CN112111556B - Method for judging whether activity detection of glucoamylase is accurate or not - Google Patents
Method for judging whether activity detection of glucoamylase is accurate or not Download PDFInfo
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- 102100022624 Glucoamylase Human genes 0.000 title claims abstract description 88
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 title claims abstract description 87
- 230000000694 effects Effects 0.000 title claims abstract description 70
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000001514 detection method Methods 0.000 title claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 47
- 102000004190 Enzymes Human genes 0.000 claims abstract description 47
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 45
- 239000008103 glucose Substances 0.000 claims abstract description 45
- 238000010561 standard procedure Methods 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims description 57
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 45
- 238000006243 chemical reaction Methods 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 238000004448 titration Methods 0.000 claims description 17
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims description 13
- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 13
- 229920002472 Starch Polymers 0.000 claims description 12
- 235000019698 starch Nutrition 0.000 claims description 12
- 239000008107 starch Substances 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 12
- 238000001816 cooling Methods 0.000 claims description 10
- 238000006911 enzymatic reaction Methods 0.000 claims description 10
- 239000007974 sodium acetate buffer Substances 0.000 claims description 10
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 10
- 239000004382 Amylase Substances 0.000 claims description 6
- 102000013142 Amylases Human genes 0.000 claims description 6
- 108010065511 Amylases Proteins 0.000 claims description 6
- 235000019418 amylase Nutrition 0.000 claims description 6
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 229910052740 iodine Inorganic materials 0.000 claims description 5
- 239000011630 iodine Substances 0.000 claims description 5
- 239000012086 standard solution Substances 0.000 claims description 5
- 238000009776 industrial production Methods 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 238000005259 measurement Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 241000228245 Aspergillus niger Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 229960002160 maltose Drugs 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
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- C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
- G01N2333/934—Glucoamylase
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Abstract
The invention discloses a method for judging whether the enzyme activity detection of glucoamylase is accurate or not, which combines a national standard method with a glucose kit method, and divides the enzyme activities detected by the two methods to obtain a ratio, wherein the ratio is a fixed value; in the process of producing and applying the glucoamylase, two measuring methods are combined to obtain the enzyme activity ratio. When the activity of the glucoamylase is measured in industrial production, the accuracy of each enzyme activity detection can be judged according to the ratio; thereby meeting the requirement of accurately detecting the activity of the glucoamylase and overcoming the problem of overlarge error in the traditional method for detecting the activity of the glucoamylase.
Description
Technical Field
The invention discloses a method for judging whether the activity detection of glucoamylase is accurate or not, which utilizes a national standard method and a glucose kit method to measure the activity of the glucoamylase in a combined way, and belongs to the field of biochemical analysis.
Background
Glucoamylase (Glucoamylase EC 3.2.1.3) is also known as a saccharifying enzyme. Mainly exists in filamentous fungi and yeasts such as Aspergillus niger, aspergillus oryzae, rhizopus and the like, and also exists in human saliva, animal pancreas and bacteria. The reported fungal microorganisms producing glucoamylase have 23 species of 35 species and 3 species of bacteria, and the strains used for industrial production are mainly Aspergillus niger. Glucoamylase is an exo-glycosidase that hydrolyzes the alpha-1, 4 glycosidic bond sequentially from the non-reducing end of starch, mainly to glucose. The glucoamylase is widely applied to industrial production of alcohol, white spirit, antibiotics, amino acid, organic acid, glycerin, starch sugar and the like, and is one of enzyme preparation products with the largest yield in China.
At present, in the production process, the enzyme activity is generally detected by using the national standard method GB1886.174-2016 (hereinafter referred to as national standard method), but the national standard method adopts a titration method to detect the enzyme activity, the method has complicated operation, and the color change needs to be observed by naked eyes during titration, so that the error is larger. Different individuals measure the same glucoamylase and different enzyme activity results are obtained. Thus, the accuracy of the judgment result is not known. The glucose kit method is relatively accurate in measuring the activity of the glucoamylase, but has small errors. The cost of glucoamylase is a significant part of the cost of industrial production. Inaccurate enzyme activity detection can lead to a great increase in production cost.
When the detection is carried out by the national standard method, the detection indexes comprise reducing sugars such as maltose, maltobiose and the like. When the activity of the glucoamylase is measured by a glucose kit method, the detection index is glucose. For the same glucoamylase, the ratio of the activity of the glucoamylase measured by the national standard method to the activity measured by the glucose kit method is a fixed value, and the accuracy of the detection of the activity of the glucoamylase can be judged according to the ratio.
The invention aims to establish a national standard method and a glucose kit for combined use to obtain the ratio of the enzyme activities of the glucoamylase measured by the two methods, and the accuracy of enzyme activity detection is judged by using the ratio so as to guide the production and application of the glucoamylase.
Disclosure of Invention
The invention discloses a method for judging whether the enzyme activity detection of glucoamylase is accurate, which solves the problem of overlarge error in the traditional glucoamylase determination method and provides a basis for detecting the accuracy of the glucoamylase activity.
The invention relates to a method for judging whether the activity detection of glucoamylase is accurate or not, which is realized by the following technical scheme:
1) Determination of the enzyme Activity of glucoamylase Using national Standard method
(1) Taking 2 conical bottles, wherein one conical bottle is a sample bottle, the other conical bottle is a blank control bottle, adding 25mL of starch solution with mass concentration of 20g/L and 5mL of acetic acid-sodium acetate buffer with pH of 4.6 into each bottle respectively, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
(2) respectively adding 2mL of glucoamylase to be detected into the sample bottle, shaking uniformly, and carrying out water bath reaction for 30min at 40 ℃;
(3) adding 0.2mL of NaOH solution with the mass concentration of 200g/L into each bottle, shaking uniformly, rapidly cooling with water, and stopping the enzyme reaction; adding 2mL of glucose amylase to be detected into a blank control bottle;
(4) 5mL of the reaction solution respectively sucked from the sample bottle and the blank control bottle in the step (3) are added into an iodometric bottle, 5mL of iodine standard solution with the concentration of 0.1M is added, 15mL of NaOH solution with the concentration of 0.1M is added, the iodometric bottle is put into the dark for reaction for 15min, the iodometric bottle is taken out, and 2mL of H with the concentration of 2M is added 2 SO 4 Titration was performed with 0.05M sodium thiosulfate;
the enzyme activity X of the glucoamylase sample to be tested was calculated according to the following formula (1) 1 The unit is U/mL or U/g:
wherein:
a: when a blank control is titrated, the standard titration solution of sodium thiosulfate is consumed in volume, and the unit is mL;
b: when the titration sample is, the standard titration solution of consumed sodium thiosulfate is volume, and the unit is mL;
C 1 : accurate concentration of sodium thiosulfate standard titration solutionThe unit is mol/L;
n: dilution factor;
constant 90.05: molar mass of glucose equivalent to 1.00mL of sodium thiosulfate standard titration solution in g/mol (=90.05);
constant 32.2: the total volume of the reaction solution is in mL;
constant 5: sucking the volume of the reaction solution;
constant 1/2: an amount converted into 1mL of enzyme solution;
constant 2: reacting for 30min, and converting into an enzyme activity coefficient of 1 h;
2) Determination of the enzyme Activity of glucoamylase Using the glucose kit method
(1) Taking 2 test tubes, wherein one test tube is a sample tube, the other test tube is a blank control tube, respectively adding 2.5mL of starch solution with mass concentration of 20g/L and 0.5mL of acetic acid-sodium acetate buffer with pH of 4.6 into each test tube, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
(2) adding 0.2mL of glucoamylase to be detected into the sample tube respectively, shaking uniformly, and carrying out water bath reaction for 30min at 40 ℃;
(3) adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking uniformly, rapidly cooling with water, and stopping the enzyme reaction; adding 0.2mL of the glucose amylase to be detected into the blank control tube;
(4) the glucose content of the reaction solution was measured by using a glucose measuring kit, and the enzyme activity X of the glucoamylase sample to be measured was calculated according to the following formula (2) 2 The unit is U/mL or U/g:
wherein:
C glucose content : the unit of the glucose content calculated by the glucose kit is mmol/L;
n: dilution factor;
constant 180: glucose molar mass in g/mol;
constant 3.22: the total volume of the reaction solution is in mL;
constant 1/0.2, amount converted into 1mL enzyme solution;
constant 2: reacting for 30min, and converting into an enzyme activity coefficient of 1 h;
3) Calculating the enzyme activity ratio R of the glucoamylase measured by the national standard method and the glucose kit method according to the formula (3):
4) Judgment standard: the R value corresponds to the constant value for each glucoamylase. In practical application, the accuracy of the detection of the activity of the glucoamylase is judged by the R value.
The preparation of the reagent and the dilution of the enzyme liquid to be tested refer to national standard/food additive// saccharifying enzyme preparation (GB 1886.174-2016) of the people's republic of China, china standard Press.
The invention has the positive effects that:
the method for judging the accuracy of the detection of the activity of the glucoamylase is provided, a national standard method is combined with a glucose kit method, and the enzyme activities measured by the two methods are divided to obtain a ratio, wherein the ratio is a fixed value; in the process of producing and applying the glucoamylase, two measuring methods are combined to obtain an enzyme activity ratio; when the activity of the glucoamylase is measured in industrial production, the accuracy of each enzyme activity detection can be judged according to the ratio; thereby meeting the requirement of accurately detecting the activity of the glucoamylase and overcoming the problem of overlarge error in the traditional method for detecting the activity of the glucoamylase.
Detailed Description
The present invention will be described in detail with reference to examples, which are not intended to limit the scope of the invention.
Example 1
1) Determination of the Activity of Glucoamylase Using national Standard method
(1) Taking 3 sample bottles and 1 blank control bottle, respectively adding 25mL of starch solution with mass concentration of 20g/L and 5mL of acetic acid-sodium acetate buffer with pH of 4.6 into each bottle, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
(2) 2mL of the to-be-detected JieNeiaceae glucoamylase A is respectively added into the 3 sample bottles, and the mixture is uniformly shaken and subjected to water bath reaction at 40 ℃ for 30min;
(3) respectively adding 0.2mL of NaOH solution with the mass concentration of 200g/L into 3 sample bottles, shaking uniformly, rapidly cooling with water, and stopping enzyme reaction; 2mL of Jenergic glucoamylase A was added to a blank bottle;
(4) 5mL of the reaction solution respectively sucked from the sample bottle and the blank control bottle in the step (3) are added into an iodometric bottle, 5mL of iodine standard solution with the concentration of 0.1M is added, 15mL of NaOH solution with the concentration of 0.1M is added, the iodometric bottle is put into the dark for reaction for 15min, the iodometric bottle is taken out, and 2mL of H with the concentration of 2M is added 2 SO 4 Titration was performed with 0.05M sodium thiosulfate;
the enzyme activity X of the Glucoamylase of Jenergy A to be tested was calculated according to the following formula (1) 1 The unit is U/mL;
ten parallel experiments were performed on jie-able glucoamylase a, as described above, and averaged to obtain x1= 107576;
2) Enzyme activity determination of glucoamylase using glucose kit
(1) 2 test tubes were taken, one of which was a sample tube and the other was a blank control tube, and 2 were added to each tube. 5mL of starch solution with the mass concentration of 20g/L and 0.5mL of acetic acid-sodium acetate buffer solution with the pH of 4.6, shaking up, and preheating in a water bath at the temperature of 40 ℃ for 5-10 min;
(2) adding 0.2mL of the to-be-detected Jieable glucoamylase A into a sample tube respectively, shaking uniformly, and carrying out water bath reaction for 30min at 40 ℃;
(3) adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking uniformly, rapidly cooling with water, and stopping the enzyme reaction; adding 0.2mL of the to-be-detected Jie-Neurospora glucoamylase A into a blank control tube;
(4) the glucose content of the reaction solution was measured using a glucose measuring kit, and the enzyme activity X of the glucoamylase A of the Gemceae to be measured was calculated according to the following formula (2) 2 The unit is U/mL:
ten parallel experiments were performed on jie-able glucoamylase a, as described above, and averaged to obtain x2= 87450;
3) Calculating R value:
according to the formula (3), the ratio R of the enzyme activities of the glucoamylase by the national standard method and the glucose kit method is 1.23. In actual production, the activity of the glucoamylase A of the Gemceae family was measured and the R value was determined. If the R value is 1.23, the measurement is accurate. If the R value is not 1.23, the Jenergy glucoamylase A activity needs to be re-measured until the R value is 1.23, at which point the measured result is correct.
Example 2
1) Determination of the enzyme Activity of glucoamylase Using national Standard method
(1) Taking 3 sample bottles and 1 blank control bottle, respectively adding 25mL of starch solution with mass concentration of 20g/L and 5mL of pH4.6 acetic acid-sodium acetate buffer solution into each bottle, shaking up, and preheating in water bath at 40 ℃ for 5-10 min;
(2) 2mL of the Baistine glucoamylase to be detected is added into the 3 sample bottles respectively, and the mixture is uniformly shaken and subjected to water bath reaction at 40 ℃ for 30min;
(3) respectively adding 0.2mL of NaOH solution with the mass concentration of 200g/L into 3 sample bottles, shaking uniformly, rapidly cooling with water, and stopping enzyme reaction; 2mL of Pasteur glucoamylase was added to the blank bottle;
(4) respectively sucking out from the sample bottle and the blank control bottle in the step (3)Adding 5mL of the reaction solution into an iodometric bottle, adding 5mL of iodine standard solution with the concentration of 0.1M, adding 15mL of NaOH solution with the concentration of 0.1M, placing the iodometric bottle into the dark for reaction for 15min, taking out the iodometric bottle, and adding 2mL of H with the concentration of 2M 2 SO 4 Titration was performed with 0.05M sodium thiosulfate;
the enzyme activity X of the measured Pasteur glucoamylase was calculated according to the following formula (1) 1 The unit is U/mL:
ten parallel experiments were performed on the busjie glucoamylase as described above, and the average was taken to give x1= 129516;
2) Method for measuring activity of glucoamylase by utilizing glucose kit
(1) 2 test tubes were taken, one of which was a sample tube and the other was a blank control tube, and 2 were added to each tube. 5mL of starch solution with the mass concentration of 20g/L and 0.5mL of acetic acid-sodium acetate buffer solution with the pH of 4.6, shaking up, and preheating in a water bath at the temperature of 40 ℃ for 5-10 min;
(2) respectively adding 0.2mL of the to-be-detected Pasteur glucose amylase into the sample tube, shaking uniformly, and carrying out water bath reaction for 30min at 40 ℃;
(3) adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking uniformly, rapidly cooling with water, and stopping the enzyme reaction; adding 0.2mL of the Jie-Neurospora glucoamylase to be detected into a blank control tube;
(4) the glucose content of the reaction solution was measured using a glucose measuring kit, and the enzyme activity X of the measured Pasteur glucose amylase was calculated according to the following formula (2) 2 The unit is U/mL:
ten parallel experiments were performed on the busjie glucoamylase according to the above method, and the average was calculated to obtain x2=106480;
3) Calculating R value:
according to the formula (3), the ratio R of the enzyme activities of the glucoamylase by the national standard method and the glucose kit method is 1.22. In actual production, the activity of the Pasteur glucoamylase was measured and the R value was determined. If the R value is 1.22, this indicates that the measurement is accurate. If the R value is not 1.22, the re-measurement of the Pasteur glucoamylase activity is required until the R value is 1.22, at which point the measurement is correct.
Example 3
1) Determination of the Activity of Glucoamylase Using national Standard method
(1) Taking 3 sample bottles and 1 blank control bottle, respectively adding 25mL of starch solution with mass concentration of 20g/L and 5mL of pH4.6 acetic acid-sodium acetate buffer solution into each bottle, shaking up, and preheating in water bath at 40 ℃ for 5-10 min;
(2) adding 2mL of to-be-detected JieNeiaceae glucoamylase B into 3 sample bottles respectively, shaking uniformly, and carrying out water bath reaction for 30min at 40 ℃;
(3) respectively adding 0.2mL of NaOH solution with the mass concentration of 200g/L into 3 sample bottles, shaking uniformly, rapidly cooling with water, and stopping enzyme reaction; 2mL of Jenergic glucoamylase B was added to a blank bottle;
(4) 5mL of the reaction solution respectively sucked from the sample bottle and the blank control bottle in the step (3) are added into an iodometric bottle, 5mL of iodine standard solution with the concentration of 0.1M is added, 15mL of NaOH solution with the concentration of 0.1M is added, the iodometric bottle is put into the dark for reaction for 15min, the iodometric bottle is taken out, and 2mL of H with the concentration of 2M is added 2 SO 4 Titration was performed with 0.05M sodium thiosulfate;
the enzyme activity X of the test Jack B glucoamylase was calculated according to the following formula (1) 1 The unit is U/mL:
ten parallel experiments were performed on jie-able glucoamylase B as described above, and the average was taken to give x1= 102646;
2) Method for measuring activity of glucoamylase by utilizing glucose kit
(1) 2 test tubes were taken, one of which was a sample tube and the other was a blank control tube, and 2 were added to each tube. 5mL of starch solution with the mass concentration of 20g/L and 0.5mL of acetic acid-sodium acetate buffer solution with the pH of 4.6, shaking up, and preheating in a water bath at the temperature of 40 ℃ for 5-10 min;
(2) adding 0.2mL of to-be-detected JieNeiaceae glucoamylase B into the sample tube respectively, shaking uniformly, and carrying out water bath reaction for 30min at 40 ℃;
(3) adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking uniformly, rapidly cooling with water, and stopping the enzyme reaction; adding 0.2mL of the Jacobian B glucoamylase to be tested to a blank control tube;
(4) the glucose content of the reaction solution was measured using a glucose measuring kit, and the enzyme activity X of the Glucoaginaceae B glucoamylase to be measured was calculated according to the following formula (2) 2 The unit is U/mL:
ten parallel experiments were performed on jie-able glucoamylase B as described above, and the average was taken to give x2= 83452;
3) Calculating R value:
according to the formula (3), the ratio R of the enzyme activities of the glucoamylase by the national standard method and the glucose kit method is 1.23. In actual production, the activity of the glucoamylase B of the Gemceae family was measured and the R value was determined. If the R value is 1.23, the measurement is accurate. If the R value is not 1.23, the Jenergy glucoamylase B activity needs to be re-measured until the R value is 1.23, at which point the measured result is correct.
Claims (1)
1. The method for judging whether the activity detection of the glucoamylase is accurate is characterized by comprising the following steps:
1) Determination of the enzyme Activity of glucoamylase Using national Standard method
(1) Taking 2 conical bottles, wherein one conical bottle is a sample bottle, the other conical bottle is a blank control bottle, adding 25mL of starch solution with mass concentration of 20g/L and 5mL of acetic acid-sodium acetate buffer with pH of 4.6 into each bottle respectively, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
(2) respectively adding 2mL of glucoamylase to be detected into the sample bottle, shaking uniformly, and carrying out water bath reaction for 30min at 40 ℃;
(3) adding 0.2mL of NaOH solution with the mass concentration of 200g/L into each bottle, shaking uniformly, rapidly cooling with water, and stopping the enzyme reaction; adding 2mL of glucose amylase to be detected into a blank control bottle;
(4) 5mL of the reaction solution respectively sucked from the sample bottle and the blank control bottle in the step (3) are added into an iodometric bottle, 5mL of iodine standard solution with the concentration of 0.1M is added, 15mL of NaOH solution with the concentration of 0.1M is added, the iodometric bottle is put into the dark for reaction for 15min, the iodometric bottle is taken out, and 2mL of H with the concentration of 2M is added 2 SO 4 Titration was performed with 0.05M sodium thiosulfate;
the enzyme activity X of the glucoamylase sample to be tested was calculated according to the following formula (1) 1 The unit is U/mL or U/g:
wherein:
a: when a blank control is titrated, the standard titration solution of sodium thiosulfate is consumed in volume, and the unit is mL;
b: when the titration sample is, the standard titration solution of consumed sodium thiosulfate is volume, and the unit is mL;
C 1 : the accurate concentration of the sodium thiosulfate standard titration solution is expressed in mol/L;
n: dilution factor;
constant 90.05: molar mass of glucose equivalent to 1.00mL of sodium thiosulfate standard titration solution in g/mol (=90.05);
constant 32.2: the total volume of the reaction solution is in mL;
constant 5: sucking the volume of the reaction solution;
constant 1/2: an amount converted into 1mL of enzyme solution;
constant 2: reacting for 30min, and converting into an enzyme activity coefficient of 1 h;
2) Determination of the enzyme Activity of glucoamylase Using the glucose kit method
(1) Taking 2 test tubes, wherein one test tube is a sample tube, the other test tube is a blank control tube, respectively adding 2.5mL of starch solution with mass concentration of 20g/L and 0.5mL of acetic acid-sodium acetate buffer with pH of 4.6 into each test tube, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
(2) adding 0.2mL of glucoamylase to be detected into the sample tube respectively, shaking uniformly, and carrying out water bath reaction for 30min at 40 ℃;
(3) adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking uniformly, rapidly cooling with water, and stopping the enzyme reaction; adding 0.2mL of the glucose amylase to be detected into the blank control tube;
(4) the glucose content of the reaction solution was measured by using a glucose measuring kit, and the enzyme activity X of the glucoamylase sample to be measured was calculated according to the following formula (2) 2 The unit is U/mL or U/g:
wherein:
C glucose content : the unit of the glucose content calculated by the glucose kit is mmol/L;
n: dilution factor;
constant 180: glucose molar mass in g/mol;
constant 3.22: the total volume of the reaction solution is in mL;
constant 1/0.2, amount converted into 1mL enzyme solution;
constant 2: reacting for 30min, and converting into an enzyme activity coefficient of 1 h;
3) Calculating the enzyme activity ratio R of the glucoamylase measured by the national standard method and the glucose kit method according to the formula (3):
4) Judgment standard: the R value corresponds to a constant value for each glucoamylase; and judging the accuracy of the detection of the activity of the glucoamylase through the R value.
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| CN102286610A (en) * | 2011-07-16 | 2011-12-21 | 吉林大学 | Method for fast glucoamylase activity microdetection |
| CN103937766A (en) * | 2014-04-25 | 2014-07-23 | 南京百斯杰生物工程有限公司 | Recombination gene and method for increasing aspergillus niger expressed saccharifying enzyme |
| WO2018032797A1 (en) * | 2016-08-18 | 2018-02-22 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain and applications thereof |
| CN110018158A (en) * | 2019-04-23 | 2019-07-16 | 南京同凯兆业生物技术有限责任公司 | The detection method of amylase enzyme activity in a kind of Penicillium citrinum fermentation liquid |
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| CN103937766A (en) * | 2014-04-25 | 2014-07-23 | 南京百斯杰生物工程有限公司 | Recombination gene and method for increasing aspergillus niger expressed saccharifying enzyme |
| WO2018032797A1 (en) * | 2016-08-18 | 2018-02-22 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain and applications thereof |
| CN110018158A (en) * | 2019-04-23 | 2019-07-16 | 南京同凯兆业生物技术有限责任公司 | The detection method of amylase enzyme activity in a kind of Penicillium citrinum fermentation liquid |
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