CN101586146A - A kind of analytical procedure that detects activity of soil xylanase - Google Patents
A kind of analytical procedure that detects activity of soil xylanase Download PDFInfo
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- CN101586146A CN101586146A CNA2008100115391A CN200810011539A CN101586146A CN 101586146 A CN101586146 A CN 101586146A CN A2008100115391 A CNA2008100115391 A CN A2008100115391A CN 200810011539 A CN200810011539 A CN 200810011539A CN 101586146 A CN101586146 A CN 101586146A
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Abstract
The present invention relates to a kind of analytical procedure that detects cellulase activity in the soil: the soil air-dry sample n part that 1) takes by weighing screening is propped up in the thick test tube in n, in each test tube, add hac buffer, use the vibration of vortex vibrator, earth suspension to 96 microwell plate (occupying n micropore) fetches earth under oscillating condition, in n-1 hole, add 4-MUB-7-β-D-cellobioside substrate solution, adding waits water gaging to contrast as no substrate in another hole, in n+1 hole, add the equivalent substrate solution and wait water gaging to contrast shaking culture under the constant temperature as no soil; 2) cultivate the end back and in microwell plate, add the NaOH termination reaction; 3) multi-functional microplate reader is carried out fluorometric assay to reaction product; 4) calculate cellulase activity.Advantage of the present invention is: 1) compare with traditional method, shortened incubation time, omitted schedule of operation such as filtration, simplified operation steps; 2) fluorescent substance in the microwell plate can obtain determination data simultaneously by multi-functional microplate reader in 15s, allows a large amount of samples to measure simultaneously; 3) the accuracy height is easy to operate; 4) result is reliable and stable, favorable reproducibility.
Description
Technical field
The present invention relates to the mensuration of cellulase activity in the soil, specifically a kind of analytical procedure that detects cellulase activity in the soil.
Background technology
The carbohydrate that enters and be accumulated in the soil is to participate in Carbon cycle under the effect of soil carbohydrase, Mierocrystalline cellulose and hemicellulose are the main kinds that constitutes carbohydrate, Mierocrystalline cellulose is to the hydrolysis of monomer whose-glucose, be under the effect of the different enzymes of the multienzyme system of cellulase complex body, carry out through some stages.Cellulase complex body in the soil is made up of four kinds of carbohydrases: 1, and 4-beta-glucan restriction endonuclease, 1,4-beta-glucan excision enzyme, 1,4-β-glucoside excision enzyme and cellobiase.They participate in the different steps that Mierocrystalline cellulose decomposes respectively.Usually the activity of soil xylanase that records is the summation of the effect of this kind combined enzyme agent.Cellulase activity usually is used as the index of indicating soil fertility, because it participates in the decomposition of the soil organism, in addition, exist dependency between the activity of cellulase and content of humic substances, between the formation of cellulosic decomposition of soil and soil ulmin, exist certain getting in touch.Generally speaking, the active reaction of soil xylanase the intensity and the soil fertility level of the bioclimate that forms of soil and ecological condition, soil biochemistry process.Activity of soil xylanase is subjected to the influence of edaphic condition, farming operation system, control measures and Different Crop, when carrying out fertility evaluation, the cellulolytic activity that usually needs to be determined under the different condition describes, therefore, setting up a kind of detection method tool of cellulase activity relatively fast and accurately has very important significance.
About the mensuration of activity of soil xylanase substantially all be with reference to Zhou Likai 1984 show method in " soil enzymology " book (this method is translated from the method that Pancholy and Rice were published among the SoilScience Society of American Proceedings in 1973).This kind method steps is loaded down with trivial details, and the substance that show color instability, it is relatively poor to measure circulation ratio, and then Schinner and van Mersi improve measuring method in the monograph Methods in Soil Biology that was shown in 1996, substance that show color is more stable, circulation ratio is better, but similarly, it measures the process more complicated, institute's time-consuming is longer, and required reagent potassium cyanide is poisonous reagent in process color, is difficult to acquisition, and has increased the danger to operator in the mensuration process.
Summary of the invention
The object of the present invention is to provide a kind of analytical procedure of checking cellulase activity in the soil.This method is on the principle basis of using for reference forefathers' measuring method, the determination step of cellulase activity and the detection method of end product are improved, thereby reduce the complicacy of sample preparation and testing sequence, make analytical results more accurately reliable, it is better to analyze circulation ratio.Its principle produces fluorescent substance for using fluorescence conjugated material as measuring substrate by enzymolysis, by multi-functional microplate reader this product is carried out fluoroscopic examination, and then calculates the cellulase activity that participates in this reaction.
For achieving the above object, the technical solution used in the present invention is:
1) the MUB solution with known series concentration is standard substance, adopts microplate reader to carry out fluoroscopic examination, and wavelength of transmitted light 365nm measures optical wavelength 470nm; As the typical curve of measuring; Make the working standard curve with the series concentration of MUB standardized solution and the light absorption value A` of mensuration, obtain slope b;
2) take by weighing the soil air-dry sample that n part 3-5g crosses 1-2mm sieve respectively and prop up in the thick test tube in n, n 〉=3 add 30-50ml 0.45-0.55molL in each test tube
-1The hac buffer of pH5.0-6.0, using micro-feed liquor rifle to get 100 μ l soil supensions under the vortex oscillating condition from thick test tube adds in the microwell plate, occupy n micropore, substrate solution (the 4-MUB-7-β-D-cellobioside that in n-1 hole wherein, adds 10mM, MUB is fluorescent substance 4-methylol-7-tonka bean camphor) 100 μ l, add 100 μ l water in another hole and contrast as no substrate; Adding 100 μ l substrate solutions and 100 μ l water contrast as no soil in n+1 hole; Shaking culture 2.5-3.5h under 25 ℃ of-30 ℃ of conditions; In the hole, add 100 μ l 2-2.5molL respectively
-1The NaOH termination reaction;
3) microwell plate is inserted multi-functional microplate reader and carry out fluoroscopic examination, wavelength of transmitted light 365nm measures optical wavelength 470nm; Measuring fluorescence on multi-functional microplate reader is A;
4), calculate the concentration S of the MUB in the filtrate that surveys, S=A*b according to light absorption value A and slope b;
5) amount according to MUB calculates enzymic activity; Calculation formula is: x=(S*40)/(V*b*T)
In the formula: x is a cellulase activity (unit: μ mol MUBg
-1H
-1), S is the concentration of the fluorescent substance that step 4 obtained, and V is the overall solution volume (unit/ml) in the reaction system, and b is that (unit: g), T is an incubation time (unit: h) to the soil weight that takes by weighing.
The making of described typical curve and mensuration process are as follows, 1. prepare 0.5molL
-1The pH5.5 acetate buffer solution; 2. use 0.5molL
-1PH5.5 acetate buffer solution compound concentration is 0,0.01,0.05,0.1,0.25, and 1.0molL
-1The MUB reference liquid, compound method: take by weighing MUB 0,0.176,0.881,1.762,4.404 and 1.762g be settled to 100ml, shake up and get final product, this liquid needs preparation on the same day; 3. in micropore, add this serial solution of 100 μ l and 100 μ l water, 100 μ l 2-2.5molL
-1Carry out fluorometric assay behind the NaOH; Contain MUB0 in this serial solution, 0.1,0.5,1.0,2.5,10 μ mol; 4. each point of typical curve repeats for 3-5 time; 5. according to the MUB of concentration known amount and obtain the working standard curve by the fluoroscopic examination data.
The present invention uses fluorescence conjugated material 4-MUB-7-β-D-cellobioside as measuring substrate, this material produces fluorescent substance MUB after by enzymic hydrolysis, MUB excites at the 365nm place, can detect fluorescence at the 470nm place, and the use of this kind substrate is the improved key of this testing method.Described microwell plate is generally 96 microwell plates, uses 96 microwell plates as the reaction system carrier, and measuring when its application makes a large amount of pedotheque becomes possibility, improves detection efficiency.
The improved foundation of the inventive method
The measurement result of the cellulase in the soil is 1,4-beta-glucan restriction endonuclease, 1,4-beta-glucan excision enzyme, 1, the general name of 4-β-glucoside excision enzyme and cellobiase, be to discharge, be accumulated in extracellular enzyme in the soil by value-added and cracked microorganism cells, they all participate in cellulosic decomposition and inversion process in the soil.Enzymic activity all is that the growing amount with the reduction of substrate and product characterizes, tradition spectrophotometry activity of soil xylanase is to use Xylo-Mucine as measuring substrate, product glucose behind the enzymolysis goes out its absorbancy under the 690nm condition by spectrophotometric determination after using chromogenic reagent, the required test duration of this kind method is grown (reaction system incubation time, enzymolysis product extraction etc.), operation is complicated, and labor capacity is bigger.Be based on a kind of fluoroscopic examination means and originally improve one's methods, this detection method is highly sensitive, demand to liquid to be measured is less, so reaction system (soil supension, substrate and corresponding damping fluid) placed in the microwell plate get final product, the reaction back can obtain the amount of the fluorescent substance in all micropores simultaneously on microplate reader, in addition, because of soil amount and amount of substrate less, so only need short incubation time can finish reaction, this method step that simplifies the operation improves determination efficiency greatly.But compare with traditional method and to have the shortcoming bigger equally the instrument dependency.
Compare with the analytical procedure in past, advantage of the present invention mainly contains:
1) the reaction system incubation time is shorter, extraction step such as need not to filter or centrifugal after the cultivation, directly adding terminator carries out measuring on multi-functional microplate reader after the termination reaction, and the fluorescent substance in the micropore can obtain determination data simultaneously in 15s, allow a large amount of samples to measure simultaneously, increase work efficiency;
2) simple to operate, analytical results is reliable and stable, favorable reproducibility;
3) required varieties of reagent reduces, and easily obtains, and the mensuration process need not poisonous reagent, improves the security of mensuration process.
Embodiment
The reagent preparation:
1.0.5molL
-1PH 5.5 acetate buffer solutions: dissolving 41g anhydrous sodium acetate is settled to 1l in distilled water; Draw 60ml ice second (vinegar) acid in the 500ml volumetric flask, use the distilled water constant volume.The anhydrous second of 1l (vinegar) acid sodium solution and the sour diluent of 45ml ice second (vinegar) are mixed, with second (vinegar) acid for adjusting pH to 5.5;
2.10mmolL
-1Ethylene glycol monomethyl ether solution: take by weighing the 0.98g ethylene glycol monomethyl ether in 1l constant bottle, with being settled to scale after the 500ml water dissolution;
3.10mmolL
-1Substrate (4-MUB-β-D-cellobioside) solution: 0.5184g substrate (4-MUB-β-D-cellobioside) is in the 100ml volumetric flask, with reagent 2 dissolving and be settled to scale;
4.2molL
-1NaOH solution: 80g NaOH is dissolved in the 800ml water, is transferred to constant volume in the 1l volumetric flask.
Operation steps:
1) making of typical curve and mensuration process are as follows, 1. prepare 0.5molL
-1The pH5.5 acetate buffer solution; 2. use 0.5molL
-1PH5.5 acetate buffer solution compound concentration is 0,0.01,0.05,0.1,0.25, and 1.0molL
-1The MUB reference liquid, compound method: take by weighing MUB 0,0.176,0.881,1.762,4.404 and 1.762g be settled to 100ml, shake up and get final product, this liquid needs preparation on the same day; 3. in micropore, add this serial solution of 100 μ l and 100 μ l water, 100 μ l 2molL
-1Carry out fluorometric assay behind the NaOH; Contain MUB0 in this serial solution, 0.1,0.5,1.0,2.5,10 μ mol; 4. each point of typical curve repeats for 3 times; 5. according to the MUB of concentration known amount and obtain the working standard curve by the fluoroscopic examination data.
2) take by weighing the soil air-dry sample that n part 3-5g crosses 1-2mm sieve respectively and prop up in the thick test tube in n, n 〉=3 add 30-50ml 0.5molL in each test tube
-1The hac buffer of pH5.5 uses micro-feed liquor rifle to get 100 μ l soil supensions under the vortex oscillating condition and adds 96 microwell plates (occupying n micropore), adds 10mmolL in n-1 hole
-1Substrate solution (4-MUB-7-β-D-xyloside, MUB is fluorescent substance 4-methylol-7-tonka bean camphor) 100 μ l, add 100 μ l water in another hole and contrast as no substrate, adding 100 μ l substrate solutions and 100 μ l water contrast as no soil in n+1 hole; Shaking culture 3h under 30 ℃ of conditions;
3) in the hole, add 100 μ l 2molL respectively
-1The NaOH termination reaction;
4) microwell plate is inserted multi-functional microplate reader and carry out fluoroscopic examination, wavelength of transmitted light 365nm measures optical wavelength 470nm;
5) measuring fluorescence on multi-functional microplate reader is A
6) make the working standard curve with the series concentration of MUB standardized solution and the light absorption value A` of mensuration, obtain slope b;
7), calculate the concentration S of the MUB in the filtrate that surveys, S=A*b according to light absorption value A and slope b;
8) amount according to MUB calculates enzymic activity;
Calculation formula is: x=(S*40)/(V*b*T)
In the formula: x is a cellulase activity (unit: μ mol MUBg
-1H
-1), S is the amount of the fluorescent substance that step 6 obtained, and V is the overall solution volume (unit/ml) in the reaction system, and b is that (unit: g), T is an incubation time (unit: h) to the soil weight that takes by weighing
Embodiment 1
The employed soil of present embodiment picks up from An Zhen town, Wuxi City, Jiangsu Province year surplus farm (31 ° of 37 ' N, 120 ° of 28 ' E) rice-Mai crop rotation FACE (Free-air Carbon Elevation open C O
2Concentration increases) system platform, platform has 3 FACE test circles and 5 contrast circles, and FACE test circle keeps CO
2The high 200 μ molmol of concentration ratio contrast circle
-1Soil type is that water is ploughed Anthrosol's (being commonly called as yellow soil).Two processing are CO
2Concentration increases and contrasts, CO
2Concentration increases handles 3 repetitions, contrasts 5 repetitions, detects activity of soil xylanase in the sampling of the ripening stage of wheat growth and with aforesaid method.
The concrete analysis step of every kind of soil is as follows:
1) takes by weighing soil air-dry sample that 3 parts of 3g cross 2mm sieve respectively in thick test tube, in each test tube, add 40ml 0.5molL
-1The hac buffer of pH5.5 uses micro-feed liquor rifle to get 100 μ l soil supensions under the vortex oscillating condition and adds microwell plate (occupying n micropore), adds 10mmolL in n-1 hole
-1Substrate solution (4-MUB-β-D-cellobioside, MUB is fluorescent substance 4-methylol-7-tonka bean camphor) 100 μ l, adding 100 μ l water in another hole contrasts as no substrate, adding 100 μ l substrate solutions and 100 μ l water contrast shaking culture 3h under 30 ℃ of conditions as no soil in n+1 hole;
2) in the hole, add 100 μ l 2molL respectively
-1The NaOH termination reaction;
3) microwell plate is inserted multi-functional microplate reader and carry out fluoroscopic examination, wavelength of transmitted light 365nm measures optical wavelength 470nm;
4) measuring fluorescence on multi-functional microplate reader is A
5) make the working standard curve with the series concentration of MUB standardized solution and the light absorption value A` of mensuration, obtain slope b;
6), calculate the concentration S of the MUB in the filtrate that surveys, S=A*b according to light absorption value A and slope b;
7) amount according to MUB calculates enzymic activity;
Calculation formula is: x=(S*40)/(V*b*T)
In the formula: x is a cellulase activity (unit: μ mol MUBg
-1H
-1), S is the amount of the fluorescent substance that step 6 obtained, and V is the overall solution volume (unit/ml) in the reaction system, and b is that (unit: g), T is an incubation time (unit: h) to the soil weight that takes by weighing
Test-results is as follows:
By data in the table as can be seen, less standard difference shows that the deviation between this analytical procedure result is less, and tolerance range is higher, favorable reproducibility.
Embodiment 2
The employed three kinds of black earth of present embodiment are all adopted in Suihua City Helen county, Heilongjiang Province: handling 1 soil is the soybean continuous cropping, and handling 2 soil is the paddy rice continuous cropping, and handling 3 soil is the corn continuous cropping.The soil of three kinds of different planting systems is measured after all crossing the 2mm sieve.Concrete steps are with embodiment 1.
Test-results is as follows:
Data have shown the stability and the higher precision of analytical results equally in the table.
Claims (3)
1. analytical procedure that detects cellulase activity in the soil is characterized in that:
1) the MUB solution with known series concentration is standard substance, adopts microplate reader to carry out fluoroscopic examination, and wavelength of transmitted light 365nm measures optical wavelength 470nm; As the typical curve of measuring; Make the working standard curve with the series concentration of MUB standardized solution and the light absorption value A` of mensuration, obtain slope b;
2) take by weighing the soil air-dry sample that n part 3-5g crosses 1-2mm sieve respectively and prop up in the thick test tube in n, n be 〉=3 positive integer, adding 30-50ml 0.45-0.55molL in each test tube
-1The hac buffer of pH5.0-6.0, using micro-feed liquor rifle to get 100 μ l soil supensions under the vortex oscillating condition adds in the microwell plate, occupy n micropore, in n-1 hole, add 10mM 4-MUB-β-D-cellobioside 100 μ l, add 100 μ l water in another hole and contrast as no substrate, adding 100 μ l substrate solutions and 100 μ l water contrast as no soil in n+1 hole; Shaking culture 2.5-3.5h under 25 ℃ of-30 ℃ of conditions; In the hole, add 100 μ l 2-2.5molL respectively
-1The NaOH termination reaction;
3) microwell plate is inserted multi-functional microplate reader and carry out fluoroscopic examination, wavelength of transmitted light 365nm measures optical wavelength 470nm; Measuring fluorescence on multi-functional microplate reader is A;
4), calculate the concentration S of the MUB in the filtrate that surveys, S=A*b according to light absorption value A and slope b;
4) measuring fluorescence on multi-functional microplate reader is A
5) amount according to MUB calculates enzymic activity; Calculation formula is: x=[S* (30-50)]/(V*b*T)
In the formula: x is a cellulase activity (unit: μ mol MUBg
-1H
-1), S is the concentration of the fluorescent substance that step 4 obtained, and V is the overall solution volume (unit/ml) in the reaction system, and b is that (unit: g), T is an incubation time (unit: h) to the soil weight that takes by weighing.
2. analytical procedure according to claim 1 is characterized in that: the making of described typical curve and mensuration process are as follows, 1. prepare 0.5molL
-1The pH5.5 acetate buffer solution; 2. use 0.5molL
-1PH5.5 acetate buffer solution compound concentration is 0,0.01,0.05,0.1,0.25, and 1.0molL
-1The MUB reference liquid, compound method: take by weighing MUB 0,0.176,0.881,1.762,4.404 and 1.762g be settled to 100ml, shake up and get final product, this liquid needs preparation on the same day; 3. treat in micropore, to add this serial solution of 100 μ l and 100 μ l water, 100 μ l 2-2.5molL after pedotheque is cultivated end
-1Carry out fluorometric assay behind the NaOH; Contain MUB0 in this serial solution, 0.1,0.5,1.0,2.5,10 μ mol; 4. each point of typical curve repeats for 3-5 time; 5. according to the MUB of concentration known amount and obtain the working standard curve by the fluoroscopic examination data.
3. analytical procedure according to claim 1 is characterized in that: described microwell plate is 96 microwell plates, uses 96 microwell plates as the reaction system carrier, and measuring when its application makes a large amount of pedotheque becomes possibility, improves detection efficiency.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796806A (en) * | 2012-07-27 | 2012-11-28 | 深圳华大基因研究院 | Method for determining cellulase activity of sample |
| CN103776779A (en) * | 2014-01-24 | 2014-05-07 | 上海市农业科学院 | Detection method for cellulase in compound feed |
| CN108507958A (en) * | 2018-04-04 | 2018-09-07 | 黑龙江大学 | A kind of assay method of soil xylanase |
| CN110632052A (en) * | 2019-10-25 | 2019-12-31 | 山西师范大学 | Fluorescence spectrum detection method for activity of soil arylsulfatase |
| CN111551528A (en) * | 2020-01-19 | 2020-08-18 | 南京林业大学 | Method for detecting activity of litter acid phosphatase |
| CN111705109A (en) * | 2020-01-19 | 2020-09-25 | 南京林业大学 | Method for detecting activity of exoglucanase of litters |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3614647A1 (en) * | 1986-04-30 | 1987-11-05 | Euratom | 7-PHENYL ACETIC ACID-4-ALKYL-COUMARINYLAMIDES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE IN METHODS FOR THE FLUOROMETRIC DETERMINATION OF THE ACTIVITY OF HYDROLASES, ESPECIALLY PENICILLIN G ACYLASE |
| CN100494979C (en) * | 2006-06-02 | 2009-06-03 | 中国科学院沈阳应用生态研究所 | Analytical method for detecting hydroxyl amine reducing enzyme activity in soil |
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2008
- 2008-05-23 CN CN2008100115391A patent/CN101586146B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796806A (en) * | 2012-07-27 | 2012-11-28 | 深圳华大基因研究院 | Method for determining cellulase activity of sample |
| CN103776779A (en) * | 2014-01-24 | 2014-05-07 | 上海市农业科学院 | Detection method for cellulase in compound feed |
| CN108507958A (en) * | 2018-04-04 | 2018-09-07 | 黑龙江大学 | A kind of assay method of soil xylanase |
| CN110632052A (en) * | 2019-10-25 | 2019-12-31 | 山西师范大学 | Fluorescence spectrum detection method for activity of soil arylsulfatase |
| CN111551528A (en) * | 2020-01-19 | 2020-08-18 | 南京林业大学 | Method for detecting activity of litter acid phosphatase |
| CN111705109A (en) * | 2020-01-19 | 2020-09-25 | 南京林业大学 | Method for detecting activity of exoglucanase of litters |
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