CN111187343B

CN111187343B - Peony 2S albumin and extraction method and application thereof

Info

Publication number: CN111187343B
Application number: CN202010182396.1A
Authority: CN
Inventors: 李聪; 杨菊转; 申烨华; 陈邦
Original assignee: Northwest University
Current assignee: Northwest University
Priority date: 2020-03-16
Filing date: 2020-03-16
Publication date: 2022-02-25
Anticipated expiration: 2040-03-16
Also published as: CN111187343A

Abstract

The invention discloses peony 2S albumin, an extraction method and application thereof, wherein the amino acid sequence of the peony 2S albumin is shown in SEQ ID NO.1, and the peony 2S albumin is obtained by using peony seeds or peony seed meal as a raw material and performing degreasing treatment, PBS (phosphate buffered saline) leaching, ammonium sulfate precipitation method crude protein extraction and DEAE (DEAE) anion exchange chromatography purification. The extraction process is simple, the purity of the obtained peony 2S albumin can reach more than 95%, the peony 2S albumin has higher homology with 2S albumin family, the obtained peony 2S albumin has good reduction capability on FRAP, has good cleaning capability on ABTS free radicals, hydroxyl free radicals and DPPH free radicals, shows good antioxidant activity, and can be used as an antioxidant.

Description

Peony 2S albumin and extraction method and application thereof

Technical Field

The invention belongs to the technical field of protein, and particularly relates to peony 2S albumin, and an extraction method and application thereof.

Background

Peony is deciduous shrub of Paeonia of Paeoniaceae, widely distributed in Henan, Shandong, Anhui, Sichuan and Zhejiang, etc., and its root bark, flower, leaf and seed can be used. The peony seed is a peony seed, is a common Chinese medicinal material, contains various active ingredients such as paeoniflorin and the like, and has various pharmacological effects of resisting bacteria, diminishing inflammation, calming, easing pain, reducing blood sugar, resisting allergy, regulating immunity and the like. The peony seeds have rich oil content, paeonia ostii and paeonia rockii seed oil are approved by the national ministry of health in 3 months in 2011 to be new resource food, the planting area of oil peonies in China is sharply enlarged, the yield of the peony seeds is increased year by year, a large amount of peony seed meal is produced after oil and fat are extracted, and the protein content of the peony seed meal is about 25 percent. At present, the peony seed meal is mainly used as a feed raw material or a base material of an edible fungus culture medium, and the comprehensive development and utilization level is low.

2S albumin is a water-soluble seed storage widely distributed in dicotyledonous plantsProtein, rich in arginine, glutamine, asparagine and cysteine. Generally, the molecular weight of 2S albumin is between 12 and 15kDa, the molecules mainly consist of two polypeptide chains, the molecular weight of the shorter peptide chain is between 3 and 5kDa, and the molecular weight of the longer peptide chain is between 8 and 10 kDa. Although 2S albumins differ in subunit structure and synthesis, they are considered to be structurally homologous, compact globular proteins, most of which have a backbone of 8 conserved cysteine residues (CX)_nCX_nCCX_nCXCX_nCX_nC) 4 disulfide bonds, including 2 interchain disulfide bonds and 2 intrachain disulfide bonds, are formed, making the structure of these proteins very stable and compact. Apart from the conserved cysteines, the homology of the amino acid sequence of 2S albumin is relatively low within and between plants. In vitro studies show that the 2S albumin family members have multiple functions, such as translation inhibition, antibiosis, digestive enzyme inhibitor, plant defense function and the like. In addition to having biochemical activity, 2S albumin has been used as a carrier for the synthesis of bioactive peptides and to improve the nutritional properties of food crops by increasing the content of essential amino acids.

Disclosure of Invention

The invention aims to provide peony 2S albumin, an extraction method of the protein and a new application of the protein.

Aiming at the purposes, the amino acid sequence of the peony 2S albumin provided by the invention is shown in SEQ ID NO.1, and the extraction method comprises the following steps:

1. degreasing treatment

After the peony seeds or peony seed meal are crushed into powder, normal hexane is added according to the material-liquid ratio of 1: 4-1: 6g/mL for degreasing, and peony seed degreased powder or peony seed meal degreased powder is obtained.

2. Leaching with PBS buffer solution

Adding 0.01-0.02 mol/L PBS buffer solution with pH of 7.0-7.5 into the peony seed defatted powder or the peony seed meal defatted powder obtained in the step 1 according to the material-liquid ratio of 1: 6-1: 16g/mL, extracting for 10-14 h at 0-4 ℃, homogenizing for 60-180 s after extraction, and performing centrifugal separation to obtain a supernatant to obtain a protein extracting solution.

3. Fractional precipitation of crude protein by ammonium sulfate method

Adding ammonium sulfate into the protein extracting solution obtained in the step 2 until the saturation degree is 20%, standing for 1.5-2 h at 0-4 ℃, and then performing centrifugal separation to obtain a supernatant; continuously adding ammonium sulfate into the supernatant until the saturation degree is 40%, standing at 0-4 ℃ for 1.5-2 h, and then centrifuging to obtain supernatant; and continuously adding ammonium sulfate into the supernatant until the saturation degree is 60%, standing at 0-4 ℃ for 1.5-2 h, and centrifugally separating to remove the supernatant, thereby obtaining the crude protein.

4. Dialysis desalination of crude protein

Dialyzing the crude protein obtained in the step 3 by using 0.01-0.04 mol/L PBS buffer solution with pH of 6.5-7.5 at the temperature of 0-4 ℃ for desalting, and then freezing and drying in vacuum.

5. DEAE anion exchange chromatography

Dissolving the protein freeze-dried in the step 4 by using 0.01-0.04 mol/L PBS buffer solution with pH of 7.0-7.5, filtering by using a 0.45 mu m filter membrane, and purifying by using a DEAE anion exchange chromatographic column, wherein the purification conditions are as follows: the method comprises the steps of firstly balancing a chromatographic column by 3-4 times of column volume of 0.01-0.04 mol/L PBS buffer solution with pH 7.0-7.5, then carrying out gradient elution, wherein a mobile phase A is 0.01-0.04 mol/L PBS buffer solution with pH 7.0-7.5, a mobile phase B is 0.01-0.04 mol/L PBS buffer solution with pH 7.0-7.5 and contains 0.5-1.0 mol/L NaCl, the elution gradient is selected from A: B from 100:0 to 85:15 to 0:100, collecting eluent with A: B being 85:15, desalting and concentrating by using a 3000-3500 Da ultrafiltration tube, and carrying out vacuum freeze drying to obtain the peony 2S albumin.

In the step 2, preferably, 0.015mol/L PBS buffer solution with pH of 7.0 is added into the peony seed defatted powder or the peony seed meal defatted powder obtained in the step 1 according to the material-liquid ratio of 1: 8-1: 10g/mL, extraction is carried out for 12 hours at 4 ℃, homogenate is carried out for 80-140 seconds after extraction is finished, and supernatant is obtained through centrifugal separation, so as to obtain protein extracting solution.

In the step 4, preferably, the crude protein obtained in the step 3 is dissolved by 0.01-0.04 mol/L PBS buffer solution with pH of 7.0-7.5, the solution is transferred into a dialysis bag with molecular weight of 3000-3500 Da, and the solution is dialyzed in 0.01-0.04 mol/L PBS buffer solution with pH of 7.0-7.5 for 12-24 h, wherein the dialysis temperature is 0-4 ℃, and the buffer solution is changed for 3-4 times in the dialysis process; the dialyzed and desalted protein was then vacuum freeze dried.

In the step 5, preferably, the lyophilized protein in the step 4 is dissolved in 0.01 to 0.02mol/L PBS buffer solution with pH of 7.0 to 7.5, filtered through a 0.45 μm filter membrane, and then purified by a DEAE anion exchange chromatography column under the following conditions: the method comprises the steps of firstly balancing a chromatographic column by 3-4 times of column volume of 0.01-0.02 mol/L PBS buffer solution with pH of 7.0-7.5, then carrying out gradient elution, wherein a mobile phase A is 0.01-0.02 mol/L PBS buffer solution with pH of 7.0-7.5, a mobile phase B is 0.01-0.02 mol/L PBS buffer solution with pH of 7.0-7.5 and contains 0.8-1.0 mol/L NaCl, the elution gradient is selected from A: B from 100:0 to 85:15 to 0:100, collecting eluent with A: B being 85:15, desalting and concentrating by using a 3000-3500 Da ultrafiltration tube, and carrying out vacuum freeze drying.

The peony 2S albumin has certain antioxidant activity and can be used as an antioxidant.

The invention separates and purifies peony 2S albumin (Ps-2S-Alb) with high purity based on ammonium sulfate salting-out, centrifugation, dialysis and DEAE anion exchange chromatography. Preliminarily determining the molecular weight and purity of the protein by SDS-PAGE electrophoresis; simultaneously, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is adopted to further determine the molecular weight; and De Novo Sequencing is adopted to perform De Novo Sequencing on the target protein, multiple enzymolysis is adopted, the specific enzyme digestion sequence comprises trypsin, chymotrypsin, staphylococcal protease and endoprotease in sequence, and meanwhile, high-resolution mass spectrometry (Obitrap Fusion Lumos) is adopted, and the 100% full sequence splicing of the protein molecules is realized by utilizing the complementarity between different enzyme digestion peptide segments. Database search and matching find that the protein has high homology (27-32%) with the 2S albumin family and is completely matched with 8 conserved cysteines of the 2S albumin family members.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention adopts mild conditions to extract protein, and the operation process does not need protein denaturation, thereby keeping good activity of the protein.

2. The invention adopts simple chromatographic separation and purification technology, the process is simple, and the purity of the peony 2S albumin obtained by purification is very high and can reach more than 95%.

3. The peony 2S albumin has certain antioxidant activity, has certain scavenging capacity on DPPH free radicals, hydroxyl free radicals and ABTS free radicals, is increased along with the increase of concentration, and has the scavenging capacity on the hydroxyl free radicals which is obviously stronger than that of ascorbic acid with the same concentration.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of each eluate obtained by anion exchange chromatography purification of the crude protein and DEAE in example 1, wherein M is the Mark molecular weight range (10-180 kDa) and 1 is the crude protein after dialysis desalting; 2 is a peak eluted with B100: 0, 3 is a peak eluted with B85: 15, and 4 is a peak eluted with B0: 100.

FIG. 2 is a mass spectrum of peony 2S albumin.

FIG. 3 is a total ion flow chromatogram of peony 2S albumin.

FIG. 4 is an MS/MS spectrum of the QQQQCAQQVQRQDLALCEQFLNQDHQQQ peptide fragment.

FIG. 5 is an MS/MS map of the EQFLNQDHQQQLRLRS peptide fragment.

FIG. 6 is an MS/MS map of the SKGGNQQQAEEL peptide fragment.

FIG. 7 is an MS/MS map of the GQCCQQLSQLRDHGCRCAALSQLVRYQVE peptide fragment.

FIG. 8 is an MS/MS spectrum of the AEQLPVMCQVGQACELGL peptide fragment.

FIG. 9 is an MS/MS map of a peptide fragment of LGLGLGGA.

FIG. 10 shows the results of sequence matching between peony family 2S albumin and 2S albumin family.

FIG. 11 shows DPPH radical scavenging efficiency of peony 2S albumin.

FIG. 12 shows the hydroxyl radical scavenging rate of peony 2S albumin.

FIG. 13 shows the ABTS radical clearance by peony 2S albumin.

FIG. 14 shows the FRAP-reducing ability of peony 2S albumin.

Detailed Description

The invention will be described in more detail below with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

Example 1

1. Degreasing treatment

Crushing the peony seed meal into powder by using a high-speed crusher, adding n-hexane into the powder according to the material-liquid ratio of 1g to 5mL for degreasing, stirring the mixture for 30min, performing suction filtration, repeating the steps twice, combining filtrates, performing reduced pressure distillation to recover the n-hexane, and drying the obtained peony seed meal degreased powder at 25 ℃.

2. Leaching with PBS buffer solution

Taking 20g of peony seed meal defatted powder, adding 160mL of 0.015mol/L PBS buffer solution with the pH value of 7.0, extracting for 12h at 4 ℃, homogenizing for 120s by using a high-speed homogenizer after extraction, centrifuging for 30min at 4000r/min, and taking supernatant to obtain a protein extracting solution, wherein the extraction rate of protein is 38.57%.

3. Fractional precipitation of crude protein by ammonium sulfate method

Adding ammonium sulfate into the protein extract obtained in the step 2 until the saturation degree is 20%, standing for 2h at 4 ℃, and then centrifuging at 12000r/min for 20min to obtain supernatant; continuously adding ammonium sulfate into the supernatant until the saturation degree is 40%, standing at 4 deg.C for 2h, and centrifuging at 12000r/min for 20min to obtain supernatant; adding ammonium sulfate into the supernatant until the saturation degree is 60%, standing at 4 deg.C for 2 hr, centrifuging at 12000r/min for 20min, and discarding the supernatant to obtain crude protein.

4. Dialysis desalination of crude protein

Dissolving the crude protein obtained in step 3 with 0.02mol/L PBS buffer solution with pH7.0, transferring into a dialysis bag with molecular weight of 3000Da, dialyzing in 0.02mol/L PBS buffer solution with pH7.0 for 24h at the dialysis temperature of 4 ℃, and replacing the buffer solution 1 time every 8h in the dialysis process, and replacing the buffer solution 3 times in total. The dialyzed and desalted protein was then vacuum freeze dried.

5. DEAE anion exchange chromatography

Dissolving the protein freeze-dried in the step 4 by using 0.02mol/L PBS buffer solution with pH7.0, and filtering by using a 0.45 mu m filter membrane; then purifying by using a DEAE anion exchange chromatographic column under the following purification conditions: the method comprises the steps of firstly balancing a chromatographic column by 3-4 times of column volume of 0.02mol/L PBS buffer solution with pH7.0, then carrying out gradient elution, wherein the mobile phase A is 0.02mol/L PBS buffer solution with pH7.0, the mobile phase B is 0.02mol/L PBS buffer solution with pH7.0 and contains 0.8mol/L NaCl, the elution gradient is selected from the group consisting of A: B from 100:0 to 85:15 to 0:100, collecting each eluent respectively, and carrying out SDS-PAGE (sodium dodecyl sulfate electrophoresis) electrophoresis test on the eluates, and the result is shown in figure 1. As can be seen from fig. 1, the protein purity of part a: B: 85:15 is very high, and can reach 95% or more. The eluent with the ratio of A to B being 85 to 15 is desalted and concentrated by a 3000Da ultrafiltration tube, and is frozen and dried in vacuum, so that 0.86g of target protein is obtained.

The target protein band in lane 3 of FIG. 1 is dotted, and its molecular weight is determined by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) under the following conditions: an electrospray ion source ESI positive ion mode positive mode, the acceleration voltage is 20.00KV, and the mass scanning range is 10-60 KDa; resolution is 20000, and the molecular weight of the target protein is 12.75KDa, and the result is shown in figure 2.

In order to obtain the molecular information of the 12.75KDa protein, the mass spectrum identification is carried out by adopting an LC-MS-MS method, and the specific determination method comprises the following steps:

(1) enzymolysis

The gel strip of the target protein is subjected to enzymolysis by trypsin, then the enzymolysis solution is dissolved in a sample dissolving solution (aqueous solution containing 0.1 vol% formic acid and 2 vol% acetonitrile), the solution is fully oscillated and vortexed, the solution is centrifuged at 13200rpm and 4 ℃ for 10min, 15mL of supernatant is taken and transferred to a loading tube, and mass spectrometry is performed.

(2) LC-MS-MS analysis

The chromatographic conditions are as follows: selecting a capillary high performance liquid chromatograph, wherein a mobile phase A: aqueous solution containing 0.1 vol% formic acid and 2 vol% acetonitrile, mobile phase B: aqueous solution containing 0.1 vol% formic acid and 80 vol% acetonitrile, flow rate: 300 nL/min; the gradient elution conditions were: 0 → 1min, 6% B; 2 → 8min, 9% B; 9 → 24min, 14% B; 25 → 60min, 30% B; 61 → 75min, 40% B; 76 → 78min, 95% B.

The mass spectrum conditions are as follows: a positive ion mode; the reference mass-to-charge ratio is 350-1800 mz; the resolution was 70000. The data are collected by the mass spectrum to generate a raw file, the raw file is opened by using an Xcaliibur, a total ion current chromatogram can be obtained as shown in figure 3, and as can be seen from the figure, the number and abundance of proteins identified in the mass spectrum are large after the whole sample is subjected to efficient separation of liquid chromatogram. The original mass spectrum file is searched by using Maxquant (1.6.2.10) to search a Uniprot database, and compared with BLAST, the identification result is shown in Table 1, the Uniprot database search finds that the highest score is the heat shock protein of a peony library, the molecular weight is 71.253kDa, but the coverage rate is too low and is only 4 percent, and the molecular weight is too different from the target protein, so that the result is not matched. The polyubiquitin protein in the peony library has the same score as the heat shock protein, the molecular weight is 18.588KDa, the coverage rate is 19.3 percent, but the abundance is 0, so the matching result is not credible. The reliable protein is not matched in the database by searching the database, and the protein is not included in the database, so that the target protein is further identified by de novo sequencing.

TABLE 2 protein LC-MS/MS identification

Performing multiple enzymolysis treatment on a target protein, performing liquid chromatography (RPLC) and high-resolution Mass Spectrometry (Obitrap Fusion Lumos) on a peptide fragment subjected to the enzymolysis treatment, performing data analysis by using protein sequence analysis Software PEAKS Mass Spectrometry Software, analyzing overlapped polypeptides by a de novo sequencing method, automatically assembling a protein sequence from the analyzed polypeptides, and realizing 100% full-sequence splicing of protein molecules. The specific method comprises the following steps:

(1) multiple enzymolysis

And (3) decoloring colloidal particles: cutting the target protein strip into 1mm³The colloidal particles of (4) were put into 1.5mL of EP tubes, respectively. And (3) decoloring by taking a mixed solution of acetonitrile and 50mmol/L ammonium bicarbonate water solution in a volume ratio of 1:1 as a decoloring solution, sucking out the decoloring solution after decoloring for 10-30 min, and repeating the operation until the colloidal particles are colorless.

And (3) dehydrating colloidal particles: adding 100 μ L acetonitrile into colorless colloidal particles, standing for 30min until the colloidal particles are white, discarding acetonitrile, and standing at room temperature for drying.

Reductive alkylation: adding 10mmol/L DTT into the dehydrated colloidal particles according to 100. mu.L/tube, reducing in 56 deg.C water bath for 1h, sucking out DTT and discarding. Adding 55mmol/L IAA according to 100 μ L/tube, reacting at room temperature in dark for 1h, sucking out IAA, discarding, washing with decolorizing solution, sucking out decolorizing solution, discarding, adding 100 μ L acetonitrile, standing for 30min until the colloidal particles are white, discarding acetonitrile, and standing at room temperature for drying.

Trypsin (Trypsin) digestion: adding 15 ng/mu L of trypsin solution (diluted by 25mmol/L of ammonium bicarbonate aqueous solution) into the reduced and alkylated colloidal particles according to the amount of 10 mu L/tube, putting the mixture into a refrigerator at 4 ℃ for incubation for 40min, taking out the mixture, adding 10 mu L of 25mmol/L of ammonium bicarbonate aqueous solution into each tube, sealing the mixture, and placing the mixture in a water bath at 37 ℃ for enzyme digestion for 16 h. Adding extractive solution (mixed solution of trifluoroacetic acid, acetonitrile and water at volume ratio of 5:50: 45) into EP tube at 100 μ L/tube, water bathing at 37 deg.C for 1 hr, ultrasonic treating for 5min, centrifuging for 5min, transferring the extractive solution into another new EP tube, repeatedly extracting, mixing extractive solutions, and vacuum centrifuging and drying. The peptide fragment was dissolved in a sample solution (aqueous solution containing 0.1 vol% formic acid and 2 vol% acetonitrile), vortexed thoroughly, centrifuged at 13200rpm at 4 ℃ for 10min, and the supernatant was transferred to a sample tube for mass spectrometry.

Chymotrypsin (chytrixin): adding 25 ng/mu L of chymotrypsin solution (diluted by 100mmol/L of Tris-HCl buffer solution with pH 8.0) into the trypsinized EP tube according to the amount of 10 mu L/tube, putting the trypsinized EP tube into a refrigerator at 4 ℃ for incubation for 40min, taking out, supplementing 10 mu L of 100mmol/L Tris-HCl buffer solution with pH 8.0 into each tube, sealing and placing the tube in a water bath at 37 ℃ for enzyme digestion for 16 h. Adding extractive solution (mixed solution of trifluoroacetic acid, acetonitrile and water at volume ratio of 5:50: 45) into EP tube at 100 μ L/tube, water bathing at 37 deg.C for 1 hr, ultrasonic treating for 5min, centrifuging for 5min, transferring the extractive solution into another new EP tube, repeatedly extracting, mixing extractive solutions, and vacuum centrifuging and drying. The peptide fragment was dissolved in a sample solution (aqueous solution containing 0.1 vol% formic acid and 2 vol% acetonitrile), vortexed thoroughly, centrifuged at 13200rpm at 4 ℃ for 10min, and the supernatant was transferred to a sample tube for mass spectrometry.

Trypsin (Trypsin) + staphylococcal protease (Glu-C) + chymotrypsin (chyotrypin): adding 15 ng/mu L of trypsin solution (diluted by 25mmol/L of ammonium bicarbonate aqueous solution) into the EP tube after the chymotrypsin enzyme digestion according to the amount of 10 mu L/tube, putting the EP tube into a refrigerator at 4 ℃ for incubation for 40min, taking out, adding 10 mu L of 25mmol/L of ammonium bicarbonate aqueous solution into each tube, sealing and placing the tube in a water bath at 37 ℃ for enzyme digestion for 16 h. Adding extractive solution (mixed solution of trifluoroacetic acid, acetonitrile and water at volume ratio of 5:50: 45) into EP tube at 100 μ L/tube, water bathing at 37 deg.C for 1 hr, ultrasonic treating for 5min, centrifuging for 5min, transferring the extractive solution into another new EP tube, repeatedly extracting, mixing extractive solutions, and vacuum centrifuging and drying. Dissolving the enzyme digestion product in 50mmol/L ammonium bicarbonate water solution again, adding staphylococcal protease according to the mass ratio of 1:40 of staphylococcal protease to the substrate, carrying out enzyme digestion at 37 ℃ for 4 hours, continuously adding the staphylococcal protease according to the mass ratio of 1:40, carrying out enzyme digestion reaction at 37 ℃ for 16 hours, adding an extracting solution (a mixed solution of trifluoroacetic acid, acetonitrile and water in a volume ratio of 5:50: 45) into an EP tube according to a 100 mu L/tube, carrying out water bath at 37 ℃ for 1 hour, carrying out ultrasonic treatment for 5min, centrifuging for 5min, transferring the extracting solution into another new EP tube, repeatedly extracting once, combining the extracting solutions, and carrying out vacuum centrifugal drying. Adding 25 ng/mu L of chymotrypsin solution (diluted by 100mmol/L of Tris-HCl buffer solution with pH 8.0) according to the amount of 10 mu L/tube, putting the mixture into a refrigerator at 4 ℃ for incubation for 40min, taking out, supplementing 10 mu L of 100mmol/L Tris-HCl buffer solution with pH 8.0 into each tube, sealing, and placing the tube in a water bath at 37 ℃ for enzyme digestion for 16 h. Adding an extracting solution (a mixed solution of trifluoroacetic acid, acetonitrile and water in a volume ratio of 5:50: 45) into an EP tube according to a volume ratio of 100 mu L/tube, carrying out water bath at 37 ℃ for 1h, carrying out ultrasonic treatment for 5min, centrifuging for 5min, transferring the extracting solution into another new EP tube, repeatedly extracting once, combining the extracting solutions, carrying out vacuum centrifugal drying, dissolving a peptide fragment with a sample dissolving solution (an aqueous solution containing 0.1 vol% of formic acid and 2 vol% of acetonitrile), fully oscillating and vortexing, centrifuging at 13200rpm and 4 ℃ for 10min, transferring a supernatant into a sample loading tube, and carrying out mass spectrometry.

Endoprotease (Asp-N) cleavage: adding 15 ng/microliter Asp-N enzyme (diluted with 25 mmol/microliter ammonium bicarbonate water solution) according to the amount of 10 microliter/tube, incubating in a refrigerator at 4 deg.C for 40min, taking out, adding 10 microliter 25 mmol/microliter ammonium bicarbonate water solution to each tube, sealing, and enzyme-digesting in water bath at 37 deg.C for 16 hr. Adding 100 μ L/tube of extractive solution (mixed solution of trifluoroacetic acid, acetonitrile and water at volume ratio of 5:50: 45), water bathing at 37 deg.C for 1 hr, ultrasonic treating for 5min, centrifuging for 5min, transferring the extractive solution into another new EP tube, repeatedly extracting once, mixing extractive solutions, and vacuum centrifuging and drying. The peptide fragment was dissolved in a sample solution (aqueous solution containing 0.1 vol% formic acid and 2 vol% acetonitrile), vortexed thoroughly, centrifuged at 13200rpm at 4 ℃ for 10min, and the supernatant was transferred to a sample tube for mass spectrometry.

Random fragmentation: adding 50 mu L of 100mmol/L ammonium bicarbonate aqueous solution into a sample, adding DTT solution to make the final concentration of the solution be 10mmol/L, and reducing the solution in 56 ℃ water bath for 1 h; then, IAA solution was added thereto to give a final concentration of 50mmol/L, and the mixture was reacted for 40min with exclusion of light. Random fragmentation was performed using physical methods, desalted using a self-packed desalting column, and vacuum dried at 45 ℃.

(2) LC-MS detection

The peptide fragments after enzymolysis treatment are subjected to liquid chromatography (RPLC) and high-resolution mass spectrometry (Obitrap Fusion Lumos).

An Ultimate 3000 capillary high performance liquid chromatograph and a Thermo Scientific electrospray-combined ion trap Orbitrap mass spectrometer are adopted; the obtained peptide fragment is subjected to gradient elution by a high performance liquid chromatograph, and the mobile phase A: aqueous solution containing 0.1 vol% formic acid and 2 vol% acetonitrile, mobile phase B: aqueous solution containing 0.1 vol% formic acid and 80 vol% acetonitrile, flow rate: 300nL/min, gradient elution conditions were: 0 → 1min, 6% B; 2 → 8min, 9% B, 9 → 24min, 14% B; 25 → 60min, 30% B; 61 → 75min, 40% B; 76 → 78min, 95% B.

The mass spectrum detection conditions are as follows: a positive ion mode; primary mass spectrum parameters: the resolution is 70000, the AGCtarget accommodated ion number is 3 multiplied by 106, and the reference mass-to-charge ratio is 350-1800 mz; secondary mass spectrum parameters: resolution is 75000, AGCtarget holds an ion number of 1X 106, NCE/steppedNCE: 27.

(3) data analysis

The method comprises the steps of utilizing protein sequence analysis Software PEAKS Mass Spectrometry Software to analyze data, analyzing overlapped polypeptides by a de novo sequencing method, utilizing Peaks Software to carry out de novo analysis on primary and secondary spectrograms, and utilizing complementarity among different enzyme digestion peptide segments to realize splicing of 100% complete sequences of protein molecules, wherein the amino acid sequence of the protein molecules is shown as a sequence table SEQ ID No. 1. After each peptide segment is analyzed by secondary mass spectrometry (MS/MS), an MS/MS spectrum with basically complete main fragment ions (bn or yn) is obtained. MS/MS of seven peptide fragments detected by mass spectrometry and analysis results thereof are shown in FIGS. 4 to 9. The complete sequence of the protein is searched and analyzed in NCBI database, and the protein is found to have higher homology with the 2S albumin family, and the sequence matching result is shown in FIG. 10. The target protein was therefore designated peony 2S albumin (Ps-2S-Alb).

Example 2

In the embodiment, peony seed meal in the embodiment 1 is replaced by peony seeds, and other steps are the same as the embodiment 1, so that 0.98g of target protein peony 2S albumin (Ps-2S-Alb) with the purity of more than 95% is obtained.

Example 3 antioxidant Activity of peony 2S Albumin extracted in example 1

In this example, four methods of DPPH scavenging ability, hydroxyl radical scavenging ability, ABTS scavenging ability and FRAP reducing ability were used to evaluate the antioxidant activity of purified peony 2S albumin in vitro, and simultaneously compared with the activity of ascorbic acid as a positive control.

(1) DPPH radical scavenging Capacity determination

Taking 3.0mL of protein water solution with a series of concentrations, adding 3.0mL of 100 mu mol/L DPPH-ethanol solution, reacting for 30min in a dark place at room temperature, and measuring the absorbance value A of the reaction mixed solution at 517 nm; replacing the protein sample with distilled water, and measuring the absorbance value A0 under the same condition; absorbance value B determined under the same conditions, using an ethanol solution instead of DPPH. And ascorbic acid (Vc) was used as a positive control, and each sample was run in triplicate. The radical clearance (dpph.sc) was calculated as follows:

(2) measurement of hydroxyl radical scavenging ability

Taking 2.0mL of protein water solution with a series of concentrations, adding 0.6mL of 6mmol/L H₂O₂Aqueous solution, 0.6mL of 6mmol/L FeSO₄Mixing the water solution, 0.6mL of 6mmol/L salicylic acid water solution and 4.2mL of distilled water, shaking, reacting at 37 deg.C for 30min, measuring absorbance at 510nm, wherein A0 is the absorbance of blank control without protein sample, A is the absorbance of sample, B is the absorbance without color developmentAgent H₂O₂The value of time. Ascorbic acid (Vc) was used as a positive control, and each sample was run in triplicate. The hydroxyl radical clearance (oh.sc) is calculated as follows:

(3) ABTS free radical scavenging Capacity assay

Mixing 7.4mmol/L ABTS aqueous solution with 2.6mmol/L K₂S₂O₈The aqueous solution is mixed in equal volume and is reacted for 12 hours in a dark place, and is diluted by 50 times by 1.0mmol/L PBS buffer solution with pH 7.4 to ensure that A_734nm0.7 ± 0.02. Adding 200 μ L protein water solution with different concentrations, adding 4.8mL BTS, mixing well, keeping out of the sun at room temperature for 10min, measuring the ultraviolet absorption A1 at 734nm, and measuring A0 with distilled water as blank control. Ascorbic acid (Vc) was used as a positive control, and each sample was run in triplicate. ABTS free radical clearance calculation formula is as follows:

(4) FRAP reducing power measurement

The determination principle is as follows: fe³⁺TPTZ can be reduced to Fe by the sample²⁺TPTZ and exhibits a distinct blue color with a maximum absorption at 593nm, the greater the absorbance, the greater the reducing power of the sample. 0.2mol/L sodium acetate buffer solution (prepared by dissolving 5.1g sodium acetate in 20mL glacial acetic acid, diluting with water and diluting to a constant volume of 250 mL), 10mmol/L TPTZ solution (prepared by diluting 31.233mg TPTZ sample with 40mmol/L hydrochloric acid solution to a constant volume of 10 mL), and 20mmol/L FeCl₃The TPTZ working solution is prepared by mixing the aqueous solution according to the volume ratio of 10:1: 1.2 mL of protein aqueous solution with different concentrations is taken, 3mL of TPTZ working solution is added, the mixture is uniformly mixed and reacts for 10min at 37 ℃, and the absorbance A is measured at 593 nm.

The test results are shown in FIGS. 11 to 14. As can be seen from the figure, in a certain concentration range, the peony 2S albumin has certain antioxidant activity, has certain scavenging capacity on DPPH free radicals, hydroxyl free radicals and ABTS free radicals, the scavenging capacity is increased along with the increase of the concentration, and the scavenging capacity on the hydroxyl free radicals is obviously stronger than that of ascorbic acid with the same concentration.

Sequence listing

<110> northwest university

<120> peony 2S albumin and extraction method and application thereof

<141> 2020-03-16

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 112

<212> PRT

<213> Paeonia suffruticosa

<400> 1

Gly Gly Gly Gly Cys Ala Gly Gly Val Gly Ala Gly Ala Leu Ala Leu

1 5 10 15

Cys Gly Gly Pro Leu Ala Gly Ala His Gly Gly Gly Leu Ala Leu Ala

20 25 30

Ser Leu Gly Gly Ala Gly Gly Gly Ala Gly Gly Leu Gly Gly Cys Cys

35 40 45

Gly Gly Leu Ser Gly Leu Ala Ala His Gly Cys Ala Cys Ala Ala Leu

50 55 60

Ser Gly Leu Val Ala Thr Gly Val Gly Met Gly Gly Ala Gly Gly Gly

65 70 75 80

Leu Leu Ala Gly Pro Val Gly Gly Leu Ala Leu Met Ala Gly Gly Leu

85 90 95

Pro Val Met Cys Gly Val Gly Gly Ala Cys Gly Leu Gly Leu Gly Ala

100 105 110

Claims

1. A peony 2S albumin, which is characterized in that: the amino acid sequence of the protein is shown as SEQ ID NO. 1.

2. The method for extracting peony 2S albumin according to claim 1, wherein:

(1) degreasing treatment

After crushing peony seeds or peony seed meal into powder, adding normal hexane into the powder according to the material-liquid ratio of 1: 4-1: 6g/mL for degreasing to obtain peony seed degreased powder or peony seed meal degreased powder;

(2) leaching with PBS buffer solution

Adding 0.01-0.02 mol/L PBS buffer solution with pH of 7.0-7.5 into the peony seed defatted powder or the peony seed meal defatted powder obtained in the step (1) according to the material-liquid ratio of 1: 6-1: 16g/mL, extracting for 10-14 h at 0-4 ℃, homogenizing for 60-180 s after extraction, and performing centrifugal separation to obtain a supernatant to obtain a protein extracting solution;

(3) fractional precipitation of crude protein by ammonium sulfate method

Adding ammonium sulfate into the protein extracting solution obtained in the step (2) until the saturation degree is 20%, standing at 0-4 ℃ for 1.5-2 h, and then centrifuging to obtain a supernatant; continuously adding ammonium sulfate into the supernatant until the saturation degree is 40%, standing at 0-4 ℃ for 1.5-2 h, and then centrifuging to obtain supernatant; continuously adding ammonium sulfate into the supernatant until the saturation degree is 60%, standing at 0-4 ℃ for 1.5-2 h, and centrifugally separating to remove the supernatant to obtain crude protein;

(4) dialysis desalination of crude protein

Dialyzing the crude protein obtained in the step (3) by using 0.01-0.04 mol/L PBS buffer solution with pH of 6.5-7.5 at 0-4 ℃ for desalting, and then carrying out vacuum freeze drying;

(5) DEAE anion exchange chromatography

Dissolving the protein freeze-dried in the step (4) by using 0.01-0.04 mol/L PBS buffer solution with pH of 7.0-7.5, filtering by using a 0.45 mu m filter membrane, and purifying by using a DEAE anion exchange chromatographic column, wherein the purification conditions are as follows: the method comprises the steps of firstly balancing a chromatographic column by 3-4 times of column volume of 0.01-0.04 mol/L PBS buffer solution with pH 7.0-7.5, then carrying out gradient elution, wherein a mobile phase A is 0.01-0.04 mol/L PBS buffer solution with pH 7.0-7.5, a mobile phase B is 0.01-0.04 mol/L PBS buffer solution with pH 7.0-7.5 and contains 0.5-1.0 mol/L NaCl, the elution gradient is selected from A: B from 100:0 to 85:15 to 0:100, collecting eluent of A: B =85:15, desalting by using a 3000-3500 Da ultrafiltration tube, and carrying out vacuum freeze drying to obtain the peony 2S albumin.

3. The method for extracting peony 2S albumin according to claim 2, wherein: in the step (2), 0.015mol/L PBS buffer solution with pH of 7.0 is added into the peony seed defatted powder or the peony seed meal defatted powder obtained in the step (1) according to the material-liquid ratio of 1: 8-1: 10g/mL, extraction is carried out for 12 hours at 4 ℃, homogenate is carried out for 80-140 seconds after extraction is finished, and supernatant is obtained through centrifugal separation, so as to obtain protein extract.

4. The method for extracting peony 2S albumin according to claim 2, wherein: in the step (4), dissolving the crude protein obtained in the step (3) by using 0.01-0.04 mol/L PBS buffer solution with pH of 7.0-7.5, transferring into a dialysis bag with the molecular weight of 3000-3500 Da, dialyzing for 12-24 h in 0.01-0.04 mol/L PBS buffer solution with pH of 7.0-7.5, wherein the dialysis temperature is 0-4 ℃, and buffer solution is changed for 3-4 times in the dialysis process; the dialyzed and desalted protein was then vacuum freeze dried.

5. The method for extracting peony 2S albumin according to claim 2, wherein: in the step (5), the protein freeze-dried in the step (4) is dissolved by 0.01-0.02 mol/L PBS buffer solution with pH of 7.0-7.5, and then filtered by a 0.45 μm filter membrane, and then purified by a DEAE anion exchange chromatographic column, wherein the purification conditions are as follows: the method comprises the steps of firstly balancing a chromatographic column by 3-4 times of column volume of 0.01-0.02 mol/L PBS buffer solution with pH of 7.0-7.5, then carrying out gradient elution, wherein a mobile phase A is 0.01-0.02 mol/L PBS buffer solution with pH of 7.0-7.5, a mobile phase B is 0.01-0.02 mol/L PBS buffer solution with pH of 7.0-7.5 and contains 0.8-1.0 mol/L NaCl, the elution gradient is selected from A: B from 100:0 to 85:15 to 0:100, collecting eluent of A: B =85:15, desalting and concentrating by using a 3000-3500 Da ultrafiltration tube, and carrying out vacuum freeze drying.

6. Use of the peony 2S albumin of claim 1 as an antioxidant for non-disease diagnostic and therapeutic purposes.