CN112111556A - 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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- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 title claims abstract description 91
- 102100022624 Glucoamylase Human genes 0.000 title claims abstract description 90
- 230000000694 effects Effects 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 42
- 238000001514 detection method Methods 0.000 title claims abstract description 20
- 239000008103 glucose Substances 0.000 claims abstract description 44
- 108090000790 Enzymes Proteins 0.000 claims abstract description 42
- 102000004190 Enzymes Human genes 0.000 claims abstract description 42
- 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 42
- 238000010561 standard procedure Methods 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims description 59
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 238000012360 testing method Methods 0.000 claims description 25
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 20
- 229910052740 iodine Inorganic materials 0.000 claims description 20
- 239000011630 iodine Substances 0.000 claims description 20
- 238000004448 titration Methods 0.000 claims description 16
- 229920002472 Starch Polymers 0.000 claims description 12
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims description 12
- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 12
- 235000019698 starch Nutrition 0.000 claims description 12
- 239000008107 starch Substances 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
- 239000012295 chemical reaction liquid Substances 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 2
- 238000009776 industrial production Methods 0.000 abstract description 5
- 229940088598 enzyme Drugs 0.000 description 29
- 238000012935 Averaging Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 102000013142 Amylases Human genes 0.000 description 5
- 108010065511 Amylases Proteins 0.000 description 5
- 239000004382 Amylase Substances 0.000 description 4
- 235000019418 amylase Nutrition 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 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 2
- 241000228245 Aspergillus niger Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 244000140786 Brassica hirta Species 0.000 description 2
- 235000011371 Brassica hirta Nutrition 0.000 description 2
- 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 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 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
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000001014 amino acid Nutrition 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
- 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 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940111205 diastase Drugs 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
- -1 maltose disaccharide Chemical class 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 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
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- 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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- G—PHYSICS
- 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, combining a national standard method and a glucose kit method, and dividing enzyme activity measured 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 determination methods are combined to obtain the enzyme activity ratio. When the activity of the glucoamylase is measured in industrial production, the accuracy of enzyme activity detection of each time 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, which utilizes a national standard method and a glucose kit method to determine the activity of glucoamylase in a combined manner and belongs to the field of biochemical analysis.
Background
Glucoamylase (Glucoamylase EC3.2.1.3) is also known as Glucoamylase. It is mainly present in filamentous fungi such as Aspergillus niger, Aspergillus oryzae, Rhizopus, etc., and yeast, and also in human saliva, animal pancreas, and bacteria. There are 35 species of 23 genus glucoamylase-producing fungal microorganisms and 3 species of 3 genus bacteria, and the strain used for industrial production is mainly Aspergillus niger. Glucoamylase is an exo-glycosidase that hydrolyzes the alpha-1, 4 glycosidic bond from the non-reducing end of starch in sequence, producing primarily glucose. The glucoamylase is widely applied to industrial production of alcohol, white spirit, antibiotics, amino acids, organic acids, glycerol, starch sugar and the like, and is one of enzyme preparation products with the largest yield in China.
Currently, in the production process, enzyme activity detection is generally carried out by using a Chinese national standard method GB1886.174-2016 (hereinafter referred to as a national standard method), but the enzyme activity is detected by using a titration method in the national standard method, the method is complicated to operate, color change needs to be observed by naked eyes during titration, and the error is large. Different people can obtain different enzyme activity results by testing the same glucoamylase. Therefore, the accuracy of the determination result is not relied upon. The glucose kit method is relatively accurate in activity determination of the glucoamylase, but has a small error. The cost of glucoamylase represents a large part of the cost of industrial production. The enzyme activity detection is inaccurate, and the production cost is greatly increased.
When the detection is carried out by a national standard method, the detection indexes comprise reducing sugars such as maltose, maltose disaccharide and the like. When the glucose kit method is used for measuring the activity of the glucoamylase, 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 detection accuracy of the glucoamylase activity can be judged according to the ratio.
The invention aims to establish a national standard method and a glucose kit to be combined to obtain the ratio of the enzymatic activity of the glucoamylase measured by the two methods, and judge the accuracy of enzymatic activity detection by using the ratio 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 overcomes the problem of overlarge error of the traditional glucoamylase detection method and provides a basis for detecting the activity accuracy of glucoamylase.
The method for judging whether the activity detection of the glucoamylase is accurate is realized by the following technical scheme:
1) determination of enzyme activity of glucoamylase by national standard method
Taking 2 erlenmeyer flasks, wherein one erlenmeyer flask is a sample flask, the other erlenmeyer flask is a blank control flask, adding 25mL of starch solution with the mass concentration of 20g/L and 5mL of acetic acid-sodium acetate buffer solution with the pH value of 4.6 into each erlenmeyer flask, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
adding 2mL of glucoamylase to be detected into the sample bottles respectively, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.2mL of NaOH solution with the mass concentration of 200g/L into each bottle, shaking up, rapidly cooling by water, and terminating the enzyme reaction; adding 2mL of glucoamylase to be detected into the blank control bottle;
fourthly, respectively sucking 5mL of reaction liquid taken out from the sample bottle and the blank control bottle in the third step into an iodine measuring bottle, adding 5mL of iodine standard liquid with the concentration of 0.1M, adding 15mL of NaOH solution with the concentration of 0.1M, putting the iodine measuring bottle into the dark for reaction for 15min, taking out the iodine measuring bottle, adding 2mL of H with the concentration of 2M2SO4With 0.05M sodium thiosulfateTitration;
calculating the enzyme activity X of the glucoamylase sample to be tested according to the following formula (1)1The unit is U/mL or U/g:
in the formula:
a: when a blank control is titrated, the standard titration solution consuming sodium thiosulfate is volume, and the unit is mL;
b: when the titration sample is, the consumption of sodium thiosulfate standard titration solution is volume, and the unit is mL;
C1: the accurate concentration of the sodium thiosulfate standard titration solution is in mol/L;
n: dilution times;
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 mL;
constant 5: sucking the volume of the reaction solution;
constant 1/2: converting into 1mL enzyme solution;
constant 2: reacting for 30min, and converting into 1h of enzyme activity coefficient;
2) method for determining enzyme activity of glucoamylase by using glucose kit method
Taking 2 test tubes, wherein one test tube is a sample tube, the other test tube is a blank control tube, adding 2.5mL of starch solution with the mass concentration of 20g/L and 0.5mL of acetic acid-sodium acetate buffer solution with the pH value of 4.6 into each test tube, shaking up, and preheating in a 40 ℃ water bath for 5-10 min;
secondly, respectively adding 0.2mL of glucoamylase to be detected into the sample tubes, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking up, rapidly cooling by water, and terminating the enzyme reaction; adding 0.2mL of glucoamylase to be detected into the blank control tube;
fourthly, determining the glucose content of the reaction solution by using a glucose determination kit, and calculating the enzyme activity X of the glucoamylase sample to be determined according to the following formula (2)2The unit is U/mL or U/g:
in the formula:
Cglucose content: calculating the glucose content by using a glucose kit, wherein the unit is mmol/L;
n: dilution times;
constant 180: the molar mass of glucose is g/mol;
constant 3.22: the total volume of the reaction solution is mL;
the constant is 1/0.2, which is converted into the amount of 1mL enzyme solution;
constant 2: reacting for 30min, and converting into 1h of enzyme activity coefficient;
3) calculating the enzyme activity ratio R of the glucoamylase measured by a national standard method and a glucose kit method according to the formula (3):
4) and (4) judging the standard: the R-value is constant for each glucoamylase. In practical application, the detection accuracy of the glucoamylase activity is judged according to the R value.
For the preparation of the reagents and the dilution of the enzyme solution to be tested, refer to national standards of the people's republic of China/food additives// diastase preparations (GB 1886.174-2016), China Standard Press.
The invention has the positive effects that:
the method for judging the activity detection accuracy of the glucoamylase is provided, a national standard method and a glucose kit method are combined, the enzyme activity measured by the two methods is divided to obtain a ratio, and the ratio is a fixed value; in the process of producing and applying the glucoamylase, two determination methods are combined to obtain the enzyme activity ratio; when the activity of the glucoamylase is measured in industrial production, the accuracy of enzyme activity detection of each time 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 is described in detail below with reference to examples, but the present invention is not limited thereto.
Example 1
1) Determination of glucoamylase activity by national standard method
Taking 3 sample bottles and 1 blank reference bottle, respectively adding 25mL of starch solution with the mass concentration of 20g/L and 5mL of acetic acid-sodium acetate buffer solution with the pH value of 4.6 into each bottle, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
adding 2mL of glucose amylase A of the Jenecidae to be detected into 3 sample bottles respectively, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.2mL of NaOH solution with the mass concentration of 200g/L into 3 sample bottles, shaking up, rapidly cooling by water, and terminating the enzyme reaction; 2mL of Jennoke glucoamylase A was added to the blank control bottle;
fourthly, respectively sucking 5mL of reaction liquid from the sample bottle and the blank control bottle in the third step into an iodine measuring bottle, adding 5mL of iodine standard liquid with the concentration of 0.1M, then adding 15mL of NaOH solution with the concentration of 0.1M, putting the iodine measuring bottle into the dark for reaction for 15min, taking out the iodine measuring bottle, adding 2mL of H with the concentration of 2M2SO4Titration with 0.05M sodium thiosulfate;
calculating the enzyme activity X of the Jencaceae A glucoamylase to be detected according to the following formula (1)1The unit is U/mL;
performing ten parallel experiments on the Jenecaceae glucoamylase A according to the method, and averaging to obtain X1= 107576;
2) method for measuring enzyme activity of glucoamylase by using glucose kit
Taking 2 test tubes, wherein one test tube is a sample tube, and the other test tube is a blank control tube, and adding 2 test tubes into each test 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 value of 4.6 are shaken up and preheated in water bath at the temperature of 40 ℃ for 5-10 min;
② adding 0.2mL of glucose amylase A of the Jefferidae to be detected into the sample tube respectively, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking up, rapidly cooling by water, and terminating the enzyme reaction; adding 0.2mL of glucoamylase A of the Jennergaceae to be detected into the blank control tube;
fourthly, the glucose content of the reaction solution is determined by using a glucose determination kit, and the enzyme activity X of the glucose amylase A of the Jenecaceae to be detected is calculated according to the following formula (2)2The unit is U/mL:
performing ten parallel experiments on the Jenecaceae glucoamylase A according to the method, and averaging to obtain X2= 87450;
3) calculating the R value:
calculated according to the formula (3), the enzyme activity ratio R of the glucoamylase prepared by the national standard method and the glucose kit method is 1.23. In actual production, the vitality of the Jencaceae glucoamylase A is determined, and the R value is determined. If the R value is 1.23, the measurement is accurate. If the R value is not 1.23, the Jergo glucoamylase A activity needs to be retested until the R value is 1.23, at which point the results are correct.
Example 2
1) Determination of enzyme activity of glucoamylase by national standard method
Taking 3 sample bottles and 1 blank reference bottle, respectively adding 25mL of starch solution with the mass concentration of 20g/L and 5mL of acetic acid-sodium acetate buffer solution with the pH value of 4.6 into each bottle, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
adding 2mL of the Pasteur glucoamylase to be detected into 3 sample bottles respectively, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.2mL of NaOH solution with the mass concentration of 200g/L into 3 sample bottles, shaking up, rapidly cooling by water, and terminating the enzyme reaction; add 2mL of Pasteur glucoamylase to the blank control bottle;
fourthly, respectively sucking 5mL of reaction liquid from the sample bottle and the blank control bottle in the third step into an iodine measuring bottle, adding 5mL of iodine standard liquid with the concentration of 0.1M, then adding 15mL of NaOH solution with the concentration of 0.1M, putting the iodine measuring bottle into the dark for reaction for 15min, taking out the iodine measuring bottle, adding 2mL of H with the concentration of 2M2SO4Titration with 0.05M sodium thiosulfate;
calculating the enzyme activity X of the Besturgeon glucoamylase to be detected according to the following formula (1)1The unit is U/mL:
performing ten parallel experiments on the Baijie glucoamylase according to the method, and averaging to obtain X1= 129516;
2) method for measuring activity of glucoamylase by using glucose kit
Taking 2 test tubes, wherein one test tube is a sample tube, and the other test tube is a blank control tube, and adding 2 test tubes into each test 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 value of 4.6 are shaken up and preheated for 5-10 min in a water bath at 40 ℃;
adding 0.2mL of the Bestev glucoamylase to be detected into the sample tubes respectively, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking up, rapidly cooling by water, and terminating the enzyme reaction; adding 0.2mL of glucoamylase of the Jenecaceae to be detected into the blank control tube;
fourthly, the glucose content of the reaction solution is determined by utilizing a glucose determination kit, and the enzyme activity X of the Pasteur glucoamylase to be determined is calculated according to the following formula (2)2The unit is U/mL:
making ten parallel experiments on Baijie glucoamylase according to the method, and averaging to obtain X2= 106480;
3) calculating the R value:
the glucoamylase enzyme activity ratio R of the national standard method and the glucose kit method is 1.22 according to the calculation of the formula (3). In actual production, the activity of the Pasteur glucoamylase is measured, and the R value is determined. If the R value is 1.22, the measurement is accurate. If the R value is not 1.22, the Pasteur glucoamylase activity needs to be retested until the R value is 1.22, at which point the test results are correct.
Example 3
1) Determination of glucoamylase activity by national standard method
Taking 3 sample bottles and 1 blank reference bottle, respectively adding 25mL of starch solution with the mass concentration of 20g/L and 5mL of acetic acid-sodium acetate buffer solution with the pH value of 4.6 into each bottle, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
adding 2mL of Jennonidae glucoamylase B to be detected into 3 sample bottles respectively, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.2mL of NaOH solution with the mass concentration of 200g/L into 3 sample bottles, shaking up, rapidly cooling by water, and terminating the enzyme reaction; 2mL of Jenenaceae glucoamylase B was added to the blank control bottle;
fourthly, respectively sucking 5mL of reaction liquid from the sample bottle and the blank control bottle in the third step into an iodine measuring bottle, adding 5mL of iodine standard liquid with the concentration of 0.1M, then adding 15mL of NaOH solution with the concentration of 0.1M, putting the iodine measuring bottle into the dark for reaction for 15min, taking out the iodine measuring bottle, adding 2mL of H with the concentration of 2M2SO4Titration with 0.05M sodium thiosulfate;
calculating the enzyme activity X of the glucoamylase B of the Jencaceae to be detected according to the following formula (1)1The unit is U/mL:
making ten parallel experiments on Jenengke glucoamylase B according to the method, and averaging to obtain X1= 102646;
2) method for measuring activity of glucoamylase by using glucose kit
Taking 2 test tubes, wherein one test tube is a sample tube, and the other test tube is a blank control tube, and adding 2 test tubes into each test 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 value of 4.6 are shaken up and preheated in water bath at the temperature of 40 ℃ for 5-10 min;
adding 0.2mL of glucose amylase B to be detected in the Jenconidae into the sample tubes respectively, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking up, rapidly cooling by water, and terminating the enzyme reaction; adding 0.2mL of Jenconidae B glucoamylase to be detected into the blank control tube;
fourthly, the glucose content of the reaction solution is determined by utilizing a glucose determination kit, and the enzyme activity X of the glucoamylase B of the family Gejieke to be detected is calculated according to the following formula (2)2The unit is U/mL:
making ten parallel experiments on Jenengke glucoamylase B according to the method, and averaging to obtain X2= 83452;
3) calculating the R value:
calculated according to the formula (3), the enzyme activity ratio R of the glucoamylase prepared by the national standard method and the glucose kit method is 1.23. In actual production, the vitality of the Jencaceae glucoamylase B is determined, and the R value is calculated. If the R value is 1.23, the measurement is accurate. If the R value is not 1.23, the Jergo glucoamylase B activity needs to be retested until the R value is 1.23, at which point the results are correct.
Claims (1)
1. A method for judging whether the activity detection of glucoamylase is accurate or not is characterized by comprising the following steps:
1) determination of enzyme activity of glucoamylase by national standard method
Taking 2 erlenmeyer flasks, wherein one erlenmeyer flask is a sample flask, the other erlenmeyer flask is a blank control flask, adding 25mL of starch solution with the mass concentration of 20g/L and 5mL of acetic acid-sodium acetate buffer solution with the pH value of 4.6 into each erlenmeyer flask, shaking up, and preheating in a water bath at 40 ℃ for 5-10 min;
adding 2mL of glucoamylase to be detected into the sample bottles respectively, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.2mL of NaOH solution with the mass concentration of 200g/L into each bottle, shaking up, rapidly cooling by water, and terminating the enzyme reaction; adding 2mL of glucoamylase to be detected into the blank control bottle;
fourthly, respectively sucking 5mL of reaction liquid taken out from the sample bottle and the blank control bottle in the third step into an iodine measuring bottle, adding 5mL of iodine standard liquid with the concentration of 0.1M, adding 15mL of NaOH solution with the concentration of 0.1M, putting the iodine measuring bottle into the dark for reaction for 15min, taking out the iodine measuring bottle, adding 2mL of H with the concentration of 2M2SO4Titration with 0.05M sodium thiosulfate;
according to the followingCalculating the enzyme activity X of the glucoamylase sample to be detected by the formula (1)1The unit is U/mL or U/g:
in the formula:
a: when a blank control is titrated, the standard titration solution consuming sodium thiosulfate is volume, and the unit is mL;
b: when the titration sample is, the consumption of sodium thiosulfate standard titration solution is volume, and the unit is mL;
C1: the accurate concentration of the sodium thiosulfate standard titration solution is in mol/L;
n: dilution times;
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 mL;
constant 5: sucking the volume of the reaction solution;
constant 1/2: converting into 1mL enzyme solution;
constant 2: reacting for 30min, and converting into 1h of enzyme activity coefficient;
2) method for determining enzyme activity of glucoamylase by using glucose kit method
Taking 2 test tubes, wherein one test tube is a sample tube, the other test tube is a blank control tube, adding 2.5mL of starch solution with the mass concentration of 20g/L and 0.5mL of acetic acid-sodium acetate buffer solution with the pH value of 4.6 into each test tube, shaking up, and preheating in a 40 ℃ water bath for 5-10 min;
secondly, respectively adding 0.2mL of glucoamylase to be detected into the sample tubes, shaking up, and carrying out water bath reaction at 40 ℃ for 30 min;
③ respectively adding 0.02mL of NaOH solution with the mass concentration of 200g/L into each tube, shaking up, rapidly cooling by water, and terminating the enzyme reaction; adding 0.2mL of glucoamylase to be detected into the blank control tube;
fourthly, determining the glucose content and the glucose radical of the reaction solution by utilizing a glucose determination kitCalculating the enzyme activity X of the glucoamylase sample to be tested according to the following formula (2)2The unit is U/mL or U/g:
in the formula:
Cglucose content: calculating the glucose content by using a glucose kit, wherein the unit is mmol/L;
n: dilution times;
constant 180: the molar mass of glucose is g/mol;
constant 3.22: the total volume of the reaction solution is mL;
the constant is 1/0.2, which is converted into the amount of 1mL enzyme solution;
constant 2: reacting for 30min, and converting into 1h of enzyme activity coefficient;
3) calculating the enzyme activity ratio R of the glucoamylase measured by a national standard method and a glucose kit method according to the formula (3):
4) and (4) judging the standard: the R value is constant for each glucoamylase; and judging the detection accuracy of the glucoamylase activity 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 |
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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 |
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