CN111690035A - Method for improving yield of antibacterial peptide - Google Patents
Method for improving yield of antibacterial peptide Download PDFInfo
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- CN111690035A CN111690035A CN202010507204.XA CN202010507204A CN111690035A CN 111690035 A CN111690035 A CN 111690035A CN 202010507204 A CN202010507204 A CN 202010507204A CN 111690035 A CN111690035 A CN 111690035A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the related technical field of biological products, and particularly relates to a method for improving the yield of antibacterial peptide. The invention is mainly obtained by grinding and filtering fresh radish to obtain an extracting solution, adding cellulase, a promoter and a sage extract into the extracting solution, further concentrating, passing through a column and drying. The method provided by the invention can obviously improve the yield of the antibacterial peptide, and the concentrated antibacterial peptide and the cellulase form a fusion protein which is easy to separate out subsequently, thereby effectively reducing the operation difficulty and providing a powerful guarantee for the acquisition and the effective application of the antibacterial peptide.
Description
Technical Field
The invention belongs to the related technical field of biological products, and particularly relates to a method for improving the yield of antibacterial peptide.
Background
Antimicrobial peptides are an important component of the innate immune system of organisms, and they are isolated from a wide range of organisms, including bacteria, plants and animals. In the past 20 years, with the continuous discovery of antibacterial peptides, the number of antibacterial peptides is rapidly increasing, and the broad-spectrum antibacterial activity of the antibacterial peptides is also intensively studied. The plant antibacterial peptide is separated from the plant, and has many differences from other antibiotic peptides in a life form. Common types of plant antimicrobial peptides include thionine, plant defensins, hevein, Knottins, lipocalins, and Snakins. Because of the increasing resistance of pathogenic microorganisms to antibiotics, there is an urgent need to find agents that can replace traditional antibiotics. Plant antimicrobial peptides have a wide range of biological activities, including antibacterial, antifungal, antiviral, insecticidal, and anticancer, and therefore they hold promise as candidates for drugs to treat infections caused by pathogens in animals and humans. The mechanism of the plant antibacterial peptide for exerting the bioactivity is an important direction of research, and is helpful for finding a new therapy for treating diseases.
Meanwhile, the plant antibacterial peptide is a small molecular polypeptide which has the inhibiting or killing effect on bacteria, fungi and other microorganisms, can be induced by bacteria, fungi or physical and chemical stimulation, and some antibacterial peptides can be constitutively expressed even in plants. From the chemical structure, the plant antibacterial peptide mainly comprises thionine, plant defensin, lipocalin, rubber substance and the like, has strong antibacterial capability, better heat resistance and unique antibacterial mechanism, and has wide application prospect in the fields of agriculture, medicine, food and the like.
At present, most researchers at home and abroad are obtained by cell culture and further separation, the method has the defects of complex preparation process and high cost, and the antibacterial peptide is separated by the cells, so that the antibacterial peptide cannot be successfully separated out completely, a large amount of antibacterial peptide is wasted, the finally obtained antibacterial peptide is low in purity, and the sterilization effect is easy to reduce, so that further research is still needed on how to improve the yield of the antibacterial peptide and ensure the purity of the antibacterial peptide.
Based on this, chinese patent application CN110105437A discloses a method for separating plant antimicrobial peptide, plant antimicrobial peptide and use of plant antimicrobial peptide, which is obtained by subjecting black sesame to ultrasonic treatment and liquid chromatography separation, although the stability of the antimicrobial peptide is improved, the yield of the obtained antimicrobial peptide is still not high, and the purity of the antimicrobial peptide thus extracted is low, which affects the bactericidal effect of the antimicrobial peptide.
In conclusion, the defects of low yield, complex preparation process, high cost, low purity of the antibacterial peptide and low bactericidal effect generally exist in the prior art.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a method for improving the yield of antibacterial peptide. The method can obviously improve the yield of the antibacterial peptide, and the concentrated antibacterial peptide and the cellulase form a fusion protein which is easy to separate out subsequently, thereby effectively reducing the operation difficulty and providing a powerful guarantee for the acquisition and the effective application of the antibacterial peptide.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for increasing the yield of antimicrobial peptides, comprising the steps of:
s1, taking 75-100 g of fresh radish, mashing with a blender, filtering with gauze to remove impurities, and collecting filtrate to obtain radish extract;
s2, adding the accelerant and the sage extract into the extracting solution obtained in the step S1, and uniformly stirring the mixture by using a glass rod to obtain a mixture I;
s3, adding an acetic acid solution into the mixture I obtained in the step S2, performing ultrasonic treatment in an ultrasonic generator for 25-30 min, filtering with gauze, and collecting filtrate to obtain an extracting solution;
s4, heating the extracting solution prepared in the step S3 in a water bath at 65-75 ℃ for 15-25 min, then reducing the temperature to 25 ℃ and centrifuging, and taking supernate to obtain a mixed solution I;
s5, dialyzing the mixed solution I obtained in the step S4, concentrating to obtain a reversed phase chromatographic sample, and further separating by a reversed phase high performance liquid chromatography.
Preferably, the accelerator in step S2 is prepared from sucrose fatty acid ester, cellulase and biotin in a weight ratio of 1-1.5: 10-13: 3 to 5.
Preferably, the accelerator in step S2 is prepared from sucrose fatty acid ester, cellulase and biotin in a weight ratio of 1.3: 12: 4.
Preferably, the addition amount of each component in step S2 is: 0.5-0.8 g of accelerant and 0.3-0.5 g of sage extract.
Preferably, the volume fraction of the acetic acid solution in the step S3 is 8-10%, and the addition amount is 350-450 mL.
Preferably, the ultrasonic processing in step S3 is performed by: and adjusting the power of the ultrasonic generator to 350-400W and the frequency to 50kHZ, and carrying out ultrasonic treatment.
Preferably, the centrifugation process in step S4 is centrifugation for 10-15 min at 9000-10000 rpm.
Preferably, the dialysis time in step S5 is 36-58 h.
Preferably, the specific process of further separating by reverse phase high performance liquid chromatography described in step S5 is:
(1) carrying out reversed-phase high performance liquid chromatography separation on the obtained reversed-phase chromatographic sample by utilizing a reversed-phase chromatographic column, wherein the reversed-phase chromatographic column is a C4 silica gel column;
(2) after separation, eluting the C4 silica gel column by double distilled water containing trifluoroacetic acid and acetonitrile containing trifluoroacetic acid to obtain an elution sample;
(3) and (3) concentrating the elution sample obtained in the step (2) under reduced pressure to obtain the product.
Preferably, in the double distilled water containing trifluoroacetic acid in the step (2), the content of trifluoroacetic acid is 0.2-0.3%, and in the acetonitrile containing trifluoroacetic acid, the content of trifluoroacetic acid is 2-3% and the content of acetonitrile is 97-98%.
Weighing 1mL of the obtained antibacterial peptide sample, dissolving in a hydrolysis bottle, treating with performic acid, adding 1mL of hydrochloric acid, hydrolyzing at 110 ℃ for 24h, and determining the hydrolysate by UPLC to find that the antibacterial peptide is As-AFP2, wherein the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO. 1.
QRPSGTWSGVCGNNNACKNQCIRLEKARHG-SCNYVFPAHKCICYFPC(SEQ ID NO.1)
According to the invention, the accelerant is added in the process of extracting the antibacterial peptide from the fresh radish, and in the extraction process, the cellulase and the antibacterial peptide form a fusion protein, so that the antibacterial peptide is separated in the form of the fusion protein; the sucrose fatty acid ester, the cellulose and the biotin are mixed according to a certain weight ratio, so that the purity of the finally obtained antibacterial peptide is effectively improved, and the antibacterial performance of the finally obtained antibacterial peptide is greatly improved.
Compared with the prior art, the method for improving the yield of the antibacterial peptide has the following advantages:
(1) according to the method for improving the yield of the antibacterial peptide, the accelerant is added, so that the yield of the antibacterial peptide is improved, the purity of the antibacterial peptide is effectively improved, and the antibacterial performance of the antibacterial peptide is improved;
(2) the method for improving the yield of the antibacterial peptide provided by the invention has the advantages that the preparation process is simple, and the antibacterial peptide in the fusion protein is easy to separate;
(3) the method for improving the yield of the antibacterial peptide provided by the invention provides a new method for extracting the antibacterial peptide from plants, effectively reduces the production cost and provides a powerful guarantee for further application of the antibacterial peptide.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1A method for increasing the production of antimicrobial peptides
The method for improving the yield of the antibacterial peptide comprises the following steps:
s1, taking 75g of fresh radish, mashing with a blender, filtering with gauze to remove impurities, and collecting filtrate to obtain radish extract;
s2, adding 0.5g of accelerant and 0.3g of sage extract into the extracting solution obtained in the step S1, and uniformly stirring the mixture by using a glass rod to obtain a mixture I; sucrose fatty acid ester, cellulose and biotin are mixed according to the weight ratio of 1: 10: 3, preparing a composition;
s3, adding 350mL of 8% acetic acid solution in volume fraction into the mixture I obtained in the step S2, performing ultrasonic treatment in an ultrasonic generator (power 350W and frequency 50KHZ) for 25min, filtering with gauze, and collecting filtrate to obtain an extracting solution;
s4, heating the extracting solution prepared in the step S3 in a water bath at 65 ℃ for 15min, then reducing the temperature to 25 ℃, centrifuging the extracting solution for 10min at the rotating speed of 9000rpm, and taking supernatant to obtain a mixed solution I;
s5, dialyzing the mixed solution I obtained in the step S4 for 36 hours, concentrating to obtain a reversed phase chromatographic sample, and further separating by a reversed phase high performance liquid chromatography to obtain the product;
the specific process of further separating by the reversed phase high performance liquid chromatography is as follows:
(1) carrying out reversed-phase high performance liquid chromatography separation on the obtained reversed-phase chromatographic sample by utilizing a reversed-phase chromatographic column, wherein the reversed-phase chromatographic column is a C4 silica gel column;
(2) after separation, the C4 silica gel column is eluted by double distilled water containing trifluoroacetic acid with the content of 0.2 percent and acetonitrile containing 2 percent of trifluoroacetic acid to obtain an elution sample;
(3) concentrating the elution sample obtained in the step (2) under reduced pressure to obtain the final product; through detection, the purity of the antibacterial peptide finally prepared by the method is 96.1%; the yield can reach 2718 AU/mL.
Example 2A method for increasing the yield of antimicrobial peptides
The method for improving the yield of the antibacterial peptide comprises the following steps:
s1, smashing 100g of fresh radish by using a smashing device, filtering by using gauze to remove impurities, and collecting filtrate to obtain radish extract;
s2, adding 0.8g of accelerant and 0.5g of sage extract into the extracting solution obtained in the step S1, and uniformly stirring the mixture by using a glass rod to obtain a mixture I; the promoter is prepared from sucrose fatty acid ester, cellulose and biotin according to a weight ratio of 1.5: 13: 5, preparing a composition;
s3, adding 450mL of 10% volume fraction acetic acid solution into the mixture I obtained in the step S2, performing ultrasonic treatment in an ultrasonic generator (power 400W, frequency 50KHZ) for 30min, filtering with gauze, and collecting filtrate to obtain an extracting solution;
s4, heating the extracting solution prepared in the step S3 in a water bath at 75 ℃ for 25min, then reducing the temperature to 25 ℃ at room temperature, centrifuging for 15min at the rotating speed of 9000-10000 rpm, and taking supernatant to obtain a mixed solution I;
s5, dialyzing the mixed solution I obtained in the step S4 for 58 hours, concentrating to obtain a reversed-phase chromatographic sample, and further separating by using a reversed-phase high performance liquid chromatography to obtain the product;
the specific process of further separating by the reversed phase high performance liquid chromatography is as follows:
(1) carrying out reversed-phase high performance liquid chromatography separation on the obtained reversed-phase chromatographic sample by utilizing a reversed-phase chromatographic column, wherein the reversed-phase chromatographic column is a C4 silica gel column;
(2) after separation, the C4 silica gel column is eluted by double distilled water containing trifluoroacetic acid with the content of 0.3 percent and acetonitrile containing 3 percent of trifluoroacetic acid to obtain an elution sample;
(3) concentrating the elution sample obtained in the step (2) under reduced pressure to obtain the final product; through detection, the purity of the antibacterial peptide finally prepared by the method is 97.8%; the yield can reach 2903 AU/mL.
Example 3A method for increasing the yield of antimicrobial peptides
The method for improving the yield of the antibacterial peptide comprises the following steps:
s1, taking 85g of fresh radish, mashing the fresh radish by using a blender, filtering the mashed fresh radish by using gauze to remove impurities, and collecting filtrate to obtain radish extract;
s2, adding 0.6g of accelerant and 0.4g of sage extract into the extracting solution obtained in the step S1, and uniformly stirring the mixture by using a glass rod to obtain a mixture I; the promoter is prepared from sucrose fatty acid ester, cellulose and biotin according to a weight ratio of 1.3: 12: 4, preparing a composition;
s3, adding 400mL of 9% acetic acid solution in volume fraction into the mixture I obtained in the step S2, performing ultrasonic treatment in an ultrasonic generator (power 380W and frequency 60KHZ) for 28min, filtering with gauze, and collecting filtrate to obtain an extract;
s4, heating the extracting solution prepared in the step S3 in water bath at 70 ℃ for 20min, then reducing the temperature to 25 ℃, centrifuging the solution at 9500rpm for 13, and taking supernatant to obtain a mixed solution I;
s5, dialyzing the mixed solution I obtained in the step S4 for 48 hours, concentrating to obtain a reversed-phase chromatographic sample, and further separating by a reversed-phase high performance liquid chromatography to obtain the product;
the specific process of further separating by the reversed phase high performance liquid chromatography is as follows:
(1) carrying out reversed-phase high performance liquid chromatography separation on the obtained reversed-phase chromatographic sample by utilizing a reversed-phase chromatographic column, wherein the reversed-phase chromatographic column is a C4 silica gel column;
(2) after separation, the C4 silica gel column is eluted by double distilled water containing trifluoroacetic acid with the content of 0.25 percent and acetonitrile containing 2.5 percent of trifluoroacetic acid to obtain an elution sample;
(3) concentrating the elution sample obtained in the step (2) under reduced pressure to obtain the final product; through detection, the purity of the antibacterial peptide finally prepared by the method is 99.4%; the yield can reach 3125 AU/mL.
Comparative example 1 production method of antibacterial peptide
The production method of the antibacterial peptide is similar to that of example 3;
the difference from example 3 is that comparative example 1 does not contain sage extract; through detection, the purity of the antibacterial peptide finally prepared by the method is 72.3%; the yield was only 1753 AU/mL.
Comparative example 2 production method of antimicrobial peptide
The production method of the antibacterial peptide is similar to that of example 3;
the difference from example 3 is that the accelerator in comparative example 2 is prepared from sucrose fatty acid ester, cellulase and biotin in a weight ratio of 1: 1: 1. The purity of the antibacterial peptide finally prepared by the method is only 65.1 percent; the yield was only 2015 AU/mL.
Test examples antibacterial peptide sterilization test
1. Test samples: the methods of producing antibacterial peptides provided in examples 1-3 and comparative examples 1-2; escherichia coli ATCC 25922; staphylococcus aureus CVCC 1882; streptococcus ATCC 55121;
2. the test method comprises diluting the antibacterial peptide with sterile water to 0.5mg/mL by Oxford cup method, and culturing the strains with culture medium to concentration of 1.2 × 106cfu/mL, 200. mu.L of antimicrobial peptide was added to each Oxford cup, and sterilized water was used as a negativeAnd (4) performing sexual contrast, standing at 4 ℃ for 24h, transferring to a constant-temperature incubator, culturing at 37 ℃ for 24h, measuring the diameter of the bacteriostatic ring by using a vernier caliper after culturing, and judging the strength of the bacteriostatic activity according to the diameter.
3. And (3) test results: the specific test results are shown in table 1.
TABLE 1 comparison of the zone of inhibition diameters for different test samples
As can be seen from table 1, compared with the negative control group, the antibacterial peptides obtained by the methods of the embodiments 1 to 3 of the present invention all have significant bactericidal effects, especially, the groups of the embodiment 3 have the strongest inhibitory effect on 3 kinds of bacteria, so the group of the embodiment 3 is the best embodiment of the present invention; the antibacterial effect of the antibacterial peptide obtained by the formula method of the comparative examples 1-2 is reduced to a certain degree; particularly, in the group of comparative example 2, the diameter of the inhibition zone is only about 5mm, and the antibacterial activity is greatly reduced.
Finally, it should be noted that the above-mentioned embodiments are merely illustrative of the principles of the present invention and its efficacy, and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
<110> Guangzhou Yangyuyu Biotech Co., Ltd
<120> a method for increasing the yield of antimicrobial peptides
<130>2020.5.26
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>47
<212>PRT
<213> Amino acid sequence of antibacterial peptide As-AFP2 (Amino acid sequence of antibacterial peptide As-AFP2)
<400>1
Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly Asn Asn Asn Ala
1 5 10 15
Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg His Gly Ser Cys
20 25 30
Asn Tyr Val Phe ProAla His Lys Cys Ile Cys Tyr Phe Pro Cys
35 40 45
Claims (10)
1. A method for increasing the yield of antimicrobial peptides, comprising the steps of:
s1, taking 75-100 g of fresh radish, mashing with a blender, filtering with gauze to remove impurities, and collecting filtrate to obtain radish extract;
s2, adding the accelerant and the sage extract into the extracting solution obtained in the step S1, and uniformly stirring the mixture by using a glass rod to obtain a mixture I;
s3, adding an acetic acid solution into the mixture I obtained in the step S2, performing ultrasonic treatment in an ultrasonic generator for 25-30 min, filtering with gauze, and collecting filtrate to obtain an extracting solution;
s4, heating the extracting solution prepared in the step S3 in a water bath at 65-75 ℃ for 15-25 min, then reducing the temperature to 25 ℃ and centrifuging, and taking supernate to obtain a mixed solution I;
s5, dialyzing the mixed solution I obtained in the step S4, concentrating to obtain a reversed phase chromatographic sample, and further separating by a reversed phase high performance liquid chromatography.
2. The method for increasing the yield of the antibacterial peptide according to claim 1, wherein the accelerator in step S2 is prepared from sucrose fatty acid ester, cellulose and biotin in a weight ratio of 1-1.5: 10-13: 3 to 5.
3. The method for increasing the production of antimicrobial peptide according to claim 2, wherein the accelerator in step S2 is prepared from sucrose fatty acid ester, cellulase and biotin in a weight ratio of 1.3: 12: 4.
4. The method for increasing the yield of antimicrobial peptide according to claim 1, wherein the addition amount of each component in step S2 is: 0.5-0.8 g of accelerant and 0.3-0.5 g of sage extract.
5. The method for increasing the yield of antibacterial peptide according to claim 1, wherein the volume fraction of the acetic acid solution in step S3 is 8-10%, and the addition amount is 350-450 mL.
6. The method for increasing the yield of antimicrobial peptide according to claim 1, wherein the sonication in step S3 comprises: and adjusting the power of the ultrasonic generator to 350-400W and the frequency to 50kHZ, and carrying out ultrasonic treatment.
7. The method for increasing the yield of antibacterial peptide according to claim 1, wherein the centrifugation process in step S4 is performed at 9000-10000 rpm for 10-15 min.
8. The method for increasing the production of antimicrobial peptide according to claim 1, wherein the dialysis time in step S5 is 36-58 h.
9. The method for increasing the yield of antimicrobial peptide according to claim 1, wherein the specific process of further separating by reverse phase high performance liquid chromatography in step S5 is:
(1) carrying out reversed-phase high performance liquid chromatography separation on the obtained reversed-phase chromatographic sample by utilizing a reversed-phase chromatographic column, wherein the reversed-phase chromatographic column is a C4 silica gel column;
(2) after separation, eluting the C4 silica gel column by double distilled water containing trifluoroacetic acid and acetonitrile containing trifluoroacetic acid to obtain an elution sample;
(3) and (3) concentrating the elution sample obtained in the step (2) under reduced pressure to obtain the product.
10. The method for increasing the yield of the antimicrobial peptide according to claim 9, wherein the trifluoroacetic acid content in the double distilled water containing trifluoroacetic acid in the step (2) is 0.2-0.3%, the trifluoroacetic acid content in the acetonitrile containing trifluoroacetic acid is 2-3%, and the acetonitrile content is 97-98%.
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