AU2020102399A4 - An Analytical method for detecting activity of soil glutamine synthetase - Google Patents
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- 239000002689 soil Substances 0.000 title claims abstract description 67
- 102000005396 glutamine synthetase Human genes 0.000 title claims abstract description 27
- 108020002326 glutamine synthetase Proteins 0.000 title claims abstract description 27
- 230000000694 effects Effects 0.000 title claims abstract description 25
- 238000004458 analytical method Methods 0.000 title claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 238000002835 absorbance Methods 0.000 claims abstract description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000006228 supernatant Substances 0.000 claims abstract description 10
- 238000005303 weighing Methods 0.000 claims abstract description 8
- 150000001408 amides Chemical class 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 6
- 239000002245 particle Substances 0.000 claims abstract description 5
- 239000007787 solid Substances 0.000 claims abstract description 5
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 49
- WTKZEGDFNFYCGP-UHFFFAOYSA-N Pyrazole Chemical compound C=1C=NNC=1 WTKZEGDFNFYCGP-UHFFFAOYSA-N 0.000 claims description 17
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims description 16
- CODWMKANXWBHSR-VKHMYHEASA-N NC([C@H](CCC(O)=O)NO)=O Chemical compound NC([C@H](CCC(O)=O)NO)=O CODWMKANXWBHSR-VKHMYHEASA-N 0.000 claims description 13
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 claims description 10
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 claims description 10
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 claims description 8
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 8
- 239000004223 monosodium glutamate Substances 0.000 claims description 8
- -1 hydroxyglutamin amide Chemical compound 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000011550 stock solution Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 2
- 239000012089 stop solution Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 6
- 244000005700 microbiome Species 0.000 description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- 229930195712 glutamate Natural products 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- AHWNEEBQYYTAIR-VKHMYHEASA-N N[C@@H](CCC(=O)NO)C(=O)N Chemical compound N[C@@H](CCC(=O)NO)C(=O)N AHWNEEBQYYTAIR-VKHMYHEASA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- MKGIQRNAGSSHRV-UHFFFAOYSA-N 1,1-dimethyl-4-phenylpiperazin-1-ium Chemical compound C1C[N+](C)(C)CCN1C1=CC=CC=C1 MKGIQRNAGSSHRV-UHFFFAOYSA-N 0.000 description 1
- DJHGAFSJWGLOIV-UHFFFAOYSA-K Arsenate3- Chemical compound [O-][As]([O-])([O-])=O DJHGAFSJWGLOIV-UHFFFAOYSA-K 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- AEFLONBTGZFSGQ-VKHMYHEASA-N L-isoglutamine Chemical compound NC(=O)[C@@H](N)CCC(O)=O AEFLONBTGZFSGQ-VKHMYHEASA-N 0.000 description 1
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa 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
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229940000489 arsenate Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- YVGZXTQJQNXIAU-VKHMYHEASA-N glutamine hydroxamate Chemical compound OC(=O)[C@@H](N)CCC(=O)NO YVGZXTQJQNXIAU-VKHMYHEASA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- GVPLVOGUVQAPNJ-UHFFFAOYSA-M potassium;hydron;trioxido(oxo)-$l^{5}-arsane Chemical compound [K+].O[As](O)([O-])=O GVPLVOGUVQAPNJ-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000003971 tillage Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/251—Colorimeters; Construction thereof
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
- C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/24—Earth materials
- G01N33/245—Earth materials for agricultural purposes
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- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention relates to an analytical method for detecting the activity of glutamine
synthetase in soil, which comprises the following steps : weighing an air-dried soil sample
and fumigating with chloroform; adding 1 gram of each sample into n (n is more than or
equal to 6) borosilicate test tubes, where 0.3-0.5ml of reaction solution is added into n/2
tubes, and 0.3-0.5ml of control solution is added into the other n/2 tubes; adding glutamine
solution into all test tubes and oscillating the test tubes at room temperature by using a
reciprocating oscillator then adding 4.0-4.8ml of termination solution to each; standing until
solid particles are precipitated, then centrifuging the supernatant; carrying out colorimetric
determination on the supernatant with a spectrophotometer; obtaining an absorbance value,
and calculating the concentration of 5-N-hydroxyglutamyl amide in a measured solution
according to the absorbance value and the slope. There are 4 advances about this invention:
1) first time determining the method for determining the glutamine synthetase in soil; 2)
reducing the dependence on equipment requirements; 3) the method is high in accuracy and
easy to operate; and 4) the result is stable and reliable and good in reproducibility.
Description
PATENTS ACT 1990
An Analytical method for detecting activity of soil glutamine synthetase
The invention is described in the following statement:-
An Analytical method for detecting activity of soil glutamine synthetase
The invention relates to determination of glutamine synthetase activity in soil, in particular to an analytical method for detecting the glutamine synthetase activity in soil.
Glutamine synthetase in soil catalyzes the reaction between ammonia and glutamate to produce glutamine:
(M)2+
Glutamate+NH3+ATP , glutamine+ADP+Pi
Nitrogen is the most important nutrient element for the growth and reproduction of microorganisms in soil, and bacteria and fungi can utilize a wide variety of organic and inorganic nitrogen compounds. The utilization ratio of organic nitrogen and inorganic nitrogen by microorganisms strongly affects the circulation of nitrogen in soil and the competition of microorganisms and plants for nitrogen. NH 4*is an inorganic nitrogen form which can be directly utilized in soil, and microorganisms have two mechanisms for utilizing the inorganic nitrogen, both resulting in the synthesis of glutamate by amination of alpha-ketoglutaric. One of these pathways is catalyzed by glutamine synthetase (E.C.6.3.1.2). Glutamine synthetase has stronger capture ability to NH 4* and stronger affinity (smaller Km value), so the enzyme is more effective when the concentration of NH4 * is lower. The determination of the activity of the enzyme is helpful for understanding the strength of microorganisms in different utilization ways of inorganic nitrogen, and has important significance for exploring the form of soil nitrogen.
The invention aims to provide an analytical method for detecting the activity of glutamine synthetase in soil. The method is the first time to determine the activity of the soil glutamyl aminase, and the determination of different soil samples shows that the analysis result is accurate and reliable, and the analysis reproducibility is better.
In order to achieve the purpose, the technical scheme adopted by the invention is as follow:
The invention relates to an analytical method for detecting the activity of glutamine synthetase in soil,
1) Weighing an air-dried soil sample with a sieve of 2-4 mm, and fumigating the air-dried soil sample with chloroform;
2) Weighing 1 gram of fumigated soil in n (n >=6) borosilicate test tubes, wherein 0.3-0.5 ml of reaction solution is added to each of the n/2, and 0.3-0.5 ml of control solution is added to the other n/2;
3) Adding 1.5-1.8 ml of glutamyl amide solution (0.2 M) to all tubes.
4) Oscillating the test tubes at room temperature for 50 -70 minutes by using a reciprocating oscillator;
5) Adding 4.0 - 4.8 ml of termination solution after oscillating;
6) Standing for 3 minutes; taking 1.3 ml of supernatant for centrifugation after the solid particles are precipitated;
7) Taking 1ml of supernatant obtained after centrifugation;
8) Colorimetric at 540 nm with a spectrophotometer;
9) So as to obtain an absorbance value A;
10) Calculating the concentration S of 5-N-hydroxyglutamyl amide in the measured solution according to the absorbance value A and the slope b, S=A*b, wherein the concentration in the sample is defined A Si, and the concentration in the control is defined as S2;
11) The activity of glutamine synthetase in soil was calculated in units of mmol hydroxyglutamyl amide kg dry soil h-i.
The calculation formula is [ (S-S2)*V/1000]*V/W. T=mmol hydroxyglutamyl amide kg dry soil h 1 .
Wherein Si is the concentration (mmol/L) of the hydroxyglutamyl amide in the sample, S2 is the concentration (mmol/L) of the hydroxyglutamyl amide in the control, V is the total volume (mL) of the leaching solution, W is the weight (g) of the soil sample, 1000 is the unit conversion coefficient, and T is the culture time (h) of the soil sample.
The method of making the work standard curve comprises the following steps: 1) preparing a standard solution: weigh 1.621 g of 5-N-hydroxyglutaminamide and dissolve it in 1000 ml of pyrazole solution to obtain a standard stock solution with the concentration of 10 mmol/L; 2) suck 0, 1, 2, 3, 4 and 5 ml of the standard stock solution into a cuvette, and add 4.0 ml - 4.8 ml of termination solution, then fill all samples into 10 ml to obtain a series of concentration solutions with the concentration of 0, 1, 2, 3, 4 and 5 mmol/L, colorimetric reaction under the condition of 540 nm afterwards, then make the standard curve based on the absorbance and sample concentration to obtain the slope b.
The reaction solution as stated consists of 10.5 ml of pyrazole buffer (pH 7.15), 0.9 ml of hydroxylamine hydrochloride (800 Mm), 0.1 ml of MnCL2 1-4H 20 (100 mM), 1.4 ml of KH 2AsO 4(280 mM); 0.14 ml of adenosine diphosphate 5'monosodium glutamate (40 mM), and the control solution is made of 11.95 ml of pyrazole buffer (pH 7.15), 0.9 ml of hydroxylamine hydrochloride, 0.1 ml of MnCL2-4H20, 0.14 ml of adenosine diphosphate 5' monosodium glutamate while the stop solution is made of 21 ml of hydrochloric acid, 20 g of trichloroformic acid and 55 g of FeClr6H20 dissolved in I L of deionized water.
The measuring principle of the method
In soil, besides the function explained above, glutamine synthetase catalyzes a conversion reaction, which is insensitive to adenosine acylation in the presence of Mn2' and stable in enzyme activity, and easy to determine. The product of the reaction, hydroxyglutamyl amide, reacts with ferric chloride to form a red chemical substance, which can be colorimetric determined at 540 nm. Based on this conversion reaction, the activity of glutamine synthetase is determined.
ADP, arsenate, Mn2+,
Glutamate+hydroxylamine , 5-N-hydroxyglutamine+NH3
Glutamine synthetase
1. Reagent preparation:
1) Reaction solution: 10.5 ml of pyrazole buffer (pH 7.15), 0.9 ml of hydroxylamine hydrochloride, 0.1 ml of MnCL2-4H2O, 1.4 ml of KH2AsO 4 , 0.14 ml of adenosine diphosphate 5'monosodium glutamate (this reagent is sufficient to measure 25 samples);
2) Control solution: 11.95 ml of pyrazole buffer (pH 7.15), 0.9 ml of hydroxylamine hydrochloride, 0.1 ml of MnCL2-4H20, 0.14 ml of adenosine diphosphate 5'monosodium glutamate (this reagent is sufficient to measure 25 samples);
3) The termination solution: 21 ml of hydrochloric acid, 20 g of trichloroformic acid and 55 g of FeCl3-6H20 are dissolved in IL of deionized water;
4)Pyrazole (whether this should be pyrazole or not) buffer (100 mM): 6.82 g of pyrazole is weighed and dissolved in 1 L of water;
5)Hydroxylamine hydrochloride (800Mm): 55.6 g of hydroxylamine hydrochloride is weighed and dissolved in 1 L of pyrazole solution;
6) MnCl (100 mM): 19.8 g of MnCl-4H20 is weighed and dissolved in 1 L of pyrazole solution;
7) Potassium hydrogen arsenate solution (280 mM): 50 g of KH2AsO 4 is weighed and dissolved in 1 L of pyrazole solution;
8) Adenosine diphosphate 5' monosodium glutamate solution (40mM): 17.01g of adenosine diphosphate 5'monosodium glutamate is weighed and dissolved in IL of pyrazole solution;
9) L-glutamyl amide solution (0.2 M): 29.3 g of L-glutamine are dissolved in 1 L of pyrazole solution.
2. Standard curve is made: Standard solution: 1.621g of 5-N-hydroxyglutamyl amide is weighed and dissolved in 1000 ml of pyrazole solution to obtain a standard stock solution with a concentration of 10 mmol/L. 1, 2, 3, 4 and 5 milliliters of the solution are absorbed and added into a cuvette and 4.0 to 4.8 milliliters of the termination solution is added afterwards, then fill all the samples to 10 milliliters and a series of concentration solutions with the concentration of 0, 1, 2, 3, 4 and 5 ml/ml are obtained. Colorimetric determination is carried out under the condition of 540 nanometers and a standard curve of the obtained absorbance and the concentration of the samples is drawn, and the slope b is obtained.
The operation steps are as follows:
1) Sieving soil sample with 2 - 4 mm sieve, weighing n grams of soil sample, and fumigating with chloroform for 4 hours;
2) Weighing and adding n portions of fumigated soil in n borosilicate test tubes (16*100mm) and each tube is Ig, wherein three of the tubes are added with 0.5 ml of reaction solution, and the other three are added with 0.5ml of control solution;
3) 1.7 ml of glutamyl amide solution is added to all tubes;
4) The test tube is oscillated for one hour at room temperature by using a reciprocating oscillator;
5)4.4 ml of termination solution is added after oscillating;
6) After settling for a few minutes to have the solid particles precipitated, 1.3 ml of supernatant is centrifuged (15000 rev min-1 , 1 min);
7) Taking 1ml of supernatant obtained after centrifugation;
8) Colorimetric at 540 nm by using a spectrophotometer;
9) So as to obtain an absorbance value A;
10) Calculating the concentration S of 5-N-hydroxyglutamamide in the determined solution according to the absorbance value A and the slope b, S=A*b, wherein the concentration in the sample is defined as Sl, and the concentration in the control is defined as S2
11) The activity of glutamine synthetase in soil was calculated in units of mmol hydroxyglutamyl amide kg dry soil h-1 .
The calculation formula is [(S-S2)*V/1000]/W.T=mmol hydroxyglutaminamide kg dry soil h 1 .
Wherein Si is the concentration (mmol/L) of the hydroxyglutamyl amide in the sample, S2 is the concentration (mmol/L) of the hydroxyglutamyl amide in the control, V is the total volume (mL) of the leaching solution, W is the weight (g) of the soil sample, 1000 is the unit conversion coefficient, and T is the culture time (h) of the soil sample.
Embodiment 1
The black soil used in this embodiment is collected from Helen ecological test station of the Chinese Academy of Sciences, and is provided with three different treatments: No. 1 is a control. It is the common soil which is cultured for more than 24 hours at 25°Cwithout any treatment and additive; No. 2 is the soil being added with a nitrification inhibitor DCD and cultured for more than 24 hours at 25°C; and No. 3 soil is added with a nitrification inhibitor DMPP and cultured for more than 24 hours at 25°C. The soil water content was 20% (air-dried soil weight). No.2 and No.3 soil are supplemented with nitrification inhibitors, both of which were 50 ppm. Glutamine synthetase activity in the three soils are tested by the means above.
The specific analytical steps of each soil are as follows:
1) Sieving soil sample with 2 - 4 mm sieve, weighing 6g soil sample, and fumigating with chloroform for 4hr;
2) Weighing and adding 6 portions of fumigated soil in 6 borosilicate test tubes (16*100mm), and each portion is Ig, wherein three of the six borosilicate test tubes are added with 0.5 ml of reaction solution, and the other three are added with 0.5ml of control solution);
3) 1.7 ml of glutamyl amide solution is added to all tubes;
4) The test tube is oscillated for one hour at room temperature by using a reciprocating oscillator;
5) 4.4ml of the termination solution is added after oscillating;
6) After settling for a few minutes to have the solid particles precipitated, 1.3 ml of supernatant is centrifuged (15000 rev min-1 , 1 min);
7) Taking 1ml of supernatant obtained after centrifugation;
8) Colorimetric at 540 nm by using a spectrophotometer;
9) So as to obtain an absorbance value A;
10) Calculating the concentration S of 5-N-hydroxyglutamamide in the determined solution according to the absorbance value A and the slope b, S=A*b, wherein the concentration in the sample is defined as S1, and the concentration in the control is defined as S2;
11) The activity of glutamine synthetase in soil was calculated in the unit of mmol hydroxyglutamyl amide kg dry soil 1 .
The test results are as follows:
Glutamine synthetase activity
(mmol of hydroxyglutamyl Difference
Dispose of Repeat amide kg of dry soil h) Stn significance deviation Measured (P<0.05) Mean value value
Repeat 1 2.2415
1 Repeat 2 2.3979 2.3334 0.0817 a
Repeat 3 2.3608
Repeat 1 4.1254
2 Repeat 2 4.1432 4.1748 0.0707 b
Repeat 3 4.2558
Repeat 1 3.5089
3 Repeat 2 3.6356 3.5600 0.0668 b
Repeat 3 3.5356
It can be seen from the data in the table that the activity of glutamine synthetase in soil significantly increased by adding nitrification inhibitor, and there was no significant difference between the second and third types of soil before adding nitrification inhibitor, which indicated that different types of nitrification inhibitor had little influence on the activity of glutamine synthetase in soil. The smaller standard difference showed that the deviation between the results of the analytical method was smaller, so as to the accuracy was higher and the reproducibility was good.
Embodiment 2
The three types of brown soil used for planting crops in this embodiment was collected at the Shenyang ecological experiment station of the Chinese Academy of
Sciences, wherein the No. 4 soil had been planted with rice, and the No. 5 soil had been planted with corn, while the No. 6 soil had been planted with soybean. The activity of glutamine synthetase was determined in the soil of three different tillage systems under the conditions of water content of 20% (air-dried soil weight) and temperature of 25°C for more than 24 hours. The detailed implementing process was the same as Embodiment 1.
The test results are as follows:
Glutamine synthetase activity
(mmol of hydroxyglutamyl Difference Standard Dispose of Repeat amide kg of dry soil h-i) . significance deviation Measured (P<0.05) Mean value value
Repeat 1 4.8842
1 Repeat 2 4.9987 4.9339 0.0587 B
Repeat 3 4.9189
Repeat 1 4.2879
2 Repeat 2 4.3147 4.2879 0.0267 c
Repeat 3 4.2613
Repeat 1 5.1546
3 Repeat 2 5.2209 5.1811 0.0351 a
Repeat 3 5.1678
The data in the table also shows the stability and high precision of the analysis results. It also showed that different farming systems had great effect on the activity of glutamine synthetase.
Claims (11)
1. An analytical method for detecting the activity of glutamine synthetase in soil is characterized in:
1) Weighing an air-dried soil sample with a sieve of 2-4 mm and fumigating the sample with chloroform;
2) Adding 1 gram of fumigated soil in n (n >=6) borosilicate test tubes, where 0.3 0.5 ml of reaction solution is added to each of the n/2, and 0.3-0.5 ml of control solution is added to each of the other n/2;
3) Adding 1.5-1.8 ml of glutamine solution (0.2 M) to all tubes.
4) Oscillating the test tube at room temperature for 50-70 minutes by using a reciprocating oscillator;
5) Adding 4.0-4.8 ml of termination solution after oscillating;
6) Standing for 3 minutes; taking 1.3 ml of supernatant for centrifugation after the solid particles are precipitated;
7) Taking 1 ml of supernatant obtained after centrifugation;
8) Colorimetric at 540 nm by using a spectrophotometer;
9) So as to obtain an absorbance value A;
10) Calculating the concentration S of 5-N-hydroxyglutamyl amide in the measured solution according to the absorbance value A and the slope b, S=A*b, wherein the concentration in the sample is defined as S1, and the concentration in the control is defined as S2;
11) The activity of glutamine synthetase in soil was calculated in units of mmol hydroxyglutamyl amide kg dry soil h 1 .
The calculation formula is [(S1-S2)*V/1000]*V/W.T=mmol hydroxyglutamyl amide kg dry soil h 1 .
Wherein, Sl is the concentration (mmol/L) of the hydroxyglutamyl amide in the sample; S2 is the concentration (mmol/L) of the hydroxyglutamyl amide in the control; V is the total volume (mL) of the leaching solution; W is the weight (g) of the soil sample; 1000 is the unit conversion coefficient; T is the culture time (h) of the soil sample.
2. The features of the analytical method according to claim 1 lie in the process to make a work standard curve: 1) preparing a standard solution: weigh 1.621 g of 5-N hydroxyglutamin amide and dissolve it in 1000 ml of pyrazole solution to get a standard stock solution with the concentration of 10 mmol/L; 2) suck 0, 1, 2, 3, 4 and 5 ml of the standard stock solution into a cuvette, and add 4.0 ml-4.8 ml of termination solution in, then fill all samples into 10 ml to obtain a series of concentration solutions with the concentration of 0, 1, 2, 3, 4 and 5 mmol/L, and colorimetric under the condition of 540 nm afterwards, then make the standard curve based on the absorbance and sample concentration to obtain the slope b.
3. The features of the analytical method according to claim 1 or 2 lie in the solutions, wherein the reaction solution is made of 10.5 ml of pyrazole buffer solution (pH 7.15), 0.9 ml of hydroxylamine hydrochloride (800 Mm), 0.1 ml of MnCL2 1. 4H20 (100 mM), 1.4 ml of KH2AsO 4 (280 mM) and 0.14 ml of adenosine diphosphate 5'monosodium glutamate (40 mM); the control solution is made of 11.95 ml of pyrazole buffer solution (pH 7.15), 0.9 ml of hydroxylamine hydrochloride, 0.1 ml of MnCL2-4H 20 and 0.14 ml of adenosine diphosphate 5'monosodium glutamate; the stop solution is made of 21 ml of hydrochloric acid, 20 g of trichloroformic acid, and 55 g of FeCl3. 6H 20 dissolved in 1 L of deionized water.
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