CN111474127A - Improved nitrite reductase assay - Google Patents
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- 108010025915 Nitrite Reductases Proteins 0.000 title claims abstract description 26
- 238000003556 assay Methods 0.000 title description 2
- 239000002689 soil Substances 0.000 claims abstract description 57
- 238000000034 method Methods 0.000 claims abstract description 36
- RUFPHBVGCFYCNW-UHFFFAOYSA-N alpha-aminonaphthalene Natural products C1=CC=C2C(N)=CC=CC2=C1 RUFPHBVGCFYCNW-UHFFFAOYSA-N 0.000 claims abstract description 16
- 230000000694 effects Effects 0.000 claims abstract description 16
- 238000005259 measurement Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 83
- 239000000523 sample Substances 0.000 claims description 62
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000012496 blank sample Substances 0.000 claims description 18
- 238000002835 absorbance Methods 0.000 claims description 17
- 239000013068 control sample Substances 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 17
- 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 description 15
- 239000008103 glucose Substances 0.000 claims description 15
- 239000012153 distilled water Substances 0.000 claims description 12
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 239000012224 working solution Substances 0.000 claims description 8
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 238000007605 air drying Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 239000012047 saturated solution Substances 0.000 claims description 2
- WPUINVXKIPAAHK-UHFFFAOYSA-N aluminum;potassium;oxygen(2-) Chemical compound [O-2].[O-2].[Al+3].[K+] WPUINVXKIPAAHK-UHFFFAOYSA-N 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 19
- 230000008569 process Effects 0.000 abstract description 15
- 230000006837 decompression Effects 0.000 abstract description 13
- 238000007254 oxidation reaction Methods 0.000 abstract description 9
- 238000012545 processing Methods 0.000 abstract description 4
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 230000008859 change Effects 0.000 abstract description 2
- 238000006911 enzymatic reaction Methods 0.000 abstract description 2
- 239000000852 hydrogen donor Substances 0.000 abstract description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- 229910002651 NO3 Inorganic materials 0.000 description 11
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 11
- 238000000605 extraction Methods 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- 238000005086 pumping Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000005484 gravity Effects 0.000 description 4
- VKJKEPKFPUWCAS-UHFFFAOYSA-M potassium chlorate Chemical compound [K+].[O-]Cl(=O)=O VKJKEPKFPUWCAS-UHFFFAOYSA-M 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 3
- 108090000913 Nitrate Reductases Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 2
- AZFNGPAYDKGCRB-XCPIVNJJSA-M [(1s,2s)-2-amino-1,2-diphenylethyl]-(4-methylphenyl)sulfonylazanide;chlororuthenium(1+);1-methyl-4-propan-2-ylbenzene Chemical compound [Ru+]Cl.CC(C)C1=CC=C(C)C=C1.C1=CC(C)=CC=C1S(=O)(=O)[N-][C@@H](C=1C=CC=CC=1)[C@@H](N)C1=CC=CC=C1 AZFNGPAYDKGCRB-XCPIVNJJSA-M 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000004304 potassium nitrite Substances 0.000 description 2
- 235000010289 potassium nitrite Nutrition 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 229950000244 sulfanilic acid Drugs 0.000 description 2
- 230000001360 synchronised effect Effects 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 229910017912 NH2OH Inorganic materials 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 1
- 235000011126 aluminium potassium sulphate Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000009096 changqing Substances 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229940050271 potassium alum Drugs 0.000 description 1
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Substances [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
Classifications
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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
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N21/11—Filling or emptying of cuvettes
-
- 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
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
- G01N21/274—Calibration, base line adjustment, drift correction
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- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Mathematical Physics (AREA)
- Theoretical Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an improved method for determining nitrite reductase in soil, which comprises the steps of reacting nitrite with sulfanilic acid- α -naphthylamine reagent in the presence of hydrogen donor under anaerobic conditions, and determining NO before and after enzymatic reaction2 ‑-the amount of change in N to indicate nitrite reductase activity. The vacuum drier is adopted to replace a decompression triangular flask to serve as a culture container, so that the culture environment can be guaranteed to be anaerobic, the operation process can be simplified, and data errors caused by culture condition differences are reduced. Simultaneously, a certain amount of KClO is added during the preparation of a test curve and a standard curve3Can inhibit NO2 ‑Oxidation to NO3 ‑Reduction of NO2 ‑The data accuracy is improved. The method has high reproducibility, is simple and feasible, reduces errors, ensures that the stability of the measurement result between parallel processing is higher, and the measurement result is closer to a true value.
Description
Technical Field
The invention relates to the technical field of soil enzyme activity determination, in particular to an improved determination method of nitrite reductase.
Background
Under the action of nitrate reductase, nitrite reductase and hydroxyamine reductase, nitrate nitrogen in soil can be reduced into ammonia. The strength of deamination in soil nitrogen conversion can be understood by measuring the activity of these enzymes in soil. In addition, the research on the activity of nitrite reductase in soil has important significance for reasonably applying fertilizer and reducing the loss of nitrogen in soil.
In the traditional nitrite reductase measuring method, in order to ensure that the soil culture condition is an anaerobic environment, the soil is cultured in a decompression triangular flask, and the decompression triangular flask is subjected to air extraction treatment during culture. In the actual operation process, under the condition that the number of samples is large, the air exhaust process consumes long time, the experiment progress is delayed, and the error of the experiment result can be caused due to asynchronous sample treatment. And the tightness of the decompression triangular flask is difficult to guarantee, which is also one of the causes of experimental error. Meanwhile, the enzyme to be detected is NO in soil2 -Conversion to NH2Catalytic enzyme of OH, NO2 -The amount of (b) represents the activity of nitrite reductase, and NO2 -Is easily oxidized into NO3 -(NO2 -+0.5O2→NO3 -) So that there is a large deviation between the measured results. Therefore, the final detection result has poor repeatability, the determination method of the nitrite reductase needs to be improved, the experimental error is reduced, the deviation between the measured results is reduced, and the experimental repeatability is increased.
Disclosure of Invention
In view of the above-mentioned prior art, it is an object of the present invention to provide an improved method for measuring nitrite reductase. Through the improvement of the test means and the test process, the test error is reduced, the deviation between the measured results is reduced, and the repeatability of the detection method is good.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an improved method for determining nitrite reductase, which comprises the following steps:
(1) sample culture: adding CaCO into soil sample to be tested3And KNO with the mass fraction of 5%2Uniformly mixing the solution, and adding a glucose solution with the mass fraction of 10% as a sample to be detected; taking a soil sample with equal mass to be measured, adding CaCO3And KNO with the mass fraction of 5%2Mixing the solution, adding distilled water instead of glucose solution as a control sample; mixing CaCO3And KNO with the mass fraction of 5%2Uniformly mixing the solutions, adding a glucose solution with the mass fraction of 10% as a soilless blank sample, simultaneously placing the three samples in the same vacuum drier for vacuumizing, and placing the vacuum drier in a constant-temperature incubator for culturing;
(2) and (3) detection: after the culture is finished, deionized water, an Alkalite solution and KClO are added into the three samples3Standing the solution for 20min, mixing uniformly, filtering, absorbing the filtrate, adding distilled water and sulfanilic acid- α -naphthylamine solution, fixing the volume after developing, carrying out color comparison at 520nm after fixing the volume, and respectively obtaining the absorbance of a soilless blank sample, a control sample and a sample to be detected;
(3) drawing a standard curve: NaNO gradient to different concentrations2Adding sulfanilic acid- α -naphthylamine solution and KClO into the working solution3And (3) carrying out colorimetric determination on the solution at the wavelength of 520nm after constant volume, drawing a standard curve by taking the absorbance as the ordinate and the concentration as the abscissa, and obtaining the concentration (C) of the soilless blank sample according to the absorbances of the soilless blank sample, the control sample and the sample to be detected obtained in the step (2)Air conditioner) Concentration of control sample (C)To pair) And the concentration of the sample to be measured (C)Sample (A))；
(4) And (3) calculating the result: activity of nitrite reductase [ mgNO ] in soil2 --N/g/24h(d)]＝C0Vf/dwt
In the formula:
C0(NO in sample solution)2 -Change in concentration) ([ C ]Air conditioner-(CSample (A)-CTo pair)]；
V is the volume of the liquid to be detected;
f is a division multiple;
and dwt is the mass of the air-dried soil sample.
Preferably, in the step (1), the soil sample to be measured needs to be air-dried for 72 hours before measurement, and the water content of the soil sample to be measured before air-drying is 45%.
Preferably, in step (1), the soil sample to be tested is mixed with CaCO3、KNO2The adding amount ratio of the solution to the glucose solution is 1 g: 20 mg: 1m L: 1m L, CaCO added to the soilless blank sample, the control sample and the sample to be detected3Same mass of KNO added2The concentration and the volume of the solution are the same, the concentration and the volume of the glucose solution added into the soilless blank sample and the sample to be detected are the same, and the reference sample replaces the glucose solution with distilled water with equal mass.
Preferably, in the step (1), the temperature of the constant temperature incubator is 30 ℃, and the incubation time is 24 h.
Preferably, in the step (1), the pressure for vacuumizing is 0.1MPa, and the time is 20 min.
Preferably, in the step (2), the potassium alum solution is a saturated solution.
Preferably, in step (2), KClO3The ratio of the addition amount of the solution to the addition amount of the soil sample to be detected is 1m L: 1g, and the KClO3The concentration of the solution was 3.2 g/L.
Preferably, in step (2), the ionic water, the solution of Alkalite and KClO3The volume ratio of the solution is 50:1:1, and the volume ratio of the suction filtrate, the distilled water and the sulfanilic acid- α -naphthylamine solution is 1:5: 4.
Preferably, in step (3), KClO3The addition amount and the concentration of the solution are the same as those of the KClO in the step (2)3The amount and concentration of the added solution were the same.
Preferably, in the step (4), the division multiple is 54m L/1 m L.
The invention has the beneficial effects that:
(1) in the invention, the samples are placed in the same vacuum drier at the same time for synchronous air extraction treatment to replace the one-by-one air extraction treatment of the vacuum triangular flasks, so that the consistency of the culture conditions of the samples is ensured, the operation process is simplified, the air extraction time is shortened, and the experimental error is reduced. And the air is synchronously pumped, so that the difference of experimental results caused by the processing time difference is reduced, and the data accuracy is improved. The vacuum triangular bottle sealing process is difficult to operate, the air leakage phenomenon is easy to occur, and the air valve is closed after the vacuum dryer finishes pumping air, so that the experimental operation is simplified, and the experimental time is shortened.
(2) In the process of adding the reagent subsequently after the sample culture is finished, the triangular flask for containing the sample needs to be opened, so that air is mixed, and NO can be generated by oxygen in the air2 -Oxidation to NO3 -. In the invention, KClO with low concentration is added in the measuring process of nitrite reductase and the drawing process of a standard curve3Inhibition of NO2 -Oxidation to NO3 -Thereby reducing NO in the leaching solution2 -The consumption of the device enables the experimental result to be more accurate and the stability of the measurement result between parallel processing to be higher.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, nitrite reductase activity varies widely with vacuum, and therefore is subject to wide systematic errors during operation. And easily mix air and NO in the test operation process2 -Oxidation to NO3 -So that the measured value is larger than the true value.
Based on this, it was an object of the present invention to provide an improved method for the determination of nitrite reductase. The improvement is carried out from two aspects of a test device and a test process, factors which may cause inaccurate test data are improved, the measured result is closer to a true value, the test repeatability is improved, and the error of the test result is reduced.
Nitrate in soil is converted into ammonia under the action of nitrate reductase, nitrite reductase and hydroxyamine reductase, and the process is as follows:
NO3 -→NO2 -→NH2OH→NH4 +
determination of NO before and after enzymatic reaction by reaction of nitrite with sulfanilic acid- α -naphthylamine solution in the presence of hydrogen donor under anaerobic conditions2 -The amount of change in N, which is indicative of nitrite reductase activity.
The invention adopts the mode that the samples are placed in the same vacuum drier at the same time to carry out synchronous air extraction treatment to replace the one-by-one air extraction treatment of the decompression triangular bottles, thereby ensuring the consistency of the culture conditions of the samples, simplifying the operation process, reducing the air extraction time, reducing the experimental error and shortening the experimental time.
In the process of adding the reagent subsequently after the sample is cultured, the triangular flask for containing the sample needs to be opened, so that air is mixed, and NO can be generated by oxygen in the air2 -Oxidation to NO3 -. This is why NO is present in the soil2 -Has an oxidation rate far greater than that of NO2 -The reduction rate of (2). KClO3Is a strong oxidant, and NO is in soil2 -The contents of the components are different, and after repeated experiments, the KClO is found when the contents are the same3When the ratio of the addition amount of the KClO to the amount of the test soil is 1m L: 1g, the ratio of the addition amount of the KClO to the amount of the test soil is3Not only can not remove NO in soil2 -Oxidation to NO3 -And also effective in inhibiting NO2 -Oxidation to NO3 -Therefore, KClO is added at a low concentration during the measurement of nitrite reductase3The test proves that when KClO is used as3At a concentration of 3.2 g/L, it inhibits NO2 -The effect of oxidation is best. In order to keep the consistency of the test in the process of drawing the standard curve, KClO with equal volume and concentration in the detection process is added3Solution, thereby reducing NO in the leach liquor2 -The consumption can reduce the deviation of the test result and improve the test repeatability.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Example (b):
(1) preparation of test solutions:
and (3) preparing the potassium chlorate solution, namely accurately weighing 0.320g of potassium chlorate, dissolving the potassium chlorate in 60m L deionized water, transferring the potassium chlorate solution to a 100m L volumetric flask, and metering the volume by using the deionized water.
Preparing a potassium nitrite solution: 5.0g analytically pure KNO2Dissolved in deionized water and diluted to 1L.
Preparation of glucose solution 10.0g of analytically pure glucose was dissolved in deionized water and diluted to 1L.
Preparing a potassium nitrite standard solution: accurately weighing analytically pure KNO21.500g of the solution was dissolved in 10m L of water and diluted 100 times before use to prepare a working solution (1m L working solution contained 0.001 mgNO)2 -)。
Preparing 0.1 percent α -naphthylamine solution (a) and 0.5 percent p-aminobenzenesulfonic acid solution (b) from acetic acid with the specific gravity of 1.04, respectively, mixing the equal volumes of the solutions (a) and (b) before use, preparing acetic acid with the specific gravity of 1.04 (which is equivalent to 35 percent by weight of acetic acid), taking 175m L100 percent of acetic acid, and fixing the volume to 500m L, namely 35 percent, weighing 0.2g α -naphthylamine from the acetic acid with the specific gravity of 1.04, dissolving the acetic acid with the specific gravity of 35 percent, and fixing the volume to 200m L, wherein the solution (a) is obtained, weighing 1.0g p-aminobenzenesulfonic acid, dissolving the acetic acid with the weight percent of 35 percent, and fixing the volume to 200m L, wherein the solution (b) is obtained.
(2) Naturally air-drying the soil with the water content of 45% for 72 hours, preparing three 10m L common triangular flasks, marking one flask without soil sample as a soilless blank sample, respectively adding 1g of air-dried soil sample into the other two flasks as a control sample and a sample to be detected, and adding 20mg of CaCO into the three common triangular flasks3，1mL 5％KNO2Mixing the solution, adding 1m L glucosePutting all the triangular flasks in the same vacuum drier at the same time, using a miniature air pump to pump air to ensure that the culture environment is anaerobic, closing an air valve after the air pumping is finished, putting the vacuum drier in a 30 ℃ constant temperature incubator to culture for 24 hours, and respectively adding 50m L deionized water, 1m L Alkalite solution and 1m L KClO with the concentration of 3.2 g/L into the three triangular flasks after the culture is finished3Standing the solution for 20min, uniformly mixing, filtering, sucking 1m L filtrate into a 50m L volumetric flask, adding 5m L distilled water and 4m L sulfanilic acid- α -naphthylamine solution, performing color development, fixing the volume, performing color comparison at 520nm after fixing the volume, and respectively determining the absorbance of a blank sample, a control sample and a sample to be detected.
(3) Drawing a standard curve, wherein NaNO of 1-10m L0.001.001 g/L is respectively taken2The solution is subjected to constant volume in a 50m L volumetric flask to prepare NaNO with different concentration gradients2Working solution, 1m L sulfanilic acid- α -naphthylamine solution and 1m L KClO with concentration of 3.2 g/L are respectively added into each working solution3Fixing the volume of the solution after 15 minutes, measuring the absorbance at the wavelength of 520nm, drawing a standard curve by taking the absorbance as the ordinate and the concentration as the abscissa, and inputting the ordinate according to the absorbance of the blank sample, the control sample and the sample to be measured obtained in the step (1) to obtain an abscissa blank sample (C)Air conditioner) Control sample (C)To pair) And a sample to be tested (C)Sample (A)) The concentration of (c).
(4) And (3) calculating the result:
activity of nitrite reductase [ mgNO ] in soil2 --N/g/24h(d)]＝C0Vf/dwt formula:
C0(NO in sample solution)2 -Change in concentration) ([ C ]Air conditioner-(CSample (A)-CTo pair)]；
CAir conditioner: obtaining NO from the blank light absorption value of the soil2 -Concentration;
Csample (A): calculating NO from standard curve of light absorption value of sample2 -Concentration;
Cto pair: obtaining NO from standard curve with soil and without substrate light absorption value2 -Concentration;
v is the volume of the solution to be measured (50m L);
f is a division multiple (54m L/1 m L);
and dwt is the quality of the air-dried soil.
The above experiment was repeated three times to obtain an average value as the final activity value of the soil nitrite reductase of the sample.
Test examples
Surface soil (0-20cm) is collected from one wheat field in Changqing district of Jinan city and taken back to a laboratory for preculture for one week, and the water content of the soil is adjusted to 45%. Sieving the soil with a 2mm sieve to pick out impurities such as stones, plant residues and the like. And (5) air-drying the soil for 72 times to obtain a soil sample to be detected. The soil sample is subjected to the following three treatments:
group A, 15 decompression triangular flasks are taken, wherein the 125m L decompression triangular flasks with the serial numbers of 1-1,1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9 and 1-10 are respectively added with 1g of soil sample to be tested, the decompression triangular flasks with the serial numbers of 1-11,1-12,1-13,1-14 and 1-15 are not added with the soil sample, and the 15 decompression triangular flasks are respectively added with 20mg of CaCO31m L5% KNO2Solution and 1m L KClO3And (3) uniformly mixing the solution, adding 1m L10% glucose solution into a decompression triangular flask with the number of 1-1,1-2,1-3,1-4,1-5,1-11,1-12,1-13,1-14 and 1-15, adding equivalent distilled water into the decompression triangular flask with the number of 1-6,1-7,1-8,1-9 and 1-10 to replace a substrate, pumping the 15 decompression triangular flasks one by one for 3 minutes, sealing, shaking the decompression triangular flask, and culturing in a 30 ℃ constant temperature incubator for 24 hours.
Group B: taking 15 common triangular bottles with the numbers of 2-1,2-2,2-3,2-4,2-5,2-6,2-7,2-8,2-9 and 2-10, respectively adding 1g of soil sample to be detected, and adding no soil sample into the common triangular bottles with the numbers of 2-11,2-12,2-13,2-14 and 2-15. 20mg of CaCO is respectively added into 15 common triangular flasks3And 1m L5% KNO2And (3) uniformly mixing the solution, adding 1m L10% of glucose solution into a common triangular flask with the number of 2-1,2-2,2-3,2-4,2-5,2-11,2-12,2-13,2-14 and 2-15, and adding equivalent distilled water into the common triangular flask with the number of 2-6,2-7,2-8,2-9 and 2-10 to replace the substrate.
Group C: taking 15 common triangular flasks numbered 3-1,3-2,3-3,3-4,3-5,3-6,3-7,3-8,3-9 and 3-101g of soil sample to be detected is added into the triangular flask, and the soil sample is not added into the common triangular flasks with the numbers of 3-11,3-12,3-13,3-14 and 3-15. 20mg of CaCO is respectively added into 15 common triangular flasks31m L5% KNO2And (3) uniformly mixing the solution, adding 1m L10% of glucose solution into a common triangular flask with the number of 3-1,3-2,3-3,3-4,3-5,3-11,3-12,3-13,3-14 and 3-15, and adding equivalent distilled water into the common triangular flask with the number of 3-6,3-7,3-8,3-9 and 3-10 to replace a substrate.
All the common triangular flasks of the group B and the group C are placed in the same vacuum drier at the same time, the culture environment is guaranteed to be anaerobic by pumping air through a miniature air pump after the cover is closed, and the air valve is closed after the pumping air is finished. The vacuum drier is placed in a 30 ℃ constant temperature incubator for 24 h.
After the incubation was completed, 50m L deionized water and 1m L Alkalite solution were added to each of the three groups (KClO with 1m L concentration of 3.2 g/L was also added to group C)3Solution), standing for 20min, mixing uniformly, filtering, sucking 1m L filtrate into a 50m L volumetric flask, adding 5m L distilled water and 4m L sulfanilic acid- α -naphthylamine solution, fixing the volume after color development, carrying out color comparison at 520nm after the fixing the volume, and obtaining 5 absorbances of a soilless blank sample, a control sample and a sample to be detected in each group.
Respectively taking 1-10m L0.001 g/L NaNO2The solution is subjected to constant volume in a 50m L volumetric flask to prepare NaNO with different concentration gradients2Working solution, 1m L sulfanilic acid- α -naphthylamine solution and 1m L KClO with concentration of 3.2 g/L are respectively added into each working solution3The solution was fixed to a constant volume after 15 minutes, and the absorbance was measured at a wavelength of 520nm, and a standard curve was drawn with the absorbance as the ordinate and the concentration as the abscissa. The formula of the obtained standard curve is as follows: y is 1.192x + 0.131; y is the absorbance of the measured solution at 520nm, and x is NO in the solution2 -The concentration of (c).
Inputting the absorbance of each group of blank samples, the absorbance of the control sample and the absorbance of the sample to be detected into a vertical coordinate to obtain each group of blank samples (C) of a horizontal coordinateAir conditioner) Control sample (C)To pair) And a sample to be tested (C)Sample (A)) Each of the blank samples (C) was obtained at 5 concentrationsAir conditioner) Concentration and control sample (C)To pair) Average of concentrationAnd (4) average value. 5 samples to be tested (C)Sample (A)) The concentrations of (A) and (B) are respectively input into the activity of the nitrite reductase [ mgNO ] of the soil2 --N/g/24h(d)]＝C0Vf/dw, the results obtained are shown in Table 1, for 5 values of soil nitrite reductase activity per group.
Table 1: value of Activity of nitrite reductase in soil
As can be seen from the data in Table 1, the standard error of group A is larger than that of group B and group C, which indicates that the vacuum drier can provide a more consistent culture environment as a culture container, improve the stability of data, and improve the experimental precision. The activity of the nitrite reductase in the soil sample of the group B is slightly higher than that of the soil sample of the group C, which indicates that KClO is added3Inhibit NO in the sample2 -Is oxidized to NO3 -The test result is closer to real NO2 -And (4) reducing the amount. Therefore, the standard error of the group C is the minimum, and the group C, namely the method disclosed by the invention, can reduce the experimental error and reduce the deviation between the measured results, so that the stability of the measured results between parallel processing is higher.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. An improved method for determining nitrite reductase, said method comprising the steps of:
(1) sample culture: adding CaCO into soil sample to be tested3And KNO with the mass fraction of 5%2Uniformly mixing the solution, and adding a glucose solution with the mass fraction of 10% as a sample to be detected; taking a soil sample with equal mass to be measured, adding CaCO3And KNO with the mass fraction of 5%2Mixing the solution, adding distilled water instead of glucose solutionThe solution was used as a control sample; mixing CaCO3And KNO with the mass fraction of 5%2Uniformly mixing the solutions, adding a glucose solution with the mass fraction of 10% as a soilless blank sample, simultaneously placing the three samples in the same vacuum drier for vacuumizing, and placing the vacuum drier in a constant-temperature incubator for culturing;
(2) and (3) detection: after the culture is finished, deionized water, an Alkalite solution and KClO are added into the three samples3Standing the solution for 20min, mixing uniformly, filtering, absorbing the filtrate, adding distilled water and sulfanilic acid- α -naphthylamine solution, fixing the volume after developing, carrying out color comparison at 520nm after fixing the volume, and respectively obtaining the absorbance of a soilless blank sample, a control sample and a sample to be detected;
(3) drawing a standard curve: NaNO gradient to different concentrations2Adding sulfanilic acid- α -naphthylamine solution and KClO into the working solution3And (3) carrying out colorimetric determination on the solution at the wavelength of 520nm after constant volume, drawing a standard curve by taking the absorbance as the ordinate and the concentration as the abscissa, and obtaining the concentration (C) of the soilless blank sample according to the absorbances of the soilless blank sample, the control sample and the sample to be detected obtained in the step (2)Air conditioner) Concentration of control sample (C)To pair) And the concentration of the sample to be measured (C)Sample (A))；
(4) And (3) calculating the result: activity of nitrite reductase [ mgNO ] in soil2 --N/g/24h(d)]＝C0Vf/dwt
In the formula:
C0(NO in sample solution)2 -Change in concentration) ([ C ]Air conditioner-(CSample (A)-CTo pair)]；
V is the volume of the liquid to be detected;
f is a division multiple;
and dwt is the mass of the air-dried soil sample.
2. The method according to claim 1, wherein in the step (1), the soil sample to be measured needs to be air-dried for 72 hours before measurement, and the water content of the soil sample to be measured before air-drying is 45%.
3. According to claimThe method according to claim 1, wherein in the step (1), the soil sample to be tested is contacted with CaCO3、KNO2The ratio of the added amount of the solution to the added amount of the glucose solution is 1g to 20mg to 1m L to 1m L.
4. The method according to claim 1, wherein in the step (1), the temperature of the incubator is 30 ℃ and the incubation time is 24 hours.
5. The method according to claim 1, wherein in the step (1), the pressure of the evacuation is 0.1MPa for 20 min.
6. The method according to claim 1, wherein in the step (2), the solution of potassium aluminate is a saturated solution.
7. The method according to claim 1, wherein KClO is used in the step (2)3The ratio of the addition amount of the solution to the addition amount of the soil sample to be detected is 1m L: 1g, and the KClO3The concentration of the solution was 3.2 g/L.
8. The method according to claim 1, wherein in the step (2), the ionic water, the Alkalite solution and KClO3The volume ratio of the solution is 50:1:1, and the volume ratio of the suction filtrate, the distilled water and the sulfanilic acid- α -naphthylamine solution is 1:5: 4.
9. The method according to claim 1, wherein KClO is used in the step (3)3The addition amount and the concentration of the solution are the same as those of the KClO in the step (2)3The amount and concentration of the added solution were the same.
10. The method of claim 1, wherein in the step (4), the division multiple is 54m L/1 m L.
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