CN101586146B - An analytical method for detecting soil cellulase activity - Google Patents
An analytical method for detecting soil cellulase activity Download PDFInfo
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- CN101586146B CN101586146B CN2008100115391A CN200810011539A CN101586146B CN 101586146 B CN101586146 B CN 101586146B CN 2008100115391 A CN2008100115391 A CN 2008100115391A CN 200810011539 A CN200810011539 A CN 200810011539A CN 101586146 B CN101586146 B CN 101586146B
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- 239000002689 soil Substances 0.000 title claims abstract description 57
- 230000000694 effects Effects 0.000 title claims abstract description 30
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 19
- 229940106157 cellulase Drugs 0.000 title claims abstract description 19
- 238000004458 analytical method Methods 0.000 title claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 37
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 28
- 239000000758 substrate Substances 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims abstract description 16
- 238000004364 calculation method Methods 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims description 16
- 238000005303 weighing Methods 0.000 claims description 12
- 230000031700 light absorption Effects 0.000 claims description 8
- 239000008351 acetate buffer Substances 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 6
- 230000003287 optical effect Effects 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 4
- 239000012467 final product Substances 0.000 claims description 4
- 238000007421 fluorometric assay Methods 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 abstract description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract 3
- 239000007853 buffer solution Substances 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 238000005259 measurement Methods 0.000 abstract 1
- 238000011017 operating method Methods 0.000 abstract 1
- 239000000725 suspension Substances 0.000 abstract 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 229920002498 Beta-glucan Polymers 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 235000021419 vinegar Nutrition 0.000 description 4
- 239000000052 vinegar Substances 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 230000035558 fertility Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000009335 monocropping Methods 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- GUBGYTABKSRVRQ-QRZGKKJRSA-N beta-cellobiose 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-QRZGKKJRSA-N 0.000 description 2
- 108010089934 carbohydrase Proteins 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 230000007096 poisonous effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 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
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000004177 carbon cycle Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012916 chromogenic reagent Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000003967 crop rotation Methods 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明涉及一种检测土壤中纤维素酶活性的分析方法：1)称取筛分的土壤风干样品n份于n支粗试管中，向各试管中加入醋酸缓冲溶液，使用旋涡振荡器振荡，在振荡条件下取土壤悬液至96微孔板(占有n个微孔)，向n-1个孔中加入4-MUB-7-β-D-纤维二糖苷底物溶液，另一孔中加入等量水作为无底物对照，向第n+1个孔中加入等量底物溶液和等量水作为无土壤对照，恒温条件下振荡培养；2)培养结束后向微孔板中加入NaOH终止反应；3)多功能酶标仪对反应产物进行荧光测定；4)计算纤维素酶活性。本发明的优点为：1)与传统方法相比，减短了培养时间，省略了过滤等操作程序，简化了操作步骤；2)微孔板内的荧光物质可通过多功能酶标仪在15s内同时获得测定数据，允许大量样品同时进行测定；3)准确度高，易操作；4)结果稳定可靠，重现性好。The invention relates to an analysis method for detecting cellulase activity in soil: 1) Take n parts of sieved soil air-dried samples in n thick test tubes, add acetic acid buffer solution to each test tube, vibrate with a vortex oscillator, Take the soil suspension to a 96-microwell plate (occupying n microwells) under shaking conditions, add 4-MUB-7-β-D-cellobioside substrate solution to n-1 wells, and add 4-MUB-7-β-D-cellobioside substrate solution to another well Add an equal amount of water as a no-substrate control, add an equal amount of substrate solution and an equal amount of water into the n+1th well as a no-soil control, and culture with shaking under constant temperature conditions; 2) Add NaOH terminates the reaction; 3) Fluorescent determination of the reaction product by a multifunctional microplate reader; 4) Calculation of cellulase activity. The advantages of the present invention are: 1) Compared with the traditional method, the culture time is shortened, operating procedures such as filtration are omitted, and the operation steps are simplified; The measurement data can be obtained at the same time in the same time, allowing a large number of samples to be measured at the same time; 3) High accuracy and easy operation; 4) The results are stable and reliable, and the reproducibility is good.
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 glucide that gets into and be accumulated in the soil is under the effect of soil carbohydrase, to participate in Carbon cycle; Mierocrystalline cellulose and semicellulose are the main kinds that constitutes glucide; The hydrolysis of Mierocrystalline cellulose to 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 cellose.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 receives the influence of edaphic condition, farming operation system, control measures and Different Crop; When carrying out fertility evaluation; The cellulolytic activity that usually need be determined under the different condition describes; Therefore, set up a kind of detection method of cellulase activity relatively fast and accurately and have very important meaning.
About the mensuration of activity of soil xylanase basically 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 substance that show color is unstable, and it is relatively poor to measure circulation ratio; Then Schinner and van Mersi improve measuring method in the monograph Methods in Soil Biology that was shown in 1996, and substance that show color is more stable, and circulation ratio is better; But likewise, it measures the process more complicated, and institute's time-consuming is longer; And required reagent Potssium Cyanide is poisonous reagent in process color, is difficult to obtain, 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 through enzymolysis, through multi-functional ELIASA this product is carried out fluoroscopic examination, and then calculates the cellulase activity of participating in this reaction.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
1) the MUB solution with known series concentration is standard substance, adopts ELIASA 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 under the vortex oscillating condition, from thick test tube, to get 100 μ l soil supensions adds in the microwell plate; Occupy n micropore; In n-1 hole wherein, add substrate solution (4-MUB-7-β-D-cellobioside, MUB are fluorescent substance 4-methylol-7-tonka bean camphor) the 100 μ l of 10mM, 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 ELIASA and carry out fluoroscopic examination, wavelength of transmitted light 365nm measures optical wavelength 470nm; On multi-functional ELIASA, measuring fluorescence is A;
4), calculate the concentration S of the MUB in the filtrating 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 said typical curve and mensuration process are following, 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 are settled to 100ml with 1.762g, shake up and get final product, and this liquid needed prepared 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 through 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.Said 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 mensuration 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 cellose is to discharge, be accumulated in the extracellular enzyme in the soil by value-added with the cracked microorganism cells, and 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 through spectrophotometric determination after using chromogenic reagent, and 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, and is less to the demand of liquid to be measured; 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 ELIASA, in addition; Because of soil amount and amount of substrate less; So only need short incubation time can accomplish 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 on multi-functional ELIASA, measuring 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, increases work efficiency;
2) simple to operate, analytical results is reliable and stable, favorable reproducibility;
3) required varieties of reagent reduces, and is prone to obtain, 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 zero(ppm) water; Draw 60ml ice second (vinegar) acid in the 500ml volumetric flask, use the zero(ppm) 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
-1EGME solution: take by weighing the 0.98g EGME 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 following, 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 are settled to 100ml with 1.762g, shake up and get final product, and this liquid needed prepared 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 through 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 under the vortex oscillating condition, to get 100 μ l soil supensions and adds 96 microwell plates (occupying n micropore), in n-1 hole, adds 10mmolL
-1Substrate solution (4-MUB-7-β-D-xyloside, MUB are fluorescent substance 4-methylol-7-tonka bean camphor) 100 μ l add 100 μ l water and contrast as no substrate in another hole, 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 ELIASA and carry out fluoroscopic examination, wavelength of transmitted light 365nm measures optical wavelength 470nm;
5) on multi-functional ELIASA, measuring fluorescence 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 filtrating 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 following:
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 under the vortex oscillating condition, to get 100 μ l soil supensions and adds microwell plate (occupying n micropore), in n-1 hole, adds 10mmolL
-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 ELIASA and carry out fluoroscopic examination, wavelength of transmitted light 365nm measures optical wavelength 470nm;
4) on multi-functional ELIASA, measuring fluorescence 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 filtrating 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 following:
Can find out that by data in the table less standard difference shows that the deviation between this analytical procedure result is less, tolerance range is higher, favorable reproducibility.
Embodiment 2
The employed three kinds of black earth of present embodiment are all adopted in the Heilongjiang Province Suihua City Helen county: 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 following:
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 ELIASA 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 under the vortex oscillating condition, to get 100 μ l soil supensions 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 ELIASA and carry out fluoroscopic examination, wavelength of transmitted light 365nm measures optical wavelength 470nm; On multi-functional ELIASA, measuring fluorescence is A;
4), calculate the concentration S of the MUB in the filtrating 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* (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 the soil weight unit that takes by weighing: g, and T is incubation time unit: h, said MUB is 4-methylol-7-tonka bean camphor.
2. analytical procedure according to claim 1 is characterized in that: the making of said typical curve and mensuration process are following, 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 MUB0,0.176,0.881,1.762,4.404 are settled to 100ml with 17.62g, shake up and get final product, and this liquid needed prepared 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 through the fluoroscopic examination data.
3. analytical procedure according to claim 1 is characterized in that: said 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.
Priority Applications (1)
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| 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 Spectroscopic Detection of Soil Arylsulfatase Activity |
| CN111705109A (en) * | 2020-01-19 | 2020-09-25 | 南京林业大学 | A method for detecting exoglucanase activity in litter |
| CN111551528A (en) * | 2020-01-19 | 2020-08-18 | 南京林业大学 | Method for detecting activity of litter acid phosphatase |
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| EP0247373A2 (en) * | 1986-04-30 | 1987-12-02 | Europäische Atomgemeinschaft (Euratom) | 7-(Phenylacetamido)-4-alkyl coumarins, process for their preparation as well as their application in processes for the fluorometric determination of the activity of hydrolases, especially of penicilline G acylase |
| CN101082567A (en) * | 2006-06-02 | 2007-12-05 | 中国科学院沈阳应用生态研究所 | Analytical method for detecting hydroxyl amine reducing ferment activity in soil |
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| EP0247373A2 (en) * | 1986-04-30 | 1987-12-02 | Europäische Atomgemeinschaft (Euratom) | 7-(Phenylacetamido)-4-alkyl coumarins, process for their preparation as well as their application in processes for the fluorometric determination of the activity of hydrolases, especially of penicilline G acylase |
| CN101082567A (en) * | 2006-06-02 | 2007-12-05 | 中国科学院沈阳应用生态研究所 | Analytical method for detecting hydroxyl amine reducing ferment activity in soil |
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