Method for extracting, purifying and identifying soybean agglutinin

Abstract

This invention discloses a method for the extraction, purification, and activity identification of soybean lectins based on isoelectric point precipitation and fractional ammonium sulfate precipitation, belonging to the field of plant-derived bioactive protein preparation technology. This method utilizes the difference in solubility characteristics between soybean storage proteins and soybean lectins under isoelectric point conditions. Through isoelectric point precipitation, two-step ammonium sulfate precipitation, dialysis desalting, and secondary purification, the method achieves efficient enrichment of soybean lectins. Purity and activity are identified using SDS-PAGE electrophoresis and rabbit erythrocyte hemagglutination assay. Compared with existing chromatographic purification techniques, this invention eliminates the need for expensive chromatographic equipment, is simple in process, low in cost, and has good reproducibility. It effectively preserves the natural biological activity of soybean lectins and is suitable for laboratory preparation, pilot-scale amplification, and animal experimental research. The obtained soybean lectins can be widely used in research on anti-nutritional factors, animal nutrition, and the development of bioactive proteins.

Description

Technical Field

[0001] This invention relates to the field of feed raw material deep processing and bioactive protein preparation technology, specifically to a method for extracting, purifying and identifying the activity of soybean lectins. Background Technology

[0002] Soybean lectin (SBA) is an N-acetyl-D-galactosamine / D-galactose-specific lectin widely found in soybean seeds and is one of the main anti-nutritional factors in soybeans. SBA can specifically recognize cell surface glycan structures and induce erythrocyte aggregation, thus it is widely used in glycobiology research, immunological detection, cell recognition, animal nutrition regulation, and evaluation of feed anti-nutritional factors.

[0003] Currently, the acquisition of SBA mainly relies on commercially available standards or separation techniques such as affinity chromatography, ion exchange chromatography, and gel filtration chromatography. While these methods can yield SBA with high purity, they generally suffer from high equipment investment, complex operating procedures, long purification cycles, high preparation costs, and difficulty in meeting the needs of large-scale animal experiments. Furthermore, some purification methods can easily disrupt the native conformation of SBA, thereby affecting its sugar-binding and hemagglutination activities.

[0004] Therefore, developing an extraction and purification method for SBA that is simple, low-cost, reproducible, and can maintain the natural biological activity of SBA is of great significance for carrying out basic research and industrial applications related to SBA. Summary of the Invention

[0005] This invention aims to provide a method for the extraction, purification, and activity identification of soybean lectin. By employing a synergistic purification strategy of isoelectric point precipitation and fractional ammonium sulfate precipitation, the method achieves efficient enrichment and purification of soybean lectin. Simultaneously, it establishes a dual identification system using SDS-PAGE and rabbit erythrocyte hemagglutination assay, thereby obtaining an SBA product with high purity and good biological activity.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for extracting, purifying, and identifying the activity of soybean lectins, comprising the following steps: Step 1: Raw material pretreatment Soybeans are ground, sieved, and then defatted to obtain defatted soybean flour.

[0007] Preferably: The particle size is 250–500 μm; Preferably 350 μm (60 mesh); Degreasing was performed using n-hexane; The material-to-liquid ratio is 1:8 to 1:12; The preferred ratio is 1:10; Degreasing sessions 2-4 times; Selected 3 times.

[0008] Step 2: Isoelectric point precipitation Defatted soybean flour is mixed with distilled water to form a protein dispersion.

[0009] Preferably: The material-to-liquid ratio is 1:8 to 1:15; The preferred ratio is 1:12; Adjust the pH to 4.2–5.0; The preferred pH is 4.6; Let stand at 2–8℃ for 8–24 hours; It is preferable to let it stand at 4℃ for 12 to 16 hours.

[0010] Collect the supernatant after removing the precipitate.

[0011] Step 3: Fractionated ammonium sulfate precipitation Ammonium sulfate was added to the supernatant obtained in step 2 to perform two-stage salting out.

[0012] Primary salting out: ammonium sulfate concentration 200–350 g / L; preferably 300 g / L; Secondary salting out: ammonium sulfate concentration 200–350 g / L; preferably 270 g / L.

[0013] Collect the precipitate to obtain crude SBA extract.

[0014] Step 4: Dialysis desalination The crude SBA extract was dissolved in distilled water and dialyzed using a dialysis bag with a molecular weight cutoff of 8–14 kDa.

[0015] Preferred: Molecular weight cutoff: 10–12 kDa; Temperature 4℃; Time: 18–36 hours; 24 hours is preferred.

[0016] Step 5: Secondary purification Adjust the pH of the dialysate back to 4.2–5.0, preferably 4.6.

[0017] Add ammonium sulfate for secondary precipitation: 40-70 g / 100 mL; preferably 56 g / 100 mL.

[0018] Collect the precipitate by centrifugation.

[0019] Step 6: Freeze-drying preparation The precipitate was dissolved using phosphate buffer.

[0020] Preferred: Buffer concentration: 0.01–0.1 mol / L; The preferred concentration is 0.05 mol / L; pH 5.5–7.0; Option 6.1 is preferred.

[0021] SBA powder was then obtained by freeze-drying.

[0022] Activity identification methods SDS-PAGE identification Use: 10%–15% separating adhesive; preferably 12.5%.

[0023] When a characteristic band appears in the 28–32 kDa position of a sample, it is determined to be SBA.

[0024] Blood coagulation activity identification Rabbit erythrocyte agglutination assay was used.

[0025] Preferred concentration: 1%–4% red blood cell concentration; preferably 2%.

[0026] Blood coagulation titer was determined using a two-fold serial dilution method.

[0027] Compared with the prior art, the present invention has the following advantages: 1. It eliminates the need for expensive equipment such as affinity chromatography columns, ion exchange chromatography columns, and FPLC systems, significantly reducing preparation costs; 2. By employing a synergistic purification strategy of isoelectric point precipitation and fractional ammonium sulfate precipitation, effective enrichment of SBA and removal of impurity proteins can be achieved. 3. The entire process uses low-temperature and mild conditions to preserve the natural conformation and biological activity of SBA to the greatest extent. 4. Establish a dual identification system of SDS-PAGE and blood coagulation activity to ensure product purity and functional activity; 5. The process is stable and has good repeatability, making it suitable for laboratory preparation, pilot-scale amplification, and animal experiment production of SBA; 6. The prepared SBA has high purity and good rabbit erythrocyte agglutination activity, and can be widely used in anti-nutritional factor research, animal nutrition research and bioactive protein development. Attached Figure Description

[0028] Figure 1 for Figure 1 SDS-PAGE electrophoresis image of SBA sample; Figure 2 This is the result of the blood coagulation activity test for SBA.

[0029] in, Figure 1Column 1 is the protein molecular weight standard marker, column 2 is the SBA standard, and columns 3–6 are the extracted SBA samples; Figure 2 The first three wells in the first column are the control group before dilution, the last three wells in the last column are the control group after dilution, and the middle four columns, a total of 12 wells, are the results of serial dilution of SBA samples. The results show that SBA can induce agglutination of rabbit erythrocytes, with the fifth well (dilution factor of 32-fold, 2^5) showing the best agglutination effect. Detailed Implementation

[0030] The present invention will now be described with reference to specific embodiments. The following examples are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0031] Example: The specific steps of the method of the present invention are as follows: 1. Materials and Methods 1.1 Materials and Reagents The defatted soybean flour used in the experiment was defatted with hexane and flash-evaporated to remove solvent, with a protein water dispersibility of ≥85%; rabbit blood (containing Alsever's solution) was stored at 4°C for no more than 2 weeks; the main reagents included ammonium sulfate, concentrated hydrochloric acid, 0.9% physiological saline, pH 6.1~7.4 phosphate buffer (PBS), protein molecular weight standards (10–250 kDa), 5×SDS loading buffer, Coomassie brilliant blue staining solution, destaining solution, etc.; experimental consumables included 10~12 kDa dialysis bags, qualitative filter paper, etc.

[0032] 1.2 Main Instruments The main instruments used in the experiment include a high-speed refrigerated centrifuge, a magnetic stirrer, a pH meter, a vacuum filtration device, a vertical electrophoresis tank, an electrophoresis apparatus, a gel imaging system, a constant temperature water bath, a freeze dryer, a blood coagulation U-shaped reaction plate, and micropipettes.

[0033] 1.3 Extraction and purification of soybean lectins 1.3.1 Protein dispersion and isoelectric point precipitation Soybean meal was pulverized and passed through a 60-mesh (350μm) sieve. 500mL of n-hexane was added, and the mixture was stirred at room temperature for 2 hours, then allowed to stand. The supernatant was discarded, and the process was repeated three times with fresh n-hexane. After treatment, the mixture was air-dried in a fume hood for later use. 50g of defatted soybean flour was weighed and slowly added to 600mL of distilled water at a ratio of 1:12. The mixture was magnetically stirred for 30 minutes to form a uniform dispersion. The pH of the dispersion was slowly adjusted to 4.6 with concentrated hydrochloric acid, and the mixture was allowed to stand overnight (12-16 hours) at 4°C to allow the main soybean protein to precipitate. The clear supernatant was collected, and the remaining turbid liquid was centrifuged at 12000 rpm at 4°C for 20 minutes. The two supernatants were combined to obtain the initial supernatant, and the bottom precipitate was discarded.

[0034] 1.3.2 Fractional ammonium sulfate salting-out For the first-stage salting out, ammonium sulfate was added at a concentration of 300 g / L to the initial supernatant while stirring continuously. Stirring continued for 30 minutes at room temperature until completely dissolved. After standing for 30 minutes, the solution was vacuum filtered, and the filtrate was collected, discarding the filter residue. For the second-stage salting out, ammonium sulfate was added at a concentration of 270 g / L to the filtrate from the first salting out. After stirring for 30 minutes until completely dissolved, the solution was allowed to stand overnight (12-16 hours) at 4°C. The next day, the solution was centrifuged at 12000 rpm for 20 minutes at 4°C. The supernatant was discarded, and the pale yellow flocculent precipitate was collected, which is the crude SBA precipitate.

[0035] 1.3.3 Dissolution and Dialysis Desalination Add 200 mL of distilled water to the crude SBA precipitate and stir at 4℃ for 1-2 h to completely dissolve it, obtaining a crude SBA solution. Place this solution into a dialysis bag pre-activated with distilled water and dialyze at 4℃ for 24 h, changing the distilled water twice until the conductivity of the dialysate matches that of the distilled water. After dialysis, centrifuge the solution in the bag at 4℃, 12000 r / min for 20 min, discarding a small amount of insoluble precipitate and retaining the supernatant. 1.3.4 Secondary Precipitation Purification The supernatant of the dialyzed SBA was adjusted to pH 4.6 dropwise with hydrochloric acid. Ammonium sulfate was added at a ratio of 56 g / 100 mL, and the mixture was stirred for 30 min to ensure complete dissolution. The solution was then allowed to stand at 4 °C for 2 h. Subsequently, the solution was centrifuged at 12000 rpm for 20 min at 4 °C, and the precipitate was collected.

[0036] 1.3.4 Dissolving and Lyophilizing The resulting secondary precipitate was dissolved in 100 mL of 0.05 M phosphate buffer (pH 6.1) at 4 °C with stirring for 1 h to obtain a pure SBA solution. This solution was then freeze-dried in a freeze-drying flask for 24 h to obtain SBA powder, which was then sealed and stored at -20 °C.

[0037] 1.4 SDS-PAGE Purity Identification Prepare an 8 mg / mL SBA powder solution with physiological saline. Mix 20 μL of the sample with 5 μL of 5×SDS-PAGE loading buffer and load the sample. Prepare a 12.5% ​​separating gel and a 4.5% stacking gel using a rapid gel preparation kit (Yamei). After mounting the gels, add 1× electrophoresis buffer. Add 10 μL of SBA sample to each well and 5 μL of standard protein marker. Perform electrophoresis at a constant voltage of 200V for approximately 50 min. Stain with Coomassie Brilliant Blue for 1–2 h until the background is transparent and the bands are clear. If a clear main band appears at approximately 30 kDa with few impurities, the SBA purity is considered high. If only a small number of low molecular weight bands are present, they can be considered SBA isoenzymes and will not affect subsequent experiments.

[0038] 1.5 SBA blood coagulation activity assay 1.5.1 Rabbit red blood cell washing treatment Take 6 50mL centrifuge tubes and aliquot them with 10mL of PBS buffer. Add 500μL of fresh rabbit whole blood to each tube. Gently invert to mix and centrifuge at 4℃ and 313×g for 10min. Remove the supernatant and repeat the washing process 4 times. The first 3 centrifugations were performed at 1500×g for 10min, and the last centrifugation was performed at 2500×g for 10min. After washing, discard the supernatant and retain about 100μL of PBS to infiltrate the red blood cell pellet to prevent the cells from drying out.

[0039] 1.5.2 Preparation of 2% rabbit red blood cell suspension Add 9.8 mL of PBS solution to a 50 mL centrifuge tube. Pipette 200 μL of compacted red blood cell pellet into the buffer solution. Gently invert and mix 10–15 times to prepare a 2% (v / v) red blood cell suspension. Observe the solution's condition after preparation; the suspension should be a uniform red color, without clots or hemolysis. A bright red color indicates hemolysis and requires re-preparation. Store at 4°C protected from light after preparation and use within two weeks. Shake well before use.

[0040] 1.5.3 Hemagglutination test to determine agglutination activity Add 25 μL of PBS buffer to each well of the hemagglutination U-shaped reaction plate. Add 25 μL of the 8 mg / mL SBA stock solution to the first well, mix well, and then perform serial dilutions up to the 12th well. Aspirate 25 μL of waste solution from the last well. Set up three blank negative controls with only PBS before dilution, and add three control groups after dilution. Add 25 μL of 2% red blood cell suspension to all reaction wells, mix gently manually for 30 seconds, and incubate at 25°C for 45–60 min. Interpret the results: red blood cells spreading out into a film indicates positive agglutination, while cells agglomerating into dots indicates negative agglutination. Calculate the hemagglutination titer based on the highest dilution well showing agglutination.

[0041] 2. Results 2.1 Extraction and purification results After isoelectric point precipitation, fractional salting out, dialysis, secondary precipitation, and lyophilization, a white or pale yellow SBA powder was obtained. It exhibits good solubility and remains stable for up to 6 months when stored sealed at -20°C. The entire process was conducted at a low temperature of 4°C, effectively preventing protein denaturation and loss of activity.

[0042] 2.2 SDS-PAGE purity determination results like Figure 1As shown, the first column represents the protein molecular weight standard marker, the second column represents the SBA standard, and the last four columns represent the extracted SBA samples. The results show that the SBA standard exhibits a clear characteristic band at approximately 30 kDa, consistent with the molecular weight range of the SBA subunits. All four lanes of the extracted samples show a distinct main band at approximately 30 kDa, and the band position is largely consistent with that of the SBA standard, indicating that the main protein component in the extracted samples is SBA. Simultaneously, only a small number of weak contaminant bands are present in the sample lanes, indicating that after acid precipitation, ammonium sulfate precipitation, dialysis, and secondary purification, contaminating proteins have been largely removed, and SBA has been effectively enriched. Overall, the extracted SBA samples have high purity and exhibit band characteristics consistent with the standard, demonstrating that this purification method has good separation efficiency.

[0043] 2.3 Results of blood coagulation activity test like Figure 2 As shown, the first three wells in the first column represent the control group before dilution, where no significant agglutination of red blood cells was observed. The last three wells in the last column represent the control group after dilution, where no significant agglutination was also observed, indicating that the experimental system was stable and that spontaneous agglutination of red blood cells did not occur under the control conditions. The middle four columns, totaling 12 wells, represent the results of serial dilutions of the SBA sample. The results show that the SBA sample can induce agglutination of rabbit red blood cells treated with trypsin at different dilution factors. Clear agglutination was still observed in well number 5 (1:32), which is the dilution with the highest agglutination activity. According to the definition of hemagglutination titer, the hemagglutination titer of SBA is 32. As the dilution factor increases, red blood cell agglutination gradually weakens, indicating that the hemagglutination activity of SBA is positively correlated with its concentration. In summary, the lowest agglutination dilution of SBA sample is 32-fold (2^5), indicating that purified SBA still retains good native hemagglutination activity.

[0044] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention. All such modifications and substitutions should be covered within the scope of the claims of the present invention and are within the scope of protection of the present invention.

Claims

1. A method for the extraction, purification, and identification of soybean lectins, characterized in that, Includes the following steps: S1. Preparation of defatted soybean flour; S2. Isoelectric point precipitation removes soybean storage protein; S3, graded ammonium sulfate salting-out enrichment of soybean lectins; S4, Dialysis desalination; S5. Secondary salting-out purification; S6. Freeze-dry to obtain soybean lectin powder; S7. Activity was identified using SDS-PAGE and rabbit erythrocyte hemagglutination assay.

2. The method for extraction, purification, and identification of soybean lectins according to claim 1, characterized in that, In step (2), the isoelectric point precipitation pH is 4.2 to 5.0, preferably 4.

6.

3. The method for extraction, purification, and identification of soybean lectins according to claim 1, characterized in that, In step (3), soybean lectins are enriched and purified by graded ammonium sulfate precipitation, wherein the final concentration of ammonium sulfate in the first-stage precipitation is 200-350 g / L and the final concentration of ammonium sulfate in the second-stage precipitation is 200-350 g / L.

4. The method for extraction, purification, and identification of soybean lectins according to claim 1, characterized in that, In step (4), dialysis is performed using a dialysis bag with a molecular weight cutoff of 8 to 14 kDa, preferably a dialysis bag with a molecular weight cutoff of 10 to 12 kDa.

5. The method for extraction, purification, and identification of soybean lectins according to claim 1, characterized in that, In step (5), the secondary salting out uses 40-70 g / 100 mL of ammonium sulfate, preferably 56 g / 100 mL.

6. The method for extraction, purification, and identification of soybean lectins according to claim 1, characterized in that, In step (7), the hemagglutination activity is evaluated using a rabbit erythrocyte agglutination test.

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