CN114891081B - Dragon cypress She Kangdong protein and preparation method thereof - Google Patents
Dragon cypress She Kangdong protein and preparation method thereof Download PDFInfo
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 82
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 82
- 241000218691 Cupressaceae Species 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 238000001814 protein method Methods 0.000 title description 2
- 238000001962 electrophoresis Methods 0.000 claims abstract description 19
- 238000002386 leaching Methods 0.000 claims abstract description 13
- 230000006698 induction Effects 0.000 claims abstract description 12
- 230000006920 protein precipitation Effects 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000001742 protein purification Methods 0.000 claims abstract description 6
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- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
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- ALCAUWPAMLVUDB-FXQIFTODSA-N Glu-Gln-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ALCAUWPAMLVUDB-FXQIFTODSA-N 0.000 description 1
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- 239000004471 Glycine Substances 0.000 description 1
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- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- HUORUFRRJHELPD-MNXVOIDGSA-N Ile-Leu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HUORUFRRJHELPD-MNXVOIDGSA-N 0.000 description 1
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- BABSVXFGKFLIGW-UWVGGRQHSA-N Leu-Gly-Arg Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N BABSVXFGKFLIGW-UWVGGRQHSA-N 0.000 description 1
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 1
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 1
- 241000209082 Lolium Species 0.000 description 1
- WLXGMVVHTIUPHE-ULQDDVLXSA-N Lys-Phe-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O WLXGMVVHTIUPHE-ULQDDVLXSA-N 0.000 description 1
- ONGCSGVHCSAATF-CIUDSAMLSA-N Met-Ala-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O ONGCSGVHCSAATF-CIUDSAMLSA-N 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- CQZNGNCAIXMAIQ-UBHSHLNASA-N Pro-Ala-Phe Chemical compound C[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O CQZNGNCAIXMAIQ-UBHSHLNASA-N 0.000 description 1
- FUOGXAQMNJMBFG-WPRPVWTQSA-N Pro-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 FUOGXAQMNJMBFG-WPRPVWTQSA-N 0.000 description 1
- OHKFXGKHSJKKAL-NRPADANISA-N Ser-Glu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OHKFXGKHSJKKAL-NRPADANISA-N 0.000 description 1
- WGDYNRCOQRERLZ-KKUMJFAQSA-N Ser-Lys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)N WGDYNRCOQRERLZ-KKUMJFAQSA-N 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- RKDFEMGVMMYYNG-WDCWCFNPSA-N Thr-Gln-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O RKDFEMGVMMYYNG-WDCWCFNPSA-N 0.000 description 1
- IMULJHHGAUZZFE-MBLNEYKQSA-N Thr-Gly-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IMULJHHGAUZZFE-MBLNEYKQSA-N 0.000 description 1
- IGXLNVIYDYONFB-UFYCRDLUSA-N Tyr-Phe-Arg Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C1=CC=C(O)C=C1 IGXLNVIYDYONFB-UFYCRDLUSA-N 0.000 description 1
- BZMIYHIJVVJPCK-QSFUFRPTSA-N Val-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N BZMIYHIJVVJPCK-QSFUFRPTSA-N 0.000 description 1
- LKUDRJSNRWVGMS-QSFUFRPTSA-N Val-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N LKUDRJSNRWVGMS-QSFUFRPTSA-N 0.000 description 1
- 108010069490 alanyl-glycyl-seryl-glutamic acid Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 108091009704 ice binding proteins Proteins 0.000 description 1
- 238000011419 induction treatment Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 201000009240 nasopharyngitis Diseases 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000001304 sample melting Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a hinoki She Kangdong protein and a preparation method thereof, belonging to the technical field of antifreeze proteins. Crude dragon cypress She Kangdong protein is obtained through cold induction, leaching, centrifugation and protein precipitation, and electrophoresis pure dragon cypress She Kangdong protein is obtained through protein purification and secondary protein precipitation; the amino acid sequence of the polypeptide comprises a fragment shown as SEQ ID No. 1. The method can simply and rapidly extract and separate the electrophoresis pure antifreeze protein with better thermal stability from the leaves of the Chinese juniper, lays valuable earlier work for further purifying the Chinese juniper She Kangdong protein and researching the structure, the function and the antifreeze mechanism and the application in frozen foods, and expands resources for researching the plant antifreeze protein.
Description
Technical Field
The invention relates to a protein of sabina She Kangdong and a preparation method thereof, which are used for successfully extracting and separating antifreeze protein in leaves of sabina, and belong to the technical field of antifreeze protein.
Background
Antifreeze proteins are some of the proteins produced by organisms living in sub-zero environments that resist freezing threats, and which are capable of binding ice crystals to modify ice crystal morphology, reduce the freezing point of the solution, and inhibit ice crystal recrystallization, and are therefore also referred to as ice binding proteins. Since the discovery of antifreeze glycoproteins in the blood of antarctic fish at the end of the 60 s of the 20 th century, various types of antifreeze proteins have been discovered in many organisms. Wherein plant AFPs have better ability to inhibit ice recrystallization than other AFPs, which enables them to survive at lower temperature environments, reducing ice crystal damage to tissue cells. Based on the characteristics, the plant antifreeze protein has good application prospect in frozen foods. However, research on plant antifreeze proteins is still less developed by researchers than by the vast number of freeze tolerant plant species, which may limit research and industrial applications of plant antifreeze proteins.
The Thuja occidentalis is a cultivar of Thuja occidentalis of Thuja of Cupressaceae. The Chinese juniper is widely planted in various places of the whole country after introduction for many years, is a common cold-resistant tree species, can survive in the environment below zero in the north of China, keeps evergreen, can be used as a medicine on branches and leaves, has the effects of promoting blood circulation and promoting urination, and dispelling wind and cold, and is a plant resource with important economic value. The invention provides an extraction and separation method of the antifreeze protein of the leaves of the Chinese juniper, which can rapidly and simply obtain the electrophoretic pure Chinese juniper She Kangdong protein with better thermal stability, thereby laying a valuable early-stage work for further purifying the Chinese juniper She Kangdong protein, researching the structure and the antifreeze mechanism of the protein and the application of the protein in frozen foods and expanding the source for researching the antifreeze protein of plants.
Disclosure of Invention
The invention aims to overcome the defects, and provides the dragon cypress She Kangdong protein with better thermal stability, and the electrophoresis pure antifreeze protein with better thermal stability can be simply and rapidly extracted and purified from the dragon cypress leaves by the method.
According to the technical scheme, the protein of the dragon cypress She Kangdong comprises a fragment shown as SEQ ID No.1 in the amino acid sequence.
Further, the amino acid fragment exhibits antifreeze activity.
Further it has a high content of hydrophilic amino acid residues and hydrophobic amino acid residues, and an aliphatic amino acid content of up to 75%.
The preparation method of the dragon cypress She Kangdong protein comprises the steps of obtaining crude dragon cypress She Kangdong protein through cold induction, leaching, centrifugation and protein precipitation, and obtaining electrophoresis pure dragon cypress She Kangdong protein through protein purification and secondary protein precipitation.
The method comprises the following specific steps:
(1) Cold induction: picking fresh leaves of the dragon juniper with good growth vigor in winter, cleaning, and then carrying out cold induction and standing treatment at a low temperature; placing the leaves of the dragon juniper after cold induction in a pulverizer, pouring liquid nitrogen, and breaking the leaves;
(2) Leaching: pouring 2-3 times of Tris-HCl buffer solution into the leaf blade obtained in the step (1), stirring and leaching for 1-2 hours, and filtering to obtain leaching solution;
(3) And (3) centrifuging: centrifuging the extract obtained in the step (2) for 25-35 minutes at the temperature of 4-10 ℃ and the rotating speed of 10000-15000 g, and taking supernatant;
(4) Protein precipitation: pouring 4-5 times of pre-ice-cold acetone into the supernatant obtained in the step (3), precipitating for 10-15 h at the temperature of minus 20 ℃, centrifuging for 15-25 min at the temperature of 4-10 ℃ and the rotating speed of 10000-15000 g, and taking the precipitate; lyophilizing to obtain crude herba Saussureae Involueratae She Kangdong protein;
(5) Protein purification: re-dissolving the crude dragon cypress She Kangdong protein prepared in the step (4) in Tris-HCl buffer solution, and heating in a water bath at 60-100 ℃ for 10-30 minutes to obtain electrophoresis pure dragon cypress She Kangdong protein with better thermal stability;
(6) Protein precipitation: pouring 4-5 times of pre-ice-cold acetone into the heat-treated hinoki She Kangdong protein solution obtained in the step (5), precipitating for 10-15 hours at the temperature of minus 20 ℃, centrifuging for 15-25 minutes at the temperature of 4-10 ℃ and the rotating speed of 10000-15000 g, and taking the precipitate; freeze-drying to obtain the electrophoresis pure hinoki She Kangdong protein.
Further, the Tris-HCl buffer solution is specifically 60-65 mM buffer solution with pH value of 6-8.
Further, the pre-cooling acetone temperature is between-10 ℃ and-20 ℃.
Further, the cold induction is specifically carried out by standing at a low temperature of-20 ℃ for more than 4 weeks.
Protein isomers and derivatives comprising the protein of hinoki She Kangdong or fragments shown in SEQ ID No. 1.
The application of the protein of the dragon cypress She Kangdong, and the food containing the protein show the frost resistance.
The invention has the beneficial effects that: the method can simply and rapidly extract and separate the electrophoresis pure antifreeze protein with better thermal stability from the leaves of the Chinese juniper, lays valuable earlier work for further purifying the Chinese juniper She Kangdong protein and researching the structure, the function and the antifreeze mechanism and the application in frozen foods, and expands resources for researching the plant antifreeze protein.
Drawings
FIG. 1 shows SDS-PAGE gel electrophoresis of the antifreeze protein of the leaves of the Thuja occidentalis.
1 is a standard protein marker,2 is crude sabina She Kangdong protein, and 3 is sabina She Kangdong protein after heat treatment.
FIG. 2 is a second-order mass spectrum of the electrophoresis-pure protein of hinoki She Kangdong.
FIG. 3 shows the matching of the protein of sabina She Kangdong with the peptide chain sequence of the protein cytochrome f.
FIG. 4 is a three-dimensional structural model of the protein of hinoki She Kangdong.
Detailed Description
The following examples were obtained from the university campus of Henan Industrial university in winter; acetone, hydrochloric acid, zhengzhou pavilion, chemical engineering limited; SDS-PAGE gel preparation kit, glycine, PAGE mucin trace recovery kit, rainbow 130 broad-spectrum protein marker, beijing Soy Bao technology Co., ltd; tris (hydroxymethyl) aminomethane, beijing bootta technologies limited; sodium dodecyl sulfate, shanghai microphone Biochemical technology Co., ltd.
Example 1
(1) Cold induction: collecting leaves of the juniper branches of a campus of Henan industrial university in winter each year, and performing cold induction treatment in a refrigerator at the temperature of minus 20 ℃ for about one month; taking 100g of leaves of the dragon cypress, putting the leaves into a pulverizer, pouring a proper amount of liquid nitrogen, crushing the leaves at a low temperature, and pouring the crushed leaves into a beaker;
(2) Leaching: adding 200mL of Tris-HCl (62.5 mM, pH 6.8) into the beaker in the step (1), stirring and leaching for 2 hours, and filtering to obtain leaching liquor;
(3) And (3) centrifuging: centrifuging the leaching solution obtained in the step (2) for 30 minutes at the temperature of 4 ℃ and the rotating speed of 12500g, and taking supernatant;
(4) Protein precipitation: pouring four times of pre-ice-cold acetone into supernatant, precipitating at-20deg.C for 12 hr, centrifuging at 4deg.C and 12500g for 20 min, collecting precipitate, and lyophilizing at-45deg.C to obtain crude herba Cypress She Kangdong protein;
(5) Protein purification: re-dissolving the crude dragon cypress She Kangdong protein obtained in the step (4) in Tris-HCl (62.5 mM, pH 6.8), and heating in a water bath at 75 ℃ for 20 minutes to obtain the electrophoresis-pure dragon cypress She Kangdong protein with better heat stability;
(6) Protein precipitation: pouring 4 times of pre-ice-cold acetone into the heat-treated protein solution, precipitating at-20deg.C for 12 hr, centrifuging at 4deg.C and 12500g for 20 min, collecting precipitate, and lyophilizing at-45deg.C to obtain electrophoresis pure herba Saussureae Involueratae She Kangdong protein.
Example 2 detection of the protein Dragon's arborvitae She Kangdong
SDS-PAGE electrophoresis
Protein electrophoresis was performed on the protein of hinoki She Kangdong extracted in example 1, and SDS-PAGE of the anti-freeze proteins of hinoki leaves was performed using 5% of the gel concentrate and 12% of the gel separator. The gel concentration voltage is 30V during electrophoresis, the gel separation voltage is 60V, and after electrophoresis, the gel is dyed with Coomassie brilliant blue R-250 for 2 hours, decolorized and photographed.
The specific results are shown in FIG. 1. In the figure, 1 is a standard protein marker,2 is crude sabina She Kangdong protein, and 3 is sabina She Kangdong protein after heat treatment.
As can be seen from FIG. 1, crude hinoki She Kangdong protein obtained by acetone precipitation has 4 protein bands in total, and is concentrated between 25kDa and 55kDa, wherein the protein near 25kDa is less, the protein near 33kDa and 55kDa is most abundant, and two protein bands with similar molecular weights are present at 33 kDa. After the crude dragon cypress She Kangdong protein is subjected to heat treatment, protein bands near 25kDa disappear, and protein bands near 55kDa are obviously shallower, which indicates that the two proteins are degraded after heat treatment. The non-reduction electrophoresis of the heat-treated hinoki leaf antifreeze protein only has one protein band at 33kDa, which is probably due to intermolecular disulfide bonds existing in target proteins near 33kDa, and the intermolecular disulfide bonds are broken to form two proteins with different molecular weights after the reduction treatment.
2. Mass spectrometry analysis
The band of the electrophoresis protein of lane 3 was cut and subjected to MALDI-TOF-MS-MS mass spectrometry (Shanghai Biotechnology Co., ltd.) whose secondary mass spectrum is shown in FIG. 2. And (5) searching and comparing the sequence fragments obtained according to the secondary mass spectrum in a protein database. According to protein score, the theoretical peptide of the conifer leaf anti-freeze protein matching protein cytochrome f (chloroflast) [ Juniperus chinensis ] (Accession No. QZI 85440.1) is obtained after screening, and 19 segments of the theoretical peptide are matched. The sequence coverage situation is shown in fig. 3: the black color is protein cytochrome f sequence, the red color is anti-freeze protein and repeated part thereof, and the sequence coverage rate is 54%.
The amino acid composition of the protein hinoki She Kangdong was analyzed, wherein the hydrophobic amino acid residue was 44.86% and the hydrophilic amino acid residue was 55.14%, similar to the amino acid composition analysis of barley antifreeze protein. According to the previous research, when the proportion of hydrophobic residues in the ice binding site of the antifreeze protein is 39% on average and the proportion of hydrophilic residues is more than 40%, the antifreeze mechanism of the antifreeze protein accords with an anchoring binding water model of an adsorption-inhibition mechanism. In addition, the content of aliphatic amino acid in the antifreeze protein is 75%, the content of aromatic amino acid is 15%, the content of heterocyclic amino acid is 10%, and the content of aliphatic amino acid is high generally, so that the protein has better heat resistance, which is consistent with the heat stability result of the dragon cypress She Kangdong protein in the extraction process.
3. Thermal hysteresis activity analysis: see Table 1
10mg of the sample was placed in an aluminum crucible with reference to an empty pan. The initial temperature is set to be 10 ℃, the temperature is reduced to-25 ℃ at the speed of 1 ℃/min, the temperature stays for 5min until the sample is completely frozen, and then the temperature is increased to 10 ℃ at the speed of 1 ℃/min. Record sample melting point T m And the system fusion enthalpy delta H m . Then cooling to-25deg.C at a rate of 1deg.C/min, standing for 5min, and heating to a certain temperature (retention temperature, T) at which the sample system is in a partially molten state at a rate of 1deg.C/min h ) Stay for 5min, and continue cooling to-25 ℃. Obtain at different T h Freezing onset temperature T of lower sample System 0 And melting enthalpy at partial freezing ΔH f . The ice crystal content (Φ) and THA of the samples at different retention temperatures were calculated according to formulas (2-1) and (2-2).
Said Φ= (1- Δh) f /ΔH m )×100％；THA＝T 0 -T h 。
In order to detect the freezing resistance activity of the hinoki leaf antifreeze protein, the invention adopts a differential scanning calorimeter to measure the thermal hysteresis activity of the hinoki She Kangdong protein solution in a partial melting state.
TABLE 1 thermal hysteresis Activity of the protein Dragon's arborvitae She Kangdong
Table 1 shows the THA values of the protein of hinokitiol She Kangdong at various retention temperatures during extraction and purification. The ice crystal content of the system is less when the retention temperature of the crude dragon cypress She Kangdong protein solution is 0.2 ℃, at this time, the THA value of the dragon cypress She Kangdong protein is as high as 3.70 ℃, when the retention temperature is 0.1 ℃ and 0.0 ℃, the ice crystal content of the system is 12.3% and 17.3%, the thermal hysteresis value of the protein is 0.40 ℃ and 0.33 ℃, according to the prior study, when the ice crystal content is more than 10%, the obtained THA value is more accurate, so the highest THA value of the crude dragon cypress She Kangdong protein is about 0.40 ℃. The heat-treated protein solution of the sabina chinensis She Kangdong has ice crystal content of 11.8% and 20% when the retention temperature is 0.3 ℃ and 0.2 ℃, and the protein thermal hysteresis value is 0.44 ℃ and 0.35 ℃, which shows that the protein of the sabina chinensis She Kangdong still has better THA and is about 0.44 ℃. The non-reduced electrophoresis protein band of the heat-treated hinoki leaf anti-freeze protein is subjected to trace recovery to obtain electrophoresis pure hinoki She Kangdong protein, and when the retention temperature is 0.5 ℃ and 0.4 ℃, the ice crystal content of the system is 7.8% and 16.5%, and the thermal hysteresis value is 0.51 ℃ and 0.43 ℃ respectively, which shows that the THA of the electrophoresis pure hinoki She Kangdong protein is about 0.43 ℃.
Example 3D Structure prediction
The three-dimensional structure prediction of the hinoki leaf antifreeze protein is shown in figure 4. Until now, the crystal structure of plant AFPs is known only from ryegrass AFP, which is composed of 8 flat beta-sheets, of which 4 are long beta-sheets and 4 are short beta-sheets, and two flat beta-sheet planes are called "a" plane and "b" plane, and it has been proved that only the "a" plane participates in ice crystal binding according to mutation experiments. According to previous studies, it was found that AFPs from different plants can form β -sheet, α -helix and random structures after using homologous modeling predictions on a plurality of plant AFPs. The invention constructs 5 ScAFP three-dimensional structures, wherein two long-chain alpha-helices exist at two ends of the ScAFP molecule in 5 model structures, and a plurality of beta-sheets exist inside the molecule. Since AFPs in different species are almost independently evolved and their sequences and structures are quite different, a large number of samples are required to find common sequences or structural features in order to explore the sequence or structural features of anti-freeze proteins with anti-freeze properties.
The novel plant antifreeze protein is prepared and the 3D structure is obtained through prediction, so that a database is expanded for the research on the antifreeze mechanism of the plant AFPs.
The structure and the property of the protein of the sabina She Kangdong are predicted and analyzed, and the isoelectric point of the protein of the sabina She Kangdong is 9.06 and the relative molecular mass is 35655; 15.89% of the alpha-helix structure, 19.31% of the beta-sheet structure, 64.8% of the random coil are present in the secondary structure; the accessibility of the surface solvent is high; the three-dimensional structure of the material has two long-chain alpha-helices and a plurality of beta-sheet structures.
Sequence listing
<110> university of Henan industry
<120> a protein of hinoki She Kangdong and its preparation method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 95
<212> PRT
<213> Dragon cypress She Kangdong protein (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
Glu Ser Gly Val Ile Asn Glu Gln Asn Ile Ser Glu Ser Lys Phe Ser
1 5 10 15
Ile Leu Glu Thr Gly Ile Lys Phe Val Gln Ala Gly Ser Glu Val Ser
20 25 30
Ala Leu Leu Gly Arg Ser Ala Pro Ala Phe Thr Gln Leu Asp Ile Lys
35 40 45
Val Ala Leu Val Tyr Gly Gln Met Asn Glu Pro Pro Gly Ala Arg Val
50 55 60
Gly Leu Thr Ala Leu Thr Met Ala Glu Tyr Phe Arg Val Ile Asp Thr
65 70 75 80
Gly Gly Pro Leu Ser Val Pro Val Gly Glu Thr Thr Leu Gly Arg
85 90 95
Claims (6)
1. A preparation method of a hinoki She Kangdong protein is characterized by comprising the following steps: crude dragon cypress She Kangdong protein is obtained through cold induction, leaching, centrifugation and protein precipitation, and then electrophoresis pure dragon cypress She Kangdong protein is obtained through protein purification and secondary protein precipitation; the method comprises the following specific steps:
(1) Cold induction: picking fresh leaves of the dragon juniper with good growth vigor in winter, cleaning, and then carrying out cold induction and standing treatment at a low temperature; placing the leaves of the dragon juniper after cold induction in a pulverizer, pouring liquid nitrogen, and breaking the leaves;
(2) Leaching: pouring 2-3 times of Tris-HCl buffer solution into the leaf blade obtained in the step (1), stirring and leaching for 1-2 hours, and filtering to obtain leaching solution;
(3) And (3) centrifuging: centrifuging the extract obtained in the step (2) for 25-35 minutes at the temperature of 4-10 ℃ and the rotating speed of 10000-15000 g, and taking supernatant;
(4) Protein precipitation: pouring 4-5 times of pre-ice-cold acetone into the supernatant obtained in the step (3), precipitating for 10-15 h at the temperature of minus 20 ℃, centrifuging for 15-25 minutes at the temperature of 4-10 ℃ and the rotating speed of 10000-15000 g, and taking the precipitate; lyophilizing to obtain crude herba Saussureae Involueratae She Kangdong protein;
(5) Protein purification: re-dissolving the crude dragon cypress She Kangdong protein prepared in the step (4) in Tris-HCl buffer solution, and heating in a water bath at 60-100 ℃ for 10-30 minutes to obtain electrophoresis-pure dragon cypress She Kangdong protein with good thermal stability;
(6) Protein precipitation: pouring 4-5 times of pre-ice-cooled acetone into the heat-treated hinoki She Kangdong protein solution obtained in the step (5), precipitating for 10-15 hours at the temperature of minus 20 ℃, centrifuging for 15-25 minutes at the temperature of 4-10 ℃ and the rotating speed of 10000-15000 g, and taking the precipitate; freeze-drying to obtain the electrophoresis pure hinoki She Kangdong protein.
2. The method for preparing the protein of the phellodendron She Kangdong, as set forth in claim 1, characterized in that: the Tris-HCl buffer solution is specifically 60-65 mM buffer solution with pH value of 6-8.
3. The method for preparing the protein of the phellodendron She Kangdong, as set forth in claim 1, characterized in that: the temperature of the pre-ice-cooled acetone is minus 10 to minus 20 ℃.
4. The method for preparing the protein of the phellodendron She Kangdong, as set forth in claim 1, characterized in that: the cold induction is specifically carried out by placing at a low temperature of-20 ℃ for more than 4 weeks.
5. The protein of hinoki She Kangdong prepared by the method of claim 1, which is characterized in that: the amino acid fragment prepared by the method shows the anti-freezing activity.
6. The use of the protein of hinokitiol She Kangdong prepared by the method of any one of claims 1 to 4, characterized in that: food products containing the above proteins exhibit freeze-resistant properties.
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| CN111471082A (en) * | 2019-01-24 | 2020-07-31 | 华东师范大学 | Method for extracting radish seedling apoplast antifreeze protein |
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| CN111471082A (en) * | 2019-01-24 | 2020-07-31 | 华东师范大学 | Method for extracting radish seedling apoplast antifreeze protein |
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| 燕麦抗冻蛋白的分离纯化及对冻藏面团品质的影响;张艳杰;中国博士学位论文全文数据库 工程科技Ⅰ辑(第03期);B024-31 * |
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