CN101819138A - Measuring method of activity of beta-glucanase - Google Patents
Measuring method of activity of beta-glucanase Download PDFInfo
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- CN101819138A CN101819138A CN201010144060A CN201010144060A CN101819138A CN 101819138 A CN101819138 A CN 101819138A CN 201010144060 A CN201010144060 A CN 201010144060A CN 201010144060 A CN201010144060 A CN 201010144060A CN 101819138 A CN101819138 A CN 101819138A
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- 238000000034 method Methods 0.000 title claims abstract description 51
- 101710130006 Beta-glucanase Proteins 0.000 title claims abstract description 42
- 230000000694 effects Effects 0.000 title claims abstract description 28
- 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 50
- 239000008103 glucose Substances 0.000 claims abstract description 50
- 238000002835 absorbance Methods 0.000 claims abstract description 30
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 claims abstract description 26
- 229920002498 Beta-glucan Polymers 0.000 claims abstract description 26
- -1 potassium ferricyanide Chemical compound 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 29
- PHOLIFLKGONSGY-CSKARUKUSA-N (e)-(3-methyl-1,3-benzothiazol-2-ylidene)hydrazine Chemical compound C1=CC=C2S\C(=N\N)N(C)C2=C1 PHOLIFLKGONSGY-CSKARUKUSA-N 0.000 claims description 26
- 102000004190 Enzymes Human genes 0.000 claims description 25
- 108090000790 Enzymes Proteins 0.000 claims description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- 239000003153 chemical reaction reagent Substances 0.000 claims description 23
- XGGLLRJQCZROSE-UHFFFAOYSA-K ammonium iron(iii) sulfate Chemical compound [NH4+].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O XGGLLRJQCZROSE-UHFFFAOYSA-K 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 238000006460 hydrolysis reaction Methods 0.000 claims description 10
- 239000012086 standard solution Substances 0.000 claims description 10
- 238000003556 assay Methods 0.000 claims description 9
- 230000007062 hydrolysis Effects 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 4
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 4
- IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid Chemical compound NS(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 239000001384 succinic acid Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- OEZPVSPULCMUQB-VRTOBVRTSA-N hydron;(e)-(3-methyl-1,3-benzothiazol-2-ylidene)hydrazine;chloride Chemical compound Cl.C1=CC=C2S\C(=N\N)N(C)C2=C1 OEZPVSPULCMUQB-VRTOBVRTSA-N 0.000 abstract 3
- 239000002253 acid Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000000758 substrate Substances 0.000 abstract 1
- 239000000872 buffer Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
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- 230000035484 reaction time Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 235000007319 Avena orientalis Nutrition 0.000 description 1
- 244000075850 Avena orientalis Species 0.000 description 1
- 235000007558 Avena sp Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 235000007238 Secale cereale Nutrition 0.000 description 1
- 244000082988 Secale cereale Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 125000000218 acetic acid group Chemical class C(C)(=O)* 0.000 description 1
- 108010050181 aleurone Proteins 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000012817 gel-diffusion technique Methods 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
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a measuring method of the activity of beta-glucanase. The method comprises the following steps of: (A) manufacturing a standard correspondence curve of a glucose absorbance value measured by using an MBTH (3-methyl-2-benzothiazolinone hydrazone HCl) method and a corresponding concentration thereof; and (B) detecting an absorbance value equivalent to the glucose generated after the beta-glucanase to be measured hydrolyzes beta-glucan by using the MBTH method; and determining the content of reducing sugar equivalent to the glucose generated within a unit time in an enzymolysis product of the beta-glucanase to be measured according to the standard correspondence curve, which is manufactured in the step (A), between the glucose concentration and the measured corresponding absorbance value so as to calculate the activity of the beta-glucanase. The detection limit and the working concentration range for measuring the glucose in the method are both obviously lower than that of a DNS (3'5-dinitrosalicylic acid) method, a potassium ferricyanide method and a Somogyi-Nelson method, thus the sensitivity of the method is obviously higher than that of the three methods. Moreover, in the method, the dosage of the beta-glucan used as a substrate is greatly reduced, thus the experiment cost can be greatly reduced.
Description
Technical field
What the present invention relates to is a kind of method of measuring activity of beta-glucanase, is specifically related to relate in particular to the method for MBTH method mensuration activity of beta-glucanase the selection of key parameter in the mensuration process.
Background technology
Beta glucan is the non-starch based polysaccharide of a class, it is by β-1, and 3-glucoside bond and/or β-1, the chain polysaccharide that the 4-glucoside bond is formed, be the constituent of plant cell wall, be present in the aleurone and albuminous cell wall of cereal class (comprising barley, oat, rye and wheat etc.).1,4 beta-glucanase has important hydrolytic action to the glycosidic bond in the beta glucan.At present, this enzyme has been widely used in industries such as beer, feed, weaving, papermaking, medicine.The detection method of this enzyme activity has viscosimetry, fluorescence method, chromogenic substrate method, gel diffusion method, reducing sugar test method etc., and wherein the method for normal employing is DNS method, potassium ferricyanide method and a Somogyi-nelson method in the reducing sugar test method.Because these three kinds of method sensitivity are lower, be difficult to measure the initial velocity of enzyme digestion reaction, therefore, accuracy is relatively poor.
The present invention is the vigor that developer is surveyed 1,4 beta-glucanase with MBTH (3-methyl-2-[4-morpholinodithio ketone hydrazone) reagent.This method is not subjected to the interference of low-concentration acetic acid salt and succinate damping fluid, and gained enzyme concentration curve and enzyme digestion reaction speed are linear dependence in the enzyme concentration scope of broad.
Summary of the invention
At the deficiency of prior art, the invention provides a kind of assay method of activity of beta-glucanase, this method adopts 3-methyl-2-[4-morpholinodithio ketone hydrazone (MBTH) method to measure the vigor of 1,4 beta-glucanase.
Below introduce the assay method of a kind of activity of beta-glucanase of the present invention in detail, A makes the glucose absorbance that records with the MBTH method and the standard corresponding relation curve of its respective concentration; B detects the absorbance that is equivalent to glucose that produces behind the beta glucan enzyme hydrolysis beta glucan to be measured with the MBTH method; Determine the content of the reducing sugar that is equivalent to glucose that in the unit interval, produced in the enzymolysis product of 1,4 beta-glucanase to be measured according to the standard corresponding relation curve between rapid concentration of glucose of making of previous step and the corresponding absorbance that records, calculate the vigor of 1,4 beta-glucanase with this.
Optimally; The concrete steps of above-mentioned steps A are; Glucose absorbance and its respective concentration standard corresponding relation curve that effect MBTH method records; Get the glucose standard solution 0.6mL of 6-15 group variable concentrations (0-30 μ g/mL) respectively, the NaOH solution that adds the 0.6mL0.5 equivalent, mixing, add MBTH reagent 0.6mL again and add ammonium ferric sulfate reagent 1.2mL while hot behind 75-85 ℃ of water-bath heating 5-20min, the absorbance of glucose is measured in room temperature cooling back in wavelength 620-680nm place; Each concentration is made three parallel samples; Above dosage can increase and decrease in proportion; According to concerning drawing standard corresponding relation curve between every group of concentration of glucose and the corresponding absorbance that records;
Optimally; The concrete steps of above-mentioned steps B are; Detect the vigor of 1,4 beta-glucanase with the MBTH method; With concentration is the beta glucan solution of measuring temperature that is preheated to of 1-5mg/mL, be added to be preheated in the beta glucan enzyme solutions of measuring temperature and carry out enzyme digestion reaction, measuring temperature is 25-40 ℃, hydrolysis time is in the 60min, get hydrolyzate rapidly with the abundant mixing of NaOH solution of isopyknic 0.5 equivalent with cessation reaction (can in course of reaction time segment sampling detect), getting 1.2mL joins in the test tube and (makees three parallel samples), add MBTH reagent 0.6mL again and behind 75-85 ℃ of water-bath heating 5-20min, add ammonium ferric sulfate reagent 1.2mL while hot, absorbance is measured in room temperature cooling back in wavelength 620-680nm place, above dosage can increase and decrease in proportion.Determine the vigor of 1,4 beta-glucanase to be measured according to the standard corresponding relation curve between the concentration of the rapid glucose of making of previous step and the corresponding absorbance that records; The unit of activity of 1,4 beta-glucanase can be defined as under these conditions, and in every milliliter of reaction system, it is an enzyme activity unit that per minute generation 1nmol is equivalent to the required enzyme amount of glucose.Wherein the method for making of MBTH reagent is that the 3-methyl-2-[4-morpholinodithio ketone hydrogencarbonate aqueous solution of 3mg/mL and the dithiothreitol (DTT) solution equal-volume of 1mg/mL mix promptly, and matching while using is effective in one day.
Optimally; The method for making of above-mentioned ammonium ferric sulfate reagent is 0.5% (FeNH
4(SO
4)
2) 12H
2O, 0.5% sulfaminic acid, 0.5 equivalent hydrochloric acid.
Optimally; Above-mentioned beta glucan is a solvent with succinic acid buffer solution or the hac buffer of 50mM.
Optimally; The optimal wavelength of said determination absorbance is 650nm.
The quantity of the advantage of the present invention glucose end group that to be the present invention produced after to beta glucan enzyme hydrolysis beta glucan with the MBTH method detects the vigor of determining 1,4 beta-glucanase with this; Glucose and oligomeric beta glucan are the products of beta glucan enzyme hydrolysis, and therefore the sensitivity that the glucose endgroup content is detected is directly determining the sensitivity to the activity of beta-glucanase detection.This method is easier, quick, accurate, inexpensive than turbidimetry.Detectability and working concentration scope all are starkly lower than DNS method and potassium ferricyanide method, so the sensitivity of this method is apparently higher than DNS method and potassium ferricyanide method.
Embodiment
Embodiment 1:
Key instrument: constant temperature water bath oscillator, SHZ-82 type, state China Electrical Appliances Co., Ltd; Electric-heated thermostatic water bath, HH-4 type, state China Electrical Appliances Co., Ltd; Digital ph, Mettler Toledo Inc.; The UV2100 ultraviolet spectrophotometer, Shanghai UNICO Instr Ltd.; 1,4 beta-glucanase, Ningxia jade of the He family Bioisystech Co., Ltd; Beta glucan, hundred special pure big molecule Science and Technology Ltd.s; Pipettor, Finnpipette Finland thunder is vigorous.
Solution allocation
MBTH develop the color the 3-methyl-2-[4-morpholinodithio ketone hydrogencarbonate aqueous solution of liquid: 3mg/mL and 1mg/mL dithiothreitol (DTT) solution mixed in equal amounts promptly, matching while using, in one day effectively.
Glucose standard solution (30 μ g/mL): accurately take by weighing the glucose 0.3000g that is dried to permanent quality, with the dissolving of the hac buffer of 50mM pH5.5, shift and be settled to 100mL, be made into the glucose standard solution of 3.00mg/mL; Therefrom get the 1mL glucose solution, shift and be settled to 100mL, promptly get the glucose standard solution of 30 μ g/mL.
The method for making of ammonium ferric sulfate reagent is 0.5% (FeNH
4(SO
4)
2) 12H
2O, 0.5% sulfaminic acid, 0.5 equivalent hydrochloric acid.
Steps A is made the glucose absorbance that records with the MBTH method and the standard corresponding relation curve of its respective concentration;
The glucose standard solution 0.5mL that adds 8 groups of variable concentrations respectively, the NaOH solution that adds the 0.5mL0.5 equivalent, mixing, add MBTH reagent 0.5mL again and add ammonium ferric sulfate reagent 1mL while hot behind 80 ℃ of water-bath heating 11-13min, the absorbance of glucose is measured in room temperature cooling back in wavelength 650nm place; Each concentration is made three parallel samples; According to concerning drawing standard corresponding relation curve between every group of glucose solution concentration and the corresponding absorbance that records;
B is with the activity of beta-glucanase of MBTH method detection;
With its concentration of 5mL beta glucan solution is 2mg/mL, the pH value is 5.5, join in the conical flask preheating 10min in 30 ℃ shaking bath, add the beta glucan enzyme solutions 5mL to be measured reaction that is hydrolyzed, for determining that suitable beta glucan enzyme concentration to be measured can become 1,4 beta-glucanase solution dilution to be measured the variable concentrations gradient to detect one by one, hydrolysis time is in the 60min, get hydrolyzate 2mL (can in course of reaction time segment sampling detect), rapidly in the NaOH solution of adding 2mL 0.5 equivalent (making three parallel samples), mixing, getting 1mL joins in the test tube, add MBTH reagent 0.5mL again and behind 80 ℃ of water-bath heating 11-13min, add ammonium ferric sulfate reagent 1.2mL while hot, absorbance is measured in room temperature cooling back in wavelength 650nm place, determine the amount of the reducing sugar that is equivalent to glucose that contained in the enzymolysis product of 1,4 beta-glucanase to be measured according to the standard corresponding relation curve between the concentration of glucose of the rapid making of previous step and the corresponding absorbance that records.The unit of activity of 1,4 beta-glucanase can be defined as under these conditions, and to produce the enzyme amount that 1nmol is equivalent to the amount of glucose be an enzyme activity unit to per minute in every milliliter of reaction system, thereby define the vigor of calculating wooden 1,4 beta-glucanase according to enzyme activity.
Embodiment 2:
Key instrument: electric-heated thermostatic water bath, HH-4 type, state China Electrical Appliances Co., Ltd; Digital ph, Mettler Toledo Inc.; The UV2100 ultraviolet-visible spectrophotometer, UNICO(Shanghai) Instruments Co., Ltd.; 1,4 beta-glucanase, Xinhuayang Biological Co., Ltd., Wuhan; Beta glucan, hundred special pure big molecule Science and Technology Ltd.s; Pipettor, Finnpipette Finland Lei Bo group.
Solution allocation
MBTH develop the color the 3-methyl-2-[4-morpholinodithio ketone hydrogencarbonate aqueous solution of liquid: 3mg/mL and 1mg/mL dithiothreitol (DTT) solution mixed in equal amounts promptly, matching while using, in one day effectively.
Glucose standard solution (30 μ g/mL): accurately take by weighing the glucose 0.3000g that is dried to permanent quality, with the dissolving of the hac buffer of 50mM pH4.0, shift and be settled to 100mL, be made into the glucose standard solution of 3.00mg/mL; Therefrom get the 1mL glucose solution, as the operation of preceding method and be settled to 100mL, promptly get the glucose standard solution of 30 μ g/mL.
The method for making of ammonium ferric sulfate reagent is 0.5% (FeNH
4(SO
4)
2) 12H
2O, 0.5% sulfaminic acid, 0.5 equivalent hydrochloric acid.
Steps A is made the glucose absorbance that records with the MBTH method and the standard corresponding relation curve of its respective concentration;
The glucose standard solution 1mL that adds 8 groups of variable concentrations respectively, the NaOH solution that adds 1mL 0.5 equivalent, mixing, add MBTH reagent 1mL again and add ammonium ferric sulfate reagent 2mL while hot behind 80 ℃ of water-bath heating 11-13min, the absorbance of glucose is measured in room temperature cooling back in wavelength 650nm place; Each concentration is made three parallel samples; According to concerning drawing standard corresponding relation curve between every group of glucose solution concentration and the corresponding absorbance that records;
B is with the vigor of MBTH method detection 1,4 beta-glucanase;
(its concentration is 2mg/mL with 0.25mL beta glucan solution, the pH value is 5.5), add in the test tube preheating 10min in 30 ℃ water-bath, add the beta glucan enzyme solutions 0.25mL that the is preheated to 30 ℃ to be measured reaction that is hydrolyzed, for determining that suitable beta glucan enzyme concentration to be measured can become 1,4 beta-glucanase solution dilution to be measured the variable concentrations gradient to detect one by one, hydrolysis time is 30 minutes, add the abundant mixing of NaOH solution of 0.5mL 0.5 equivalent rapidly with cessation reaction, add 0.5mL MBTH reagent, abundant mixing, after 80 ℃ of water-bath heating, add ammonium ferric sulfate reagent 1mL while hot, absorbance is measured in room temperature cooling back in wavelength 650nm place, make three parallel samples as method, determine the amount of the reducing sugar that is equivalent to glucose that contained in the enzymolysis product of 1,4 beta-glucanase to be measured according to the standard corresponding relation curve between rapid concentration of glucose of making of previous step and the corresponding absorbance that records.The unit of activity of 1,4 beta-glucanase can be defined as under these conditions, and per minute produces 1nmol to be equivalent to the required enzyme amount of glucose be an enzyme activity unit in every milliliter of reaction system, thereby defines the vigor of calculating wooden 1,4 beta-glucanase according to enzyme activity.
Certainly, above-mentioned explanation is not to be limitation of the present invention, and the present invention also is not limited to aforesaid operations, and all variations of making in essential scope of the present invention, remodeling, interpolation or replacement all should belong to protection scope of the present invention.
Claims (6)
1. the assay method of an activity of beta-glucanase is characterized in that;
A makes the absorbance of the glucose that records with the MBTH method and the standard corresponding relation curve of its respective concentration;
B detects the absorbance that is equivalent to glucose that produces behind the beta glucan enzyme hydrolysis beta glucan to be measured with the MBTH method; Determine the content of the reducing sugar that is equivalent to glucose that in the unit interval, produced in the enzymolysis product of 1,4 beta-glucanase to be measured according to the standard corresponding relation curve between rapid concentration of glucose of making of previous step and the corresponding absorbance that records, calculate the vigor of 1,4 beta-glucanase with this.
2. the assay method of activity of beta-glucanase according to claim 1 is characterized in that; The concrete steps of above-mentioned steps A are; Get the glucose standard solution 0.6mL that the 6-15 group has variable concentrations respectively, concentration range is between the 0-30 mcg/ml, the NaOH solution that adds 0.6mL 0.5 equivalent, mixing, add MBTH reagent 0.6mL again and add ammonium ferric sulfate reagent 1.2mL while hot behind 75-85 ℃ of water-bath heating 8-20min, absorbance is measured in room temperature cooling back in wavelength 620-680nm place; Each concentration is made three parallel samples; Above dosage can increase and decrease in proportion; According to concerning drawing standard corresponding relation curve between every group of concentration of glucose and the absorbance that records;
3. the assay method of activity of beta-glucanase according to claim 1 and 2 is characterized in that; The concrete steps of above-mentioned steps B are; With final concentration is the beta glucan solution of measuring temperature that is preheated to of 1-5mg/mL, join in the beta glucan enzyme solutions that is preheated to relevant temperature and begin hydrolysis reaction, hydrolysis temperature is 25-40 ℃, hydrolysis time is in the 60min, this enzymolysis liquid is mixed with cessation reaction rapidly with the NaOH solution of isopyknic 0.5 equivalent, can in course of reaction, take a sample stage by stage, therefrom getting 1.2mL joins and makees three parallel samples in the test tube, add MBTH reagent 0.6mL again and behind 75-85 ℃ of water-bath heating 5-20min, add ammonium ferric sulfate reagent 1.2mL while hot, absorbance is measured in room temperature cooling back in wavelength 620-680nm place, the concentration of reduced sugar that is equivalent to glucose that standard corresponding relation curve between concentration of glucose of making according to steps A and the corresponding absorbance that records is determined in the enzymolysis product to be contained is calculated the vigor of 1,4 beta-glucanase with this; The unit of activity of 1,4 beta-glucanase can be defined as under these conditions, and it is an enzyme activity unit that every milliliter of reaction system per minute produces the required enzyme amount of amount that 1nmol is equivalent to glucose; Wherein the method for making of MBTH reagent is that the 3-methyl-2-[4-morpholinodithio ketone hydrogencarbonate aqueous solution of 3mg/mL and the dithiothreitol (DTT) solution equal-volume of 1mg/mL mix promptly, and matching while using is effective in one day.
4. the assay method of activity of beta-glucanase according to claim 3 is characterized in that; The method for making of above-mentioned ammonium ferric sulfate reagent is 0.5%FeNH
4(SO
4)
212H
2O, 0.5% sulfaminic acid, 0.5 equivalent hydrochloric acid.
5. the assay method of activity of beta-glucanase according to claim 3 is characterized in that; Above-mentioned beta glucan enzyme solutions is a solvent with succinic acid or the acetum of 50mM.
6. the assay method of activity of beta-glucanase according to claim 3 is characterized in that; The optimal wavelength of said determination absorbance is 650nm.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010144060A CN101819138A (en) | 2010-04-12 | 2010-04-12 | Measuring method of activity of beta-glucanase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010144060A CN101819138A (en) | 2010-04-12 | 2010-04-12 | Measuring method of activity of beta-glucanase |
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| Publication Number | Publication Date |
|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506059A (en) * | 2016-01-18 | 2016-04-20 | 黑龙江大学 | Method for determining activity of beta-mannase |
| CN109632787A (en) * | 2019-02-21 | 2019-04-16 | 浙江省农业科学院 | A kind of quick method for measuring of barley grain beta glucan extract purity |
-
2010
- 2010-04-12 CN CN201010144060A patent/CN101819138A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506059A (en) * | 2016-01-18 | 2016-04-20 | 黑龙江大学 | Method for determining activity of beta-mannase |
| CN109632787A (en) * | 2019-02-21 | 2019-04-16 | 浙江省农业科学院 | A kind of quick method for measuring of barley grain beta glucan extract purity |
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