Compound and method for improving activity of glucose oxidase, reagent, biosensor, and use of compound

Abstract

Provided are a compound and method for improving the activity of glucose oxidase, a reagent, a biosensor, and a use of the compound. A sulfonyl sweetener and glucose oxidase are compounded, and a better compounding ratio is provided, so that the enzyme activity of glucose oxidase, the affinity of glucose oxidase to glucose and the catalytic efficiency of glucose oxidase for glucose in unit time can be significantly improved, and therefore, it is expected that the application effect of glucose oxidase in the fields of biosensors, food industry, medicines and the like can be improved.

Description

A complex, method, reagent, and biosensor for enhancing glucose oxidase activity, and their applications. Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a complex, method, reagent, and biosensor for enhancing glucose oxidase activity and their applications. Background Technology

[0002] Glucose oxidase (GOD) is a highly specific oxidoreductase that catalyzes the conversion of β-D-glucose to gluconic acid, producing hydrogen peroxide. As an important biocatalyst, glucose oxidase has wide applications in various fields. In the food industry, glucose oxidase is widely used in wine, beer, juice, milk powder, flour improvement, and prevention of food browning. Its main function is deoxygenation, that is, by consuming oxygen in food while catalyzing the conversion of glucose to gluconic acid, it reduces the oxygen level in the food. In this way, glucose oxidase not only helps prevent food oxidation and spoilage but also effectively prevents browning, maintaining the original color and flavor of the food. In clinical medicine, the catalytic process of glucose by glucose oxidase not only consumes glucose in the tumor region but also removes oxygen from the tumor microenvironment, creating a hypoxic and acidic environment, which helps inhibit the growth of tumor cells and improve the efficacy of certain chemotherapy drugs. In the field of biosensors, when glucose is present, it is catalyzed by glucose oxidase to hydrogen peroxide, leading to a potential change and forming a current on the electrode surface for monitoring blood glucose levels. In the feed industry, hydrogen peroxide produced by glucose oxidase catalyzing glucose has a broad-spectrum bactericidal effect, inhibiting the growth and reproduction of pathogenic microorganisms such as Escherichia coli and Salmonella. Glucose oxidase also consumes oxygen in the intestines, creating an anaerobic environment that promotes the proliferation of beneficial bacteria, thereby inhibiting the survival of pathogenic microorganisms and improving the animal's immunity. In the textile industry, bleaching using hydrogen peroxide produced by glucose oxidase catalyzing glucose eliminates the need for additional bleaching agents. This method causes less damage to fibers, results in less loss of fiber strength, and reduces bleaching energy consumption, making it an ideal and environmentally friendly bleaching method. Therefore, it is necessary to provide a method to improve glucose oxidase activity.

[0003] CN113528476A discloses a glucose oxidase mutant through gene-specific modification, which improves its applicable temperature and pH ranges. Genetic modification to construct glucose oxidase variants is a common method in the industry to improve the application performance of glucose oxidase, but this method is time-consuming and prone to errors. CN110885802A discloses a composition containing catalase and glucose oxidase, which, when mixed in a certain proportion, can slightly increase glucose oxidase activity. When this composition is applied to broiler feed, it only increases dry matter digestibility by 0.9636%. Summary of the Invention

[0004] To address the problem of low enzyme activity of glucose oxidase in existing technologies, this invention utilizes a compound of sulfonyl sweeteners and glucose oxidase, which can significantly improve the enzyme activity of glucose oxidase. This is expected to improve the application effect of glucose oxidase in biotechnology, food industry, medicine and other fields at low cost and high efficiency.

[0005] The technical solution adopted in this invention is: a complex for improving glucose oxidase activity, comprising: a sulfonyl sweetener and glucose oxidase. Preferably, the sulfonyl sweetener is selected from at least one of sodium o-benzoylsulfonylimide, potassium acesulfame potassium, and sodium cyclohexylsulfamate.

[0006] Preferably, the concentration of glucose oxidase is 0.0001–0.0003 mg / mL, and the concentration of sulfonyl sweetener is 0.01–0.2 mol / L. Preferably, the concentration of glucose oxidase is 0.0002 mg / mL, and the concentration of sulfonyl sweetener is 0.02–0.1 mol / L.

[0007] The present invention also provides a method for improving glucose oxidase activity, comprising: dissolving glucose oxidase and sulfonyl sweetener in PBS buffer; wherein the concentration of glucose oxidase is 0.0001-0.0003 mg / mL and the concentration of sulfonyl sweetener is 0.01-0.2 mol / L.

[0008] Preferably, the sulfonyl sweetener is selected from at least one of sodium o-benzoylsulfonamide, potassium acesulfame potassium, and sodium cyclohexylaminosulfonate.

[0009] Preferably, the concentration of glucose oxidase is 0.0002 mg / mL, and the concentration of sulfonyl sweetener is 0.02–0.1 mol / L.

[0010] The present invention also provides products containing the said complex. Preferably, the products are selected from food additives, pharmaceuticals, biosensors, feed, and bleaching agents.

[0011] The present invention also provides a biosensor for detecting glucose in a sample, the biosensor comprising the complex described herein.

[0012] The present invention also provides a reagent for detecting glucose in a sample, the reagent comprising the complex described herein.

[0013] The present invention also provides the application of the composite in the preparation of a biosensor, wherein the composite is fixed on the working electrode of the biosensor during the preparation process.

[0014] This invention also provides the application of the aforementioned complex in the preparation of food additives, which include antioxidants, flour improvers, anti-browning agents, antibacterial agents, acidulants, pH adjusters, and preservatives. In the food industry, glucose oxidase specifically catalyzes the reaction of β-D-glucose with oxygen to produce gluconic acid and hydrogen peroxide, exhibiting antioxidant, browning-inhibiting, and food texture-improving effects. In terms of food antioxidation and preservation, glucose oxidase consumes oxygen in food, thereby reducing the risk of food oxidation and maintaining food freshness. Simultaneously, hydrogen peroxide has a certain bactericidal effect, helping to extend the shelf life of food. Furthermore, since polyphenol oxidase requires oxygen to catalyze the oxidation of phenolic compounds to quinone compounds, glucose oxidase can indirectly inhibit the activity of polyphenol oxidase, reducing enzymatic browning in food. The combination of sulfonyl sweeteners and glucose oxidase can serve as a novel food additive, further reducing the oxygen content in the food system and increasing the content of gluconic acid and hydrogen peroxide, thus better promoting food preservation, extending shelf life, and maintaining food color. In improving food texture, the hydrogen peroxide produced by glucose oxidase catalyzing glucose can oxidize the -SH groups in gluten proteins to -SS- groups, helping to form a better protein network structure between gluten proteins. The combination of sulfonyl sweeteners and glucose-like oxidase can help improve the stability and strength of the gluten network, thus improving the taste of food. In improving gluconic acid production efficiency, gluconic acid produced by glucose oxidase is an important food additive that can be used as an acidulant, pH adjuster, and preservative. The combination of sulfonyl sweeteners and glucose-like oxidase can increase the gluconic acid production rate in the food industry.

[0015] This invention also provides the application of the aforementioned complex in the preparation of a biosensor. The method of application includes: immobilizing the complex on the working electrode of the biosensor, catalyzing the oxidation of glucose to gluconic acid and hydrogen peroxide. The generated hydrogen peroxide can be directly oxidized by the working electrode to generate a current signal. Alternatively, a current can be generated on the working electrode through a redox medium (such as ferricyanide, ferrocene derivatives, osmium complexes, ruthenium complexes, etc.), thereby achieving quantitative detection of glucose. Alternatively, a current signal can be generated through direct electron transfer between the active site (FAD) of glucose oxidase and the electrode surface. The combination of sulfonyl sweeteners and glucose-like oxidase helps to improve the bioactivity of glucose oxidase in the sensor, thereby increasing its electron transfer rate and improving the current response and electrode sensitivity of the biosensor. In this invention, the biosensor can be a traditional biosensor for measuring analytes such as glucose in bodily fluids such as blood, requiring prior extraction of the bodily fluid, which is then added to the biosensor to measure the analyte; the biosensor can also be a biosensor for continuous analyte detection, where at least a portion of the biosensor is implanted into the skin before measurement, allowing continuous detection of the analyte concentration level in bodily fluids over a period of time.

[0016] This invention also provides the application of the aforementioned complex in the preparation of pharmaceuticals. Glucose oxidase catalyzes the production of gluconic acid and hydrogen peroxide from glucose, exhibiting antibacterial and deoxygenating functions. In regulating the digestive tract microenvironment, glucose oxidase consumes oxygen through the catalytic oxidation of glucose, reducing the oxygen content in the intestine and creating a relatively anaerobic environment. This environment is conducive to the proliferation of beneficial bacteria in the intestine and unfavorable to the growth of aerobic pathogens, thus helping to regulate the balance of intestinal flora. The combination of sulfonyl sweeteners and glucose-like oxidase can accelerate the formation of an anaerobic environment in the digestive tract, thereby regulating the intestinal flora. In protecting the integrity of the intestinal epithelium, sulfonyl sweeteners enhance the ability of glucose oxidase to catalyze oxidation reactions and scavenge excess free radicals, protecting intestinal epithelial cells, preventing the invasion of pathogens and coccidia, and maintaining intestinal health. In lowering the pH of the gastrointestinal tract, sulfonyl sweeteners enhance the ability of glucose oxidase to produce gluconic acid, lowering the pH in the gastrointestinal tract, which is beneficial for inhibiting the growth of pathogens, while simultaneously promoting intestinal enzyme secretion and improving digestibility.

[0017] This invention also provides the application of the aforementioned complex in feed preparation. Glucose oxidase, as an aerobic dehydrogenase, specifically catalyzes β-D-glucose, consuming molecular oxygen, which promotes the growth of beneficial anaerobic microorganisms (such as lactobacilli and bifidobacteria) in the digestive tract, while simultaneously producing hydrogen peroxide, inhibiting the growth of harmful microorganisms such as Escherichia coli and Salmonella. Through these effects, glucose oxidase can improve animal intestinal health, enhance immunity, reduce stress response, and improve stress resistance. Sulfonyl sweeteners can protect the activity of glucose oxidase, making its biological activity more stable in animals. The combination of sulfonyl sweeteners and glucose-like oxidase as a food additive requires relatively small amounts, which can reduce feed production costs.

[0018] The beneficial effects of this invention are:

[0019] This invention utilizes a sulfonyl sweetener in combination with glucose oxidase, and provides an optimal combination ratio that can significantly improve the enzyme activity of glucose oxidase, the affinity of glucose oxidase for glucose, and the catalytic efficiency of glucose oxidase per unit time. This is expected to improve the application effect of glucose oxidase in biotechnology, food industry, medicine and other fields at low cost and high efficiency. Attached Figure Description

[0020] Figure 1 shows the standard curve of hydrogen peroxide, the product in the example.

[0021] Figure 2 shows the effects of different concentrations of sodium o-benzoylsulfonylimide, potassium acesulfame potassium, and sodium cyclohexylsulfamate on glucose oxidase activity in the examples.

[0022] Figure 3 shows the redox kinetic curves of glucose oxidase and glucose in the sodium o-benzoylsulfonylimide series formulations.

[0023] Figure 4 shows the redox kinetic curves of glucose oxidase and glucose in the potassium acesulfame potassium series formulations.

[0024] Figure 5 shows the redox kinetic curves of glucose oxidase and glucose in the sodium cyclohexylsulfamate series formulations.

[0025] Figure 6 shows the change of the apparent redox rate constant with the concentration of sulfonyl sweetener in the formulation. Detailed Implementation

[0026] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0027] It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of this invention are conventional methods, and the reagents used are commercially available.

[0028] Experimental materials:

[0029] Glucose oxidase: Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No.: 9001-37-0, EC No.: 232-601-0;

[0030] Peroxidase (POD): Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No.: 9003-99-0, EC No.: 232-668-6;

[0031] β-D-glucose (D-(+)-Glucose): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 50-99-7;

[0032] Sodium saccharin: Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 82385-42-0;

[0033] Acesulfame potassium: Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 55589-62-3;

[0034] Sodium Cyclamate: Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 139-05-9;

[0035] Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 13472-35-0;

[0036] Disodium hydrogen phosphate (Na2HPO4): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 7558-79-4;

[0037] 4-Aminoantipyrine (4-AA): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 83-07-8;

[0038] N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 82692-93-1;

[0039] Hydrogen peroxide solution (H2O2): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 7722-84-1.

[0040] Formulation and method for determining the standard curve of hydrogen peroxide in the product:

[0041] A certain amount of 30% H2O2 was serially diluted with ultrapure water (0, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, and 0.05 mmol·L⁻¹). -1 A standard H₂O₂ solution was obtained. PBS (3.95 mL), 4-AA (0.45 mL), TOOS (0.45 mL), and POD (0.10 mL) were mixed to prepare a reaction solution. Equal volumes of the reaction solution and the hydrogen peroxide standard solution were added to a 96-well plate and reacted at 37 °C for 3 minutes. The change in absorbance (ΔOD) at 555 nm was measured. A standard curve was plotted and fitted with the concentration of the added hydrogen peroxide standard solution on the x-axis and the absorbance at 555 nm on the y-axis (Figure 1).

[0042] Example 1: Formulation of a sulfonyl sweetener-glucose oxidase complex

[0043] Weigh 15.601 g of NaH₂PO₄·2H₂O and dissolve it in 900 mL of ultrapure water. Stir magnetically for 2 hours at room temperature until fully dissolved, then bring the volume to 1000 mL with ultrapure water. Weigh 14.196 g of Na₂HPO₄ into a clean beaker and prepare it using the same method as buffer solution A. Mix 725 mL of NaH₂PO₄·2H₂O solution and 275 mL of Na₂HPO₄ solution separately and stir on a magnetic stirrer for 2 hours to obtain 0.1 mol·L⁻¹ solution. -1 The solution used was phosphate-buffered saline (PBS) with a pH of 6.5. Unless otherwise specified, all solvents used in the experiments were this PBS buffer.

[0044] Accurately weigh 0.001 g of glucose oxidase into an appropriate amount of the above PBS buffer, vortex for 3 min, and then bring the volume to 50 mL. Store at 4°C and use immediately. Separately weigh certain amounts of the three sulfonyl sweeteners (sodium o-benzoylsulfonamide, potassium acesulfame potassium, and sodium cyclohexylaminosulfamate) and dissolve them in the buffer solution. Stir for at least 2 h until the final concentration of each sweetener is 0.1 mol·L⁻¹. -1 Three sulfonyl sweetener stock solutions were prepared. The corresponding stock solutions were transferred to volumetric flasks, diluted with buffer, and brought to volume to obtain concentrations of 0, 0.02, 0.04, 0.06, 0.08, and 0.1 mol·L⁻¹. -1 Different sulfonyl sweetener solutions were prepared by accurately transferring 4.95 mL of each solution of various concentrations into different black-capped bottles, and adding a certain amount of glucose oxidase solution to each bottle to achieve an enzyme concentration of 0.0002 mg / mL in the mixture. -1 .

[0045] Control group 1:5 ml reaction system, glucose oxidase concentration 0.0002 mg / mL -1 The concentration of sulfonyl sweetener is 0 mol·L⁻¹ -1 .

[0046] (1) Sodium o-benzoylsulfonylimide series formulations:

[0047] Formula 1: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of sodium o-benzoylsulfonylimide is 0.02 mol·L⁻¹. -1 .

[0048] Formula 2: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of sodium o-benzoylsulfonylimide was 0.04 mol·L⁻¹. -1 .

[0049] Formula 3: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of sodium o-benzoylsulfonylimide was 0.06 mol·L⁻¹. -1 .

[0050] Formula 4: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of sodium o-benzoylsulfonylimide was 0.08 mol·L⁻¹. -1 .

[0051] Formula 5: 5ml reaction system, glucose oxidase concentration is 0.0002mg / mL -1 The concentration of sodium o-benzoylsulfonylimide is 0.1 mol·L⁻¹. -1.

[0052] (2) Acesulfame potassium series formulations:

[0053] Formula 1: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of acesulfame potassium was 0.02 mol·L⁻¹. -1 .

[0054] Formula 2: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of acesulfame potassium was 0.04 mol·L⁻¹. -1 .

[0055] Formula 3: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of acesulfame potassium is 0.06 mol·L⁻¹. -1 .

[0056] Formula 4: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of acesulfame potassium is 0.08 mol·L⁻¹. -1 .

[0057] Formula 5: 5ml reaction system, glucose oxidase concentration is 0.0002mg / mL -1 The concentration of acesulfame potassium is 0.1 mol·L⁻¹. -1 .

[0058] (3) Sodium cyclohexylsulfamate series formulations:

[0059] Formula 1: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of sodium cyclohexylsulfamate was 0.02 mol·L⁻¹. -1 .

[0060] Formula 2: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of sodium cyclohexylsulfamate was 0.04 mol·L⁻¹. -1 .

[0061] Formula 3: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1 The concentration of sodium cyclohexylsulfamate was 0.06 mol·L⁻¹. -1 .

[0062] Formula 4: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg / mL -1The concentration of sodium cyclohexylsulfamate was 0.08 mol·L⁻¹. -1 .

[0063] Formula 5: 5ml reaction system, glucose oxidase concentration is 0.0002mg / mL -1 The concentration of sodium cyclohexylsulfamate is 0.1 mol·L⁻¹. -1 .

[0064] [Performance Testing]

[0065] 1. Determine the activity of glucose oxidase in the formula.

[0066] The reaction solution formulation for enzyme activity assay: 2.1 mmol·L⁻¹ -1 4-Aminoantipyrine (4-AA), 3.0 mmol·L -1 N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) and 1.0 mg·mL -1 All peroxidase (POD) solutions were prepared with ultrapure water and stored in a refrigerator at 4°C in the dark.

[0067] When determining the effect of different concentrations of artificial sweeteners on glucose oxidase activity, glucose solution was used instead of PBS buffer in the standard curve, and glucose oxidase stock solution was used instead of hydrogen peroxide standard solution in the standard curve for glucose oxidase activity determination.

[0068] The enzyme activity (U) of glucose oxidase is defined as the amount of hydrogen peroxide produced per minute per milligram of glucose oxidase from the substrate under the conditions of 37°C and pH = 6.5. The results are shown in Figure 2 and Table 1.

[0069] The specific formula for calculating the relative activity of glucose oxidase using relative enzyme activity mapping is as follows:

[0070] U0 and U1 represent the activity of glucose oxidase in buffer or sulfonyl sweetener formulations, respectively.

[0071] As shown in Figure 2 and Table 1, the formulations of sodium o-benzoylsulfonamide, potassium acesulfame potassium, and the potassium acesulfame potassium series are within the addition range (0.02-0.1 mol·L⁻¹). -1 They all promote glucose oxidase activity. Furthermore, the rate of promotion of glucose oxidase activity by these additives all showed an increasing trend with increasing concentration. Among them, sodium o-benzoylsulfonylimide and potassium acesulfame potassium series formulations showed the best effect on promoting glucose oxidase activity at a concentration of 0.1 mol·L⁻¹. -1The activity of glucose oxidase can reach around 40% in all cases, while the sodium cyclohexylsulfamate series formulations have a relatively weaker promoting effect. This indicates that sodium benzoylsulfonylimide, acesulfame potassium, and acesulfame potassium series formulations can enhance glucose oxidase activity in certain scenarios, with the sodium benzoylsulfonylimide series formulations being the best. More preferably, 0.1 mol·L -1 Sodium o-benzoylsulfonylimide and 0.0002 mg·mL -1 Glucose oxidase is the optimal choice.

[0072] Table 1. Statistical table of relative activities of glucose oxidase in different formulations

[0073] 2. Determination of the enzyme activity mechanics of glucose oxidase in the formula.

[0074] During the test, glucose oxidase was used with a sulfonyl sweetener (0.05 mol·L⁻¹). -1 Buffer preparation for glucose oxidase. The concentration of glucose oxidase was fixed, and its effect on different concentrations of glucose (10, 20, 40, 60, 80, 90, and 100 mmol / mL) was determined. -1 The enzymatic reaction rate of glucose oxidase was determined. A double reciprocal curve was plotted using the reciprocal of glucose concentration (1 / [Glucose]) and the reciprocal of reaction rate (1 / [v]) as the x and y axes, respectively, according to the Lineweaver-Burk equation (6). Glucose oxidase activity was determined using the same method as in (II) 2., and a double reciprocal curve was plotted using the reciprocal of glucose concentration (1 / [Glucose]) and the reciprocal of reaction rate (1 / [v]) as the x and y axes, respectively, according to the Lineweaver-Burk equation:

[0075] In the Lineweaver-Burk equation, V max K represents the maximum rate of the enzymatic reaction. m Let V be the Michaelis constant. The calculated reaction rate V... max The catalytic constant K of glucose oxidase is calculated in conjunction with the enzyme concentration [E] using the following formula. cat :

[0076] Table 2 summarizes the effects of sodium o-benzoylsulfonylimide, potassium acesulfame potassium, and sodium cyclohexylsulfamate on the kinetic parameters of glucose oxidase. It shows that all three formulations reduced the Michaelis constant K of glucose oxidase-glucose kinetics. m This increased the reaction rate V max and the catalytic constant K catThe results indicate that sodium benzoylsulfonylimide, potassium acesulfame, and the potassium acesulfame series formulations all improved the affinity of glucose oxidase for glucose and the catalytic efficiency of glucose oxidase per unit time, with the sodium benzoylsulfonylimide series formulations being the best.

[0077] Table 2. Enzyme kinetic parameters of glucose oxidase in different sulfonyl sweetener formulations

[0078] 3. Determine the apparent redox rate between glucose oxidase and glucose.

[0079] The apparent redox rate between glucose oxidase and glucose in the formulation was determined by stop-flow fluorescence spectroscopy. During the test, glucose oxidase solutions prepared separately with buffer or sulfonyl sweetener solution were added to tube I after removing air bubbles using an injection. The glucose solution (100 mmol / L) was then added. -1 Add it to tube II.

[0080] The fluorescence-time kinetic trajectory obtained from the scan was fitted using the exponential equation in the instrument's built-in Pro-Date software: Y=c+∑ N Ai exp(-k i t)

[0081] In the formula, Y represents the change in UV intensity of glucose oxidase at 450 nm; c represents the intercept; Ai represents the magnitude of the change; t is the reaction time; and ki is the apparent rate constant obtained from the fitting, which is denoted as k here. e express.

[0082] By fitting the absorption spectral kinetics of the FAD cofactor of glucose oxidase during the reaction with glucose in a series of formulations containing sodium benzoylsulfonylimide, potassium acesulfame potassium, and sodium cyclohexylsulfamate, as shown in Figures 3 to 6, it was found that the apparent redox rate constant of the reaction process increases with the increase of the concentration of sulfonyl sweetener in the series of formulations, and the promoting effect shows a trend of sodium benzoylsulfonylimide > potassium acesulfame potassium ≈ sodium cyclohexylsulfamate. This indicates that the sodium benzoylsulfonylimide, potassium acesulfame potassium, and potassium acesulfame potassium series of formulations all improve the redox rate between glucose oxidase and glucose, with the sodium benzoylsulfonylimide series of formulations being the best.

[0083] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A complex for enhancing glucose oxidase activity, characterized in that, This includes sulfonyl sweeteners and glucose oxidase.

2. The complex according to claim 1, characterized in that, The concentration of glucose oxidase is 0.0001–0.0003 mg / mL, and the concentration of sulfonyl sweetener is 0.01–0.2 mol / L.

3. The complex according to claim 2, characterized in that, The concentration of glucose oxidase is 0.0002 mg / mL, and the concentration of sulfonyl sweetener is 0.02–0.1 mol / L.

4. The complex according to claim 1, characterized in that, The sulfonyl sweetener is selected from at least one of sodium o-benzoylsulfonamide, potassium acesulfame potassium, and sodium cyclohexylaminosulfonate.

5. A method for increasing glucose oxidase activity, characterized in that, include: Glucose oxidase and sulfonyl sweetener are dissolved in PBS buffer; wherein the concentration of glucose oxidase is 0.0001-0.0003 mg / mL and the concentration of sulfonyl sweetener is 0.01-0.2 mol / L.

6. The method as described in claim 5, characterized in that, The sulfonyl sweetener is selected from at least one of sodium o-benzoylsulfonamide, potassium acesulfame potassium, and sodium cyclohexylaminosulfonate.

7. The method as described in claim 5, characterized in that, The concentration of glucose oxidase is 0.0002 mg / mL, and the concentration of sulfonyl sweetener is 0.02–0.1 mol / L.

8. A biosensor for detecting glucose in a sample, characterized in that, The biosensor comprises the complex according to any one of claims 1 to 3.

9. The application of the complex according to any one of claims 1 to 3 in the preparation of biosensors, characterized in that, During the preparation process, the composite is immobilized on the working electrode of the biosensor.

10. A reagent for detecting glucose in a sample, characterized in that, The reagent includes the complex according to any one of claims 1 to 3.

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