Efficient determination method suitable for activity of carbon-nitrogen-phosphorus cycle-related enzymes in desert soil

Abstract

The invention provides an efficient determination method suitable for the activity of a carbon-nitrogen-phosphorus cycle-related enzyme in desert soil. The method comprises the following steps: 1) collecting and storing a soil sample; (2) preparation of a reagent: preparing a 4-MUB (4-MUB)-alpha-D-glucose solution, a 4-MUB (4-MUB)-beta-D-glucose solution, a 4-MUB-N-acetamide-beta-D-glucose saminide solution, a Urea (Urea) solution, a 4-MUB-phosphate solution, a 4-MUB (4-MUB) solution, a buffer solution and a NaOH (sodium hydroxide) solution; (3) preparing soil supernate; 4) sample determination; and 5) calculating a result. According to the method, the defects of insufficient sensitivity, long sample treatment time, limited substrate selection, poor environmental condition control and poor data standardization and comparability of a traditional enzyme activity determination technology in a desert ecosystem are overcome; the method is high in accuracy, high in sensitivity, quick in response, high in specificity and good in data standardization in the determination of the activity of the enzymes related to decomposition of carbon, nitrogen and phosphorus in a desert ecosystem.

Description

Technical Field

[0001] The present invention belongs to the technical field of soil enzyme activity determination. Specifically, the present application relates to a high-efficiency determination method applicable to the enzyme activities related to carbon, nitrogen, and phosphorus cycling in desert soil. Background Art

[0002] Enzyme activity determination is an important tool for evaluating soil microbial functions and ecosystem processes, and is widely used in fields such as ecology, environmental science, agriculture, and forestry. Existing studies have shown that extracellular enzymes (such as α-1,4-glucosidase, β-1,4-glucosidase, β-1,4-N-acetylglucosaminidase, urease, acid phosphatase, etc.) play a key role in the mineralization and transformation processes of carbon, nitrogen, and phosphorus. For example, Ajwa et al. (1999) found that nitrogen fertilizer application significantly affected the activities of β-glucosidase and acid phosphatase, while Sinsabaugh et al. (2002) pointed out that nitrogen deposition changed the distribution of enzyme activities during litter decomposition, promoting the decomposition of carbohydrates but inhibiting the oxidation of lignin. Traditional determination methods such as spectrophotometry and colorimetry are applicable to relatively mild environments such as temperate forests, farmlands, and grasslands, but are difficult to cope with extreme conditions such as deserts. In recent years, the application of fluorescence substrate methods, electrochemical sensing technologies, and high-throughput sequencing has significantly improved the sensitivity and accuracy of determination, but still faces challenges in application in desert environments.

[0003] The characteristics of the desert environment are low water content, high salinity, extreme temperature fluctuations, and low microbial activity, which make it difficult to effectively apply traditional enzyme activity determination methods. In addition, the enzyme activities in desert ecosystems are usually at extremely low levels, further increasing the difficulty of determination. Traditional methods are often limited by problems such as insufficient sensitivity, longer sample processing time, limited substrate selection, insufficient environmental condition control, poor data standardization and comparability under extremely arid conditions, and it is difficult to provide accurate results. Therefore, it is particularly important to develop a highly sensitive, rapid, and standardized enzyme activity determination method suitable for the desert environment. Summary of the Invention

[0004] In order to overcome the deficiencies of the existing methods for measuring the activities of enzymes related to carbon, nitrogen, and phosphorus decomposition in desert ecosystems, the present invention significantly improves the sensitivity and accuracy of detection through a series of innovative measures. Specifically, we introduce the technique of simultaneous multi-enzyme determination, simplify the experimental process, and make the operation more convenient and rapid; optimize the pH value and temperature of the reaction to simulate the real conditions of the desert environment; shorten the reaction time and improve the detection efficiency; select highly specific and sensitive fluorescent substrates, determine the minimum substrate concentration for the enzymatic reaction, which can not only reduce costs but also produce obvious fluorescent signals, reduce the influence of background noise, and improve the detection limit; use high-speed centrifugation to quickly separate the enzymes in the soil; and at the same time adopt a unified calculation formula. These improvements enable the present invention to effectively detect the trace enzyme activities in desert soil.

[0005] On the one hand, the present application provides an efficient method for measuring the activities of enzymes related to the carbon, nitrogen, and phosphorus cycles in desert soil, and the method includes:

[0006] 1) Collection and storage of soil samples;

[0007] 2) Reagent preparation: Prepare 4-MUB-α-D-glucoside solution, 4-MUB-β-D-glucoside solution, 4-MUB-N-acetyl-β-D-glucosaminide solution, Urea solution, 4-MUB-phosphate solution, 4-MUB solution, buffer solution, and NaOH solution;

[0008] 3) Preparation of soil supernatant;

[0009] 4) Sample determination;

[0010] 5) Result calculation.

[0011] Further, in step 2), the concentration of the 4-MUB-α-D-glucoside solution is 200 μM, the concentration of the 4-MUB-β-D-glucoside solution is 200 μM, the concentration of the 4-MUB-N-acetyl-β-D-glucosaminide solution is 200 μM, the concentration of the Urea solution is 200 μM, and the concentration of the 4-MUB-phosphate solution is 200 μM.

[0012] Further, in step 2), the concentration of the 4-MUB solution is 10 μM, the buffer solution is a 50 mM Tris-base buffer solution with a pH of 8.0, and the concentration of the NaOH solution is 1 M.

[0013] Further, step 4) is carried out on a microplate.

[0014] The orifice plate can be of existing commercially available or customized types, including but not limited to 44, 48, 96-well plates, etc. Those skilled in the art can appropriately combine orifice plates according to the number of rows and columns of the orifice plate to match different sample numbers and detection instruments.

[0015] Further, in step 4), a blank well, a standard fluorescence well, a sample control well, a standard control well, a substrate control well, and a sample fluorescence well are set up, and dark constant temperature culture, termination of the reaction, and measurement on the machine are carried out;

[0016] Further, buffer solution is added to the blank well;

[0017] Buffer solution and 4-MUB solution are added to the standard fluorescence well;

[0018] Soil supernatant and buffer solution are added to the sample control well;

[0019] Soil supernatant and 4-MUB solution are added to the standard control well;

[0020] There are 5 substrate control wells, which are respectively added with 4-MUB-α-D-glucoside solution, 4-MUB-β-D-glucoside solution, 4-MUB-N-acetyl-β-D-glucosaminide solution, Urea solution, 4-MUB-phosphate solution, and buffer solution;

[0021] The sample fluorescence wells are in groups of 5. Each group is added with 4-MUB-α-D-glucoside solution, 4-MUB-β-D-glucoside solution, 4-MUB-N-acetyl-β-D-glucosaminide solution, Urea solution, 4-MUB-phosphate solution, and soil supernatant.

[0022] The number of sample fluorescence wells can be set according to the number of samples to be detected. For example, for detecting two samples, 2 groups of 10 wells can be set, and for 7 samples, 7 groups of 35 wells can be set. To ensure accuracy, multiple groups of substrate control wells can also be set, preferably one group of 5 for each orifice plate.

[0023] Further, 250 μl of buffer solution is added to the blank well;

[0024] 200 μl of buffer solution and 50 μl of 4-MUB solution are added to the standard fluorescence well;

[0025] 200 μl of soil supernatant and 50 μl of buffer solution are added to the sample control well;

[0026] 200 μl of soil supernatant and 50 μl of 4-MUB solution are added to the standard control well;

[0027] The substrate control wells are 5, each added with 50 μl of 4-MUB-α-D-glucoside solution, 50 μl of 4-MUB-β-D-glucoside solution, 50 μl of 4-MUB-N-acetyl-β-D-glucosaminide solution, 50 μl of Urea solution, 50 μl of 4-MUB-phosphate solution and 200 μl of buffer;

[0028] The sample fluorescence wells are in groups of 5, and each group is added with 50 μl of 4-MUB-α-D-glucoside solution, 50 μl of 4-MUB-β-D-glucoside solution, 50 μl of 4-MUB-N-acetyl-β-D-glucosaminide solution, 50 μl of Urea solution, 50 μl of 4-MUB-phosphate solution and 200 μl of soil supernatant.

[0029] Further, in step 4), it is placed in an incubator at 20 °C, 40 °C or 27 °C and cultured for 2.5 h under dark conditions; the microplate is taken out 1 min before measurement, and 10 μl of 1 mol L -1 NaOH solution is added to terminate the reaction; the fluorescence is measured under the conditions of an excitation wavelength of 365 nm and an emission wavelength of 450 nm.

[0030] Further, step 5) includes:

[0031]

[0032] Further, step 1) includes:

[0033] Soil sample collection: Collect fresh soil samples;

[0034] On-site treatment: Mix the soil samples evenly, pass through a 2-mm sieve to remove impurities;

[0035] Sample storage: Immediately after sieving, put the samples into a sealed bag and transport them in an ice box;

[0036] Refrigerated storage: Store the soil samples in a refrigerator at 4 °C and complete the enzyme activity measurement within one week

[0037] Further, step 3) includes:

[0038] Weigh the soil sample: Weigh 1.00 ± 0.001 g of soil sample into a 250-ml shaking flask;

[0039] Add buffer: Add 100 ml of 50 mmol L -1 Tris-base buffer;

[0040] Constant temperature oscillation: Add small glass beads and oscillate at 180 rpm for 0.5 h in a constant temperature shaker at 20 °C, 40 °C or 27 °C;

[0041] High-speed centrifugation: Centrifuge at 3000 g for 15 min to obtain the supernatant.

[0042] Furthermore, before adding the soil supernatant in step 4), place it on a magnetic stirrer and stir at 900 rpm.

[0043] The present invention provides a new efficient method for measuring the activities of enzymes related to carbon, nitrogen and phosphorus decomposition in desert ecosystems. Compared with the original method, the most important technical effects are as follows: introducing the technology of simultaneous measurement of multiple enzymes, which is simple and fast to operate; higher sensitivity and lower detection limit; introducing specific fluorescent substrates; optimizing the reaction temperature, pH and substrate concentration; shortening the operation time; improving data standardization and comparability. The specific improvements are reflected in the following aspects: using fluorescent substrates of different enzymes to simultaneously measure the activities of 3 enzymes in the same microplate, which greatly saves time and operational convenience. This method can obtain the activity data of multiple enzymes through one-time processing and analysis steps, not only significantly shortening the experimental period, but also greatly simplifying the experimental process, making the operation more simple and fast. In addition, it reduces the usage of samples and reagents, lowers the experimental cost, and at the same time ensures that all measurements are carried out under consistent conditions, thus improving the reliability and repeatability of the results.

[0044] Using specific fluorescent substrates for enzyme activity determination significantly improves the detection sensitivity and reduces the detection limit. And the minimum substrate concentration required to analyze the potential enzyme activities of enzymes related to carbon, nitrogen and phosphorus decomposition in desert soil is determined to be 200 μM, which enables us to measure at a lower substrate concentration, thereby reducing the sample usage and experimental cost. In addition, the fluorescence signal has good stability and low background noise, further improving the reliability and accuracy of the data.

[0045] In order to ensure that the results of enzyme activity determination can more accurately reflect the actual situation of seasonal changes in the Gurbantunggut Desert environment, we adjusted the experimental conditions based on the data of the climate observation system in the study area. Specifically, the experimental temperature in spring is set at 20 °C to simulate the relatively mild climate in this season; in summer, it is increased to 40 °C to reflect the actual situation under high-temperature environment; in autumn, 27 °C is adopted to simulate the moderate temperature conditions in this season. However, since the temperature in winter is too low and not suitable for enzyme activity determination, the experimental temperature is not set in this season. At the same time, since desert soil is alkaline soil, Tris-base with pH = 8 is used as the buffer to simulate the alkaline environment of the desert, ensuring that the enzyme activity determination conditions can accurately simulate the characteristics of the desert environment and reducing the errors caused by environmental differences.

[0046] To improve the experimental efficiency, the shaking time of the soil suspension was adjusted to 0.5 h, which could not only fully mix the enzymes and substrates in the soil sample, but also significantly shorten the pretreatment time and improve the experimental efficiency. The shaken soil suspension was centrifuged at 3000 g for 15 min to obtain the supernatant, thereby effectively separating the solid and liquid phases and extracting a relatively pure enzyme solution, reducing impurity interference and facilitating subsequent enzyme activity determination. At the same time, according to the time gradient experiment, the incubation time of the microplate was set to 2.5 h. At this time, the potential enzyme activities of the enzymes related to carbon, nitrogen, and phosphorus decomposition in desert soil had reached a plateau, which was sufficient for the enzymes to fully react with the fluorescent substrates, generating enough fluorescent signals for detection, while avoiding side reactions or signal saturation caused by too long incubation time, thus shortening the overall experimental cycle.

[0047] To unify the calculation method and enzyme activity unit and facilitate the comparison of different experimental results, a unified calculation formula was formulated to convert the fluorescence signal intensity into enzyme activity units. All experimental data were calculated according to this formula, ensuring the consistency and comparability of the data; a standard enzyme activity unit (n mol / h / g) was defined, ensuring a high degree of consistency and comparability between different experimental results. Brief Description of the Drawings

[0048] Figure 1 It is a schematic diagram of a black 96-well microplate.

[0049] Figure 2 It is a diagram of the sample addition sequence of a black 96-well microplate.

[0050] Figure 3 Showing the comparison of the activities of enzymes related to carbon, nitrogen, and phosphorus decomposition in desert soil under the traditional colorimetric method and the method of the present invention. Detailed Embodiments

[0051] The following examples are convenient for better understanding of the present invention, but are not limited thereto. These examples are only for illustrative purposes and in no way limit the protection scope of the present invention.

[0052] Based on the principle of enzyme-catalyzed hydrolysis of fluorescent substrates, specific fluorescent substrates produce fluorescent products after being hydrolyzed by the target enzymes. The change in fluorescence intensity is monitored in real time by a high-sensitivity fluorescence detection instrument to quantitatively determine the activities of enzymes related to carbon, nitrogen, and phosphorus decomposition in desert ecosystems. The optimized reaction conditions and standardized operation procedures further improve the accuracy and comparability of the detection.

[0053] Example 1 Sample Collection and Storage

[0054] Soil sample collection: Fresh soil samples were collected at the in-situ test site on the southern edge of the Gurbantunggut Desert in Xinjiang on March 25, 2024, using the five-point sampling method;

[0055] Field treatment: Mix the soil samples from the same treatment and soil layer evenly, pass them through a 2-mm sieve, and remove impurities;

[0056] Sample storage: Immediately after sieving, put the samples into a sealed bag and transport them in an ice box;

[0057] Refrigerated storage: Store the soil samples in a refrigerator at 4°C and complete the enzyme activity determination within one week.

[0058] Example 2 Reagent Preparation and Soil Supernatant Preparation

[0059] Reagent 1: Preparation of 100 ml of 200 μM 4-MUB-α-D-glucoside solution. Weigh 6.7662 mg of 4-MUB-α-D-glucoside, dissolve it in deionized water, and make up the volume to 100 ml;

[0060] Reagent 2: Preparation of 100 ml of 200 μM 4-MUB-β-D-glucoside solution. Weigh 6.7662 mg of 4-MUB-β-D-glucoside, dissolve it in deionized water, and make up the volume to 100 ml;

[0061] Reagent 3: Preparation of 100 ml of 200 μM 4-MUB-N-acetyl-β-D-glucosaminide solution. Weigh 7.5872 mg of 4-MUB-N-acetyl-β-D-glucosaminide, dissolve it in deionized water, and make up the volume to 100 ml;

[0062] Reagent 4: Preparation of 100 ml of 200 μM Urea solution. Weigh 1.2012 mg of Urea, dissolve it in deionized water, and make up the volume to 100 ml;

[0063] Reagent 5: Preparation of 100 ml of 200 μM 4-MUB-phosphate solution. Weigh 5.1230 mg of 4-MUB-phosphate, dissolve it in deionized water, and make up the volume to 100 ml;

[0064] Reagent 6: Preparation of 100 ml of 10 μM 4-MUB solution. First, prepare 100 ml of 100 μM 4-MUB stock solution. Weigh 1.7620 mg of 4-MUB with a one-millionth balance, dissolve it in deionized water, and make up the volume to 100 ml in a volumetric flask. Store it in a -20°C refrigerator. Before use, pipette 10 ml of the 100 μM 4-MUB stock solution and make up the volume to 100 ml;

[0065] Reagent 7: 2 L of 50 mmol L -1Preparation of Tris-base buffer: weigh 12.1140 g Tris-base on a 1 / 10,000 balance, dissolve in deionized water and make up to 2 L, pH = 8;

[0066] Reagent 8: 100ml 1mol L -1 To prepare the NaOH solution, weigh 4 g of NaOH, dissolve it in deionized water and make up to 100 ml.

[0067] Soil supernatant preparation:

[0068] Weigh soil samples: weigh 1.00±0.001g soil sample from each soil layer under each treatment into a 250ml shaking bottle;

[0069] Add buffer: add 100ml 50mmol L -1 Tris-base buffer;

[0070] Constant temperature shaking: add small glass beads and shake on a constant temperature shaker at 20°C and 180 rpm for 0.5 h;

[0071] High-speed centrifugation: Centrifuge at 3000g for 15 minutes to obtain the supernatant.

[0072] Example 3 Sample determination

[0073] Stirring the sample: Place the sample supernatant on a magnetic stirrer and stir continuously at 900 rpm;

[0074] Sample addition conditions Figure 2 As shown:

[0075] Add clear solution: black 96-well microplate as Figure 1 Place horizontally, starting from row B, add 200 μl of the supernatant of the first sample to each well, add the second sample to row C, and then add the seventh sample to row H, and then repeat the above steps for the next plate, adding 2 plates in total;

[0076] Add buffer and substrate: first add 200μl buffer to row A, then add 50μl buffer to column 1, add 50μl MUB to column 2, add 50μl substrate 1 to columns 3-5, add 50μl substrate 2 to columns 6-8, add 50μl substrate 3 to columns 9-11, and start timing. Repeat the above steps for the second plate, but add 50μl substrate 4 to columns 3-5 and 50μl substrate 5 to columns 6-8, and then start timing.

[0077] Dark constant temperature culture: Place in a 20℃ incubator for 2.5h under dark conditions;

[0078] Stop the reaction: Take out the microplate 1 min in advance and add 10 μl of 1 mol L -1 NaOH solution terminates the reaction;

[0079] On-machine determination: Immediately measure the fluorescence on an A615 fluorescence multi-functional microplate reader under the conditions of an excitation wavelength of 365 nm and an emission wavelength of 450 nm.

[0080] Calculation formula:

[0081]

[0082]

[0083] Example 4: Study on the influence of substrate concentration and incubation time on the detection effect

[0084] By setting four groups of substrate concentration gradients of 50 μM, 100 μM, 200 μM, and 400 μM, it was found that when the substrate concentration was lower than the critical threshold of 200 μM, the fluorescence signal intensity of enzymes related to carbon, nitrogen, and phosphorus decomposition in desert soil (such as β-1,4-glucosidase) decreased significantly, resulting in a substantial decrease in detection sensitivity (the detection limit increased to more than 5 times the original level), and it was difficult to accurately quantify the actual enzyme activity. Further experiments showed that when the substrate concentration was increased to 200 μM, the fluorescence signal showed a high linear correlation with the substrate concentration (R 2 > 0.95), and at the same time, the fluorescence quenching phenomenon caused by excessive substrate (such as the competitive binding of high-concentration substrate and fluorescent product to the excitation light) was effectively avoided. Under this threshold, the background noise of the detection system was suppressed to an ideal level, and the dynamic enzyme activity could be stably reflected.

[0085] Under the incubation time gradient of 0.5 - 4 hours, the enzyme activity in desert soil reached a plateau at 2.5 hours (the increase rate < 5%). If the incubation time was insufficient (such as 1.5 hours), the activity of some enzymes (such as β-1,4-N-acetylglucosaminidase) was underestimated by more than 30% due to incomplete reactions; after the incubation time exceeded 3 hours, side reactions caused abnormal increases in fluorescence values, and at the same time, the background signal increased significantly due to spontaneous hydrolysis of the substrate. The experiment finally determined that the optimal incubation time was 2.5 hours. At this time, the enzymatic reaction was fully completed, and the background interference was at a controllable level, ensuring the reliability and repeatability of the detection results.

[0086] Example 5: Comparison of the activities of enzymes related to carbon, nitrogen, and phosphorus decomposition in desert soil under the traditional colorimetric method and the method of the present invention

[0087] The traditional colorimetric method is based on the pNP chromogenic substrate, reacts at a high substrate concentration (≥400 μM) in a universal buffer (pH 6.5), and is detected by colorimetry at a wavelength of 405 nm after termination with strong acid. However, there are significant limitations when applied to desert soils: the pNP chromogenic sensitivity is relatively low, and the signal response to low-activity enzymes is insufficient; the acidic termination solution is prone to induce precipitation in alkaline soils, and the universal buffer does not match the pH of the desert environment; in addition, the detection of carbon, nitrogen, and phosphorus degrading enzymes requires separate and independent operations, and the process is complex and time-consuming. In contrast, the method of the present invention significantly overcomes the above defects through system optimization (4-MUB fluorescent substrate, pH 8.0 adapted buffer, multi-enzyme synchronous detection design, and alkaline termination solution). As Figure 3 shown, the detected activity values of α-1,4-glucosidase, β-1,4-glucosidase, β-1,4-N-acetylglucosaminidase, urease, and alkaline phosphatase in the spring soil of the Gurbantunggut Desert are all significantly improved compared with the traditional method, indicating that the advantages of the present invention in sensitivity and anti-interference can more truly reflect the actual activity levels of carbon, nitrogen, and phosphorus degrading enzymes in desert soils.

Claims

1. An efficient method for determining the activity of enzymes related to the carbon, nitrogen and phosphorus cycles in desert soils, characterized in that: The method comprises: 1) Soil sample collection and storage; 2) Reagent preparation: prepare 4-MUB-α-D-glucoside solution, 4-MUB-β-D-glucoside solution, 4-MUB-N-acetyl-β-D-glucosaminide solution, Urea solution, 4-MUB-phosphate solution, 4-MUB solution, buffer solution, and NaOH solution; 3) Preparation of soil supernatant; 4) Sample determination; 5) Calculation of results.

2. The method according to claim 1, wherein in step 2), the concentration of the 4-MUB-α-D-glucoside solution is 200 μM, the concentration of the 4-MUB-β-D-glucoside solution is 200 μM, the concentration of the 4-MUB-N-acetyl-β-D-glucosaminide solution is 200 μM, the concentration of the Urea solution is 200 μM, and the concentration of the 4-MUB-phosphate solution is 200 μM.

3. The method according to claim 1, wherein in step 2), the concentration of the 4-MUB solution is 10 μM, the buffer is 50 mM Tris-base buffer at pH 8.0, and the concentration of the NaOH solution is 1 M.

4. The method according to claim 1, wherein step 4) is performed on a well plate.

5. The method according to claim 4, wherein step 4) comprises setting blank wells, standard fluorescence wells, sample control wells, standard control wells, substrate control wells and sample fluorescence wells; and performing dark constant temperature incubation, terminating the reaction and measuring on a machine.

6. The method according to claim 5, wherein a buffer is added to the blank well; Add buffer and 4-MUB solution into the standard fluorescence well; The sample control wells are added with soil supernatant and buffer; The standard control wells were added with soil supernatant and 4-MUB solution; There are 5 substrate control wells, into which 4-MUB-α-D-glucoside solution, 4-MUB-β-D-glucoside solution, 4-MUB-N-acetyl-β-D-glucosaminide solution, Urea solution, 4-MUB-phosphate solution and buffer solution are added respectively; The sample fluorescence wells are divided into groups of 5, and 4-MUB-α-D-glucoside solution, 4-MUB-β-D-glucoside solution, 4-MUB-N-acetyl-β-D-glucosaminide solution, Urea solution, 4-MUB-phosphate solution and soil supernatant are added to each group.

7. The method according to claim 6, wherein 250 μl of buffer is added to the blank well; 200 μl of buffer and 50 μl of 4-MUB solution were added to the standard fluorescence wells; 200 μl of soil supernatant and 50 μl of buffer were added to the sample control well; The standard control wells were added with 200 μl of soil supernatant and 50 μl of 4-MUB solution; The substrate control wells consisted of five wells, each of which was added with 50 μl of 4-MUB-α-D-glucoside solution, 50 μl of 4-MUB-β-D-glucoside solution, 50 μl of 4-MUB-N-acetyl-β-D-glucosaminide solution, 50 μl of Urea solution, 50 μl of 4-MUB-phosphate solution, and 200 μl of buffer; The sample fluorescence wells are divided into groups of 5, and 50 μl of 4-MUB-α-D-glucoside solution, 50 μl of 4-MUB-β-D-glucoside solution, 50 μl of 4-MUB-N-acetyl-β-D-glucosaminide solution, 50 μl of Urea solution, 50 μl of 4-MUB-phosphate solution and 200 μl of soil supernatant are added to each group.

8. The method according to claim 5, wherein in step 4), the microplate is placed in a 20°C, 40°C or 27°C incubator for 2.5 h under dark conditions; 1 min before the measurement, the microplate is taken out and 10 μl of 1 mol L -1 The reaction was terminated with NaOH solution; fluorescence was measured at an excitation wavelength of 365 nm and an emission wavelength of 450 nm.

9. The method according to claim 1, wherein step 5) comprises:

10. The method according to claim 1, wherein step 1) comprises: Soil sample collection: Collect fresh soil samples; On-site treatment: Mix the soil sample evenly and pass it through a 2mm sieve to remove impurities; Sample storage: After screening, immediately put the sample into a sealed bag and place it in an ice box for transportation; Refrigerated storage: Store soil samples in a refrigerator at 4°C and complete enzyme activity assay within one week. Step 3) includes: Weigh soil sample: weigh 1.00±0.001g soil sample into a 250ml shaking bottle; Add buffer: add 100ml 50mmol L -1 Tris-base buffer; Constant temperature shaking: add small glass beads, shake at 180 rpm for 0.5 h at 20℃, 40℃ or 27℃ in a constant temperature shaker; High-speed centrifugation: Centrifuge at 3000g for 15 minutes to obtain the supernatant. Before adding the soil supernatant in step 4), the soil supernatant was stirred at 900 rpm on a magnetic stirrer.

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