Biotechnology Advances 27 (2009) 489–501

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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

⁎ Corresponding author. Tel.: +91 22 24145616; fax:E-mail address: laxmi@udct.org (L. Ananthanarayan

0734-9750/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2009.04.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 12 December 2008Received in revised form 25 March 2009Accepted 7 April 2009Available online 15 April 2009

Keywords:Glucose oxidaseCatalseOptimization of fermentation parametersDownstream processingImmobilizationApplication in biosensors

Glucose oxidase (β-D-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) catalyzes the oxidation of β-D-glucose togluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogenperoxide.Microbial glucoseoxidase is currently receivingmuch attentiondue to itswide applications in chemical,pharmaceutical, food, beverage, clinical chemistry, biotechnology and other industries. Novel applications ofglucose oxidase in biosensors have increased the demand in recent years. Present review discusses theproduction, recovery, characterization, immobilization and applications of glucose oxidase. Production of glucoseoxidase by fermentation is detailed, alongwith recombinantmethods. Various purification techniques for higherrecoveryof glucoseoxidase aredescribedhere. Issues of enzymekinetics, stability studies and characterization areaddressed. Immobilized preparations of glucose oxidase are also discussed. Applications of glucose oxidase invarious industries and as analytical enzymes are having an increasing impact on bioprocessing.

© 2009 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4901.1. Glucose oxidase reaction mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4901.2. Composition of glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4901.3. Characteristics of glucose oxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4911.4. Analysis of glucose oxidase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491

2. Fermentative production of GOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4912.1. Microbial strains producing glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4912.2. Parameters affecting enzyme production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491

2.2.1. Carbon source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4912.2.2. Nitrogen source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4922.2.3. Calcium carbonate as an inducer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4922.2.4. Other medium components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4932.2.5. Effect of aeration and agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4932.2.6. Effect of culture pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4932.2.7. Effect of growth temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4932.2.8. Fed-batch culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

2.3. Optimization by statistical methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4942.4. Mathematical model for glucose oxidase kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

3. Genetic expression for glucose oxidase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4954. Downstream processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4955. Immobilization of glucose oxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4966. Characterization of glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

6.1. Substrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4976.2. pH optima and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4976.3. Optimum temperature and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4976.4. Variation of the initial rate with enzyme concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4976.5. Kinetic parameters variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4976.6. Storage stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

+91 22 24145614.).

l rights reserved.

490 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501

7. Applications of glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4987.1. Glucose biosensor for diabetes monitoring. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4987.2. Biofuel cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4987.3. Food and beverage additive. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4987.4. Low alcohol wine production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4987.5. Oral hygiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4997.6. Gluconic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4997.7. Textile industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499

8. Concluding remark . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499

1. Introduction

Soil, organic waste and plant cell material are a few of the diverseenvironments inwhich filamentous fungi are known to flourish. Fungiproduce a wide range of different enzymes which enables them to usemany organic compounds as nutrient sources (Gouka et al., 1997).Among non-hydrolytic enzymes of fungal origin, glucose oxidase (EC1.1.3.4) has seen large-scale technological applications since the early1950′s (Fiedurek and Gromada, 1997). Glucose oxidase (GOD) hasbeen purified from a range of different fungal sources, mainly from thegenus Aspergillus (Kalisz et al., 1991; Hatzinikolaou et al., 1996) andPenicillium (Eryomin et al., 2004; Sukhacheva et al., 2004), of whichA. niger is the most commonly utilized for the production of GOD(Pluschkell et al., 1996). GOD from Penicillium species has been shownto exhibit more advantageous kinetics for glucose oxidation than thatfrom 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 asan electron acceptor with simultaneous production of hydrogenperoxide (H2O2) (Hatzinikolaou and Macris 1995). GOD has foundseveral commercial applications including glucose removal from driedegg; improvement of color, flavor, and shelf life of food materials;oxygen removal from fruit juices, canned beverages; and frommayonnaise to prevent rancidity. It has also been used in an automaticglucose assay kit in conjunction with catalase (Hatzinikolaou andMacris 1995) and chiefly in biosensors for the detection andestimation of glucose in industrial solutions and in body fluids suchas blood and urine (Petruccioli et al., 1999). GOD is reported to havethe best antagonistic effect against different food-borne pathogenssuch 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 oftoothpaste (Petruccioli et al., 1999), for the production of gluconicacid, and as a food preservative (Pluschkell et al., 1996). Implantableglucose 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-

Fig. 1. Representation of the GOD

erable interest despite the abundant availability of commercial GOD(Rando et al., 1997).

1.1. Glucose oxidase reaction mechanism

GOD is a flavoprotein which catalyses the oxidation of β-D-glucoseto D-glucono-δ-lactone and H2O2 using molecular oxygen as anelectron 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 oxidationof β-D-glucose to D-glucono-δ-lactone, which is non-enzymaticallyhydrolyzed to gluconic acid. Subsequently the flavine adeninedinucucleotide (FAD) ring of GOD is reduced to FADH2 (Witt et al.,2000). In the oxidative half reaction, the reduced GOD is reoxidizedby oxygen to yield H2O2. The H2O2 is cleaved by catalase (CAT) toproduce water and oxygen. Witteveen et al. (1992) found the enzymelactonase (EC 3.1.1.17) in A. niger to be responsible for catalyzing thehydrolysis of glucono-δ-lactone to gluconic acid.

1.2. Composition of glucose oxidase

GOD from ascomycetes is a dimeric glycoprotein consisting of twoidentical polypeptide chain subunits that are covalently linkedtogether via disulfide bonds (Rando et al., 1997; Kalisz et al., 1997).Fig. 2 depicts the FAD moiety and the key conserved active siteresidues of a GOD subunit from P. amagasakiense (Wohlfahrt et al.,1999). The structure of the P. amagasakiense GOD shows each of itssubunit to contain onemole of tightly bound, but not covalently linkedFAD moiety as co-factor (Rando et al., 1997; Witt et al., 2000). GODfrom 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 andHis-563 (Fig. 2) (Witt et al., 2000). Witt et al. (2000) concluded thatArg-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

reaction (Witt et al., 2000).

Fig. 2. Glucose oxidase from P. amagasakiense showing the FAD moiety, indicating the key conserved active site residues (Wohlfahrt et al., 1999).

491S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501

aromatic residues Tyr-73, Phe-418 and Trp-430were important for thecorrect orientation of the substrate as well as for the maximal velocityof glucose oxidation. His-520 and His-563 form hydrogen bonds withthe 1-OH of glucose during the reaction.

1.3. Characteristics of glucose oxidase

The molecular weight of GOD ranges from approximately 130 to175 kDa (Kalisz et al., 1997). GOD is highly specific for the β-anomer ofD-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 studieson the GOD of A. niger and P. amagasakiense, and found their molec-ular weights to be 152 and 150 kDa, respectively. GOD produced byboth the strains had similar carbohydrates, which consisted mainly ofglucose, mannose and hexosamine. A. niger GOD contained moremannose and hexosamine than that of P. amagasakiense, but lessglucose. The overall carbohydrate content was found to be 16% for A.niger and 11% for P. amagasakiense. The amino acid content of twoenzymes 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. nigerand 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 rangethan that from P. amagasakiense GOD (Nakamura and Fujiki, 1968).

1.4. Analysis of glucose oxidase activity

Literature depicts various analytical methods for determination ofGOD. Tongbu et al. (1996) used titrimetric method for determinationof the GOD. In this method, enzyme solution was added to sodiumacetate buffer containing 2% β-D-glucose and the reactionwas stoppedby adding sodium hydroxide solution. The resulting mixture wastitrated with standard hydrochloric acid solution to calculate volumeof added standard HCl and thereby to calculate GOD activity.

Most researchers use an analytical method for GOD that is basedon the principle that GOD oxidizes β-D-glucose in the presence ofoxygen to β-D-glucono-δ-lactone and H2O2. The H2O2 is then utilizedto oxidize a chromogenic substrate in a secondary reaction withhorseradish peroxidase (HRP) with a resultant color change that

Scheme 1. GOD reaction with ABTS as a chromagenic dye (Witt et al., 1998).

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 (Bergmeyeret al., 1974). ABTS forms a greenish-blue oxidized product that is mea-sured spectrophotometrically at 420 nm. The reactions involved arerepresented 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) usedbenzoquinone for spectrophotometric measurement of GOD activity.Reaction mixtures containing 1 M glucose, 0.1% benzoquinone, and0.1 M Na-citrate buffer, pH 5.0 were preincubated at 25 °C and thereactionwas initiated by adding the enzyme solution. Themethodwasbased on enzymatic reduction of benzoquinone by hydroquinonewhich 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 themethod was useful to study the kinetic properties of GOD since thesubstrate 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 thefeasibility of following the reaction by quantifying δ-gluconolactoneformation.

2. Fermentative production of GOD

2.1. Microbial strains producing glucose oxidase

The most common microbial sources for fermentative productionof GOD are Aspergillus, Penicillium, and Saccharomyces species. Most ofthe commercially produced GOD is isolated from mycelium of Asper-gillus niger, grown principally for the production of gluconic acid or itssalts such as sodium gluconate or calcium gluconate. Accordingly, theenzyme is obtained essentially as a by-product or co-product ofgluconate production. Table 1 compiles detailed information on GODproduction, production media and different assay methods used byvarious researchers.

2.2. Parameters affecting enzyme production

2.2.1. Carbon sourceDuring 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).

Table 1Various microorganisms producing GOD.

Microorganism Media composition (g/l) Assay method Yield (Unit) References

Penicillium variabile P16 Glucose,80; NaNO3,5; KCl,0.5; KH2PO4,1; FeSO4.7H2O,0.01;mycological peptone, 1; CaCO3 35.

Reduction of benzoquinone by hydroquinonemeasured by the rate of absorbance increase at290 nm

5.52 U ml−1 Petruccioli et al. (1999).Petruccioli et al. (1995b)

A. niger glucose oxidasegene expressed in S.cerevisiae

Yeast peptone dextrose (YPD) medium: Yeast extract, 10;peptone,20; glucose,20.

Plate assay: with o-dianisidine Coupled coupledo-dianisidine-peroxidase reaction.

125 U ml−1 Hodgkins et al. (1993).Malherbe et al. (2003).

Aspergillus niger (BTL) Sucrose,70; (NH4)2HPO4,0.4; KH2PO4,0.2; MgSO4.7H2O,0.2;peptone,10; CaCO3,35.

Coupled o-dianisidine-peroxidase reaction. 7.5 Uml−1 Hatzinikolaou and Macris(1995).

RecombinantSaccharomycescerevisiae

Yeast extract,40; hycas, 5; glucose, 20; galactose,30. Coupled ABTS reaction 3.43 U mg−1

dry cell massKapat et al. (2001).

Aspergillus niger AM111 Glucose,80; peptone,30; NaNO3,0.5; KH2PO4,1; CaCO3,35. Coupled reaction assay 2.5 U ml−1 Fiedurek and Gromada(2000).

Penicillium pinophilumDSM 11428

Sucrose,40; Na2PO4.2H2O,4.45; KH2PO4,1.5; NaNO3, 1.9;MgSO4.7H2O,0.2; CaCl2.2H2O, 0.02; malt extract,10; yeastextract,5; Trace element,10; vitamine,10.

Coupled ABTS -peroxidase reaction. 1.9 U ml−1 Rando et al. (1997).

Aspergillus niger ZBY-7 Glucose,150; inorganic salts,0.35; metal caronate,35; Titrimetric 6 U ml−1 Tongbu et al. (1996).

492 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501

the synthesis of polysaccharide and as energy source. The rate atwhich carbon source is metabolized can often influence the formationof biomass or production of primary or secondary metabolites. Fastgrowth due to high concentration of rapidly metabolized sugars isoften associatedwith high productivity of growth-associated productsor primary metabolites (Stanbury et al., 1997).

Hatzinikolaou and Macris (1995) investigated the effects ofdifferent carbon sources on growth and total GOD activity for A.niger. Although A. niger grew on all the carbon sources that theytested, 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 byinvertase) was the principal inducer for the transcription of the GODgene. Kona et al. (2001) used sucrose as carbon sourcewhen they usedeconomical nutrient containing corn steep liquor for A. nigerfermentation. Petruccioli et al. (1993) studied GOD production by84 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 sourceproduced GOD activity ranging from 0.61 U/ml to 5.45 U/ml. Thestrains mentioned were investigated for their ability to oxidizeglucose, fructose, mannose, galactose, arabinose and xylose. Onlyone of the P. italicum strains (NRRL 983) displayed enhanced oxidizingactivity towards mannose, galactose, and xylose being 32.38%, 17.90%and 26.40% compared to glucose (100%), respectively (Petruccioli etal., 1993). Petruccioli et al. (1997) investigated the effect of 10 differentcarbon sources on growth and GOD production of P. variabile mutantM80.10, and found cultivation with only glucose and mannose toproduce 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 withRogalski et al. (1988) who also reported 8% glucose to be optimal forGOD in the A. niger G-13 mutant. Higher glucose concentrationsdecreased the mycelial mass, culture pH and GOD concentrations.Kusai et al. (1960) reported sucrose to be the best carbon source forthe production of GOD by P. amagasakiense; although if the pH ofgrowth medium was maintained during cultivation, glucose was thecarbon source of choice.

2.2.2. Nitrogen sourceInorganic nitrogen is supplied as ammonia gas, ammonium salts or

nitrates. Ammonia has been used for pH control. Ammonium saltssuch as ammonium sulphate usually produces acidic conditions as theammonium ion gets utilized and the free acid is liberated. On the otherhand, nitrates will normally cause an alkaline drift as they getmetabolized. Ammonium nitrate will first cause an acid drift as the

ammonium ion is utilized, and nitrate assimilation is repressed. Whenthe ammonium ion gets exhausted, there is an alkaline drift as thenitrate is then used as an alternative nitrogen source (Morton andMacMillan, 1954). Rogalski et al. (1988) showed that when cultivatingA. niger mutant G-13 and supplementing the growth media with 3%peptone there was 36% and 42% increase in GOD activity and biomassproduction, respectively. Hatzinikolaou and Macris (1995) investi-gated different nitrogen sources on the growth and total GOD activityof A. niger cultivated on sucrose and molasses as sole carbon sources.They found that the peptone concentration had a marked effect on thetotal 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 steepliquor 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 to640 Uml−1, and that other nitrogen sources did not further improvedthe enzyme synthesis.

2.2.3. Calcium carbonate as an inducerPetruccioli et al. (1995a) found that the addition of calcium car-

bonate to growthmedium in shake flasks and in fermenters preventedpH drop during cultivation, which is necessary for optimal GOD pro-duction. Rogalski et al. (1988) showed that the synthesis of GOD wassensitively influenced by increasing concentrations of calciumcarbonate (0–4.5%) with maximal GOD activity at approximately3.5%. Hatzinikolaou and Macris (1995) reported calcium carbonate tobe a strong inducer of GOD in A. niger and demonstrated it to beessential for increased levels of GOD production. Optimum calciumcarbonate of 4 and 5% was observed for GOD production using sucroseand molasses respectively. Bankar et al. (2008) found 3% of CaCO3 tobe 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 calciumcarbonate (optimum 4%), which also maintained the pH of thecultivation media between 6.5 and 6.8. They showed that the activityof the glycolytic enzyme, glucose-6-phosphate isomerase, was higherin growth media without calcium carbonate, while that of GOD andcatalase (CAT) were quite low. Inclusion of calcium carbonateincreased the GOD and CAT activities with a simultaneous decreasein the glucose-6-phosphate isomerase activity. They suggested thatthe addition of calcium carbonate in the growth media might cause ametabolic shift from glycolysis to the pentose phosphate pathway,thereby increasing GOD levels. Addition of calcium carbonate in thegrowth 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

493S.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 carbonateproduced high levels of 6-PFK and low amounts of G-6-PDH, GODand CAT. Addition of calcium carbonate to the growth mediumincreased the production of GOD and CAT, and decreased the synthesisof 6-PFK. Therefore, this observation might indicate that the inclusionof calcium carbonate is accompanied by a metabolic shift from theglycolytic pathway (EMP) to direct oxidation of glucose by GOD(Fig. 3).

2.2.4. Other medium componentsGOD 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 soybeanoil, respectively. This is attributed to an increase in the efficiency ofenzyme synthesis in the cells by n-dodecane and n-hexadecane, andan increase in biomass on addition of soybean oil. Fiedurek et al.(1996) investigated the effect of different medium components andmetabolic inhibitors on GOD production in A. niger, and found thatsubstituting ammonium phosphate with sodium nitrate in their basalsalt medium significantly increased GOD activity by 269.6%. Further-more, intracellular GOD activities increased by 68.3% in the presenceof sodium orthovanadate (1 mM), and extracellular GOD activitiesincreased in the presence of hematin (1 mM), choline (40 mM) andTween 80 (0.1%). The extracellular increase in GOD activity obtainedwas between 31.4–53.9%.

2.2.5. Effect of aeration and agitationGas–liquid transfer is known to be a limiting factor inmany aerobic

fermentations. One of the main reasons for this is the low solubility ofoxygen in fermentation media when compared to the solubility ofcarbon, nitrogen sources and other nutrients. Oxygen supply is furtherhindered 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 aerobicfermentation processes. Aeration of growing aerobic cultures fulfillsthe requirements of oxygen supply and also removes gaseous wasteproducts. Oxygen for growth and production in fungal cultures isensured by the aeration and agitation of the mycelial culture (Zetelakiand Vas, 1968).

Filamentous fungi grow as dense aggregated mycelial mats re-sulting in decreased accessibility of oxygen to the actively growingcells. These problems are overcome by agitation of broth cultures(Zetelaki, 1970), the use of H2O2 as the sole oxygen source (Fiedurekand Gromada, 2000), and the use of pure oxygen over air. Agitationincreases the efficiency of aeration by forcing the supplied air bubblesto disintegrate into smaller bubbles resulting in an increased interfacebetween the gas and the liquid (Zetelaki and Vas, 1968). Increasedagitator 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.

of dissolved oxygen resulting in a faster growth rate and increasedGOD production (Zetelaki and Vas, 1968; Zetelaki, 1970). Fiedurek andGromada (2000) as stated previously, identified a novel method ofincreasing dissolved oxygen in the growth media by adding H2O2 asthe sole oxygen source. CAT produced by the organism catalyzed thedecomposition of H2O2 to water and free oxygen; in this case, H2O2

acted as both the electron donor and acceptor in the reaction. Theyalso investigated some of the factors that affect oxygenation of theculture. Maximal oxygen concentration occurred in 50 ml of themedium 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 ofA. niger in submerged cultures. The growth rate increased from 61 mgmycelial dry weight/100 ml/h (aerated with air) to 95 mg mycelialdry weight/100 ml/h (aerated with pure oxygen). Comparison of theGOD activities of 24 h old cultures of A. niger agitated at 460, 700 and900 rpm, respectively, showed the activity of the culture agitated at700 rpm to be approximately 20–24% higher than those agitated at460 rpm. Further increase in agitator speed did not improve thegrowth rate or GOD production. Oxygenated culture reached a highermaximum mycelial yield 8 h earlier than in the aerated culture. It isnoted that autolysis was higher in the oxygenated culture medium. Inaddition, the viscosity of oxygenated culture was approximately 50%lower than that of the aerated culture. Cell walls of the aeratedcultures were found to be thicker and more rigid than that of theoxygenated culture, thereby causing the oxygenated cells to be lessresistant to mechanical agitation. This in turn could have lowered theviscosity that is observed in the oxygenated culture. At the same time,the GOD activity of the oxygenated culture was twice that of theaerated culture (Zetelaki and Vas, 1968). The only disadvantage of useof pure oxygen would be the large-scale financial implications (Kleinet al., 2002).

2.2.6. Effect of culture pHAmong the physical parameters, pH of the growth medium plays

an important role by inducing morphological change in the organismand in enzyme secretion. pH is a significant factor that influencesthe physiology of a microorganism by affecting nutrient solubilityand uptake, enzyme activity, cell membrane morphology, by productformation and oxidative reductive reactions. The pH change observedduring the growth of organism also affects product stability in themedium. Culture pH can have profound effects on both the rate ofproduction and the synthesis of enzymes. The appropriate pH formaximum production of the GOD can differ from that for optimumgrowth. Most of the strains used commercially for the productions ofGOD have an optimumpH between 6.0 and 7.0 for growth and enzymeproduction. In many of the processes, the buffering capacity of somemedia constituents such as CaCO3 and other phosphates sometimeseliminates the need for pH control. It is reported that A. nigeraccumulated GOD in the mycelia when grown in presence of CaCO3 ascompared to that in the absence of CaCO3 (Hatzinikolaou and Macris,1995; Rogalski et al., 1988).

2.2.7. Effect of growth temperatureThe internal temperature of the microorganism must be equal to

that of its environment and, the microbial activity is well known to besensitive 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 withmesophilic fungi within the temperature range of 25–37 °C. Optimumyields of GODwere achieved at 27–37 °C for A. niger. Caridis et al. (1991)studied the simultaneous production of GOD and CAT by Alternariaalternate, and revealed that GOD had its optimum temperature at32.3 °C and CAT at 18.1 °C. Hatzinikolaou and Macris (1995) examinedthe effect of growth temperature on total GOD production at 22.5 to32 °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 cultureThe aim of fed-batch study is to determine the degree of im-

provement in enzyme production over that of batch culture, and to getan insight in to the utilization of the carbon source. Sophisticated feedback controlled fed-batch systems are also available. Fed-batchcultivation for GOD production is reported by Kapat et al. (1998)who showed that the addition of part of the carbon source at a laterstage could improve GOD formation. Out of four different feedingstrategies tested by them for the production of extracellularrecombinant GOD from Saccharomyces cerevisiae, constant feeding ofgalactose on exhaustion of initial glucose gave the highest yield of154 U/ml, which was 62% above the yield achieved in batch operation(95 U/ml).

2.3. Optimization by statistical methods

The conventional ‘one variable at-a-time’ approach is time con-suming and often leads to confusion in the understanding of processparameters. Use of statistical methods enables easy selection of im-portant parameters from a large number of factors, and also explainsthe interactions between important variables. A number of statisticalexperimental designs have been used for optimizing fermentationvariables. Plackett–Burman design (Plackett and Burman, 1946) iswell known and is a widely used statistical technique for screeningand selection of most significant culture variables, while central com-posite design provides important information regarding the optimumlevel of each variable along with its interactions with other variablesand their effects on product yield (Pardeep and Satyanarayana, 2006).

Plackett–Burman saturated orthogonal designs work at two levels,and can be constructed on the basis of fractional replication of a fullfactorial design. This design allows reliable short listing of a smallnumber of ingredients for further optimization and allows one toobtain unbiased estimates of linear effects of all the factors withmaximum accuracy for a given number of observations, the accuracybeing the same for all effects (Krishnan et al., 1998). Since this designis a preliminary optimization technique which tests only two levels ofeach medium component, it cannot provide the optimal quantity ofeach component required in the medium. This technique, however,provides indications of how each component tends to affect enzymeproduction (Yu et al., 1997). Bankar et al. (2008) highlighted theimportance of Plackett–Burman experiments for optimizing culturevariables in attaining higher GOD titers. Among the six variableswhich were expected to play a critical role in enhancing GOD produc-tion, three factors (calcium carbonate, proteose peptone, and magne-sium sulphate) significantly affected enzyme production.

Response surface methodology (RSM) by central composite design(CCD) or by Box-behnken are the tools specifically used in presenttimes in fermentation technology to find out the optimum concentra-tion of the most effective variables for getting higher enzyme titersand to study their interactions. Various statistical software packagesare available for statistical optimization of variables. Liu et al. (2003)applied RSM to optimize the speed of agitation and rate of aeration formaximum production of GOD by A. niger. They found aeration to havea more negative effect on GOD production than agitation. Significantnegative interaction existed between agitation and aeration. RSM wasalso successfully employed for determination of optimum concentra-tion of media components by Bankar et al. (2008). They found maxi-mum GOD production at 3.08% calcium carbonate, 0.97% peptone and0.1% magnesium sulphate.

2.4. Mathematical model for glucose oxidase kinetics

It is generally accepted that the GOD is a primary metabolite, but itis difficult for even Luedeking and Piret model (Luedeking and Piret,1959; Crueger and Crueger, 1990) to explain the levels of GOD pro-duced in the medium as alone. These levels depend on microbial

production, as well on the processes that affect the enzyme once it hasbeen released, amongst which the most noteworthy is its deactivationby the H2O2 produced by the enzymatic action itself. However, themicroorganism also produces catalase, which breaks H2O2, thusopposing the previous effect and helping to preserve GOD (Mironet al., 2002).

Growth of the microorganism and the production of GOD bothshow the characteristics of a diauxic process. This diauxic nature, withlogistic and linear phases, is also evident in the disappearance ofglucose. The disappearance of glucose in the first phase of the culture,although partly due to microbial consumption, is mainly a conse-quence of its conversion to gluconic acid as a result of the action ofGOD produced in the culture. Once the glucose is exhausted, themicroorganism begins to use gluconic acid that accumulates duringthe first stage as a carbon source, with basically linear kinetics. GOD isconsidered as a semi-constitutive enzyme with a moderate rate ofbiosynthesis that remains constant in the absence of inducers, andincrease in the presence of inducers (Miron et al., 2002).

Microbial processes do not necessarily follow the classical kineticmodel of substrate-limited biomass growth and product formationas proposed by Monod in 1949. Therefore, the logistic equation, asubstrate-independent model, is used as an alternative empiricalfunction. In many fermentation systems, cell growth has been charac-terized by the logistic equation. The logistic equation (Eq. (1)) can bedescribed as follows:

dXdt

= μm 1− XXm

� �X ð1Þ

where X is amount of biomass formed at time t, Xm is maximumbiomass formed; µm is maximum specific growth rate and dX

dt is therate of biomass formation (Wang et al., 2006).

Logistic equations are a set of equations that characterize growth interms of carrying capacity (maximum cell mass; X∞). The usualapproach is based on formulation in which specific growth rate isrelated to the amount of unused carrying capacity (Shuler and Kargi,2002).

μg = k 1− XX∞

� �

where X∞is maximum cell mass-produced and k is carrying capacitycoefficient.

Thus,

dXdt

= kX 1− XX∞

� �ð2Þ

Integrating Eq. (2) with boundry conditions X (0)=Xo yields thelogistic curve.

X =Xoe

k;t

1− XoX∞

1− ek;t� �

Eq. (2) can be rewritten as

1XdXdt

= k 1− XX∞

� �

or

k =1XdXdt

= 1− XX∞

� �

The kinetics of product formation is based on the Luedeking–Piretequation (Luedeking and Piret, 1959). According to this model, the

495S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501

product formation rate (rP) depends on both the instantaneousbiomass concentration and the growth rate in a linear manner.

rp =dpdt

= αdXdt

+ βX ð3Þ

where α and β are the product formation constants and may differunder different fermentation conditions.

Miron et al. (2002) found the kinetics of the GOD to be affected bysubstrate inhibition, competitive inhibition by gluconic acid, decreaseof the reaction rate due to diffusional restrictions determined by theviscosity of the gluconic acid, and decrease in the reaction rate due toenzyme deactivation by H2O2. The last mentioned is a feature withsome phenomenological resemblance to true substrate inhibition, andwhich disappears when catalase is present.

Liu et al. (2003) proposed a simple model using the Logisticequation for growth, the Luedeking–Piret equation for GOD produc-tion and Luedeking–Piret-like equation for glucose consumption. Theyshowed the biosynthesis of GOD to be strongly linearly related to thegrowth, and that the correlation coefficient was very high. Theseresults 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 ofapproximately 150–170 kDa containing two tightly, but non-cova-lently bound FAD cofactors, and about 11–13% carbohydrate moietyof 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 theseproblems, use of genetically modified microorganisms rather thannatural sources for the expression of this enzyme has been stronglysuggested (Park et al., 2000).

Despite the abundant availability of commercial GOD, there is stillconsiderable interest in its new forms with useful properties forspecial applications in biotechnology. With a view to improve theproperties of enzyme by protein engineering, the GOD gene of A. nigerwas 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 GODin S. cerevisiae and evaluated the transformants for lower alcoholproduction and inhibition of wine spoilage organisms such as aceticacid bacteria and lactic acid bacteria during fermentation. They devel-oped tailored strains of S. cerevisiae for biopreserved wines with loweralcohol content. To test this novel concept, an antimicrobial yeaststarter culture system, on selective agar plate and in liquid assay wasdone. The yeast transformants displayed antimicrobial activity in aplate 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 resultedin 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 productionsystems and suggested for heterologous GOD production; but furtherstudies showed hyperglycosylation with yeast which may lead toserious limitations of usage (Romanos et al., 1992). Expression ofrecombinant 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. cerevisiaewas hypergly-cosylated and thus characterized by reduced substrate binding capacityand catalytic activity (Kapat et al., 1998). Crognale et al. (2006) usedmethylotrophic yeast Pichia pastoris as host for expression and secretion

of recombinant GOD of the filamentous fungus P. variabile P16. Theytransformed the gene to P. pastorisX33, a strain largely used for selectionon zeocin and large scale growth studies. They demonstrated P. pastoristo be an efficient host for expression of both secreted and intracellularheterologous proteins. Fermentation in 3 l fermenter lead to GODproduction of up to 50 U/ml that represents approximately four timesincrease in the production as compared to P. variabile P16 cultivatedunder optimized conditions. Kriechbaum et al. (1989) described thecloning and sequencing of the GOD gene of A. nigerNRRL-3 including 5′and 3′ flanking regions. They also represented the DNA-derived aminoacid sequence of GOD and showed its identity with peptide sequencesdetermined 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 effectiveindustrial production. Recombinant yeast expression plasmids havebeen constructed by Frederick et al. (1990) containing a hybrid yeastalcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogen-ase promoter, either the yeast α factor pheromone leader or the GODpresequence, and the mature GOD coding sequence. When trans-formed into yeast, these plasmids direct the synthesis and secretion ofbetween 75 and 400 μg/ml of active GOD. Analysis of the yeast-derived enzymes showed that they are of comparable specific activityand have more extensive N-linked glycosylation than the A. nigerprotein.

Kriechbaum et al. (1989) constructed a library of the A. nigerNRRL-3 genome in the phage A substitution vector EMBL3. Theyisolated one hybridizing clone which contained an insert of 15 kbp,from 12000 recombinant plaques with the nick-translated 800 bpcDNA fragment. The phage DNA was cleaved with SalI and theresulting fragments were subcloned into pBluescript SK (+). Theyused hybridization techniques and the shotgun sequencing methodfor identification of 1.8 kbp and 2.0 kbp SalI fragment containing thecoding region of GOD as well as small 5′ and longer 3′ untranslatedregions.

4. Downstream processing

Development of new and efficient separation processes is basedon effectively exploiting differences in the actual physicochemicalproperties 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 forpurification of GOD.

GOD has been purified for commercial application from differentfungi including A. niger (Hatzinikolaou et al., 1996; Kalisz et al., 1991;SwobodaandMassey,1965) andPenicillium species suchasP. pinophilum(Rando et al., 1997), P. amagasakiense (Kusai et al., 1960; Kalisz et al.,1997), P. chrysogenum (Eriksson et al., 1987), P. notatum (Gorniak andKączkowski, 1974), and P. funiculosum (Eryomin et al., 2004).

GOD is known to be produced intracellularly or extracellularly orsometimes as mycelia-associated enzyme. Hence cells have to bedisrupted for complete release of GOD into the broth. The intra- orextracellular location of the enzyme of A. niger and Penicillium specieshas been the subject of numerous discussions. In the meantime, theperiplasmic location of the A. niger GOD was clearly demonstrated(Witteveen et al., 1992), which is in agreement with the presence of asignal 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 bymechanical and physical forces, e.g. agitation and/or sonication.

Fig. 4. General protocol for purification of glucose oxidase.

496 S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501

Various methods of cell disruption have been used for filamentousfungi, including homogenization (Hatzinikolaou et al., 1996; Fiedurekand Gromada, 1997), sonication (Lu et al., 1996) and a combination ofboth (Hatzinikolaou and Macris, 1995). A comprehensive study ofdifferent methods for the disruption of two filamentous fungi, Gano-derma applanatum and Pycnoporus cinnabarinus was performed byTaubert et al. (2000). They concluded that fungal cells were parti-cularly resistant to some of the disintegration methods commonlyused 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 GODinto the liquid broth, various methods like sonication, bead mill,homogenizer and freeze-thawingwere applied. After the disruption ofthe cells, GOD is released in the fermentation broth which may beseparated from the cells either by differential centrifugation or byfiltration.

Various precipitation techniques have been used for purification ofGOD including ammonium sulphate (Kalisz et al., 1997; Kusai et al.,1960; Swoboda and Massey, 1965), uranyl acetate (Gorniak andKączkowski, 1974), potassium hexacyanoferrate and copper sulphate(Eriksson et al., 1987). Ammonium sulphate precipitation has beensuccessfully employed to precipitate both intra- and extracellular GODwith different percent cut of ammonium sulphate. The differences inammonium sulphate precipitation characteristics for intra- andextracellular 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% carbohydratedescribed as the high-mannose type (Kusai et al., 1960; Erikssonet al., 1987; Nakamura and Fujiki, 1968).

Precipitation is followed by chromatographic separation techni-ques such as ion exchange chromatography. On an average, the pI ofGODhasbeen found in the rangeof pH4 to5 (Eriksson et al.,1987;Kaliszet al.,1997;Kusai et al.,1960).Hence ananionexchange chromatographyis commonly used for its purification (Kalisz et al., 1997; Swoboda andMassey,1965; Dai et al., 2002; Hatzinikolaou et al., 1996). GOD ismainlyeluted with salt gradients using NaCl (Hatzinikolaou et al., 1996; Randoet al., 1997; Dai et al., 2002), althoughmixed pH and salt gradients havepreviously 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 CATcould therefore be exploited toassist in the purification of GOD to ensure that, it is free from CAT byusing anion exchange chromatography, since the separation with thismethod is based on differences in pI differences. GOD from P.amagasakiense has been reported to contain 4 different isoenzymes ofpI 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 pIbeing 4–5. It is therefore adsorbed at the beginning of the column.On eluting with the elution buffer (pH 3.6), GOD becomes positivelycharged and get desorbed from the anionic exchange resin. Thedifferent eluted fractions of GOD carries different amounts of nega-tive charges and this separates them from each other. The firstseparated part, GOD A has less charge than GOD B in the purificationprocess; therefore influence of charge impulse to the conformationof GOD A is small and has a higher enzyme activity. GOD B carriesmore negative charge than GOD A, and therefore it can only beeluted after GOD A and also destroys the native conformation anddecreases the enzyme activity (Dai et al., 2002). Rando et al. (1997)used a very efficient and elaborate procedure for the purification ofGOD from P. pinophilum. They purified GOD to apparent homo-geneity with a yield of 74% by including an efficient extraction stepof the mycelium mass at pH 3.0 and ion-exchange chromatographyfollowed by gel filtration.

5. Immobilization of glucose oxidase

Enzyme immobilization has attracted awide range of interest fromfundamental academic research to many different industrial applica-tions. To date, several immobilized enzyme-based processes haveproved to be economical. They have been implemented on a largerscale, mainly in the food industry, where they replace free enzyme-catalyzed processes, and in the manufacture of fine specialtychemicals and pharmaceuticals, particularly where asymmetric syn-thesis or resolution of enantiomers to produce optically pure productsare 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 activityoffered by materials having relatively high surface areas, porous glassand cellulose have been the most popular supports. For example, thepreparation and to improve the storage stability of GOD on porousglass by γ-amino-propyltriethoxysilane (APTES) was used by variousresearchers for silanization of the glass; prior to immobilization ofGOD. Immobilized GOD on both solid and porous glass was alsoperformed 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 canbe coupled by thiophosgene method, but the screen offers littlesurface area (Herring et al., 1972). GOD is difficult to crosslink with anagent like glutaraldehyde, but does crosslink rather easily in the pre-sence of certain proteins that have high number of reactive aminogroups 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 andTelefoncu 1992; Godjevargova et al., 2004; Podual et al., 2000).Ozyilmaz and Tukel (2007) used inorganic and porous magnesiumsilicate (Florisil) as a support for simultaneous co-immobilization ofGOD and CAT. Basic property of carrier may play a special role inpartial neutralization of gluconic acid produced by bound GOD in thepores of the carrier. This prevents a dramatic decrease in pH ofmicroenvironment 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 functionalgroups in the enzymes, which are not essential for catalytic activity.But, it is known that immobilization decreases enzyme activity due toblocking of the active site or due to changes in the enzyme geometryat the end of the coupling procedure. Addition of a substrate or acompetitive inhibitor to the coupling mixture protects the active siteon the enzyme against loss of activity (Srere et al., 1976). Severalstudies are available in literature on immobilization of enzymes in thepresence of their substrates.

497S.B. Bankar et al. / Biotechnology Advances 27 (2009) 489–501

To protect the active sites of GOD and CAT, co-immobilizationwas carried out in the presence of the glucose which is a substrate ofGOD. During the coupling period, GOD oxidizes glucose to produceH2O2 which is a substrate of CAT. Thus, co-immobilization is carriedout in the presence of both glucose and H2O2, which are substratesof GOD and CAT, respectively (Ozyilmaz and Tukel, 2007). Ozyilmazand 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 oftheir substrates significantly improves the activity and reusability ofboth enzymes. GOD is inactivated by H2O2, the concentration ofwhich increases during the catalytic turnover (Kleppe, 1966). H2O2

inactivation can be reduced with CAT which reduces H2O2 andeventually removes it from the system. By this reaction, someoxygen is recovered and made available for the oxidation of glucose(Fig. 5). Buchholz and Godelmann (1978) explained the reactionmechanism and deactivational parameters such as H2O2 deactivationand conversion of reduced GOD into oxidized GOD (Fig. 5). CAT plays akey role to prevent GOD from H2O2 deactivation by converting H2O2

into H2O and O2. Although CAT has preventive action in GOD/CATsystem, its concentration has little effect on the activity of the GOD onthe support.

Due to superior bonding properties of CAT and higher concentra-tion of CAT in the immobilizing solution, CAT activity is considerablyhigher in co-immobilized system than GOD. The clear dependence ofmeasured activity on particle size is due to both external film andinternal diffusional resistances. Experiments in which GOD and CATadsorbed directly onto the supports without pretreatment with γ-APTES or glutaraldehyde indicated activities up to 10 and 40% of theglutaraldehyde-bonded system were obtained for GOD and CATsystem, respectively (Markey et al., 1974).

Bouin et al. (1976) reported optimal coupling of GOD to occurabove pH 6. Below pH 3, the immobilized GOD completely andirreversibly lost activity. The effect of pH over the range from 4.5 to 8.0on immobilization of CAT has also been evaluated. An increase in pHup to pH increased the activity of CAT. It appeard that CAT activity hadlittle effect on the activity of GOD on the support. CAT concentrationwas 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 marginalactivities with other sugars. β-D-glucose gets oxidized in the presenceof molecular oxygen at C-1 position to δ -glucono-1, 5-lactone, whichis, in turn is spontaneously hydrolyzed to D-gluconic acid. Thesestructural features and the regioselectivity of the reaction allows aclear distinction between GOD and pyranose oxidase, which is a

Fig. 5. Simplified scheme for transport and reaction processes in the heterogeneoussystem (Buchholz and Godelmann, 1978).

homotetramer, and oxidizes D-glucose at the C-2 position (Danneelet 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 theamino acids in the active site, pH plays an important role inmaintaining the proper conformation of an enzyme. Most proteinsare 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 ofGOD vary from 5.0 to 7.0. GOD from most fungi and yeast have pHoptima in the acidic to neutral range such as A. niger and P. chrysogenumshowspHoptimaof 5.0 to 6.0. (Kalisz et al.,1991; Eriksson et al.,1987). Incontrast, the GOD obtained from P. funiculosum 433 and P. canescensshow 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. Therelationship between reaction rate of an enzyme and temperature isexponential. For every 10 °C rise in temperature, the rate of an enzymereaction doubles. At temperature range between 40 °C and 70 °C mostenzymes get denaturated and lose their activity. Enzymes are knownto display maximal activity at a temperature known as the optimumtemperature of the enzyme (Wilson and Walker, 1995). The lowestoptimum temperature for GOD is reported to be 25–30 °C from P.funiculosum 433 (Sukhacheva et al., 2004) and the highest of 40–60 °Cfrom 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 velocityof catalysis of an enzyme varied hyperbolically with substrateconcentration (Voet and Voet, 1995). The initial rate increases withan increase in substrate concentration to a point where it would reachmaximumvelocity (Vmax). At low substrate concentrations, initial rateis proportional to the substrate concentration, referred to as first orderkinetics. At high substrate concentrations the initial rate is indepen-dent of substrate concentration, referred to as saturation or zero orderkinetics. GOD at 0.2 U/ml could be accurately determined using theGOD dye binding assay (Bergmeyer et al., 1974). At higher GODactivities, the initial rates begin to decrease which lead to inconsistentactivity determinations.

6.5. Kinetic parameters variability

TheMichaelis constant (Km) and themaximal limiting rate velocity(Vmax) for GODwere calculated by various researchers and they foundslight differences in their values. The Km value of the GOD from T. favusis 10.9 mM (Kim et al., 1990) and is 33 mM from A. niger (Swobodaand Massey 1965). P. amagasakiense ATCC 28686 and P. funiculosum433 shows lower Km value of 5.7 mM and 3.3 mM respectively (Wittet al., 1998; Sukhacheva et al., 2004). It should also be noted thatreestimations of the Km values of glycosylated and deglycosylatedP. amagasakiense GOD gave values of 3.4 mM and 2.7 mM respectively(Kim and Schmid 1991). Vmax value of GOD ranges between 450to 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. ImmobilizedGOD would be more effective for applications at 37 °C. Polyhydricalcohols including ethylene glycol, glycerol, erythritol, xylitol, sorbitol

Fig. 6. Preparation of low alcohole wine from GOD/CAT treated grape juice (Pickeringet al., 1998).

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 stablefor a minimum of 6 months at −20 °C.

7. Applications of glucose oxidase

GOD has gained considerable commercial importance in the lastfew years due to its multitude of applications in the chemical,pharmaceutical, food, beverage, clinical chemistry, biotechnology andother industries. GOD is the most widely used enzyme as an analyticalreagent for determination of glucose due to its relatively low cost andgood stability. Its uses range from a glucose biosensor for the controlof diabetes, to a food preservative and color stabilizer. With co-immobilized enzymes, improved stability, reusability, continuousoperation, possibility of better control of reactions, high purity andproduct 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 theirblood glucose levels in order to detect fluctuations in glucose levelthat could lead to hyperglycemia (high blood glucose levels) andhypoglycemia (low blood glucose levels) so as to control the disease.Currently, such monitoring is done using finger-prick blood samplesand a portable meter several times a day. Biosensors are beingdeveloped to measure blood glucose levels. GOD is one of the possibleenzymes that can be used in biosensor. Biosensors work by keepingtrack of the number of electrons that pass through an enzyme byconnecting 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 softdrinks (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 smallpower sources to sustain operations. Biofuel cells convert biochemicalenergy into electrical energy using a biocatalyst. One type of biofuelcell uses enzymes as a biocatalyst. For example, GOD and micro-peroxidase-8 can be used on the cathode, where the H2O2 producedby GOD oxidizes microperoxidase-8 to directly accept electrons fromthe carbon rod electrode. Biofuel cells consist of a two electrode set (ofany stable and electrically conducting material) modified by biocata-lytic enzymes to specifically oxidize/reduce substrates. One approachtowards the design of an implantable, membraneless and biocompa-tible biofuel cell consists of catalyzing the oxidation of glucose at theanode using either GOD or glucose dehydrogenase enzymes. Theseenzymes are coupled to the reduction of dioxygen at the cathode by adioxygen-reducing enzyme such as laccase, bilirubin oxidase orcytochrome oxidase (Chen et al., 2001; Mano et al., 2002; Soukharevet al., 2004). Electron transfer to/from the biocatalytic active sites canbe mediated by polymer bound or entrapped redox complexes

(Barton et al., 2004). Both water-soluble fuel molecules (glucoseand O2) are found in body fluids and in blood at 10 and 0.1 mM,respectively. Besides, these molecules are converted at the electrodesinto naturally occurring degradation molecules in low concentration(gluconolactone and water). The maximum theoretical electromotiveforce (emf) allowed by the thermodynamics of glucose oxidation anddioxygen reduction at physiological pH is approximately 1 V.

7.3. Food and beverage additive

GOD has been used successfully to remove residual glucose andoxygen in foods and beverages in order to prolong their shelf life. TheH2O2 produced by the enzyme acts as a good bactericide, and can laterbe removed using a second enzyme, CAT that converts H2O2 to oxygenand water. GOD/CAT is used to remove glucose during themanufacture of egg powder, preventing browning during dehydrationcaused by the Maillard reaction for use in baking industry, providingslight 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 bottledbeverages before they are sealed. GOD/CAT system is shown to controlnon-enzymatic browning during fruit processing and puree storage.The scavenging of the oxygen by the enzyme system had a stabilizingeffect. In addition, GOD is used to prevent color and flavor loss as wellas to stabilize color and flavor in beer, fish, tinned foods and soft drinksby removing oxygen from foods and beverages (Crueger and Crueger,1990). For example, they are used to reduce the discolorationoccurring in wines and mayonnaises. GOD/CAT enzyme system canbe used to retard the lipid oxidation in mayonnaise stored at 5 °C and25 °C, in mayonnaises containing pure soybean oil, and where up tohalf the vegetable oil had been supplemented with fish oil. Theenzyme system was responsible for scavenging the oxygen duringglucose oxidation thereby decreasing the availability of the oxygen forlipid metabolism (Isaksen and Adler-Nissen,1997). Bonet et al. (2006)studied the effect of GOD on dough rheology and bread quality andshowed the strengthening of wheat dough and an improvement inbread quality on addition of GOD. However, the enzyme level must bevery carefully added, since adverse effects were obtained on additionof excessive enzyme.

7.4. Low alcohol wine production

GOD has potential for use in the wine industry, where it can lowerthe alcohol content of wine through the removal of some of theglucose (by converting it to δ-glucono-1, 5-lactone), which wouldotherwise be converted to alcohol. Tests showed that the GODtreatment of wine-must could reduce the potential alcohol content ofwine by about 2%. In addition, GOD is able to inhibit wine spoilagethrough its bactericidal effect on acetic acid bacteria and lactic acid

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bacteria during the fermentation process. The bactericidal effect ofan 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 carbonateprior to the enzymatic treatment.

7.5. Oral hygiene

GOD as well as lactoperoxidase can be used as anti-microbialagents in oral care products (Afseth and Rolla, 1983). The oral cavityhouses several species of Streptococci such as Streptococcus mutans,which is a significant contributor to tooth decay and is carried byvirtually everyone. The H2O2 produced by GOD acts as a usefulbacteriocide. The ability of GOD to kill S. mutans appears to beenhanced 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, whichcan be produced by the hydrolysis of δ-glucono-1, 5-lactone, the end-product of GOD catalysis. Gluconic acid has been used as a foodadditive to act as an acidity regulator, in sterilization solution orbleaching in food manufacturing, and as a salt in chemical compo-nents for medication. Gluconic acid is also used as a mild acidulant inthe metal and leather industries. It has even been used in theconstruction industry as an additive in cement to increase its resis-tance and stability under extreme weather conditions. It occursnaturally in honey, fruit and wine (Crueger and Crueger, 1990; Nakaoet al., 1997; Klein et al., 2002).

7.7. Textile industry

GOD has found applications in the textile industry as a method forproducing H2O2 for bleaching. Tzanov et al. (2002) covalentlyimmobilized GOD on alumina and glass supports resulting in higherrecoveries. Maximum H2O2 concentrations of 0.35 g/l and 0.24 g/lwere reached after 450 min for GOD immobilized on the glass andalumina supports respectively. The H2O2 produced was tested forbleaching scoured woven cotton fabric and was found to be com-parable to standard bleaching processes. No stabilizers were neededsince the gluconic acid produced acted as a stabilizing agent (Tzanovet al., 2002). A new concept has been developed byOpwis et al. (2008)which is based on simultaneous application of GOD and peroxidase.Starting with glucose as a substrate for GOD, H2O2 was generated insitu. The freshly formed H2O2 was immediately used by the PODoxidizing colored compounds in dyeing baths. These enzymes areused in decoloration process and bleaching of natural fibers in textileindustries.

8. Concluding remark

As evident from the foregoing review, glucose oxidase is among themost important enzymes used in industrial processes. It is potentiallyuseful in food, pharmaceutical, biotechnology and flavoring industries.Commercially viable processes are needed for large-scale productionof the enzyme by fermentation. Only few reports address fermentativeproduction of glucose oxidase at large scale with economical processand its subsequent uses in glucose determination or glucose removalin various analytical methods as well as industries. Although, numberof microbial sources exists for the efficient production of this enzyme,commercial production of this enzyme has been limited to only a few

selected strains of fungi and yeast. Further, there arises a need formore efficient glucose oxidase in various sectors, which can beachieved either by chemical modification of the existing enzymes orthrough protein engineering. In the light of modern biotechnology,glucose oxidase is now gaining importance in biopharmaceuticalapplications. Although glucose oxidase has long been used in variousindustries, technological innovation such as the use of immobilizationsupports and continuous-flow systems have been considered recently.However, satisfactory results in the application of immobilizedglucose oxidase without any limitations have rarely been achieved.Problems such as diffusional constraints and decrease in the enzymeactivity after immobilization need to be overcome for greater benefits.Similarly, research efforts on the optimization and design of a suitablereactor are still required before considering future commercialapplications.

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