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Method for quickly and accurately discovering, identifying and preparing proteolysis provenance antibacterial peptide

A protein hydrolyzate and antimicrobial peptide technology, which is applied in the field of preparation and purification of bioactive peptides, can solve the problems of industrialization difficulties, high cost of chemical synthesis, and high cost, and achieve the effect of reducing production costs and reducing environmental pollution

Active Publication Date: 2012-07-04
JIANGNAN UNIV
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  • 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

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  • Method for quickly and accurately discovering, identifying and preparing proteolysis provenance antibacterial peptide
  • Method for quickly and accurately discovering, identifying and preparing proteolysis provenance antibacterial peptide
  • Method for quickly and accurately discovering, identifying and preparing proteolysis provenance antibacterial peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of Affinity Cell Membrane Stationary Phase

[0024] 500 ml of the E. coli culture cultured to the logarithmic phase was centrifuged at 4000 r / min for 15 min to obtain the bacteria, which were washed 10 times with 25 ml of Tris-EDTA buffer (pH 7.4) to remove medium residues. The cleaned cells were reconstituted with Tris-EDTA buffer, and frozen at -30°C for 4h, then removed and thawed in a 37°C water bath. After repeated freezing and thawing 5 times, centrifuged at 3000r / min for 15min. The frozen and thawed bacteria were subjected to cell lysis treatment at 600W ultrasonic power, and the ultrasonic wave was irradiated for 4 seconds each time, with an interval of 4 seconds, and a total time of 60 minutes. The precipitate was obtained by low-speed centrifugation. The precipitate was the cell membrane of E. coli. 10ml of suspension prepared by reconstituted cell membrane was placed in a centrifuge tube, then 0.5g (3-7μm) of activated macroporous spherical...

Embodiment 2

[0025] Example 2 Preparation of protein antibacterial hydrolysate

[0026] Taking Jatropha curcas seed meal protein as an example, take a protein sample of 10 g, with a purity of 92.26%, add 100 mL of deionized water, and use different hydrolytic proteases for hydrolysis. The optimal conditions for protease hydrolysis are shown in Table 1. After the protein solution is hydrolyzed by different proteases to different degrees of hydrolysis (7%, 9%, 11%, 13%, 15% and 17%), the turbid solution is adjusted to pH 7.5 at 45°C, and water bathed at 100°C. Enzyme for 10 minutes, then centrifuged at 4000r / min for 15 minutes, the supernatant was obtained, lyophilized, and reconstituted into a 1mg / ml sample solution, and the antibacterial ability test was performed respectively to find the component with the strongest antibacterial ability and cool to dry. Name this sample Fh13.

[0027] Table 1 Optimal working conditions of protease

[0028]

Embodiment 3

[0029] Example 3 Affinity extraction combined with high performance liquid phase and mass spectrometry technology to accurately and quickly discover and identify antimicrobial peptides

[0030] Place the affinity stationary phase of Example 1 and the medium Fh13 of Example 2 in a 10 ml centrifuge tube, shake and combine at 37° C. for 1 hour, and centrifuge the supernatant. The precipitation was washed 7 times with buffer solution, the washing solution and supernatant were combined, cooled to dryness and named Fh13-1. Reconstitute Fh13 and Fh13-1, and use high performance liquid-mass spectrometry to detect their fingerprints and analyze the difference peaks (as shown in the accompanying drawings image 3 Peaks 1, 2 and 3), prepare the difference peaks and test the antibacterial ability. Under these conditions, it was quickly discovered that a protein hydrolysate-derived antibacterial peptide was prepared. The series of this peptide is CAILTHKR, and the minimum inhibitory solubilit...

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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. Resistance to existing antibiotics (penicillin, macrolides, trimethoprim, sulfamethoxazole, tetracyclines, fluoroquinolones, chloramphenicol, vancomycin) has been found in humans and animals ) resistant strains. In the past two decades, the infection of drug-resistant strains has also been on the rise year by year, seriously threatening human health. The problem of increasing resistance to conventional antibiotics has become a glo...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/22G01N27/62
Inventor 张晖肖建辉郭晓娜钱海峰王立齐希光
Owner JIANGNAN UNIV
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