Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Jatropha curcas seed protein hydrolysate-derived antibacterial peptide and preparation method thereof

A technology of protein hydrolyzate and jatropha seeds, applied in the field of preparation and purification of bioactive peptides, can solve the problems of high cost of chemical synthesis, difficult industrialization, weak anti-virus ability, etc., to reduce environmental pollution and reduce production costs Effect

Active Publication Date: 2014-03-26
JIANGNAN UNIV
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently the following problems in the production of antimicrobial peptides: natural antimicrobial peptide resources are limited, and the direct extraction process is complicated and expensive; chemical synthesis is too expensive, industrialization is difficult, and it is difficult to ensure the biological activity of synthetic peptides; Although the engineering method makes it possible to obtain a large amount of cheap antimicrobial peptides, it is found that the obtained antibacterial peptides have weak antibacterial and antiviral abilities in actual operation, causing recipient cells to "suicide" and it is difficult to use commonly used bacteria and viruses as expression systems. Gene expression yield is not high
However, there are no related literature reports on the separation and purification of antimicrobial peptides by cell membrane chromatography at home and abroad.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Jatropha curcas seed protein hydrolysate-derived antibacterial peptide and preparation method thereof
  • Jatropha curcas seed protein hydrolysate-derived antibacterial peptide and preparation method thereof
  • Jatropha curcas seed protein hydrolysate-derived antibacterial peptide and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The preparation of embodiment 1 affinity cell membrane stationary phase

[0023] Centrifuge 500ml of Escherichia coli cultured to the logarithmic phase at 4000r / min for 15min to obtain the bacteria. The bacteria were washed 10 times with 25ml of Tris-EDTA buffer (pH7.4) to remove the medium residue. The cleaned bacterial cells were redissolved with Tris-EDTA buffer, frozen in a -30°C refrigerator for 4 hours, taken out and thawed in a 37°C water bath, and then centrifuged at 3000r / min for 15min after repeated freezing and thawing 5 times. The frozen-thawed bacteria were subjected to cell lysis treatment at 600W ultrasonic power. The ultrasonic wave was irradiated for 4s each time, with an interval of 4s, and a total time of 60min. The pellet was obtained by low-speed centrifugation, which was the cell membrane of Escherichia coli. Put 10ml of the suspension prepared by reconstitution of the obtained cell membrane into a centrifuge tube, then add 0.5g (3-7μm) of activate...

Embodiment 2

[0024] The preparation of embodiment 2 protein antibacterial hydrolyzate

[0025] Taking Jatropha curcas seed meal protein as an example, take 10g of protein sample with a purity of 92.26%, add 100mL of deionized water, and hydrolyze with different proteases. The optimal conditions for protease hydrolysis are shown in Table 1. After the protein solution is hydrolyzed by different proteases into different degrees of hydrolysis (7%, 9%, 11%, 13%, 15% and 17%), adjust the pH of the turbid solution to 7.5 at 45°C, and inactivate the enzyme in a 100°C water bath 10min, then centrifuged at 4000r / min for 15min, obtained the supernatant, freeze-dried, reconstituted into 1mg / ml sample solution, and tested the antibacterial ability separately to find out the component with the strongest antibacterial ability, and cooled to dry. name this sample

[0026] Named as Fh13. Table 1 Optimum Action Conditions for Protease

[0027]

Embodiment 3

[0028] Example 3 Affinity Extraction Combined with High Performance Liquid Chromatography and Mass Spectrometry for Accurate and Rapid Discovery and Identification of Antimicrobial Peptides

[0029] The affinity stationary phase in Example 1 and the Fh13 in Example 2 were placed in a 10 ml centrifuge tube, and after shaking and binding at 37° C. for 1 hour, the supernatant obtained was centrifuged. The precipitate was washed 7 times with buffer solution, and the washing solution and supernatant were combined, cooled to dryness and named as Fh13-1. Redissolve Fh13 and Fh13-1, and use high performance liquid chromatography-mass spectrometry to detect their fingerprints and analyze differential peaks (see attached image 3 No. 1, 2 and 3 peaks in ), prepare differential peaks and carry out antibacterial ability test. Under this condition, a protein hydrolyzate-derived antimicrobial peptide was rapidly discovered and prepared. The series of this peptide is CAILTHKR, and its minim...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for quickly and accurately discovering, identifying and preparing a proteolysis provenance antibacterial peptide, belonging to the field of the preparation and the purification of biologically-active peptides. According to the method, for example, Jatropha curcas seed protein, an ingredient containing the antibacterial peptide is prepared through hydrolysis by using a variety of proteases, and the antibacterial peptide is quickly discovered, identified and prepared and purified by using a technology which combines cell membrane affinity chromatography with a high-performance liquid phase fingerprint atlas and a mass spectrum. According to the method, the separation and the purification are divided into two steps, i.e. affinity extraction and BR-HPLC / MS / MS separation and identification, the traditional steps (usually involving 5-7 steps, such as ultrafiltration, anion and cation exchange, macroporous resin adsorption, desalting, gel chromatography and BR-HPLC) for purifying the antibacterial peptide are greatly shortened, and all of the obtained samples reach chromatographic purity. Visibly, a way with simplicity, convenience and fastness in operation, for the large-scale preparation, the accurate discovery and the fast purification of biological active substances in less content, such as the antibacterial peptide, is provided due to the establishment of the method provided by the invention.

Description

technical field [0001] The invention relates to a process for rapidly and accurately discovering, identifying and preparing protein hydrolyzate-derived antibacterial peptides by using cell membrane affinity chromatography combined with high-performance liquid phase fingerprinting and mass spectrometry, and belongs to the field of preparation and purification of biologically active peptides. Background technique [0002] Due to the severe residues caused by the abuse of antibiotics, the resistance of microorganisms in nature continues to increase. Humans and animals have been found to be resistant to existing antibiotics (penicillin, macrolides, trimethoprim, sulfamethoxine, etc.) azoles, tetracyclines, fluoroquinolones, chloramphenicol, vancomycin) drug-resistant strains, in the past two decades, the infection of drug-resistant strains has been increasing year by year, seriously threatening human health, the resistance of conventional antibiotics The increase problem has bec...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/22C12P21/06G01N27/62
Inventor 张晖肖建辉郭晓娜钱海峰王立齐希光
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com