CN111850092A - Soil biological activity and productivity evaluation method based on soil enzyme activity determination - Google Patents
Soil biological activity and productivity evaluation method based on soil enzyme activity determination Download PDFInfo
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Abstract
The invention discloses a soil biological activity and productivity evaluation method based on soil enzyme activity determination, which comprises the steps of selecting a soil sample, extracting and determining the activities of urease, phosphatase, beta-glucosidase, arylsulfatase and N-acetyl-beta-D-glucosaminidase in the soil sample; evaluating the circulation and migration capacities of carbon, nitrogen, phosphorus and sulfur in the soil sample according to the activity of 5 soil enzymes, and further evaluating the biological activity and productivity of the soil; the conformity of culture conditions, substrate types and concentrations, soil sample storage modes and the like to the 5-soil enzyme activity measurement is determined. The invention selects 5 enzymes which can represent the circulation of organic source nutrient elements such as carbon, nitrogen, phosphorus, sulfur and the like in soil to evaluate the soil quality, provides a rapid, effective, accurate and stable measuring method of corresponding enzyme activity, enables the soil quality to be evaluated according to a uniform enzyme activity measuring method, realizes the comparability of experimental data, is convenient for data processing and comparison, and can be popularized and applied.
Description
Technical Field
The invention belongs to the technical field of soil detection, and particularly relates to a soil biological activity and productivity evaluation method based on soil enzyme activity determination.
Background
Soil acts as a "life-like body" in that almost all biochemical reactions are driven by enzymes. The soil enzyme activity can be used as important indexes of soil biological activity and productivity such as soil fertility, soil quality, soil health and the like. Soil enzymes promote the formation of soil humus, and the conversion of organic substances and organic residues entering the soil into soil humus is a very complex biochemical process which cannot be catalyzed by various enzymes.
The soil enzymes catalyze the biogeochemical cycle of organic nutrient elements such as carbon, nitrogen, phosphorus, sulfur and the like. Various nutrient elements such as carbon, nitrogen, phosphorus, sulfur and the like in the plant residues or animal residues enter the soil in the form of organic matters, and then are decomposed into small molecular organic substances and inorganic ions through complex biochemical transformation under the catalysis of various enzymes such as oxidoreductase and hydrolase in the soil, and the small molecular organic substances and the inorganic ions are passively absorbed by the plants and assimilated in vivo to form various organic compounds, thereby completing the circulation of biosphere-soil ring-biosphere. The soil enzyme is one of the most active organic components in soil components, and the activity of the soil enzyme not only can reflect the vigorous degree of energy metabolism of soil substances, but also can be used as an important index for evaluating the soil fertility and the ecological environment quality.
In the domestic research process, according to the method introduced in soil enzyme and its research method in 1986, although there are many methods for testing enzyme activity, there is still no unified and standardized method for testing soil enzyme activity, and at the same time, there are many pretreatment items before enzyme activity measurement, which results in messy test data of each research institution, difficult comparison and difficult application. At present, the research on the soil enzyme activity determination mainly focuses on limited enzyme types, the research on methods for determining the activities of urease, arylsulfatase and phosphatase is few, particularly, the research on methods for detecting soil beta-glucosidase and N-acetyl-beta-D-glucosaminidase is not reported, and no stable and available method for determining the enzyme activity exists. Soil enzyme activity measurement is affected by various factors such as sample treatment (storage method, temperature, sterilization method), measurement conditions (temperature, buffer type, pH value, substrate type, ionic strength), etc., and therefore it is very difficult to quantitatively extract and measure soil enzyme activity, and it is generally required to perform under strictly controlled conditions such as a certain temperature, pH buffer system, substrate concentration, etc. If the determination conditions are changed, the obtained results can be greatly different, the efficiency and accuracy of the soil enzyme activity determination are reduced, and the assessment of the soil quality is directly influenced. The technology aims to establish the standard of soil enzyme activity determination, and has important significance for the research work of soil enzymology, the improvement of soil ecological environment and the improvement of soil fertility.
Disclosure of Invention
In view of the deficiencies noted in the background above, the present invention provides a soil biological activity and productivity evaluation method based on soil enzyme activity assay, which aims to solve the problems of the prior art in the background above.
In order to achieve the purpose, the invention adopts the technical scheme that:
soil biological activity and productivity evaluation method based on soil enzyme activity determination comprises selecting soil sample, extracting and determiningThe activity of urease, phosphatase, beta-glucosidase, arylsulfatase, and N-acetyl-beta-D-glucosaminidase in the soil sample; 1g of NH in the soil after 24h3-the milligrams of N represent the soil urease activity; the activity of the soil phosphatase is expressed in milligrams of phenol released in 1g of soil after 24 h; under alkaline condition, the p-nitrophenol generated by hydrolyzing the p-nitrophenol sulfate solution as a substrate represents the activity of the soil aryl sulfatase; carrying out enzymolysis by taking p-nitrophenyl beta-D-glucoside as a substrate, wherein the amount of p-nitrophenol released after hydrolysis of the substrate represents the activity of the soil beta-glucosidase; carrying out enzymolysis by taking p-nitrophenyl-N-acetyl-beta-D-glucosaminide as a substrate, wherein the amount of p-nitrophenol released after hydrolysis of the substrate represents the activity of the soil N-acetyl-beta-D-glucosaminidase; and (3) evaluating the circulating and migrating capacities of carbon, nitrogen, phosphorus and sulfur in the soil sample according to the activity of urease, phosphatase, beta-glucosidase, arylsulfatase and N-acetyl-beta-D-glucosaminidase, and further evaluating the biological activity and productivity of the soil.
Preferably, the urease, phosphatase, beta-glucosidase, arylsulfatase and N-acetyl-beta-D-glucosaminidase activity are measured by using fresh soil stored at-20 ℃ or 4 ℃ as a soil sample.
Preferably, the specific method for measuring the urease activity is as follows: placing fresh soil into a triangular flask, adding 10% urea solution and a pH 6.7 citrate buffer solution, uniformly shaking, plugging a cork stopper, culturing in a 37 ℃ thermostat for 24h, taking out, adding a 1.0mol/L KCl +0.01mol/L HCl solution, oscillating for 30min at a rotating speed of 160r/min, wherein the volume ratio of the mass of the fresh soil to the 10% urea solution, the pH 6.7 citrate buffer solution and the 1.0mol/L KCl +0.01mol/L HCl is 5 g: 10 ml: 20 ml; centrifuging and filtering the oscillated soil turbid liquid, taking 3ml of filtrate, injecting the filtrate into a 50ml volumetric flask, adding 4ml of sodium phenolate solution and 3ml of sodium hypochlorite solution, shaking up along with adding, developing after 20min, fixing the volume, using a 1cm cuvette within 1h, carrying out color comparison at the position with the wavelength of 578nm on a spectrophotometer, comparing the color comparison with a standard series for quantification, and measuring NH by using a detection method3the-N amount characterizes the soil urease activity.
Preferably, the specific method for determining phosphatase activity is: placing fresh soil into a triangular flask, adding toluene into a fume hood, adding a plug, slightly shaking, standing for 15min, and adding disodium phenyl phosphate with the mass concentration of 0.5%, wherein the volume ratio of the mass of the fresh soil to the toluene to the disodium phenyl phosphate is 2 g: 0.5 ml: 20 ml; forcibly shaking to uniformly mix the soil sample and the solution, then putting the mixture into a constant-temperature incubator at 37 ℃ for culturing for 12 hours, taking out the sample after the culture is finished, adding 0.3% of aluminum sulfate solution, filtering, putting 3mL of filtrate into a 50mL colorimetric tube, adding a buffer solution and 4 drops of chlorodibromo-p-benzoquinone imine reagent, an acetate buffer solution for acid phosphatase, a citrate buffer solution for neutral phosphatase and a borate buffer solution for alkaline phosphatase; and (3) shaking while adding, diluting to a scale after color development, after 30min, carrying out color comparison on the 660nm wavelength on an ultraviolet visible spectrophotometer by using a 1cm cuvette, and calculating the phenol content to obtain the activity of the soil phosphatase.
Preferably, the specific method for measuring arylsulfatase activity is as follows: placing fresh soil into a triangular flask, adding toluene, 0.5mol/L acetic acid buffer solution with the pH value of 5.8 and 0.02mol/L p-nitrobenzene potassium sulfate solution, shaking to uniformly mix the fresh soil and the p-nitrobenzene potassium sulfate solution, wherein the pH value is controlled to be 7-11, and the volume ratio of the mass of the fresh soil to the toluene, the 0.5mol/L acetic acid buffer solution and the 0.02mol/L p-nitrobenzene potassium sulfate solution is 2 g: 0.5 ml: 8 ml: 2 ml; adding cover, culturing in 37 deg.C incubator for 1 hr, removing cover, adding 0.5mol/L CaCL2And 0.5mol/L NaOH solution, shaking up, filtering, carrying out colorimetric determination on the filtrate at 400nm, and calculating the p-nitrophenol quantity to represent the activity of the soil arylsulfatase.
Preferably, the specific method for measuring the activity of the beta-glucosidase is as follows: placing fresh soil into a triangular flask, adding toluene into a fume hood, slightly shaking with a plug, standing for 15min, adding MUB solution and PNG solution, strongly shaking to uniformly mix a soil sample with the solution, wherein the pH value is controlled to be 4.0-6.0, and the volume ratio of the mass of the fresh soil to the toluene, the MUB solution and the PNG solution is 2 g: 0.5 ml: 8 ml: 2 ml; then culturing in a 37 deg.C constant temperature incubator for 1h, adding CaCL2Mixing with Tris buffer solution with pH value of 12.0, shaking, rapidly filtering with rapid filter paper, comparing color of the filtrate at 400nm in spectrophotometer, and calculating The amount of nitrophenol represents the activity of the soil beta-glucosidase.
Preferably, the specific method for measuring the activity of the N-acetyl-beta-D-glucosaminidase is as follows: placing fresh soil into a triangular flask, adding toluene, taking 100mmol/L acetic acid buffer solution with pH value of 5.5 and 10mmol/L pNP-beta-D-GlcNAc as substrates, shaking to uniformly mix the fresh soil and the substrates, and sealing, wherein the volume ratio of the mass of the fresh soil to the toluene to the 100mmol/L acetic acid buffer solution to the mass of the fresh soil to the mass of the pNP-beta-D-GlcNAc to the substrate is 1 g: 0.25 ml: 4 ml: 1 ml; culturing at 35 deg.C for 1 hr, adding 0.5mol/L CaCL2And (3) stopping the reaction with 0.5mol/L NaOH, filtering, carrying out colorimetric determination on the filtrate at 400nm, and calculating the p-nitrophenol amount to show the activity of the soil N-acetyl-beta-D-glucosaminidase.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects:
the invention selects 5 enzymes which can represent biogeochemical cycles of organic nutrient elements such as carbon, nitrogen, phosphorus, sulfur and the like in soil for research, and realizes the consistency of the biological activity and productivity evaluation of the soil by measuring the activity of the 5 representative soil enzymes. And the best culture condition of the soil enzyme, the substrate type and the concentration thereof in the soil enzyme test and the conformity of the air-dried soil and the fresh soil to the enzyme activity determination are determined, 5 a rapid, effective and accurate determination method for the soil enzyme activity is provided, the bottleneck problem in the soil enzyme activity determination is solved, the comparability of experimental data can be realized according to a unified enzyme activity determination method when the soil quality is evaluated, a rapid, simple and stable detection method is formed, the comparison among data and the data processing are convenient, and the method can be popularized and applied to the determination of the soil enzyme activity.
Drawings
FIG. 1 is a graph showing the results of substrate concentration on urease activity provided by the examples of the present invention.
FIG. 2 is a graph showing the results of the effect of buffer solution volume on urease activity provided by the examples of the present invention.
FIG. 3 is a graph showing the results of the effect of incubation temperature and time on urease activity provided by the examples of the present invention.
FIG. 4 is a graph showing the results of the effect of substrate concentration on phosphatase activity provided by the examples of the present invention.
FIG. 5 is a graph showing the results of the effect of the incubation temperature on phosphatase activity, which is provided in the examples of the present invention.
FIG. 6 is a graph showing the results of the effect of buffer volume on phosphatase activity provided by the examples of the present invention.
FIG. 7 is a graph showing the results of pH effect on arylsulfatase stability provided by examples of the invention.
FIG. 8 is a graph showing the effect of temperature on the thermal stability of arylsulfatase according to examples of the present invention.
FIG. 9 is a graph showing the results of various temperature and time effects on phosphatase activity provided by examples of the present invention.
FIG. 10 is a graph showing the results of pH stability of β -glucosidase provided in the examples of the present invention.
FIG. 11 is a graph showing the results of the effect of temperature on N-acetyl-. beta. -D-glucosaminidase activity provided in the examples of the present invention.
FIG. 12 is a graph showing the results of the effect of pH on N-acetyl-. beta. -D-glucosaminidase activity provided in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The soil biological activity and productivity evaluation method based on soil enzyme activity determination has the advantages that the existing soil enzymes are various in types, the activity determination methods are more, but a unified method is not provided, meanwhile, the pretreatment items before the enzyme activity determination are more, the operation standards are not consistent enough, and the soil biological activity and productivity cannot be effectively evaluated in a standardized mode. According to the influence degree of different soil enzymes on soil biological activity and productivity such as soil fertility, soil quality and the like, the method determines the method capable of catalyzing carbon, nitrogen, phosphorus, sulfur and the like in soilThe research is carried out on 5 enzymes of the biogeochemical cycle of organic source nutrient elements, wherein the 5 soil enzymes are urease, phosphatase, beta-glucosidase, arylsulfatase and N-acetyl-beta-D-glucosaminidase, and NH in 1g soil after 24h3-the milligrams of N represent the soil urease activity; the activity of the soil phosphatase is expressed in milligrams of phenol released in 1g of soil after 24 h; under alkaline condition, the p-nitrophenol generated by hydrolyzing the p-nitrophenol sulfate solution as a substrate represents the activity of the soil aryl sulfatase; carrying out enzymolysis by taking p-nitrophenyl beta-D-glucoside as a substrate, wherein the amount of p-nitrophenol released after hydrolysis of the substrate represents the activity of the soil beta-glucosidase; carrying out enzymolysis by taking p-nitrophenyl-N-acetyl-beta-D-glucosaminide as a substrate, wherein the amount of p-nitrophenol released after hydrolysis of the substrate represents the activity of the soil N-acetyl-beta-D-glucosaminidase; through researches on extraction, culture, activity determination and the like of 5 soil enzymes, 5 soil enzymes are provided with corresponding rapid, simple, stable and reliable activity determination methods, and the circulation and migration capacities of carbon, nitrogen, phosphorus and sulfur in soil samples are evaluated according to the activity of urease, phosphatase, beta-glucosidase, arylsulfatase and N-acetyl-beta-D-glucosaminidase, so that the biological activity and the productivity of the soil are effectively and accurately evaluated.
In the soil enzyme activity measurement process, it was found that the results of soil enzyme activity measurement are greatly affected by various factors such as sample treatment (storage method, temperature, sterilization method), measurement conditions (temperature, buffer type and pH, substrate type, ionic strength), etc., it is difficult to determine how these factors affect the enzyme activity, and a skilled person cannot know what measurement conditions are used, what buffers and substrates are used, etc., and can measure the enzyme activity quickly and accurately. Therefore, it is very difficult to quantitatively extract and measure the soil enzyme activity, and it is generally required to perform under strictly controlled conditions such as a certain temperature, pH buffer system, substrate concentration, etc. If the determination conditions are changed, the obtained results are greatly different, so that the soil quality cannot be uniformly and contrastively evaluated, and the efficiency and the accuracy of the soil enzyme activity determination are reduced under different determination conditions, so that the assessment of the soil quality is directly influenced. The technology aims to establish the standard of soil enzyme activity determination, and has important significance for the research work of soil enzymology, the improvement of soil ecological environment and the improvement of soil fertility.
The invention provides a method for rapidly and accurately measuring the enzyme activity by researching factors such as the type of soil to be tested, the storage of a soil sample, the culture time, an inhibitor, the concentration of a substrate, the volume of a buffer solution, the culture temperature, the culture time, the volume of the inhibitor and the like, changing the conditions of various influencing factors, measuring the change of the enzyme activity and analyzing the influence of various factors on the enzyme activity.
Example 2
(1) Method for measuring urease activity
Removing foreign matters (leaves, stones and the like) in the collected soil sample, using a ceramic mortar for pressing, uniformly stirring, sieving by a 1mm sieve, and storing in a refrigerator at 4 ℃ for later use. Placing 5g fresh soil into a 50mL triangular flask, adding 10mL 10% urea solution and 20mL citrate buffer solution with the pH value of 6.7, shaking uniformly, plugging a cork stopper into a 37 ℃ incubator, culturing for 24h, taking out, adding 20mL (1.0mol/L KCl +0.01mol/L HCl) solution, oscillating for 30min at the rotating speed of 160r/min, centrifuging the agitated soil suspension at the rotating speed of 4000r/min for 5min, filtering, taking 3mL filtrate, injecting into a 50mL volumetric flask, adding 4mL sodium phenate solution and 3mL sodium hypochlorite solution, shaking uniformly with the addition, developing and fixing the volume after 20min, using a 1cm cuvette within 1h, carrying out color comparison at the wavelength of 578nm on a spectrophotometer, comparing the quantitative determination with the standard series, and measuring NH by using a measuring method3the-N amount characterizes the soil urease activity. Meanwhile, no substrate and no soil treatment are carried out as a control group.
And (4) calculating a result: 1g of NH in the soil after 24h3The number of milligrams of-N represents the soil urease activity (Ure).
In the formula: a: determination of NH of the sample from the calibration curve3-N milligrams;
b: determination of the NH of the soil-free control from the standard curve 3-N milligrams;
c: determination of NH without matrix control from the calibration curve3-N milligrams;
v: the volume of the color developing solution is mL;
k: dividing times (volume of leachate mL/volume of absorption solution mL);
m: the weight g of the fresh soil was converted into the weight g of the air-dried soil sample.
(2) Substrate concentration optimization
Urea solutions with mass concentrations of 2%, 5%, 10%, 15%, and 20% were prepared, and urease activities were measured as described above, and the results of the experiments are shown in FIG. 1. From the results in FIG. 1, it is clear that the urease activity was highest at a substrate concentration of 10%, and the enzyme activity was maintained substantially unchanged by increasing the urea concentration, and was highest at a 10% urea concentration. Therefore, 10% urea concentration was selected for urease determination.
(3) Buffer volume optimization
The volumes of the citric acid buffer solution having a pH of 6.7 added to the flask were set to 10mL, 15mL, 20mL, 25mL and 30mL, respectively, and the urease activity was measured in the same manner as described above, and the measurement results are shown in FIG. 2, and it is preferable to select 20mL as the volume of the buffer solution.
(4) Effect of incubation temperature and time on urease Activity
The reaction system was cultured in an incubator at 20 ℃ 30 ℃, 37 ℃, 50 ℃ and 60 ℃ respectively, and after 24 hours of culture, samples were taken to determine changes in urease activity, and the results of the determination are shown in FIG. 3 (a). It is found that the culture temperature for measuring urease activity is preferably 37 ℃. The reaction system is respectively cultured in an incubator at 37 ℃ for 8h, 16h, 24h, 32h and 48h, and the activity change of urease is analyzed by sampling at regular time, and the result is shown in figure 3(b), and as can be seen from figure 3(b), the enzyme activity is obviously increased along with the prolonging of the culture time, the enzyme activity is slowly increased after 24h, and 24h is most suitable for selection from the aspects of time saving and pursuit of the optimal enzyme activity.
(5) Effect of soil sample preservation mode on urease Activity
The conventional preservation modes of soil samples for measuring the enzyme activity comprise fresh soil preservation at-20 ℃, fresh soil preservation at 4 ℃ and air-dried soil preservation at room temperature, in order to verify the preservation temperature and preservation time suitable for urease, 3 kinds of sandy soil, loam and clay soil are respectively taken to measure the activity of urease which is preserved for 3d and 30d under the 3 preservation modes, and the measurement results are shown in the following table 1. From the results in table 1, the priority order of the 3 storage methods is: fresh soil is more than fresh soil at-20 ℃ and 4 ℃ and air-dried soil.
TABLE 1 Effect of soil sample storage on urease Activity
A KCl solution is often selected as an extracting agent for urease activity detection, a non-toluene method is adopted in the experiment, 4 extracting agents with different concentrations are adopted, the concentrations of the KCl extracting agents are set to be 0.5, 1.0, 1.5 and 2.0moL/L respectively, 0.01moL/L HCl is used as an inhibitor, and urease activity detection is carried out on sandy soil, loam and clay soil by changing the concentrations of the KCl solution under the experiment condition in order to determine the optimal concentration of the extracting agents.
The reason for adding KCl is that the soil colloidal particles are negatively charged and can easily adsorb NH generated by urea hydrolysis4+And N, if the ammonia nitrogen adsorbed on the soil particles is directly filtered after the culture is finished, the ammonia nitrogen cannot be filtered out, so that the urease determination result is smaller. At this time, a KCl solution is added, then K +Will remove NH in the soil4+And (4) replacing. The addition of HCl can significantly enhance urease activity, perhaps due to the addition of inhibitors that increase the leaching rate, or HCl can effectively inhibit further hydrolysis of urea, thereby increasing urease activity.
From the above experimental results, the optimal conditions for measuring urease activity in the laboratory are as follows: the soil samples were selected from fresh soil stored at-20 ℃ or 4 ℃ and the measurements were completed as soon as possible within 30 days of storage. When 5g of soil sample is weighed, 10mL of 10% urea solution is adopted as a substrate, 20mL of citrate buffer solution with pH 6.7 is adopted as a buffer solution, the culture temperature is 37 ℃, the culture time is 24h, and 20mL of 1.0mol/L KCl +0.01mol/L HC solution is selected as an extractant. From this, it can be found that, in the measurement of urease activity, the volume ratio of the mass of fresh soil to 10% urea solution, pH 6.7 citrate buffer solution and 1.0mol/L KCl +0.01mol/L HCl is 5 g: 10 ml: 20 ml.
Example 3
(1) Method for measuring phosphatase activity
Removing foreign matters (leaves, stones and the like) in the collected soil sample, using a ceramic mortar for pressing, uniformly stirring, sieving by a 1mm sieve, and storing in a refrigerator at 4 ℃ for later use. Placing 2g of fresh soil into a 200mL triangular flask, storing a fresh soil sample at-20 ℃ or 4 ℃, adding 0.5mL of toluene into a fume hood, slightly shaking with a plug, standing for 15min, adding 20mL of disodium phenylphosphate with the mass concentration of 0.5%, arranging a soil control (20 mL of water is used for replacing a disodium phenylphosphate solution substrate) and a soilless control (2 mL of water is used for replacing the soil sample), strongly shaking to mix the soil sample and the solution, then placing the mixture into a 37 ℃ constant temperature incubator for culturing for 12h, taking out a sample after the culture is finished, adding 40mL of 0.3% aluminum sulfate solution, filtering, placing 3mL of filtrate into a 50mL colorimetric tube, adding 5mL of pH 9.4 buffer solution and 4 drops of chloro-dibromo-p-benzoquinone imine reagent, shaking while adding, diluting to a scale after color development, using a 1cm dish after 30min, carrying out color comparison at the wavelength of 660nm on an ultraviolet visible spectrophotometer, and calculating the phenol content to obtain the activity of the soil phosphatase.
And (4) calculating a result: the soil phosphatase activity was expressed in mg of phenol released from 1g of soil after 12 h.
In the formula: a: obtaining the milligrams of phenol of the sample from the standard curve;
b: obtaining the milligrams of the soilless contrast phenol by a standard curve;
c: determining the number of milligrams of the non-matrix control phenol according to a standard curve;
v: the volume of the color developing solution is mL;
k: dividing times (volume of leachate mL/volume of absorption solution mL);
m: the weight g of the fresh soil was converted into the weight g of the air-dried soil sample.
(2) Effect of substrate on phosphatase Activity
Disodium phenyl phosphate solutions with mass concentrations of 0.2%, 0.4%, 0.5%, 0.8% and 1.0% are prepared respectively, and the phosphatase activity is determined according to the method, and the experimental result is shown in fig. 4. From the results of FIG. 4, it is understood that the phosphatase activity is highest at a concentration of 0.5% in the disodium phenylphosphate solution, and the enzyme activity is not increased by increasing the concentration of disodium phenylphosphate, and that the enzyme activity is at the maximum reaction rate at 0.5% in the disodium phenylphosphate solution. Therefore, the phosphatase assay was performed with a 0.5% disodium phenyl phosphate concentration.
(3) Effect of incubation temperature on phosphatase Activity
The reaction system was incubated in an incubator at 20 ℃, 30 ℃, 37 ℃, 43 ℃ and 50 ℃ respectively, and after 12 hours of incubation, samples were taken and the change in phosphatase activity was measured, and the results of the measurement are shown in FIG. 5, and it is preferable to select the incubation temperature for the phosphatase activity measurement at 37 ℃.
(4) Buffer volume optimization
The volumes of the buffer solutions added to the triangular flask were set to 2mL, 4mL, 5mL, 8mL, and 10mL, respectively, and the phosphatase activity was measured by the above-described method, and the measurement results are shown in FIG. 6, indicating that 5mL is preferable as the buffer volume.
From the above experimental results, the optimal conditions for measuring phosphatase activity in the laboratory are: the soil sample is preferably fresh soil stored at the temperature of minus 20 ℃ or 4 ℃, when 2g of the soil sample is weighed, 0.5mL of toluene is required to be added, 20mL of disodium phenyl phosphate with the mass concentration of 0.5% is adopted as a substrate, the culture temperature is 37 ℃, and the culture time is 24 hours. From this, it was found that the volume ratio of the mass of fresh soil to toluene and 0.5 mass% disodium phenylphosphate was 2 g: 0.5 ml: 20ml, respectively, in the phosphatase activity measurement. In addition, when the soil phosphatase activity is investigated, whether the phosphatase is an acid phosphatase, an alkaline phosphatase or a neutral phosphatase is selected depending on the soil pH. Acetate buffer for acid phosphatase; citrate buffer for neutral phosphatase; the alkaline phosphatase was buffered with borate. The selection of an appropriate buffer solution makes the results of the study more representative.
Example 4
(1) Method for measuring activity of arylsulfatase
Removing the collected soil sample from the soil sampleThe foreign matters (leaves, stones, etc.) are crushed and mixed evenly by a ceramic mortar, and then the mixture is sieved by a 1mm sieve and stored in a refrigerator at 4 ℃ for standby. Placing 2g of fresh soil into a 50mL triangular flask, adding 0.5mL of toluene, 8mL of 0.5mol/L acetic acid buffer solution (pH value of 5.8) and 2mL of 0.02mol/L p-nitrobenzene potassium sulfate solution, shaking to mix uniformly, controlling the pH value to be 7-11, covering, culturing in a 37 ℃ constant temperature incubator for 1h, removing the cover, adding 2mL of 0.5mol/L CaCL2And 8mL0.5mol/L NaOH solution, shaking up, filtering, carrying out colorimetric determination on the filtrate at 400nm, and calculating the p-nitrophenol amount to show the activity of the soil aryl sulfatase. A large number of experiments show that the activity of the arylsulfatase can be accurately and rapidly determined under the condition that the volume ratio of the mass of the fresh soil to the toluene, the 0.5mol/L acetic acid buffer solution and the 0.02mol/L p-nitrobenzene potassium sulfate solution is 2 g: 0.5 ml: 8 ml: 2 ml.
And (4) calculating a result:
in the formula: a: solving the microgram number of the p-nitrophenol of the sample from the standard curve;
b: obtaining microgram of soilless contrast p-nitrophenol from a standard curve;
c: solving the microgrammes of the non-matrix control paranitrophenol from the standard curve;
v: the volume of the color developing solution is mL;
k: dividing times (volume of leachate mL/volume of absorption solution mL);
m: the weight g of the fresh soil was converted into the weight g of the air-dried soil sample.
(2) pH stability of the enzyme
FIG. 7 shows the influence of different pH environments on the stability of arylsulfatase, and it can be seen from FIG. 7 that when the enzyme solution is placed in an acidic environment with a pH of 3-7, the stability of the enzyme activity is poor, and when the enzyme solution is placed in an alkaline environment with a pH of 7-11, the enzyme activity is relatively stable and can be maintained at about 90%, indicating that the enzyme has a wide pH adaptation range, but when the pH is increased to 12, the stability of the enzyme is rapidly reduced.
(3) Thermostability of the enzyme
The method comprises the steps of removing foreign matters (leaves, stones and the like) in a soil sample, using a ceramic mortar for pressing, uniformly stirring, screening by a 1mm sieve, and storing the soil sample at 4 ℃, 25 ℃, 35 ℃, 45 ℃ and 55 ℃ for different times respectively to obtain the arylsulfatase activity shown in figure 8, wherein the arylsulfatase activity is basically kept unchanged under the storage condition of 4 ℃, the enzyme activity starts to decline after the storage time is more than 1.5 days at 25 ℃, and the enzyme activity declines obviously along with the prolonging of the time when the soil sample is stored at 35 ℃, 45 ℃ and 55 ℃, so that the enzyme is not suitable for being stored in an environment above room temperature for a long time, and therefore, the optimal storage temperature of the enzyme below the room temperature (such as 4 ℃) can be selected.
Example 5
(1) Method for measuring beta-glucosidase activity
Currently, many methods for measuring β -glucosidase activity are available, and in summary, fluorescence methods, spectrophotometry methods, and the like are mainly used. The Barush and Swiain methods which take salicin as an enzyme reaction substrate detect that the substrate is hydrolyzed by beta 2 glucosidase to generate trace glucose, the reaction is easy to interfere, and the detection sensitivity is not high. The fluorescence method is originally used for monitoring beta-glucosidase in higher animal tissues with low enzyme content, has high sensitivity and is rapid, but the current application is not many due to the complex operation and poor reproducibility of the experimental process. The method for measuring the activity of the beta-glucosidase in the invention comprises the following steps:
removing foreign matters (leaves, stones and the like) in the collected soil sample, using a ceramic mortar for pressing, uniformly stirring, sieving by a 1mm sieve, and storing in a refrigerator at 4 ℃ for later use. Placing 2g fresh soil in a 50mL triangular flask, adding 0.5mL toluene in a fume hood, adding a plug, shaking gently, standing for 15min, adding 8mL MUB solution and 2mL PNG solution, shaking vigorously to mix the soil sample and the solution, controlling pH value at 4.0-6.0, culturing in a 37 ℃ constant temperature incubator for 1h, adding 2mL CaCL2And 8mL of Tris buffer solution with the pH value of 12.0, shaking up, quickly filtering by using quick filter paper, carrying out color comparison on the filtrate at 400nm on a spectrophotometer, and calculating The p-nitrophenol content represents the activity of the soil beta-glucosidase. A large number of experiments show that the activity of the beta-glucosidase can be accurately and rapidly determined under the condition that the volume ratio of the mass of the fresh soil to the toluene, the MUB solution and the PNG solution is 2 g: 0.5 ml: 8 ml: 2 ml.
And (3) calculating the result:
the absorbance of the test solution and the blank solution was measured at 400nm by UV-visible spectrophotometry. The beta-glucosidase activity was calculated according to the following formula:
in the formula: a: solving the microgram number of the p-nitrophenol of the sample from the standard curve;
b: obtaining microgram of soilless contrast p-nitrophenol from a standard curve;
c: solving the microgrammes of the non-matrix control paranitrophenol from the standard curve;
v: the volume of the color developing solution is mL;
k: dividing times (volume of leachate mL/volume of absorption solution mL);
m: the weight g of the fresh soil was converted into the weight g of the air-dried soil sample.
(2) Selection of characteristic absorption wavelength
After the reaction system is respectively carried out for 40min and 60min, the changes of the light absorption values under different wavelengths are shown in table 2, and as can be seen from table 2, the maximum absorption values at 410nm are obtained after the reaction is carried out for 40min and 60min, and the light absorption values under other wavelengths are lower. Therefore, 410nm can be selected as the wavelength for spectrophotometric measurement of β -glucosidase activity using PNG as the substrate.
TABLE 2 selection of the wavelength of the beta-glucosidase activity assay
(3) Effect of incubation temperature and time on beta-glucosidase Activity
The concentration change of p-nitrophenol in the reaction system is shown in FIG. 9 after enzymatic reaction for 0min, 30min, 60min, 90min and 120min at four temperatures of 30 deg.C, 37 deg.C, 44 deg.C and 53 deg.C, respectively. As can be seen from FIG. 9, the reaction was carried out at 30 ℃ and 37 ℃ for 120min, the enzymatic reaction was linear, and the rate of formation of the product was the greatest at 37 ℃ than at the other temperatures. At 44 ℃ and 53 ℃ the enzymatic reaction is less linear and the rate of product formation at 53 ℃ is slow. The activity of the beta-glucosidase is shown to be stable at 30 ℃ and 37 ℃, and the beta-glucosidase is more suitable for the enzymatic reaction at 37 ℃. As can be seen, the beta-glucosidase activity assay is preferably selected to be 37 ℃.
(4) Study of pH stability of beta-glucosidase
Adding the beta-glucosidase solution into different pH buffer solutions, standing for 24h at 4 ℃, and measuring the enzyme activity. The results are shown in FIG. 10, in which the pH stability curve was obtained using 100% of the enzyme activity of the enzyme solution that had not been allowed to stand under the corresponding conditions. The stability range of the beta-glucosidase is narrow, the beta-glucosidase is stable between 4.0 and 6.0, the relative enzyme activity is over 80 percent, and the stability of the beta-glucosidase is poor when the pH is lower than 4 or higher than 6.
Example 6
(1) Method for determining activity of N-acetyl-beta-D-glucosaminidase
Removing foreign matters (leaves, stones and the like) in the collected soil sample, using a ceramic mortar for pressing, uniformly stirring, sieving by a 1mm sieve, and storing in a refrigerator at 4 ℃ for later use. Placing 1g fresh soil in a triangular flask, adding 0.25mL toluene, using 4mL100mmol/L acetate buffer (pH 5.5) and 1mL 10mmol/L pNP-beta-D-GlcNAc as substrates, shaking to mix well, sealing, culturing at 35 deg.C for 1h, adding 1mL 0.5mol/L CaCL after culturing2And 4mL of 0.5mol/L NaOH to terminate the reaction, filtering, carrying out colorimetric determination on the filtrate at 400 nm, and calculating the p-nitrophenol amount to represent the activity of the soil N-acetyl-beta-D-glucosaminidase. A large number of experiments show that the activity of the N-acetyl-beta-D-glucosaminidase can be accurately and rapidly determined under the condition that the volume ratio of the mass of the fresh soil to the toluene, the 100mmol/L acetic acid buffer solution and the 10 mmol/LpNP-beta-D-GlcNAc is 1 g: 0.25 ml: 4 ml: 1 ml.
And (3) calculating the result: the activity of N-acetyl-beta-D-glucosaminidase is expressed by p-nitrophenol measured by 1g soil sample after being cultured for 1 h.
In the formula: a: solving the microgram number of the p-nitrophenol of the sample from the standard curve;
b: obtaining microgram of soilless contrast p-nitrophenol from a standard curve;
c: solving the microgrammes of the non-matrix control paranitrophenol from the standard curve;
v: the volume of the color developing solution is mL;
k: dividing times (volume of leachate mL/volume of absorption solution mL);
m: the weight g of the fresh soil was converted into the weight g of the air-dried soil sample.
(2) Effect of incubation temperature on N-acetyl-beta-D-glucosaminidase Activity
In a system for measuring activity of 0.1mol/L phosphate buffer solution with pH of 5.5, 20 mu L of enzyme solution (the enzyme concentration is 0.5mg/mL) is added to measure the initial speed of the enzyme catalytic reaction after reacting for 1h at different temperatures, and the influence of the temperature on the activity of the N-acetyl-beta-D-glucosaminidase is studied. The results are shown in FIG. 11, which shows that the enzyme activity is higher at 25-45 deg.C, and the enzyme activity is highest when the temperature is 35 deg.C. The enzyme activity gradually decreases along with the increase of the temperature, and when the temperature exceeds 50 ℃, the enzyme activity is greatly influenced and gradually lost. From this, it was found that the optimum reaction temperature of the enzyme was 35 ℃.
(3) Effect of pH on N-acetyl-beta-D-glucosaminidase Activity
In an enzyme activity system at 35 ℃, the initial speed of an enzymatic reaction under different pH values is measured, and the influence of the pH value on the initial speed of the hydrolysis reaction of an enzymatic substrate p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNp-beta-D-GlcNAc) is researched. The results are shown in FIG. 12, which shows that the activity of N-acetyl-beta-D-glucosaminidase is in mask-like relation with pH, and the optimum pH is 5.5.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. A soil biological activity and productivity evaluation method based on soil enzyme activity determination is characterized in that a soil sample is selected, and the activities of urease, phosphatase, beta-glucosidase, arylsulfatase and N-acetyl-beta-D-glucosaminidase in the soil sample are extracted and determined; 1g of NH in the soil after 24h3-the milligrams of N represent the soil urease activity; the activity of the soil phosphatase is expressed in milligrams of phenol released in 1g of soil after 24 h; under alkaline condition, the p-nitrophenol generated by hydrolyzing the p-nitrophenol sulfate solution as a substrate represents the activity of the soil aryl sulfatase; carrying out enzymolysis by taking p-nitrophenyl beta-D-glucoside as a substrate, wherein the amount of p-nitrophenol released after hydrolysis of the substrate represents the activity of the soil beta-glucosidase; carrying out enzymolysis by taking p-nitrophenyl-N-acetyl-beta-D-glucosaminide as a substrate, wherein the amount of p-nitrophenol released after hydrolysis of the substrate represents the activity of the soil N-acetyl-beta-D-glucosaminidase; and (3) evaluating the circulating and migrating capacities of carbon, nitrogen, phosphorus and sulfur in the soil sample according to the activity of urease, phosphatase, beta-glucosidase, arylsulfatase and N-acetyl-beta-D-glucosaminidase, and further evaluating the biological activity and productivity of the soil.
2. The method for evaluating the biological activity and productivity of soil based on the determination of the activity of soil enzyme according to claim 1, wherein the urease, phosphatase, β -glucosidase, arylsulfatase and N-acetyl- β -D-glucosaminidase activity are determined by using fresh soil stored at-20 ℃ or 4 ℃ as a soil sample.
3. The soil biological activity and productivity evaluation method based on soil enzyme activity assay according to claim 2, wherein the concrete method of urease activity assay is: placing fresh soil in a triangular flask, adding 10% urea solution and citrate buffer solution with pH value of 6.7, shaking upPlugging a cork stopper, culturing for 24 hours in a constant temperature box at 37 ℃, taking out, adding a solution of 1.0mol/LKCl +0.01mol/L HCl, oscillating for 30 minutes at the rotating speed of 160r/min, wherein the volume ratio of the mass of the fresh soil to the 10% urea solution, the pH value of 6.7 citrate buffer solution and the volume ratio of 1.0mol/L KCl +0.01mol/L HCl are 5 g: 10 ml: 20ml respectively; centrifuging and filtering the oscillated soil turbid liquid, taking 3ml of filtrate, injecting the filtrate into a 50ml volumetric flask, adding 4ml of sodium phenolate solution and 3ml of sodium hypochlorite solution, shaking up along with adding, developing after 20min, fixing the volume, using a 1cm cuvette within 1h, carrying out color comparison at the position with the wavelength of 578nm on a spectrophotometer, comparing the color comparison with a standard series for quantification, and measuring NH by using a detection method 3the-N amount characterizes the soil urease activity.
4. The soil biological activity and productivity evaluation method based on soil enzyme activity assay according to claim 2, wherein the specific method of phosphatase activity assay is: placing fresh soil into a triangular flask, adding toluene into a fume hood, adding a plug, slightly shaking, standing for 15min, and adding disodium phenyl phosphate with the mass concentration of 0.5%, wherein the volume ratio of the mass of the fresh soil to the toluene to the disodium phenyl phosphate is 2 g: 0.5 ml: 20 ml; forcibly shaking to uniformly mix the soil sample and the solution, then putting the mixture into a constant-temperature incubator at 37 ℃ for culturing for 12 hours, taking out the sample after the culture is finished, adding 0.3% of aluminum sulfate solution, filtering, putting 3mL of filtrate into a 50mL colorimetric tube, adding a buffer solution and 4 drops of chlorodibromo-p-benzoquinone imine reagent, an acetate buffer solution for acid phosphatase, a citrate buffer solution for neutral phosphatase and a borate buffer solution for alkaline phosphatase; and (3) shaking while adding, diluting to a scale after color development, after 30min, carrying out color comparison on the 660nm wavelength on an ultraviolet visible spectrophotometer by using a 1cm cuvette, and calculating the phenol content to obtain the activity of the soil phosphatase.
5. The soil biological activity and productivity evaluation method based on soil enzyme activity assay according to claim 2, wherein the specific method of arylsulfatase activity assay is: placing fresh soil in a triangular flask, adding toluene, 0.5mol/L acetic acid buffer solution with pH value of 5.8 and 0.02mol/L p-nitrobenzene potassium sulfate solution, and shaking Uniformly mixing the fresh soil and the solution, wherein the pH value is controlled to be 7-11, and the mass ratio of the fresh soil to the toluene, the 0.5mol/L acetic acid buffer solution and the 0.02mol/L p-nitrobenzene potassium sulfate solution is 2 g: 0.5 ml: 8 ml: 2 ml; adding cover, culturing in 37 deg.C incubator for 1 hr, removing cover, adding 0.5mol/LCaCL2And 0.5mol/L NaOH solution, shaking up, filtering, carrying out colorimetric determination on the filtrate at 400nm, and calculating the p-nitrophenol quantity to represent the activity of the soil arylsulfatase.
6. The method for evaluating the biological activity and productivity of soil based on the determination of the activity of soil enzyme according to claim 2, wherein the specific method for determining the activity of β -glucosidase is as follows: placing fresh soil into a triangular flask, adding toluene into a fume hood, slightly shaking with a plug, standing for 15min, adding MUB solution and PNG solution, strongly shaking to uniformly mix a soil sample with the solution, wherein the pH value is controlled to be 4.0-6.0, and the volume ratio of the mass of the fresh soil to the toluene, the MUB solution and the PNG solution is 2 g: 0.5 ml: 8 ml: 2 ml; then culturing in a 37 deg.C constant temperature incubator for 1h, adding CaCL2And Tris buffer solution with the pH value of 12.0, shaking up, quickly filtering by using quick filter paper, carrying out color comparison on the filtrate at 400nm on a spectrophotometer, and calculating the p-nitrophenol content to represent the activity of the soil beta-glucosidase.
7. The soil biological activity and productivity evaluation method based on soil enzyme activity assay according to claim 2, wherein the specific method for N-acetyl- β -D-glucosaminidase activity assay is: placing fresh soil into a triangular flask, adding toluene, taking 100mmol/L acetic acid buffer solution with pH value of 5.5 and 10mmol/L pNP-beta-D-GlcNAc as substrates, shaking to uniformly mix the fresh soil and the substrates, and sealing, wherein the volume ratio of the mass of the fresh soil to the toluene to the 100mmol/L acetic acid buffer solution to the mass of the fresh soil to the mass of the pNP-beta-D-GlcNAc to the substrate is 1 g: 0.25 ml: 4 ml: 1 ml; culturing at 35 deg.C for 1 hr, adding 0.5mol/LCaCL2And (3) stopping the reaction with 0.5mol/L NaOH, filtering, carrying out colorimetric determination on the filtrate at 400nm, and calculating the p-nitrophenol amount to show the activity of the soil N-acetyl-beta-D-glucosaminidase.
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