Glucose oxidase

Class of enzymes

If you are looking for more details, kindly visit our website.

Chemical compound

The glucose oxidase enzyme (GOx or GOD) also known as notatin (EC number 1.1.3.4) is an oxidoreductase that catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone. This enzyme is produced by certain species of fungi and insects and displays antibacterial activity when oxygen and glucose are present.[2]

Glucose oxidase is widely used for the determination of free glucose in body fluids (medical testing), in vegetal raw material, and in the food industry. It also has many applications in biotechnologies, typically enzyme assays for biochemistry including biosensors in nanotechnologies.[3][4] It was first isolated by Detlev Müller in from Aspergillus niger.[5]

Function

Several species of fungi and insects synthesize glucose oxidase, which produces hydrogen peroxide, which kills bacteria.[2]

Notatin, extracted from antibacterial cultures of Penicillium notatum, was originally named Penicillin A, but was renamed to avoid confusion with penicillin.[6] Notatin was shown to be identical to Penicillin B and glucose oxidase, enzymes extracted from other molds besides P. notatum;[7] it is now generally known as glucose oxidase.[8]

Early experiments showed that notatin exhibits in vitro antibacterial activity (in the presence of glucose) due to hydrogen peroxide formation.[6][9] In vivo tests showed that notatin was not effective in protecting rodents from Streptococcus haemolyticus, Staphylococcus aureus, or salmonella, and caused severe tissue damage at some doses.[9]

Glucose oxidase is also produced by the hypopharyngeal glands of honeybee workers and deposited into honey where it acts as a natural preservative. GOx at the surface of the honey reduces atmospheric O2 to hydrogen peroxide (H2O2), which acts as an antimicrobial barrier.[10]

Structure

GOx is a dimeric protein, the 3D structure of which has been elucidated. The active site where glucose binds is in a deep pocket. The enzyme, like many proteins that act outside of cells, is covered with carbohydrate chains. GOx is a glucose oxidising enzyme with a molecular weight of 160 kDa. It is a dimeric glycoprotein consisting of two subunits each weighing 80 kDa. Flavinadenine dinucleotide (FAD) in the active site is buried approximately 1.5 nm inside the protein shell and acts as the initial electron acceptor.[11]

Mechanism

At pH 7, glucose exists in solution in cyclic hemiacetal form as 63.6% β-D-glucopyranose and 36.4% α-D-glucopyranose, the proportion of linear and furanose form being negligible. The glucose oxidase binds specifically to β-D-glucopyranose and does not act on α-D-glucose. It oxidises all of the glucose in solution because the equilibrium between the α and β anomers is driven towards the β side as it is consumed in the reaction.[3]

Glucose oxidase catalyzes the oxidation of β-D-glucose into D-glucono-1,5-lactone, which then hydrolyzes into gluconic acid.

In order to work as a catalyst, GOx requires a coenzyme, flavin adenine dinucleotide (FAD). FAD is a common component in biological oxidation-reduction (redox) reactions. Redox reactions involve a gain or loss of electrons from a molecule. In the GOx-catalyzed redox reaction, FAD works as the initial electron acceptor and is reduced to FADH&#;.[12] Then FADH&#; is oxidized by the final electron acceptor, molecular oxygen (O2), which can do so because it has a higher reduction potential. O2 is then reduced to hydrogen peroxide (H2O2).

Applications

Glucose monitoring

Glucose oxidase is widely used coupled to peroxidase reaction that visualizes colorimetrically the formed H2O2, for the determination of free glucose in sera or blood plasma for diagnostics, using spectrometric assays manually or with automated procedures, and even point-of-use rapid assays.[3][8]

Similar assays allows the monitoring of glucose levels in fermentation, bioreactors, and to control glucose in vegetal raw material and food products.[citation needed] In the glucose oxidase assay, the glucose is first oxidized, catalyzed by glucose oxidase, to produce gluconate and hydrogen peroxide. The hydrogen peroxide is then oxidatively coupled with a chromogen to produce a colored compound which may be measured spectroscopically. For example, hydrogen peroxide together with 4 amino-antipyrene (4-AAP) and phenol in the presence of peroxidase yield a red quinoeimine dye that can be measured at 505 nm. The absorbance at 505 nm is proportional to concentration of glucose in the sample.

Enzymatic glucose biosensors use an electrode instead of O2 to take up the electrons needed to oxidize glucose and produce an electronic current in proportion to glucose concentration.[13] This is the technology behind the disposable glucose sensor strips used by diabetics to monitor serum glucose levels.[14]

Food preservation

In manufacturing, GOx is used as an additive thanks to its oxidizing effects: it prompts for stronger dough in baking, replacing oxidants such as bromate.[15] It is also used as a food preservative to help remove oxygen and glucose from food when packaged such as dry egg powder to prevent unwanted browning and undesired taste.[16]

Wound treatment

Wound care products, such as "Flaminal Hydro" make use of an alginate hydrogel containing glucose oxidase and other components as an oxidation agent.

Clinical trials

A nasal spray from a bag-on-valve device that mixes glucose oxidase with glucose has undergone clinical trials in for the prevention and treatment of the common cold.[17][18][19]

See also

References

What then enables the activity of the entrapped GOx at all? The gold matrix is porous with a hierarchical structure. The smallest building blocks are gold nanocrystals of around 16 nm (determined from XRD analysis13), which are tightly aggregated into sub-micron structures, which are further aggregated into micron-size particles, as can be seen in the HR-SEM imaging in Fig. 5. The surface area is&#;~&#;30 m2/g, which is typical for porous materials, the porosity of which is interstitial. This porous structure allows diffusion of substrate molecules to the buried enzyme molecules, and diffusion of product molecules out, but the tight aggregation around the entrapped enzyme molecules prevents their leaching out (there is zero activity of the supernatant solutions). That property of efficient entrapment on one hand, and free molecular diffusion on the other hand is possible because the cage walls are perforated with interstitial pores which are too small for the enzyme to leach out, as illustrated in Fig. 6 (roughly, with an Au nanocrystallite average size of 16 nm, one gets interstitial pores to be around 4 nm, while GOx diameter is around 8 nm33). Adding to this physical entrapment property are the strong interactions of the enzyme with gold and CTAB, detailed below. The known cost of entrapment within a porous matrix is the diffusional limitation which slows the reaction rate, but then the gains are the enhanced stability (Fig. 3), the recyclability (Fig. 4), the ability to construct a device, and the prospect of modifying the activity.

HR-SEM images of GOx/CTAB@Au (bars: left 500 nm, right: 5 µm).

Full size image

Left: illustration of the interactions which affect a suggested channel opening of the entrapped enzyme in gold with CTAB; right: zoom-in on the CTAB bilayer formed between the gold and GOx (RCSB ID: 1GPE).

Full size image

In general, affecting and widening of enzyme activity has been achieved by two types of approach: tampering with the primary structure of the protein by various enzyme-engineering and mutation methods, which, from that point of view can be considered as forming new enzymes34,35,36; or, affecting the tertiary and quaternary conformational structure of the original native enzyme, leaving the sequence of amino acids untouched. Such conformational changes have been induced mainly by adsorptive interactions37,38. The main target of both approaches has been, as expected, the active site36,38,39, but far less attention has been devoted to affecting the channel leading to the active site. The rationale in approaching the (dynamic) channel is that it acts like a highly stereoselective separation column, allowing entrance to the active site only for a substrate that fits the stereoselective screening. Thus, changing the conformation of the channel&#;particularly widening that entrance&#;may lead to alteration of the selectivity; this, we propose below, is the main mechanism that explains the observations of this report. Affecting the channel was reported by enzyme engineering methods40, but affecting it by conformational changes that do not involve changes in the primary sequence, to the best of our knowledge, has not been reported.

The conformational changes that GOx undergoes in its entrapped state are due to three types of powerful interactions, each of which has been separately well documented, operating all together in the GOx/CTAB@Au system. The first is the interaction of proteins with gold. These interactions are with functional groups of most amino acids on the outer surface of the protein41,42, and particularly with the SH groups of the cysteine residues43, with the S of methionine side chain44, and with the exposed S&#;S moieties of cystine45,46 (Figs. S8, S9, Supplementary Material). Furthermore, since the buffered pH of the entrapped GOx&#;pH 5.1&#;is higher than its pI (4.247) rendering the enzymes interface negatively charged, one should also consider interactions with the anionic aspartate and glutamate41.

The second interaction to consider is that of CTAB with gold. It is a well-known strong interaction48,49, which has been used, for instance, to control the morphology and shape of gold nano-structures49,50,51,52,53. A special feature of the CTAB-gold interaction, first proposed by El-Sayed et al.49 and confirmed by numerous subsequent studies, is the formation of an adsorbed CTAB double layer on the gold surface49,54,55. The proposed structure of that bilayer is the following (Fig. 6, right): in the first layer the cationic (Me3)R&#;N+ faces the gold surface through a bromide bridge54: Au&#;Br&#;&#;N+. Supporting that proposition is that fact that the bromide anion is known to adsorb strongly on gold (particularly on 111 planes but also on lower index plains53), and it has also strong attraction to CTA+53. The cetyl chain of CTAB, perpendicular to the gold surface, accepts the second layer through cetyl&#;cetyl hydrophobic interactions and a double-layer forms, much like in liposomes55: Au&#;Br&#;&#;N+&#;R&#;R&#;N+. That second layer terminal N+ of CTA+ is then free to interact with the negatively charged protein56,57, releasing the bromide. We assume that the bilayer structure is not perfect inside the gold cage, and that there are also isolated CTAB molecules adsorbed on the gold surface which do not take part in a bilayer&#;these may add hydrophobic interactions between the alkyl tail of the CTAB and the residues of the hydrophobic amino acids on the surface of the protein58,59, such as valine and leucine59. That holding of the enzyme inside the cage more rigidly by these combined types of interactions, is expressed by the major increase in the thermal stability (Fig. 3), and by the ability to recycle the entrapped enzyme (Fig. 4). But how do these interactions also affect the conversion of GOx into a general oxidase? This is discussed next:

The proposed induced conformational change is based on the fact that GOx is a homodimeric oligomer of which the two monomeric units are held by non-covalent bonds60. As common in many homodimeric enzymes, the interface between these two units forms the channel leading to the two active sites, a channel that evolved also to act as a stereoselective filter for substrates which can enter and reach the active site61,62. Unlike conformational changes which occur by 2D adsorption, which is a non-isotropic process, the 3D gold cage in our case, pulls apart the protein in an isotropic manner, that is, in all directions (Fig. 6). Since the interface between the two monomeric units is not held together by strong covalent bonds, it constitutes an initial &#;crack&#; that already exists in the enzyme, and which can be further affected. Thus, based on the activity analysis, we propose that a reasonable interpretation is that the combined pull of the direct protein-gold interaction and the gold-CTAB double layer interactions, opens that crack to a degree that the channel loses its evolutionary built strict stereoselective gate-keeper property, tailored for d-glucose exclusively. This, in turn, allows all substrates described in this report, to diffuse to the two active sites and reach them.

Further confirmation to this proposed mechanism comes from the comparison of the activity of GOx when entrapped with or without entrapment CTAB (a comparison possible for the monosaccharides which can be entrapped without CTAB). It is seen&#;Figs. 3, 4 and Table 2&#;that GOx/CTAB@Au greatly outperforms GOx@Au in any parameter, including even the performance on the native d-glucose. This comparison allows the two types of pulling effects&#;with pure gold, and through CTAB. In fact, that additional pull, overshadows the pull of the gold itself, as seen in Table 2. The observation that the partial widening of activity with gold only is generalized by co-entrapping CTAB, strengthens the proposed conformational opening the access to the active site. The focus on the channel is also highlighted by discussing what do these observations mean from the point of view of the active site&#;next.

Our observations indicate that once the access to the active site is opened to saccharides other than d-glucose, the active site is capable of oxidizing non-specifically other saccharides. That is, our observations indicate that selectivity of GOx is not a sole feature of the active site. Furthermore, these observations also suggest an interpretation that the active site of GOx (the peptide fragment containing Glu412, His516 and His) resembles an early-evolutionary preserved structure of this biocatalyst, and that the strict stereospecificity evolved over the ages with the build-up of the encompassing protein and its dimerization, to form the exclusive activity towards d-glucose of the modern enzyme we know. That non-specificity of the active site towards the stereochemistry of the saccharide CH&#;OH is particularly evident by the ability of the entrapped GOx to oxidize methyl glucoside and sucrose, two molecules in which the d-glucose analogous cyclic β-C(1)H-OH is blocked. That free GOx is capable of oxidizing methyl glucoside at all, utilizing air oxygen and releasing H2O2 is evident from the low but detectable activity in solution&#;Fig. 7. The efficient activity of the exposed active site is also compatible with the observation&#;Table 1&#;that the higher apparent KM values observed of methyl glucoside, raffinose, sucrose and glucose-6-phosphate, are accompanied by higher Vmax values: the lower affinity (higher KM) means shorter residence time at the active site, and that increases Vmax if the oxidation step is fast.

Activity of free GOx on methylglucoside: GOx@Au (red), GOx/CTAB@Au (black) and free GOx (blue) on methylglucoside (right).

Full size image

In conclusion, &#;Au my GOx&#;: we have been able to convert GOx into a general sugar oxidase, including saccharides which lack natural specific oxidases. High-specificity and wide scope activity of enzymes are two desirable properties, which are complementary, and answer different needs in biotechnology, medicine, pharmaceutics, and enzymatic devices. Enzymes in their native form and environments usually answer the high-specificity requirement. And since this is the starting point, widening the scope of activity requires manipulation of the native enzyme. Here we have shown how adsorptive-induced conformational changes can be utilized for that purpose. As GOx is common and robust, and since most of the saccharides employed in this study are of high-volume industrial use but lack specific oxidases, our study opens new directions to be considered. For instance, one could envisage that GOx/air/d-glucose fuel cells64 can now be generalized to other common sugars or their mixtures, using GOx only65. An added advantage of the entrapment in gold developed in this study is that the protective heterogenization, renders the use of the general oxidative GOx easier to implement in the variety of potential uses.

The company is the world’s best Glucose Oxidase supplier. We are your one-stop shop for all needs. Our staff are highly-specialized and will help you find the product you need.