MINI-REVIEW

Glucose oxidase: natural occurrence, function, propertiesand industrial applications

Chun Ming Wong & Kwun Hei Wong & Xiao Dong Chen

Received: 7 July 2007 /Revised: 8 February 2008 /Accepted: 8 February 2008 / Published online: 11 March 2008# Springer-Verlag 2008

Abstract Glucose oxidase (GOX) from Aspergillus niger isa well-characterised glycoprotein consisting of two identical80-kDa subunits with two FAD co-enzymes bound. Both theDNA sequence and protein structure at 1.9 Ǻ have beendetermined and reported previously. GOX catalyses theoxidation of D-glucose (C6H12O6) to D-gluconolactone(C6H10O6) and hydrogen peroxide. GOX is producednaturally in some fungi and insects where its catalyticproduct, hydrogen peroxide, acts as an anti-bacterial andanti-fungal agent. GOX is Generally Regarded As Safe, andGOX from A. niger is the basis of many industrialapplications. GOX-catalysed reaction removes oxygen andgenerates hydrogen peroxide, a trait utilised in foodpreservation. GOX has also been used in baking, dry eggpowder production, wine production, gluconic acid produc-tion, etc. Its electrochemical activity makes it an importantcomponent in glucose sensors and potentially in fuel cellapplications. This paper will give a brief background on thenatural occurrence, functions as well as the properties ofglucose oxidase. A good coverage on the diverse uses ofglucose oxidase in the industry is presented with a briefoutline on the working principles in the various settings.Furthermore, food grade GOX preparations are relativelyaffordable and widely available; the readers may beencouraged to explore other potential uses of GOX. One

example is that GOX-catalysed reaction generates significantamount of heat (∼200 kJ/mol), and this property has beenmostly neglected in the various applications described so far.

Keywords Glucose oxidase . Industrial applications .

Aspergillus niger . Food processing . Additive . Enzyme .

Properties . Occurrence . Functions

Introduction

Glucose-1-oxidase (GOX) (beta-D-glucose:oxygen-1-oxidoreductase, EC 1.1.3.4) is a well-characterised enzyme(Table 1), which catalyses the oxidation of beta-D-glucose(Eq. 1) to D-gluconolactone and hydrogen peroxide(Kleppet 1966; Wilson and Turner 1992). Both hydrogenperoxide (Eq. 2) and D-gluconolactone (Eq. 3) breaks downspontaneously and catalytically. Despite this, GOX’senzymatic activity is reduced when hydrogen peroxideaccumulates and inactivates the enzyme (Kleppet 1966); thebreakdown product of D-gluconolactone, gluconic acid(C6H12O7) accumulates, reducing pH of the solution. Notsurprisingly, both gluconic acid (Miron et al. 2004) andhydrogen peroxide (Bao et al. 2001, 2003) can result inproduct inhibition of GOX.

"‐D‐glucose

þO2�!glucose oxidase D‐gluconolactoneþ H2O2

2H2O2�!spontaneous=catalase 2H2Oþ O2 ð2Þ

D‐gluconolactone

þH2O�!spontaneous=lactonase gluconic acid

Appl Microbiol Biotechnol (2008) 78:927–938DOI 10.1007/s00253-008-1407-4

C. M. Wong (*) :X. D. Chen (*)Department of Chemical Engineering,Biotechnology & Food Engineering Group, Monash University,Melbourne, Victoria 3800, Australiae-mail: eric.wong@eng.monash.edu.au

K. H. WongP.O. Box 109175, Newmarket,Auckland 1149, New Zealand

e-mail: dong.chen@eng.monash.edu.au

(1)

(3)

Glucose oxidase was first discovered by Muller (1928)in Aspergillus niger extracts. DNA sequence of the GOXgene (Kriechbaum et al. 1989) as well as structure at 1.9 Ǻ(Wohlfahrt et al. 1999) had subsequently been determinedand published. Structurally, as shown in Fig. 1, glucoseoxidase is a homodimer made up of two identical 80-kDasubunits and two non-covalently bound flavin adeninedinucleotides (FAD). The FAD coenzyme acts as anelectron carrier during catalysis (Wilson and Turner 1992).The most commonly available form of the enzyme comesfrom A. niger contains about 20 wt.% amino sugar andabout 16–19 wt.% carbohydrate in which 80 wt.% is N- orO-glycosidically linked mannose molecules (Swoboda andMassey 1965; Nakamura and Fujiki 1968; Tsuge et al.1975). Further details on glucose oxidase’s history, chem-

ical and physical characteristics can be found in a reviewpaper by Wilson and Turner (1992).

Natural occurrence and function

Glucose oxidase is a naturally produced enzyme by somefungi and insects as listed in Table 2. As one would expect,its natural function is closely related to its catalytic activity(Eqs. 1–3). The main function of GOX is to act as anti-bacterial and anti-fungal agent through the production ofhydrogen peroxide. Permanent oxidative stress through themaintenance of hydrogen peroxide at low concentration byGOX’s continued catalytic activity has been reported inliterature (Tiina and Sandholm 1989; Dobbenie et al. 1995;Wu et al. 1995) to be very effective against bacterial orfungal growth, especially if the micro-organism does notproduce hydrogen peroxide scavengers such as catalase.Breakdown of hydrogen peroxide by the micro-organism’sintrinsic catalase may protect it against hydrogen peroxide’santi-bacterial or anti-fungal effect. It is, however, not aseffective if hydrogen peroxide is constantly produced byGOX. Previous experiments have demonstrated that in thepresence of catalase, milli-molar level of hydrogen peroxideis required to inhibit cell growth. Whereas in the presenceof GOX, the micro-molar level of hydrogen peroxide that isconstantly maintained by the catalytic activity of GOX isalready sufficient to inhibit cell growth (Leiter et al. 2004).Other interesting natural functions of GOX include assist-ing in plant infection, lignin degradation, lowering pH ofthe environment, etc. (see Table 2).

Table 1 Properties of glucose oxidase from A. niger

Properties Values

Molecular weight 150–186 kDa (Swoboda and Massey 1965; Nakamura and Fujiki 1968; Tsuge et al. 1975)UV–VIS absorption (Tsuge et al. 1975) 280: 380: 450 (nm)

11.5: 1: 1.03 (ratio)Extinction coefficient (M−1 cm−1) 270,000 (at 280 nm; Solomon et al. 1977)

25,180–28,200 (at 450 nm; Swoboda and Massey 1965; Johnson et al. 1989)21,600 (at 452 nm; Nakamura and Fujiki 1968)

Specific activity (pH 5.6, 25–37°C) 80–172 μmol glucose/min/mg enzyme (Swoboda and Massey 1965; Tsuge et al. 1975)Km (Michaelis constant, with respect to glucose) 198–248 mM (pH 5–7, 20–30°C, oxygen; Bao et al. 2001)

110–120 mM (pH 5.6, 0–38°C, oxygen; Gibson et al. 1964)50–74 mM (pH 5.5, 15–30°C, oxygen; Nakamura and Ogura 1968b)33 mM (pH 5.6, 25°C, oxygen; Swoboda and Massey 1965)41.8 mM (pH 6.86, 25°C, benzoquinone; Bohmhammel et al. 1993b)

Temperature range 20–50°C (Gouda et al. 2003)pH range 4–7 (Keilin and Hartree 1947; Nakamura and Fujiki 1968; Bao et al. 2001)Inhibitors Ag+, Hg2+ and Cu2+ ions (μmol; Nakamura and Ogura 1968a; Toren and Burger 1968)

Arsenite, p-chloromercuribenzoate, phenymercuric acetate (mmol; Nakamuraand Ogura 1968a) and others

Isoelectric point (pI) 4.2 (Pazur and Kleppe 1964)

Fig. 1 Glucose oxidase with its two subunits depicted as dark andlight blue, while the FAD coenzyme is depicted as pink. Image byGoodsell (2006), the Scripps Research Institute

928 Appl Microbiol Biotechnol (2008) 78:927–938

While GOX from A. niger is well characterised,properties of GOX from many organisms have also beendetermined. Molecular weight, carbohydrate content, pHoptimum, enzyme inhibitors, Km, absorption spectrum andother properties of GOX from other species have beendetermined experimentally and referenced in Table 2.

Substrates utilised by glucose oxidase

Glucose oxidase is capable of oxidising monosaccharides,nitroalkanes and hydroxyl compounds (Wilson and Turner1992). Using the reaction rate of glucose as reference(100%), only 2-deoxy-D-glucose (20–30%), 4-O-methyl-D-glucose (15%) and 6-deoxy-D-glucose (10%) are oxidised byglucose oxidase from A. niger at a significant rate (Pazur andKleppe 1964; Leskovac et al. 2005). The activities ofglucose oxidase against other substrates are typically poor,with reaction rates lower than 2% of glucose’s (Keilin andHartree 1947; Pazur and Kleppe 1964; Leskovac et al. 2005).

Like any other enzymes, properties of GOX, such assubstrate specificity, relative activity against glucose,optimal pH and temperature, inhibitors are species/sourcespecific and display significant variation to the commonlystudied GOX from A. niger. Details of these properties canbe found in the various papers (see Table 2), e.g. Shin et al.

(1993) tabulated GOX information from Pleurotus ostreatus;Keilin and Hartree (1947), Leskovac et al. (2005) and Pazurand Kleppe (1964) listed the substrates catalysed by GOXfrom A. niger and their relative reaction rates to glucose.

In addition to the reductant or electron donor, which isthe sugar molecules, glucose oxidase also requires anoxidant or electron acceptor to complete the enzymaticcatalysis. The natural electron acceptor, dissolved oxygengives the highest enthalpy (Bohmhammel et al. 1993a) inthe 263 enzymatic reactions listed in the database ofGoldberg et al. (2004), freely available from air and non-toxic. In comparison, other potential electron acceptors(Wilson and Turner 1992; Bohmhammel et al. 1993a;Leskovac et al. 2005) including quinones, generally havelower reaction rate and redox potential compared to oxygen(Gerd et al. 2004). Detailed explanation of the catalyticmechanisms is available and can be found elsewhere(Michael et al. 1998; Gerd et al. 2004; Leskovac et al.2005; Klinman 2007).

Applications of glucose oxidase

Detailed below are many industrial and commercialapplications where glucose oxidase may be used in. Theusefulness of GOX in diverse fields had triggered the

Table 2 Natural occurrence and functions of glucose oxidase

Natural occurrence Functions

Insect Honey bee (Apis mellifera L.)—salivary secretion fromhypopharyngeal gland (Ohashi et al. 1999; Santos et al. 2005;AbuSara 2006)

Honey preservation (AbuSara 2006)

Larva (Helicoverpa zea (Eichenseer et al. 1999), Helicoverpaarmigera, Helicoverpa assulta (Zong and Wang 2004)—salivarysecretion from labial glands

Disinfect ingested food and gastrointestinal function (Eichenseeret al. 1999), suppress plant defence (Musser et al. 2002; Zongand Wang 2004; Musser et al. 2005)

Grass hopper (Schistocerca americana)—cuticle (Candy 1979) Antibacterial, hardening of cuticle (Candy 1979)Larva (Spodoptera exigua, Mamestra configurata)—salivarysecretion from labial glands (Merkx-Jacques and Bede 2004,2005)

NA

Fungi Talaromyces flavus (Kim et al. 1990; Murray et al. 1997) Anti-fungal, i.e. antibiosis (Kim et al. 1990; Murray et al. 1997)Pleurotus ostreatus (Shin et al. 1993) Generate H2O2 for H2O2 dependent lignin degradation by

extracellular peroxidase (Kang et al. 1993; Shin et al. 1993)Aspergillus niger (Muller 1928; Kriechbaum et al. 1989;Hatzinikolaou et al. 1996)—mycelium cell wall (Witteveenet al. 1992)

Lower pH of environment (Magnuson and Lasure 2004)

Penicillium spp.—Penicillium amagasakiense (Wohlfahrt et al.1999), Penicillium canescens (Simpson 2006), Penicilliumnotatum (Coulthard et al. 1945; Bright and Appleby 1969),Penicillium pinophilum (Rando et al. 1997), Penicilliumfuniculosum (Sukhacheva et al. 2004), Penicillium variabile(Pulci et al. 2004), Penicillium chrysogenum (Leiter et al. 2004),Penicillium adametzii (Eremin et al. 2006)

Antibacterial and anti-fungal (Leiter et al. 2004)

Phanerochaete chrysosporium (Ramasamy et al. 1985) Assist lignin degradation (Ramasamy et al. 1985)Botrytis cinerea (Liu et al. 1998) Assist in infection of plants (Edlich et al. 1989)

Appl Microbiol Biotechnol (2008) 78:927–938 929

search for new sources of GOX (listed in Table 2) fromother species of fungi (including A. niger; Hatzinikolaouet al. 1996) and insects to satisfy the demand for improvedproperties such as higher catalytic activity (Fiedurek andGromada 1997). The following discussion will attempt tocover the major applications together with the respectiveworking principle utilised.

Food processing—additive

Glucose oxidase has Generally Regarded As Safe (GRAS)status under FDA classification (FDA/CFSAN 2002a, b)and is available in bulk for use in food industry as anadditive in liquid (e.g. 5L package) or powder (e.g. 2kg bag)form. It is often classified with antioxidant, preservativeand stabiliser properties. There are many food products inwhich glucose oxidase can be used. Some of them areelaborated below, and a detailed list can be found in CodexAlimentarius Commission’s database (2007b). The foodgrade GOX preparation used typically contains a mixtureof GOX and catalase because the two enzymes are foundnaturally together in the mycelium cell wall (Witteveen etal. 1992). Separation of GOX from catalase is costly and notessential in food grade preparations. Furthermore, catalaseassists in the breakdown of hydrogen peroxide produced byGOX, thereby reducing inhibition and deactivation byhydrogen peroxide (Bao et al. 2001, 2003).

Dairy and the lactoperoxidase system

One of the most important applications of glucose oxidasein food processing industry is food preservation. Thelactoperoxidase (LP) system, when used in conjunctionwith glucose oxidase, is a very useful anti-microbial agent.Lactoperoxidase system is part of the immune system’sinnate defence mechanism against foreign micro-organismsand can be found in mammalian secretions such as milk, tearsand saliva. This system consists of three components—lactoperoxidase, thiocyanate (SCN−) and hydrogen perox-ide. LP system activation occurs only in the presenceof thiocyanate and hydrogen peroxide. Catalysis bylactoperoxidase generates active intermediates, which hasanti-microbial properties and is completely safe to humans.The presence of GOX and its substrate (glucose) allowshydrogen peroxide required by LP system to be continu-ously generated and replenished (Seifu et al. 2005).

For the transportation and/or storage of raw milk, the useand activation of the LP system is effective against spoilage,and it is recommended to be used when refrigeration isunavailable or as a complement to refrigeration. Experimentshave demonstrated that the shelf-life of milk with active LPenzyme almost doubled compared to milk with inactivated LP(Marks et al. 2001). In addition, activation of the LP system is

also suggested as a pre-treatment for dairy products toenhance bacterial deactivation including mastitis pathogens(Sandholm et al. 1988) and to allow lower temperaturetreatments during pasteurisation (Seifu et al. 2005).

The same LP–GOX system mentioned previously can alsobe used in cheese production. The hydrogen peroxideproduced by GOX is utilised by the LP system for cold, i.e.room temperature sterilization, while the gluconic acidproduced is used for direct acidification (Fox and Stepaniak1993). It should be noted that this LP–GOX anti-microbialsystem is not limited to food and has been used in toothpaste(Biotene 2006; National Library of Medicine 2007a), lotions(National Library of Medicine 2007b), shampoos, cosmetics,meat processing (Food standards Australia New Zealand2002) and fish farming (Seifu et al. 2005).

Breadmaking

Maturing/oxidising agents are an essential additive to flour.One of its purposes is to strengthen gluten, thereby improvethe bread’s final texture. This is achieved through theoxidation of two proteins within flour, gliadin and glutenento allow more bonds to form when gluten develops. Glutenforms when gliadin and glutenen are in contact with waterand its maturation is assisted by the actions of yeast(Corriher 2001). Only a small amount of maturing agents inthe level of parts per million is needed in this process andtraditionally, potassium bromate (KBrO3) was used (Figoni2003). However, it has been recognised that bromate iscarcinogenic, causing DNA damage in vitro and in vivothat may contribute towards cancer (Moore and Chen2006). As a result, most countries have prohibited the useof bromate in food, and an alternative such as glucoseoxidase is used in bakery (Enzyme Technical Association2001).

GOX is an effective oxidant to produce bread withimproved texture and increased loaf volume (Vemulapalliet al. 1998; Rasiah et al. 2005). The basis of oxidation byGOX has been validated to be a result of the hydrogenperoxide produced, which yields dough that is more elasticand viscous than the control without GOX (Vemulapalliet al. 1998). In addition, GOX also causes a drying effecton dough that is attributed to gel formation of water solublepentosans (Vemulapalli and Hoseney 1998). As it isrecognised that potassium bromate does not cause thisdrying effect, it is postulated that this effect is induced byglucose oxidase (Vemulapalli et al. 1998). Although theexact mechanisms by which hydrogen peroxide producedby glucose oxidase improves the dough properties are notcompletely understood, more work are in progress, andsome theories have been proposed (Rasiah et al. 2005;Franziska Hanft 2006). Nevertheless, GOX is known tocause cross-linking of dough protein (Rasiah et al. 2005)

930 Appl Microbiol Biotechnol (2008) 78:927–938

and exert effects such as reducing the sulfhydryl content aswell as increasing viscosity in the water soluble portion ofdough (Vemulapalli and Hoseney 1998).

Dry egg powder

Maillard non-enzymatic browning is a result of reactionbetween the amino group of proteins or amino acid andreducing sugars. In the production of dried egg powder, thisreaction causes undesirable browning and formation ofunwanted flavour. Therefore, the glucose present (∼4 g/L)in liquid egg is typically removed before spray drying(Sisak et al. 2006). Glucose removal also has the addedbenefit of longer shelf-life and enhanced microbial toler-ance. Besides that, hydrogen peroxide produced in thereaction can also kill or inhibit growth of micro-organismscommonly present in liquid egg (Dobbenie et al. 1995).One of the ways to remove glucose from egg is by addingglucose oxidase before spray drying takes place, as allowedunder FDA regulations. This is typically a batch processand contains the enzyme as an impurity in the product.Through immobilisation, continuous process is possible,and the enzyme can also be retained and recycled instead asbeing an impurity in the product. Hence, there were studiesinto the viability of reactors for desugaring based onimmobilised glucose oxidase (Sisak et al. 2006). It shouldbe noted that undesirable browning caused by Maillardreaction is not only unique to eggs. Potato products alsosuffer the same issue, and GOX can be employed to reduceglucose content, thereby reducing browning (Low et al.1989).

Antioxidant/preservative (oxygen scavenger)

The presence of oxygen is a problem in many foodproducts. In high-fat foods such as mayonnaise andsalad dressing, lipid oxidation can cause deteriorationand rancid taste (Isaksen and Adler-Nissen 1997). Thesame is true for beverage such as wine and beer, keepingoxygen out of the drink helps to maintain taste andflavour (McLeod and Ough 1970; Labuza and Breene1989). In canned/bottled/packaged food, oxygen alsopromotes bacterial growth, hence, it is desirable to removeoxygen from the headspace to maintain an anaerobicenvironment (Kirk et al. 2002). In puree processing,oxygen contributes to maillard non-enzymatic browning,therefore appropriate controls must be in place (Parpinelloet al. 2002).

The overall reaction (Eq. 4) catalyzed by glucoseoxidase involves the consumption of two glucose moleculesand one oxygen molecule to produce two gluconic acidmolecules. This reaction consumes oxygen, a trait thatallows GOX to be used as an active oxygen scavenger,

antioxidant and preservative in the various food applica-tions described in the previous paragraph. Detailed analysison using GOX as oxygen scavenger can be found in Labuzaand Breene (1989). Moreover, the catalytic product,gluconic acid, is safe for human consumption, and WHOhas not specified a limit on its acceptable daily intake. This,combined with the demand from consumers to replacechemical antioxidant and oxygen scavenger with naturalcompounds, makes GOX an ideal candidate in foodpreservation.

"‐D‐glucoseþ 1

2O2�!glucose oxidase gluconic acid ð4Þ

As a preservative, GOX has been demonstrated to beuseful to extend the shelf-life of some seafood. Fillets orwhole fish dipped with GOX/glucose solution before beingrefrigerated could be stored for up to 21 vs. 15 days forcontrol (Field et al. 1986), and similarly treated shrimpcould also be stored for up to 11 vs. 6 days for control(Dondero et al. 1993). This phenomenon is most likely dueto growth inhibition of the spoilage bacteria such asPseudomonas fragi, which is commonly present in fish(Yoo and Rand 1995); Pseudomonas fluorescens, which isassociated with shrimp (Kantt et al. 1993); enterotoxicbacteria Escherichia coli and Salmonella derby (Massaet al. 2001).

Other than the food industry, the “natural” advantage ofGOX has also triggered interests from the pharmaceuticalcompany to pursue the use of glucose oxidase to replacetraditional antioxidant in their formulations (Uppoor et al.2001).

Reduced alcohol wine

Sugar is an important ingredient when it comes to alcoholproduction through fermentation because it is the primarysubstrate used by Saccharomyces cerevisiae to producealcohol. Hence, reduction of glucose in the must, i.e. thejuice used for wine fermentation, can result in lower alcoholcontent. As there are demands for reduced alcohol wines,partly driven by its lower tax and tariffs, there wereinvestigations on the feasibility of using various technologies(Gary 2000). One of them is to use glucose oxidase to reducethe amount of glucose available, subsequently yielding loweralcohol content.

One of the easiest ways to do this is to add glucoseoxidase to the must before fermentation. Glucose oxidaseconsumes some of the glucose present, making themunavailable for alcohol fermentation, thereby resulting inwine with reduced alcohol (Pickering et al. 1998, 1999a, b, c).At the same time, hydrogen peroxide generated may reducethe activity or growth of the S. cerevisiae used for alcoholfermentation. Nevertheless, experiments had shown that the

Appl Microbiol Biotechnol (2008) 78:927–938 931

must containing GOX completed fermentation in 10 days,whereas the control required 12 days (Pickering et al.1999a). Another approach examined was to geneticallyengineer the S. cerevisiae used during the fermentationprocess to express glucose oxidase. This approach wasconsidered viable but requires more work to be done beforeit can reach the market (Malherbe et al. 2003). One addedadvantage of the use of glucose oxidase is that the hydrogenperoxide produced acts as a bactericide, helping to act aspreservative for the wine (Malherbe et al. 2003).

Gluconic acid production

Gluconic acid and its derivative salts are GRAS and can beused in a wide range of industries (Ramachandran et al.2006) including textile dying, metal surface cleaning, foodadditives, detergents, concrete, cosmetics (Yu and Scott1997) and pharmaceuticals (BACAS 2004). As a foodadditive, it can be used as an acidity regulator, raisingagent, colour stabiliser, antioxidant and chelating agent inbread, feed, beverage, etc (Brookes et al. 2005; CodexAlimentarius Commission 2007a). Industrially, gluconicacid is mostly produced from fermentation (Singh et al.2005), with an estimated global production of about50,000–100,000 ton/year (BACAS 2004; EuropaBio andESAB 2005). Further information regarding the properties,applications and microbial production of gluconic acid havebeen described by Ramachandran et al. (2006).

As with all fermentation processes, there are somedisadvantages. Cultures require various added nutrientsand at least a few days to grow and perform bioconversion.In addition, culture solutions produce and contain unwantedby-products, need downstream purifications and consumesubstrates prohibiting high conversion efficiency. Hence,the use of enzyme-based conversion is considered a viablemethod to reduce production cost and time (Nakao et al.1997). For example, during 1997 and 2003, there werepatents filed making claims of glucose-oxidase-basedprocess that is capable of almost 100% conversionefficiency, require less time than fermentation and do notcontain impurities (Vroemen and Beverini 1999; Lanteroand Shetty 2004). Bioreactor using immobilised glucoseoxidase is one of the preferred setups being investigated(Godjevargova and Turmanova 2004). Immobilisationallows the enzymes to be recycled, reduces cost as well aspermitting relatively easier design and construction ofreactor to produce and remove of the desired product,gluconic acid continuously. Despite these potential advan-tages, the lack of industrial adoption implies that there aremajor hurdles to overcome before mass adoption ofenzyme-based bioconversion process occurs. This is evi-dent, as there is patent covering industrial scale productionof gluconic acid from glucose using glucose oxidase dating

decades old (Bergmeyer and Jaworek 1976). Nevertheless,electrodeionisation-based separative bioreactor (a technologyused to make deionised water; Arora et al. 2007) backed bythe US Department of Energy Biomass Program seemspromising and may be adopted by the industry in the nearfuture.

Glucose sensor/assay

Newman and Turner (2005) recently estimated the world’smarket value of biosensors to be about $5 billion dollars,and 85% is attributed to glucose biosensors. Many glucosesensors available in the market are based on immobilisedglucose oxidase, and more information about glucosesensors available in the market can be found in the reviewarticle by Newman and Turner (2005).

Glucose oxidase is commonly used to construct amper-ometric biosensors for medical (Wilkins and Atanasov1996; Newman and Turner 2005) and food industry (Melloand Kubota 2002). A constant electric potential is appliedbetween working and reference electrode, promoting thecatalytic reaction, which drive the current flow that isproportional to the concentration of the target molecule(Terry et al. 2005). Such amperometric glucose sensorsbased on glucose oxidase can be divided into threegenerations according to its principle of operation andhistorical development (Wilkins and Atanasov 1996;Newman and Turner 2005; Park et al. 2006). In the firstgeneration (Fig. 2a), the concentration of oxygen orhydrogen peroxide, as measured by an appropriateelectrode, is used as an indicator of glucose concentration.Depletion of oxygen or production of hydrogen peroxideoccurs when glucose is present and oxidised by glucoseoxidase. Interference by other redox species is a problemwith this design. In the second generation (Fig. 2b),mediators are used to shuttle electrons back and forthbetween the enzyme and the electrode. The mediatorreplaces oxygen, which is necessary and often in limita-tion in the first generation. However, the mediator usedmust be present in close proximity to the electrode, i.e.insoluble and have high turn-over rate. Unfortunately, itstill suffers to a smaller extent than first generation fromthe interferences due to other redox species such asoxygen. Therefore, to eliminate interference, in the thirdgeneration (Fig. 2c), direct electron transfer was employedto transfer electron directly from the enzyme to theelectrode via a series of “wired” relay centres. Recently,there are also investigations into immobilising GOXonto carbon nanotubes (Tsai et al. 2005) or nanofibres(Vamvakaki et al. 2006), thereby eliminating the electronrelay centres as the immobilising carbon nanofibres/nanotubes matrix are electrically conductive and are alsoacting as the electrode/transducer.

932 Appl Microbiol Biotechnol (2008) 78:927–938

The oxidation of glucose, as catalysed by glucoseoxidase (Eq. 4), yields a significant amount of heat. Severalpapers have cited theoretical enthalpy value at 180 kJ/mol(Muehlbauer et al. 1989; Bataillard et al. 1993; Zhang andTadigadapa 2004), while experimental measurementsreported value as high as 223 kJ/mol (Bohmhammel et al.1993a; Huttl et al. 1993). Hence, the heat released can beused as the basis for thermistor-based (Harborn et al. 1997;Ramanathan and Danielsson 2001) or thermopile-based(Muehlbauer et al. 1989; Bataillard et al. 1993; Zhang andTadigadapa 2004) glucose sensors. However, such enthalpy-based glucose sensors are not common compared toamperometric biosensors described above. Thermistor is avery sensitive thermometer whose electrical resistance varieswith temperature, while thermopile consists of a number ofthermocouples connected in series, which generate a voltagedue to the temperature difference at the junction, a result ofthermoelectric effect. These sensors work on the principlethat the amount of heat released is proportional to the molarenthalpy and the amount of substrates reacted. The measure-ment is also dependent on the heat capacity of the solventused. Organic solvent, which has two to three times lower

heat capacity than aqueous solvent, often gives bettersensitivity and detection limit. In depth details in thermalsensors are covered by Ramanathan and Danielsson (2001).

In the medical industry, use of glucose oxidase inglucose sensors is not only limited to the traditional‘fingerpricking’ blood glucose measurement devices buthas also been investigated to be used in continuousmonitoring of glucose in vivo (Wilson and Hu 2000;Klonoff 2005) such as fluorescent-based glucose sensing(Brown et al. 2005, 2006; Pickup et al. 2005; Brown andMcShane 2006; Yang et al. 2006). Fluorescent-basedglucose sensing has some advantages over fingerprickingsensors, for example, extreme sensitivity, non-invasive, etc.Glucose oxidase can be utilised in several ways (Pickupet al. 2005) including the change in GOX fluorescence dueto the different oxidative state of FAD within GOX anddetection of the oxygen consumption or hydrogen peroxideproduction via an appropriately coupled fluorophore. As thefluorescent of FAD is weak, fluorescent change whenglucose binds to deactivated GOX with its FAD removedand covalently bound to molecules such as 8-anilino-1-naphthalene sulphonic acid, has also been investigated.

When used in a continuous monitoring system, theincreased data points per sampling time period fromthe frequent measurements allows a better representation ofthe glycemic history and status of the patient. If thisinformation is used appropriately, such as that in conjunctionwith insulin pump, which can supply basal and larger dosesof insulin, tighter control against hypo- and hyperglycermiacan be achieved in diabetic individuals (Newman and Turner2005). However, one of the problems with glucose oxidasein such applications is the inherent instability of the enzymeand the potential loss of activity when the enzyme issubjected to thermal and chemical denaturation duringproduction, storage and the application of the glucose sensor.Consequently, quality control in the reproducibility of theenzyme-based sensors is problematic (Park et al. 2006).

It should be noted that the use of glucose oxidase inglucose sensors are not limited to those described above.For example, glucose oxidase immobilised at the tip ofmicrocantilever can be used to measure glucose concentra-tion (Pei et al. 2004). The degree of microcantileverdeflection was shown to be related to the concentration ofthe immobilised enzyme as well as the concentration ofglucose solution being measured possibly due to the stresscaused by the enzymatic reaction.

In the laboratory (Giampietro et al. 1982), accurateglucose measurement is typically performed using glucoseassay kits. These kits are widely available in the market(e.g. from Sigma Aldrich) and typically also make use ofglucose oxidase’s specificity against glucose to produce acoloured product, which can be accurately measured usingabsorption spectrometry. The coloured product is typically

GOX[FAD]

GOX[FAD]

GOX[FADH2]

GOX[FADH2]

Glucose

Glucose

Gluconic acid

Gluconic acid

O2

H2O2

O2

or H

2O2

prob

e

GOX[FAD]

Glucose

Gluconic acid

Relay centre

Ele

ctro

de

Ele

ctro

de

e- e-

e-

Mediator [oxidized]

Mediator [reduced]

1/2

a

b

cFig. 2 Principle of operation for first (a), second (b) and third(c) generation glucose sensor (Newman and Turner 2005; Park et al.2006)

Appl Microbiol Biotechnol (2008) 78:927–938 933

produced as a result of hydrogen peroxide reacting witho-dianisidine in the presence of peroxidase.

Fuel cell

The use of glucose oxidase in fuel cell is not a recent trend.Investigations on glucose-oxidase-based fuel cells havebeen on-going since the 1960s (Davis and Yarbrough 1962;Yahiro et al. 1964). Despite these efforts and the ability toproduce cells with near 100% current (faradic) efficiency(Weibel and Dodge 1975), biofuel cells are still not readyfor applications outside the laboratory. As pointed out inmany literatures (CalabreseBarton et al. 2004; Bullen et al.2006; Davis and Higson 2006), two main hurdles of biofuelcells are the limited lifetime and limited power output of thecells. Higher enzyme stability is needed to improve lifetimeof the cells from days/months to years, while higherenzyme catalytic rate is needed to improve power outputby several orders of magnitude. One viable approach totackle these problems is by using directed evolution. Forexample, by using glucose oxidase detection assay, whichmeasures the amount of NADPH produced from thedownstream catalysis of glucose oxidase end-products at340 nm, Zhu et al. (2006) obtained glucose oxidase mutantE4, which displays a higher catalytic activity.

Glucose oxidase are typically used in the anode ofbiofuel cells to oxidise glucose, i.e. extract electrons andtransfer it to the anode electrode from which the electronswill flow through the load in the circuit to the cathode atwhich the electron will be used to reduce molecules, e.g.oxygen to water (Weibel and Dodge 1975). At the sametime, ions such as proton, i.e. H+, will diffuse from theanode compartment through the separating semi-permeablemembrane to the cathode compartment to complete thecircuit. Typically, this semi-permeable membrane is necessaryto prevent mixing of materials in the two compartments,which may cause interference to the operation of the cell.However, when glucose oxidase is immobilised onto theelectrode, the efficient interfacial bioelectrocatalytic processesmake such interference insignificant, therefore permitting theabsence of separations (Katz et al. 1999; Chen et al. 2001). Insuch configurations (Fig. 3), electrons can pass directly fromthe enzyme, e.g. glucose oxidase to the electrode via linkedredox centres. This is termed direct electron transfer (DET),but it should be noted that DET does not mean thatseparating membrane would be absent (Willner et al.1998). Whereas in mediated electron transfer, there are aconstant recycling of oxidised and reduced mediators thattransport electrons to and from the enzyme to the electrodesurface via diffusion (Weibel and Dodge 1975). Moredetailed information on fuel cells such as its history andrecent advances can be found in Bullen et al. (2006) andDavis and Higson (2006).

Other uses and concluding remarks

Use of glucose oxidase is not limited to the applicationsdescribed above. In the textile industry, there are considerableinterests to replace chemical bleaching with environmentallyfriendly bio-bleaching processes. Chemical bleaching requiresaround pH 10.5–11 and near boiling temperature, whereasbio-bleaching can be conducted at lower temperature andaround neutral pH. This means significant cost saving inenergy and effluent treatment. Research conducted so far haveshown promising results in using glucose oxidase to producehydrogen peroxide for bleaching, utilising the glucosegenerated from the upstream desizing and bioscouringprocesses (Buschle-Diller et al. 2002; Tzanov et al. 2002).Although it was noted that the cost of the enzyme is tooexpensive for textile processing (Hamlyn 2000), a patent byNovozyme North America, Inc. (Salmon et al. 2006) can befound, which covers the use of carbohydrate oxidase,including glucose oxidase to bleach textiles. In other words,it would be reasonable to speculate that the use of glucoseoxidase in textile industry should be economically viable inthe near future. While bio-bleaching can be performed in thefactories, it is also possible to do the same in the everydaylaundry by adding glucose oxidase to laundry detergentpreparations (Pramod 1999).

In the laboratory, glucose oxidase also has diverse uses.For example, glucose oxidase can be used in variousimmunoassays and/or staining procedures as well asremoval of excess glucose (Rathlev 1983; Porter and Porter1984; Pfreundschuh et al. 1988; Blais and Yamazaki 1992;Dosch et al. 1998; Megazyme 2003). Whereas, in real-timefluorescent microscopy for biological samples, glucoseoxidase/catalase is often used for oxygen scavenging toreduce photodamage (Desai et al. 1999). In geochemical

Fig. 3 An example of a non-compartmented biofuel cell usingglucose oxidase at the anode (Katz et al. 1999)

934 Appl Microbiol Biotechnol (2008) 78:927–938

prospecting, heap leaching, pollution studies, etc., glucoseoxidase can be used to prepare mineral leaching solutions asboth hydrogen peroxide and gluconic acid produced facili-tates leaching (Clark 1995, 1996). In genetic engineering,expression of GOX in plants can provide resistance tobacterial infection (Wu et al. 1995).

Obviously, there is no restriction to the number ofpotential applications that glucose oxidase may beemployed in. Other than the known uses mentioned in thisarticle, there seems to be plenty of room for new novelapplications. For instance, when GOX is used as preserva-tive in packaged food, changes in pH due to glucosehydrolysis can potentially be monitored using a pH stripvisible outside the package. As the GOX-catalysed reactionis oxygen limited, if the package is broken and air leaks in,it will provide the necessary oxygen for GOX to hydrolyseglucose into gluconic acid (Eq. 4), causing pH to drop.Alternatively, a specially designed container could permitsome degree of air penetration, providing the necessaryoxygen for GOX to generate and maintain low level ofhydrogen peroxide for microbial inhibition.

Enzymes, including glucose oxidase, are gaining impor-tance and popularity in the industry as an environmental-friendly alternative to the traditional chemical treatments,especially when it becomes more cost effective to produce.With the enzyme market predicted to have annual growthrate at 7.6% per annum and market value increase from$4.1 billion to $6 billion by 2011 (The Freedonia Group2007), we would expect to see more breakthroughs,investments as well as innovations in the applications ofGOX in the future.

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