CN104237145A - Method for rapidly determining activity of glucose oxidase - Google Patents
Method for rapidly determining activity of glucose oxidase Download PDFInfo
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- CN104237145A CN104237145A CN201410409560.2A CN201410409560A CN104237145A CN 104237145 A CN104237145 A CN 104237145A CN 201410409560 A CN201410409560 A CN 201410409560A CN 104237145 A CN104237145 A CN 104237145A
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- hydrogen peroxide
- glucose oxidase
- dianisidine
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Abstract
The invention relates to the field of analytic chemistry, and in particular relates to a method for rapidly determining activity of glucose oxidase. The method comprises the following steps: (1) drawing a hydrogen peroxide concentration-dianisidine color developing agent light absorption value standard curve; (2) preprocessing a to-be-determined glucose oxidase sample; and (3) calculating the activity of the glucose oxidase in the test sample by utilizing the hydrogen peroxide concentration-light absorption value standard curve. The method is rapid, reliable, simple to operate, sensitive and accurate. According to the principle of the detection method, under the effect of the glucose oxidase, glucose reacts with oxygen to generate gluconic acid and hydrogen peroxide, hydrogen peroxide and the colorless reduction dianisidine react to generate water and brown oxidized dianisidine under the effect of the peroxidase oxidase, the brown oxidized dianisidine reacts with sulfuric acid solution to turn pink, and the activity of the glucose oxidase can be rapidly determined in a colorimetric manner at the position of 540nm of the maximum absorption wavelength.
Description
Technical field
The present invention relates to analytical chemistry field, be specifically related to a kind of method of Fast Measurement glucose oxidase enzyme activity.
Background technology
Glucose oxidase (Glucose oxidase, E.C.1.1.3.4 are called for short GOD) is a kind of aerobic dehydrogenase, and its systematic naming method is β-D-Glucose oxidoreducing enzyme.This enzyme is separated early than nineteen twenty-eight by M ǖ ller and is reported from aspergillus niger, and Pazur obtains pure glucose oxidase in nineteen sixty-five.When there being oxygen to exist, glucose oxidase can oxidation of beta-D-Glucose in specific manner, generate gluconic acid and hydrogen peroxide, have a wide range of applications in gluconate manufacture, food additives, animal feed additive, biochemistry detection and biology sensor etc.
At present, the detection method that glucose oxidase enzyme activity is main has titrimetry, electrochemical process and spectrophotometric method.Titration ratio juris is that glucose oxidase enzymatic glucose generates glucuronic acid, then indirect determination can go out the output of glucuronic acid by acid base titration, then calculates the enzyme activity of GOD according to the generation of glucuronic acid.Although the method is simple, cost is low, and the method exists the shortcoming that metrical error is large and sensitivity is low, and needs to carry out complicated to move liquid titration operation, and workload is larger.Galvanochemistry ratio juris measures oxidation-reduction quality enzyme activity according to the change of the voltage in enzymatic reaction or electric current, measure with oxygen electrode, but this method is easily subject to the interference detecting dissolved oxygen and other electroactive substances in liquid, thus affect experimental result.Spectrophotometric ratio juris is when aerobic, and glucose oxidase enzymatic β-D-Glucose produces gluconic acid and hydrogen peroxide.Subsequently, horseradish peroxidase (HRP) catalyzing hydrogen peroxide and chromogenic substrate react, and then adopt spectrophotometer to detect the coloring matter generated.The method utilizing this principle to carry out the detection of GOD enzyme activity at present mainly contains isatin Fading Spectrophotometic Method, quinone imines method, diaminobenzidine and o-dianisidine method etc.Although spectrophotometric method is very sensitive, but regrettably above method also exists substance that show color instability in the document reported, data redundancy is bad, the typical curve range of linearity is narrower and detect the lower situation of GOD enzyme activity result, still rest on the basis of qualitative analysis, be not suitable for carrying out promoting and formulation standard.Therefore, set up quick, accurate, sensitive and that testing cost is low GOD enzyme activity detection method is the target that research worker lays siege to always.
In order to overcome the shortcoming of GOD enzyme activity detection system, design fast and reliable, simple to operate, sensitive and accurate detection method, widen the scope of application of detection system, correct irrational factor, the invention provides a kind of method that quantitative and qualitative analysis measures glucose oxidase enzyme activity, by repeatedly verifying, establish comparatively practical enzyme activity detection system reliably.
Summary of the invention
The object of this invention is to provide a kind of method that quantitative and qualitative analysis measures glucose oxidase enzyme activity.
Method according to Fast Measurement glucose oxidase enzyme activity of the present invention comprises the following steps:
(1) concentration of hydrogen peroxide-dianisidine developer light absorption value typical curve is drawn, first draw Hydrogen peroxide standard solution 0.25mL respectively, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL and 0.75mL, 25mL is settled to respectively with phosphate buffer, be made into the concentration of hydrogen peroxide gradient series that final concentration is respectively 24 μ g/mL ~ 72 μ g/mL, 2.5mL dianisidine dilution is added successively in test tube, 0.3mL glucose solution, 0.1mL horseradish peroxidase solution, the each 0.1mL of above-mentioned variable concentrations Hydrogen peroxide standard solution is added again respectively after mixing, the sulfuric acid solution of 2mL 2mol/L is added after mixing, light absorption value is measured at 540nm place with 1cm cup after shaken well.Take light absorption value as horizontal ordinate, concentration of hydrogen peroxide is ordinate drawing standard curve;
(2) carry out pre-service to glucose oxidase sample to be measured, with phosphate buffer dilute sample, in the sample after dilution, glucose oxidase enzyme activity controls between 0.8 ~ 2.8U/mL;
(3) during reaction, 2.5mL dianisidine dilution is added successively in test tube, 0.3mL glucose solution, 0.1mL horseradish peroxidase solution, after shaken well after set point of temperature water-bath 5min, measure pipe and add the enzyme sample solution 0.1mL diluted, after set point of temperature reaction 3min, add the sulfuric acid solution 2mL cessation reaction of 2mol/L, shaken well after being cooled to room temperature, light absorption value is measured with 1cm cup at 540nm place, blank is done with the enzyme liquid of heat inactivation, concentration of hydrogen peroxide-light absorption value typical curve is utilized to calculate glucose oxidase enzyme activity in test specimen.
The Cleaning Principle of GOD enzyme activity is as follows:
Whole process comprises three steps:
The first step, in aqueous, under the existence of GOD, 1mol β-D-Glucose and the reaction of 1mol molecular oxygen generate the hydrogen peroxide of 1mol gluconic acid and 1mol;
Second step, hydrogen peroxide and colourless reduced form dianisidine, under the effect of horseradish peroxidase, generate water and brown oxidized o-Dianisidine;
3rd step, brown oxidized o-Dianisidine and sulfuric acid solution react and generate peach oxidized o-Dianisidine.
Above-mentioned three-step reaction all carries out in same glass test tube, and operation is very easy.
According to the specific embodiment of the present invention, before mensuration enzyme activity, first to prepare the 0.1mol/L phosphate buffer of regulation pH, then the glucose solution of buffer 180g/L, by the dianisidine storage liquid of methyl alcohol preparation 1%, with phosphate buffer dilution before using, namely 0.1mL dianisidine methyl alcohol storage liquid joins in 12mL phosphate buffer and mixes.Also need the horseradish peroxidase solution and the 2mol/L H that prepare 100U/mL
2sO
4solution.Finally, note to use KMnO in advance before preparation 2400mg/L Hydrogen peroxide standard solution
4demarcate 30% superoxol.Concrete implementation step is as follows:
(1) drafting of concentration of hydrogen peroxide-dianisidine developer light absorption value typical curve will before the reaction, first be carried out.First draw Hydrogen peroxide standard solution 0.25mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL and 0.75mL respectively, be settled to 25mL with phosphate buffer respectively, be made into the concentration of hydrogen peroxide gradient series that final concentration is respectively 24 μ g/mL ~ 72 μ g/mL.Selecting the concentration of hydrogen peroxide gradient series of 24 μ g/mL ~ 72 μ g/mL, is because through repetition test, and the enzyme activity size ability of light absorption value size corresponding within the scope of this and GOD is linear.
(2) in test tube, add 2.5mL dianisidine dilution, 0.3mL glucose solution, 0.1mL horseradish peroxidase solution successively, the each 0.1mL of above-mentioned variable concentrations Hydrogen peroxide standard solution is added again respectively after mixing, add the sulfuric acid solution of 2mL 2mol/L after mixing, after shaken well, measure light absorption value at 540nm place with 1cm cup.Take light absorption value as horizontal ordinate, concentration of hydrogen peroxide is ordinate drawing standard curve.
(3) pre-service is carried out to testing sample, solid sample generally takes about quality 1g, be accurate to 0.001g, be placed in conical flask, add 100mL phosphate buffer, magnetic agitation or shaking table vibration 20min, then be diluted to suitable multiple with phosphate buffer, fluid sample is directly diluted to suitable multiple with phosphate buffer solution.The amount of glucose in reaction system not only will be made far away excessive, the size of the size of GOD enzyme activity and light absorption value also will be made linear, in the sample therefore after regulation dilution, glucose oxidase enzyme activity controls between 0.8 ~ 2.8U/mL.
(4) during reaction, 2.5mL dianisidine dilution, 0.3mL glucose solution, 0.1mL horseradish peroxidase solution is added successively in test tube, after shaken well after set point of temperature water-bath 5min, measure pipe and add the enzyme sample solution 0.1mL being diluted to suitable multiple, after set point of temperature reaction 3min, add the sulfuric acid solution 2mL cessation reaction of 2mol/L, shaken well also, after being cooled to room temperature, measures light absorption value at 540nm place with 1cm cup.Do blank with the enzyme liquid of heat inactivation, utilize concentration of hydrogen peroxide-light absorption value typical curve to calculate glucose oxidase enzyme activity in test specimen.Reaction time is set as 3min, is because through repetition test, finds that the reaction of GOD catalysis glucose is linear catalytic reaction at 3min, namely enzymic catalytic reaction speed and enzyme concentration size linear.And 540nm is the maximum absorption wavelength of this color solution.When measuring glucose oxidase enzyme activity, generally choose the optimal reaction pH of this enzyme and optimal reactive temperature respectively as the pH value of required damping fluid during reaction and the design temperature of water-bath.
GOD enzyme activity is defined as, 1g solid enzyme powder (or 1mL liquid enzymes), under uniform temperature and pH value condition, 1min catalysis β-D-Glucose is oxidized the enzyme amount produced needed for 0.1 μ g hydrogen peroxide, be 1 enzyme activity unit, represent with U/g (or U/mL).
What the present invention adopted is a kind of new spectrophotometric method, and the method fast and reliable is simple to operate, sensitive and accurate.This detection method principle is under the effect of glucose oxidase, glucose and oxygen reaction, generate gluconic acid and hydrogen peroxide, hydrogen peroxide and colourless reduced form dianisidine are under the effect of peroxidase, generate water and brown oxidized o-Dianisidine, brown oxidized o-Dianisidine and sulfuric acid solution react and become pink, can in maximum absorption wavelength 540nm place rapid colorimetric determination.
The present invention has following characteristics:
1, the novel colourimetry that the present invention sets up has the feature of rapid sensitive, react by brown oxidized o-Dianisidine and sulfuric acid solution the light absorption value generating pink material to the mensuration of concentration of hydrogen peroxide to realize, and the whole enzymic catalytic reaction time is 3min, than the colourimetry reported, there is the shorter reaction time.
2, in the present invention, reaction conditions is gentle, and generally not by the interference of other impurity in sample, the test sample scope of application is wide in range.The dianisidine solution concentration of preparation is low, if in fuming cupboard operation during preparation, then can not produce any injury to human body.The horseradish peroxidase amount needed in detection is few, and cost is low, has easily bought.
3, according to the GOD enzyme activity appraisement system of method establishment of the present invention, only need water-bath and visible spectrophotometer, do not need exact instrument and complicated operation steps, be applicable to vast enterprises and institutions, scientific research institution, hospital and school etc. generally use.
Accompanying drawing explanation
Fig. 1 shows the concentration of hydrogen peroxide-dianisidine developer light absorption value typical curve measured in the embodiment of the present invention, and condition determination is temperature 37 DEG C, pH6.0.
Fig. 2 shows the relation curve that in the embodiment of the present invention 1, GOD enzyme activity changes with solution ph.To remain most high enzymatic activity for 100%, the percentage that the enzyme activity under other conditions accounts for most high enzymatic activity is the enzyme activity of this enzyme under this pH.
Fig. 3 shows the relation curve that in the embodiment of the present invention 1, GOD enzyme activity changes with temperature of reaction.To remain most high enzymatic activity for 100%, the percentage that the enzyme activity under other conditions accounts for most high enzymatic activity is this enzyme enzyme activity at this temperature.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Embodiment 1
Glucose oxidase fluid sample is raised Biological Co., Ltd. by Xinhua and is provided, and measures it at pH6.0, the glucose oxidase enzyme activity at temperature 37 DEG C.Fluid sample is directly diluted to suitable multiple with 0.1mol/L pH6.0 phosphate buffer.Then, in test tube, 2.5mL dianisidine dilution, 0.3mL glucose solution, 0.1mL horseradish peroxidase solution is added successively, shaken well.After 37 DEG C of water-bath 5min, measure pipe and add the sample enzyme solutions 0.1mL being diluted to suitable multiple, after 37 DEG C of reaction 3min, add the sulfuric acid solution 2mL cessation reaction of 2mol/L, shaken well also, after being cooled to room temperature, measures light absorption value at 540nm place with 1cm cup.Do blank with the enzyme liquid of heat inactivation, last glucose oxidase enzyme activity is calculated by formula 1:
X＝[(A-A
0)×K+C
0]×N/T/10 (1)
In formula: the glucose oxidase enzyme activity in X-sample, U/mL;
The absorbance of A-enzyme reaction group;
A
0the absorbance of-blank group;
The slope of K-typical curve;
C
othe intercept of-typical curve;
Total extension rate of N-sample;
T-reaction time 3min;
10-the enzyme amount produced needed for 0.1 μ g hydrogen peroxide is 1 enzyme activity unit.
The relative error of same sample three replicate determination values is no more than 5.0%, and the mean value of three is final enzyme activity determination value, repetitive operation twice.Sample enzyme activity testing result is in table 1.
Embodiment 2
Glucose oxidase powder samples is provided by Wuhan Ding Guo Bioisystech Co., Ltd, indicates enzyme activity scope 300 ~ 350U/mg, measures it at pH6.0, the glucose oxidase enzyme activity at temperature 37 DEG C.Accurately take solid powder with per mille balance and be about 1g, be placed in conical flask, and record the quality of pulvis.Then add 100mL0.1mol/L pH6.0 phosphate buffer, leave standstill to layering after shaking table vibration 20min, then Aspirate supernatant 1ml, be diluted to suitable multiple with 0.1mol/L pH6.0 phosphate buffer.Subsequent operation is carried out according to the step described in embodiment 1.Last glucose oxidase enzyme activity is calculated by formula 2:
X＝[(A-A
0)×K+C
0]×N/(T×M)/10 (2)
In formula: the glucose oxidase enzyme activity in X-sample, U/g;
The absorbance of A-enzyme reaction group;
A
0the absorbance of-blank group;
The slope of K-typical curve;
C
othe intercept of-typical curve;
Total extension rate of N-sample;
T-reaction time 3min;
The quality of M-sample, g;
10-the enzyme amount produced needed for 0.1 μ g hydrogen peroxide is 1 enzyme activity unit.
The relative error of same sample three replicate determination values is no more than 5.0%, and the mean value of three is final enzyme activity determination value, repetitive operation twice.Sample enzyme activity testing result is in table 1.
Table 1 sample enzyme activity testing result
Note: the enzyme slip-knot fruit detected in table is mean+SD (n=3).
Claims (1)
1. a method for Fast Measurement glucose oxidase enzyme activity, is characterized in that, said method comprising the steps of:
(1) concentration of hydrogen peroxide-dianisidine developer light absorption value typical curve is drawn, first draw Hydrogen peroxide standard solution 0.25mL respectively, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL and 0.75mL, 25mL is settled to respectively with phosphate buffer, be made into the concentration of hydrogen peroxide gradient series that final concentration is respectively 24 μ g/mL ~ 72 μ g/mL, 2.5mL dianisidine dilution is added successively in test tube, 0.3mL glucose solution, 0.1mL horseradish peroxidase solution, the each 0.1mL of above-mentioned variable concentrations Hydrogen peroxide standard solution is added again respectively after mixing, the sulfuric acid solution of 2mL 2mol/L is added after mixing, light absorption value is measured at 540nm place with 1cm cup after shaken well.Take light absorption value as horizontal ordinate, concentration of hydrogen peroxide is ordinate drawing standard curve;
(2) carry out pre-service to glucose oxidase sample to be measured, with phosphate buffer dilute sample, in the sample after dilution, glucose oxidase enzyme activity controls between 0.8 ~ 2.8U/mL;
(3) during reaction, 2.5mL dianisidine dilution is added successively in test tube, 0.3mL glucose solution, 0.1mL horseradish peroxidase solution, after shaken well after set point of temperature water-bath 5min, measure pipe and add the enzyme sample solution 0.1mL diluted, after set point of temperature reaction 3min, add the sulfuric acid solution 2mL cessation reaction of 2mol/L, shaken well after being cooled to room temperature, light absorption value is measured with 1cm cup at 540nm place, blank is done with the enzyme liquid of heat inactivation, concentration of hydrogen peroxide-light absorption value typical curve is utilized to calculate glucose oxidase enzyme activity in test specimen.
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| CN201410409560.2A CN104237145B (en) | 2014-08-19 | A kind of method of quick mensuration glucoseoxidase vigor |
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|---|---|---|---|
| CN201410409560.2A CN104237145B (en) | 2014-08-19 | A kind of method of quick mensuration glucoseoxidase vigor |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105911005A (en) * | 2016-06-06 | 2016-08-31 | 长春理工大学 | Hydrogen peroxide photometric method adopting DhHP-6 mimic enzyme |
| CN108195782A (en) * | 2018-01-25 | 2018-06-22 | 江南大学 | It is a kind of to be simple and efficient the method for measuring beta amylase vigor |
| CN108336322A (en) * | 2017-12-28 | 2018-07-27 | 广州倬粤动力新能源有限公司 | The preparation method of pole plate creme |
| CN108336325A (en) * | 2017-12-28 | 2018-07-27 | 广州倬粤动力新能源有限公司 | Pole plate creme |
| CN108336362A (en) * | 2017-12-28 | 2018-07-27 | 广州倬粤动力新能源有限公司 | The preparation method of alloy-coated material |
| CN108336323A (en) * | 2017-12-28 | 2018-07-27 | 广州倬粤动力新能源有限公司 | Unleaded cladding material |
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| CN109001189A (en) * | 2017-06-06 | 2018-12-14 | 中粮营养健康研究院有限公司 | Detect the method and its application of the amylase content range in the sugar products of sugarcane |
| CN109280693A (en) * | 2018-11-12 | 2019-01-29 | 济南百斯杰生物工程有限公司 | A kind of method of Fast Evaluation glucose oxidase acid producing ability |
| CN110923291A (en) * | 2019-12-13 | 2020-03-27 | 山东隆科特酶制剂有限公司 | Method for detecting activity of glucose oxidase for feed |
| CN111398434A (en) * | 2019-01-02 | 2020-07-10 | 东莞市东阳光生物合成药有限公司 | Glucose oxidase activity determination method |
| CN112301092A (en) * | 2020-10-30 | 2021-02-02 | 大连大学 | Colorimetric analysis method for determining activity of glucose oxidase |
| CN113720961A (en) * | 2021-08-17 | 2021-11-30 | 武汉新华扬生物股份有限公司 | Method for detecting activity of glucose oxidase |
| CN115791665A (en) * | 2023-01-09 | 2023-03-14 | 北京挑战农业科技有限公司 | Method for measuring activity of glucose oxidase |
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| CN105911005A (en) * | 2016-06-06 | 2016-08-31 | 长春理工大学 | Hydrogen peroxide photometric method adopting DhHP-6 mimic enzyme |
| CN109001189A (en) * | 2017-06-06 | 2018-12-14 | 中粮营养健康研究院有限公司 | Detect the method and its application of the amylase content range in the sugar products of sugarcane |
| CN108336322A (en) * | 2017-12-28 | 2018-07-27 | 广州倬粤动力新能源有限公司 | The preparation method of pole plate creme |
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| CN108195782B (en) * | 2018-01-25 | 2020-01-21 | 江南大学 | Method for measuring activity of beta-amylase |
| CN108195782A (en) * | 2018-01-25 | 2018-06-22 | 江南大学 | It is a kind of to be simple and efficient the method for measuring beta amylase vigor |
| CN109280693A (en) * | 2018-11-12 | 2019-01-29 | 济南百斯杰生物工程有限公司 | A kind of method of Fast Evaluation glucose oxidase acid producing ability |
| CN111398434A (en) * | 2019-01-02 | 2020-07-10 | 东莞市东阳光生物合成药有限公司 | Glucose oxidase activity determination method |
| CN111398434B (en) * | 2019-01-02 | 2023-12-08 | 宜昌东阳光生化制药有限公司 | Glucose oxidase activity determination method |
| CN110923291A (en) * | 2019-12-13 | 2020-03-27 | 山东隆科特酶制剂有限公司 | Method for detecting activity of glucose oxidase for feed |
| CN110923291B (en) * | 2019-12-13 | 2023-06-09 | 山东隆科特酶制剂有限公司 | Method for detecting activity of glucose oxidase for feed |
| CN112301092A (en) * | 2020-10-30 | 2021-02-02 | 大连大学 | Colorimetric analysis method for determining activity of glucose oxidase |
| CN113720961A (en) * | 2021-08-17 | 2021-11-30 | 武汉新华扬生物股份有限公司 | Method for detecting activity of glucose oxidase |
| CN115791665A (en) * | 2023-01-09 | 2023-03-14 | 北京挑战农业科技有限公司 | Method for measuring activity of glucose oxidase |
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Address after: No.98, guangguba Road, Donghu New Technology Development Zone, Wuhan City, Hubei Province Patentee after: Wuhan Sunhy Biology Co., Ltd. Address before: 430074, No. 5, Ling Nan Road, East Lake New Technology Development Zone, Wuhan, Hubei, China Patentee before: Wuhan Sunhy Biology Co., Ltd. |