CN115791665A - Method for measuring activity of glucose oxidase - Google Patents
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Abstract
The invention relates to a method for measuring the activity of glucose oxidase, which adopts a spectrophotometry to measure the activity of the glucose oxidase, the pretreatment time is short (about 30 min), the reaction is carried out in a water bath kettle at 37 ℃, the reaction temperature at 37 ℃ is closer to the body temperature of animals, the reaction time (about 10 min) is more reasonable, after the reaction timing is finished, a terminator SDS solution is added to terminate the reaction, then a spectrophotometer is used to measure the OD value, and the enzyme activity value is calculated. The reaction efficiency is high, and the enzyme reaction is carried out in a water bath, so that the detection of several enzymes can be carried out simultaneously. The precision of the detection method meets the requirement, and the repeatability of the detection result is good. The method is suitable for a feed additive glucose oxidase product, and the minimum detection amount of a sample is 5.0U/g.
Description
Technical Field
The invention relates to detection of enzyme activity, in particular to a method for determining activity of glucose oxidase.
Background
At present, the main detection methods of the activity of the glucose oxidase comprise a titration method and a spectrophotometry method. The spectrophotometry is very sensitive, but the chromogenic substance is unstable, the data repeatability is poor, the detection enzyme activity is low, and the method is not suitable for popularization and standard establishment. The titration method has low cost, but has the defects of difficult titration end point determination, low temperature for detecting the enzyme activity, low sensitivity, large error and long detection process time. Therefore, it is highly desirable to establish a rapid, sensitive and low-cost method for detecting GOD.
The method for determining the local standard DB 13/T1444-2011 feed additive glucose oxidase in Hebei province is suitable for using specific enzyme (glucose oxidase)Penicillium notatum) Is a glucose oxidase preparation produced by fermentation of a production strain. At present, most of glucose oxidase producing strains in feed are pichia pastorisPichia pastoris) Or Aspergillus nigerAspergillus niger) The enzyme activity of a mainstream product of the glucose oxidase which is not suitable for a feed additive market is found by actually measuring the activity of the glucose oxidase. In addition, the activity of the glucose oxidase is measured by using a titration method according to the local standard DB 13/T1444-2011 in Hebei province, and a glucose oxidase sample is added with water, stirred uniformly and leached for 15-16h at the temperature of 4-8 ℃ to be used as an enzyme solution to be measured. Adding the substrate glucose solution, carrying out oscillation reaction at the temperature of 30 ℃ and the speed of 150rpm of a shaking table for 60 minutes, taking out, adding 0.1mol/L sodium hydroxide solution to terminate the reaction, adding 1 drop of phenolphthalein indicator, and titrating the residual sodium hydroxide by using 0.1mol/L hydrochloric acid. The total reaction time is 17-18h, the detection time is long, and the efficiency is not high enough.
The existing glucose oxidase detection method lacks national standards, in a spectrophotometry method commonly used by part of enterprises, the difference between the reaction temperature of 30 ℃ and the actual body temperature of animals is large, meanwhile, the reaction is continuously timed for 6min, the operation time is too short, large experimental errors are easily caused, the reaction is to preheat and mix reaction solution in a water bath kettle, then add the reaction solution into a cuvette, and after the reaction is finished, read the absorbance value A at the moment 1 Through A 1 -A 0 And (4) performing calculation. The reaction is carried out in a cuvette without temperature control measures, and the operation time of 6min is too short, so that the real enzyme activity condition cannot be reflected.
Based on the situation, a set of standard glucose oxidase activity determination method needs to be established, and particularly, the glucose oxidase activity determination method is suitable for the production of pichia and aspergillus niger, and is more suitable for glucose oxidase, particularly glucose oxidase preparations in feeds. The method can meet the requirement of simultaneously measuring a plurality of samples, and has the advantages of accurate enzyme activity detection, simple operation and good repeatability of detection results.
Disclosure of Invention
The invention aims to provide a method for determining the activity of glucose oxidase.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a method for measuring glucose oxidase activity, comprising the steps of:
(1) Preparing a sample to be detected;
(2) Preparing four test tubes, wherein one test tube is a sample blank tube, and three test tubes are sample tubes; respectively adding 0.1mL of samples to be detected into the four test tubes, and balancing in a water bath at 37 ℃ for 1min; adding 300 mu L of 100g/L SDS solution into a sample blank tube, and carrying out vortex oscillation for 3 to 5s; then adding 3mL of balanced determination solution, and reacting for 10min at 37 ℃; respectively adding 3mL of balanced determination solution into the three sample tubes, reacting for 10min at 37 ℃, and then respectively adding 300 mu L of 100g/L SDS solution into the three sample tubes to terminate the reaction; adjusting to zero with water, and measuring the absorbance of a blank sample tube and the absorbance of a sample tube at 500nm by using a 10mm cuvette; calculating the activity of glucose oxidase by substituting formula (I):
In the formula:
X D : the activity of the glucose oxidase in the sample to be detected is U/mL, and X is more than or equal to 0.05U/mL D ≤0.20U/mL；
A: average absorbance values of the three sample tubes;
A 0 : absorbance of the sample blank tube;
10: reaction time in min;
1: the thickness of the cuvette is in cm;
12.88: millimolar absorption coefficient in L.mmoL of quinone imine as chromogenic substance -1 ·cm -1 ；
2: h for each 1 mole of quinoneimine formed 2 O 2 The amount of the substance;
3.4: the total volume of the reaction mixed solution is mL;
0.1: the volume of the sample to be detected is mL;
(3) Mixing X D Calculating the activity of the glucose oxidase in the sample by substituting formula (II):
In the formula:
x: the activity of the glucose oxidase in the sample is U/g or U/mL;
X D : the activity of the glucose oxidase in the sample to be detected is U/mL, and X is more than or equal to 0.05U/mL D ≤0.20U/mL；
n: total dilution of the sample.
Further, the preparation method of the assay solution comprises: 20.0mL of phosphate buffer solution A, 6.0mL of substrate glucose solution, 1.0mL of horseradish peroxidase solution, 1.0mL of 4-aminoantipyrine solution and 2.0mL of phenol solution were aspirated and mixed together in a total of 30.0mL to prepare a measurement solution, and the measurement solution was equilibrated in a water bath at 37 ℃ for 5min.
Wherein the concentration of the phosphate buffer solution A is 0.1mol/L and the pH value is 6.0 (the preparation method comprises the steps of mixing 0.1mol/L disodium hydrogen phosphate solution and 0.1mol/L sodium dihydrogen phosphate solution according to a certain proportion, adjusting the pH value to 6.0 by using the sodium dihydrogen phosphate solution if the pH value is higher than 6.0 after mixing, and adjusting the pH value to 6.0 by using the disodium hydrogen phosphate solution if the pH value is lower than 6.0 after mixing);
the concentration of the substrate glucose solution is 1.0mol/L;
the concentration of the horseradish peroxidase solution is 0.5mg/mL;
the concentration of the 4-aminoantipyrine solution is 4mg/mL;
the concentration of the phenol solution is 1.5mg/mL, and the phenol solution is prepared by the phosphate buffer solution A.
The preparation method of the sample to be detected in the step (1) comprises the following steps:
(a) For solid samples
Weighing 0.1-5.0 g of solid sample, accurately weighing the solid sample to 0.001g, placing the solid sample in a 100mL volumetric flask, adding 40mL of phosphate buffer solution B, stirring or oscillating (200 r/min) for 30min at 20 ℃, then diluting the volume of the sample with the phosphate buffer solution B, shaking up, centrifuging at 4000r/min for 5min, taking the supernatant, diluting the supernatant with the phosphate buffer solution B, and controlling the activity of glucose oxidase in the sample to be detected to be within the range of 0.05-0.20U/mL after dilution.
The corresponding relationship between the enzyme activity of the sample and the suggested sample weighing amount is shown in Table 1:
TABLE 1 correspondence table of enzyme activity and recommended sample weighing
(b) For liquid samples
And (3) directly diluting the liquid sample with a phosphate buffer solution B, fixing the volume, controlling the activity of glucose oxidase in the diluted sample to be detected within the range of 0.05U/mL-0.20U/mL and the pH to 6.0 (if the pH of the diluted enzyme solution deviates from 6.0, adjusting the pH to 6.0 by using 0.1mol/L disodium hydrogen phosphate solution or 0.1mol/L sodium dihydrogen phosphate solution, and then properly diluting and fixing the volume by using the phosphate buffer solution B).
Wherein the phosphate buffer solution B is a phosphate buffer solution A containing 0.1% of Tween-20.
In the present invention, the sample is derived from a glucose oxidase preparation produced by fermentation with a strain having glucose oxidase-producing ability.
The strain comprises but are not limited to specific enzymes (A), (B), (C)Penicillium notatum) Pichia pastoris (A), (B)Pichia pastoris) Or Aspergillus nigerAspergillus niger）。
In a second aspect, the invention provides a glucose oxidase activity assay kit, which comprises a phosphate buffer solution A, a phosphate buffer solution B, a 1.0mol/L glucose solution, a 0.5mg/mL horseradish peroxidase solution, a 4mg/mL 4-aminoantipyrine solution, a 1.5mg/mL phenol solution and a 100g/L SDS solution.
Wherein the concentration of the phosphate buffer solution A is 0.1mol/L, and the pH value is 6.0;
the phosphate buffer solution B is a phosphate buffer solution A containing 0.1% of Tween-20.
By means of the technical scheme, the invention at least has the following advantages and beneficial effects:
the method adopts a spectrophotometry to measure the activity of the glucose oxidase, the pretreatment time is short (only about 30 min), the reaction is carried out in a water bath kettle at 37 ℃, the reaction temperature at 37 ℃ is closer to the body temperature of animals, the reaction time is 10min, the reaction time is more reasonable, after the reaction timing is finished, a terminator SDS solution is added to terminate the reaction, then a spectrophotometer is used to measure the OD value, and the enzyme activity value is calculated. The reaction efficiency is high, and the enzyme reaction is carried out in a water bath, so that the detection of several enzymes can be carried out simultaneously.
And secondly, the glucose oxidase determination method is suitable for determining the activity of all glucose oxidases and has wide application range. DB 13/T1444-2011 adopts an acid-base titration method to measure the activity of the glucose oxidase, has high requirements on the technical level of operators, and causes large experimental errors easily due to inaccurate titration end point judgment. The method for measuring the enzyme activity by using the spectrophotometry is simple to operate and has low requirements on the technical level of operators.
And DB 13/T1444-2011 sample pretreatment time is 15-16h, and the pretreatment time is only about 30min, so that the sample pretreatment time is greatly shortened.
And (IV) although part of enterprises adopt a spectrophotometry method to determine the activity of the glucose oxidase at present, the enzyme activity reaction of the sample is actually carried out in a cuvette of a spectrophotometer without temperature control measures, the reaction temperature is influenced by room temperature, and the reaction temperature directly influences the detection value of the enzyme activity. Within a certain range, the lower the reaction temperature is, the lower the enzyme activity detection value is. And the reaction time in the cuvette is 6min, the reaction time is too short, operation errors are easily caused, the reproducibility of a detection result is poor, and the enzyme activity cannot be truly reflected due to too short reaction time.
And fifthly, the precision of the detection method meets the requirement, and the repeatability of the detection result is good.
And sixthly, the method is suitable for the feed additive glucose oxidase product, and the minimum detection amount of the sample is 5.0U/g.
Detailed Description
The present invention utilizes spectrophotometry to determine the glucose oxidase activity of a sample.
The principle of the detection method is as follows:
glucose oxidase catalyzes oxidation of glucose to generate hydrogen peroxide (H) 2 O 2 ），H 2 O 2 Under the catalysis of peroxidase, 4-aminoantipyrine and phenol can be oxidized and condensed to generate pink quinoneimine, and the basic reaction is as follows:
beta-D-glucose + O 2 Glucose oxidase D-gluconic acid + H 2 O 2
2H 2 O 2 + 4-aminoantipyrine + phenol Peroxidase enzymes Quinolimine +4H 2 O
In the first step of the reaction, the glucose substrate is required to be in absolute excess, so that the catalytic reaction is a quasi-first order reaction, and the reaction rate is proportional to the amount of enzyme added, regardless of the substrate concentration. The purpose of the second reaction is to accurately determine the H liberated in the first reaction 2 O 2 The amount of peroxidase relative to H required for this reaction is 2 O 2 In absolute excess, the H produced 2 O 2 Quickly participate in the reaction so as to achieve the aim of measuring the reaction rate of the first step in real time. The color depth of the reaction solution is in direct proportion to the amount of quinoneimine generated by peroxidase catalysis, and the generation amount of quinoneimine is in direct proportion to H 2 O 2 Proportional to the amount of production, H 2 O 2 The amount of the product is proportional to the activity of glucose oxidase in the reaction solution. Therefore, the intensity of the color of the reaction solution can be measured by measuring the change in absorbance at 500nm with a spectrophotometer, and the inverse can be calculatedThe activity of glucose oxidase in the reaction solution.
Definition of enzymatic activity unit of glucose oxidase: oxidizing 1 μmol glucose per minute to gluconic acid and H under the conditions of 37 deg.C, pH6.0, and glucose concentration of 0.2mol/L 2 O 2 The amount of enzyme required is defined as 1 enzyme activity unit (U).
The enzyme sample to be detected is required to be diluted to 0.05U/mL-0.20U/mL by using the buffer solution, namely the activity of the glucose oxidase in the enzyme solution to be detected after dilution is controlled to be 0.05U/mL-0.20U/mL, and the enzyme activity is determined again after the dilution times are recommended to be adjusted.
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.
The instruments and equipment used in the following examples:
analytical balance: the sensory quantity is 0.001 g.
A pH meter: to the nearest 0.01.
A magnetic stirrer or a constant temperature oscillator.
An electromagnetic oscillator or a vortex mixer.
A centrifuge: 3000 r/min is higher than the prior art.
A constant-temperature water bath: the temperature control range is 20-60 ℃ and the precision is 0.1 ℃.
A stopwatch: the error per hour does not exceed 5s.
A liquid transfer device: accuracy of 1
。
Visible spectrophotometer: can detect the absorbance range of 350-800 nm.
Reagents and solutions used in the following examples:
the reagents used in the invention are analytical pure reagents except for special instructions, and water is distilled water or deionized water (secondary water) which meets the requirements of GB/T6682.
(1) Disodium hydrogen phosphate solution with concentration of 0.1mol/L
14.196g of anhydrous disodium hydrogen phosphate (M = 141.96) was weighed, dissolved in water, and the volume was adjusted to 1L.
(2) Sodium dihydrogen phosphate solution with concentration of 0.1mol/L
24g of anhydrous sodium dihydrogen phosphate (M = 119.98) was weighed, dissolved in water, and the volume was set to 2L.
(3) Phosphate buffer A, concentration 0.1mol/L, pH6.0
Mixing the prepared disodium hydrogen phosphate solution and sodium dihydrogen phosphate solution according to a certain proportion, and adjusting the pH value to 6.0 by using the sodium dihydrogen phosphate solution if the pH value is higher than 6.0 after mixing; after mixing, the pH value is adjusted to 6.0 by using disodium hydrogen phosphate solution.
(4) Phosphate buffer B with concentration of 0.1mol/L and pH of 6.0, containing 0.1% Tween-20
The preparation method is the same as phosphate buffer solution A, and meanwhile, tween-20 needs to be added into the phosphate buffer solution B, and the addition amount of the Tween-20 is 1L of buffer solution and 1g of Tween-20.
(5) Substrate glucose solution with concentration of 1.0mol/L
Weighing 19.8g of dextrose monohydrate (or 18g of anhydrous glucose), adding water to dissolve, diluting to 100mL, standing at room temperature for more than 2h (or overnight at 4 ℃) after preparation, storing at 4 ℃, and prolonging the effective period to 3 days.
(6) Horse radish peroxidase solution with concentration of 0.5mg/mL
25.0mg of peroxidase (250U/mg) was weighed, dissolved in water precooled with ice, to a constant volume of 50mL, and the brown reagent bottle was stored in a refrigerator at 4 ℃ for an expiration period of 2 weeks.
(7) 4-aminoantipyrine solution with concentration of 4mg/mL
Weighing 0.4g 4-aminoantipyrine, adding water to dissolve, fixing the volume to 100mL, and storing a brown reagent bottle in a refrigerator at 4 ℃ for 2 weeks.
(8) Phenol solution at a concentration of 1.5mg/mL
Weighing 0.15g of redistilled phenol, dissolving in phosphate buffer solution A, fixing the volume to 100mL, and storing a brown reagent bottle in a refrigerator at 4 ℃ for 2 weeks.
(9) Sodium Dodecyl Sulfate (SDS) solution with concentration of 100g/L
About 10g of SDS was dissolved in about 80mL of distilled water, and the solution was dissolved by heating to 100mL. After the preparation is finished, the mixture is placed at normal temperature, and the validity period is 3 months.
Example 1 determination of glucose oxidase Activity
1. Preparation of enzyme solution for reaction
1.1 Preparation of enzyme solution for reaction of solid sample
Weighing 0.2g of sample, accurately weighing 0.001g of sample, placing the sample in a 100mL volumetric flask, adding about 40mL of phosphate buffer B, magnetically stirring or shaking by a constant temperature oscillator (200 r/min) for 30min at room temperature (20 ℃), taking out, fixing the volume by using the phosphate buffer B, and shaking up. And (3) centrifuging the shaken storage solution in a centrifuge at 4000r/min for 5min, and then taking the supernatant and properly diluting the supernatant by using a phosphate buffer solution B (the activity of glucose oxidase in the enzyme solution to be tested is controlled between 0.05U/mL and 0.20U/mL after dilution).
1.2 Preparation of enzyme solution for reaction of liquid sample
The liquid sample can be directly diluted by phosphate buffer solution B and subjected to constant volume (the activity of glucose oxidase in the enzyme solution to be detected after dilution is controlled between 0.05U/mL-0.20U/mL). If the pH of the diluted enzyme solution deviates from 6.0, the pH can be adjusted to 6.0 by using a disodium hydrogen phosphate solution or a sodium dihydrogen phosphate solution, and then the enzyme solution is appropriately diluted by using a phosphate buffer solution B to fix the volume.
It is recommended to add a reference sample of glucose oxidase with known activity to the sample to be tested, so as to test whether the whole operation process has deviation.
2. Measurement procedure
20.0mL of phosphate buffer solution A, 6.0mL of substrate glucose solution, 1.0mL of horseradish peroxidase solution, 1.0mL of 4-aminoantipyrine solution and 2.0mL of phenol solution are sucked and mixed uniformly to obtain a total of 30.0mL of the solutions, and the solution is put into a small test tube in a proper amount and is balanced in a water bath at 37 ℃ for 5min.
Preparing four test tubes (one sample blank tube and three sample tubes), respectively adding 0.1mL of appropriately diluted enzyme solution into the four test tubes, balancing in a 37 ℃ water bath for 1min, selecting one test tube from the four test tubes as the sample blank tube, adding 300 mu L of 100g/L SDS solution, and performing vortex oscillation for 3 to 5s. Then 3mL of equilibrated assay was added and the reaction was timed accurately at 37 ℃ for 10min. Respectively adding 3mL of balanced determination solution into the three sample tubes, accurately timing the reaction at 37 ℃ for 10min, and then quickly and accurately adding 300 mu L of 100g/L SDS solution into the three sample tubes to stop the enzymolysis reaction. The absorbance of the sample blank tube solution was measured at 500nm using a 10mm cuvette (A) with water adjusted to zero (zero) 0 ) And the absorbance (A) of the sample solution in the sample tube.
3. Calculation of results
Activity of glucose oxidase in sample dilution by X D Expressed in units of enzyme activity units (U/mL), calculated according to formula (I); the activity of glucose oxidase in the sample is expressed by X, and the unit is the unit of enzyme activity (U/g or U/mL), and is calculated according to the formula (II):
In the formula:
X D : the activity of the glucose oxidase in the sample to be detected is U/mL, and the unit is more than or equal to 0.05U/mL and less than or equal to X D ≤0.20U/mL；
A: average absorbance values of the three sample tubes;
A 0 : absorbance of the sample blank tube;
10: reaction time in min;
1: the thickness of the cuvette is in cm;
12.88: millimolar absorption coefficient in L.mmoL of quinone imine as chromogenic substance -1 ·cm -1 ；
2: h for each 1 mole of quinoneimine formed 2 O 2 The amount of the substance;
3.4: the total volume of the reaction mixed solution is mL;
0.1: the volume of the sample to be detected is mL;
X D the value should be between 0.05U/mL-0.20U/mL, if not, the dilution of the enzyme solution should be reselected and then the assay is performed.
In the formula:
x: the activity of the glucose oxidase in the sample is U/g or U/mL;
n: total dilution of the sample.
4. Repeatability of
The relative deviation of 2 parallel measured values of the same sample is not more than 8.0 percent, and the average value of the two values is the final enzyme activity measured value.
EXAMPLE 2 screening of terminator
The screening process is as follows: glucose oxidase catalyzes oxidation of glucose to generate hydrogen peroxide (H) 2 O 2 ），H 2 O 2 Under the catalysis of peroxidase, 4-aminoantipyrine and phenol can be oxidized and condensed to generate pink quinoneimine, the depth of the color of the reaction solution is in direct proportion to the amount of quinoneimine generated by the catalysis of peroxidase, and the amount of quinoneimine generated is in direct proportion to H 2 O 2 Proportional to the amount of production, H 2 O 2 The amount of the produced glucose oxidase is proportional to the activity of the glucose oxidase in the reaction solution. Therefore, the intensity of the color of the reaction solution can be measured by measuring the change of the absorbance at 500nm by a spectrophotometer, and the activity of the glucose oxidase in the reaction solution can be calculated.
The terminating agents for the enzyme preparation reaction are typically acid solutions, alkali solutions, and therefore, a 3M hydrochloric acid solution, a 1. Finally, 100g/L Sodium Dodecyl Sulfate (SDS) solution was selected as the terminator.
TABLE 2 screening of terminators
Example 3 Effect of reaction temperature on enzyme Activity detection accuracy
The enzyme activity of different glucose oxidase samples is respectively measured by a conventional spectrophotometry (namely a continuous spectrophotometry without adding a terminator and without temperature control measures) and the measuring method of the invention.
Conventional spectrophotometry: preheating the determination solution (see step 2 in example 1) and the enzyme solution to be determined (see step 1 in example 1) in a water bath kettle at 37 ℃, then transferring to a cuvette of a spectrophotometer to perform enzyme reaction, timing for 10min, selecting the absorbance difference between the 6 th min and the 2 nd min, and substituting into a formula to calculate. In the conventional determination method, since there is no constant temperature condition, the actual temperature of the enzyme reaction is far lower than 37 ℃, and the specific temperature has uncertainty, which is greatly influenced by the room temperature, and the lower the room temperature is, the lower the detection result is.
The enzyme reaction temperature of the method is constant at 37 ℃, the measured solution and the enzyme solution to be measured are reacted in a constant-temperature water bath kettle at 37 ℃, the reaction time is 10min, after the reaction is accurately timed, a terminator SDS solution is added to terminate the reaction, then a spectrophotometer is used to measure the OD value of a sample tube and the OD value of a blank tube, and the enzyme activity value is calculated (table 3).
TABLE 3 influence of reaction temperature on enzyme activity detection accuracy
As can be seen from Table 3, the enzyme activity detection value of the same glucose oxidase sample by using the method of the invention is 1.15-1.29 times of that of the conventional method, the reaction temperature is constant at 37 ℃, the enzyme activity is consistent with the definition of enzyme activity, and the temperature is closer to the actual body temperature of animals. The enzyme activity detection method has the advantages that the enzyme activity detection method can react at 37 ℃ in a constant-temperature water bath kettle, the reaction temperature is fixed, the influence of the ambient room temperature is avoided, the enzyme activity detection value is higher, and the enzyme activity detection result is more stable.
Example 4 comparison of three glucose oxidase detection methods
A comparison of the three glucose oxidase detection methods is shown in Table 4.
TABLE 4 comparison of three glucose oxidase detection methods
The glucose oxidase determination method is suitable for determining the activity of all glucose oxidases and has wide application range. For example, 10000U/g glucose oxidase samples produced by fermentation of specific penicillium, pichia pastoris strains, aspergillus niger and the like are used for detecting the enzyme activity by the method, and the enzyme activity detection values are all more than or equal to 10000U/g, so that the product label enzyme activity is achieved. DB 13/T1444-2011 is not suitable for determining the activity of glucose oxidase produced by pichia pastoris strains, for example, if 1 ten thousand glucose oxidase samples produced by pichia pastoris are detected, the enzyme activity value detected by adopting a DB 13/T1444-2011 titration method is 872U/g, and the enzyme activity value determined by adopting the method disclosed by the invention is 13833U/g. DB 13/T1444-2011 adopts an acid-base titration method to measure the activity of the glucose oxidase, has high requirements on the technical level of operators, and causes large experimental errors easily due to inaccurate titration end point judgment. The method for measuring the enzyme activity by using the spectrophotometry is simple to operate and has low requirements on the technical level of operators. The pre-treatment time of the DB 13/T1444-2011 sample is 15-16h, the pre-treatment time of the method is only about 30min, and the pre-treatment time of the sample is greatly shortened. Although part of enterprises adopt a spectrophotometry method to determine the activity of the glucose oxidase at present, the enzyme activity reaction of a sample is actually carried out in a cuvette of a spectrophotometer without temperature control measures, the reaction temperature is influenced by room temperature, and the reaction temperature directly influences the detection value of the enzyme activity. Within a certain range, the lower the reaction temperature is, the lower the enzyme activity detection value is. Moreover, the reaction time in the cuvette is 6min, the reaction time is too short, operation errors are easily caused, the reproducibility of the detection result is poor, and the enzyme activity cannot be reflected truly due to the too short reaction time.
The method adopts a spectrophotometry method to measure the activity of the glucose oxidase, has short pretreatment time, and only needs 35min (wherein a sample is dissolved for 30min and centrifuged for 5 min). The reaction is carried out in a water bath kettle, the reaction temperature is 37 ℃, the temperature is closer to the animal body temperature at 37 ℃, the reaction time is 10min, the reaction time is more reasonable, after the reaction timing is finished, a terminator SDS solution is added to stop the reaction, then the OD value is measured by a spectrophotometer, and the enzyme activity value is obtained by calculation. The reaction efficiency is high, and the enzyme reaction is carried out in a water bath, so that the detection of several enzymes can be carried out simultaneously. The precision of the detection method meets the requirement, and the detection result has good repeatability.
EXAMPLE 5 determination of the concentration Range of the test solutions
The enzyme sample to be detected is required to be diluted to 0.05U/mL-0.20U/mL by using the buffer solution, namely the activity of the glucose oxidase in the enzyme solution to be detected after dilution is controlled to be 0.05U/mL-0.20U/mL, and the enzyme activity is determined again after the dilution times are recommended to be adjusted.
Weighing and dissolving 2000U/g of a glucose oxidase sample, diluting the sample by different dilution times, and controlling the activity of the glucose oxidase in the enzyme solution to be detected to be 0.05-0.20U/mL, wherein the detection result is shown in Table 5:
TABLE 5 influence of different enzyme solution concentrations to be tested on enzyme activity
The result shows that the activity of the glucose oxidase in the enzyme solution to be detected is controlled to be 0.05U/mL-0.20U/mL, the variation coefficient is 2.05%, and the repeatability of the enzyme activity detection result is good.
Example 6 precision examination of the test methods
By adopting the method, 10000U/g of a glucose oxidase standard sample and 7 parallel tests are carried out by an experimenter on the same day, and the activity of the glucose oxidase is shown in table 6:
TABLE 6 results of enzyme activity of glucose oxidase in multiple assays
The method provided by the invention is adopted to detect the enzyme activity of the glucose oxidase, the coefficient of variation is 1.09%, and the repeatability is good. The detection precision is better than that of a control method 2 (DB 13/T1444-2011) and a control method 3 (a spectrophotometry method adopted by part of enterprises at present).
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (6)
1. The method for measuring the activity of the glucose oxidase is characterized by comprising the following steps of:
(1) Preparing a sample to be detected;
(2) Preparing four test tubes, wherein one test tube is a sample blank tube, and three test tubes are sample tubes; respectively adding 0.1mL of samples to be detected into the four test tubes, and balancing in a water bath at 37 ℃ for 1min; adding 300 mu L of 100g/L SDS solution into a sample blank tube, and carrying out vortex oscillation for 3 to 5s; then adding 3mL of balanced determination solution, and reacting for 10min at 37 ℃; respectively adding 3mL of balanced determination solution into the three sample tubes, reacting for 10min at 37 ℃, and then respectively adding 300 mu L of 100g/L SDS solution into the three sample tubes to terminate the reaction; adjusting to zero with water, and measuring the absorbance of a blank sample tube and the absorbance of a sample tube at 500nm by using a 10mm cuvette; calculating the activity of glucose oxidase in place of formula (I):
In the formula:
X D : the activity of the glucose oxidase in the sample to be detected is U/mL, and X is more than or equal to 0.05U/mL D ≤0.20U/mL；
A: average absorbance values of the three sample tubes;
A 0 : absorbance of a sample blank tube;
10: reaction time in min;
1: the thickness of the cuvette is in cm;
12.88: millimolar absorption coefficient in L.mmoL of quinone imine as chromogenic substance -1 ·cm -1 ；
2: h required for each 1 mole of quinoneimine formed 2 O 2 The amount of the substance;
3.4: the total volume of the reaction mixed solution is mL;
0.1: the volume of the sample to be detected is mL;
(3) Mixing X D Calculating the activity of the glucose oxidase in the sample by substituting formula (II):
In the formula:
x: the activity of the glucose oxidase in the sample is U/g or U/mL;
X D : the activity of the glucose oxidase in the sample to be detected is U/mL, and X is more than or equal to 0.05U/mL D ≤0.20U/mL；
n: total dilution of the sample.
2. The method according to claim 1, wherein the preparation method of the assay solution comprises: 20.0mL of phosphate buffer solution A, 6.0mL of substrate glucose solution, 1.0mL of horseradish peroxidase solution, 1.0mL of 4-aminoantipyrine solution and 2.0mL of phenol solution are sucked and mixed uniformly to obtain a total of 30.0mL of measuring solution, and the measuring solution is balanced in a water bath at 37 ℃ for 5min;
wherein the concentration of the phosphate buffer solution A is 0.1mol/L, and the pH value is 6.0;
the concentration of the substrate glucose solution is 1.0mol/L;
the concentration of the horseradish peroxidase solution is 0.5mg/mL;
the concentration of the 4-aminoantipyrine solution is 4mg/mL;
the concentration of the phenol solution is 1.5mg/mL, and the phenol solution is prepared by the phosphate buffer solution A.
3. The method according to claim 2, wherein the method for preparing the sample to be tested in step (1) comprises:
(a) For solid samples
Weighing 0.1-5.0 g of solid sample, accurately weighing the solid sample to 0.001g, placing the solid sample in a 100mL volumetric flask, adding 40mL of phosphate buffer solution B, stirring or shaking the solid sample for 30min at 20 ℃, then fixing the volume by using the phosphate buffer solution B and shaking the mixture evenly, centrifuging the mixture for 5min at 4000r/min, taking supernatant liquid and diluting the supernatant liquid by using the phosphate buffer solution B, and controlling the activity of glucose oxidase in a sample to be tested to be in the range of 0.05-0.20U/mL after dilution;
(b) For liquid samples
Directly diluting the liquid sample with a phosphate buffer solution B, fixing the volume, controlling the activity of glucose oxidase in the diluted sample to be detected within the range of 0.05U/mL-0.20U/mL, and controlling the pH to 6.0;
wherein the phosphate buffer solution B is a phosphate buffer solution A containing 0.1% of Tween-20.
4. The method of claim 3, wherein the sample is derived from a glucose oxidase preparation produced by fermentation with a species having glucose oxidase production capacity.
5. The method of claim 4, wherein the bacterial species is a specific enzyme(s) (QIAGEN)Penicillium notatum) Pichia pastoris (A), (B)Pichia pastoris) Or Aspergillus nigerAspergillus niger）。
6. The glucose oxidase activity determination kit is characterized by comprising a phosphate buffer solution A, a phosphate buffer solution B, a 1.0mol/L glucose solution, a 0.5mg/mL horseradish peroxidase solution, a 4mg/mL 4-aminoantipyrine solution, a 1.5mg/mL phenol solution and a 100g/L SDS solution;
wherein the concentration of the phosphate buffer solution A is 0.1mol/L, and the pH value is 6.0;
the phosphate buffer solution B is a phosphate buffer solution A containing 0.1% of Tween-20.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0121254A2 (en) * | 1983-04-02 | 1984-10-10 | Wako Pure Chemical Industries, Ltd. | Process for determining substrate or enzymatic activity |
| CN101655493A (en) * | 2008-08-20 | 2010-02-24 | 中国科学院成都有机化学有限公司 | Colorimetric analysis method for measuring content of glucose and activity of glucose oxidase |
| CN102382773A (en) * | 2011-10-26 | 2012-03-21 | 江南大学 | Aspergillus niger strain capable of producing glucose oxidase and application thereof |
| CN104237145A (en) * | 2014-08-19 | 2014-12-24 | 武汉新华扬生物股份有限公司 | Method for rapidly determining activity of glucose oxidase |
-
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- 2023-01-09 CN CN202310024360.4A patent/CN115791665B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0121254A2 (en) * | 1983-04-02 | 1984-10-10 | Wako Pure Chemical Industries, Ltd. | Process for determining substrate or enzymatic activity |
| CN101655493A (en) * | 2008-08-20 | 2010-02-24 | 中国科学院成都有机化学有限公司 | Colorimetric analysis method for measuring content of glucose and activity of glucose oxidase |
| CN102382773A (en) * | 2011-10-26 | 2012-03-21 | 江南大学 | Aspergillus niger strain capable of producing glucose oxidase and application thereof |
| CN104237145A (en) * | 2014-08-19 | 2014-12-24 | 武汉新华扬生物股份有限公司 | Method for rapidly determining activity of glucose oxidase |
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| 刘砚韬等: "过氧化氢酶活性测定的新方法" * |
| 李丕武等: "两种葡萄糖氧化酶活力测定方法的比较" * |
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