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CAS Number: 9001-37-0
EC Number: 1.1.3.4
Glucose Oxidase = GOD = Glucose Oxyhydrase
PRODUCT DESCRIPTION
Glucose Oxidase SBE-01GO is produced by submerged fermentation of Aspergillus niger followed by purification, formulation and drying.
The product is able to whiten flour, strengthen gluten and improve dough handling properties and is often used for various baked products.
MECHANISM
Glucose Oxidase can specifically catalyze β-D-glucose to form gluconic acid and hydrogen peroxide at the presence of oxygen, which promotes the formation of gluten network, and thereafter the dough handling properties and sensory properties of baked products.
A C6H12O6+ O2 + H2O → C6H12O7 + H2O2
B C6H12O6+ 12 O2 →C6H12O7
REACTION PARAMETERS
PARAMETERS
RANGE
Activity Temperature
20℃-50℃
Optimum Temperature
20℃-35℃
Activity pH
4.0-6.5
Optimum pH
5.0-6.0
PRODUCT SPECIFICATION
Items
Description
Declared Activity
2700 ug
Physical Form
Powder
Color
Light yellow
Odour
Normal microbial fermentation odour.
APPLICATION RECOMMENDATION
For baking industry: The recommended dosage is 2-40g per ton of flour.
The dosage has to be optimized based on each application, the raw material specifications, product expectation and processing parameters.
Glucose Oxidase is better to begin the test with the convenient volume.
SAFE HANDLING PRECAUTIONS
Enzyme preparations are proteins that may induce sensitization and cause allergic type of symptoms in susceptible individuals.
Prolonged contact may cause minor irritation for skin, eyes or nasal mucosa.
Any direct contact with human body should be avoided.
Glucose Oxidase irritation or allergic response for skin or eyes develops, please consult a doctor.
WARNINGS
Keep sealed after use every time to avoid microbial infections and inactivation of enzymes until its finish.
PACKAGE AND STORAGE
Ø Package： 25kgs/drum; 1,125kgs/drum.
Ø Storage： Keep sealed in a dry and cool place and avoid direct sunlight
Ø Shelf life: 12 months in a dry and cool place.
Glucose oxidase is a subset of oxidoreductase enzymes that catalyzes the transfer of electrons from an oxidant to a reductant.
Glucose oxidases use oxygen as an external electron acceptor that releases hydrogen peroxide (H2 O2 ).
Glucose oxidase has many applications in commercial processes, including improving the color and taste, increasing the persistence of food materials, removing the glucose from the dried egg, and eliminating the oxygen from different juices and beverages.
Moreover, glucose oxidase, along with catalase, is used in glucose testing kits (especially in biosensors) to detect and measure the presence of glucose in industrial and biological solutions (e.g., blood and urine specimens).
Hence, glucose oxidase is a valuable enzyme in the industry and medical diagnostics.
Therefore, evaluating the structure and function of glucose oxidase is crucial for modifying as well as improving its catalytic properties.
Finding different sources of glucose oxidase is an effective way to find the type of enzyme with the desired catalysis.
Besides, the recombinant production of glucose oxidase is the best approach to produce sufficient amounts of glucose oxidase for various uses.
Accordingly, the study of various aspects of glucose oxidase in biotechnology and bioprocessing is crucial.
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.
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.
Glucose Oxidase also has many applications in biotechnologies, typically enzyme assays for biochemistry including biosensors in nanotechnologies.
Glucose Oxidase was first isolated by Detlev Müller in 1928 from Aspergillus niger.
Function
Several species of fungi and insects synthesize glucose oxidase, which produces hydrogen peroxide, which kills bacteria
Notatin, extracted from antibacterial cultures of Penicillium notatum, was originally named Penicillin A, but was renamed to avoid confusion with penicillin.
Notatin was shown to be identical to Penicillin B and glucose oxidase, enzymes extracted from other molds besides P. notatum; it is now generally known as glucose oxidase.
Early experiments showed that notatin exhibits in vitro antibacterial activity (in the presence of glucose) due to hydrogen peroxide formation.
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.
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.
Structure
Glucose oxidase enzyme powder from Aspergillus niger.
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.
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.
Glucose Oxidase 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.
Glucose oxidase catalyzes the oxidation of β-D-glucose into D-glucono-1,5-lactone, which then hydrolyzes into gluconic acid.
Glucose Oxidase 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.
Glucose Oxidase the GOx-catalyzed redox reaction, FAD works as the initial electron acceptor and is reduced to FADH−.
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.
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.
This is the technology behind the disposable glucose sensor strips used by diabetics to monitor serum glucose levels.
Food preservation
Glucose Oxidase manufacturing, GOx is used as an additive thanks to its oxidizing effects: it prompts for stronger dough in baking, replacing oxidants such as bromate.
Glucose Oxidase 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.
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 2016 for the prevention and treatment of the common cold.
Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) catalyzes the oxidation of β-d-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen peroxide.
Microbial glucose oxidase is currently receiving much attention due to its wide applications in chemical, pharmaceutical, food, beverage, clinical chemistry, biotechnology and other industries.
Novel applications of glucose oxidase in biosensors have increased the demand in recent years.
Present review discusses the production, recovery, characterization, immobilization and applications of glucose oxidase.
Production of glucose oxidase by fermentation is detailed, along with recombinant methods.
Various purification techniques for higher recovery of glucose oxidase are described here.
Issues of enzyme kinetics, stability studies and characterization are addressed.
Immobilized preparations of glucose oxidase are also discussed.
Applications of glucose oxidase in various industries and as analytical enzymes are having an increasing impact on bioprocessing.
Glucose oxidase (GOX) from Aspergillus niger is a well-characterised glycoprotein consisting of two identical 80-kDa subunits with two FAD co-enzymes bound.
Both the DNA sequence and protein structure at 1.9 Ǻ have been determined and reported previously.
GOX catalyses the oxidation of D-glucose (C6H12O6) to D-gluconolactone (C6H10O6) and hydrogen peroxide.
GOX is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent.
GOX is Generally Regarded As Safe, and GOX from A. niger is the basis of many industrial applications.
GOX-catalysed reaction removes oxygen and generates hydrogen peroxide, a trait utilised in food preservation.
GOX has also been used in baking, dry egg powder production, wine production, gluconic acid production, etc.
Its electrochemical activity makes it an important component in glucose sensors and potentially in fuel cell applications.
This paper will give a brief background on the natural occurrence, functions as well as the properties of glucose oxidase.
A good coverage on the diverse uses of glucose oxidase in the industry is presented with a brief outline on the working principles in the various settings.
Furthermore, food grade GOX preparations are relatively affordable and widely available; the readers may be encouraged to explore other potential uses of GOX.
One example is that GOX-catalysed reaction generates significant amount of heat (∼200 kJ/mol), and this property has been mostly neglected in the various applications described so far.
Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) catalyzes the oxidation of beta-D-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen peroxide.
Microbial glucose oxidase is currently receiving much attention due to its wide applications in chemical, pharmaceutical, food, beverage, clinical chemistry, biotechnology and other industries.
Novel applications of glucose oxidase in biosensors have increased the demand in recent years.
Present review discusses the production, recovery, characterization, immobilization and applications of glucose oxidase.
Production of glucose oxidase by fermentation is detailed, along with recombinant methods.
Various purification techniques for higher recovery of glucose oxidase are described here.
Issues of enzyme kinetics, stability studies and characterization are addressed.
Immobilized preparations of glucose oxidase are also discussed.
Applications of glucose oxidase in various industries and as analytical enzymes are having an increasing impact on bioprocessing.
Glucose Oxidase
Glucose oxidase measures blood glucose level in biosensors
Glucose oxidase with FAD in red.
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Diabetes is a worldwide health problem affecting hundreds of millions of people.
Fortunately, with careful management of diet and medication, the many complications of diabetes can be reduced.
Part of this treatment includes the monitoring of glucose levels in the blood, so that proper action may be taken if levels get too high.
The enzyme glucose oxidase has made glucose measurement fast, easy, and inexpensive.
Chemical Defense
Glucose oxidase, shown here from PDB entry 1gpe , is a small, stable enzyme that oxidizes glucose into glucolactone, converting oxygen into hydrogen peroxide in the process.
Glucose Oxidase normal biological function appears to be centered on the peroxide that is formed: hydrogen peroxide is a toxic compound that can be used to kill bacteria. For instance, glucose oxidase is found on the surface of fungi, where it helps protect against bacterial infection, and it is also found in honey, where it acts as a natural preservative.
Biotech Bonanza
Glucose Oxidase is used as the heart of biosensors that measure the amount of glucose in blood.
The trick to these biosensors is that the enzyme takes something that is difficult to measure--glucose--and creates something that is easy to measure--hydrogen peroxide.
A typical laboratory glucose meter includes some of the enzyme trapped inside a membrane.
Glucose enters the sensor and is converted to glucolactone.
Glucose Oxidase the process, hydrogen peroxide is formed, which is then sensed by a platinum electrode.
Glucose OxidaseThe more glucose there is in the sample, the more peroxide is formed, and the stronger the signal at the electrode.
Controlling Costs
Of course, platinum electrodes are expensive, so newer biosensors use a serendipitous property of glucose oxidase to make even more affordable glucose meters.
Glucose oxidase is very specific for glucose in the initial oxidation reaction, but can use many different compounds as the final electron acceptor, not just oxygen.
So, biotech researchers have developed different "mediator" molecules, such as ferrocene, to work in place of oxygen.
Glucose Oxidase molecules pick up electrons from the glucose reaction, and then deliver them directly to a cheaper type of electrode.
Glucose Oxidase method is used to create single-use, disposable test strips that have glucose oxidase, the mediator, and the electrode compound all printed on the surface.
Two bacterial glucose dehydrogenases.
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Choices for Biosensors
Other enzymes that oxidize glucose are also used as glucose monitors.
Two glucose dehydrogenases that are being explored for use in biosensors are shown here.
At the top is an enzyme that uses NAD as the cofactor to perform the oxidation reaction (PDB entry 1gco ), and the enzyme at the bottom (PDB entry 1cq1 ) uses an unusual pyrroloquinoline quinone cofactor.
General description
Mass of glucose oxidase (GOX), a flavoprotein ranges from approximately 130 to 175 kDa. Some fungi and insects are capable of producing GOX.
Molecular Weight: 160 kDa (gel filtration)
pI: 4.2
Extinction coefficient: E1% = 16.7 (280 nm)
Glucose oxidase from Aspergillus niger is a dimer consisting of 2 equal subunits with a molecular mass of 80 kDa each.
Each subunit contains one flavin adenine dinulceotide moiety and one iron.
The enzyme is a glycoprotein containing ~16% neutral sugar and 2% amino sugars. The enzyme also contains 3 cysteine residues and 8 potential sites for N-linked glycosylation.
Glucose oxidase is capable of oxidizing D-aldohexoses, monodeoxy-D-glucoses, and methyl-D-glucoses at varying rates.
Glucose OxidaseGlucose oxidase is specific for β-D-glucose with a KM of 33-110 mM.
Glucose oxidase does not require any activators, but it is inhibited by Ag+, Hg2+, Cu2+, phenylmercuric acetate, and p-chloromercuribenzoate. It is not inhibited by the nonmetallic SH reagents: N-ethylmaleimide, iodoacetate, and iodoacetamide.
Glucose oxidase can be utilized in the enzymatic determination of D-glucose in solution.
As glucose oxidase oxidizes β-D-glucose to D-gluconolactate and hydrogen peroxide, horseradish peroxidase is often used as the coupling enzyme for glucose determination.
Although glucose oxidase is specific for β-D-glucose, solutions of D-glucose can be quantified as α-D-glucose will mutorotate to β-D-glucose as the β-D-glucose is consumed by the enzymatic reaction.
We are committed to bringing you Greener Alternative Products, which adhere to one or more of The 12 Principles of Greener Chemistry.
This product has been enhanced for energy efficiency and waste prevention when used in fuel cell research.
For more information see the article in biofiles.
Application
Glucose Oxidase from Aspergillus niger has been used:
in the (glucose oxidase) GO reagent to measure the glucose content by the glucose oxidase (GO) method
to activate the human renal carcinoma cell line for constructing the oxidative stress model
to study its influence in the paste on the analytical performance of the bioelectrode
Glucose oxidase is widely used in the food and pharmaceutical industries as well as a major component of glucose biosensors.
Packaging
10000, 50000, 250000, 1000000, 2500000 units in poly bottle
Biochem/physiol Actions
Glucose oxidase (GOX) oxidizes D-glucose to D-gluconolactone and hydrogen peroxide.
Hydrogen peroxide, the catalytic product of GOX, serves as an anti-bacterial and anti-fungal agent.
Glucose Oxidase is safe and can be used in several industrial applications like baking, dry egg powder production, wine production, gluconic acid production, etc.
Glucose Oxidase possesses electrochemical activity, which makes it extremely important in glucose sensors and in fuel cell applications.
Glucose oxidase catalyses the oxidation of β-d-glucose to d-glucono-β-lactone and hydrogen peroxide, with molecular oxygen as an electron acceptor.
Quality
May contain traces of amylase, maltase, glycogenase, invertase, and galactose oxidase.
Unit Definition
One unit will oxidize 1.0 μmole of β-D-glucose to D-gluconolactone and H2O2 per min at pH 5.1 at 35 °C, equivalent to an O2 uptake of 22.4 μl per min.
Glucose Oxidase the reaction mixture is saturated with oxygen, the activity may increase by up to 100%.
Analysis Note
Protein determined by biuret
Fungal glucose oxidase (GOD) is widely employed in the different sectors of food industries for use in baking products, dry egg powder, beverages, and gluconic acid production.
GOD also has several other novel applications in chemical, pharmaceutical, textile, and other biotechnological industries.
Glucose Oxidase electrochemical suitability of GOD catalyzed reactions has enabled its successful use in bioelectronic devices, particularly biofuel cells, and biosensors.
Other crucial aspects of GOD such as improved feeding efficiency in response to GOD supplemental diet, roles in antimicrobial activities, and enhancing pathogen defense response, thereby providing induced resistance in plants have also been reported.
Moreover, the medical science, another emerging branch where GOD was recently reported to induce several apoptosis characteristics as well as cellular senescence by downregulating Klotho gene expression.
These widespread applications of GOD have led to increased demand for more extensive research to improve its production, characterization, and enhanced stability to enable long term usages.
Currently, GOD is mainly produced and purified from Aspergillus niger and Penicillium species, but the yield is relatively low and the purification process is troublesome.
Glucose Oxidase is practical to build an excellent GOD-producing strain.
Therefore, the present review describes innovative methods of enhancing fungal GOD production by using genetic and non-genetic approaches in-depth along with purification techniques.
The review also highlights current research progress in the cost effective production of GOD, including key advances, potential applications and limitations. Therefore, there is an extensive need to commercialize these processes by developing and optimizing novel strategies for cost effective GOD production.
Overview
Glucose oxidase (GOD; β-D-glucose:oxygen 1-oxidoreductase; glucose aerodehydrogenase; is a very important oxidoreductase enzyme (flavoprotein). Structurally, GOD is a holoenzyme consisting of two identical 80 kDa subunits at the active site that containing a cofactor flavin adenine dinucleotide (FAD).
These subunits act as a redox carrier in catalysis.
GOD belongs to the glucose/methanol/choline (GMC) oxidoreductase family, which incorporates numerous other industrially imperative catalysts, particularly in the field of diagnostics, for example, cholesterol oxidase, choline oxidase, methanol oxidase, alcohol oxidase, amino acid oxidase, and pyranose oxidase (Ferri et al., 2011).
These members of the GMC oxidoreductase family share a homologous structural backbone, including an adenine-dinucleotide-phosphate binding βαβ-fold close to their amino terminus and five other segments of conserved sequences dispersed throughout their primary sequences.
GOD catalyzes the oxidation of β-D-glucose into D-glucono-δ-lactone at its first hydroxyl group using atomic oxygen (O2) as the electron acceptor with the synchronous generation of hydrogen peroxide (H2O2).
Both end products break down spontaneously and catalytically.
Specifically, D-glucono-δ-lactone is subsequently hydrolyzed slowly by enzyme lactonase to D-gluconic acid (GA), while the generated H2O2 is broken down to O2 and water (H2O) by catalase (CAT). GOD catalyzes the oxidation of glucose, according to a ping-pong mechanism.
The overall reaction is given below:
GOD (FAD) + β−D-Glucose → GOD (FADH2) + D-Glucono-δ-lactone
GOD (FADH2) + O2 → GOD (FAD) + H2O2
β−D-Glucose + GOD (FAD) + O2 → GA + GOD (FADH2) + H2O2
GOD is profoundly particularly for the β-anomer of D-glucose, while the α-anomer does not seem, by all accounts, to be a reasonable substrate . Thus, GOD shows bring down exercises while using 2-deoxy-D-glucose, D-mannose, and D-galactose as substrates.
Among enzymes currently known to oxidize glucose; GOD is the best-known because of its high degree of specificity.
GOD can be obtained from a large number of different sources, including red algae, citrus fruits, insects, bacteria, plants, animals, and fungi. The regular capacity of GOD in these organic frameworks is to act primarily as an antibacterial and antifungal specialist through the generation of H2O2.
Among these sources, fungi have an eminent status and industrially fungal sources have been preferred since the early 1950s (Fiedurek and Gromada, 1997a).
For decades, fungi have been thoroughly inspected for GOD production as cell factories due to their magnificent capacities to use an assortment of carbon sources and to accumulate a large proportion of natural GOD under stressed conditions.
Because GOD from fungi has applications in a broad spectrum, it must be stable at the higher temperature and for a longer duration so that it can be used economically.
Nevertheless, the mechanisms of GOD accumulation by different fungi are not fully understood, even though many successful attempts have been made to improve and optimize fungal GOD production by using genetic modifications and other approaches.
The filamentous fungi Aspergillus and Penicillium serve as industrial producers of GOD at a large scale, among which Aspergillus niger is the most ordinarily used for the industrial yield of GOD (Pluschkell et al., 1996).
Various properties of GOD produced by A. niger are listed in Table 1, including methods that have been reported for stabilizing GOD, the use of additives, and engineering through site-directed or random mutagenesis coupled to expression in heterologous hosts
Bright and Porter (1975) have reviewed the kinetic behavior and redox states of the flavin coenzyme. Bentley (1963) has reviewed the general properties of the enzyme.
Since its discovery as an "antibiotic" (shown subsequently to be due to peroxide formation) there has been an interest in glucose oxidase, chiefly because of its utility in glucose estimation.
Following Keston's report in 1956 of coupling the reaction to peroxidase and a chromogen, (qualitative) glucose "dip-sticks" became available for screening for urine glucose.
Based on Teller's paper in the same year, Worthington offered the first quantitative enzymatic system for the colorimetric determination of glucose.
For most clinical work the crude form of the A. niger enzyme has been satisfactory.
However, it contains trace amount of polysaccharidases such as amylase, maltase, and sucrase which can contribute to falsely high glucose levels.
The purified enzyme is free of these traces and is recommended for analytical use in the presence of di- or polysaccharides.
Characteristics of Glucose Oxidase from A. niger:
Molecular weight: 160,000 (Tsuge et al. 1975).
Composition: The enzyme consists of two identical polypeptide chain subunits (80,000 daltons) covalently linked by disulfide bonds (O'Malley and Weaver 1972). Each subunit contains one mole of Fe and one mole of FAD (flavin-adenine dinucleotide).
Tsuge et al. (1975) report the molecule to be approximately 74% protein, 16% neutral sugar and 2% amino sugars.
Glucose Oxidase indicate that the FAD is replaceable with FHD (flavin-hypoxanthine dinucleotide) without loss of activity.
Optimum pH: 5.5 with broad range 4 - 7 (Bright and Appleby 1969).
See also Weibel and Bright (1971b).
Specificity: The enzyme is highly specific for β-D-glucose.
The α anomer is not acted upon. 2-deoxy-D-glucose, D-mannose and D-galactose exhibit low activities as substrate. (See Bentley 1966).
Inhibitors: Ag+, Hg2+, Cu2+ ( Nakamura and Ogura 1968).
FAD binding is inhibited by several nucleotides (Swobada 1969). See also Rogers and Brandt (1971).
Stability: Dry preparations are stable for years when stored cold.
Solutions are reasonably stable under a variety of conditions.
Chemical Name：Glucose oxidase
CAS No.:9001-37-0
Appearance: Yellow powder
Packaging:25KG/Drum
Sample: available
Categories: Catalysts & Chemical Auxiliary Agents, Food Additives
Glucose oxidase Usage
—— antioxidants, color protectors, preservatives, enzyme preparations. Flour reinforcement agent. Enhanced strength of gluten
—— glucose oxidase as a food additive with glucose conversion and removal of residual oxygen
Glucose oxidase Packaging and Shipping
Packing:25KG/Drum
Glucose oxidase Storage
Should be stored at room temp, away from heat, moisture, and direct light.
Glucose Oxidase (GOD) oxidizes glucose to -gluconolactone in presence of moleculer oxygen by forming hydrogen peroxide.
As a result of the catalysed reaction, GOD is widely used in cases where glucose or molecular oxygen should be removed to extend the shelf life of foods or used in the production of controlled hydrogen peroxide or gluconic acid.
One of the most important application areas of GOD is the construction of the glucose biosensors.
Glucose Oxidase are several studies about GOD purification, immobilization, industrial and analytical applications, so, fast and sensitive determination of GOD activity is essential for these studies.
Glucose Oxidase this study, GOD activity determination methods were reviewed mainly four approaches: determination of decrease in glucose or oxygen concentration and determination of increase in hydrogen peroxide or gluconic acid levels.
Abstract
Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells.
Glucose Oxidase is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity.
Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced.
GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (KM) and nearly four-fold greater catalytic rate (kcat).
Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability.
A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions.
Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis.
Ultimately, four mutants were identified with significantly improved catalytic activity.
A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in kcat at the expense of a 3% loss of substrate affinity (increase in apparent KM for glucose) resulting in a specify constant (kcat/KM) of 23.8 (mM−1 · s−1) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx.
Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM−1 · s−1, respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.
What is Glucose Oxidase?
Glucose oxidase, or simply GOX, is a type of enzyme which belongs to the group of oxidoreductases. It has extensive application in the food industry. GOX has become a part of flour improver mixes and dough conditioners. It is a clean label alternative to ADA, potassium bromate and other oxidizing agents that have come under public scrutiny due to health concerns.
Uses of GOX in food include:1
Oxygen scavenging / depletion in beverages to extend their shelf life
Glucose removal in egg powder processing
Bread flour improver or dough strengthener
Online glucose monitoring and control in commercial bioprocesses (fermentations)
Prevention of color loss in foods triggered by enzymatic reactions and oxygen presence
Glucose determination or quantification (analytical methods in foods)
Origin
Almost all GOX preparations available on the market are produced by Aspergillus niger and other species belonging to that fungal genus. Production of glucose oxidase is believed to be a survival mechanism for fungus under certain adverse conditions.
Function
The following table summarizes some key aspects of GOX when used in high-speed breadmaking:
GOX irreversibly oxidizes β-D-glucose to glucono-δ-lactone, which is immediately converted to gluconic acid in the presence of water. In addition to glucose, it oxidizes a number of aldose molecules such as α-D-glucose, mannose, xylose, and galactose.2
GOX addition to yeast-leavened systems creates oxidative conditions which promote the formation of a superior or more developed polymeric network through one or a combination of the following mechanisms:3
Oxidation of sulfhydryl (SH) groups and formation of higher amounts of disulphide (S–S) bonds that bring separate protein chains together.
2R–SH (2 cysteine residues from separate protein chains) + H2O2 from GOX → R–S–S–R’ (cysteine molecules link and bring protein chains together)
Non-disulphide protein cross-linking (aggregation) by formation of dityrosine bridges between separate gluten-forming chains.
This is possible due to the oxidation of tyrosine residues. This mechanism is highly appreciated in gluten-free formulations rich in egg proteins.
Oxidate gelation of beneficial WE-AXs which further strengthens the gluten-starch matrix.
Ferulic acid residues link adjacent AX chains by forming diferulic acid bridges.
When adding GOX to bread and bun formulations, the ultimate goal is to produce hydrogen peroxide H2O2 and create the necessary oxidizing conditions that provide the dough strengthening effects.
Commercial production
Large scale production of GOX relies on fermentation and downstream processing using modern biotechnology.
The enzyme is typically obtained from fungal sources, such as Aspergillus oryzae or Penicillium chrysogenum.
The following diagram summarizes the commercial production of GOX:
The commercial production of Glucose Oxidase.
Application
The addition of GOX creates a stronger dough which renders the following results:
Higher loaf volume
Reduced dough stickiness due to surface drying effect of hydrogen peroxide
Superior oven spring and gas retention
Improved (more even and tighter) crumb structure
Increased tolerance to overproofing and gas bubble coalescence due to unexpected line stoppages
Reduced use of vital wheat gluten
Slightly increased water absorption
Like any other enzyme, functionality of GOX in the bakery industry is affected by temperature, pH, ionic strength, and moisture level in the dough impacted by flour hydration.
As GOX requires both oxygen and glucose, its strengthening effect on the bulk rheology of the dough occurs mainly during mixing, where oxygen is not limited.
Glucose Oxidase later stages of the breadmaking process, such as proofing and baking, the reaction rate and GOX functionality is yet to be fully understood.
Glucose Oxidase is reasonable to assume that GOX action stops quickly after mixing as the oxygen incorporated as air during mixing is rapidly consumed by yeast.
Regulation
GOX is GRAS (Generally Recognized as Safe) and considered a food processing aid according to the Codex Alimentarius.
The FDA regulates their origin (food-compatible) and establishes limits to their use (if applicable) based on GMP.
Optimizing the electrical communication between enzymes and electrodes is critical in the development of biosensors, enzymatic biofuel cells, and other bioelectrocatalytic applications.
One approach to address this limitation is the attachment of redox mediators or relays to the enzymes. Here we report a simple genetic modification of a glucose oxidase enzyme to display a free thiol group near its active site.
Glucose Oxidase facilitates the site-specific attachment of a maleimide-modified gold nanoparticle to the enzyme, which enables direct electrical communication between the conjugated enzyme and an electrode.
Glucose oxidase is of particular interest in biofuel cell and biosensor applications, and the approach of “prewiring” enzyme conjugates in a site-specific manner will be valuable in the continued development of these systems.
Application Notes
Glucose oxidase can be utilized in the enzymatic determination of D-glucose in solution.
As glucose oxidase oxidizes β-D-glucose to D-gluconolactate and hydrogen peroxide, horseradish peroxidase is often used as the coupling enzyme for glucose determination.
Although glucose oxidase is specific for β-D-glucose, solutions of D-glucose can be quantified as α-D-glucose will mutorotate to β-D-glucose as the β-D-glucose is consumed by the enzymatic reaction. Glucose oxidase is widely used in the food and pharmaceutical industries as well as a major component of glucose biosensors.
Usage Statement
Unless specified otherwise, MP Biomedical's products are for research or further manufacturing use only, not for direct human use.
For more information, please contact our customer service department.
Key Applications
Enzymes | Inhibitors | Substrates
SKU 02195196-CF
Alternate Names β-D-Glucose:oxygen 1-oxidoreductase; G.Od.; Gox
Application Notes
Glucose oxidase can be utilized in the enzymatic determination of D-glucose in solution. As glucose oxidase oxidizes β-D-glucose to D-gluconolactate and hydrogen peroxide, horseradish peroxidase is often used as the coupling enzyme for glucose determination. Although glucose oxidase is specific for β-D-glucose, solutions of D-glucose can be quantified as α-D-glucose will mutorotate to β-D-glucose as the β-D-glucose is consumed by the enzymatic reaction. Glucose oxidase is widely used in the food and pharmaceutical industries as well as a major component of glucose biosensors.
Base Catalog Number 195196
Biochemical Physiological Actions Glucose oxidase catalyses the oxidation of β-d-glucose to d-glucono-β-lactone and hydrogen peroxide, with molecular oxygen as an electron acceptor.
CAS # 9001-37-0
EC Number 232-601-0
Extinction Coefficient 16.7 (280 nm)(Lit.)
Format Lyophilized powder
Hazard Statements H334
Isoelectric Point 4.2 (Lit.)
Melting Point 83 °C
Molecular Weight 180.156 g/mol
Personal Protective Equipment Dust mask, Eyeshields, Faceshields, Gloves
RTECS Number RQ8452000
Safety Symbol GHS08
Solubility Water Solubility1200000 mg/L (at 30 °C)
Source Aspergillus niger
Specific Activity >100 u/mg material
Unit Definition One unit of glucose oxidase is the activity which causes the liberation of 1 micromole of glucose per minute at 25 °C and pH 7.0 under the specified conditions.
Usage Statement Unless specified otherwise, MP Biomedical's products are for research or further manufacturing use only, not for direct human use. For more information, please contact our customer service department.
Synonyms
AldO,
beta-D-glucose oxidase,
beta-D-glucose oxygen-1-oxidoreductase,
beta-D-glucose/oxygen 1-oxidoreductase,
beta-D-glucose: oxygen 1-oxidoreductase,
beta-D-glucose:O2 1-oxidoreductase,
beta-D-glucose:O2-1-oxidoreductase,
beta-D-glucose:oxygen 1-oxido-reductase,
beta-D-glucose:oxygen 1-oxidoreductase,
beta-D-glucose:oxygen oxidoreductase
Glucose Oxidase food enzyme glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase; EC 1.1.3.4) is produced with a genetically modified Aspergillus niger strain ZGL by DSM Food Specialties B.V.. The genetic modifications do not give rise to safety concerns.
Glucose Oxidase food enzyme is free from viable cells of the production organism and recombinant DNA.
Glucose Oxidase glucose oxidase is intended to be used in baking processes.
Based on the maximum use levels, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.004 mg TOS/kg body weight (bw) per day.
Glucose Oxidase toxicity studies were carried out with an asparaginase from A. niger (strain ASP).
Glucose Oxidase Panel considered this enzyme as a suitable substitute to be used in the toxicological studies, because they derive from the same recipient strain, the location of the inserts are comparable, no partial inserts were present and the production methods are essentially the same.
Genotoxicity tests did not raise a safety concern.
Glucose Oxidase systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats.
Glucose Oxidase Panel identified a no observed adverse effect level (NOAEL) at the highest dose of 1,038 and 1,194 mg TOS/kg bw per day (for males and females, respectively) that, compared with the estimated dietary exposure, results in a sufficiently high margin of exposure (MoE) (of at least 260,000).
Similarity of the amino acid sequence to those of known allergens was searched and one match was found.
Glucose Oxidase Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low.
Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.
Conversion of sucrose into fructose and gluconic acid using invertase, glucose oxidase and catalase was studied by discontinuous (sequential or simultaneous addition of the enzymes) and continuous (simultaneous addition of the enzymes in a 100 kDa-ultrafiltration membrane reactor) processes.
The following parameters were varied: concentration of enzymes, initial concentration of substrates (sucrose and glucose), pH, temperature and feeding rate (for continuous process).
The highest yield of conversion (100%) was attained through the discontinuous (batch) process carried out at pH 4.5 and 37 ºC by the sequential addition of invertase (14.3 U), glucose oxidase (10,000 U) and catalase (59,000 U).
Herein, we demonstrate glucose oxidase (GOx) mediated targeted cancer-starving therapy by self-assembled vesicle of trimesic acid based biotinylated amphiphile (TMB).
The TMB vesicles entrapped GOx and selectively killed cancer cells (HeLa, B16F10), with ∼6-fold higher efficiency compared to non-cancer cells (CHO, NIH3T3), by blocking the energy supply to tumors through the oxidation of intracellular glucose.
Since its discovery in 1928, glucose oxidase (GOx) is still one of the most studied enzymes worldwide because of its various industrial applications.
GOx can be used for textile bleaching, in the food and baking industr or for the production of gluconic acid for example.
The most well-known application of glucose oxidase is its use in daily and continuous glucose sensors for diabetes management.
GOx may also find applications in glucose/O2 biofuel cell or self-power sensors.
The history of electrochemical glucose sensors is well documented and has been recently reviewed elsewhere.
The greatest benefits of glucose oxidase over other sugars enzymes for bioelectrochemical applications are its high specificity for glucose, low redox potential (~ -0.42V vs. Ag/AgCl at pH 7.4), and good thermostability.
The three major drawbacks are the production of H2O2 during the oxidative half-reaction which can decrease the stability of enzymes, the competition between O2 and redox mediators for GOx electrons and the fact that the active redox center in native GOx is deeply buried within the protein preventing direct electron transfer (DET) to electrode surfaces.
Various efforts have been put forward to solve those issues mainly focus on increasing electrode surfaces to enhance current/power densities and/or improving the electrical communication between the redox site of GOx and electrodes surfaces.
So many and diverse strategies, more or less successful, have been reported that makes impossible a summary of those methods.
Enzyme engineering has also been employed to improve the specific activity,glucose affinity and O2 sensitivity of GOx including ultrahigh-throughput screening.
But, most of those efforts were performed in homogeneous solution using O2 as an electron acceptor and are not suitable for electrochemical applications.
In addition, other parameters such as the interaction between GOx and redox mediators were not taken into account.
Very few reports describe the specific reengineering of GOx for electrochemical applications and are the subject of this review.
This review is not a review on the use of glucose oxidase in biosensors and biofuel cells neither a review on the electrode materials or redox mediators developed to make these glucose sensors/anodes more efficient.
Such topics have been summarized on various occasions.
(Table 1) Rather, this is a review on glucose oxidase and how it can be rationally, or not, engineered to better serve glucose sensors/biofuel cells.
This review will first start with a brief presentation of glucose oxidase, its property, and structure.
Then, I will discuss the few reports aiming at reengineering glucose oxidase either by rational/semi-rational or directed evolution strategies for improved electrochemical applications.
Particularly, the kind of engineering/mutations performed to increase the electron transfer rate between GOx and electrode surfaces and to decrease the O2 sensitivity of the enzyme will be presented.
Synonym(s)
Aero-Glucose Dehydrogenase
Glucose Aerodehydrogenase
Glucose Oxyhydrase
Notatin
Functional Classes
Antioxidant
glucose oxyhydrase;
corylophyline;
penatin;
glucose aerodehydrogenase;
microcid;
β-D-glucose oxidase;
D-glucose oxidase;
D-glucose-1-oxidase;
β-D-glucose:quinone oxidoreductase;
glucose oxyhydrase; deoxin-1;
GOD;
glucose oxidase enzyme; GOx; notatin;
glucose oxidase
Miscellaneous
This enzyme is widely applied for the determination of glucose in body fluids and in removing residual glucose or oxygen from foods and beverages. Furthermore, glucose oxidase-producing molds such as aspergillus and penicillium species are used for the biological production of gluconic acid.
Enzyme Activity: Other Activities
EC Number: Glucose oxidase: 1.1.3.4,
Catalase: 1.11.1.16
CAS Number: Glucose oxidase: 9001-37-0,
Catalase: 9001-05-2
Synonyms: Glucose oxidase: glucose oxidase; beta-D-glucose:oxygen 1-oxidoreductase,
Catalase: hydrogen-peroxide:hydrogen-peroxide oxidoreductase
Source: Eukaryote
Concentration: Glucose oxidase (12,000 U) plus Catalase (300,000 U) per vial
Expression: Purified from a Eukaryotic source
Specificity: Catalyses the reactions:
(1) D-Glucose + O2 + H20 = D-Gluconate + H2O2
(2) 2H2O2 = 2H2O + O2
Specific Activity: Glucose oxidase (12,000 U) plus Catalase (300,000 U) per vial
Temperature Optima: 30oC
pH Optima: 7
Application examples: For use in removal of excess glucose in conjunction with the Sucrose/Glucose Assay Kit (K-SUCGL) and the Fructan HK Assay Kit (K-FRUCHK).
Thermal inactivation of glucose oxidase (GOD; β-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives.
Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 °C.
The activation energy increased from 60.3 kcal mol–1 to 72.9, 76.1, and 88.3 kcal mol–1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal·mol–1·deg–1 in the presence of 7.1 × 10–5m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively.
Glucose Oxidase thermal unfolding of GOD in the temperature range of 25–90 °C was studied using circular dichroism measurements at 222, 274, and 375 nm.
Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 °C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 °C.
Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD.
Glucose Oxidase irreversible nature of inactivation was caused by a change in the state of association of apoenzyme.
Glucose Oxidase dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity.
Cysteine oxidation did not contribute to the nonspecific aggregation.
Glucose Oxidase stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.
Glucose oxidase (β-d-glucose:oxygen-oxidoreductase, EC 1.1.3.4) from Aspergillus niger is a flavoprotein that catalyzes the oxidation of β-d-glucose to d-glucono-δ-lactone and hydrogen peroxide, using molecular oxygen as the electron acceptor. Glucose oxidase (GOD)
Glucose Oxidase abbreviations used are: GOD, glucose oxidase; ANS, 8-anilino-1-naphthalenesulfonic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); HPLC, high performance liquid chromatography.
finds application in the food and fermentation industry apart from being an analytical tool in biosensors for medical applications and environmental monitoring .
Glucose Oxidase protein is a dimer of two identical subunits with a molecular weight of 160,000.
Glucose Oxidase dimer contains two disulfide bonds, two free sulfhydryl groups , and two FAD molecules (tightly bound) not covalently linked to the enzyme.
Glucose Oxidase dimer has a high degree of localization of negative charges on the enzyme surface and dimer interface.
Glucose Oxidase flavin cofactors are responsible for the oxidation-reduction properties of the enzyme.
Under denaturing conditions, the subunits of GOD dissociate accompanied by the loss of cofactor FAD.
Various additives such as salts, mono- and polyhydric alcohols, and polyelectrolytes were used to increase the thermal stability of GOD.
The effectiveness of additives depended on the nature of enzyme, its hydrophobic/hydrophilic character, and the degree of its interaction with the additives.
Aggregation, the main causative factor for the inactivation of glucose oxidase, could be prevented by modifying the microenvironment of the enzyme.
Glucose Oxidase thermal stability of GOD at 60 °C could be increased by incorporating lysozyme as an additive during immobilization.
Glucose Oxidase role played by the complementarity of surface charges of the enzyme and lysozyme appeared to be crucial in the stabilization of GOD .
Glucose Oxidase presence of salt ions (primarily sulfate) is known to increase the stability of the folded conformations of proteins.
Details of the mechanism are not yet completely understood, partly because of the presence of several intra- and intermolecular interactions in proteins that may or may not be stabilized by sulfate.
Light is yet to be shed on the mechanism of thermal inactivation of GOD, despite several attempts at improving its stability.
An understanding of the thermal inactivation mechanism of GOD could lead to thermostabilization of the enzyme using appropriate additives.
With this objective, experiments were carried out on the effect of some selected additives on the thermal stability of GOD.
In addition to lysozyme, found earlier by us to increase the stability of GOD, two more salts, NaCl and K2SO4, which are commonly known to stabilize enzymes through ionic and hydrophobic interactions, respectively, were selected for the thermal stability studies reported here.
The mechanism of inactivation and the effect of additives on the thermal stability of the enzyme were followed by kinetics of inactivation, spectroscopic measurements, and size exclusion chromatography.
How can Glucose Oxidase be used? Glucose Oxidase (GO) is used in liquid and powder glucose laboratory reagents, urine test strips, colorimetric blood glucose strips and biosensors for blood glucose monitoring.
What makes BBI a leading manufacturer of Glucose Oxidase?
With over 60 years’ experience supplying critical raw materials to the diagnostics industry, BBI Solutions (BBI) is renowned as one of the world’s leading providers of high quality enzymes for biosensor applications.
BBI’s Glucose Oxidase is tried, tested and proven to perform in over 5 billion test strips every year.
The high activity and stability of our GO means you use less enzyme per strip – reducing costs, while increasing the speed, accuracy and longevity of the strip, and it’s a proven raw material, which reduces validation time.
Our manufacturing procedures, which have been developed and optimised over many years, ensure a product of the highest quality, stability, and batch-to-batch consistency, providing sensor and reagent manufacturers with a range of Glucose Oxidase products with proven performance.
1. The reaction catalyzed by the glucose oxidase from Aspergillus niger was markedly inhibited by Ag+, Hg++, Cu++, PCMB and PMA, but not by non-metallic SH-reagents, such as NEM, IA and IAA. No sulfhydryl group was detected in the enzyme protein by spectrophotometric titration with PCMB and NEM. Characteristic difference spectra due to the absorption by the FAD moiety of the enzyme were obtained on the addition of Ag+, Hg++, Cu++, PCMB and PMA to enzyme solutions while no spectral change was observed on the addition of other metal ions and NEM, which did not inhibit the enzyme reaction.
2. From the studies by overall- reaction kinetics and the “flow method”, it was confirmed that Ag+ and Hg++ ions inhibit the oxidation of the reduced FAD moiety, competing with molecular oxygen used as a hydrogen acceptor.
While Ag+ ion did not inhibit the reduction of the FAD moiety, Hg++ ion more or less inhibited the reduction described above.
It was suggested that there is an interaction, probably by steric hindrance, between the substrate and the Hg++ ion for binding to the enzyme molecule.
From the kinetic data obtained in the presence of both Ag+ and Hg++ ions, it was concluded that the binding sites for these two kinds of metal ions on the enzyme molecule are different.
Abstract
The subcellular localization of glucose oxidase in Aspergillus niger N400 was investigated by (immuno)cytochemical methods.
By these methods, the bulk of the enzyme was found to be localized in the cell wall.
Glucose Oxidase addition, four different catalases were demonstrated by nondenaturing polyacrylamide gel electrophoresis of crude extracts of induced and noninduced cells.
Comparison of both protoplast and mycelial extracts indicated that, of two constitutive catalases, one is located outside the cell membrane whereas the other is intracellular.
Parallel with the induction of glucose oxidase, two other catalases are also induced, one located intracellularly and one located extracellularly.
Furthermore, lactonase activity, catalyzing the hydrolysis of glucono-δ-lactone to gluconic acid, was found to be exclusively located outside the cell membrane, indicating that gluconate formation in A. niger occurs extracellularly.
Glucose oxidase is fermented by the improved strains using a liquid submerged fermentation technique, and produced by an advanced post-treatment technique.
Glucose Oxidase is a kind of new green feed additive which protects and promotes intestinal health, promotes digestion and absorption of nutrients, inhibits mycotoxin, removes toxins, and improves the production performance of poultry and livestock.
ecently there has been increasing interest in screening programs for diabetes, and several different screening methods have been advocated.1 The results of screening programs differ considerably, depending on the age, weight, and family history of the group screened; on the type, technique, and standards of screening; on the extent of diagnostic evaluation; and on the diagnostic criteria for diabetes mellitus used in the program.1a
Although there is disagreement as to the choice of a screening procedure, certain general principles are acceptable to most authorities.1e
Glucose Oxidase is generally recognized that, while the glucose tolerance test would be the ideal procedure, it is too expensive and too complicated for most large-scale programs.
The combination of a urine sugar test and a post-prandial blood sugar test is considered to be one of the most effective methods, but this has a limited application because of the necessity of obtaining a blood specimen.
Glucose oxidase is used in the enzymatic determination of D-glucose in solution (1).
Glucose oxidase oxidizes β-D-glucose to D-gluconolactate and hydrogen peroxide. Horseradish peroxidase is then used as the coupling enzyme for glucose determination.
Although glucose oxidase is specific for β-D-glucose, solutions of D-glucose can be quantified as α-D-glucose will mutorotate to β-D-glucose as the β-D-glucose is consumed by the enzymatic reaction.
Ca. 300 U/mg protein.
Unit definition: 1 U catalyzes the oxidation of 1 µmole glucose to glucuronic acid per minute at 25 °C, pH 7 coupled with peroxidase and o-dianisidine.
Extraneous activities: Amylase, saccharase and maltase less than 0.05 %; GOD/catalase min. 2000.
Overview
Oxidoreductase that catalyzes the conversion of D-glucose to D-glucono-1,5-lactone which hydrolyzes spontanously to gluconate.
Application
Use Glucose Oxidase (GOD), Grade I for the determination of α-amylase and D-glucose or O2.
Properties
Nomenclature: β-D-glucose:oxygen 1-oxidoreductase
Molecular weight: 79 kD
Isoelectric point: 4.3
Michaelis constants (Glucose):
Acetate buffer, pH 5.0, +25°C: 3.6 x 10-2 mol/L
Potassium phosphate buffer, 0.2 mol/L, pH 7.5, +25°C: 4.8 x 10-2 mol/L
Inhibitors: Ag+, Hg2+, Cu2+, 4-choloromercuribenzoate, D-arabinose (50%). FAD binding is inhibited by several nucleotides.
pH optimum: 7.0 (see figure)
Temperature dependence: See figure
pH stability: See figure
Thermal stability: See figure
Specificity: Glucose oxidase is specific for β-D-glucose. O2 can be replaced by hydrogen acceptors such as 2,6-dichlorophenol indophenol.
Specifications
Appearance: Yellowish lyophilizate
Conductivity (1%, w/v): ≤250 μS/cm
Activity (+25°C, glucose): ≥300 U/mg lyophilizate
Contaminants (expressed as percentage of Glucose Oxidase activity):
Amylase: ≤0.01
Catalase: ≤0.5
Saccharase: ≤0.01
Stability: At +2 to +8°C within specification range for 24 months. Store dry.
Regulatory Disclaimer
For further processing only.
