阅读说明：本技术 一种从红刀豆中提取脲酶的方法 (Method for extracting urease from red sword beans ) 是由 李婷婷 朱海 贾玮玮 于 2021-11-10 设计创作，主要内容包括：本发明属于生物蛋白提取技术领域,具体涉及一种从红刀豆中提取脲酶的方法,方法包括如下步骤：称取红刀豆研磨成粉,筛分后加入提取液,将红刀豆粉末和提取液充分搅拌,在室温下离心,收集上清液A,上清液A滤纸抽滤,得到上清液B；在上清液B加入60％PEG4000,在4℃温度下静置,离心,收集沉淀；将沉淀加入重悬液,重悬溶解后离心,收集上清并抽滤,得到溶液A；先用HCl将溶液A调为pH＝5.1,再用NaOH将溶液调为pH＝6.0,得到溶液B；将溶液B加入至柠檬酸-柠檬酸钠中进行透析,在4℃温度下静置,离心,抽滤,收集上清,冻干。本发明制备流程简单,不需要在提取剂中添加丙酮溶液,反应温和,最终可得到蛋白浓度较高的脲酶。(The invention belongs to the technical field of biological protein extraction, and particularly relates to a method for extracting urease from red sword beans, which comprises the following steps: weighing semen Canavaliae, grinding into powder, sieving, adding extractive solution, stirring, centrifuging at room temperature, collecting supernatant A, and vacuum filtering with filter paper to obtain supernatant B; adding 60% PEG4000 into the supernatant B, standing at 4 deg.C, centrifuging, and collecting precipitate; adding the precipitate into a resuspension solution, carrying out centrifugation after resuspension and dissolution, collecting a supernatant, and carrying out suction filtration to obtain a solution A; adjusting the pH of the solution A to 5.1 by using HCl, and adjusting the pH of the solution to 6.0 by using NaOH to obtain a solution B; adding the solution B into citric acid-sodium citrate, dialyzing, standing at 4 deg.C, centrifuging, vacuum filtering, collecting supernatant, and lyophilizing. The preparation method has simple preparation flow, does not need to add acetone solution into the extractant, has mild reaction, and can finally obtain the urease with higher protein concentration.)

1. A method for extracting urease from red sword beans is characterized by comprising the following steps:

1) extraction: weighing and grinding the sword beans into powder, adding an extracting solution after screening, fully stirring the sword bean powder and the extracting solution at 28 ℃ to ensure that all urease in the sword bean powder is leached, then centrifuging the leaching solution at room temperature, collecting supernatant A, and carrying out suction filtration on the supernatant A by using filter paper to obtain supernatant B;

2) coarse precipitation: adding 60% PEG4000 into the obtained supernatant B, standing at 4 deg.C, centrifuging, and collecting precipitate;

3) resuspending: adding the obtained precipitate into a resuspension solution, carrying out centrifugation after resuspension and dissolution, collecting a supernatant, and carrying out suction filtration to obtain a solution A;

4) adjusting the pH value: adjusting the pH of the obtained solution A to 5.1 by using HCl, centrifuging, collecting supernatant, and adjusting the pH of the solution to 6.0 by using NaOH to obtain a solution B;

5) freeze-drying: adding the obtained solution B into citric acid-sodium citrate, dialyzing, standing at 4 deg.C, centrifuging, vacuum filtering, collecting supernatant, and lyophilizing.

2. The method of claim 1, wherein the urease is extracted from jack beans by: in the step 1), the extracting solution is 50mM citric acid-sodium citrate + 3% PEG4000 solution, and the red sword bean powder and the extracting solution are mixed according to the material-liquid ratio of 150-.

3. The method of claim 1, wherein the urease is extracted from jack beans by: in the step 1), grinding the sword beans into powder, and screening the powder by using a vibrating screen to remove insoluble impurities with larger granularity, wherein the sword bean powder and the extracting solution are stirred by magnetic force for 20-40 min; the centrifugal speed is 9000-10000rpm, and the centrifugal time is 8-12 min.

4. The method of claim 1, wherein the urease is extracted from jack beans by: in the step 2), the 60% PEG4000 solution is slowly added dropwise to a final concentration of 10%.

5. The method of claim 1, wherein the urease is extracted from jack beans by: in the step 2), the centrifugation temperature is 3-5 ℃, the centrifugation speed is 9000-10000rpm, and the centrifugation time is 18-22 min.

6. The method of claim 1, wherein the urease is extracted from jack beans by: in step 3), the components of the resuspension solution are 50mM citric acid-sodium citrate, 5mg/ml glycine, 5mg/ml glucose and 30mg/ml sorbitol.

7. The method of claim 1, wherein the urease is extracted from jack beans by: in the step 3), the centrifugation temperature is 7-9 ℃, the centrifugation speed is 9000-10000rpm, and the centrifugation time is 25-35 min.

8. The method of claim 1, wherein the urease is extracted from jack beans by: in step 4), the resulting solution a was adjusted to pH 5.1 with 10% HCl, centrifuged, and the supernatant was collected and adjusted to pH6.0 with 6M NaOH to give solution B.

9. The method of claim 1, wherein the urease is extracted from jack beans by: in the step 4), the centrifugation temperature is 16-20 ℃, the centrifugation speed is 9000-10000rpm, and the centrifugation time is 25-35 min.

10. The method of claim 1, wherein the urease is extracted from jack beans by: in the step 5), performing dialysis by using 10mM sodium citrate, and centrifuging after the dialysis is finished, wherein the centrifugation condition is 4 ℃, 9500rpm and 30 min; the supernatant was filtered with a 0.45 μ M filter membrane, and the filtrate was freeze-dried.

Technical Field

The invention belongs to the technical field of biological protein extraction, and particularly relates to a method for extracting urease from sword beans.

Background

Urease is an important biological agent and is widely distributed in plant seeds, with the highest content in soybean and sword bean. Urease is an oligomeric enzyme, can catalyze urea to hydrolyze and release ammonia and carbon dioxide, has the catalytic efficiency which is more than 1000 times higher than the rate without the existence of urease, and is widely applied to the fields of agriculture, medicine industry and the like: in korea, it is used for the treatment of emesis, abdominal edema, and lumbago related to kidney, in japan, it is effective for the treatment of pyorrhea, otitis media, furuncles and cancers, various inflammatory diseases and atopic dermatitis, in china, urease is used for the treatment of animal excreta to prevent air pollution, and urease can also treat waste water containing urea, meeting the current requirements of green chemistry in china. With the economic development of China and the progress of the immobilized urease technology, the demand on urease is more and more increased.

The bean resources of China are quite rich, and the research report aiming at urease extraction in China at present mainly focuses on extraction and purification of plants such as soybeans, soybeans and the like, and Cuiyou et al adopts acetate buffer solution to extract soybean flour in the research on urease extraction and properties of soybeans, then sodium sulfite is used for salting out, and precipitates are dissolved by phosphate buffer solution, dialyzed, subjected to DEAE-cellulose column chromatography separation, and subjected to eluent freeze drying to obtain urease products. However, the process flow is complex, the urease loss is serious, and the cost is high. Hsien-Yi Sung et al in "A Procedure for Purifying Jack Bean enzyme for Clinical Use" mentioned a method for Purifying Urease, using 20% acetone (containing 1mM EDTA and 1mM mercaptoethanol) for extraction, centrifugation, adjusting the acetone concentration of the clear liquid to 31-35%, then alkalizing with sodium hydroxide, heat treating at 40 deg.C for 5min, centrifugation, adjusting the clear liquid to near isoelectric point with citric acid, centrifuging, collecting precipitate, neutralizing with phosphate buffer solution, and freeze drying to obtain Urease. Although the method can obtain the protein with higher purity, acetone hazardous reagents are used, and the experimental process is hazardous.

Compared with the acetone extraction of the urease of the beans, the urease of the red jack beans is easy to obtain as a raw material, the red jack beans are mainly distributed in the south of China, the yield is quite rich, acetone dangerous reagents are not needed in the experimental process, and compared with the urease extraction of the soybeans and the soybeans, the urease of the red jack beans is extracted by the method, so that the process route is simple, and the purity of the extracted urease is higher. At present, the research on the extraction technology taking the sword beans as the raw material is less, the method for extracting the urease has the advantages that the raw material and the extraction reagent are easy to obtain, the purity of the extracted urease is high, and the method has the prospect of industrial production.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provides a method for extracting urease from red sword beans, which has the advantages of simple process flow, short operation time, mild conditions, no need of using dangerous reagents, high urease purity of over 80 percent, good activity, high recovery rate and stable industrial prospect.

In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:

a method for extracting urease from red sword beans comprises the following steps:

1) extraction: weighing and grinding the sword beans into powder, adding an extracting solution after screening, fully stirring the sword bean powder and the extracting solution at 28 ℃ to ensure that all urease in the sword bean powder is leached, then centrifuging the leaching solution at room temperature, collecting supernatant A, and carrying out suction filtration on the supernatant A by using filter paper to obtain supernatant B;

2) coarse precipitation: adding 60% PEG4000 into the obtained supernatant B, standing at 4 deg.C, centrifuging, and collecting precipitate;

3) resuspending: adding the obtained precipitate into a resuspension solution, carrying out centrifugation after resuspension and dissolution, collecting a supernatant, and carrying out suction filtration to obtain a solution A;

4) adjusting the pH value: adjusting the pH of the obtained solution A to 5.1 by using HCl, centrifuging, collecting supernatant, and adjusting the pH of the solution to 6.0 by using NaOH to obtain a solution B;

5) freeze-drying: adding the obtained solution B into citric acid-sodium citrate, dialyzing, standing at 4 deg.C, centrifuging, vacuum filtering, collecting supernatant, and lyophilizing.

Further, in the method for extracting urease from jack beans as described above, in step 1), the extract is a 50mM citric acid-sodium citrate + 3% PEG4000 solution, and the jack bean powder and the extract are mixed at a feed-to-liquid ratio of 150 g: 1L.

Further, in the method for extracting urease from jack beans as described above, in step 1), jack beans are ground into powder and then screened by a vibrating screen to remove insoluble impurities with larger granularity, and the jack bean powder and the extracting solution are stirred by magnetic force for 20-40 min; the centrifugal speed is 9000-10000rpm, and the centrifugal time is 8-12 min.

Further, in the method for extracting urease from red sword bean as described above, in step 2), 60% PEG4000 solution is slowly added dropwise to a final concentration of 10%.

Further, in the method for extracting urease from red sword bean as described above, in step 2), the centrifugation temperature is 3-5 ℃, the centrifugation speed is 9000-10000rpm, and the centrifugation time is 18-22 min.

Further, in the method for extracting urease from red sword bean as described above, in step 3), the composition of the heavy suspension is 50mM citric acid-sodium citrate, 5mg/ml glycine, 5mg/ml glucose, 30mg/ml sorbitol.

Further, in the method for extracting urease from red sword bean as described above, in step 3), the centrifugation temperature is 7-9 ℃, the centrifugation speed is 9000-10000rpm, and the centrifugation time is 25-35 min.

Further, in the method for extracting urease from red sword bean as described above, in step 4), the obtained solution a was adjusted to pH 5.1 with 10% HCl, centrifuged, the supernatant was collected, and the solution was adjusted to pH6.0 with 6m naoh to obtain solution B.

Further, in the method for extracting urease from red sword bean as described above, in step 4), the centrifugation temperature is 16-20 ℃, the centrifugation speed is 9000-10000rpm, and the centrifugation time is 25-35 min.

Further, the method for extracting urease from red sword bean as described above, step 5), dialyzing with 10mM sodium citrate, centrifuging after dialysis, the centrifugation condition is 4 ℃, 9500rpm, 30 min; the supernatant was filtered with a 0.45 μ M filter membrane, and the filtrate was freeze-dried.

The invention has the beneficial effects that:

1. the invention adopts the red sword beans as raw materials, the main producing area of the red sword beans is in China, the raw materials are easy to obtain, and the citric acid-sodium citrate is used as a buffer solution system, so that the buffer capacity is strong, the activity of protein is not influenced, and the stable structure of the protein can be maintained. The whole process flow has fewer steps and shorter time consumption, does not need to add acetone solution into an extracting agent, has mild reaction, and separates out most of mixed protein through polyethylene glycol precipitation and isoelectric point precipitation to finally obtain the urease with the protein concentration of 80%.

2. The method has the advantages of simple operation, short process flow, low energy consumption and mild reaction, solves the problems of reaction time and reaction danger, adopts a conventional solid-liquid separation mode in the extraction process, and is an extraction method with industrial application value.

Of course, it is not necessary for any one product that embodies the invention to achieve all of the above advantages simultaneously.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a process flow diagram of the process of the present invention;

FIG. 2 is an SDS-PAGE electrophoresis of jack bean urease according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

A method for extracting urease from red sword beans comprises the following steps:

1) extraction: weighing and grinding the sword beans into powder, adding an extracting solution after screening, fully stirring the sword bean powder and the extracting solution at 28 ℃ to ensure that all urease in the sword bean powder is leached, then centrifuging the leaching solution at room temperature, collecting supernatant A, and carrying out suction filtration on the supernatant A by using filter paper to obtain supernatant B;

2) coarse precipitation: adding 60% PEG4000 into the obtained supernatant B, standing at 4 deg.C, centrifuging, and collecting precipitate;

3) resuspending: adding the obtained precipitate into a resuspension solution, carrying out centrifugation after resuspension and dissolution, collecting a supernatant, and carrying out suction filtration to obtain a solution A;

4) adjusting the pH value: adjusting the pH of the obtained solution A to 5.1 by using HCl, centrifuging, collecting supernatant, and adjusting the pH of the solution to 6.0 by using NaOH to obtain a solution B;

5) freeze-drying: adding the obtained solution B into citric acid-sodium citrate, dialyzing, standing at 4 deg.C, centrifuging, vacuum filtering, collecting supernatant, and lyophilizing.

The specific embodiment of the invention is as follows:

example 1

Weighing 500g of jack beans, grinding the jack beans, sieving the jack beans by a 80-mesh sieve for 3 times to obtain 51.27g of crude bean powder and 440.54g of fine bean powder, sieving the crude bean powder and the fine bean powder by a vibrating sieve to remove insoluble impurities with larger granularity, weighing 300g of fine jack bean powder, pouring the fine jack bean powder into a 2L beaker, adding 1.5L of a reagent (50mM citric acid-sodium citrate pH6.0+ 3% PEG4000), uniformly stirring by a glass rod, then, magnetically stirring the jack beans powder and an extracting solution at room temperature for fully mixing for 30min to ensure that all urease in the jack beans powder is leached, then, centrifuging the extracting solution at 9500rpm for 15min at room temperature, collecting supernatant A, finding that the solution is turbid, and performing suction filtration by using filter paper to obtain 1200ml of supernatant B.

Adding 120ml of 60% PEG4000 into the filtered supernatant B to make the final concentration of the mixture in the solution be 10%, adding a stirrer, stirring for 10min, uniformly mixing, and standing for 1h at 4 ℃. After the solution is stirred evenly after standing, the solution is centrifuged for 20min at 9500rpm at 4 ℃, and precipitates are collected (in the step, the supernatant is controlled to be dry so as not to influence the subsequent dissolution).

200ml of 50mM citric acid-sodium citrate pH6.0 (5 mg/ml glycine, 5mg/ml glucose, 30mg/ml sorbitol) was added to the pellet for resuspension. After the suspension was completely dissolved, the suspension was centrifuged at 9500rpm at 18 ℃ for 1 hour, and the supernatant was collected, filtered through a 0.45 μm filter membrane, and collected after filtration.

The supernatant was adjusted to pH 5.1 by addition of 9.5ml of 10% HCl. Centrifugation was carried out at 9500rpm for 30min at 8 ℃ with the contaminating proteins in the pellet and the urease all in the supernatant. The solution was adjusted to pH6.0 using 1.3ml of 6M NaOH.

The dialysis solution was dialyzed against 10mM sodium citrate pH6.0 for 2 times of 1.5 hours each. After dialysis, centrifugation was carried out at 9500rpm for 30min at 4 ℃ for 30min, the supernatant was filtered with a 0.45 μ M filter membrane, the filtrate was frozen in a freezer at-80 ℃ and the supernatant was dried in a vacuum freeze dryer (to prevent the sample from melting as soon as possible) to obtain 6.2g of protein sample.

Example 2

The protein samples obtained in the examples were used as the subjects.

And (3) enzyme activity determination: freeze-drying a sample to prepare a 10mg/ml aqueous solution, placing the sample in a test tube with the precision amount of 30 mu l of the solution in a dry type thermostat at 37 ℃ for 2min, taking 120 mu l of 125mM urea to react for 6min, adding 100ul of color developing agent 1 (1% phenol, 0.02% sodium nitrosoferrous chloride) after the reaction is finished, adding 100ul of color developing agent 2 (0.6% sodium hydroxide and 0.6% sodium hypochlorite solution) in 1min, immediately shaking up by hand, placing the sample in the dry type thermostat, covering the sample at 37 ℃, using a timer to time the reaction for 20min, taking 200ul of the sample, placing the sample in a 96-well plate, and measuring the absorbance of the sample in a full-automatic enzyme labeling instrument at 595 nm.

Calculated according to the following formula:

the activity of the obtained urease is 37.54, and meets the urease activity standard (more than or equal to 15U).

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.