CN102108377A - Method for determining activity of soil arginine desaminase - Google Patents
Method for determining activity of soil arginine desaminase Download PDFInfo
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- CN102108377A CN102108377A CN2009102487779A CN200910248777A CN102108377A CN 102108377 A CN102108377 A CN 102108377A CN 2009102487779 A CN2009102487779 A CN 2009102487779A CN 200910248777 A CN200910248777 A CN 200910248777A CN 102108377 A CN102108377 A CN 102108377A
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Abstract
The invention relates to a method for determining the activity of soil arginine desaminase, which comprises the following steps: 1) weighting n fresh soil or precultured soil samples, placing in n conical flasks having plugs (wherein n is more than or equal to 4), respectively adding arginine substrate solution into each conical flask, uniformly mixing the arginine substrate solution with the soil, blocking each flask with a rubber plug, and then culturing for 3 hours at constant temperature; 2) after the culturing process is finished, adding a certain volume of potassium chloride solution into the flasks, oscillating, and filtering; 3) determining the quantity of generated ammonium ions by using the indophenol blue colorimetry; and 4) calculating the activity of arginine desaminase. The method for determining the activity of soil arginine desaminase has the following advantages: 1) compared with the traditional biochemical determining method, the method provided by the invention simplifies the operation steps; 2) the dependence on high equipment demand is reduced, and a common spectroscopic instrument can meet the demand; 3) the accuracy is high, and the operation can be easily performed; and 4) the result is stable and reliable, and the repeatability is good.
Description
Technical field
The present invention relates to the active mensuration of soil arginine desaminase, the active novel method of specifically a kind of detection soil arginine desaminase.
Background technology
Arginine desaminase in the soil is a kind of of arginine lyase, and it participates in arginic conversion cycles.Arginine is a kind of half necessary or necessary basic aminoacids of conditionality, hydrolysis generates ornithine and urea under the effect of arginine desaminase, the urea that forms is released in the soil for the plant absorbing utilization, this shows, arginine is a kind of important nitrogen storage, it can utilize minimum carbon in conjunction with maximum nitrogen, is the highest amino acid of N/C ratio in 18 kinds of necessary amino acid.
Therefore, the active power of arginine desaminase has influence on the nitrogen metabolism process of soil urea in the soil, and then influences the utilising efficiency of nitrogen.The activity of arginine desaminase is subjected to influencing strongly of edaphic condition such as moisture, temperature, the soil texture in the soil, also is subjected to farming operation system, control measures and different influence of planting plants simultaneously.Therefore, the active check of arginine desaminase in the soil is had very important significance.Up to the present, the relevant active mensuration of arginine desaminase does not have a kind of easy accurately and reliably measuring method, and this has limited its applied research greatly.
Summary of the invention
The object of the present invention is to provide the active analytical procedure of arginine desaminase in a kind of check soil.This method is on the principle basis of using for reference the related enzyme activity measuring method, at soil arginine desaminase in the nitrogen transformation metabolism functional performance and carried out method improvement, make analytical results more accurately reliable, it is better to analyze circulation ratio.
For achieving the above object, the technical solution used in the present invention is:
The active method of a kind of detection soil arginine desaminase,
1) taking by weighing n part 5-10g respectively crosses the bright soil of 1-2mm sieve or cultivates pedotheque in advance in the 150mL triangular flask, n 〉=4; The substrate arginine solution that adds 2～5mL, 10～12mol/L in triangular flask mixes soil and substrate solution, air-locked beyond the Great Wall soft rubber ball on triangular flask;
To wherein n-2 triangular flask constant temperature culture 170-190min in 36-38 ℃ of incubator, as sample;
2 remaining triangular flasks are constant temperature culture 170-190min under-19--21 ℃ condition, in contrast; To contrast triangular flask after cultivation finishes thaws at ambient temperature;
Other gets a triangular flask and does the reagent blank contrast simultaneously, does not promptly add soil, and all the other are handled with sample;
2) in above-mentioned triangular flask, add 40～60mL, 1.5～2mol/L KCI solution respectively, stopper beyond the Great Wall, vibration 55-65min filters;
3) draw 1～2mL filtrate respectively in the colour developing test tube, add 3～5mL, 1.5～2mol/L KCI solution, 1～3mL, 0.10～0.15mol/L sodium phenylate solution, 0.5～2mL clorox basic solution and the general solution of receiving of 1～3mL, 0.10～0.5mmol/L nitre, mix, the 50-70min that at room temperature develops the color sentences the reagent blank contrast at 630nm and is colorimetric zero point, the light absorption value A of working sample and contrast;
4) make the working standard curve, make the working standard curve, obtain slope b with the standard series concentration of ammonium nitrogen solution and the light absorption value A ' of mensuration;
5), calculate the ammonium nitrogen concentration S of the solution in the triangular flask of surveying, S=A*b according to light absorption value A and slope b;
6) according to the ammonium nitrogen concentration S of solution in the triangular flask, distinguish ammonium nitrogen content S in the calculation sample
1And average ammonium nitrogen content S in the contrast
2
7) calculate arginine desaminase activity
Calculation formula: (S
1-S
2)/W.T, the mg NH of unit
4 +-N/g dry ground 3h
In the formula: S
1Be the NH in the sample
4 +-N content (mg), S
2Be two average N H in the contrast
4 +-N content (mg), W are dry ground heavy (g), T soil sample incubation time (h).
The pH of control reaction system should be between 10.5～11.7 in the process color;
If before measuring light absorption value, contain noisy metal ion in the liquid to be measured, available EDTA sequestrant is sheltered;
Sequestering agent adds after colour developing, promptly can add sequestering agent behind the liquid colour developing 50-70min to be measured in the time of 20-30 ℃; As too early adding, can make color reaction very slow, blue on the low side; Added evening, then the precipitation of hydroxide of Sheng Chenging may be worn out and is difficult for dissolving.
Nitre Pu Na can quicken colour developing as catalyzer, strengthens blue and its stability, improves reaction sensitivity.Colorimetric after when 20 ℃ of left and right sides room temperatures, generally must placing 1h, colour developing needs 2～3h approximately fully; Sodium phenylate and clorox reagent instability must be stored in shaded bottle, leave in 4 ℃ of refrigerators, and the time spent must be warmed to room temperature.
Making working standard curve process is: draw 10ml ammonium sulfate standard reserving solution and place the 100ml volumetric flask, with 2mol/L KCI solution constant volume, shake up; Draw 0ml, 2ml, 4ml, 6ml, 8ml, 10ml, 15ml then respectively in the 100ml volumetric flask, with 2mol/L KCI solution constant volume, shake up, obtain the typical curve liquid of a series of concentration (0mg/L, 2mg/L, 4mg/L, 6mg/L, 8mg/L, 10mg/L, 15mg/L); Draw 1ml typical curve liquid respectively in the colour developing test tube, with sample colour developing and mensuration optical density, the drawing standard working curve obtains slope b.
The improved foundation of the inventive method
Arginine desaminase in the soil is under the jurisdiction of the arginine lyase, and it can participate in arginic metabolic process in the soil, and then the metabolic process of indirect adjustments and controls blood urea nitrogen.The active mensuration of arginine desaminase is mainly taken off the ammonium nitrogen amount that amine generates according to arginine in the soil, or arginic decomposition amount.The active mensuration of soil arginine desaminase is exactly ammonium nitrogen and the colouring reagents reaction that decomposites when utilizing the arginine deamination in present method, by its content of colorimetric estimation, and then the ammonium nitrogen amount that unit soil produces in the Units of Account time characterizes the activity of soil arginine desaminase, this method is simple to operate, measures accurately.
Measuring principle used in the present invention is: utilize arginine to make substrate, the ammonium nitrogen that under the effect of arginine desaminase, produces in strongly basic medium with sodium phenylate and sodium hypochlorite reaction, generate the water-soluble dye indophenol blue, the blueness of solution is very stable, at 630nm colorimetric estimation absorbance, and then the ammonium nitrogen amount that unit soil generates in the Units of Account time characterizes the activity of arginine desaminase.
Compare with other colorimetric analysis method, advantage of the present invention mainly contains:
1) required equipment is simple, has reduced experimentation cost.Colorimetric instrument among the present invention is 722 common spectrophotometers, and other is laboratory glassware commonly used then.
2) simple to operate, analytical results is reliable and stable, favorable reproducibility.The blue compound that colorimetric generates among the present invention is very stable, and absorption value does not have noticeable change in the 24h, and the accuracy height is easy to operate.
3) use nitre Pu Na as catalyzer, can improve reaction sensitivity.Alleviated the dependency to high equipment requirements, common spectroscopic instruments just can meet the demands.
Embodiment
Reagent preparation: 1. arginine substrate solution (11.5M): take by weighing the 200.33g arginine in volumetric flask, be settled to 100mL with deionized water.
2.KCI solution (2M): take by weighing 149.1g KCI in volumetric flask, be settled to 1000mL with distilled water.
3. sodium phenylate solution (0.12M): take by weighing the 13.93g sodium phenylate in volumetric flask, be settled to 1000mL with distilled water.
4. clorox basic solution: take by weighing 5.00g sodium hydroxide and 0.3722g clorox in volumetric flask, be settled to 1000mL with distilled water.
5. nitre Pu Na (0.17mM): take by weighing that 0.045g nitre is general to be contained in the volumetric flask, be settled to 1000mL with distilled water.Note nitre general receive poisonous!
6. ammonium sulfate standard reserving solution (1000mg/L, N meter): take by weighing 4.7619g ammonium sulfate in volumetric flask, be settled to 1000mL with deionized water.
Operation steps: 1) take by weighing 4 parts of bright soil of 5g of crossing the 2mm sieve in the 150mL triangular flask, in triangular flask, add the arginine substrate solution of 2mL 11.5mol/L, soil and substrate solution are mixed, air-locked soft rubber ball beyond the Great Wall; To wherein 2 triangular flask constant temperature culture 3h (sample) in 37 ± 1 ℃ of incubators, remaining triangular flask constant temperature culture 3h (contrast) under-20 ± 1 ℃ of condition; To contrast triangular flask after cultivation finishes thaws at ambient temperature.
Other gets a triangular flask and does the reagent blank contrast simultaneously, does not promptly add soil, and all the other are handled with sample;
2) in above-mentioned triangular flask, add 50mL 2mol/L KCI solution respectively, stopper beyond the Great Wall, vibration 1h filters;
3) draw 1mL filtrate in the colour developing test tube, add 3mL 2mol/L KCI solution, 2mL0.12mol/L sodium phenylate solution, 1mL clorox basic solution and the general solution of receiving of 1mL 0.17mmol/L nitre, mix, the 1h that at room temperature develops the color sentences the reagent blank contrast at 630nm and is colorimetric zero point, the light absorption value A of working sample and contrast;
4) make the working standard curve, make the working standard curve, obtain slope b with the standard series concentration of ammonium nitrogen solution and the light absorption value A ' of mensuration;
5), calculate the ammonium nitrogen concentration S of the solution in the triangular flask of surveying, S=A*b according to light absorption value A and slope b;
6) according to the ammonium nitrogen concentration S of solution in the triangular flask, distinguish ammonium nitrogen content S in the calculation sample
1And average ammonium nitrogen content S in the contrast
2
7) calculate arginine desaminase activity
Calculation formula: (S
1-S
2)/W.T, the mg NH of unit
4 +-N/g dry ground 3h
In the formula: S
1Be the NH in the sample
4 +-N content (mg), S
2Be two average N H in the contrast
4 +-N content (mg), W are dry ground heavy (g), T soil sample incubation time (h).
Embodiment
The employed brown earth of present embodiment picks up from the ecological testing station in Chinese Academy of Sciences Shenyang, cinnamon soil picks up from experimental plot, clay fertilizer station, Chaoyang, two kinds of soil are all cultivated more than the 24h at 25 ℃, and moisture content about 15% is with the arginine desaminase activity of two kinds of soil of aforesaid method detection.
It is as follows that every kind of soil must be made a concrete analysis of step:
1) takes by weighing 4 parts of bright soil of 5g of crossing the 2mm sieve in the 150mL triangular flask, in triangular flask, add the arginine substrate solution of 2mL 11.5mol/L, soil and substrate solution are mixed, air-locked soft rubber ball beyond the Great Wall; To wherein 2 triangular flask constant temperature culture 3h (sample) in 37 ℃ of incubators, remaining triangular flask constant temperature culture 3h (contrast) under-20 ℃ of conditions; To contrast triangular flask after cultivation finishes thaws at ambient temperature.Do the reagent blank contrast simultaneously, promptly do not add soil, all the other are handled with sample;
2) in above-mentioned triangular flask, add 50mL 2mol/L KCI solution respectively, stopper beyond the Great Wall, vibration 1h filters;
3) draw 1mL filtrate in the colour developing test tube, add 3mL 2mol/L KCI solution, 2mL0.12mol/L sodium phenylate solution, 1mL clorox basic solution and the general solution of receiving of 1mL 0.17mmol/L nitre, mix, the 1h that at room temperature develops the color sentences the light absorption value A that reagent is blank determination sample and contrast at 630nm.
4) make the working standard curve, make the working standard curve, obtain slope b with the standard series concentration of ammonium nitrogen solution and the light absorption value A ' of mensuration;
5), calculate the ammonium nitrogen concentration S that extracts in the solution that surveys, S=A*b according to light absorption value A and slope b;
6) ammonium nitrogen content in the calculation sample;
7) calculate arginine desaminase activity;
Test-results is as follows:
By data in the table as can be seen, result difference reaches conspicuous level between the different soils, and less standard difference shows that the deviation between this analytical procedure result is less, and tolerance range is higher, favorable reproducibility.
Claims (4)
1. one kind is detected the active method of soil arginine desaminase, it is characterized in that:
1) taking by weighing n part 5-10g respectively crosses the bright soil of 1-2mm sieve or cultivates pedotheque in advance in the 150mL triangular flask, n 〉=4; The substrate arginine solution that adds 2～5mL, 10～12mol/L in triangular flask mixes soil and substrate solution, air-locked beyond the Great Wall soft rubber ball on triangular flask;
To wherein n-2 triangular flask constant temperature culture 170-190min in 36-38 ℃ of incubator, as sample;
2 remaining triangular flasks are constant temperature culture 170-190min under-19--21 ℃ condition, in contrast; To contrast triangular flask after cultivation finishes thaws at ambient temperature;
Other gets a triangular flask and does the reagent blank contrast simultaneously, does not promptly add soil, and all the other are handled with sample;
2) in above-mentioned triangular flask, add 40～60mL, 1.5～2mol/L KCI solution respectively, stopper beyond the Great Wall, vibration 55-65min filters;
3) draw 1～2mL filtrate respectively in the colour developing test tube, add 3～5mL, 1.5～2mol/L KCI solution, 1～3 mL, 0.10～0.15mol/L sodium phenylate solution, 0.5～2 mL clorox basic solutions and the general solution of receiving of 1～3 mL, 0.10～0.5mmol/L nitre, mix, the 50-70min that at room temperature develops the color sentences the reagent blank contrast at 630nm and is colorimetric zero point, the light absorption value A of working sample and contrast;
4) make the working standard curve, make the working standard curve, obtain slope b with the standard series concentration of ammonium nitrogen solution and the light absorption value A ' of mensuration;
5), calculate the ammonium nitrogen concentration S of the solution in the triangular flask of surveying, S=A*b according to light absorption value A and slope b;
6) according to the ammonium nitrogen concentration S of solution in the triangular flask, distinguish ammonium nitrogen content S in the calculation sample
1And average ammonium nitrogen content S in the contrast
2
7) calculate arginine desaminase activity
Calculation formula: (S
1-S
2)/W.T, the mg NH of unit
4 +-N/g dry ground 3h
In the formula: S
1Be the NH in the sample
4 +-N content (mg), S
2Be two average N H in the contrast
4 +-N content (mg), W are dry ground heavy (g), T soil sample incubation time (h).
2. method according to claim 1 is characterized in that: draw 10 ml ammonium sulfate standard reserving solutions and place 100 ml volumetric flasks, with 2mol/L KCI solution constant volume, shake up; Draw 0 ml, 2 ml, 4 ml, 6 ml, 8 ml, 10 ml, 15 ml then respectively in 100 ml volumetric flasks, with 2mol/L KCI solution constant volume, shake up, obtain the typical curve liquid of a series of concentration 0 mg/L, 2 mg/L, 4 mg/L, 6 mg/L, 8 mg/L, 10 mg/L, 15 mg/L; Draw 1ml typical curve liquid respectively in the colour developing test tube, with sample colour developing and mensuration optical density, drawing standard working curve.
3. method according to claim 1 is characterized in that: the pH of control reaction system should be between 10.5～11.7 in the process color.
4. method according to claim 1 is characterized in that: if before measuring light absorption value, contain noisy metal ion in the liquid to be measured, available EDTA sequestrant is sheltered;
Sequestering agent adds after colour developing, promptly can add sequestering agent behind the liquid colour developing 50-70min to be measured in the time of 20-30 ℃; As too early adding, can make color reaction very slow, blue on the low side; Added evening, then the precipitation of hydroxide of Sheng Chenging may be worn out and is difficult for dissolving.
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|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102788791A (en) * | 2012-08-10 | 2012-11-21 | 青岛农业大学 | Fast determination method for soil acidity and alkalinity and determination device thereof |
| CN103760119A (en) * | 2014-01-14 | 2014-04-30 | 沈阳建筑大学 | Method for determining inhibition effect of plant sample on soil nitrification |
| CN107247028A (en) * | 2017-03-06 | 2017-10-13 | 四川新华扬山野生物有限公司 | A kind of method of total fiber element enzyme activity detection accuracy in raising feed |
-
2009
- 2009-12-25 CN CN2009102487779A patent/CN102108377A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102788791A (en) * | 2012-08-10 | 2012-11-21 | 青岛农业大学 | Fast determination method for soil acidity and alkalinity and determination device thereof |
| CN102788791B (en) * | 2012-08-10 | 2015-06-17 | 青岛农业大学 | Fast determination device for soil acidity and alkalinity |
| CN103760119A (en) * | 2014-01-14 | 2014-04-30 | 沈阳建筑大学 | Method for determining inhibition effect of plant sample on soil nitrification |
| CN107247028A (en) * | 2017-03-06 | 2017-10-13 | 四川新华扬山野生物有限公司 | A kind of method of total fiber element enzyme activity detection accuracy in raising feed |
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Application publication date: 20110629 |