic
acid produced.

A very sensitive and accurate method is based on

the production of hydrogen peroxide. The amount of
hydrogen peroxide is determined indirectly by the oxi-
dation of a chromogen catalyzed by a peroxidase.
Owing to the very high afﬁnity of this peroxidase
for hydrogen peroxide compared to catalase, the pre-
sence of this last enzyme in GOX preparations does
not inﬂuence the analytical result. As chromogen, o-
dianisidine, o-toluidine, and others are used, the
developed color is measured in a spectrophotometer,
and by using internal standards the amount of oxi-
dized glucose can be determined. The activity is often
expressed as IU (international units), deﬁned as
micromolar glucose oxidized per min under optimal
conditions. For the Aspergillus niger enzyme the rela-

tionship between IU and Sarrett units is determined
as 1 IU=1.12 SU (16, 18) or 1.1 SU(17). (See Annex
1 for a detailed description of a method with o-dia-
nisidine.) Methods based on the oxidation of a chro-
mogen by hydrogen peroxide are now generally used.
The relationship between IU and Sarrett units can be
different for glucose oxidation from different organ-
isms. Finally, it should be mentioned that comparison
of units in literature is extremely difﬁcult owing to
different incubation conditions, including saturation
levels of oxygen, and different unit deﬁnitions. The
best thing to do is to work with an internal standard
of GOX of known activity.

Speciﬁc Activity

By measuring the oxygen consumption of a pure, cat-
alase-free GOX preparation of A. niger, Tsuge et al.
(21) found a speciﬁc activity of 172 IU/mg protein at
308C and pH 5.6. About the same value has been
reported by Hayashi and Nakamura (22). Several
research groups have puriﬁed the A. niger enzyme by
ion-exchange chromatography and obtained pure pre-
parations with activities from 200 to 250 Sarrett units/
mg protein, equaling 180–225 IU/mg protein. Pure cat-
alase-free preparations can be obtained from compa-

Annex 1

Analysis of Glucose Oxidase

The method used by Whittington (26) is mentioned here:
Glucose solution A: 2 g/L in 0.2 M Tris phosphate buffer,

pH 7.0.

-Dianisidine solution B: 2 g/L in 0.2 M Tris phosphate

buffer pH 7.0.

Horseradish peroxidase solution C: 60 units/mL in 0.2 M

Tris phosphate buffer pH 7.0.

Glycerol.
Enzyme solution standard D: 10 IU glucose oxidase/mL.
Hydrochloric acid solution E: 5 M.
All reagents Sigma quality.
Preincubate all solutions at least 10 min at 308C.
Add to 1 mL A, 0.1 mL B, 0.1 mL C, and 0.8 mL glycerol.

Mix carefully.

Add 10–20

L of an enzyme sample containing 0–10 IU/

mL.

Incubate 30 min at 308C.
Stop reaction by adding 2 mL of 5 M hydrochloric acid

solution. Mix carefully.

Oxidized o-dianisidine is measured in a spectrophotometer

at 525 nm.

Compare with internal standard of 0–10 IU/mL enzyme.

nies selling research chemicals, or by growing a trans-
genic yeast harboring a GOX gene.

The speciﬁc activity of the puriﬁed Penicillium ama-

gasakiense

enzyme is

60% higher than that of the A.

niger

enzyme (22).

Turnover Number

Gibson et al. (11) found a turnover number of 16,200/
min. Whittington et al. (27) cloned the A. niger enzyme
in yeast and found a value of 17,000–20,000 for the
yeast-derived enzyme. V

max

values of 235/sec for the

wild-type enzyme and 500 for the yeast-derived enzyme
are found, but it remains unclear whether these differ-
ences are really signiﬁcant.

pH Activity Proﬁle

The pH activity curves for both the Penicillium and
Aspergillus

enzymes show a horizontal proﬁle between

pH 4.5 and 7.5, with a sharp decline on both sides (10,
20). There is almost no activity at < pH 3.0 or > pH
8.5. GOX is stabilized by its substrate glucose (32),
explaining why in certain applications with a pH
between 2.5 and 3.0 and with a high concentration of
glucose the enzyme is still active. Also at > pH 8.0 the
stability of the enzyme is improved by adding the
substrate.

Temperature Activity Proﬁle

In determination of the temperature activity proﬁle of
GOX, some speciﬁc complications are encountered.
GOX is sensitive to the hydrogen peroxide formed espe-
cially at higher temperatures. This means that when a
method is applied in which the hydrogen peroxide is not
removed immediately and completely, the optimum
temperature and the maximum temperature will be
low as compared to a method where this is done prop-
erly (10). Moreover, long incubation times give a lower
optimum. In the analytical method using perosidase
and a chromogen, the hydrogen peroxide reacts imme-
diately with the chromogen. This method gives an activ-
ity proﬁle which is rather constant with time between
308C and 608C. Above 608C activity goes down slowly
with still 10% activity at 708C but no activity at 808C.

Protein Properties

The molecular weights of the enzymes from both
Aspergillus

and Penicillium have been determined at

between 140 and 160 kDa (19–21). The enzyme consists

of two identical subunits with 1 molecule FAD per
subunit as coenzyme. Because of this, the color of the
oxidized protein is yellow with absorption maxima at
377 and 455 nm. Under anaerobic conditions in the
presence of glucose, the enzyme molecule is reduced
and the color disappears. When oxygen is admitted to
the system the color reappears. The FAD is not cova-
lently linked and can easily be removed by acid, urea, or
guanidine. But without these reagents, the FAD is
bound to the enzyme. The apoenzyme is not active
but activity is restored upon incubation with FAD (15).

GOX is a glycoprotein. The amount of carbohydrate

in different preparations from A. niger can vary from
10% to 18% (22, 23). Mannose is by far the most
important sugar with amounts of 70–80%. Galactose
contributes

5% and glucosamine 15% to the total

carbohydrate content. In the Penicillium enzyme Kalisz
(13) found 95 residues of mannose, 12 residues of glu-
cosamine, and ﬁve residues of galactose per molecule of
enzyme, and a total carbohydrate content of 13%.

In the A. niger enzyme the sugar molecules are N

and O linked. By selective elimination of the O-linked
sugars Takegawa (12) showed that predominantly
mannose monomers are linked to serine and threonine,
but the exact place in the molecule is not yet known.
The N-linked sugar moiety can be selectively removed
by an enzyme from Flavobacterium, resulting in the
liberation of 30% of the total carbohydrates. The
partly deglycosylated enzyme has the same kinetic
and biochemical properties and resistance to proteases
as the native enzyme except that the native enzyme
precipitates at higher concentrations of ammonium
sulfate or TCA, and the deglycosylated enzyme is less
stable in relation to pH and temperature. The same
results were obtained with the Penicillium enzyme by
Kalisz et al. (13). In this case 95% of the carbohydrate
was split off by the enzymatic treatment. Losses in pH
and temperature stability were observed as well.

The enzyme from Phanerochaete chrysosporium (24)

was found to be a ﬂavoprotein with a molecular weight
of

160 kDa and containing 2 mol FAD per mol pro-

tein, but it does not seem to contain any carbohydrate
and its speciﬁcity is lower. Although glucose is the
main substrate, considerable activity is still found on
sorbose, xylose, and maltose.

The gene of the A. niger enzyme has been isolated

and the sequence analyzed by various research groups
with identical results (25–27). This sequence is now
available in data bases. The gene codes for a signal
peptide of 22 amino acids (1–22), followed by the
583 amino acid subunit itself (–23–605). The molecular
weight of the subunit plus signal peptide is calculated

to be 65,638, and without signal peptide it is 63,250.
With

16% sugars and two molecules of FAD the

total molecular weight can be calculated to be

152

kDa. When the gene is expressed in yeast, an enzyme is
obtained with a higher glycolysation level and an
improved thermostability. This is in line with the
observed lower thermostability the deglycosylated
enzymes. The kinetic parameters of the yeast-derived
enzyme and the original one are not signiﬁcantly dif-
ferent. The yeast expression system can be used to
obtain GOX completely free of catalase. The trans-
genic yeast must not been grown on glucose since the
hydrogen peroxide formed will inactivate the enzyme
and stop growth. Instead it can be grown on sacchar-
ose, since this sugar is hydrolyzed by membrane-bound
invertase in the cell to fructose and glucose, which
are immediately metabolized without formation of glu-
conic acid and hydrogen peroxide.

Genetic Aspects

A comparison of the mature GOX sequence with
sequences present in data bases shows 26% homology
with the alcohol oxidase from Hansenula polymorpha.
The GOX’s from Aspergillus and Penicillium show
66% identity and 79% similarity. The Penicillium
enzyme consists of 587 amino acids. The two enzymes
are highly conserved in the FAD-binding and sub-
strate-binding domain, in their secondary structures
and in regions at the subunit interface. The highest
similarity with other oxidoreductases is observed in
the FAD-binding domain. Aryloxidase, a FAD-depen-
dent enzyme involved in lignin degradation, has been
cloned from Pleurotus eryngii. The enzyme is com-
posed of 593 amino acids, 27 of which form a signal
peptide. It shows 33% sequence identity with GOX
from Aspergillus niger. The predicted secondary struc-
tures of the two enzymes are very similar (28).

Three-Dimensional Structure

The three-dimensional structures of the two enzymes
of Aspergillus and Penicillium have been determined by
x-ray crystallography at 2.3 Angstrom resolution (29)
and later improved to 1.9 A˚ resolution (30). The FAD-
binding domain is very similar to other FAD-binding
proteins, and 11 amino acid residues from different
parts of the molecule are involved in this binding.
The same is true for substrate binding. The substrate
enters a deep pocket and is stabilized by 12 hydrogen
bonds and hydrophobic contacts to three aromatic
residues and to FAD. A detailed analysis of this sub-

strate-binding site explains the high speciﬁcity of GOX
described above. Part of the entrance to the pocket is
at the interface to the second subunit and is formed by
a 20-residue lid. The carbohydrate moiety attached to
Asn89 at the top of this lid forms a link between the
subunits of the dimer. In total, there are four N-glyco-
sylation sites, with an extended carbohydrate moiety at
Asn89. Starting from the 3D structure of the
Pencillium amagasakiense

enzyme, it could be shown

by mutation of key conserved active-site residues that
Arg516 is involved in the binding to the 3-OH group of
the glucose molecule (31). Replacement of this arginine
by another amino acid lowers the afﬁnity for the sub-
strate. Aromatic residues on other locations like 73,
418, and 430 are important for the correct orientation
and maximum velocity of glucose oxidation.

IV.

APPLICATIONS OF GOX IN FOOD

Looking at the reaction catalyzed by the enzyme, the
applications are linked to four different aspects:

Removal of glucose

Removal of oxygen

Production of gluconic acid

Production of hydrogen peroxide

A separate subject is the application of glucose oxidase
as an analytical agent.

Removal of glucose (10)

Glucose is a reducing sugar that can react with amino
groups in, for instance, proteins, to form colored
Maillard components that are often undesirable in
food products. Additions of GOX to the food system
in the presence of air or an oxygen donor like hydrogen
peroxide will result in the conversion of glucose to the
non-amine-reactive gluconic acid and thus prevent the
formation of these Maillard components. The best
known application is the prevention of nonenzymatic
browning in egg white powder. A detailed description
of this application is given by Scott (10). Treatment
can be done up to 508C and in the pH range from 4
to 7. Egg white is neutral at the time of laying, but the
pH rises quickly as carbon dioxide is lost, such that it is
generally close to 9 when the egg white is processed.
The pH is brought below 7 by adding citric acid; addi-
tion of hydrogen peroxide as oxygen donor is advan-
tageous. Owing to the low afﬁnity of GOX for glucose,
high quantities of enzyme and/or long incubation times
are necessary to remove the glucose almost completely,

making this application less competitive than other
systems like addition of active yeast. Canadian
researchers have shown that GOX is also efﬁcient in
the reduction of nonenzymatic browning in potato
products like chips and French fries (33, 34).
However, complete removal of glucose is also not
realized in this process. It is clear that in this type of
application only glucose is converted to a nonreducing
component; other sugars, such as maltose and lactose,
maintain their reducing characteristics. A solution can
be the application of an oxidase active on a broad
range of reducing sugars. Some enzyme candidates
are under investigation now.

Removal of Oxygen

During storage of food, oxygen can have a detrimental
effect on quality. As an example, oxidation of unsatu-
rated fatty acids can lead to rancidity of vegetable oils.
Oxidation of colored components will change the color
of beverages or wine. In addition, oxygen inﬂuences
the taste of beer in a negative way during storage.
The majority of foods contain certain quantitites of
glucose. In closed systems, the quantity of oxygen to
be removed, in order to prevent oxidation, is generally
rather low. Therefore GOX can be used to remove
oxygen. If necessary a small quantity of glucose is
added. An important aspect is the stability of the
enzyme under application conditions. Fruit juices and
wine have a very low pH of 2.5–3, conditions in which
the enzyme is neither active nor stable. However, the
high glucose concentrations in fruit juices appear to
have a stabilizing effect on the enzyme.

A large number of publications describe the positive

effect on quality of adding GOX to food. A good
review is given by Scott (10). Other publications con-
cern: prevention of rancidity in oils, fats and ﬁsh (35–
37); and prevention of off-ﬂavors and color changes in
fruit juices, fruit concentrates, white wine, and beer
(10, 38, 39). Despite all these positive results, no appli-
cations of adding GOX to food has been realized on an
industrial scale until now. The main reason seems to be
the existence of competing technologies to prevent oxi-
dation; for example, ﬂushing out of oxygen by carbon
dioxide or nitrogen in beverages, addition of antioxi-
dants as BHA, ascorbic acid, sulﬁte, etc.

Production of Gluconic Acid

Milk can be directly acidiﬁed by adding glucose, GOX,
and hydrogen peroxide as oxygen donor. A variant is
combining lactase, which hydrolyzes the milk sugar

into glucose and galactose, with GOX and hydrogen
peroxide (40). Owing to the pH drop, the milk coagu-
lates. In principle, the same can be done in cheese
manufacture, where direct acidiﬁcation is fairly com-
mon. However, in industrial practice, the addition of
an acid or gluconolactone, which under the conditions
of cheese manufacturing slowly hydrolyzes into glu-
conic acid, is preferred.

Until, now production of gluconic acid on an indus-

trial scale is done by fermentation with selected strains
of Aspergillus niger and Gluconobacter oxydans.
Several attempts have been made toward production
of gluconic acid using an enzymatic system. Until
recently complete bioconversion was only obtained
with glucose solutions of 10% and lower, which is
not interesting from an economical point of view.
Beverini and Vroemen (41) have now shown that glu-
cose concentrations as high as 40–50% can be comple-
tely converted in a fermenter at pH 5–6 and up to
308C, using low quantities of GOX rich in catalase in
a relatively short time. The high catalase content
needed is necessary owing to the low afﬁnity of this
enzyme for hydrogen peroxide. Under the conditions
mentioned, the concentration of hydrogen peroxide
remains low such that the two enzymes GOX and cat-
alase are not completely inactivated before the end of
the bioconversion. The advantages of this enzymatic
process are that no fermentation is necessary (steriliza-
tion, nutrients, time), the yield is up to 100%, and the
puriﬁcation of the ﬁnal product is much easier.

Production of Hydrogen Peroxide

Hydrogen peroxide is a potent oxidant, which can be
used as an active antimicrobial agent. Based on this
property of hydrogen peroxide, several applications
of GOX have been developed of which some are
applied on a large scale. For these applications the
GOX preparations have to be exempt or poor in cat-
alase. Unfortunately, the GOX itself risks also being
inactivated by the hydrogen peroxide formed.

Toothpaste

In the Netherlands, toothpaste containing enzymes has
been developed (42). If sufﬁcient glucose is present, the
GOX generates hydrogen peroxide, which kills plaque-
forming bacteria in the mouth. To obtain sufﬁcient
glucose,

amyloglucosidase

can be

added, which

together with the amylase present in saliva, hydrolyzes
starch into glucose. In addition, it is claimed that GOX
stimulates the lactoperoxidase system in the mouth.

Milk and Milk Products

GOX can generate hydrogen peroxide for the lactoper-
oxidase (LPO) system naturally present in milk. This
combined GOX-LPO system has been studied exten-
sively by a number of research groups to solve severe
contamination problems in milk and cheese produc-
tion (43, 44). Especially in cheese made from raw non-
pasteurized milk, there is an urgent need for a natural
antibacterial system. Although a strong effect could be
shown on a high number of pathogens, introduction
of the GOX-LPO system has not yet taken place. One
of the reasons is that it has not given an absolute
guarantee against all pathogens.

Baking

An important aspect of baking is the strength or weak-
ness of the dough. Flours with a low protein content
are characterized as weak, and the gluten is very exten-
sible under stress but does not return to its original
dimensions when the stress is released. Bakers gener-
ally prefer strong doughs because of their better rheo-
logical and handling properties, which result in a better
form and texture of the ﬁnal bread. Bakers have used
dough conditioners to strengthen the dough. These
conditioners are mostly nonspeciﬁc oxidants like bro-
mates, iodates, and ascorbic acid. In North America
and Western Europe, public opinion and legislation
are more and more opposed to the use of chemicals
in bread. Moreover, these nonspeciﬁc oxidants can
have a negative inﬂuence on the bread aroma. In the
United Kingdom bromates are no longer allowed since
1990, and in France no addition of chemicals is per-
mitted in the production of traditional bread (pain a`
tradition franc¸aise). Therefore, a need exists to replace
the nonspeciﬁc oxidants by a natural alternative.
Enzymes are judged as natural and in agreement with
‘‘clean label.’’ Already in 1957, a patent appeared (45)
which describes the addition of GOX to ﬂour to
improve the dough quality and the baking properties.
As a particular advantage, the combination with ascor-
bic acid is mentioned. This is already an indication that
GOX added gives a better result than ascorbic acid or
other nonspeciﬁc oxidants alone, which has been con-
ﬁrmed in many tests. This, together with the fact that
in that era the acceptance of chemicals was still high,
resulted in a low market penetration of the addition of
GOX to ﬂour.

The big breakthrough came in the beginning of the

1990s, when researchers of the Finnish company
Cultor discovered the synergistic effect of combining
several enzymes. The ﬁrst patent (46) concerns the

combination of GOX and sulfhydryl oxidase. The lat-
ter enzyme catalyzes the selective oxidation of sulfhy-
dryl groups to disulﬁdes by oxygen.

2 RSH

þ O

Ð RSSR þ H

ð7Þ

This leads to interprotein disulﬁde bonds. The role of
GOX is not yet completely understood. It clearly par-
ticipates in the formation of disulﬁde bonds between
gluten proteins, but most probably it also participates
in the formation of other bonds like oxidative gelation.
This is the coupling of two ferulic acid residues of
neighboring arabinoxylan chains by the hydrogen-
peroxide formed by glucose oxidase.

The second patent (47) concerns the synergistic

effect of GOX and hemicellulases, like xylanases. It is
believed that GOX makes stronger doughs, permitting
the addition of higher amounts of hemicellulases.
Addition of such amounts of hemicellulases alone
often results in softer and sometimes sticky doughs.
Especially, the latter combination of GOX and hemi-
cellulases has obtained a high market acceptance. This
‘‘Hemilox’’ preparation, produced and commercialized
by DSM Baking Ingredients under license of Cultor,
permits production of clean-label breads of high qual-
ity without addition of nonspeciﬁc oxidants or emulsi-
ﬁers (48).

Glucose Oxidase as an Analytical Agent

GOX is the most widely employed enzyme as an ana-
lytical agent, particularly for the determination of glu-
cose in clinical laboratories, fermentation media, food,
feed, etc. Different systems have been developed using
glucose oxidase in soluble form, but also in immobi-
lized form as glucose electrodes or sticks for the deter-
mination of glucose in urine, as practiced by every
physician. For a good detailed review see Raba and
Mottola (49).

ACKNOWLEDGMENTS

The author wants to thank his son Casper W. Vroemen
of Wageningen University for valuable comments on
the manuscript.

REFERENCES

B Drews, H Smalla. Brantweinwirtschaft 22, 1969.

K Zetelaki, K Vas. Biotech Bioeng 10:45, 1968.

W Franke, L Mochel, K Haye. Arch J Mikrobiol
51:323, 1965.

T Yoshimura, T Isemura. J Biochem Tokyo 1971
69:839.

4a.

S Nakamura, S Hayashi. FEBS Lett 41:327, 1974.

JP van Dijken, M Veenhuis. Eur J Appl Microbiol,
9:275, 1980.

T Yoshimura, T Isemura. J Biochem (Tokyo) 69:839,
1971.

JT Pronk, PR Levering, W Olijve, JP van Dijken.
Enzyme Microb Technol, 11:160, 1989.

D Keilin, EF Hartree. J Biochem 42:221–229, 1948.

D Scott. Enzymes in Food Processing. New York:
Academic Press, 1975, p 228.

10.

Q H Gibson, BEP Swoboda, V Massey. J Biol Chem
239:3927, 1964.

11.

K Takegawa, K Fujiwara, S Wahora. Biochem Cell
Biol 67:460, 1989.

12.

HM Kalisz, J Hendle, RD Schmid. Appl Microbiol
Biotechnol 47:502–507, 1997.

13.

D Scott, J Agric Food Chem, 1:727, 1953.

14.

Underkoﬂer.

Proc

Int

Symp

Enzyme

Chemistry,

Tokyo

and

Kyoto

1957.

London:

Pergamon Press, 1957, p 586.

15.

G Tholey, B Wurtz. Soc Biol 159:2512, 1965.

16.

Catalog PL Biochemicals 1974.

17.

Catalog Serva Biochemicals 1975.

18.

T Yoshimura, T Isemura. J Biochem (Tokyo) 69:839,
1971.

19.

S Nakamura, S Fujiki. J Biochem (Tokyo) 63:51,
1968.

20.

H Tsuge, O Natsuaki, K Ohashi. J Biochem (Tokyo)
78:835, 1975.

21.

S Hayashi, S Nakamura. Biochim Biophys Acta
438:37–48, 1976.

22.

S Hayashi, S Nakamura. Biochim Biophys Acta
657:40–51, 1981.

23.

RL Kelley, CA Reddy. J Bacteriol 166:269, 1986.

24.

M Kriechbaum et al. FEBS Lett 255:63–66, 1989.

25.

KR Frederick, S Chakraborty, et al. J Biol Chem
265:3793, 1990.

26.

H Whittington et al. Curr Genet 18:531–536, 1990.

27.

E Varela, MJ Martinet, AT Martinez. Biochim
Biophys Acta 1481:202–208, 2000.

28.

HJ Hecht et al. J Mol Biol 229:153–172, 1993.

29.

G Wohlfahrt et al. Acta Cryst D55:969–977, 1999.

30.

S Witt, G Wohlfahrt, D Schomburg, HJ Hecht, H M
Kalisz. Biochem 347:553–559, 2000.

31.

P Vaha-Vahe. Food Technol Int Eur, p 139, 1994.

32.

Z Jiang, B Ooraikul. J Food Prod Preserv 13:175–186,
1989.

33.

N Low et al. J Food Sci 54:118–121, 1989.

34.

CA Kannt et al. J Food Sci 58:104–107, 1993.

35.

YH Lin. Dissertation, University of Rhode Island,
1987.

36.

M Dedek, J Hanus, M Vedlich. Int Dairy Cong
Sydney, 1970, p 225.

37.

Ough, Mcleod. Am J Enol Viticult 26:30–36, 1975.

38.

U Schobinger, P Durr, R Waldvogel. Fluss Obst
59:586–588, 1992.

39.

AGJ Rand. J Food Sci 37:698–701, 1972.

40.

M Beverini, AJ Vroemen. European Patent.

41.

Technical Documentation Zendium. Akzo Dental
Research now Sara Lee, Veenendaal, Netherlands.

42.

M Sandholm et al. J Vet Med B 35:346–352, 1988.

43.

M Desmazeaud et al. Food Ingredients Europe. Conf
Proc Maarssen, Netherlands, 1989, pp 96–103.

44.

-. Luther, U.S. Patent 2783150, 1957.

45.

S Haarasilta, S Vaisanen, D Scott. U.S. Patent
136003, 1987.

46.

S Haarasilta, T Pullinen, S Vaisanen, I Tammersalo-
Karsten. U.S. Patent 4990343, 1991.

47.

DSM-Gist, P.O. Box 1, 2600 MA Delft, Netherlands.
Technical Documentation Hemilox.

J Raba, HA Mottola. Crit Rev Anal Chem 25:1–42,
1995.

Document Outline

Contents
- PART II. SPECIFIC ENZYMES
  - OXIDOREDUCTASES
    - Chapter 30- Glucose Oxidase

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