Extraction method of plant urease

Abstract

The invention relates to an extraction method of plant urease. According to the extraction method of the plant urease provided by the invention, after pretreatment of drying, crushing and sieving, a buffer solution is added for extraction, and finally, centrifugal filtration is performed to obtain a plant urease solution. According to the extraction method, the requirements of reducing the protein content and improving the urease activity can be met, the extraction efficiency is improved, operation is easy, safety is high, and popularization is convenient.

Description

Technical Field

[0001] The present invention relates to the technical field of sand and soil fixation materials, and particularly to a method for extracting plant urease. Background Art

[0002] The Microbial Induced Calcium Carbonate Precipitation (MICP) technology cultivates and propagates specific microorganisms, generally using urease produced by their metabolism, to rapidly generate carbonate ions by catalyzing the hydrolysis reaction of urea within a short time, and then react with externally added soluble calcium salts to generate calcium carbonate products with cementing properties, so as to realize engineering applications such as sand and soil fixation. Compared with traditional sand fixation technologies, the MICP technology has the advantages of controllable reaction rate, good cementing effect, and less toxic substance emissions, and is a sand fixation technology with low energy consumption and low pollution. Compared with the path of generating urease through bacterial metabolism, the technology of using finished urease to catalyze and induce calcium carbonate precipitation (EICP) shows higher mineralization efficiency under dry and anaerobic or less oxygen conditions due to avoiding the limitation of insufficient microbial environmental adaptability.

[0003] The urease used in the EICP technology can be roughly divided into two types. One is commercially available urease. Due to the high difficulty and cost of purifying commercial urease, the economy of the EICP technology is poor, restricting its large-scale application in practical engineering. The other is to extract crude enzyme solution from plant seeds. In order to reduce costs, researchers have successfully extracted crude plant urease from certain tissues and organs of plants (such as soybean, jack bean, watermelon seeds, etc.). Plant urease can perfectly replace commercial urease for EICP experiments. A large number of experiments have proved that the technology of inducing calcium carbonate precipitation with plant urease is simple in construction, short in construction period, and good in economy, and has been applied to various engineering fields such as foundation anti-liquefaction treatment, desert control, and slope reinforcement, achieving good results.

[0004] To further expand the application scope of the technology of inducing calcium carbonate precipitation with plant urease, the extraction method of plant urease needs to be improved urgently. Traditional plant urease purification technologies generally adopt a crude purification method. The obtained enzyme products have limited mineralization activity, and the products contain impurities such as proteins and lipids that do not participate in or even hinder the mineralization reaction process. Since these impurities undergo a series of physical and chemical reactions with inorganic salts during the mineralization process, the pores of sand particles are blocked during the sand fixation process, restricting the uniformity of subsequent mineralization cementing products. Correspondingly, researchers have adopted the extraction method with organic solvents (such as ethanol, propanol, etc.), and successfully reduced the organic matter content of the products. However, the activity of the enzyme extracted by this method has been greatly reduced, and the operation process is cumbersome. Moreover, acetone solvent is flammable and toxic, and belongs to controlled chemical drugs, which is not conducive to the extraction of a large amount of urease at the actual engineering site.

[0005] In summary, the existing technology cannot take into account both the requirements of reducing protein content and maintaining or improving urease activity. Therefore, there is an urgent need for a new method to improve the existing plant urease extraction, which can remove most of the irrelevant impurities such as proteins in the solution without affecting or slightly affecting the plant urease activity, thereby improving the application effect of plant urease induced EICP technology. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for extracting plant urease.

[0007] The plant urease extraction method provided by the present invention can overcome the problem that the prior art cannot simultaneously take into account the two requirements of reducing protein content and maintaining or improving urease activity. While reducing the protein content of the urease product, the urease activity is significantly improved compared with the traditional plant urease extraction technology. It has the advantages of simple operation, high safety, and easy promotion.

[0008] The present invention provides a method for extracting plant urease, comprising the following steps:

[0009] (1) drying and crushing the plant seeds to obtain soybean powder;

[0010] (2) extracting the soybean powder obtained in step (1) with an extraction solvent, removing the soybean powder residue after the extraction, and obtaining an extract;

[0011] (3) centrifuging the extract obtained in step (2), collecting the supernatant, and removing oil on the surface of the supernatant to obtain a plant urease solution;

[0012] The leaching solvent includes one of a Tris-HCl solution, a phosphate buffer, an acetate buffer and a citrate buffer.

[0013] In some embodiments of the present invention, the plant seeds include one or more of soybeans, red beans, and sword beans.

[0014] In some embodiments of the present invention, the baking temperature of the plant seeds is ≤40°C, such as 10°C, 20°C, 30°C, 40°C, etc.

[0015] In the present invention, the plant seeds are baked to reduce the influence of the water contained in the plant seeds on the solid-liquid ratio of the enzyme extraction suspension.

[0016] In some embodiments of the present invention, the mesh size of the soybean powder is ≥60 meshes.

[0017] In the present invention, the particle size of the bean powder after sieving is above 60 meshes, which can reduce the content of solid impurities and increase the activity of the extracted plant urease.

[0018] In some embodiments of the present invention, the extraction solvent is a Tris-HCl solution.

[0019] In some embodiments of the present invention, the concentration of the extraction solvent is 0.1 - 1 mol / L, such as 0.2 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.8 mol / L, etc.

[0020] Within the concentration range of the extraction solvent of the present invention, a stable pH environment can be maintained to protect the activity of urease, while avoiding the salting-out effect caused by too high ionic strength or the interference of impurities caused by too low ionic strength, thereby improving the extraction efficiency and enzyme stability.

[0021] In some embodiments of the present invention, the pH of the extraction solvent is 4.6 - 6.5, such as 5.6, 5.8, 6, 6.2, 6.4, etc., and preferably 5.5 - 6.5.

[0022] The environment of the buffer solution within the pH range of the present invention can reach the isoelectric point of most irrelevant proteins in soybeans, inducing the precipitation of this series of irrelevant proteins, while retaining as much soybean urease as possible in the solution, thereby improving the activity of the extracted plant urease.

[0023] In some embodiments of the present invention, the mass-volume ratio of the soybean powder to the extraction solvent is 1:(3 - 14), such as 1:3, 1:5, 1:8, 1:10, 1:12, 1:14, etc.

[0024] Within the range of the material-liquid ratio of the present invention, the extraction efficiency and raw material consumption can be balanced, so that urease can be fully dissolved and released, while avoiding the loss of enzyme activity or too high impurity concentration caused by too dilute or too concentrated solution, and optimizing the extraction effect.

[0025] In some embodiments of the present invention, the steps of extraction are: stirring for 10 - 40 min,

[0026] such as 10 min, 20 min, 30 min, 40 min, etc., or standing for 3 - 12 h after stirring for 10 - 40 min,

[0027] such as 3 h, 5 h, 7 h, 10 h, 12 h.

[0028] In some embodiments of the present invention, the temperature of the extraction is 2 - 4 °C, such as 2 °C, 3 °C,

[0029] 4 °C, etc.

[0030] In some embodiments of the present invention, the centrifugation speed is 3000 - 10000 rpm, such as 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm, etc., and preferably 5000 - 8000 rpm.

[0031] During the enzyme activity extraction process, if the centrifugation speed is lower than 2000 rpm, it will significantly affect the extraction effect: low-speed centrifugation cannot fully precipitate impurities, resulting in particulate matter remaining in the supernatant. These impurities will not only compete for enzyme binding sites, release inhibitory substances, but also increase the detection background interference.

[0032] In some embodiments of the present invention, the centrifugation time is 5 - 30 min, such as 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, etc.

[0033] The centrifugation speed and time within the scope of the present invention can effectively separate solid impurities and extract the urease component with high activity.

[0034] The urease extracted by the extraction method provided by the present invention is used in the EICP technology, including applications in geotechnical and environmental engineering such as sand consolidation, soil consolidation, and heavy metal consolidation.

[0035] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0036] The plant urease extraction method of the present invention, while reducing the content of organic impurities, significantly improves the activity of the urease product compared with the traditional plant urease extraction technology. At the same time, it shortens the extraction time, improves the urease extraction efficiency, and has the advantages of simple operation, short operation time, and high safety, which is convenient for popularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings herein are incorporated into the specification and form a part of the specification, showing embodiments consistent with the present invention and used together with the specification to explain the principles of the present invention.

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0039] Figure 1 It is a flowchart of the extraction and activity determination steps of the plant urease of the present invention;

[0040] Figure 2 It is a photo of the urease solution extracted with different pH extraction solvents;

[0041] Figure 3 It is a bar chart showing the influence of different centrifugal speeds on urease activity. Specific implementation manners

[0042] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the solution of the present invention will be further described below. It should be noted that, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0043] Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0044] Example 1

[0045] This example provides a method for extracting plant urease, and the steps are as follows:

[0046] (1) Pretreatment: Weigh an appropriate amount of soybeans and dry them in an oven at 40°C for 6 h. After drying, put them into a pulverizer and pulverize them in small amounts (about 30 g) multiple times. Pass the soybean powder through a 100-mesh sieve and place it in a refrigerator at 4°C for later use;

[0047] (2) Extraction: Mix the soybean powder obtained in step (1) with 0.1 M Tris-HCl solution (pH 4.6) at a mass-to-volume ratio of 1:10 to form a soybean powder suspension with a concentration of 100 g / L. Stir with a magnetic stirrer for 30 min. Filter the stirred soybean powder solution through a 100-mesh gauze to remove the soybean powder and obtain a light yellow soybean powder solution;

[0048] (3) Centrifugation: Divide the soybean powder solution obtained in step (2) into centrifuge tubes (pour the same mass of soybean powder solution into each centrifuge tube), balance them, and place them in a centrifuge for centrifugation at room temperature. After centrifuging at 3000 rpm for 15 min, collect the supernatant, and filter off the creamy soybean oil floating on the surface of the supernatant with a 200-mesh gauze to obtain a soybean urease solution.

[0049] Example 2

[0050] This example provides a method for extracting plant urease, and the steps are as follows:

[0051] (1) Pretreatment: Weigh an appropriate amount of soybeans and dry them in an oven at 40°C for 6 h. After drying, put them into a pulverizer and pulverize them in small amounts (about 30 g) multiple times. Pass the soybean powder through a 100-mesh sieve and place it in a refrigerator at 4°C for later use;

[0052] (2) Extraction: Mix the soybean powder obtained in step (1) with 0.1 M Tris-HCl solution (pH 6) at a volume ratio of 1:10 to form a soybean powder suspension with a concentration of 100 g / L. Stir with a magnetic stirrer for 30 min, filter the well-stirred soybean powder solution through a 100-mesh gauze to remove the soybean powder and obtain a light yellow soybean powder solution;

[0053] (3) Centrifugation: Aliquot the soybean powder solution obtained in step (2) into centrifuge tubes (pour the same mass of soybean powder solution into each centrifuge tube), balance, place in a centrifuge and centrifuge at room temperature. After centrifuging at 3000 rpm for 5 min, collect the supernatant, and filter off the creamy soybean oil floating on the surface of the supernatant with a 200-mesh gauze to obtain a soybean urease solution.

[0054] Examples 3 - 7

[0055] Examples 3 - 7 provide a method for extracting plant urease, which is different from Example 2 in that the pH of the Tris-HCl solution is 4.6, 5, 7, 8, and 9 respectively.

[0056] Example 8

[0057] This example provides a method for extracting plant urease, and the steps are as follows:

[0058] (1) Pretreatment: Weigh an appropriate amount of soybeans, dry them in an oven at 40 °C for 6 h, put them into a pulverizer and pulverize them in small amounts (about 30 g) multiple times. Pass the soybean powder through a 100-mesh sieve and store it in a 4 °C refrigerator for later use;

[0059] (2) Extraction: Mix the soybean powder obtained in step (1) with 0.1 M Tris-HCl solution (pH 4.6) at a mass-to-volume ratio of 1:10 to form a soybean powder suspension with a concentration of 100 g / L. Stir with a magnetic stirrer for 30 min, filter the well-stirred soybean powder solution through a 100-mesh gauze to remove the soybean powder and obtain a light yellow soybean powder solution;

[0060] (3) Centrifugation: Aliquot the soybean powder solution obtained in step (2) into centrifuge tubes (pour the same mass of soybean powder solution into each centrifuge tube), balance, place in a centrifuge and centrifuge at room temperature. After centrifuging at 3000 rpm for 5 min, collect the supernatant, and filter off the creamy soybean oil floating on the surface of the supernatant with a 200-mesh gauze to obtain a soybean urease solution.

[0061] Examples 9 - 18

[0062] Example 9-18 provides a method for extracting plant urease, which is different from Example 8 in that the rotation speeds for centrifugation in step (3) are 0, 1000 rpm, 2000 rpm, 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, and 10000 rpm respectively.

[0063] Example 19

[0064] This example provides a method for extracting plant urease, which is different from Example 1 in that:

[0065] In step (2), soybean powder is mixed with 1M Tris-HCl solution (pH 6.5) at a volume ratio of 1:14 and stirred for 40 min;

[0066] In step (3), centrifuge for 30 min.

[0067] Example 20

[0068] This example provides a method for extracting plant urease, which is different from Example 1 in that:

[0069] In step (2), soybean powder is mixed with 0.5M Tris-HCl solution (pH 5.5) at a volume ratio of 1:3 and stirred for 10 min.

[0070] Example 21

[0071] This example provides a method for extracting plant urease, which is different from Example 1 in that in step (2), after stirring evenly, let it stand for 3 h.

[0072] Comparative Examples 1 and 2

[0073] Comparative Examples 1 and 2 provide a method for extracting plant urease, which is different from Example 21 in that in step (2), the extraction reagents are distilled water and 30% ethanol respectively.

[0074] Comparative Example 3

[0075] This comparative example provides a method for extracting plant urease, which is different from Example 21 in that in step (2), the extraction reagent is distilled water. After stirring evenly, calcium sulfate dihydrate is added to the solution of distilled water extracting soybean powder at a ratio of 1 g calcium sulfate dihydrate / 10 g soybean powder, and continue to stir with a magnetic stirrer for 10 min.

[0076] Method for measuring urease activity and solid content

[0077] 1. Method for measuring urease activity: Mix the plant urease solutions obtained in Examples 1, 8 - 21 and Comparative Examples 1 - 3 with the urea solution, and use a conductivity meter to measure the change rate of the conductivity of the mixed solution at 30°C. The growth rate of the initial conductivity is linearly related to the urease activity, and thus the urea hydrolysis rate when urease reacts with urea can be characterized. Add 36 mL of 1.5 mol / L urea solution to 4 mL of the plant urease solution, and use a conductivity meter to measure the change value of the conductivity within the first 5 minutes. The change value of the conductivity per minute (mS·cm·min -1 ) corresponds to the urea hydrolysis amount of 11.11 mM urea min -1 . The calculation method of urease activity is the change value of the average conductivity per minute multiplied by the dilution factor 10 and then multiplied by 11.11.

[0078] 2. Determination of total solid content: Weigh the mass of the petri dish (M1), take 20 ml of the obtained soybean urease solution into the petri dish, and dry it in an oven at 105°C for 24 h until constant weight (M2). The mass difference (M2 - M1) is the content of total solids.

[0079] Table 1 Results of urease activity and solid content

[0080]

[0081]

[0082] The results are shown in Table 1 and Figures 1-3 as follows. Figure 1 This is the schematic flow chart of the extraction method of the present invention, Figure 2 This is a photo of the urease solution extracted with extraction solvents at different pH values, Figure 3 This is a bar chart showing the influence of different centrifugation speeds on urease activity.

[0083] Effect of different extraction pH values on enzyme activity: From the results of Examples 2 - 7 and Figure 2 in Table 1, it can be seen that when the extraction pH value is higher than 7, the enzyme activity of the enzyme solution shows a downward trend. At the same time, the turbidity of the extracted enzyme solution increases, and the content of irrelevant impurities such as proteins increases. When the pH of the extraction solution is 6, the extraction effect is the best. While increasing the activity of the extracted plant urease, the impurity content of the enzyme extraction solution can be reduced.

[0084] Effect of different centrifugation speeds on enzyme activity and total solid content: From the results of Examples 8 - 18 and Figure 3As can be seen from the results, centrifugation at a speed above 3000 rpm can significantly reduce the total solids content in the enzyme solution while increasing its urease activity. However, when the speed is higher than 8000 rpm, some urease may be separated into the precipitate, resulting in a decrease in urease activity in the supernatant. When the speed range is 5000 - 8000 rpm, a plant urease solution with high urease activity and low content of irrelevant impurities such as proteins can be effectively separated.

[0085] Effects of different extraction solvents on enzyme activity and total solids content: As can be seen from the comparison between Example 21 and Comparative Examples 1 - 3 in Table 1, selecting 30% ethanol, 0.1M Tris-HCl extraction reagent, and the additional addition of calcium sulfate dihydrate can all reduce most of the irrelevant impurities such as proteins in the extracted enzyme solution, resulting in a decrease in the solid impurity content of the extracted enzyme solution. However, the urease activity of the enzyme solution after extraction with 0.1M Tris-HCl in the present invention is the highest. Compared with Comparative Examples 2, 3, and 4, the enzyme activity in Example 21 is increased by 15.64%, 128%, and 27.82% respectively. While reducing the protein content, the plant urease activity obtained exceeds the traditional plant urease extraction technology. In addition, as can be seen from the comparison between Example 1 and Example 21, the extraction method provided by the present invention can achieve good extraction efficiency without standing in step (2) of the extraction process. Compared with the traditional method, it can increase the urease activity while reducing the content of impurities such as proteins.

[0086] In summary, the plant urease extraction method provided by the present invention can significantly improve the extracted urease activity while reducing the content of organic impurities such as proteins by optimizing the extraction solvent, seed pretreatment method, and centrifugation speed. It also shortens the extraction time, improves the extraction efficiency, is simple to operate, has high safety, and is convenient for promotion.

[0087] It should be noted that in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising a..." does not exclude the existence of additional identical elements in the process, method, article or device including the element.

[0088] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments described herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting plant urease, characterized in that, It includes the following steps: (1) Dry and crush plant seeds to obtain bean powder; (2) Extract the bean powder obtained in step (1) with an extraction solvent. After the extraction is completed, remove the bean powder residue to obtain an extract; (3) Centrifuge the extract obtained in step (2), collect the supernatant, and remove the grease on the surface of the supernatant to obtain a plant urease solution; The extraction solvent includes one of Tris-HCl solution, phosphate buffer solution, acetate buffer solution, and citrate buffer solution.

2. The method according to claim 1, characterized in that, The plant seeds include one or more of soybeans, red beans, and jack beans.

3. The method according to claim 1, wherein The temperature during drying is ≤40°C.

4. The method according to claim 1, characterized in that, The mesh number of the bean powder is ≥60 mesh.

5. The method according to claim 1, wherein The extraction solvent is Tris-HCl solution.

6. The method according to claim 1 or 5, characterized in that, The concentration of the extraction solvent is 0.1 - 1 mol / L; Preferably, the pH of the extraction solvent is 4.6 - 6.5, and more preferably 5.5 - 6.

5.

7. The method according to claim 1, characterized in that, The mass-volume ratio of the bean powder to the extraction solvent is 1:(3 - 14) g / mL.

8. The method according to claim 1, wherein The extraction steps are: stir for 10 - 40 min, or stir for 10 - 40 min and then stand for 3 - 12 h.

9. The method according to claim 1 or 8, characterized in that, The extraction temperature is 2 - 4°C.

10. The method according to claim 1, characterized in that, The rotation speed of the centrifugation is 3000 - 10000 rpm, preferably 5000 - 8000 rpm; Preferably, the centrifugation time is 5 - 30 min.

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