阅读说明：本技术 一种检测葡萄糖氧化酶活性的方法 (Method for detecting activity of glucose oxidase ) 是由 程瑛 张成杰 徐丽 周樱 詹志春 于 2021-08-17 设计创作，主要内容包括：本发明属于酶制剂检测技术领域,具体提供了一种检测葡萄糖氧化酶活性的方法。本发明在待测葡萄糖氧化酶液和葡萄糖及碘化钾充分反应后加入硫酸终止反应,然后使用硫代硫酸钠滴定生成的碘,从而定量的计算出葡萄糖氧化酶的酶活性。该检测方法操作简单,反应时间短,检测灵敏准确,反应过程中使用的均为常规化学试剂,检测过程安全,反应条件温和,适用于食品酿造、饲料添加、面粉改良、医药领域等,而且不需要制作过氧化氢标准曲线,避免操作误差导致结果不准确。(The invention belongs to the technical field of enzyme preparation detection, and particularly provides a method for detecting the activity of glucose oxidase. The method comprises the steps of adding sulfuric acid to stop reaction after glucose oxidase liquid to be detected fully reacts with glucose and potassium iodide, and then titrating the generated iodine by using sodium thiosulfate, so that the enzyme activity of the glucose oxidase is quantitatively calculated. The detection method is simple to operate, short in reaction time, sensitive and accurate in detection, uses conventional chemical reagents in the reaction process, is safe in detection process, mild in reaction condition, suitable for the fields of food brewing, feed addition, flour improvement, medicines and the like, does not need to make a hydrogen peroxide standard curve, and avoids inaccurate results caused by operation errors.)

1. A method for detecting the activity of glucose oxidase is characterized by comprising the following steps:

(1) diluting glucose oxidase by using a phosphate buffer solution to serve as an enzyme solution to be detected, recording the dilution multiple as N, and preheating the enzyme solution to be detected;

(2) preparing a glucose solution, a potassium iodide solution and an ammonium molybdate solution, uniformly mixing the glucose solution, the potassium iodide solution and the ammonium molybdate solution, preheating, and dividing into a plurality of reaction substrates for later use;

(3) get V₁Adding ml of preheated enzyme solution to be detected into one part of the reaction substrate in the step (2), and immediately adding a sulfuric acid solution to terminate the reaction after the enzymolysis reaction is carried out for T minutes;

(4) get V₁Adding ml of phosphate buffer solution into another part of the reaction substrate in the step (2), and immediately adding a sulfuric acid solution which is equal to that in the step (3) after T minutes to serve as a blank control group;

(5) dropwise adding an indicator into the product obtained in the step (3), uniformly mixing, titrating to be colorless by using an iodine simple substance reducing agent with the concentration of f mol/l, and recording the consumed volume of the iodine simple substance reducing agent as V₂ml; simultaneously carrying out the same titration reaction on the blank control group in the step (4), and recording the consumption volume of the iodine simple substance reducing agent as V₃ ml；

(6) Calculating the glucose oxidase activity X according to the formula

Wherein a is the conversion coefficient of the iodine simple substance reducing agent and hydrogen peroxide; 1000 is a transforming factor.

2. The method of detecting glucose oxidase activity according to claim 1, wherein: the phosphate buffer solution in the step (1) is prepared from NaH₂PO₄·2H₂O and Na₂HPO₄·12H₂O is mixed to prepare a phosphate buffer solution with the concentration of 0.05-0.25mol/l and the pH value of 3.0-7.0.

3. The method of detecting glucose oxidase activity according to claim 2, wherein: the concentration of the phosphate buffer solution is 0.1mol/l, and the pH value is 6.0.

4. The method of detecting glucose oxidase activity according to claim 1, wherein: in the step (2), the concentration of the glucose solution is 180g/l, the concentration of the potassium iodide solution is 100g/l, and the concentration of the ammonium molybdate solution is 10 g/l.

5. The method of detecting glucose oxidase activity according to claim 1, wherein: among the reaction substrates prepared in the step (2), potassium iodide solution: glucose solution: the volume ratio of the ammonium molybdate solution is 25:3: 1.

6. The method of detecting glucose oxidase activity according to claim 1, wherein: the temperature of the enzymolysis reaction in the step (3) is 20-45 ℃.

7. The method of detecting glucose oxidase activity according to claim 6, wherein: the temperature of the enzymatic reaction is 37 ℃.

8. The method of detecting glucose oxidase activity according to claim 1, wherein: the concentration of the sulfuric acid solution used in the step (3) and the step (4) is 1 mol/l.

9. The method of detecting glucose oxidase activity according to claim 1, wherein: the indicator used in the step (5) is a starch indicator.

10. The method of detecting glucose oxidase activity according to claim 1, wherein: the iodine simple substance reducing agent used in the step (5) comprises one of hydrogen sulfide, sulfur dioxide and sodium thiosulfate solution.

Technical Field

The invention belongs to the technical field of enzyme preparation detection, and particularly relates to a method for detecting the activity of glucose oxidase.

Background

Glucose oxidase is an aerobic dehydrogenase that can oxidize β -D-glucose to gluconic acid and hydrogen peroxide using molecular oxygen as a receptor. The glucose oxidase has the characteristics of high catalytic efficiency, no toxic or side effect, high specificity and the like, and can oxidize glucose into gluconic acid to achieve the effects of removing glucose, deoxidizing and sterilizing, so the glucose oxidase is widely applied to the aspects of medicine production, food processing, feed addition and the like, the global GOD annual total yield exceeds 150 hundred million yuan, and the demand is increased by 10-30% year by year. However, the activity definition and detection method of the glucose oxidase has no national standard, only individual provinces have local standards, and most production enterprises adopt own enterprise standards for determination.

At present, the main glucose oxidase activity detection methods include a titration method, an electrochemical method, a spectrophotometer method, a pressure measurement method, a gel electrophoresis method and a Fourier transform infrared spectrometry method, wherein the electrochemical method, the pressure measurement method, the gel electrophoresis method, the Fourier transform infrared spectrometry method and the continuous spectrophotometry method have the problems of complicated operation, instrument dependence, reagent shortage and the like, and are not widely popularized and used. The principle of both titration and ordinary spectrophotometers is that glucose oxidase reacts with glucose, and subsequent detection is carried out according to the reaction product. The spectrophotometer method detects the generated colored substances by using an isatin fading method, a quinoneimine method, a phthalic diamine method, an o-dianisidine method and the like, but many of the reagents belong to carcinogenic highly toxic compounds, the situation that the colored substances are unstable exists, and the hydrogen peroxide is used as a standard yeast for calculation, so that the problems of poor stability and repeatability of the standard yeast exist, the enzyme activity detection result is unstable, and the method is not suitable for popularization and standard establishment. Therefore, there is a need to find a more accurate, stable and reproducible method. The titration method belongs to an arbitration method in a plurality of detection methods, and the currently used titration method has low sensitivity and relatively large error, so the invention improves on the basis of the original titration method and designs the method for the activity of the glucose oxidase which has the advantages of rapidness, reliability, simple operation, sensitivity, accuracy, stability, higher repeatability and no toxic or side effect.

Disclosure of Invention

The invention aims to solve the problem that the activity detection reliability and safety of glucose oxidase in the prior art are low.

Therefore, the invention provides a method for detecting the activity of glucose oxidase, which is characterized by comprising the following steps:

(1) diluting glucose oxidase by using a phosphate buffer solution to serve as an enzyme solution to be detected, recording the dilution multiple as N, and preheating the enzyme solution to be detected;

(2) preparing a glucose solution, a potassium iodide solution and an ammonium molybdate solution, uniformly mixing the glucose solution, the potassium iodide solution and the ammonium molybdate solution, preheating, and dividing into a plurality of reaction substrates for later use;

(3) get V₁Adding ml of enzyme solution to be detected after water bath preheating into one part of the reaction substrate in the step (2), and immediately adding a sulfuric acid solution to terminate the reaction after the enzymolysis reaction is carried out for T minutes;

(4) get V₁Adding ml of phosphate buffer solution into another part of the reaction substrate in the step (2), and immediately adding a sulfuric acid solution which is equal to that in the step (3) after T minutes to serve as a blank control group;

(5) dropwise adding an indicator into the product obtained in the step (3), uniformly mixing, titrating to be colorless by using an iodine simple substance reducing agent with the concentration of f mol/l, and recording the consumed volume of the iodine simple substance reducing agent as V₂ml; simultaneously carrying out the same titration reaction on the blank control group in the step (4), and recording the consumption volume of the iodine simple substance reducing agent as V₃ ml；

(6) Calculating the glucose oxidase activity X according to the formula

Wherein a is the conversion coefficient of the iodine simple substance reducing agent and hydrogen peroxide; 1000 is a transforming factor.

Specifically, the phosphate buffer solution in the step (1) is composed of NaH₂PO₄·2H₂O and Na₂HPO₄·12H₂O is mixed to prepare a phosphate buffer solution with the concentration of 0.05-0.25mol/l and the pH value of 3.0-7.0.

Specifically, the concentration of the phosphate buffer solution was 0.1mol/l, and the pH was 6.0.

Specifically, in the step (2), the concentration of the glucose solution is 180g/l, the concentration of the potassium iodide solution is 100g/l, and the concentration of the ammonium molybdate solution is 10 g/l.

Specifically, in the reaction substrate prepared in the step (2), the potassium iodide solution: glucose solution: the volume ratio of the ammonium molybdate solution is 25:3: 1.

Specifically, the temperature of the enzymolysis reaction in the step (3) is 20-45 ℃.

Specifically, the temperature of the enzymatic hydrolysis reaction is 37 ℃.

Specifically, the concentration of the sulfuric acid solution used in the above step (3) and step (4) is 1 mol/l.

Specifically, the indicator used in the step (5) is a starch indicator.

Specifically, the iodine simple substance reducing agent used in the step (5) includes one of hydrogen sulfide, sulfur dioxide and a sodium thiosulfate solution.

The principle of the invention is as follows: under the action of glucose oxidase, glucose reacts with oxygen to generate gluconic acid and hydrogen peroxide, the hydrogen peroxide oxidizes potassium iodide under an acidic condition to generate quantitative iodine, and then the iodine is titrated by an iodine simple substance reducing agent, so that the content of the hydrogen peroxide can be measured, and the activity of the glucose oxidase can be calculated.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the glucose oxidase activity detection method provided by the invention is simple to operate, short in reaction time and sensitive and accurate in detection; a hydrogen peroxide standard curve does not need to be made, so that inaccurate results caused by operation errors are avoided; the used chemical reagents are conventional chemical reagents, and the detection process is safe and convenient; the reaction condition is mild, and the method is suitable for the fields of food brewing, feed addition, flour improvement, medicine and the like.

The present invention will be described in further detail below with reference to the accompanying drawings.

Drawings

FIG. 1 is a graph showing the activity of glucose oxidase as a function of reaction temperature in example 1 of the present invention.

FIG. 2 is a graph of glucose oxidase activity as a function of solution pH for example 2 of the present invention.

Detailed Description

The technical solutions in the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Although representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made thereto without departing from the scope of the invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.

The invention provides a method for measuring the activity of glucose oxidase, which comprises the following steps:

(1) diluting glucose oxidase by using a phosphate buffer solution to serve as an enzyme solution to be detected, recording the dilution multiple as N, and preheating the enzyme solution to be detected in a water bath;

wherein the phosphate buffer is composed of NaH₂PO₄·2H₂O and Na₂HPO₄·12H₂O is mixed to prepare a phosphate buffer solution with the concentration of 0.05-0.25mol/l and the pH value of 3.0-7.0; preferably, the concentration of the phosphate buffer solution is 0.1mol/l, and the pH value is 6.0;

(2) preparing a glucose solution, a potassium iodide solution and an ammonium molybdate solution, uniformly mixing the glucose solution, the potassium iodide solution and the ammonium molybdate solution, preheating in a water bath, and dividing into a plurality of reaction substrates for later use;

specifically, the concentration of the glucose solution is 180g/l, the concentration of the potassium iodide solution is 100g/l, and the concentration of the ammonium molybdate solution is 10 g/l; potassium iodide solution: glucose solution: the volume ratio of the ammonium molybdate solution is preferably 25:3: 1;

(3) get V₁Adding ml of enzyme solution to be detected after water bath preheating into one part of the reaction substrate in the step (2), and immediately adding a sulfuric acid solution to terminate the reaction after the enzymolysis reaction is carried out for T minutes;

specifically, the temperature of the enzymolysis reaction is 20-45 ℃, and preferably 37 ℃;

(4) get V₁Adding ml of phosphate buffer solution into another reaction substrate in the step (2), adding an equal amount of sulfuric acid solution as in the step (3) immediately after T minutes to serve as a blank control group；

Specifically, the concentration of the sulfuric acid solution is 1 mol/l;

(5) dropwise adding an indicator into the product obtained in the step (3), uniformly mixing, titrating to be colorless by using an iodine simple substance reducing agent with the concentration of f mol/l, and recording the consumed volume of the iodine simple substance reducing agent as V₂ml; simultaneously carrying out the same titration reaction on the blank control group in the step (4), and recording the consumption volume of the iodine simple substance reducing agent as V₃ ml；

Specifically, the indicator is a starch indicator; the iodine simple substance reducing agent comprises one of hydrogen sulfide, sulfur dioxide and sodium thiosulfate solution, and preferably sodium thiosulfate;

(6) calculating the glucose oxidase activity X according to the formula

Wherein a is the conversion coefficient of the iodine simple substance reducing agent and hydrogen peroxide, and when the iodine simple substance reducing agent is sodium thiosulfate, the conversion coefficient a is 2; 1000 is a transforming factor.

The effect of the method for detecting the activity of glucose oxidase of the present invention was examined below by way of specific examples, and the glucose oxidase used in the examples of the present invention was derived from the organism GmbH, Xinhua Yangyang, Wuhan.

The enzyme concentration after the glucose oxidase is diluted in the embodiment of the invention is controlled to be 8-12U/ml. For samples with unknown enzyme activity, after the approximate range of the enzyme activity is obtained through a plurality of preliminary experiments by adopting the method provided by the invention, the enzyme concentration is controlled to be 8-12U/ml, and then the experimental detection is carried out, wherein the errors are all less than 8 percent (the specified value of the enzyme activity detection error). When the enzyme concentration is out of the range, the dilution times are different, and the errors are all larger than 8%.

Example 1:

in this example, the enzyme activities of glucose oxidase at different temperatures were measured by the following specific steps:

(1) preparing a phosphate buffer solution: 877ml of 0.1mol/l NaH are taken₂PO₄·2H₂O and 123ml of 0.1mol/l Na₂HPO₄·12H₂Mixing O, and adding 0.1mol/l Na₂HPO₄Adjusting the pH value of the solution to 6.0 to obtain 0.1mol/l phosphate buffer solution;

diluting glucose oxidase by using a phosphate buffer solution to serve as an enzyme solution to be detected, and carrying out water bath heat preservation on the enzyme solution to be detected in a water bath kettle at 37 ℃ for 5 min;

(2) preparing a substrate reagent: 180g/l glucose solution, the preparation method is that 18g of anhydrous glucose is weighed and dissolved in 80mL of water, after the anhydrous glucose is stirred to be completely dissolved, the volume of the water is determined to be 100 mL; 100g/l potassium iodide solution, the preparation method is that 10g potassium iodide is weighed and dissolved in 80ml water, after the potassium iodide is stirred to be completely dissolved, the volume is determined to 100ml by water; 10g/l of ammonium molybdate solution, wherein the preparation method comprises the steps of weighing 1g of ammonium molybdate, dissolving the ammonium molybdate solution in 80mL of water, stirring the solution until the ammonium molybdate solution is completely dissolved, and then metering the volume to 100m l by using the water;

preparing a reaction substrate: uniformly mixing 2.5ml of potassium iodide solution, 0.3ml of glucose solution and 0.1ml of ammonium molybdate solution, and preserving the temperature in a 37 ℃ water bath kettle for 5min to serve as a reaction substrate;

(3) preparing a titration reaction reagent: 1mol/l sulfuric acid solution, the preparation method is to take 30ml concentrated sulfuric acid to fix the volume to 100 ml; the 10g/l starch indicator is prepared by adding 1g of soluble starch dissolved in a small amount of water into about 30ml of boiling water, heating and boiling for 3min, and metering to 100ml for use within 24 h; 0.01mol/l sodium thiosulfate solution, the preparation method is to weigh 26g of pentahydrate sodium thiosulfate, add 0.2g of anhydrous sodium carbonate, dissolve in 1000ml of water, boil slowly for 10min, calibrate after cooling and placing for 2 weeks, use after diluting 10 times;

experimental group 1: transferring 0.1ml of diluted N-300 times and heat-preserved enzyme solution to be detected into a reaction substrate, mixing uniformly, carrying out enzymolysis reaction for 5min at 37 ℃ in a water bath, adding 2ml of sulfuric acid solution to terminate the reaction, then adding 2 drops of starch indicator, shaking uniformly, titrating to be colorless by using sodium thiosulfate, and recording the volume of the consumed sodium thiosulfate solution as V₂＝10.5ml；

Experimental group 2: the same enzymolysis and titration method as in experiment group 1 were adopted except that the enzymolysis temperature was 20 deg.C and the dilution factor of the enzyme solution was 200 times, and digestion was recordedThe volume of the sodium thiosulfate solution consumed is V₂＝11.5ml；

Experimental group 3: the same enzymolysis and titration method as in experimental group 1 was used, except that the enzymolysis temperature was 30 ℃, the dilution factor of the enzyme solution was N-300 times, and the volume of the sodium thiosulfate solution consumed was recorded as V₂＝9.8ml；

Experimental group 4: the same enzymolysis and titration method as in experimental group 1 was used, except that the enzymolysis temperature was 45 ℃, the dilution factor of the enzyme solution was N-250 times, and the volume of the sodium thiosulfate solution consumed was recorded as V₂＝11.0ml；

Experimental group 5: the same enzymolysis and titration method as in experimental group 1 was used, except that the enzymolysis temperature was 50 ℃, the dilution factor of the enzyme solution was 220 times, and the volume of the sodium thiosulfate solution consumed was recorded as V₂＝11.9ml；

Experimental group 6: the same enzymolysis and titration method as in experimental group 1 was used, except that the enzymolysis temperature was 60 ℃, the dilution factor of the enzyme solution was N-170 times, and the volume of the sodium thiosulfate solution consumed was recorded as V₂＝10.0ml；

Experimental group 7: the same enzymolysis and titration method as in experimental group 1 was used, except that the enzymolysis temperature was 70 ℃, the dilution factor of the enzyme solution was N-100 times, and the volume of the sodium thiosulfate solution consumed was recorded as V₂＝10.6ml；

Blank control group: transferring 0.1ml phosphate buffer solution into a part of reaction substrate, mixing, reacting at 20 deg.C, 30 deg.C, 45 deg.C, 50 deg.C, 60 deg.C, 70 deg.C for 5min, adding 2ml sulfuric acid solution to terminate reaction, adding 2 drops of starch indicator, shaking, titrating with sodium thiosulfate to colorless, and consuming sodium thiosulfate solution with volume V₃＝0.06ml；

Calculating the glucose oxidase activity X according to the formula

Wherein x is the activity of glucose oxidase and the unit is u/g or u/ml;

V₁to enter intoThe volume of enzyme solution to be detected for carrying out enzymolysis reaction is ml;

V₂the volume of sodium thiosulfate consumed for the experimental group was in ml;

V₃the volume of sodium thiosulfate consumed for the blank control group was in ml;

f is the concentration of the sodium thiosulfate solution, and the unit is mol/L;

a is a conversion coefficient of sodium thiosulfate and hydrogen peroxide, 2mmol/l of sodium thiosulfate is equivalent to 1mmol/l of hydrogen peroxide, and in the case, a is 2;

1000 is conversion factor, 1mmol ═ 1000 μmol;

n is the dilution multiple of the glucose oxidase by phosphate buffer;

t is enzymolysis reaction time, and the unit is min;

the glucose oxidase activity as a function of reaction temperature is shown in FIG. 1.

Example 2:

in this example, the enzyme activities of glucose oxidase at different pH were tested by the following specific steps:

(1) preparing phosphate buffer solutions with different pH values: adding 0.1mol/l NaH₂PO₄·2H₂O and 0.1mol/l Na₂HPO₄·12H₂O is mixed uniformly and 0.1mol/l of Na is used₂HPO₄Adjusting the pH value of the solution to prepare phosphate buffer solutions with the concentration of 0.1mol/l and the pH values of 3.0, 4.0, 5.0, 5.5, 6.0, 7.0 and 8.0 respectively;

(2) preparing a substrate reagent: 180g/l glucose solution, the preparation method is that 18g of anhydrous glucose is weighed and dissolved in 80mL of water, after the anhydrous glucose is stirred to be completely dissolved, the volume of the water is determined to be 100 mL; 100g/l potassium iodide solution, the preparation method is that 10g potassium iodide is weighed and dissolved in 80ml water, after the potassium iodide is stirred to be completely dissolved, the volume is determined to 100ml by water; 10g/l of ammonium molybdate solution, wherein the preparation method comprises the steps of weighing 1g of ammonium molybdate, dissolving the ammonium molybdate solution in 80mL of water, stirring the solution until the ammonium molybdate solution is completely dissolved, and then metering the volume to 100mL by using the water;

preparing a reaction substrate: uniformly mixing 2.5ml of potassium iodide solution, 0.3ml of glucose solution and 0.1ml of ammonium molybdate solution, and preserving the temperature in a 37 ℃ water bath kettle for 5min to serve as a reaction substrate;

(3) preparing a titration reaction reagent: 1mol/l sulfuric acid solution, the preparation method is to take 30ml concentrated sulfuric acid to fix the volume to 100 ml; the 10g/l starch indicator is prepared by adding 1g of soluble starch dissolved in a small amount of water into about 30ml of boiling water, heating and boiling for 3min, and metering to 100ml for use within 24 h; 0.01mol/l sodium thiosulfate solution, the preparation method is to weigh 26g of pentahydrate sodium thiosulfate, add 0.2g of anhydrous sodium carbonate, dissolve in 1000ml of water, boil slowly for 10min, calibrate after cooling and placing for 2 weeks, use after diluting 10 times;

experimental group 1: diluting glucose oxidase as enzyme solution to be detected by using phosphate buffer solution with pH of 6.0, wherein the dilution multiple is N-300, and keeping the temperature of the enzyme solution to be detected in a water bath kettle at 37 ℃ for 5 min; transferring 0.1ml of heat-preserved enzyme solution to be detected into a reaction substrate, mixing uniformly, carrying out enzymolysis reaction for 5min in water bath at 37 ℃, adding 2ml of sulfuric acid solution to terminate the reaction, then adding 2 drops of starch indicator, shaking up, titrating to be colorless by using sodium thiosulfate, and recording the volume of the consumed sodium thiosulfate solution as V₂＝10.5ml；

Experimental group 2: the same experimental method as experimental group 1 was used except that glucose oxidase was diluted with phosphate buffer solution having pH of 3.0 as the enzyme solution to be measured at a dilution factor of N200, and the volume of the consumed sodium thiosulfate solution was recorded as V₂＝10.82ml；

Experimental group 3: the same experimental method as experimental group 1 was used except that glucose oxidase was diluted with phosphate buffer solution having pH of 4.0 as the enzyme solution to be measured at a dilution factor of N240, and the volume of the consumed sodium thiosulfate solution was recorded as V₂＝10.42ml；

Experimental group 4: the same experimental method as experimental group 1 was used, except that glucose oxidase was diluted with phosphate buffer solution having pH of 5.0 as the enzyme solution to be measured, the dilution ratio of the enzyme solution was N-250, and the volume of the consumed sodium thiosulfate solution was recorded as V₂＝10.22ml；

Experimental group 5: the same experimental method as in experimental group 1 was used except that glucose oxidase was diluted with phosphate buffer solution having pH of 5.5As the enzyme solution to be tested, the dilution factor of the enzyme solution is N-270, and the volume of the consumed sodium thiosulfate solution is recorded as V₂＝11.02ml；

Experimental group 6: the same experimental method as experimental group 1 was used, except that glucose oxidase was diluted with phosphate buffer solution having pH of 7.0 as the enzyme solution to be measured at a dilution factor of N190, and the volume of the consumed sodium thiosulfate solution was recorded as V₂＝10.02ml；

Experimental group 7: the same experimental method as experimental group 1 was used, except that glucose oxidase was diluted with phosphate buffer solution having pH of 8.0 as the enzyme solution to be measured, the dilution factor of the enzyme solution was N60, and the volume of the sodium thiosulfate solution consumed was recorded as V₂＝11.42ml；

Blank control group: transferring 0.1ml of phosphate buffer solution with pH values of 3.0, 4.0, 5.0, 5.5, 6.0, 7.0 and 8.0 into a part of reaction substrate, mixing uniformly, reacting at 37 ℃ for 5min, adding 2ml of sulfuric acid solution to stop the reaction, adding 2 drops of starch indicator, shaking uniformly, titrating to colorless by using sodium thiosulfate, and consuming the sodium thiosulfate solution with the volume V₁＝0.04ml；

(4) Calculating the glucose oxidase activity X according to the formula

Wherein x is the activity of glucose oxidase and the unit is u/g or u/ml;

V₁the volume of enzyme solution to be detected for carrying out enzymolysis reaction is ml;

V₂the volume of sodium thiosulfate consumed for the experimental group was in ml;

V₃the volume of sodium thiosulfate consumed for the blank control group was in ml;

f is the concentration of the sodium thiosulfate solution, and the unit is mol/L;

a is a conversion coefficient of sodium thiosulfate and hydrogen peroxide, 2mmol/l of sodium thiosulfate is equivalent to 1mmol/l of hydrogen peroxide, and in the case, a is 2;

1000 is conversion factor, 1mmol ═ 1000 μmol;

n is the dilution multiple of the glucose oxidase by phosphate buffer;

t is enzymolysis reaction time, and the unit is min;

the glucose oxidase activity as a function of solution pH is shown in FIG. 2.

The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.