Definition of the activity of glucose oxidase: the amount of the enzyme that catalyzes the conversion of 1 umol of glucose to gluconic acid per minute at a pH of 5.6, a temperature of 35 °C, and oxygenation, is an enzyme activity unit, expressed as Titr.
Glucose oxidase method principle: Glucose oxidase is an aerobic dehydrogenase that catalyzes the oxidation of glucose to generate gluconic acid. Neutralize the produced gluconic acid with excess sodium hydroxide, and then use the hydrochloric acid back titration method to obtain the output of gluconic acid, and the enzyme activity level is expressed according to the amount of gluconic acid produced.
(1) Preparation of sample diluent
Glucose oxidase method: first weigh 1g of the sample (accurate to 0.0001g) and place it in a mortar, add a small amount of phosphate buffer and grind for 10 minutes (grind until there are no large particles), transfer it into a 100ml volumetric flask, and grind the mortar with phosphate buffer. . Make sure that all the samples are transferred to the volumetric flask, make up to volume, and shake well (to control the concentration of the enzyme solution to about 3U/ml). The sample solution to be tested is measured within three hours after adding phosphate buffer.
(2) Operation steps
Sample determination: Take 25.00ml of 2% glucose phosphate buffer solution and put it in a 250ml conical flask, put it in a water bath to preheat at 35°C for 5 minutes, add 3.00ml of the above measurement solution accurately, immediately put it in a constant temperature water bath, and connect to constant pressure. Oxygen pipe (nozzle into the liquid surface). Wait for it to react for 20 minutes, take it out (accurate timing), take out the oxygen tube, rinse the mouth of the tube with a small amount of water (about 5ml), and immediately add 50.00ml of 0.1mol/L sodium hydroxide solution accurately, shake to stop the reaction, but the reaction solution Transfer to a 300ml beaker, wash the conical flask twice with a small amount of CO2-free distilled water (about 10-20ml), pour all the cleaning solution into the beaker, and shake it well with magnetic stirring (do not stir with other materials, such as a glass rod), use 0.1 The mol/L hydrochloric acid was titrated to the pH value of the solution to 8.0 (measured accurately by pH), and the number of milliliters of hydrochloric acid consumed was recorded.
Blank control: accurately pipette 50.00ml of 0.1mol/L sodium hydroxide solution into a 300ml beaker, accurately add 25.00ml of phosphate buffer solution and 3.00ml of enzyme solution, stir well with magnetic stirring and titrate its pH value with 0.1mol/L hydrochloric acid to 8.0 (accurately measured by PH), record the number of milliliters of hydrochloric acid consumed. The blank assay does not add substrate, oxygen, or heating.
(3) Enzyme activity determination and calculation
Glucose oxidase activity unit (Titr, u/g)=△V×N×f×1000×3/(20×3×m)
△V: volume difference of consumed HCL between sample and blank
N: concentration of HCl used for titration
f: dilution factor (dilution factor)
1000: Conversion factor between micromoles and moles
3: The amount of glucose added (umol)
20: is the reaction time, in minutes
3: Volume of enzyme solution added
m: the mass of the sample
(1) The amount of sodium hydroxide added each time is critical to the accuracy of the data. The amount added each time must be exactly the same (with a large belly pipette), and the concentration of sodium hydroxide added to the blank sample and the sample for each test is the same.
(2) The difference between the blank control of each test and the test time of the sample is not more than 2 hours.
(3) After the sample terminates the reaction, the reaction solution is titrated within 20 minutes, and the blank control solution is titrated within 20 minutes of adding the enzyme solution.
(4) Since the enzyme solution will settle during the stationary process, it must be shaken before each use to ensure that the enzyme solution is evenly mixed.
(5) Glass instruments used in the determination of glucose oxidase should be cleaned with detergent, rinsed with distilled water, and dried before use.
(6) The oxygen nozzle is kept below the liquid surface to ensure that the oxygen is in contact with the reaction liquid.
In order to ensure the accuracy and scientificity of the data, the experiment was repeated 3 times, and the average value of the data with the parallel relative range of less than 5% was taken as the calculated data respectively (blank, sample). If there is no data with a relative range of less than 5%, it must be re-measured.
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