A method for efficiently recovering active glycoprotein Patatin from potato starch processing waste juice

Abstract

The application discloses a method for recovering active glycoprotein Patatin from potato starch processing waste juice, and belongs to the technical field of protein extraction. The method is characterized in that the active Patatin protein is efficiently separated by using the combination of ammonium sulfate fractionation precipitation, anion exchange chromatography, Con A affinity chromatography and gel chromatography, and the obtained Patatin protein retains the original protein structure and biological function activity. Compared with the prior art, the method has better separation purity and higher efficiency, and the obtained Patatin protein has complete protein higher structure and function activity, and has a better application scene.

Description

Technical Field

[0001] This invention relates to a method for recovering the active glycoprotein Patatin from potato starch processing waste juice, belonging to the field of protein extraction technology. Background Technology

[0002] Potato juice is a byproduct of potato starch processing, primarily composed of potato protein. The amino acid composition of potato protein is balanced, with essential amino acids accounting for nearly half of the total amino acid content, significantly higher than the 36.0% of FAO / WHO standard protein and close to the 49.7% amino acid content of egg protein, giving it extremely high nutritional value. Furthermore, potato protein has a high absorbability and is non-allergenic, making it safer and more easily absorbed and utilized by the human body.

[0003] Currently, in industrial production, potato starch processing waste juice is often directly discharged, which not only causes certain pollution to the ecological environment, but also wastes high-quality protein resources. Therefore, recycling the protein components in potato waste juice has great economic significance.

[0004] Potato protein mainly consists of high-molecular-weight proteins, the glycoprotein Patatin, and low-molecular-weight protease inhibitors. Patatin is the main component of potato protein, accounting for approximately 40% of soluble protein. Due to variations in potato varieties, its molecular weight ranges from 40 to 45 kDa. Patatin is a high-quality plant glycoprotein with multiple physiological functions. Its essential amino acid index (EAAI) is as high as 76%, making its nutritional value far superior to soybean protein. It also has various positive effects on human health, including antioxidant activity, regulation of cholesterol and blood pressure, and reduction of acyl hydrolase activity. Furthermore, compared to other components of potato protein, Patatin possesses excellent properties for food processing, such as gelling, emulsifying, and foaming properties, allowing it to be added as a food ingredient and additive. Additionally, Patatin can be used as a clarifying agent in red wine or as a flavor enhancer in cheese to release free fatty acids and increase the content of flavor compounds.

[0005] Currently, potato protein recovery mainly employs methods such as acid-base precipitation, expanded bed adsorption, and enzymatic methods. However, these methods all exhibit certain limitations in practical applications, such as insufficient separation of different protein components (especially the difficulty in separating Patatin protein and low-molecular-weight protease inhibitors), high energy consumption, cumbersome operation, and potential protein denaturation risks. In industrial production, most companies use acid-thermal flocculation to recover potato protein. Although this method can reduce production costs and simplify equipment requirements, it often leads to the almost complete loss of the protein's functional properties and activity, which greatly limits its application in the food and pharmaceutical industries. Summary of the Invention

[0006] In view of the shortcomings and deficiencies of the existing technology, the main objective of this invention is to provide a method for efficiently recovering active glycoprotein Patatin from potato starch processing waste liquid. This method aims to solve the problem of protein inactivation during the separation and purification of potato glycoprotein Patatin, reduce the environmental pollution caused by the direct discharge of potato processing waste liquid, and increase the added value of potato product processing.

[0007] To achieve the above objectives, the following technical solution is provided:

[0008] This invention provides a method for recovering the active glycoprotein Patatin from potato starch processing waste juice, the method comprising the following steps:

[0009] (1) The pretreated potato starch processing waste juice was subjected to ammonium sulfate fractionation precipitation, and the protein precipitates of each grade were collected and placed in buffer A for dialysis. The protein solution after dialysis was centrifuged and the supernatant was collected.

[0010] (2) The supernatant obtained in step (1) is sequentially passed through an anion exchange chromatography column and a PreCap Con A affinity chromatography column to obtain the target protein eluent, and then dialyzed in ultrapure water to obtain the Patatin protein solution;

[0011] (3) The Patatin protein solution obtained in step (2) is concentrated by ultrafiltration, the concentrate is passed through a Sephadex G-75 gel chromatography column, and the target protein is eluted with ultrapure water. The eluent is collected and freeze-dried to obtain the final product.

[0012] In one embodiment, the pretreatment in step (1) specifically involves filtering the potato starch processing waste juice and then centrifuging it to remove impurities; the filter mesh size is 200~300 mesh; the centrifugation parameters are: 10000-12000 r / min, time is 10~20 min, and temperature is 4~8℃.

[0013] In one embodiment, the ammonium sulfate fractionation in step (1) specifically involves adding ammonium sulfate to potato starch processing waste juice to a saturation level of 1, then precipitating at 0-4°C for 30-60 min, centrifuging, and collecting the protein precipitate; continuing to add ammonium sulfate to the supernatant to a saturation level of 2, then precipitating at 0-4°C for 30-60 min, centrifuging, and collecting the protein precipitate; repeating this ammonium sulfate fractionation process.

[0014] In one embodiment, during the ammonium sulfate graded precipitation process, the ammonium sulfate saturation levels are 20%, 30%, 40%, 50%, 60%, and 70%, respectively; wherein saturation level 1 is 20%, saturation level 2 is 30%, and the saturation levels increase sequentially.

[0015] In one embodiment, the centrifugation parameters are: 10000~12000 r / min, time 15~20 min, and temperature 4~8℃.

[0016] In one embodiment, the buffer solution A in step (1) is 20±5 mmol / L NaH2PO4-Na2HPO4 with pH=7.2.

[0017] In one embodiment, the dialysis in step (1) specifically involves dissolving protein precipitates of various grades in buffer A, then placing them into a dialysis bag with a molecular weight cutoff of 1000-15000 Da, and dialyzing them in buffer A at 4-8°C for 6-14 h; wherein the ratio of buffer A added to dissolve the protein precipitates is 1g wet protein to 10-20 mL buffer A; the dialysis bag is preferably a dialysis bag with a molecular weight cutoff of 3500 Da; and the volume ratio of protein solution to dialysis fluid is preferably 1:50.

[0018] In one embodiment, the centrifugation parameters in step (1) are: 10000~12000 r / min, centrifuged at 4~8℃ for 10~15 min.

[0019] In one embodiment, the anion exchange chromatography column in step (2) is an IexCap Smac Q column.

[0020] In one embodiment, the separation by anion exchange chromatography column in step (2) specifically involves: loading the supernatant obtained in step (1) onto anion exchange chromatography IexCap Smac Q column, first rinsing with buffer A to remove impurities, and then eluting the target protein with buffer B using a linear elution program. The eluent is collected in separate tubes and analyzed by SDS-PAGE. Based on the analysis results, the eluent containing the target protein is collected.

[0021] In one embodiment, the rinsing volume of buffer A is 3 to 5 column volumes.

[0022] In one embodiment, the buffer solution B is 20±5 mmol / L NaH2PO4-Na2HPO4, 1 mol / L NaCl, pH=7.2.

[0023] In one embodiment, the linear elution program is specifically: 0-20 min, 0-60% buffer B, flow rate 2 mL / min; 20-25 min, 60-100% buffer B, flow rate 2 mL / min.

[0024] In one embodiment, the amount collected in the tubes is 1-4 mL / tube; preferably 2 mL / tube.

[0025] In one embodiment, the collection of eluent containing the target protein specifically refers to the eluent containing the target protein eluted when the NaCl concentration in the eluent is 50-150 mmol / L.

[0026] In one embodiment, the separation via PreCap Con A affinity chromatography column in step (2) specifically involves: dialyzing the target protein eluent collected via IexCap Smac Q chromatography column and then loading it onto the PreCap Con A affinity chromatography column. First, impurity proteins are washed with buffer C, and then the target protein is eluted with buffer D using a linear elution program. The eluent is collected in separate tubes and analyzed by SDS-PAGE. Based on the analysis results, the eluent of the target protein is collected.

[0027] In one embodiment, the target protein eluent collected by the IexCap Smac Q chromatographic column is dialyzed. Specifically, the collected target protein eluent is placed in buffer C and dialyzed at 4-8°C for 6-14 h. Buffer C consists of 20±5 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, and pH 7.4. The molecular weight range of the dialysis bag is 1000-15000 Da.

[0028] In one embodiment, the rinsing volume of the buffer solution C is 3 to 5 column volumes.

[0029] In one embodiment, the buffer solution D is 20±5 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 0.2 mol / L α-D-methylmannoside, pH 7.4.

[0030] In one embodiment, the linear elution program is specifically: 0-20 min, 0-100% buffer D, flow rate 2 mL / min.

[0031] In one embodiment, the amount collected in the tubes is 1-4 mL / tube; preferably 2 mL / tube.

[0032] In one embodiment, the collection of eluent containing the target protein specifically refers to the target protein eluent eluted when the concentration of α-D-methylmannoside in buffer D is 10-20 mmol / L.

[0033] In one embodiment, the ultrafiltration tube used for ultrafiltration concentration in step (3) has a molecular weight cutoff of 10,000 to 12,000 Da and a concentration factor of 10 to 15 times.

[0034] In one embodiment, the concentrate in step (3) needs to be filtered through a 0.45 μm water filter before passing through the column.

[0035] In one embodiment, the ultrapure water elution rate in step (3) is 1~1.5 mL / min.

[0036] In one embodiment, the collection of eluent in step (3) specifically involves: collecting the eluent in separate tubes and performing SDS-PAGE analysis on it, and then pooling all the eluent containing the target protein based on the analysis results.

[0037] In one embodiment, the standard for collecting samples in step (3) is 1~2 mL / tube; preferably 1 mL / tube.

[0038] This invention also provides the application of the above-described method in the field of potato deep processing technology.

[0039] The present invention also provides a method for improving the activity of the potato glycoprotein Patatin, the method comprising the following steps:

[0040] (1) The pretreated potato starch processing waste juice was subjected to ammonium sulfate fractionation precipitation, and the protein precipitates of each grade were collected and placed in buffer A for dialysis. The protein solution after dialysis was centrifuged and the supernatant was collected.

[0041] (2) The supernatant obtained in step (1) is sequentially passed through an anion exchange chromatography column and a PreCap Con A affinity chromatography column to obtain the target protein eluent, and then dialyzed in ultrapure water to obtain the Patatin protein solution;

[0042] (3) The Patatin protein solution obtained in step (2) is concentrated by ultrafiltration. The concentrate is passed through a Sephadex G-75 gel chromatography column and the target protein is eluted with ultrapure water. The eluent is collected in separate tubes and analyzed by SDS-PAGE. Based on the analysis results, all eluents containing the target protein are collected and freeze-dried to obtain the final product.

[0043] Beneficial effects

[0044] The method for recovering active Patatin protein from potato waste juice based on the combined use of ammonium sulfate precipitation, anion exchange chromatography, affinity chromatography, and gel chromatography provided by this invention has the following advantages compared with existing technologies:

[0045] (1) The high-efficiency adsorption of Patatin protein by anion exchange chromatography IexCap Smac Q column and affinity chromatography effectively removes non-protein impurities and some high molecular weight impurities, resulting in high-purity Patatin protein; further, gel chromatography is used to remove small molecule proteins such as protease inhibitors to obtain high-purity Patatin protein. This method is simple, has a high recovery rate and high purity of recovered protein.

[0046] (2) This invention does not use organic solvents, strong acids and strong bases, etc. The recovery process does not require high-temperature heating denaturation. The recovery method is mild. The function of potato Patatin protein is effectively preserved during the extraction process. The isolated Patatin protein has a normal protein structure and excellent antioxidant and lipase activity.

[0047] (3) The reagents used in this invention are all buffer solutions with water and salt as the main components. The consumables used can also be recycled and reused. The preparation method is sustainable and helps to promote the application of potato protein in food, cosmetics, medicine and other fields, and reduces resource waste and pollution in potato processing. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the steps for separating and purifying Patatin protein from potato starch processing waste juice according to the present invention.

[0049] Figure 2 This is an SDS-PAGE image of potato protein precipitated at different ammonium sulfate saturations in Example 1;

[0050] Figure 3 This is an SDS-PAGE image of Patatin protein separated by anion exchange chromatography column in Example 1;

[0051] Figure 4This is an SDS-PAGE image of Patatin protein separated by Con A affinity chromatography column in Example 1;

[0052] Figure 5 This is an SDS-PAGE image of Patatin protein separated by gel chromatography using Sephadex G-75 in Example 1;

[0053] Figure 6 This is a circular dichroism spectral analysis data of the Patatin protein obtained in Example 1;

[0054] Figure 7 The image shows the fluorescence spectral analysis data of the Patatin protein obtained in Example 1.

[0055] Figure 8 This is a graph showing the antioxidant activity analysis data of the Patatin protein obtained in Example 1;

[0056] Figure 9 This is a graph showing the lipase activity analysis data of the Patatin protein obtained in Example 1;

[0057] Figure 10 The image shows the SDS-PAGE of Patatin protein separated by anion exchange chromatography using IexCap Smac DEAE in Comparative Example 2.

[0058] Figure 11 SDS-PAGE image of Patatin protein isolated and purified in Comparative Example 3;

[0059] Figure 12 The image shows the SDS-PAGE of Patatin protein separated using a Sephadex G-100 gel chromatography column in Comparative Example 4. Detailed Implementation

[0060] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0061] The testing method involved in this invention:

[0062] 1. Protein content detection methods

[0063] Protein concentration was determined according to the instructions of the BCA protein assay kit. A standard curve for protein concentration determination was plotted using bovine serum albumin (BSA) as the standard protein.

[0064] The specific procedure for determining the protein concentration of the sample is as follows: Prepare an appropriate amount of BCA working solution at a volume ratio of solution A: solution B = 50:1 and mix thoroughly. Take 100 μL of the dialyzed protein sample, using 100 μL of dialysis buffer as a blank control. Add 2 mL of BCA working solution to both the protein sample and the control sample. Incubate at 37℃ for 30 min, then cool to room temperature and measure the absorbance at 562 nm. Substitute the measured absorbance value (y) into the BSA protein standard curve equation y = 0.0011x to obtain x, which is the concentration of the protein sample.

[0065] 2. Protein purity detection methods

[0066] Protein samples were collected at different stages and prepared for electrophoresis at a sample volume to loading buffer ratio of 5:1. SDS-PAGE analysis was then performed using a protein gel containing 12% acrylamide. Electrophoresis conditions were: 60 V for 20-30 min, followed by 120 V for 60-80 min, stopping electrophoresis when the bromophenol blue indicator reached the protein gel substrate. Coomassie brilliant blue staining was applied, and the protein expression yield was determined using a BCA kit.

[0067] The sources of raw materials involved in this invention are as follows:

[0068] The waste juice from potato starch processing comes from Inner Mongolia Huaou Starch Factory;

[0069] Buffer A is 20 mmol / L NaH2PO4-Na2HPO4, pH=7.2;

[0070] Buffer B is 20 mmol / L NaH2PO4-Na2HPO4, 1 M NaCl, pH=7.2;

[0071] Buffer C consists of 20 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, and pH 7.4.

[0072] Buffer D consisted of 20 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 0.2 mol / L α-D-methylmannoside, and pH 7.4.

[0073] Example 1

[0074] A method for efficiently recovering the active glycoprotein Patatin from potato starch processing waste juice includes the following steps:

[0075] (1) Take fresh potato starch processing waste juice and filter the residue through gauze (300 mesh). Then centrifuge at 12000 r / min for 10 min to remove the precipitate and obtain potato waste juice. Accurately measure 500 mL of potato waste juice into a beaker, slowly add 53 g of ammonium sulfate powder and stir to dissolve it until the saturation reaches 20%. Precipitate the protein at 4℃ for 60 min. Then centrifuge at 10000 r / min at 4℃ for 10 min and collect the protein precipitate (named 20% protein precipitate).

[0076] Pour the supernatant back into the beaker, and add 29 g of ammonium sulfate to the beaker to make it saturated to 30%. Precipitate the protein at 4°C for 60 min. Then centrifuge at 10000 r / min and 4°C for 10 min and collect the protein precipitate (named 30% protein precipitate).

[0077] Pour the supernatant back into the beaker, and repeat this process by adding 31g, 32.5g, 35g, and 37.5g of ammonium sulfate to the beaker respectively, until the saturation reaches 40%, 50%, 60%, and 70% respectively. Then, centrifuge at 10000 r / min and 4℃ for 10 min, and collect the protein precipitate. The collected protein precipitates are named 40%, 50%, 60%, and 70% protein precipitates respectively.

[0078] (2) 50 mL of buffer A (20 mmol / L NaH2PO4-Na2HPO4, pH=7.2) was added to proteins precipitated with ammonium sulfate at different saturations to resuspend and dissolve the proteins. The proteins were then centrifuged at 10000 r / min and 4℃ for 15 min to remove the insoluble protein precipitates. The supernatant after centrifugation was analyzed using SDS-PAGE. The results are as follows: Figure 2 As shown, at 20-50% ammonium sulfate saturation, most Patatin proteins can be precipitated, while some other proteins and non-protein impurities are removed.

[0079] (3) Collect potato protein precipitated with ammonium sulfate in the range of 20-50% saturation, add 100 mL of buffer A to resuspend and dissolve, then dialyze the protein solution in 5 L of buffer A at 4℃ for 12 h, centrifuge the dialyzed protein solution at 10000 r / min at 4℃ for 10 min, and collect the protein supernatant for later use.

[0080] (4) Load the IexCap Smac Q column into the column position valve of the AKTA rapid protein purification instrument. First, wash the system and column with ultrapure water, and then equilibrate with buffer A. Use a sample pump to draw up the protein supernatant obtained in step (3) and load it onto the sample. Rinse with 20 mL of buffer A to remove unbound contaminating proteins. Then, elute the target protein with buffer B (20 mmol / L NaH2PO4-Na2HPO4, 1 mol / L NaCl, pH=7.2) using a linear elution program (0-30 min, 0-100% buffer B, flow rate 2 mL / min). Monitor the protein at 280 nm UV. Collect the eluted protein at a rate of 2 mL / tube. Perform SDS-PAGE analysis on the protein supernatant, flow-through, and eluent. The results are as follows: Figure 3 As shown, Patatin protein can be eluted when the NaCl concentration in the elution buffer is 50-150 mmol / L, while other contaminating proteins are present in different salt concentration elution ranges. All Patatin proteins are collected and then dialyzed at 4°C for 12 h in 2 L of buffer C (20 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, pH 7.4).

[0081] (5) Load the PreCap Con A affinity chromatography column into the column position valve of the AKTA rapid protein purification instrument. First, wash the system and column with ultrapure water, and then equilibrate with buffer C. Load the protein solution after dialysis in step (4) into the PreCap Con A affinity chromatography column. Wash the contaminating protein with buffer C for 3 column volumes (15 mL). Then, elute the target protein with buffer D (20 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 0.2 mol / L α-D-methylmannoside, pH 7.4) using a linear elution program (0-25 min, 0-100% buffer D, flow rate 2 mL / min). Monitor the protein at 280 nm UV. Collect the eluted protein at 2 mL / tube. Perform SDS-PAGE analysis on the protein supernatant, flow-through, and eluent. The results are as follows: Figure 4 As shown, high molecular weight impurities in the sample were removed by flow-through, while a few low molecular weight protease inhibitors and Patatin protein were co-eluted in buffer D at a concentration of 10-20 mmol / L of α-D-methylmannoside. All Patatin protein in the collection tube was collected and then dialyzed into 2 L of ultrapure water and dialyzed at 4 °C for 12 h.

[0082] (6) The Patatin protein solution after dialysis in step (5) was concentrated 10 times using an ultrafiltration tube with a molecular weight cutoff of 10,000 Da to achieve a protein concentration of 10 mg / mL. The solution was then filtered through a 0.45 μm filter for later use. A Sephadex G-75 gel chromatography column was loaded into the column position valve of an AKTA rapid protein purification instrument. The system and column were washed and equilibrated with ultrapure water. The concentrated protein solution was loaded into the Sephadex G-75 gel chromatography column, and the column was washed with ultrapure water at a flow rate of 1.5 mL / min. Based on the chromatogram monitored by the UV detector at 280 nm, eluted protein was collected at a rate of 1 mL / tube. The eluted target protein was analyzed using SDS-PAGE. The results are as follows: Figure 5 As shown; the Patatin protein purified by Con A affinity chromatography contains some low molecular weight protease inhibitors, but these protease inhibitors can be effectively removed by Sephadex G-75 gel chromatography to obtain high-purity Patatin protein; the eluent containing Patatin protein is collected to obtain the final product.

[0083] Structure and activity identification

[0084] 1. The secondary structure of Patatin protein was analyzed using a far-ultraviolet CD spectrometer.

[0085] After freeze-drying the Patatin protein elution fraction obtained in Example 1, the protein powder was dissolved in ultrapure water to a final concentration of 0.02 mg / mL. Then, 200 μL was placed in a cuvette for CD analysis. The preferred CD test conditions were: scanning wavelength range of 190-260 nm, bandwidth of 1 nm, scanning step size of 1 nm, slit width of 0.02 nm, path of 1 mm, and scanning speed of 50 nm / min. Ultrapure water was used as a blank control, and the Patatin protein sample spectrum was baseline corrected by subtracting the blank control spectrum.

[0086] The results are as follows Figure 6 As shown, the isolated and extracted Patatin protein has a typical α-helix characteristic spectrum with obvious negative peaks at 208 and 220 nm, which is completely consistent with other naturally extracted and recombinantly expressed Patatin proteins, indicating that the correct secondary structure has been formed.

[0087] 2. The tertiary structure of the Patatin protein was analyzed using intrinsic fluorescence spectroscopy.

[0088] After freeze-drying the Patatin protein eluent obtained in Example 1, the protein powder was dissolved in ultrapure water to a final concentration of 0.4 mg / mL. Then, 2-4 mL of the solution was placed in a quartz cuvette and subjected to fluorescence spectroscopy analysis at an emission wavelength of 300-500 nm.

[0089] The results are as follows Figure 7 As shown, the Patatin protein exhibits the highest absorption at a wavelength of 330 nm, which is largely consistent with existing reports.

[0090] 3. The antioxidant activity of Patatin protein was analyzed using the DPPH antioxidant kit.

[0091] After freeze-drying the Patatin protein elution fraction obtained in Example 1, the protein powder was dissolved in buffer A, and then DPPH antioxidant activity was analyzed. Specifically, 400 μL of Patatin protein solution was mixed with 600 μL of DPPH working solution, and then incubated at room temperature in the dark for 30 min. After centrifugation at 4000 r / min for 5 min, the absorbance was measured at 517 nm. Vitamin E was used as a positive control, and the antioxidant activity of Patatin protein was finally converted into vitamin E equivalents.

[0092] The results are as follows Figure 8 As shown, compared with the potato protein Patatin-B2 disclosed in the prior art (DOI: 10.1016 / j.synbio.2025.01.003; 2025.02), the natural Patatin protein extracted in Example 1 has excellent antioxidant activity. At a concentration of 2 mg / mL, the DPPH scavenging rate can reach 50%. The antioxidant activity produced by a unit mass of Patatin protein is comparable to that produced by 13.5 mg of vitamin E, and it has higher antioxidant activity than Patatin-B2.

[0093] 4. Patatin protein lipase activity was analyzed using p-nitrophenylacetate (p-NPC2), butyrate (p-NPC4), hexanoate (p-NPC6), caprylate (p-NPC8), and laurate (p-NPC12) with different carbon chain lengths.

[0094] After freeze-drying the Patatin protein elution fraction obtained in Example 1, the protein powder was dissolved in buffer A to form a Patatin protein solution with a concentration of 1 mg / mL; then, lipase activity was measured.

[0095] Specifically, a series of carbon chain lengths are dissolved using buffer A. pPrepare a 10 mmol / L stock solution of p-NPC2-12 substrate. Add 20 μL of the stock solution to 160 μL of buffer A, then quickly add 20 μL of 1 mg / mL Patatin protein solution. Incubate at 37 °C for 5 min, then measure the absorbance at 405 nm. Use buffer A instead of Patatin protein solution as a blank control. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol / L p-nitrophenol per minute by breaking down p-NPC2-12. The absorbance of 1 μmol / L p-nitrophenol at 405 nm is 0.01. Patatin protein activity is expressed as U / mg.

[0096] The results are as follows Figure 9 As shown, Patatin protein p-nitrophenylacetate p -NP2 exhibited the best hydrolysis effect, and the lipase activity gradually decreased with the lengthening of the carbon chain. This result is consistent with the substrate specificity of the previously reported Patatin protein, indicating that the Patatin protein extracted in Example 1 has good lipase activity.

[0097] 5. The performance of the Patatin protein obtained in each step of Example 1 was analyzed, and the results are shown in Table 1:

[0098] Table 1. Potato glycoproteins obtained at each step

[0099]

[0100] As shown in Table 1, the purity of the potato glycoprotein Patatin obtained in Example 1 gradually increased from the original 23.4% to 95.5%. The lipase hydrolysis activity produced per unit mass of protein also increased, from 20.2 U / mg in the original potato waste juice to 71.8 U / mg.

[0101] Comparative Example 1

[0102] The difference from Example 1 is that step (4) uses a cation exchange chromatography column IexCap Smac S to separate Patatin protein. However, Patatin protein cannot be bound to the IexCap Smac S column in the pH range of 6.0-9.0. Almost all potato protein flows out in the form of flow-through liquid. Only when the pH is adjusted to below 4.0 can Patatin protein bind to the packing material in the IexCap Smac S column. However, after eluting the protein with high salt at this time, the activity of the obtained Patatin protein is severely lost and it cannot catalyze the hydrolysis of acyl groups.

[0103] Comparative Example 2

[0104] The difference from Example 1 is that in step (4), the IexCap Smac Q anion exchange column was replaced with the IexCap Smac DEAE anion exchange column, while all other parameters and conditions remained the same as in step (4); the results are as follows. Figure 10 As shown, compared with the separation effect of anion exchange chromatography IexCap Smac Q, although some low molecular weight impurities can be removed after separation by IexCap Smac DEAE, a large number of impurities still exist in the target protein component, and the separation effect is not good.

[0105] Comparative Example 3

[0106] The difference from Example 1 is that step (5) uses a Sephadex G-75 gel chromatography column for separation, and step (6) uses a PreCap Con A affinity chromatography column for separation. All other parameters and conditions are the same as in Example 1.

[0107] The results are as follows Figure 11 As shown, with Figure 5 Compared to the high-purity Patatin protein obtained after separation, if the Patatin protein is first separated using a Sephadex G-75 gel chromatography column and then separated using a PreCap Con A affinity chromatography column, the obtained Patatin protein still contains a large number of high-molecular-weight impurities and low-molecular-weight protease inhibitors, resulting in low product purity.

[0108] Comparative Example 4

[0109] The difference from Example 1 is that in step (6), the Sephadex G-75 gel chromatography column is replaced with a Sephadex G-100 gel chromatography column, while the other parameters and conditions are the same as in Example 1.

[0110] The results are as follows Figure 12 As shown, when using the Sephadex G-100 column, low molecular weight protease inhibitors were still present in the obtained Patatin protein, indicating that the Sephadex G-100 column cannot effectively separate Patatin protein and low molecular weight protease inhibitors.

[0111] Comparative Example 5

[0112] The differences in performance between the technical solution of Example 1 and existing methods are shown in Table 2:

[0113] Table 2. Recovery efficiency of different methods for potato protein Patatin

[0114]

[0115] [1] Gao Jie. Study on the recovery and properties of protein in potato starch wastewater [D]. Shaanxi University of Science and Technology, 2012.

[0116] [2]Waglay A, Karboune S, Alli I. Potato protein isolates: Recovery and characterization of their properties[J]. Food Chemistry, 2014, 142: 373-382..

[0117] [3] Liu Weicong. Research and process design of protein recovery method in wastewater from potato starch production [D]. Heilongjiang Oriental College, 2022.

[0118] As shown in Table 2, the existing technologies that use flocculation, acid-base precipitation, acid-heating, and ammonium sulfate precipitation to separate crude protein or some glycoproteins and protease inhibitors from potato processing wastewater have not achieved ideal purity, yield, and efficiency of the final Patatin protein. Furthermore, the potato protein obtained by these methods has essentially lost its biological activity, making it difficult to achieve large-scale application. In addition, the separation and purification process of these methods generates a large amount of waste pollutants.

[0119] This invention is the first to use ammonium sulfate fractionation, anion exchange chromatography, Con A affinity chromatography, and gel chromatography to achieve efficient separation of active Patatin protein. The obtained Patatin protein retains the original protein structure and biological activity. Compared with existing methods, the separation has better purity and higher efficiency. The obtained Patatin protein has complete protein higher-order structure and functional activity, and has better application scenarios.

[0120] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for the recovery of the active glycoprotein Patatin from potato starch processing waste juice, characterized in that, The method comprises the following steps: (1) The pretreated potato starch processing waste juice is subjected to ammonium sulfate fractionation precipitation, and the protein precipitates of different levels are collected and placed in buffer A for dialysis, and the protein solution after dialysis is centrifuged, and the supernatant is collected; The ammonium sulfate fractionation precipitation is specifically as follows: the potato starch processing waste juice is added with ammonium sulfate to a saturation degree of 1, then precipitated at 0-4 DEG C for 30-60 min, centrifuged, and the protein precipitate is collected; ammonium sulfate is continuously added to the supernatant to a saturation degree of 2, then precipitated at 0-4 DEG C for 30-60 min, centrifuged, and the protein precipitate is collected; the ammonium sulfate fractionation precipitation is repeatedly performed in this way; During the ammonium sulfate fractionation precipitation, the saturation degrees of ammonium sulfate are 20%, 30%, 40%, 50%, 60% and 70%, respectively; (2) The supernatant obtained in step (1) is sequentially subjected to anion exchange chromatography column and PreCap Con A affinity chromatography column to obtain target protein eluate, and then dialyzed in ultrapure water to obtain Patatin protein solution; The anion exchange chromatography column is IexCap Smac Q chromatography column; The separation by the anion exchange chromatography column is specifically as follows: the supernatant obtained in step (1) is loaded on the IexCap Smac Q chromatography column, first, the buffer A is used to wash off the impurities, and then the buffer B is used to elute the target protein by linear elution program, the eluate is collected in two parts, and SDS-PAGE analysis is performed on the eluate, and the eluate containing the target protein is collected according to the analysis results; wherein the buffer A is 20±5 mmol / L NaH2PO4-Na2HPO4, pH=7.2; the buffer B is 20±5 mmol / L NaH2PO4-Na2HPO4, 1 mol / L NaCl, pH=7.2; (3) The Patatin protein solution obtained in step (2) is ultrafiltrated and concentrated, the concentrated solution is subjected to Sephadex G-75 gel chromatography column, and the target protein is eluted with ultrapure water, the eluate is collected, and freeze-drying is performed, thereby obtaining the product.

2. The method of claim 1, wherein, The eluate containing the target protein is specifically the eluate containing the target protein eluted when the NaCl concentration in the eluate is 50-150 mmol / L.

3. The method of claim 1, wherein, The separation by the PreCap Con A affinity chromatography column in step (2) is specifically as follows: after dialysis, the target protein eluate collected from the IexCap Smac Q chromatography column is loaded into the PreCap Con A affinity chromatography column, impurity proteins are first washed with buffer C, and then the target protein is eluted with buffer D by linear elution, the eluate is collected in two parts, and SDS-PAGE analysis is performed, and the target protein eluate is collected according to the analysis results; wherein the buffer C is 20±5 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, pH 7.4; and the buffer D is 20±5 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 0.2 mol / L α-D-methylmannoside, pH 7.

4.

4. The method according to claim 3, characterized in that The eluate containing the target protein is specifically collected when the α-D-methylmannoside concentration in the eluate is 10-20 mmol / L.

5. The use of the method according to any one of claims 1-4 in the technical field of potato deep processing.

Images