Urease activity detection and analysis method suitable for EICP technology

Abstract

The invention relates to the technical field of biomineralization, in particular to a urease activity detection and analysis method suitable for an EICP technology, and the urease activity detection and analysis method comprises the following steps: preparing a urease solution, a urea solution and an ammonium ion standard solution; measuring the change of the conductivity value along with the ammonium ion concentration at the test temperature; measuring the change of the conductivity value of the urease and urea mixed solution along with time at the test temperature; and calculating the generation rate of ammonium ions in the mixed solution at the test temperature according to a measurement result, and further calculating the activity of urease. The urease activity detection and analysis method suitable for the EICP technology has the advantages of being convenient to operate, easy to analyze, wide in application range and the like compared with a common urease activity detection method at present, and can be used for rapidly determining the urease activity at different temperatures, so that the proper temperature is selected to apply the EICP technology.

Description

Technical Field

[0001] The present invention relates to the technical field of biomineralization, and specifically to a method for detecting and analyzing urease activity applicable to the EICP technology. Background Art

[0002] Biomineralization technology has received extensive attention and research from scholars around the world due to its characteristics such as being green, environmentally friendly, and highly applicable. Two commonly used biomineralization technologies are respectively the microbial-induced calcium carbonate precipitation (abbreviated as MICP) technology and the urease-induced calcium carbonate precipitation (abbreviated as EICP) technology. Compared with the MICP technology, the EICP technology can more precisely control the biomineralization process by extracting urease from plants (such as jack beans and soybeans); it can directly catalyze the reaction without relying on microbial proliferation, eliminating the risk of uncontrollable microbial reproduction and breaking through the temperature limit for microbial survival. In recent years, the EICP technology has been widely applied in many fields such as soil solidification, water quality improvement, and ancient building maintenance due to characteristics such as easy availability of materials and environmental friendliness.

[0003] The principle of the EICP technology is that urease promotes the hydrolysis of urea to produce carbonate ions, and the carbonate ions react with calcium ions in the calcium source to form calcium carbonate crystals. Urease activity is an important factor affecting the hydrolysis efficiency of urea, and the hydrolysis efficiency of urea directly affects the binding rate of carbonate ions and calcium ions, thereby affecting the entire biomineralization process. Therefore, when applying the EICP technology, how to quickly and accurately measure urease activity is an urgent problem to be solved. Currently, commonly used methods for measuring urease activity include colorimetry, acid-base titration, conductivity method, etc. The measurement results of colorimetry are greatly affected by the environment, the operation of acid-base titration is complex, and the conductivity method has become a commonly used method for measuring urease activity due to its simple operation and low cost.

[0004] When urease catalyzes the hydrolysis of urea, the conductivity of the solution continuously increases. The conductivity method is proposed based on this phenomenon, establishing a connection between the change in conductivity and the hydrolysis amount of urea. The commonly used method for calculating urease activity was proposed by Whiffin in 2004. He corresponded the change in conductivity of 1 μS / cm per minute in the mixed solution at 25°C to the urea hydrolysis amount of 11.11 μmol / (L·min), as shown in formulas (1) and (2).

[0005]

[0006] Wherein, ΔEC is the change rate of the conductivity of the mixed solution from the t1-th minute to the t2-th minute, with the unit of μS / (cm·min); EC1 is the conductivity value of the mixed solution at the t1-th minute, with the unit of μS / cm; EC2 is the conductivity value of the mixed solution at the t2-th minute, with the unit of μS / cm; A is the urease activity, with the unit of U / mg; V is the volume of the mixed solution, with the unit of L; m is the mass of urease in the mixed solution, with the unit of mg.

[0007] Although the above method is widely used, it has the following three defects. First, the calculation result of formula (1) is affected by the selected time range. When the conductivity of the mixed solution at different times is selected, there will be a large deviation in the urease activity value calculated according to formula (1). Second, formula (2) is an empirical formula established under standard conditions (i.e., 25°C). If the environmental temperature changes, the amount of urea hydrolysis corresponding to the change in conductivity of 1 μS / cm per minute in the mixed solution will also change. Finally, when most existing studies use the conductivity method to measure urease activity, they usually use the temperature compensation function built into the conductivity meter to automatically convert the conductivity value of the mixed solution in a non-standard environment into the conductivity value at 25°C. The generally adopted temperature compensation coefficient is 0.02. However, this temperature compensation coefficient is not applicable to all ions, and there will be a large error in the electrolysis rate value obtained by the temperature compensation method, which will further lead to a more serious deviation in the urease activity value calculated according to formula (1) and formula (2). Considering the deficiencies of the existing conductivity method for measuring urease activity, it is necessary to further optimize the urease activity detection and analysis method applicable to the EICP technology. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the present invention provides a urease activity detection and analysis method applicable to the EICP technology, which has the characteristics of convenient operation, simple analysis, wide application range, etc., and can be used to quickly measure the activity of urease at different temperatures, so as to select a suitable temperature for applying the EICP technology.

[0009] To achieve the above object, the present invention provides the following technical solution: A urease activity detection and analysis method applicable to the EICP technology, including the following steps,

[0010] S1. Prepare urease solution, urea solution and ammonium ion standard solutions with different concentrations;

[0011] S2. Set the temperature of the constant temperature water bath device, and place the test tubes filled with deionized water, urease solution, urea solution and ammonium ion standard solutions with different concentrations into the constant temperature water bath device for heat preservation treatment; put the thermometer probe into the test tube filled with deionized water to monitor the temperature change. When the thermometer shows the set temperature and after heat preservation for a period of time, start the conductivity test;

[0012] S3. Immerse the conductivity electrode of the conductivity meter successively in ammonium ion standard solutions with different concentrations, and measure the conductivity values of the ammonium ion standard solutions with different concentrations at the test temperature. After the test, plot the curve of the conductivity value versus the ammonium ion concentration, fit it with a linear equation, and calculate the slope of the curve. This slope value is denoted as k1.

[0013] S4. Quickly mix the urease solution and the urea solution. Then immerse the conductivity electrode of the conductivity meter in the prepared mixed solution and start timing. Record the conductivity value every 30 s until 11 min ends. After the test, plot the curve of the conductivity value versus time at the test temperature, select the linear part of the curve, fit it with a linear equation, and calculate the slope of the linear part. This slope value is denoted as k2.

[0014] S5. Calculate the formation rate of ammonium ions and the urease activity in the mixed solution in step S4. The calculation formulas are as follows:

[0015]

[0016] Preferably, in step S1, when preparing different solutions, to ensure that the reagents are fully dissolved in deionized water, place the beakers containing each solution on a magnetic stirrer and stir for at least 10 min.

[0017] Preferably, in step S1, the prepared urease solution is stored in a low-temperature environment to prevent inactivation.

[0018] Preferably, in step S1, when preparing the urea solution, the urea concentration should not be too low to ensure that the urease activity can be fully exerted.

[0019] Preferably, in step S3, when quickly mixing the urease solution and the urea solution, pour the urea solution into the test tube containing the urease solution to prevent the temperature of the urease solution from changing.

[0020] Preferably, in steps S3 - S4, when measuring the conductivity value of the solution with a conductivity meter, to measure the conductivity value of the solution at the current test temperature, do not turn on the temperature compensation function of the conductivity meter.

[0021] Preferably, in step S5, in the formula: Δc is the formation rate of ammonium ions, with the unit of mmol / min; k1 is the change rate of conductivity with respect to the ammonium ion concentration, with the unit of μS / (cm·mM); k2 is the change rate of conductivity with respect to time, with the unit of μS / (cm·min); V is the volume of the mixed solution, with the unit of L; A is the urease activity, with the unit of U / mg; m is the mass of urease in the mixed solution, with the unit of mg.

[0022] The present invention provides a method for detecting and analyzing urease activity applicable to the EICP technology, which has the following beneficial effects compared with the prior art:

[0023] Based on the theory that the ions generated by the hydrolysis of urea catalyzed by urease cause an increase in the conductivity of the solution, the present invention proposes a method for detecting and analyzing urease activity applicable to the EICP technology. By measuring the change curve of the conductivity value with the ammonium ion concentration at the test temperature and the change curve of the conductivity value of the mixed solution with time at the test temperature, the conductivity change is related to the urea hydrolysis rate. Therefore, it can be used to replace the previous method of detecting urease activity by conductivity. Compared with the existing method of measuring urease activity by conductivity, the present invention has the characteristics of simple operation, rapid analysis, wide application range, etc., and can be used to quickly measure the urease activity at different temperatures, so as to select a suitable temperature for applying the EICP technology. Description of the Drawings

[0024] The drawings described herein are used to provide a further understanding of the present application and form a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation to the present application. In the drawings:

[0025] Figure 1 It is the change curve of the conductivity value with the ammonium ion concentration at 20 °C in Example 1 of the present invention;

[0026] Figure 2 It is the change curve of the conductivity value of the mixed solution with time at 20 °C in Example 1 of the present invention;

[0027] Figure 3 It is the change curve of the conductivity value with the ammonium ion concentration at 40 °C in Example 2 of the present invention;

[0028] Figure 4 It is the change curve of the conductivity value of the mixed solution with time at 40 °C in Example 2 of the present invention;

[0029] Figure 5 It is the relationship curve of urease activity and temperature in Example 7 of the present invention. Detailed Embodiments

[0030] The following embodiments are used to detail the implementation manners of the present application, so as to fully understand how the present application uses technical means to solve technical problems and achieve the realization process of technical effects and implement accordingly.

[0031] Example 1

[0032] A method for detecting and analyzing urease activity applicable to the EICP technology includes the following steps:

[0033] S1. Prepare urease solution, urea solution and ammonium ion standard solutions with different concentrations;

[0034] S11. Weigh 6.0 mg of jack bean urease using a high-precision balance with an accuracy of 0.1 mg and place it in a beaker. Pour deionized water into the beaker to make up the volume to 300 mL to obtain a urease solution with a concentration of 20 mg / L. Place the beaker containing the urease solution in a magnetic stirrer and stir for 10 min, then place it in an incubator with a set temperature of 5 °C for storage;

[0035] S12. Weigh 27 g of urea using a high-precision balance with an accuracy of 0.1 mg and place it in a beaker. Pour deionized water into the beaker to make up the volume to 300 mL to obtain a urea solution with a concentration of 1.5 mol / L. Place the beaker containing the urea solution in a magnetic stirrer and stir for 10 min, then store it at room temperature;

[0036] S13. Weigh 106.9 mg of ammonium chloride using a high-precision balance with an accuracy of 0.1 mg and place it in a beaker. Pour deionized water into the beaker to make up the volume to 200 mL to obtain a 10 mmol / L ammonium chloride solution as the stock solution. Place the beaker containing the ammonium chloride solution in a magnetic stirrer and stir for 10 min. Use a pipette to measure 3 mL, 6 mL, 9 mL, 12 mL, and 15 mL of the 10 mmol / L ammonium chloride solution into test tubes respectively, add deionized water to make up the volume to 30 mL and shake well to obtain ammonium chloride solutions with concentrations of 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, and 5 mmol / L as ammonium ion standard solutions;

[0037] S2. Set the temperature of the constant temperature water bath equipment to 20 °C. Place test tubes containing 30 mL of deionized water, 3 mL of urease solution, 27 mL of urea solution, and 30 mL of ammonium ion standard solutions with different concentrations into the constant temperature water bath equipment for heat preservation; Place the thermometer probe into the test tube containing deionized water to monitor the temperature change. When the thermometer shows the set temperature and remains stable for 5 min, start the conductivity test;

[0038] S3. Immerse the conductivity electrode of the conductivity meter successively in ammonium ion standard solutions with different concentrations to measure the conductivity values of ammonium ion standard solutions with different concentrations at 20 °C (without turning on the temperature compensation function of the conductivity meter); After the test, plot the curve of conductivity value versus ammonium ion concentration, fit it with a linear equation and calculate the slope of the curve. This slope value is denoted as k1, as Figure 1 shown;

[0039] S4. Rapidly mix 3 mL of urease solution and 27 mL of urea solution. The urea solution should be poured into the test tube containing the urease solution. Subsequently, immerse the conductivity electrode of the conductivity meter into the prepared mixed solution and start timing. Record the conductivity value every 30 s (without turning on the temperature compensation function of the conductivity meter) until 11 min ends. After the test, plot the curve of the conductivity value versus time at the test temperature, select the conductivity values from the 30th s to the 4th min, fit them with a linear equation, and calculate the curve slope of the linear part. This slope value is denoted as k2, as Figure 2 shown;

[0040] S5. Calculate the generation rate of ammonium ions and the urease activity in the mixed solution in step S4. The calculation formulas are as follows:

[0041]

[0042] In the formula: Δc is the generation rate of ammonium ions, with the unit of mmol / min; k1 is the change rate of conductivity with respect to the ammonium ion concentration, with the unit of μS / (cm·mM); k2 is the change rate of conductivity with respect to time, with the unit of μS / (cm·min); V is the volume of the mixed solution, with the unit of L; A is the urease activity, with the unit of U / mg; m is the mass of urease in the mixed solution, with the unit of mg.

[0043] The urease activity calculated in this example at 20 °C is 48.18 U / mg.

[0044] Example 2

[0045] It is basically the same as Example 1, except that the temperature of the constant temperature water bath equipment is set to 40 °C. Among them, after the test, plot the curve of the conductivity value versus the ammonium ion concentration at 40 °C, fit it with a linear equation, and calculate the curve slope, as Figure 3 shown; plot the curve of the conductivity value of the mixed solution versus time at 40 °C, select the conductivity values from the 30th s to the 4th min, fit them with a linear equation, and calculate the curve slope, as Figure 4 shown.

[0046] The urease activity calculated in this example at 40 °C is 104.82 U / mg.

[0047] Example 3

[0048] It is basically the same as Example 2, except that the temperature of the constant temperature water bath equipment is set to 25 °C. The urease activity calculated in this example at 25 °C is 56.55 U / mg.

[0049] Example 4

[0050] Basically the same as Example 2, with the difference that the temperature of the constant temperature water bath equipment is set at 30 °C. The activity of urease at 25 °C calculated in this example is 68.93 U / mg.

[0051] Example 5

[0052] Basically the same as Example 2, with the difference that the temperature of the constant temperature water bath equipment is set at 35 °C. The activity of urease at 35 °C calculated in this example is 79.98 U / mg.

[0053] Example 6

[0054] Basically the same as Example 2, with the difference that the temperature of the constant temperature water bath equipment is set at 45 °C. The activity of urease at 45 °C calculated in this example is 124.27 U / mg.

[0055] Example 7

[0056] Statistically analyze the activities of urease at various temperatures in Examples 1-6, as shown in Table 1. According to the data in Table 1, convert the test temperature to Kelvin temperature and take its reciprocal as the abscissa, and take the logarithm of urease activity as the ordinate to plot a relationship curve, as Figure 5 shown. It can be found that the relationship between urease activity and temperature conforms to the Arrhenius equation.

[0057] Table 1 Urease activity at different temperatures

[0058] Temperature (°C) 20 25 30 35 40 45 Urease activity (U / mg) 48.18 56.55 68.93 79.98 104.82 124.27

[0059] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting and analyzing urease activity applicable to the EICP technology, characterized in that, It includes the following steps: S1. Prepare urease solution, urea solution and ammonium ion standard solutions with different concentrations; S2. Set the temperature of the constant temperature water bath equipment. Put the test tubes filled with deionized water, urease solution, urea solution and ammonium ion standard solutions with different concentrations into the constant temperature water bath equipment for heat preservation. Put the thermometer probe into the test tube filled with deionized water to monitor the temperature change. When the thermometer reading reaches the set temperature and is kept warm for a period of time, start the conductivity test; S3. Immerse the conductivity electrode of the conductivity meter into the ammonium ion standard solutions with different concentrations in turn, and measure the conductivity values of the ammonium ion standard solutions with different concentrations at the test temperature. After the test, plot the curve of the conductivity value changing with the ammonium ion concentration, fit it with a linear equation and calculate the slope of the curve. This slope value is denoted as k1; S4. Quickly mix the urease solution and the urea solution. Then immerse the conductivity electrode of the conductivity meter into the prepared mixed solution and start timing. Record the conductivity value every 30 s until 11 min ends. After the test, plot the curve of the conductivity value changing with time at the test temperature, select the linear part of the curve, fit it with a linear equation and calculate the slope of the linear part of the curve. This slope value is denoted as k2; S5. Calculate the generation rate of ammonium ions and the urease activity in the mixed solution in step S4. The calculation formulas are as follows:

2. The urease activity detection and analysis method applicable to the EICP technology according to claim 1, wherein In step S1, when preparing different solutions, to ensure that the reagents are fully dissolved in deionized water, place the beakers containing each solution on a magnetic stirrer and stir for at least 10 min.

3. A method for detecting and analyzing urease activity applicable to the EICP technique according to claim 1, characterized in that, In step S1, the prepared urease solution is stored in a low-temperature environment to prevent inactivation.

4. The urease activity detection and analysis method applicable to the EICP technology according to claim 1, characterized in that, In step S1, when preparing the urea solution, the urea concentration should not be too low to ensure the full play of urease activity.

5. A method for detecting and analyzing urease activity applicable to the EICP technology according to claim 1, characterized in that, In step S3, when quickly mixing the urease solution and the urea solution, pour the urea solution into the test tube containing the urease solution to prevent the temperature of the urease solution from changing.

6. The urease activity detection and analysis method applicable to the EICP technology according to claim 1, characterized in that, In steps S3 - S4, when measuring the conductivity value of the solution with a conductivity meter, do not turn on the temperature compensation function of the conductivity meter to measure the conductivity value of the solution at the current test temperature.

7. A method for detecting and analyzing urease activity applicable to the EICP technique according to claim 1, characterized in that, In step S5, in the formula: Δc is the generation rate of ammonium ions, with the unit of mmol / min; k1 is the change rate of conductivity with ammonium ion concentration, with the unit of μS / (cm·mM); k2 is the change rate of conductivity with time, with the unit of μS / (cm·min); V is the volume of the mixed solution, with the unit of L; A is the urease activity, with the unit of U / mg; m is the mass of urease in the mixed solution, with the unit of mg.

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