WO2022134082A1

WO2022134082A1 - Extracting solution for extracting urease active ingredient from canavalia gladiata and method for extracting urease active component

Info

Publication number: WO2022134082A1
Application number: PCT/CN2020/139654
Authority: WO
Inventors: 张梅燕; 袁振华
Original assignee: 美敦力公司; 张梅燕
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2022-06-30

Abstract

An extracting solution for extracting a urease active ingredient from Canavalia gladiata, wherein the extracting solution contains DTT with a molar concentration of 0.26 to 13 mM. In addition, also disclosed is a method for extracting a urease active ingredient from Canavalia gladiata, and a method for extracting urease from Canavalia gladiate. The extraction rate of the urease active ingredient is greatly improved by means of using the technical solution. The extracting solution has an excellent extraction performance for both Canavalia gladiate having a high activity and Canavalia gladiate having a low activity.

Description

Extraction solution for extracting urease active component from concanavali and method for extracting urease active component

technical field

The present invention relates to the extraction of adsorbent materials, in particular to the extraction of urease active components.

Background technique

Urease, also known as urease, is an adsorbent material commonly used in renal dialysis treatments that catalyzes the hydrolysis of urea to release ammonia and carbon dioxide, thereby converting urea into a nitrogen source available to the organism. Urease is easily soluble in water, insoluble in organic solvents such as alcohol, ether and acetone, and is unstable at normal temperature and pressure. In addition, urease is also very sensitive to temperature, pH and various factors that lead to protein denaturation, and is easily affected by external conditions to change its own properties.

In addition to the above-mentioned adsorbent materials for renal dialysis treatment, urease can also be used clinically to diagnose urea ammonia in serum and the like. In addition, urease can treat urea wastewater. Therefore, the market has a great demand for urease.

Methods for extracting urease from concanavalinas are known in the art. For example, in the paper "A Procedure for Purifying Jack Bean Urease for Clinical Use" by Hsien-Yi Sung et al., a method for purifying urease was mentioned using 20% acetone (containing 1 mM EDTA and 1 mM mercaptoethanol) ) was used as an extract to extract urease.

For another example, the publication number is CN104480092A, the publication date is April 1, 2015, and the title is "a method for extracting urease from concanava", which discloses the use of phosphate buffer, BTP buffer or Tris-HCL buffer A method for extracting urease using liquid as an extract.

However, compared with the technical solution to be proposed in the present invention, the extraction rate of the urease active components in the prior art is not high.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide an extract for extracting urease active components from concanavali. The inventors of the present invention have found that by using the extract in this technical solution, the urease active component can be greatly improved in the initial extraction step. The extraction rate can also be greatly improved in the end.

Based on the above purpose of the invention, the technical solution provides an extract for extracting urease active components from concanavali, the extract contains: DTT (ie DL-Dithiothreitol) with a molar concentration of 0.26-13mM.

Further preferably, the molar concentration of DTT in the extract is 5.2-13 mM.

In the prior art, concanava is used as a raw material for extracting urease. Generally speaking, the low activity concanava refers to the concanava whose activity is less than 2.5IU/mg, the high activity one refers to the concanava whose activity is greater than 3.2IU/mg, and the concanava whose activity is 2.5-3.2IU/mg is called It is medium active concanava.

However, the inventors of the present case have found through extensive research that the urease extraction method in the prior art is often suitable for high-activity concanavalina, but has poor effect on low-activity concanavalina. On this basis, the inventor found through further research that when DTT with a molar concentration of 0.26-13 mM is added to the extract, the extraction rate of urease active components will be greatly improved. This greatly improved effect is not only applicable to highly active beans, but also to low activity beans.

For example, for highly active concanava, using an extract containing 0.26 mM or more DTT can make the extraction rate of urease active components reach more than 40%, and using an extract containing 5.2 mM or more DTT can make the extraction rate of urease active components It can reach more than 50%, and even in some preferred embodiments, it can make the extraction rate of urease active components reach more than 60%. For low activity concanava, using an extract containing more than 0.26mM DTT can make the extraction rate of urease active components reach more than 30%, and in some preferred embodiments, using an extract containing more than 5.2mM DTT can achieve The extraction rate of the urease active component can reach more than 50%. Therefore, the extracting solution of the present invention has excellent extraction performance for both high activity and low activity concanava.

Further, the extracting solution of the present invention also contains a buffer solution with a molar concentration of 25mM-200mM.

Further, the pH value of the buffer solution is 6.5-8.5.

Further, the buffer solution may include a phosphate buffer solution, such as NaH ₂ PO 4 -Na ₂ HPO 4 buffer solution, or other similar phosphate buffer solutions.

In other embodiments, the buffer solution may include a Tris system buffer solution, such as Tris-HCl buffer solution, Tris-citrate buffer solution, or other similar Tris system buffer solutions.

Still further, the molarity of the phosphate buffer solution may be 50-200 mM, and further the molarity of the phosphate buffer solution may be 50 mM-150 mM.

Further, the pH value of the phosphate buffer solution may be 6.5-8.3, and the pH value of the Tris system buffer solution may be 7.1-8.5.

For example, Na2HPO4 _- citrate buffer solution, its molarity can be 25mM-200mM, its pH value can be 6.5-8.3. For another example, the KH ₂ PO4-NaOH buffer solution may have a molar concentration of 25mM-200mM, and a pH value of 6.5-8.3. For another example, the molar concentration of Tris-HCl buffer solution can be 25mM-200mM, and its pH value can be 7.1-8.5. Tris-citrate buffer solution, its molar concentration can be 25mM-200mM, and its pH value can be 7.1-8.5.

Further, the extracting solution of the present invention also contains: at least one of ethanol and acetone, wherein the mass percentage concentration of ethanol and/or acetone is 25-35%.

It should be noted that, preferably, the extraction solution of the present invention can obtain the claimed extraction rate without EDTA, while the extraction solution in the prior art often contains EDTA.

Based on the purpose of the present invention, the present invention also provides an extract for extracting urease active components from concanavali, which is composed of the following: (1) DTT; (2) buffer solution; (3) ethanol and At least one of acetone; wherein the molar concentration of DTT is 0.26-13 mM.

Further preferably, the molar concentration of DTT in the extract is 5.2-13 mM.

Further, the molar concentration of the buffer solution is 25mM-200mM.

Further, the pH value of the buffer solution is 6.5-8.5.

Further, the buffer solution may include a phosphate buffer solution, such as NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer solution, or other similar phosphate buffer solutions.

Further, in the extraction solution of the present invention, the mass percentage concentration of ethanol and/or acetone is 25-35%.

Further, using the extraction solution of the present invention, the extraction rate of the urease active component extracted from the concanavalina is ≥30%.

Further, using the extract of the present invention, the extraction rate of the urease active component extracted from the concanava is ≥40%.

Further, using the extract of the present invention, the extraction rate of the urease active component extracted from the concanavalina is ≥50%.

Further, using the extraction solution of the present invention, the extraction rate of the urease active component extracted from the concanavalina is ≥60%.

It should be noted that, in this technical solution, the extraction rate of the urease active ingredient refers to the content of the urease active ingredient in the solution containing the urease active ingredient (that is, the first supernatant liquid described below) and the raw material of the urease active ingredient. ratio of active ingredient content. Wherein, the content of the active ingredient of urease can be obtained by multiplying the concentration of the active ingredient in the solution (for example, the unit parameter is IU/ml) by the volume of the solution (for example, the unit parameter is ml), and the content of the active ingredient in the concanava can be obtained by the concave bean The activity value of (for example, the unit parameter is IU/mg) is multiplied by the mass of the bean (for example, the unit parameter is mg).

Another object of the present invention is to provide a method for extracting urease active components from concanava, which can greatly improve the extraction rate of urease.

Based on the above-mentioned object of the invention, the present invention provides a method for extracting urease active component from concanavalin, which comprises the steps:

Grind the concanavato into concanava powder;

adding concanava powder into the extract of the present invention, stirring and soaking;

A first supernatant containing the urease active component is obtained.

Further, in the method of the present invention, the temperature of the extraction solution is 18-40°C.

Further, in the method of the present invention, the temperature of the extraction solution is 31-35°C.

Further, in the method of the present invention, the stirring and soaking time is ≥1h.

Further, in the method of the present invention, the stirring and soaking time is ≥2h.

Another object of the present invention is to provide a method for extracting urease from concanavalinum. The method adopts a specific extracting solution, so that the concanavalina treated with the extracting solution has a higher urease extraction rate, so that the final prepared Urease has a high yield.

Based on the above-mentioned purpose of the invention, the present invention provides a kind of method for extracting urease from concanavali, which comprises the steps:

Grind the concanavato into concanava powder;

Obtain the first supernatant containing urease active component;

Urease was extracted from the first supernatant.

Further, the step of extracting urease from the first supernatant comprises:

(a) adding an alkaline solution (eg, NaOH solution) to the first supernatant to make the first supernatant alkaline;

(b) adding acetone and/or ethanol, stirring for a period of time at a temperature of 35-45 ° C, then cooling to room temperature, and centrifuging to obtain a second supernatant;

(c) adding an acid solution (eg, citric acid) to the second supernatant to make the second supernatant acidic, and then allowing it to stand for a period of time at a temperature of 2-8° C. to allow sufficient precipitation;

(d) extracting the precipitate after centrifugation;

(e) adding water to dissolve the precipitate, and adjusting the solution dissolved with the precipitate to neutrality with an alkaline solution;

(f) obtaining a third supernatant after centrifugation;

(g) Urease was obtained from the third supernatant.

Further, in step (a), the pH value of the first supernatant is adjusted to 7.5-9.

Further, in step (b), acetone or ethanol is added to make the mass percentage concentration reach 30-40%.

Further, in step (c), the pH value of the second supernatant is adjusted to 5.0-6.0.

Further, in step (b), the stirring time can be 30-45min.

Further, in step (c), stand for 16-20h.

Further, in step (e), the solution is adjusted to neutrality by using Tris solution as an alkaline solution.

Further, in step (g), urease is obtained by freeze-drying or chromatography.

The extraction solution of the present invention for extracting urease active components from concanavalinas adopts the above technical scheme, so that the extraction rate of urease active components is greatly improved. For example, for highly active concanava, using an extract containing 0.26 mM or more DTT can make the extraction rate of urease active components reach more than 40%, and using an extract containing 5.2 mM or more DTT can make the extraction rate of urease active components reach more than 50%. For low activity concanavalina, the extraction rate of urease active components can reach more than 30% by using the extract containing DTT above 0.26mM, and in some preferred embodiments, using DTT containing more than 5.2mM and using phosphoric acid The extraction solution of the salt buffer solution can make the extraction rate of urease active components reach more than 50%.

In addition, it should be noted that the use of different buffer solutions in this technical solution will make the extraction rate of urease active components different. For example, for the buffer solution of Tris system, the extraction rate of urease active components will vary with the buffer solution. increased with an increase in pH. However, no matter which buffer solution is used, under the premise that other extraction conditions are the same, the addition of DTT can help improve the extraction rate of urease active components under the same extraction conditions, compared with no addition of DTT, for example, The extraction rate of the active ingredient of low activity canava urease can be increased by at least 24%, and the extraction rate of active ingredient of high activity canava urease can be increased by at least 18%. Therefore, the extracting solution of the present invention has excellent extraction performance for both high activity and low activity concanava.

The method for extracting urease active components of the present invention also has the above beneficial effects.

Similarly, due to the use of the extraction solution in the technical solution, the extraction rate of the urease active component can be greatly improved in the initial extraction step, and the yield of urease can also be greatly improved in the end.

Detailed ways

The extract for extracting urease active components from concave bean according to the present invention, the method for extracting urease active components and the method for extracting urease will be further explained and explained below in conjunction with specific examples. It does not constitute an improper limitation to the technical solutions of the present invention.

Examples 1-79

In this embodiment, the following steps are used to extract urease active components, and further extract urease:

(1) Grinding the concanavato into concanava powder;

(2) Slowly adding the concanava flour into the extract (Table 1 lists the components of the specific extract in each embodiment of this case), to avoid the concanava flour agglomeration; stirring and soaking, the stirring speed can be from slow to Slightly faster, for example, 20 rpm at the beginning, with the input of concanava flour, the stirring speed can be appropriately increased to 30 rpm, but do not produce a lot of foam. During this process, control the temperature of the extract to 18-40 ℃, stir and soak time≥1h;

(3) the first supernatant containing urease active component is obtained after centrifugation;

(4) extracting urease from the first supernatant:

(4a) at room temperature, adding an alkaline solution (such as a 2M NaOH solution) to the first supernatant to adjust the pH of the first supernatant to 7.5-9;

(4b) adding acetone and/or ethanol to make its mass percentage concentration reach 30-40%, stirring at a temperature of 35-45°C for 30-45min, then cooling to room temperature, and centrifuging to obtain the second supernatant;

(4c) To the second supernatant, add an acid solution (such as citric acid with a molar concentration of 1 M) to adjust the pH of the second supernatant to 5.0-6.0, and then refrigerate it at 2-8°C Let stand for 16-20h (for example, 18h) at the temperature to make it fully precipitate;

(4d) extracting the precipitate after centrifugation;

(4e) adding water to dissolve the precipitate, and adjusting the solution with the precipitate dissolved to neutrality with a Tris solution (for example, the pH can be 8.5);

(4f) obtaining the third supernatant after centrifugation;

(4g) Urease is obtained from the third supernatant by lyophilization or chromatography.

Table 1 lists the components of the extracts employed in Examples 1-79 of the present invention.

Table 1.

Table 2 lists the process parameters of each step of extracting urease active components and urease in Examples 1-79 of the present invention.

Table 2.

As can be seen from Table 1 and Table 2: the use of the extract of the present invention for extracting urease active components from concanava can greatly improve the extraction rate of urease active components. The reason is that 0.26mM-13mM of DTT is added to the extract. The addition of 0.26mM-13mM DTT is very effective in improving the extraction rate of urease active components of low activity and high activity concanava beans, especially for the extraction rate of urease active components of low activity concanavalis. Effect. For low activity concanava, it can be seen from Table 1 that the extraction rate of urease active components can reach at least 30% or more by using the extract containing more than 0.26mM DTT, while using the extract containing more than 5.2mM DTT The extraction rate of the urease active component can also be made to reach at least 30% or more, and when a more appropriate buffer solution, such as a phosphate buffer solution, is used, the extraction rate of the urease active component can reach more than 50%. For highly active concanava, the extraction rate of urease active components can reach at least 40% by using the extract containing more than 0.26mM DTT, and using the extract containing more than 5.2mM DTT can make the extraction of urease active components The rate reaches more than 50%, while the use contains. In addition, in a more preferred embodiment, the extraction rate of the urease active component can reach more than 60% by using the DTT-containing extract.

In addition, in order to demonstrate the improvement in the extraction rate of urease active components caused by the addition of at least 0.26 mM DTT relative to no addition of DTT, Table 1 specifically lists all extraction conditions (including extraction process, buffer type, concentration, pH value) , auxiliary types and concentrations, etc.) are all the same, the extraction rate of the urease active ingredient without DTT, and the relative improvement rate of the extraction rate of the urease active ingredient due to the addition of DTT compared to the absence of DTT. As can be seen from Table 1, although the use of different buffer solutions will make the extraction rate of urease active components different, for example, for the buffer solution of Tris system, the extraction rate of urease active components will vary with the pH value of the buffer solution. rise and rise. However, no matter which buffer solution is used, the addition of DTT can help to improve the extraction rate of urease active components on the original basis, compared with no addition of DTT. For example, for low-activity concanava, adding DTT can increase the extraction rate of its urease active component by at least 24%, compared with no DTT. For highly active concanava, adding DTT can increase the extraction rate of its urease active component by at least 18%, compared with not adding DTT.

It should be noted that the prior art part in the protection scope of the present invention is not limited to the examples given in this application document, and all prior art that does not contradict the solution of the present invention, including but not limited to the prior art Patent documents, prior publications, prior publications, etc., can all be included in the protection scope of the present invention.

In addition, the combination of the technical features in this case is not limited to the combination described in the claims of this case or the combination described in the specific embodiments, and all the technical features described in this case can be freely combined or combined in any way, unless conflict with each other.

It should also be noted that the above enumeration is only a specific embodiment of the present invention, and it is obvious that the present invention is not limited to the above embodiment, and there are many similar changes. All modifications directly derived or thought of by those skilled in the art from the content disclosed in the present invention shall belong to the protection scope of the present invention.

Claims

An extraction solution for extracting active components of urease from concanavali, characterized in that the extraction solution contains: DTT with a molar concentration of 0.26-13 mM.
The extraction solution according to claim 1, characterized in that, the extraction solution further contains: a buffer solution with a molar concentration of 25mM-200mM.
The extraction solution according to claim 1, wherein the pH value of the buffer solution is 6.5-8.5.
The extraction solution according to claim 1, characterized in that, the extraction solution further contains at least one of ethanol and acetone, wherein the mass percentage concentration of ethanol and/or acetone is 25-35%.
The extraction solution of claim 1, wherein the extraction solution does not contain EDTA.
The extraction solution according to claim 1, characterized in that, the extraction solution is composed of the following: (1) DTT; (2) buffer solution; (3) at least one of ethanol and acetone; The molarity is 0.26-13 mM.
The extraction solution according to claim 2 or 6, wherein the buffer solution comprises a phosphate buffer solution or a buffer solution of a Tris system.
The extract of claim 7, wherein the phosphate buffer solution comprises one of the following: (a) NaH 2 PO 4 -Na 2 HPO 4 ; (b) KH 2 PO 4 - NaOH; (c) Na 2 HPO 4 -citric acid; the buffer solution of the Tris system includes one of the following: (d) Tris-HCl; (e) Tris-citric acid.
The extraction solution according to claim 7, wherein when the buffer solution comprises a phosphate buffer solution, its pH value is 6.5-8.3; when the buffer solution comprises a Tris system buffer solution, its pH value is is 7.1-8.5.
The extraction solution according to any one of claims 1-6, wherein the molar concentration of the DTT is 5.2-13 mM.
The extraction solution according to claim 6, wherein the molar concentration of the buffer solution is 25mM-200mM.
The extraction solution according to claim 6, wherein the mass percentage concentration of ethanol and/or acetone is 25-35%.
The extraction solution according to any one of claims 1-6, 8-9, and 11-12, characterized in that the extraction rate of the urease active component extracted from concanava is ≥30%.
The extraction solution according to claim 13, characterized in that the extraction rate of the urease active component extracted from the concanavalina is ≥50%.
A method for extracting urease active component from concanava, it is characterized in that, it comprises the steps:

Grind the concanavato into concanava powder;

The concanava flour is added to the extraction solution as described in any one of claims 1-14, stirring and soaking;

After centrifugation, a first supernatant containing urease active components was obtained.
The method of claim 15, wherein the temperature of the extraction solution is 18-40°C.
The method of claim 16, wherein the temperature of the extraction solution is 31-35°C.
The method according to any one of claims 15-17, wherein the stirring and soaking time is ≥1h.
The method of claim 18, wherein the stirring and soaking time is ≥2h.
A method for extracting urease from concanava, it is characterized in that, it comprises the steps:

(1) the steps included in the method for extracting urease active component from Concanavalinas according to any one of claims 15-19;

(2) Extracting urease from the first supernatant.
The method of claim 20, wherein the step (2) comprises:

(a) adding an alkaline solution to the first supernatant to make the first supernatant alkaline;

(b) adding acetone and/or ethanol, stirring for a period of time at a temperature of 35-45 ° C, then cooling to room temperature, and centrifuging to obtain a second supernatant;

(c) adding an acid solution to the second supernatant to make the second supernatant acidic, and then allowing it to stand for a period of time at a temperature of 2-8° C. to fully precipitate;

(d) extracting the precipitate after centrifugation;

(e) adding water to dissolve the precipitate, and adjusting the solution dissolved with the precipitate to neutrality with an alkaline solution;

(f) obtaining a third supernatant after centrifugation;

(g) Urease was obtained from the third supernatant.
The method of claim 21, wherein, in step (a), the pH value of the first supernatant is adjusted to 7.5-9.
The method of claim 21, wherein in step (b), acetone or ethanol is added to make the mass percentage concentration reach 30-40%.
The method of claim 21, wherein in step (c), the pH value of the second supernatant is adjusted to 5.0-6.0.