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

Abstract

The invention provides a compound, a method, a reagent and a biosensor for improving the activity of glucose oxidase and application thereof. The sulfonyl sweetening agent and the glucose oxidase are compounded, and a better compounding ratio is provided, so that the enzyme activity of the glucose oxidase, the affinity of the glucose oxidase to glucose and the catalytic efficiency of the glucose oxidase to the glucose in unit time can be remarkably improved; therefore, the application effect of the glucose oxidase in the fields of biosensors, food industry, medicines and the like is expected to be efficiently improved at low cost.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to a compound, a method, a reagent and a biosensor for improving the activity of glucose oxidase and applications thereof. Background Art

[0002] Glucose oxidase (GOD) is a highly specific oxidoreductase that can catalyze the conversion of β-D-glucose into gluconic acid and produce hydrogen peroxide. As an important biocatalyst, glucose oxidase has a wide range of applications in many fields. In the field of 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 and catalyzing the conversion of glucose into gluconic acid, thereby reducing the oxygen level in food. In this way, glucose oxidase not only helps prevent food from oxidative deterioration, but also effectively prevents food from browning and maintains the original color and flavor of food. In the field of clinical medicine, the catalytic process of glucose oxidase on glucose not only consumes glucose in the tumor area, but also removes oxygen in the tumor microenvironment, creating an oxygen-deficient and acidic environment, which helps inhibit the growth of tumor cells and improves the efficacy of certain chemotherapy drugs. In the field of biosensors, when glucose is present, it is catalyzed by glucose oxidase to oxidize into hydrogen peroxide, which in turn causes a change in potential and forms a current on the electrode surface for monitoring blood sugar levels. In the feed industry, hydrogen peroxide produced by glucose catalyzed by glucose oxidase has a broad-spectrum bactericidal effect, which can inhibit the growth and reproduction of pathogenic microorganisms such as Escherichia coli and Salmonella. Glucose oxidase can also consume oxygen in the intestine, create an anaerobic environment, promote the proliferation of probiotics, thereby inhibiting the survival of pathogenic microorganisms and improving the immunity of livestock. In the textile field, glucose oxidase is used to catalyze the hydrogen peroxide produced by glucose for bleaching without the need to add additional bleaching agents. This method causes less damage to the fiber, less fiber strength loss, and reduces bleaching energy consumption. It is an ideal and more environmentally friendly bleaching method. Therefore, it is necessary to provide a method for improving the activity of glucose oxidase.

[0003] CN113528476A provides a glucose oxidase mutant through gene-specific modification, and its applicable temperature range and pH range are improved. Genetic modification to construct glucose oxidase variants is a conventional means in the industry to improve the application performance of glucose oxidase, but this method is time-consuming and has a high degree of randomness. CN110885802A discloses a composition comprising catalase and glucose oxidase, which can be slightly improved by compounding in a certain proportion. The composition is applied to broiler feed, and the dry matter digestibility is only increased by 0.9636%. Summary of the invention

[0004] In order to solve the problem of low enzymatic activity of glucose oxidase in the prior art, the present invention utilizes sulfonyl sweeteners to compound with glucose oxidase, which can significantly improve the enzymatic activity of glucose oxidase, thereby hopefully improving the application effect of glucose oxidase in the fields of biotechnology, food industry, medicine, etc. at low cost and high efficiency.

[0005] The technical solution adopted by the present invention is: a compound for improving the activity of glucose oxidase, comprising: a sulfonyl sweetener and glucose oxidase. Preferably, the sulfonyl sweetener is selected from at least one of sodium o-benzoylsulfonimide, potassium acesulfame, and sodium cyclohexylaminosulfonate.

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

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

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

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

[0010] The present invention also provides a product containing the complex. Preferably, the product is selected from food additives, medicines, biosensors, feeds, and bleaching agents.

[0011] The present invention also provides a biosensor for detecting glucose in a sample, wherein the biosensor comprises the composite.

[0012] The present invention also provides a reagent for detecting glucose in a sample, wherein the reagent comprises the complex.

[0013] The present invention also provides the use of the complex in preparing a biosensor. During the preparation process, the complex is fixed on a working electrode of the biosensor.

[0014] The present invention also provides the use of the complex in the preparation of food additives, wherein the food additives include antioxidants, flour improvers, anti-browning agents, antibacterial agents, acidulants, pH regulators, and preservatives. In the field of food industry, glucose oxidase can specifically catalyze the reaction of β-D-glucose with oxygen to generate gluconic acid and hydrogen peroxide, and has the effects of anti-oxidation, inhibiting browning, and improving food texture. In terms of food anti-oxidation and anti-corrosion, glucose oxidase consumes oxygen in food, thereby reducing the risk of food oxidation and maintaining the freshness of food. At the same time, hydrogen peroxide has a certain bactericidal effect, which helps to extend the shelf life of food. In addition, since polyphenol oxidase requires oxygen to catalyze the oxidation of phenolic compounds into quinone compounds, glucose oxidase can indirectly inhibit the activity of polyphenol oxidase and reduce the enzymatic browning of food. The combination of sulfonyl sweeteners and glucose oxidase can be used as a new food additive, which is more conducive to food anti-corrosion, extending the shelf life of food and maintaining the color of food by further reducing the oxygen content in the food system and increasing the content of gluconic acid and hydrogen peroxide in the products. In terms of improving food texture, hydrogen peroxide produced by glucose catalyzed by glucose oxidase can oxidize the -SH group in gluten protein to -SS- group, which helps to form a better protein network structure between gluten proteins. Sulfonyl sweeteners combined with glucose-like oxidase can help improve the stability and strength of the gluten network and improve the taste of food. In terms of improving the efficiency of gluconic acid production, gluconic acid produced by glucose catalyzed by glucose oxidase is an important food additive that can be used as an acidulant, pH adjuster, preservative, etc. Sulfonyl sweeteners combined with glucose-like oxidase can increase the productivity of gluconic acid in the food industry.

[0015] The present invention also provides the use of the complex in the preparation of a biosensor, and the method of the use includes: fixing the complex on the working electrode of the biosensor, catalyzing the oxidation of glucose to generate 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 is generated on the working electrode through a redox medium (such as ferrocyanide, ferrocene derivatives, osmium complexes, ruthenium complexes, etc.), thereby achieving quantitative detection of glucose. Alternatively, a current signal is generated through direct electron transfer between the active center (FAD) of glucose oxidase and the electrode surface. The sulfonyl sweeteners and glucose-like oxidases are combined to help increase the biological activity of glucose oxidase in the sensor, thereby increasing its electron transfer rate, and improving the current response and electrode sensitivity of the biosensor. In the present invention, the biosensor can be a conventional biosensor for measuring analytes such as glucose in biological fluids such as blood, which requires the extraction of biological fluids in advance, and the extracted biological fluids are added to the biosensor to measure the analytes; the biosensor can also be a biosensor for continuously detecting analytes, and at least a portion of the biosensor is implanted in the skin before measurement, and the analyte concentration level in the biological fluid can be continuously detected over a period of time.

[0016] The present invention also provides the use of the complex in the preparation of medicines. Glucose oxidase has antibacterial and deoxygenation functions by catalyzing glucose to produce gluconic acid and hydrogen peroxide. In terms of regulating the digestive tract microenvironment, glucose oxidase consumes oxygen by catalyzing glucose oxidation, reduces the oxygen content in the intestine, and creates a relatively anaerobic environment for the intestine. This environment is conducive to the proliferation of beneficial bacteria in the intestine, and is not conducive to the growth of aerobic pathogens, thereby helping to regulate the balance of intestinal flora. Sulfonyl sweeteners and glucose-like oxidases can accelerate the formation of an anaerobic environment in the digestive tract, thereby playing a role in regulating intestinal flora. In terms of protecting the integrity of the intestinal epithelium, the sulfonyl sweetness improves the ability of glucose oxidase to catalyze oxidation reactions to remove excess free radicals, protects intestinal epithelial cells, prevents pathogens and coccidia from invading, and maintains intestinal health. In terms of reducing the pH value of the gastrointestinal tract, the sulfonyl sweetness improves the ability of glucose oxidase to produce gluconic acid, reduces the pH in the gastrointestinal tract, is conducive to inhibiting the growth of pathogens, and promotes intestinal enzyme secretion and improves digestibility.

[0017] The present invention also provides the application of the complex in the preparation of feed. Glucose oxidase, as an aerobic dehydrogenase, can specifically catalyze β-D-glucose, consume molecular oxygen, and is beneficial to the growth of beneficial anaerobic microorganisms (such as lactobacillus and bifidobacterium) in the digestive tract. At the same time, it produces hydrogen peroxide, which inhibits the growth of harmful microorganisms such as Escherichia coli and Salmonella. Through these effects, glucose oxidase can improve the intestinal health of animals, enhance the body's immunity, reduce stress response and enhance stress resistance. Sulfonyl sweeteners can protect the activity of glucose oxidase, making its biological activity in animals more stable. Sulfonyl sweeteners and glucose-like oxidases are combined as food additives, and the amount used is relatively small, which can reduce the cost of feed production.

[0018] Beneficial effects of the present invention:

[0019] The present invention utilizes a sulfonyl sweetener to compound with glucose oxidase and provides a relatively optimal compounding ratio, which can significantly improve the enzyme activity of glucose oxidase, the affinity of glucose oxidase to glucose and the catalytic efficiency of glucose oxidase to glucose per unit time, thereby hopefully improving the application effect of glucose oxidase in the fields of biotechnology, food industry, medicine and the like at low cost and with high efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the standard curve of the product hydrogen peroxide in the example.

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

[0022] Figure 3 Figure 2 is the fast redox kinetics of the reaction between glucose oxidase and glucose; (a)-(c) are the redox kinetic curves of glucose oxidase and glucose in a series of formulations of sodium o-benzoylsulfonyl imide, potassium acesulfame and sodium cyclohexylaminosulfonate, respectively; (d) is the change of the redox apparent rate constant with the concentration of sulfonyl sweetener in the formulation. DETAILED DESCRIPTION

[0023] The following describes the implementation of the present invention through specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific implementations, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0024] It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are all conventional methods, and the reagents used can be obtained from commercial sources.

[0025] Experimental Materials:

[0026] Glucose oxidase (Glucose Oxidase): Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No.: 9001-37-0, EC No.: 232-601-0;

[0027] Peroxidase (POD): Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No.: 9003-99-0, EC No.: 232-668-6;

[0028] β-D-glucose (D-(+)-Glucose): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 50-99-7;

[0029] Sodium Saccharin: Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 82385-42-0;

[0030] Acesulfame potassium: Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 55589-62-3;

[0031] Sodium Cyclamate: Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 139-05-9;

[0032] Sodium dihydrogen phosphate dihydrate (NaH 2 PO 4 ·2H 2 O): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 13472-35-0;

[0033] Sodium hydrogen phosphate (Na 2 HPO 4 ): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 7558-79-4;

[0034] 4-Aminoantipyrine (4-AA): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 83-07-8;

[0035] N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt (TOOS): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 82692-93-1;

[0036] Hydrogen peroxide solution (H 2 O 2 ): Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 7722-84-1.

[0037] Formula and method for determining the standard curve of product hydrogen peroxide:

[0038] A certain amount of 30% H 2 O 2 Ultrapure water was used for gradient dilution (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 ) to obtain the standard H 2 O 2 Solution. PBS (3.95 mL), 4-AA (0.45 mL), TOOS (0.45 mL) and POD (0.10 mL) were mixed to obtain 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 the absorbance value (Absorbance) at 555 nm (ΔOD) was measured. A standard curve was drawn and fitted with the concentration of the added hydrogen peroxide standard solution as the horizontal axis and the absorbance value at 555 nm as the vertical axis ( Figure 1 ).

[0039] Example 1: Sulfonyl sweetener-glucose oxidase complex formulation

[0040] Weigh 15.601 g NaH 2 PO 4 ·2H 2 O was dissolved in 900 mL of ultrapure water. After fully dissolved under magnetic stirring for 2 h at room temperature, the volume was made up to 1000 mL with ultrapure water for later use. Weigh 14.196 g of Na 2 HPO 4 In a clean beaker, prepare buffer A in the same manner and set aside. 2 PO 4 ·2H 2 O solution 725mL, Na 2 HPO 4After 275 mL of the solution was mixed, it was stirred on a magnetic stirring table for 2 h to obtain 0.1 mol·L -1 Phosphate buffered saline (PBS) with a pH of 6.5. Unless otherwise specified, the solvent used in the experiment was this PBS buffer.

[0041] Accurately weigh 0.001g of glucose oxidase in an appropriate amount of the above PBS buffer, shake for 3 minutes, dilute to 50mL and place in a 4℃ refrigerator for immediate use. Weigh a certain amount of three sulfonyl sweeteners (sodium o-benzoylsulfonimide, potassium acesulfame, sodium cyclohexylaminosulfonate) and dissolve them in the buffer, stir for more than 2 hours, and make the final concentration after dilution to 0.1mol·L -1 The mother solutions of three sulfonyl sweeteners were pipetted into volumetric flasks, diluted with buffer and fixed to volume to obtain concentrations of 0, 0.02, 0.04, 0.06, 0.08 and 0.1 mol·L -1 Accurately pipette 4.95 mL of various solutions of different concentrations into different black-capped bottles, and add a certain amount of glucose oxidase enzyme solution to make the enzyme concentration in the mixed solution 0.0002 mg·mL -1 .

[0042] 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 .

[0043] (1) Formula of sodium o-benzoylsulfonimide series:

[0044] Formula 1: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of sodium o-benzoylsulfonimide is 0.02 mol·L -1 .

[0045] Formula 2: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of sodium o-benzoylsulfonimide is 0.04 mol·L -1 .

[0046] Formula 3: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of sodium o-benzoylsulfonimide is 0.06 mol·L -1 .

[0047] Formula 4: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of sodium o-benzoylsulfonimide is 0.08 mol·L-1 .

[0048] Formula 5: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of sodium o-benzoylsulfonimide is 0.1 mol·L -1 .

[0049] (2) Acesulfame Potassium Series Formula:

[0050] Formula 1: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of acesulfame potassium is 0.02 mol·L -1 .

[0051] Formula 2: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of acesulfame potassium is 0.04 mol·L -1 .

[0052] 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 .

[0053] 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 .

[0054] Formula 5: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of acesulfame potassium is 0.1 mol·L -1 .

[0055] (3) Formula of sodium cyclohexylaminosulfonate series:

[0056] Formula 1: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of sodium cyclamate is 0.02 mol·L -1 .

[0057] Formula 2: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of sodium cyclamate is 0.04 mol·L -1 .

[0058] Formula 3: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1, the concentration of sodium cyclamate is 0.06 mol·L -1 .

[0059] Formula 4: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of sodium cyclamate is 0.08 mol·L -1 .

[0060] Formula 5: 5 ml reaction system, glucose oxidase concentration is 0.0002 mg mL -1 , the concentration of sodium cyclamate is 0.1 mol·L -1 .

[0061]

Performance test

[0062] 1. Determination of glucose oxidase activity in the formula

[0063] Reaction solution formula for enzyme activity assay: 2.1mmol·L -1 4-aminoantipyrine (4-AA), 3.0mmol·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 away from light.

[0064] When determining the effect of different concentrations of artificial sweeteners on the activity of glucose oxidase, the substrate glucose solution was used to replace the PBS buffer in the standard curve, and the glucose oxidase mother solution was used to replace the hydrogen peroxide standard solution in the standard curve to determine the activity of glucose oxidase.

[0065] The enzyme activity (U) of glucose oxidase is defined as the amount of micromoles of hydrogen peroxide generated per minute per mg of glucose oxidase catalyzed by the substrate at 37°C and pH 6.5. Figure 2 and Table 1.

[0066] Using relative enzyme activity mapping, the specific calculation formula for the relative activity of glucose oxidase is as follows:

[0067]

[0068] Among them, U 0 and U 1 Represents the activity of glucose oxidase in buffer or sulfonyl sweetener series formulations, respectively.

[0069] Depend on Figure 2As shown in Table 1, the addition range of sodium o-benzoylsulfonimide, acesulfame potassium and acesulfame potassium series formulas (0.02-0.1 mol·L -1 ) promotes glucose oxidase activity. In addition, their promotion rates of glucose oxidase activity all show a trend of increasing with the increase of added concentration. Among them, the promotion effect of o-benzoylsulfonyl imide sodium and acesulfame potassium series formulas on glucose oxidase activity is the highest at an added concentration of 0.1 mol·L -1 The results showed that sodium cyclohexylaminosulfonate series formulas can increase the activity of glucose oxidase in some scenarios, and sodium cyclohexylaminosulfonate series formulas are better. -1 Sodium o-benzoylsulfonimide and 0.0002mg·mL -1 Glucose oxidase is optimal.

[0070] Table 1. Statistics of relative activity of glucose oxidase in different formulations

[0071]

[0072] 2. Determination of the enzyme activity kinetics of glucose oxidase in the formulation

[0073] Glucose oxidase was tested with sulfonyl sweetener (0.05 mol·L -1 The concentration of glucose oxidase was fixed and its effects on glucose at different concentrations (10, 20, 40, 60, 80, 90 and 100 mmol·mL -1 ) enzyme reaction rate. The reciprocal of glucose concentration (1 / [Glucose]) and the reciprocal of reaction rate (1 / [v]) were used as horizontal and vertical coordinates, and a double reciprocal curve was drawn according to Lineweaver-Burk equation (6). Glucose oxidase activity was determined by the same method as in (II) 2., and the reciprocal of glucose concentration (1 / [Glucose]) and the reciprocal of reaction rate (1 / [v]) were used as horizontal and vertical coordinates, and a double reciprocal curve was drawn according to Lineweaver-Burk equation (6).

[0074] Lineweaver-Burk equation plots a double reciprocal curve:

[0075] In the Lineweaver-Burk equation, V max is the maximum enzymatic reaction rate; K m is the Michaelis constant. The obtained reaction rate V max The catalytic constant K of glucose oxidase was calculated by the following formula: cat :

[0076]

[0077] Table 2 summarizes the effects of sodium o-benzoylsulfonimide, potassium acesulfame and sodium cyclohexylsulfamate series formulas on the kinetic parameters of glucose oxidase. It can be seen that sodium o-benzoylsulfonimide, potassium acesulfame and sodium cyclohexylsulfamate series formulas all reduce the Michaelis constant K of glucose oxidase-glucose m , increasing the reaction rate V max and the catalytic constant K cat The results showed that sodium o-benzoylsulfonyl imide, acesulfame potassium and acesulfame potassium series formulas all increased the affinity of glucose oxidase to glucose and the catalytic efficiency of glucose oxidase to glucose per unit time, and the sodium o-benzoylsulfonyl imide series formula was the best.

[0078] Table 2. Kinetic parameters of glucose oxidase in different sulfonyl sweetener formulations

[0079]

[0080] 3. Determination of the apparent redox rate between glucose oxidase and glucose

[0081] Stopped-flow fluorescence spectroscopy was used to determine the apparent redox rate between glucose oxidase and glucose in the formula. During the test, the glucose oxidase solution prepared with buffer or sulfonyl sweetener solution was added to tube I through an injection to remove bubbles. The glucose solution (100 mmol·L -1 ) was added to tube II.

[0082] The fluorescence-time kinetic trajectory obtained by scanning was fitted using the exponential equation in the Pro-Date software provided by the instrument:

[0083] Y=c+∑ N Aiexp(-k i t)

[0084] Where, Y represents the change of UV intensity of glucose oxidase at 450nm; c represents the intercept; Ai represents the amplitude of change; t is the reaction time; ki is the apparent rate constant obtained by fitting, here k e express.

[0085] By fitting the absorption spectrum kinetic trajectory of the cofactor FAD when glucose oxidase reacts with glucose in a series of formulas of sodium o-benzoylsulfonyl imide, potassium acesulfame and sodium cyclohexylaminosulfonate, as shown in the figure. Figure 3As shown, it was found that the apparent redox rate constant of the reaction process increased with the increase of the concentration of sulfonyl sweeteners in the series of formulas, and the promotion effect showed a trend of sodium o-benzoylsulfonimide> potassium acesulfame ≈ sodium cyclohexylaminosulfonate. This shows that sodium o-benzoylsulfonimide, potassium acesulfame and potassium acesulfame series formulas all increased the redox rate between glucose oxidase and glucose, and the sodium o-benzoylsulfonimide series formula was the best.

[0086] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. A compound for improving the activity of glucose oxidase, characterized in that: Includes sulfonyl sweeteners and glucose oxidase.

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

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

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

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

6. The method according to claim 5, characterized in that The sulfonyl sweetener is selected from at least one of sodium o-benzoylsulfonimide, acesulfame potassium, and sodium cyclamate.

7. The method according to claim 5, characterized in that The concentration of the glucose oxidase is 0.0002 mg / mL, and the concentration of the 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. Use of the complex according to any one of claims 1 to 3 in the preparation of a biosensor, characterized in that: During the preparation process, the complex is immobilized on the working electrode of the biosensor.

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

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