Biotechnology Advances 27 (2009) 489–501

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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Glucose oxidase — An overview

Sandip B. Bankar, Mahesh V. Bule, Rekha S. Singhal, Laxmi Ananthanarayan ⁎
Food Engineering and Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India

a r t i c l e i n f o a b s t r a c t

Article history: Glucose oxidase (β-D-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) catalyzes the oxidation of β-D-glucose to
Received 12 December 2008 gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen
Received in revised form 25 March 2009 peroxide. Microbial glucose oxidase is currently receiving much attention due to its wide applications in chemical,
Accepted 7 April 2009
pharmaceutical, food, beverage, clinical chemistry, biotechnology and other industries. Novel applications of
Available online 15 April 2009
glucose oxidase in biosensors have increased the demand in recent years. Present review discusses the
production, recovery, characterization, immobilization and applications of glucose oxidase. Production of glucose
Keywords:
Glucose oxidase
oxidase by fermentation is detailed, along with recombinant methods. Various purification techniques for higher
Catalse recovery of glucose oxidase are described here. Issues of enzyme kinetics, stability studies and characterization are
Optimization of fermentation parameters addressed. Immobilized preparations of glucose oxidase are also discussed. Applications of glucose oxidase in
Downstream processing various industries and as analytical enzymes are having an increasing impact on bioprocessing.
Immobilization
Application in biosensors
© 2009 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
1.1. Glucose oxidase reaction mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
1.2. Composition of glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
1.3. Characteristics of glucose oxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
1.4. Analysis of glucose oxidase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
2. Fermentative production of GOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
2.1. Microbial strains producing glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
2.2. Parameters affecting enzyme production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
2.2.1. Carbon source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
2.2.2. Nitrogen source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
2.2.3. Calcium carbonate as an inducer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
2.2.4. Other medium components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
2.2.5. Effect of aeration and agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
2.2.6. Effect of culture pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
2.2.7. Effect of growth temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
2.2.8. Fed-batch culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
2.3. Optimization by statistical methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
2.4. Mathematical model for glucose oxidase kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
3. Genetic expression for glucose oxidase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
4. Downstream processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
5. Immobilization of glucose oxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
6. Characterization of glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
6.1. Substrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
6.2. pH optima and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
6.3. Optimum temperature and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
6.4. Variation of the initial rate with enzyme concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
6.5. Kinetic parameters variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
6.6. Storage stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

⁎ Corresponding author. Tel.: +91 22 24145616; fax: +91 22 24145614.

E-mail address: laxmi@udct.org (L. Ananthanarayan).

0734-9750/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2009.04.003
490 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501

7. Applications of glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

7.1. Glucose biosensor for diabetes monitoring. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
7.2. Biofuel cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
7.3. Food and beverage additive. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
7.4. Low alcohol wine production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
7.5. Oral hygiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
7.6. Gluconic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
7.7. Textile industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
8. Concluding remark . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499

1. Introduction erable interest despite the abundant availability of commercial GOD

Soil, organic waste and plant cell material are a few of the diverse
environments in which filamentous fungi are known to flourish. Fungi
produce a wide range of different enzymes which enables them to use
many organic compounds as nutrient sources (Gouka et al., 1997).
Among non-hydrolytic enzymes of fungal origin, glucose oxidase (EC
1.1.3.4) has seen large-scale technological applications since the early
1950′s (Fiedurek and Gromada, 1997). Glucose oxidase (GOD) has
been purified from a range of different fungal sources, mainly from the
genus Aspergillus (Kalisz et al., 1991; Hatzinikolaou et al., 1996) and
Penicillium (Eryomin et al., 2004; Sukhacheva et al., 2004), of which
A. niger is the most commonly utilized for the production of GOD
(Pluschkell et al., 1996). GOD from Penicillium species has been shown
to exhibit more advantageous kinetics for glucose oxidation than that
from A. niger (Kusai et al., 1960).
GOD (β-D-glucose:oxygen 1-oxidoreductase) catalyzes the oxida-
tion of β-D-glucose to gluconic acid by utilizing molecular oxygen as
an electron acceptor with simultaneous production of hydrogen
peroxide (H2O2) (Hatzinikolaou and Macris 1995). GOD has found
several commercial applications including glucose removal from dried
egg; improvement of color, flavor, and shelf life of food materials;
oxygen removal from fruit juices, canned beverages; and from
mayonnaise to prevent rancidity. It has also been used in an automatic
glucose assay kit in conjunction with catalase (Hatzinikolaou and
Macris 1995) and chiefly in biosensors for the detection and
estimation of glucose in industrial solutions and in body fluids such
as blood and urine (Petruccioli et al., 1999). GOD is reported to have
the best antagonistic effect against different food-borne pathogens
such as Salmonella infantis, Staphylococcus aureus, Clostridium perfrin-
gens, Bacillus cereus, Campylobacter jejuni and Listeria monocytogens
(Kapat et al., 1998). GOD has also been used as an ingredient of
toothpaste (Petruccioli et al., 1999), for the production of gluconic
acid, and as a food preservative (Pluschkell et al., 1996). Implantable
glucose sensors have found applications for diabetes patients
(Gerritsen et al., 2001). GOD in new formulations with useful prop-
erties for applications in biotechnology continues to be of consid-
(Rando et al., 1997).
1.1. Glucose oxidase reaction mechanism
GOD is a flavoprotein which catalyses the oxidation of β-D-glucose
to D-glucono-δ-lactone and H2O2 using molecular oxygen as an
electron acceptor (Pluschkell et al., 1996; Hatzinikolaou et al., 1996).
This reaction can be divided in to a reductive and an oxidative step
(Fig. 1). In the reductive half reaction, GOD catalyzes the oxidation
of β-D-glucose to D-glucono-δ-lactone, which is non-enzymatically
hydrolyzed to gluconic acid. Subsequently the flavine adenine
dinucucleotide (FAD) ring of GOD is reduced to FADH2 (Witt et al.,
2000). In the oxidative half reaction, the reduced GOD is reoxidized
by oxygen to yield H2O2. The H2O2 is cleaved by catalase (CAT) to
produce water and oxygen. Witteveen et al. (1992) found the enzyme
lactonase (EC 3.1.1.17) in A. niger to be responsible for catalyzing the
hydrolysis of glucono-δ-lactone to gluconic acid.
1.2. Composition of glucose oxidase
GOD from ascomycetes is a dimeric glycoprotein consisting of two
identical polypeptide chain subunits that are covalently linked
together via disulfide bonds (Rando et al., 1997; Kalisz et al., 1997).
Fig. 2 depicts the FAD moiety and the key conserved active site
residues of a GOD subunit from P. amagasakiense (Wohlfahrt et al.,
1999). The structure of the P. amagasakiense GOD shows each of its
subunit to contain one mole of tightly bound, but not covalently linked
FAD moiety as co-factor (Rando et al., 1997; Witt et al., 2000). GOD
from P. amagasakiense is glycosylated with a mannose-rich carbohy-
drate content of approximately 11–13% (Nakamura and Fujiki, 1968;
Kusai et al., 1960).
The key conserved active-site residues of GOD from P. amagasa-
kiense are Tyr-73, Phe-418, Trp-430, Arg-516, Asn-518, His-520 and
His-563 (Fig. 2) (Witt et al., 2000). Witt et al. (2000) concluded that
Arg-516 was the most critical amino acid for the efficient binding of β-
D-glucose by GOD, while Asn-518 contributed to a lesser extent. The

Fig. 1. Representation of the GOD reaction (Witt et al., 2000).

 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501
Fig. 2. Glucose oxidase from P. amagasakiense showing the FAD moiety, indicating the key conserved active site residues (Wohlfahrt et al., 1999).
aromatic residues Tyr-73, Phe-418 and Trp-430 were important for the
correct orientation of the substrate as well as for the maximal velocity
of glucose oxidation. His-520 and His-563 form hydrogen bonds with
the 1-OH of glucose during the reaction.
1.3. Characteristics of glucose oxidase
The molecular weight of GOD ranges from approximately 130 to
175 kDa (Kalisz et al., 1997). GOD is highly specific for the β-anomer of
D-glucose, while α-anomer does not appear to be a suitable substrate.
Low GOD activities were exhibited when utilizing 2-deoxy-D-glucose,
D-mannose and D-galactose as substrates. Inhibitors of GOD include p-
chloromecuribenzoate, Ag+, Hg2+, Cu2+, hydroxylamine, hydrazine,
phenylhydrazine, dimedone and sodium bisulphate (Kusai et al.,
1960). Nakamura and Fujiki (1968) performed comparative studies
on the GOD of A. niger and P. amagasakiense, and found their molec-
ular weights to be 152 and 150 kDa, respectively. GOD produced by
both the strains had similar carbohydrates, which consisted mainly of
glucose, mannose and hexosamine. A. niger GOD contained more
mannose and hexosamine than that of P. amagasakiense, but less
glucose. The overall carbohydrate content was found to be 16% for A.
niger and 11% for P. amagasakiense. The amino acid content of two
enzymes revealed that the A. niger GOD contained more histidine,
arginine and tyrosine and less lysine and phenylalanine than the P.
amagasakiense GOD. The optimum pH ranges for GOD from A. niger
and P. amagasakiense were shown to be 3.5–6.5 and 4.0–5.5, res-
pectively. It is evident that GOD from A. niger has a broader pH range
than that from P. amagasakiense GOD (Nakamura and Fujiki, 1968).
1.4. Analysis of glucose oxidase activity
Literature depicts various analytical methods for determination of
GOD. Tongbu et al. (1996) used titrimetric method for determination
of the GOD. In this method, enzyme solution was added to sodium
acetate buffer containing 2% β-D-glucose and the reaction was stopped
by adding sodium hydroxide solution. The resulting mixture was
titrated with standard hydrochloric acid solution to calculate volume
of added standard HCl and thereby to calculate GOD activity.
Most researchers use an analytical method for GOD that is based
on the principle that GOD oxidizes β-D-glucose in the presence of
oxygen to β-D-glucono-δ-lactone and H2O2. The H2O2 is then utilized
to oxidize a chromogenic substrate in a secondary reaction with
horseradish peroxidase (HRP) with a resultant color change that
Scheme 1. GOD reaction with ABTS as a chromagenic dye (Witt et al., 1998).
491
is monitored spectrophotometrically. Two of the chromogenic sub-
strates used for the GOD reaction are: 2, 2′-Azino-di-[3-ethylbenzthia-
zolin-sulfonate] (ABTS) (Witt et al., 1998) and o-dianisidine (Bergmeyer
et al., 1974). ABTS forms a greenish-blue oxidized product that is mea-
sured spectrophotometrically at 420 nm. The reactions involved are
represented in Schemes 1 and 2.
Oxidation of o-dianisidine forms a quinoneimine dye that is mea-
sured spectrophotometrically at 500 nm. Petruccioli et al. (1999) used
benzoquinone for spectrophotometric measurement of GOD activity.
Reaction mixtures containing 1 M glucose, 0.1% benzoquinone, and
0.1 M Na-citrate buffer, pH 5.0 were preincubated at 25 °C and the
reaction was initiated by adding the enzyme solution. The method was
based on enzymatic reduction of benzoquinone by hydroquinone
which was measured by the rate of increase of absorbance at 290 nm.
A new assay for GOD using Fourier transform infrared spectro-
scopy, was developed by Karmali et al. (2004), who concluded that the
method was useful to study the kinetic properties of GOD since the
substrate and product of the reaction absorbs at different frequencies.
Distinct advantages over coupled assays were the speed of assay,
requirement of smaller amounts of substrate and enzyme, and the
feasibility of following the reaction by quantifying δ-gluconolactone
formation.
2. Fermentative production of GOD
2.1. Microbial strains producing glucose oxidase
The most common microbial sources for fermentative production
of GOD are Aspergillus, Penicillium, and Saccharomyces species. Most of
the commercially produced GOD is isolated from mycelium of Asper-
gillus niger, grown principally for the production of gluconic acid or its
salts such as sodium gluconate or calcium gluconate. Accordingly, the
enzyme is obtained essentially as a by-product or co-product of
gluconate production. Table 1 compiles detailed information on GOD
production, production media and different assay methods used by
various researchers.
2.2. Parameters affecting enzyme production
2.2.1. Carbon source
During microbial fermentations, the carbon source not only acts as
a major constituent for building of cellular material, but is also used in
Scheme 2. GOD reaction with o-dianisidine as a chromagenic dye (Bergmeyer et al.,
1974).
492 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501
Table 1
Various microorganisms producing GOD.
Microorganism Media composition (g/l)
Penicillium variabile P16 Glucose,80; NaNO3,5; KCl,0.5; KH2PO4,1; FeSO4.7H2O,0.01;
mycological peptone, 1; CaCO3 35.
A. niger glucose oxidase Yeast peptone dextrose (YPD) medium: Yeast extract, 10;
gene expressed in S. peptone,20; glucose,20.
cerevisiae
Aspergillus niger (BTL) Sucrose,70; (NH4)2HPO4,0.4; KH2PO4,0.2; MgSO4.7H2O,0.2; Coupled o-dianisidine-peroxidase reaction.
peptone,10; CaCO3,35.
Recombinant Yeast extract,40; hycas, 5; glucose, 20; galactose,30. Coupled ABTS reaction
Saccharomyces
cerevisiae
Aspergillus niger AM111 Glucose,80; peptone,30; NaNO3,0.5; KH2PO4,1; CaCO3,35.
Penicillium pinophilum Sucrose,40; Na2PO4.2H2O,4.45; KH2PO4,1.5; NaNO3, 1.9;
DSM 11428 MgSO4.7H2O,0.2; CaCl2.2H2O, 0.02; malt extract,10; yeast
extract,5; Trace element,10; vitamine,10.
Aspergillus niger ZBY-7 Glucose,150; inorganic salts,0.35; metal caronate,35;
the synthesis of polysaccharide and as energy source. The rate at
which carbon source is metabolized can often influence the formation
of biomass or production of primary or secondary metabolites. Fast
growth due to high concentration of rapidly metabolized sugars is
often associated with high productivity of growth-associated products
or primary metabolites (Stanbury et al., 1997).
Hatzinikolaou and Macris (1995) investigated the effects of
different carbon sources on growth and total GOD activity for A.
niger. Although A. niger grew on all the carbon sources that they
tested, significant levels of GOD were only obtained using glucose,
sucrose and molasses. Furthermore, Hatzinikolaou and Macris (1995)
stated that glucose (pure or as a product of sucrose hydrolysis by
invertase) was the principal inducer for the transcription of the GOD
gene. Kona et al. (2001) used sucrose as carbon source when they used
economical nutrient containing corn steep liquor for A. niger
fermentation. Petruccioli et al. (1993) studied GOD production by
84 strains of the genus Penicillium and reported that P. expansum
(1 strain), P. italicum (1 strain), P. chrysogenum (3 strains) and P.
variabile (3 strains), when cultivated on glucose as the carbon source
produced GOD activity ranging from 0.61 U/ml to 5.45 U/ml. The
strains mentioned were investigated for their ability to oxidize
glucose, fructose, mannose, galactose, arabinose and xylose. Only
one of the P. italicum strains (NRRL 983) displayed enhanced oxidizing
activity towards mannose, galactose, and xylose being 32.38%, 17.90%
and 26.40% compared to glucose (100%), respectively (Petruccioli et
al., 1993). Petruccioli et al. (1997) investigated the effect of 10 different
carbon sources on growth and GOD production of P. variabile mutant
M80.10, and found cultivation with only glucose and mannose to
produce high levels of GOD(Petruccioli et al., 1995b). Petruccioli et al.
(1997) also determined that optimal production of GOD in P. variabile
(M-80.10) to be 8% (w/v). Their findings were in agreement with
Rogalski et al. (1988) who also reported 8% glucose to be optimal for
GOD in the A. niger G-13 mutant. Higher glucose concentrations
decreased the mycelial mass, culture pH and GOD concentrations.
Kusai et al. (1960) reported sucrose to be the best carbon source for
the production of GOD by P. amagasakiense; although if the pH of
growth medium was maintained during cultivation, glucose was the
carbon source of choice.
2.2.2. Nitrogen source
Inorganic nitrogen is supplied as ammonia gas, ammonium salts or
nitrates. Ammonia has been used for pH control. Ammonium salts
such as ammonium sulphate usually produces acidic conditions as the
ammonium ion gets utilized and the free acid is liberated. On the other
hand, nitrates will normally cause an alkaline drift as they get
metabolized. Ammonium nitrate will first cause an acid drift as the
Assay method Yield (Unit) References
Reduction of benzoquinone by hydroquinone 5.52 U ml− 1 Petruccioli et al. (1999).
measured by the rate of absorbance increase at Petruccioli et al. (1995b)
290 nm
Plate assay: with o-dianisidine Coupled coupled 125 U ml− 1 Hodgkins et al. (1993).
o-dianisidine-peroxidase reaction. Malherbe et al. (2003).
7.5 Uml− 1 Hatzinikolaou and Macris
(1995).
−1
3.43 U mg Kapat et al. (2001).
dry cell mass
Coupled reaction assay 2.5 U ml− 1 Fiedurek and Gromada
(2000).
Coupled ABTS -peroxidase reaction. 1.9 U ml− 1 Rando et al. (1997).
Titrimetric 6 U ml− 1 Tongbu et al. (1996).
ammonium ion is utilized, and nitrate assimilation is repressed. When
the ammonium ion gets exhausted, there is an alkaline drift as the
nitrate is then used as an alternative nitrogen source (Morton and
MacMillan, 1954). Rogalski et al. (1988) showed that when cultivating
A. niger mutant G-13 and supplementing the growth media with 3%
peptone there was 36% and 42% increase in GOD activity and biomass
production, respectively. Hatzinikolaou and Macris (1995) investi-
gated different nitrogen sources on the growth and total GOD activity
of A. niger cultivated on sucrose and molasses as sole carbon sources.
They found that the peptone concentration had a marked effect on the
total GOD production. With sucrose and molasses as carbon sources,
maximum GOD activity was achieved at 1–2% and 0.2–0.3% peptone,
respectively. Kona et al. (2001) investigated the effect of corn steep
liquor as the sole nutrient source on the production of GOD from A.
niger and found it to increase the GOD activity from 550 Uml− 1 to
640 Uml− 1, and that other nitrogen sources did not further improved
the enzyme synthesis.
2.2.3. Calcium carbonate as an inducer
Petruccioli et al. (1995a) found that the addition of calcium car-
bonate to growth medium in shake flasks and in fermenters prevented
pH drop during cultivation, which is necessary for optimal GOD pro-
duction. Rogalski et al. (1988) showed that the synthesis of GOD was
sensitively influenced by increasing concentrations of calcium
carbonate (0–4.5%) with maximal GOD activity at approximately
3.5%. Hatzinikolaou and Macris (1995) reported calcium carbonate to
be a strong inducer of GOD in A. niger and demonstrated it to be
essential for increased levels of GOD production. Optimum calcium
carbonate of 4 and 5% was observed for GOD production using sucrose
and molasses respectively. Bankar et al. (2008) found 3% of CaCO3 to
be optimum for highest GOD production. Hatzinikolaou et al. (1996)
cultivated A. niger using optimized cultivation media of Rogalski et al.
(1988) and demonstrated the induction of GOD production by calcium
carbonate (optimum 4%), which also maintained the pH of the
cultivation media between 6.5 and 6.8. They showed that the activity
of the glycolytic enzyme, glucose-6-phosphate isomerase, was higher
in growth media without calcium carbonate, while that of GOD and
catalase (CAT) were quite low. Inclusion of calcium carbonate
increased the GOD and CAT activities with a simultaneous decrease
in the glucose-6-phosphate isomerase activity. They suggested that
the addition of calcium carbonate in the growth media might cause a
metabolic shift from glycolysis to the pentose phosphate pathway,
thereby increasing GOD levels. Addition of calcium carbonate in the
growth medium caused changes in GOD, CAT, 6-phosphofructokinase
(6-PFK) and glucose-6-phosphate dehydrogenase (G-6-PDH) activ-
ities. 6-PFK is a key regulatory enzyme of Embden–Meyerhof–Parnas
 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501
(EMP) pathway in most living cells (Liu et al., 2001). Liu et al. (2001)
further showed that cells grown in media without calcium carbonate
produced high levels of 6-PFK and low amounts of G-6-PDH, GOD
and CAT. Addition of calcium carbonate to the growth medium
increased the production of GOD and CAT, and decreased the synthesis
of 6-PFK. Therefore, this observation might indicate that the inclusion
of calcium carbonate is accompanied by a metabolic shift from the
glycolytic pathway (EMP) to direct oxidation of glucose by GOD
(Fig. 3).
2.2.4. Other medium components
GOD concentration in A. niger could be increased by the inclusion
of various hydrocarbons during cultivation. Li and Chen (1994)
reported an increase in maximum intracellular GOD activity by 43%,
110% and 31% on addition of n-dodecane, n-hexadecane and soybean
oil, respectively. This is attributed to an increase in the efficiency of
enzyme synthesis in the cells by n-dodecane and n-hexadecane, and
an increase in biomass on addition of soybean oil. Fiedurek et al.
(1996) investigated the effect of different medium components and
metabolic inhibitors on GOD production in A. niger, and found that
substituting ammonium phosphate with sodium nitrate in their basal
salt medium significantly increased GOD activity by 269.6%. Further-
more, intracellular GOD activities increased by 68.3% in the presence
of sodium orthovanadate (1 mM), and extracellular GOD activities
increased in the presence of hematin (1 mM), choline (40 mM) and
Tween 80 (0.1%). The extracellular increase in GOD activity obtained
was between 31.4–53.9%.
2.2.5. Effect of aeration and agitation
Gas–liquid transfer is known to be a limiting factor in many aerobic
fermentations. One of the main reasons for this is the low solubility of
oxygen in fermentation media when compared to the solubility of
carbon, nitrogen sources and other nutrients. Oxygen supply is further
hindered in viscous media which occur at high concentrations of cells,
and are typically seen in fungal fermentations (Klein et al., 2002).
Aeration and agitation are both therefore important factors for aerobic
fermentation processes. Aeration of growing aerobic cultures fulfills
the requirements of oxygen supply and also removes gaseous waste
products. Oxygen for growth and production in fungal cultures is
ensured by the aeration and agitation of the mycelial culture (Zetelaki
and Vas, 1968).
Filamentous fungi grow as dense aggregated mycelial mats re-
sulting in decreased accessibility of oxygen to the actively growing
cells. These problems are overcome by agitation of broth cultures
(Zetelaki, 1970), the use of H2O2 as the sole oxygen source (Fiedurek
and Gromada, 2000), and the use of pure oxygen over air. Agitation
increases the efficiency of aeration by forcing the supplied air bubbles
to disintegrate into smaller bubbles resulting in an increased interface
between the gas and the liquid (Zetelaki and Vas, 1968). Increased
agitator speed corresponds to an increase in the concentration
Fig. 3. Metabolic pathway of glucose in the absence and presence of CaCO3 by Asper-
gillus niger.
493
of dissolved oxygen resulting in a faster growth rate and increased
GOD production (Zetelaki and Vas, 1968; Zetelaki, 1970). Fiedurek and
Gromada (2000) as stated previously, identified a novel method of
increasing dissolved oxygen in the growth media by adding H2O2 as
the sole oxygen source. CAT produced by the organism catalyzed the
decomposition of H2O2 to water and free oxygen; in this case, H2O2
acted as both the electron donor and acceptor in the reaction. They
also investigated some of the factors that affect oxygenation of the
culture. Maximal oxygen concentration occurred in 50 ml of the
medium containing 0.2 g wet mycelium and 0.2% glucose at pH 5.0
(Fiedurek and Gromada, 2000).
Pure oxygen was also shown to be beneficial to the growth rate of
A. niger in submerged cultures. The growth rate increased from 61 mg
mycelial dry weight/100 ml/h (aerated with air) to 95 mg mycelial
dry weight/100 ml/h (aerated with pure oxygen). Comparison of the
GOD activities of 24 h old cultures of A. niger agitated at 460, 700 and
900 rpm, respectively, showed the activity of the culture agitated at
700 rpm to be approximately 20–24% higher than those agitated at
460 rpm. Further increase in agitator speed did not improve the
growth rate or GOD production. Oxygenated culture reached a higher
maximum mycelial yield 8 h earlier than in the aerated culture. It is
noted that autolysis was higher in the oxygenated culture medium. In
addition, the viscosity of oxygenated culture was approximately 50%
lower than that of the aerated culture. Cell walls of the aerated
cultures were found to be thicker and more rigid than that of the
oxygenated culture, thereby causing the oxygenated cells to be less
resistant to mechanical agitation. This in turn could have lowered the
viscosity that is observed in the oxygenated culture. At the same time,
the GOD activity of the oxygenated culture was twice that of the
aerated culture (Zetelaki and Vas, 1968). The only disadvantage of use
of pure oxygen would be the large-scale financial implications (Klein
et al., 2002).
2.2.6. Effect of culture pH
Among the physical parameters, pH of the growth medium plays
an important role by inducing morphological change in the organism
and in enzyme secretion. pH is a significant factor that influences
the physiology of a microorganism by affecting nutrient solubility
and uptake, enzyme activity, cell membrane morphology, by product
formation and oxidative reductive reactions. The pH change observed
during the growth of organism also affects product stability in the
medium. Culture pH can have profound effects on both the rate of
production and the synthesis of enzymes. The appropriate pH for
maximum production of the GOD can differ from that for optimum
growth. Most of the strains used commercially for the productions of
GOD have an optimum pH between 6.0 and 7.0 for growth and enzyme
production. In many of the processes, the buffering capacity of some
media constituents such as CaCO3 and other phosphates sometimes
eliminates the need for pH control. It is reported that A. niger
accumulated GOD in the mycelia when grown in presence of CaCO3 as
compared to that in the absence of CaCO3 (Hatzinikolaou and Macris,
1995; Rogalski et al., 1988).
2.2.7. Effect of growth temperature
The internal temperature of the microorganism must be equal to
that of its environment and, the microbial activity is well known to be
sensitive to environmental temperatures. The influence of tempera-
ture on GOD production is related to the growth of the organism.
Among the fungi, most GOD production studies have been done with
mesophilic fungi within the temperature range of 25–37 °C. Optimum
yields of GOD were achieved at 27–37 °C for A. niger. Caridis et al. (1991)
studied the simultaneous production of GOD and CAT by Alternaria
alternate, and revealed that GOD had its optimum temperature at
32.3 °C and CAT at 18.1 °C. Hatzinikolaou and Macris (1995) examined
the effect of growth temperature on total GOD production at 22.5 to
32 °C, and found 27.5 °C as the optimum.
494 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501

2.2.8. Fed-batch culture production, as well on the processes that affect the enzyme once it has
The aim of fed-batch study is to determine the degree of im- been released, amongst which the most noteworthy is its deactivation
provement in enzyme production over that of batch culture, and to get by the H2O2 produced by the enzymatic action itself. However, the
an insight in to the utilization of the carbon source. Sophisticated feed microorganism also produces catalase, which breaks H2O2, thus
back controlled fed-batch systems are also available. Fed-batch opposing the previous effect and helping to preserve GOD (Miron
cultivation for GOD production is reported by Kapat et al. (1998) et al., 2002).
who showed that the addition of part of the carbon source at a later Growth of the microorganism and the production of GOD both
stage could improve GOD formation. Out of four different feeding show the characteristics of a diauxic process. This diauxic nature, with
strategies tested by them for the production of extracellular logistic and linear phases, is also evident in the disappearance of
recombinant GOD from Saccharomyces cerevisiae, constant feeding of glucose. The disappearance of glucose in the first phase of the culture,
galactose on exhaustion of initial glucose gave the highest yield of although partly due to microbial consumption, is mainly a conse-
154 U/ml, which was 62% above the yield achieved in batch operation quence of its conversion to gluconic acid as a result of the action of
(95 U/ml). GOD produced in the culture. Once the glucose is exhausted, the
microorganism begins to use gluconic acid that accumulates during
2.3. Optimization by statistical methods the first stage as a carbon source, with basically linear kinetics. GOD is
considered as a semi-constitutive enzyme with a moderate rate of
The conventional ‘one variable at-a-time’ approach is time con- biosynthesis that remains constant in the absence of inducers, and
suming and often leads to confusion in the understanding of process increase in the presence of inducers (Miron et al., 2002).
parameters. Use of statistical methods enables easy selection of im- Microbial processes do not necessarily follow the classical kinetic
portant parameters from a large number of factors, and also explains model of substrate-limited biomass growth and product formation
the interactions between important variables. A number of statistical as proposed by Monod in 1949. Therefore, the logistic equation, a
experimental designs have been used for optimizing fermentation substrate-independent model, is used as an alternative empirical
variables. Plackett–Burman design (Plackett and Burman, 1946) is function. In many fermentation systems, cell growth has been charac-
well known and is a widely used statistical technique for screening terized by the logistic equation. The logistic equation (Eq. (1)) can be
and selection of most significant culture variables, while central com- described as follows:
posite design provides important information regarding the optimum
level of each variable along with its interactions with other variables  
dX X
and their effects on product yield (Pardeep and Satyanarayana, 2006). = μm 1 − X ð1Þ
dt Xm
Plackett–Burman saturated orthogonal designs work at two levels,
and can be constructed on the basis of fractional replication of a full
factorial design. This design allows reliable short listing of a small where X is amount of biomass formed at time t, Xm is maximum
number of ingredients for further optimization and allows one to biomass formed; µm is maximum specific growth rate and dX dt
is the
obtain unbiased estimates of linear effects of all the factors with rate of biomass formation (Wang et al., 2006).
maximum accuracy for a given number of observations, the accuracy Logistic equations are a set of equations that characterize growth in
being the same for all effects (Krishnan et al., 1998). Since this design terms of carrying capacity (maximum cell mass; X∞). The usual
is a preliminary optimization technique which tests only two levels of approach is based on formulation in which specific growth rate is
each medium component, it cannot provide the optimal quantity of related to the amount of unused carrying capacity (Shuler and Kargi,
each component required in the medium. This technique, however, 2002).
provides indications of how each component tends to affect enzyme  
X
production (Yu et al., 1997). Bankar et al. (2008) highlighted the μg = k 1 −
X∞
importance of Plackett–Burman experiments for optimizing culture
variables in attaining higher GOD titers. Among the six variables
where X∞is maximum cell mass-produced and k is carrying capacity
which were expected to play a critical role in enhancing GOD produc-
coefficient.
tion, three factors (calcium carbonate, proteose peptone, and magne-
Thus,
sium sulphate) significantly affected enzyme production.
Response surface methodology (RSM) by central composite design  
dX X
(CCD) or by Box-behnken are the tools specifically used in present = kX 1 − ð2Þ
dt X∞
times in fermentation technology to find out the optimum concentra-
tion of the most effective variables for getting higher enzyme titers
Integrating Eq. (2) with boundry conditions X (0) = Xo yields the
and to study their interactions. Various statistical software packages
logistic curve.
are available for statistical optimization of variables. Liu et al. (2003)
applied RSM to optimize the speed of agitation and rate of aeration for Xo e k;t
maximum production of GOD by A. niger. They found aeration to have X= Xo

1− X∞ 1 − e k;t
a more negative effect on GOD production than agitation. Significant
negative interaction existed between agitation and aeration. RSM was
Eq. (2) can be rewritten as
also successfully employed for determination of optimum concentra-
tion of media components by Bankar et al. (2008). They found maxi-  
1 dX X
mum GOD production at 3.08% calcium carbonate, 0.97% peptone and = k 1−
X dt X∞
0.1% magnesium sulphate.
or
2.4. Mathematical model for glucose oxidase kinetics
 
1 dX X
k= = 1−
It is generally accepted that the GOD is a primary metabolite, but it X dt X∞
is difficult for even Luedeking and Piret model (Luedeking and Piret,
1959; Crueger and Crueger, 1990) to explain the levels of GOD pro- The kinetics of product formation is based on the Luedeking–Piret
duced in the medium as alone. These levels depend on microbial equation (Luedeking and Piret, 1959). According to this model, the
 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501
product formation rate (rP) depends on both the instantaneous
biomass concentration and the growth rate in a linear manner.
dp dX
rp = =α + βX
dt dt
where α and β are the product formation constants and may differ
under different fermentation conditions.
Miron et al. (2002) found the kinetics of the GOD to be affected by
substrate inhibition, competitive inhibition by gluconic acid, decrease
of the reaction rate due to diffusional restrictions determined by the
viscosity of the gluconic acid, and decrease in the reaction rate due to
enzyme deactivation by H2O2. The last mentioned is a feature with
some phenomenological resemblance to true substrate inhibition, and
which disappears when catalase is present.
Liu et al. (2003) proposed a simple model using the Logistic
equation for growth, the Luedeking–Piret equation for GOD produc-
tion and Luedeking–Piret-like equation for glucose consumption. They
showed the biosynthesis of GOD to be strongly linearly related to the
growth, and that the correlation coefficient was very high. These
results showed that the biosynthesis of GOD could be growth asso-
ciated. The model provided a reasonable description for various ki-
netic model parameters during the growth phase.
3. Genetic expression for glucose oxidase production
As explained in section 1, the fungal GOD are homodimers of
approximately 150–170 kDa containing two tightly, but non-cova-
lently bound FAD cofactors, and about 11–13% carbohydrate moiety
of the high-mannose type. Typical problems that are usually encoun-
tered during their production are either low productivity or con-
comitant production of other enzymes such as CAT. To overcome these
problems, use of genetically modified microorganisms rather than
natural sources for the expression of this enzyme has been strongly
suggested (Park et al., 2000).
Despite the abundant availability of commercial GOD, there is still
considerable interest in its new forms with useful properties for
special applications in biotechnology. With a view to improve the
properties of enzyme by protein engineering, the GOD gene of A. niger
was characterized (Frederick et al., 1990; Kriechbaum et al., 1989;
Hatzinikolaou et al., 1996) and its crystal structure was elucidated
(Hecht et al., 1993).
Malherbe et al. (2003) expressed the A. niger gene encoding GOD
in S. cerevisiae and evaluated the transformants for lower alcohol
production and inhibition of wine spoilage organisms such as acetic
acid bacteria and lactic acid bacteria during fermentation. They devel-
oped tailored strains of S. cerevisiae for biopreserved wines with lower
alcohol content. To test this novel concept, an antimicrobial yeast
starter culture system, on selective agar plate and in liquid assay was
done. The yeast transformants displayed antimicrobial activity in a
plate assay due to production of H2O2, a final product of GOD enzy-
matic reaction and also a known antimicrobial agent. Production of δ-
glucono-1, 5-lactone and gluconic acid from glucose by GOD resulted
in wines containing 1.8–2.0% less alcohol.
In the last few years, yeasts such as Hansenula polymorpha and S.
cerevisiae have been investigated as promising high-yield production
systems and suggested for heterologous GOD production; but further
studies showed hyperglycosylation with yeast which may lead to
serious limitations of usage (Romanos et al., 1992). Expression of
recombinant GOD using E. coli (Witt et al., 1998) and S. cerevisiae
(Frederick et al., 1990; Ko et al., 2002) has always shown limitations.
In the case of E. coli, 60% of the recombinant protein was inactive,
whereas the recombinant GOD expressed in S. cerevisiae was hypergly-
cosylated and thus characterized by reduced substrate binding capacity
and catalytic activity (Kapat et al., 1998). Crognale et al. (2006) used
methylotrophic yeast Pichia pastoris as host for expression and secretion
495
of recombinant GOD of the filamentous fungus P. variabile P16. They
transformed the gene to P. pastoris X33, a strain largely used for selection
on zeocin and large scale growth studies. They demonstrated P. pastoris
to be an efficient host for expression of both secreted and intracellular
ð3Þ
heterologous proteins. Fermentation in 3 l fermenter lead to GOD
production of up to 50 U/ml that represents approximately four times
increase in the production as compared to P. variabile P16 cultivated
under optimized conditions. Kriechbaum et al. (1989) described the
cloning and sequencing of the GOD gene of A. niger NRRL-3 including 5′
and 3′ flanking regions. They also represented the DNA-derived amino
acid sequence of GOD and showed its identity with peptide sequences
determined for parts of this protein.
The yeast has potential to perform many post-translational modi-
fications, typically associated with higher eukaryotes such as proces-
sing of signal sequences, folding, formation of disulfide bridges,
certain types of lipid addition, and O- and N-linked glycosylation.
Since yeast requires minimal salt media, it contributes to cost effective
industrial production. Recombinant yeast expression plasmids have
been constructed by Frederick et al. (1990) containing a hybrid yeast
alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogen-
ase promoter, either the yeast α factor pheromone leader or the GOD
presequence, and the mature GOD coding sequence. When trans-
formed into yeast, these plasmids direct the synthesis and secretion of
between 75 and 400 μg/ml of active GOD. Analysis of the yeast-
derived enzymes showed that they are of comparable specific activity
and have more extensive N-linked glycosylation than the A. niger
protein.
Kriechbaum et al. (1989) constructed a library of the A. niger
NRRL-3 genome in the phage A substitution vector EMBL3. They
isolated one hybridizing clone which contained an insert of 15 kbp,
from 12000 recombinant plaques with the nick-translated 800 bp
cDNA fragment. The phage DNA was cleaved with SalI and the
resulting fragments were subcloned into pBluescript SK (+). They
used hybridization techniques and the shotgun sequencing method
for identification of 1.8 kbp and 2.0 kbp SalI fragment containing the
coding region of GOD as well as small 5′ and longer 3′ untranslated
regions.
4. Downstream processing
Development of new and efficient separation processes is based
on effectively exploiting differences in the actual physicochemical
properties of the product such as surface charge / titration curve,
surface hydrophobicity, molecular weight, biospecificity towards cer-
tain ligands (e.g. metal ions, dyes), isoelectric point (pI) and stability,
compared to those of the contaminant components in the crude broth
(Asenjo, 1993). The crucial step after completion of successful fermen-
tation is recovery of GOD. Fig. 4 illustrates a general protocol for
purification of GOD.
GOD has been purified for commercial application from different
fungi including A. niger (Hatzinikolaou et al., 1996; Kalisz et al., 1991;
Swoboda and Massey,1965) and Penicillium species such as P. pinophilum
(Rando et al., 1997), P. amagasakiense (Kusai et al., 1960; Kalisz et al.,
1997), P. chrysogenum (Eriksson et al., 1987), P. notatum (Gorniak and
Kączkowski, 1974), and P. funiculosum (Eryomin et al., 2004).
GOD is known to be produced intracellularly or extracellularly or
sometimes as mycelia-associated enzyme. Hence cells have to be
disrupted for complete release of GOD into the broth. The intra- or
extracellular location of the enzyme of A. niger and Penicillium species
has been the subject of numerous discussions. In the meantime, the
periplasmic location of the A. niger GOD was clearly demonstrated
(Witteveen et al., 1992), which is in agreement with the presence of a
signal sequence preceding the A. niger GOD gene (Kriechbaum et al.,
1989; Frederick et al., 1990). As a consequence of peripheral location,
the release of the enzyme from mycelium may be facilitated by
mechanical and physical forces, e.g. agitation and/or sonication.
496 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501
Fig. 4. General protocol for purification of glucose oxidase.
Various methods of cell disruption have been used for filamentous
fungi, including homogenization (Hatzinikolaou et al., 1996; Fiedurek
and Gromada, 1997), sonication (Lu et al., 1996) and a combination of
both (Hatzinikolaou and Macris, 1995). A comprehensive study of
different methods for the disruption of two filamentous fungi, Gano-
derma applanatum and Pycnoporus cinnabarinus was performed by
Taubert et al. (2000). They concluded that fungal cells were parti-
cularly resistant to some of the disintegration methods commonly
used for yeasts and bacteria as well as the mechanical and non-
mechanical cell disruption methods described by Christi and Moo-
Young (1986). For release of intracellular as well as cell-bound GOD
into the liquid broth, various methods like sonication, bead mill,
homogenizer and freeze-thawing were applied. After the disruption of
the cells, GOD is released in the fermentation broth which may be
separated from the cells either by differential centrifugation or by
filtration.
Various precipitation techniques have been used for purification of
GOD including ammonium sulphate (Kalisz et al., 1997; Kusai et al.,
1960; Swoboda and Massey, 1965), uranyl acetate (Gorniak and
Kączkowski, 1974), potassium hexacyanoferrate and copper sulphate
(Eriksson et al., 1987). Ammonium sulphate precipitation has been
successfully employed to precipitate both intra- and extracellular GOD
with different percent cut of ammonium sulphate. The differences in
ammonium sulphate precipitation characteristics for intra- and
extracellular GOD may be attributed to the fact that GOD from Peni-
cillium species are known to be glycosylated. GOD from P. amagasa-
kiense is a glycoprotein which contains 11–13% carbohydrate
described as the high-mannose type (Kusai et al., 1960; Eriksson
et al., 1987; Nakamura and Fujiki, 1968).
Precipitation is followed by chromatographic separation techni-
ques such as ion exchange chromatography. On an average, the pI of
GOD has been found in the range of pH 4 to 5 (Eriksson et al.,1987; Kalisz
et al.,1997; Kusai et al.,1960). Hence an anion exchange chromatography
is commonly used for its purification (Kalisz et al., 1997; Swoboda and
Massey, 1965; Dai et al., 2002; Hatzinikolaou et al., 1996). GOD is mainly
eluted with salt gradients using NaCl (Hatzinikolaou et al., 1996; Rando
et al., 1997; Dai et al., 2002), although mixed pH and salt gradients have
previously been used (Kalisz et al., 1997). The pI of CAT from P.
chrysogenum was reported to be 6.5 (Eriksson et al., 1987). The differ-
ences in pI values between GOD and CAT could therefore be exploited to
assist in the purification of GOD to ensure that, it is free from CAT by
using anion exchange chromatography, since the separation with this
method is based on differences in pI differences. GOD from P.
amagasakiense has been reported to contain 4 different isoenzymes of
pI values 4.37; 4.42; 4.46 and 4.51 (Kalisz et al., 1997).
GOD has a negative charge in double distilled water due to its pI
being 4–5. It is therefore adsorbed at the beginning of the column.
On eluting with the elution buffer (pH 3.6), GOD becomes positively
charged and get desorbed from the anionic exchange resin. The
different eluted fractions of GOD carries different amounts of nega-
tive charges and this separates them from each other. The first
separated part, GOD A has less charge than GOD B in the purification
process; therefore influence of charge impulse to the conformation
of GOD A is small and has a higher enzyme activity. GOD B carries
more negative charge than GOD A, and therefore it can only be
eluted after GOD A and also destroys the native conformation and
decreases the enzyme activity (Dai et al., 2002). Rando et al. (1997)
used a very efficient and elaborate procedure for the purification of
GOD from P. pinophilum. They purified GOD to apparent homo-
geneity with a yield of 74% by including an efficient extraction step
of the mycelium mass at pH 3.0 and ion-exchange chromatography
followed by gel filtration.
5. Immobilization of glucose oxidase
Enzyme immobilization has attracted a wide range of interest from
fundamental academic research to many different industrial applica-
tions. To date, several immobilized enzyme-based processes have
proved to be economical. They have been implemented on a larger
scale, mainly in the food industry, where they replace free enzyme-
catalyzed processes, and in the manufacture of fine specialty
chemicals and pharmaceuticals, particularly where asymmetric syn-
thesis or resolution of enantiomers to produce optically pure products
are involved (Krajewska, 2004).
Varieties of supports have been used for immobilization of en-
zymes such as cellulose, solid glass particles, porous glass particles,
and nickel screen. Because of the advantages in catalytic activity
offered by materials having relatively high surface areas, porous glass
and cellulose have been the most popular supports. For example, the
preparation and to improve the storage stability of GOD on porous
glass by γ-amino-propyltriethoxysilane (APTES) was used by various
researchers for silanization of the glass; prior to immobilization of
GOD. Immobilized GOD on both solid and porous glass was also
performed by some researchers (Bouin et al., 1976; Herring et al., 1972;
Sreedivya et al., 1998; Wasilewska et al., 1987; Wassrman and Hultin,
1980). In addition, nickel oxide screen can be silanized and GOD can
be coupled by thiophosgene method, but the screen offers little
surface area (Herring et al., 1972). GOD is difficult to crosslink with an
agent like glutaraldehyde, but does crosslink rather easily in the pre-
sence of certain proteins that have high number of reactive amino
groups such as polyethyleneimine (Bouin et al., 1976).
Co-immobilization of GOD and CAT were studied by several inves-
tigators (Tarhan and Telefoncu, 1990; Blandino et al., 2002; Tarhan and
Telefoncu 1992; Godjevargova et al., 2004; Podual et al., 2000).
Ozyilmaz and Tukel (2007) used inorganic and porous magnesium
silicate (Florisil) as a support for simultaneous co-immobilization of
GOD and CAT. Basic property of carrier may play a special role in
partial neutralization of gluconic acid produced by bound GOD in the
pores of the carrier. This prevents a dramatic decrease in pH of
microenvironment of bound enzymes, which may hinder their dena-
turation and thereby enhance the stability of co-immobilized GOD/
CAT.
Enzymes are covalently linked to the support through functional
groups in the enzymes, which are not essential for catalytic activity.
But, it is known that immobilization decreases enzyme activity due to
blocking of the active site or due to changes in the enzyme geometry
at the end of the coupling procedure. Addition of a substrate or a
competitive inhibitor to the coupling mixture protects the active site
on the enzyme against loss of activity (Srere et al., 1976). Several
studies are available in literature on immobilization of enzymes in the
presence of their substrates.
 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501
To protect the active sites of GOD and CAT, co-immobilization
was carried out in the presence of the glucose which is a substrate of
GOD. During the coupling period, GOD oxidizes glucose to produce
H2O2 which is a substrate of CAT. Thus, co-immobilization is carried
out in the presence of both glucose and H2O2, which are substrates
of GOD and CAT, respectively (Ozyilmaz and Tukel, 2007). Ozyilmaz
and Tukel (2007) further reported the maximum activities of co-
immobilized GOD and CAT in the presence of 15 and 20 mM glucose,
respectively. Co-immobilization of GOD and CAT in the presence of
their substrates significantly improves the activity and reusability of
both enzymes. GOD is inactivated by H2O2, the concentration of
which increases during the catalytic turnover (Kleppe, 1966). H2O2
inactivation can be reduced with CAT which reduces H2O2 and
eventually removes it from the system. By this reaction, some
oxygen is recovered and made available for the oxidation of glucose
(Fig. 5). Buchholz and Godelmann (1978) explained the reaction
mechanism and deactivational parameters such as H2O2 deactivation
and conversion of reduced GOD into oxidized GOD (Fig. 5). CAT plays a
key role to prevent GOD from H2O2 deactivation by converting H2O2
into H2O and O2. Although CAT has preventive action in GOD/CAT
system, its concentration has little effect on the activity of the GOD on
the support.
Due to superior bonding properties of CAT and higher concentra-
tion of CAT in the immobilizing solution, CAT activity is considerably
higher in co-immobilized system than GOD. The clear dependence of
measured activity on particle size is due to both external film and
internal diffusional resistances. Experiments in which GOD and CAT
adsorbed directly onto the supports without pretreatment with γ-
APTES or glutaraldehyde indicated activities up to 10 and 40% of the
glutaraldehyde-bonded system were obtained for GOD and CAT
system, respectively (Markey et al., 1974).
Bouin et al. (1976) reported optimal coupling of GOD to occur
above pH 6. Below pH 3, the immobilized GOD completely and
irreversibly lost activity. The effect of pH over the range from 4.5 to 8.0
on immobilization of CAT has also been evaluated. An increase in pH
up to pH increased the activity of CAT. It appeard that CAT activity had
little effect on the activity of GOD on the support. CAT concentration
was never high enough to interfere significantly with GOD coupling
(Ramchandran and Perlmutter, 1976).
6. Characterization of glucose oxidase
6.1. Substrate specificity
GOD is highly specific for β-D-glucose and show only marginal
activities with other sugars. β-D-glucose gets oxidized in the presence
of molecular oxygen at C-1 position to δ -glucono-1, 5-lactone, which
is, in turn is spontaneously hydrolyzed to D-gluconic acid. These
structural features and the regioselectivity of the reaction allows a
clear distinction between GOD and pyranose oxidase, which is a
Fig. 5. Simplified scheme for transport and reaction processes in the heterogeneous
system (Buchholz and Godelmann, 1978).
497
homotetramer, and oxidizes D-glucose at the C-2 position (Danneel
et al., 1993; Hatzinikolaou et al., 1996; Pluschkell et al., 1996).
6.2. pH optima and stability
Since enzyme activity is dependent on the ionization state of the
amino acids in the active site, pH plays an important role in
maintaining the proper conformation of an enzyme. Most proteins
are only active within a narrow pH range, usually in the range of 5–9
(Wilson and Walker, 1995; Voet and Voet, 1995). The pH optima of
GOD vary from 5.0 to 7.0. GOD from most fungi and yeast have pH
optima in the acidic to neutral range such as A. niger and P. chrysogenum
shows pH optima of 5.0 to 6.0. (Kalisz et al.,1991; Eriksson et al.,1987). In
contrast, the GOD obtained from P. funiculosum 433 and P. canescens
show slightly alkaline pH optima of 6 to 8.6 (Sukhacheva et al. (2004).
6.3. Optimum temperature and stability
Enzymes are known to be sensitive to changes in temperature. The
relationship between reaction rate of an enzyme and temperature is
exponential. For every 10 °C rise in temperature, the rate of an enzyme
reaction doubles. At temperature range between 40 °C and 70 °C most
enzymes get denaturated and lose their activity. Enzymes are known
to display maximal activity at a temperature known as the optimum
temperature of the enzyme (Wilson and Walker, 1995). The lowest
optimum temperature for GOD is reported to be 25–30 °C from P.
funiculosum 433 (Sukhacheva et al., 2004) and the highest of 40–60 °C
from A. niger and P. amagasakiense ATCC 28686 (Kalisz et al., 1991,
1997).
6.4. Variation of the initial rate with enzyme concentration
Michaelis and Menten determined that the initial rate or velocity
of catalysis of an enzyme varied hyperbolically with substrate
concentration (Voet and Voet, 1995). The initial rate increases with
an increase in substrate concentration to a point where it would reach
maximum velocity (Vmax). At low substrate concentrations, initial rate
is proportional to the substrate concentration, referred to as first order
kinetics. At high substrate concentrations the initial rate is indepen-
dent of substrate concentration, referred to as saturation or zero order
kinetics. GOD at 0.2 U/ml could be accurately determined using the
GOD dye binding assay (Bergmeyer et al., 1974). At higher GOD
activities, the initial rates begin to decrease which lead to inconsistent
activity determinations.
6.5. Kinetic parameters variability
The Michaelis constant (Km) and the maximal limiting rate velocity
(Vmax) for GOD were calculated by various researchers and they found
slight differences in their values. The Km value of the GOD from T. favus
is 10.9 mM (Kim et al., 1990) and is 33 mM from A. niger (Swoboda
and Massey 1965). P. amagasakiense ATCC 28686 and P. funiculosum
433 shows lower Km value of 5.7 mM and 3.3 mM respectively (Witt
et al., 1998; Sukhacheva et al., 2004). It should also be noted that
reestimations of the Km values of glycosylated and deglycosylated
P. amagasakiense GOD gave values of 3.4 mM and 2.7 mM respectively
(Kim and Schmid 1991). Vmax value of GOD ranges between 450
to 1000 U/mg. A. niger shows Vmax value of 458 U/mg while P.
amagasakiense ATCC 28686 exhibited 925 U/mg (Witt et al., 1998;
Kalisz et al., 1991).
6.6. Storage stability
GOD has half-life of approximately 30 min at 37 °C. Immobilized
GOD would be more effective for applications at 37 °C. Polyhydric
alcohols including ethylene glycol, glycerol, erythritol, xylitol, sorbitol
498 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501
and polyethylene glycol have shown stabilizing effect on GOD from A.
niger (Ye et al., 1988). The lyophilized GOD preparation remains stable
for a minimum of 6 months at −20 °C.
7. Applications of glucose oxidase
GOD has gained considerable commercial importance in the last
few years due to its multitude of applications in the chemical,
pharmaceutical, food, beverage, clinical chemistry, biotechnology and
other industries. GOD is the most widely used enzyme as an analytical
reagent for determination of glucose due to its relatively low cost and
good stability. Its uses range from a glucose biosensor for the control
of diabetes, to a food preservative and color stabilizer. With co-
immobilized enzymes, improved stability, reusability, continuous
operation, possibility of better control of reactions, high purity and
product yields and economical process can be expected (Kleppe,
1966). Some of its current applications in industry are described here.
7.1. Glucose biosensor for diabetes monitoring
People with diabetes mellitus need to constantly monitor their
blood glucose levels in order to detect fluctuations in glucose level
that could lead to hyperglycemia (high blood glucose levels) and
hypoglycemia (low blood glucose levels) so as to control the disease.
Currently, such monitoring is done using finger-prick blood samples
and a portable meter several times a day. Biosensors are being
developed to measure blood glucose levels. GOD is one of the possible
enzymes that can be used in biosensor. Biosensors work by keeping
track of the number of electrons that pass through an enzyme by
connecting it to an electrode and measuring the resultant charge.
Alternatively, some biosensors use sensitive fluorescence measure-
ments, monitoring changes in the intrinsic FAD fluorescence of GOD
(Wilson and Turner, 1992).
Various GOD based biosensors are as listed below:
1. On line glucose monitoring for fermentations (Vodopivec et al.,
2000).
2. Fibre optic biosensor for analyzing glucose concentrations in soft
drinks (Chudobova et al., 1996).
3. Disposable strip-type biosensor for blood and serum monitoring
(Cui et al., 2001).
4. Strip type biosensor for blood (GOD-HPR-dye) (Kim et al., 2001).
5. Miniaturized thermal biosensor for whole blood (Harborn et al.,
1997).
6. Glucose sensor for whole blood (Santoni et al., 1997).
7. Glucose biosensor for serum from human blood (Zhu et al., 2002).
7.2. Biofuel cells
Bio-electronic devices are energy demanding, requiring small
power sources to sustain operations. Biofuel cells convert biochemical
energy into electrical energy using a biocatalyst. One type of biofuel
cell uses enzymes as a biocatalyst. For example, GOD and micro-
peroxidase-8 can be used on the cathode, where the H2O2 produced
by GOD oxidizes microperoxidase-8 to directly accept electrons from
the carbon rod electrode. Biofuel cells consist of a two electrode set (of
any stable and electrically conducting material) modified by biocata-
lytic enzymes to specifically oxidize/reduce substrates. One approach
towards the design of an implantable, membraneless and biocompa-
tible biofuel cell consists of catalyzing the oxidation of glucose at the
anode using either GOD or glucose dehydrogenase enzymes. These
enzymes are coupled to the reduction of dioxygen at the cathode by a
dioxygen-reducing enzyme such as laccase, bilirubin oxidase or
cytochrome oxidase (Chen et al., 2001; Mano et al., 2002; Soukharev
et al., 2004). Electron transfer to/from the biocatalytic active sites can
be mediated by polymer bound or entrapped redox complexes
(Barton et al., 2004). Both water-soluble fuel molecules (glucose
and O2) are found in body fluids and in blood at 10 and 0.1 mM,
respectively. Besides, these molecules are converted at the electrodes
into naturally occurring degradation molecules in low concentration
(gluconolactone and water). The maximum theoretical electromotive
force (emf) allowed by the thermodynamics of glucose oxidation and
dioxygen reduction at physiological pH is approximately 1 V.
7.3. Food and beverage additive
GOD has been used successfully to remove residual glucose and
oxygen in foods and beverages in order to prolong their shelf life. The
H2O2 produced by the enzyme acts as a good bactericide, and can later
be removed using a second enzyme, CAT that converts H2O2 to oxygen
and water. GOD/CAT is used to remove glucose during the
manufacture of egg powder, preventing browning during dehydration
caused by the Maillard reaction for use in baking industry, providing
slight improvements to the crumb properties in bread and croissants
(Rasiah et al., 2005; Crueger and Crueger, 1990).
GOD can also be used to remove oxygen from the top of bottled
beverages before they are sealed. GOD/CAT system is shown to control
non-enzymatic browning during fruit processing and puree storage.
The scavenging of the oxygen by the enzyme system had a stabilizing
effect. In addition, GOD is used to prevent color and flavor loss as well
as to stabilize color and flavor in beer, fish, tinned foods and soft drinks
by removing oxygen from foods and beverages (Crueger and Crueger,
1990). For example, they are used to reduce the discoloration
occurring in wines and mayonnaises. GOD/CAT enzyme system can
be used to retard the lipid oxidation in mayonnaise stored at 5 °C and
25 °C, in mayonnaises containing pure soybean oil, and where up to
half the vegetable oil had been supplemented with fish oil. The
enzyme system was responsible for scavenging the oxygen during
glucose oxidation thereby decreasing the availability of the oxygen for
lipid metabolism (Isaksen and Adler-Nissen, 1997). Bonet et al. (2006)
studied the effect of GOD on dough rheology and bread quality and
showed the strengthening of wheat dough and an improvement in
bread quality on addition of GOD. However, the enzyme level must be
very carefully added, since adverse effects were obtained on addition
of excessive enzyme.
7.4. Low alcohol wine production
GOD has potential for use in the wine industry, where it can lower
the alcohol content of wine through the removal of some of the
glucose (by converting it to δ-glucono-1, 5-lactone), which would
otherwise be converted to alcohol. Tests showed that the GOD
treatment of wine-must could reduce the potential alcohol content of
wine by about 2%. In addition, GOD is able to inhibit wine spoilage
through its bactericidal effect on acetic acid bacteria and lactic acid
Fig. 6. Preparation of low alcohole wine from GOD/CAT treated grape juice (Pickering
et al., 1998).
 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501
bacteria during the fermentation process. The bactericidal effect of
an enzyme means fewer preservatives need to be added to the wine
(Malherbe et al., 2003). Pickering et al. (1998) reduced the fermen-
tative alcohol potency by pre-treating the grape juice with the GOD/
CAT enzyme system to convert the available glucose to gluconic acid
(Fig. 6). They achieved 87% of glucose conversion with this system.
The low pH of the grape juice was determined to be a limiting factor,
which was subsequently overcome by the use of calcium carbonate
prior to the enzymatic treatment.
7.5. Oral hygiene
GOD as well as lactoperoxidase can be used as anti-microbial
agents in oral care products (Afseth and Rolla, 1983). The oral cavity
houses several species of Streptococci such as Streptococcus mutans,
which is a significant contributor to tooth decay and is carried by
virtually everyone. The H2O2 produced by GOD acts as a useful
bacteriocide. The ability of GOD to kill S. mutans appears to be
enhanced by the fusion of the enzyme with heavy chain antibodies
(Etemadzadeh et al., 1985).
7.6. Gluconic acid
GOD is also used as a commercial source of gluconic acid, which
can be produced by the hydrolysis of δ-glucono-1, 5-lactone, the end-
product of GOD catalysis. Gluconic acid has been used as a food
additive to act as an acidity regulator, in sterilization solution or
bleaching in food manufacturing, and as a salt in chemical compo-
nents for medication. Gluconic acid is also used as a mild acidulant in
the metal and leather industries. It has even been used in the
construction industry as an additive in cement to increase its resis-
tance and stability under extreme weather conditions. It occurs
naturally in honey, fruit and wine (Crueger and Crueger, 1990; Nakao
et al., 1997; Klein et al., 2002).
7.7. Textile industry
GOD has found applications in the textile industry as a method for
producing H2O2 for bleaching. Tzanov et al. (2002) covalently
immobilized GOD on alumina and glass supports resulting in higher
recoveries. Maximum H2O2 concentrations of 0.35 g/l and 0.24 g/l
were reached after 450 min for GOD immobilized on the glass and
alumina supports respectively. The H2O2 produced was tested for
bleaching scoured woven cotton fabric and was found to be com-
parable to standard bleaching processes. No stabilizers were needed
since the gluconic acid produced acted as a stabilizing agent (Tzanov
et al., 2002). A new concept has been developed by Opwis et al. (2008)
which is based on simultaneous application of GOD and peroxidase.
Starting with glucose as a substrate for GOD, H2O2 was generated in
situ. The freshly formed H2O2 was immediately used by the POD
oxidizing colored compounds in dyeing baths. These enzymes are
used in decoloration process and bleaching of natural fibers in textile
industries.
8. Concluding remark
As evident from the foregoing review, glucose oxidase is among the
most important enzymes used in industrial processes. It is potentially
useful in food, pharmaceutical, biotechnology and flavoring industries.
Commercially viable processes are needed for large-scale production
of the enzyme by fermentation. Only few reports address fermentative
production of glucose oxidase at large scale with economical process
and its subsequent uses in glucose determination or glucose removal
in various analytical methods as well as industries. Although, number
of microbial sources exists for the efficient production of this enzyme,
commercial production of this enzyme has been limited to only a few
499
selected strains of fungi and yeast. Further, there arises a need for
more efficient glucose oxidase in various sectors, which can be
achieved either by chemical modification of the existing enzymes or
through protein engineering. In the light of modern biotechnology,
glucose oxidase is now gaining importance in biopharmaceutical
applications. Although glucose oxidase has long been used in various
industries, technological innovation such as the use of immobilization
supports and continuous-flow systems have been considered recently.
However, satisfactory results in the application of immobilized
glucose oxidase without any limitations have rarely been achieved.
Problems such as diffusional constraints and decrease in the enzyme
activity after immobilization need to be overcome for greater benefits.
Similarly, research efforts on the optimization and design of a suitable
reactor are still required before considering future commercial
applications.
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