CN104892723B - Preparation method of hairtail liver antibacterial peptide - Google Patents

Abstract

The invention discloses a preparation method of hairtail liver antibacterial peptide. During the preparation, hairtail liver is used as a raw material, and the antibacterial peptide is obtained through liver crude protein extraction, enzymatic hydrolysis, ultrafiltration and chromatography. The amino acid sequence of the antimicrobial peptide is Lys‑Val‑Phe‑Trp‑Gly‑Leu‑His‑Pro, and the molecular weight detected by ESI‑MS is 983.15 Da. The antibacterial peptide Lys-Val-Phe-Trp-Gly-Leu-His-Pro prepared by the present invention is effective against Gram-negative bacteria, Pseudomonas fluorescens, Pseudomonas aeruginosa and Gram-positive bacteria, Bacillus megaterium, Luccinia cambogia Sarcinus has a strong inhibitory effect.

Description

Preparation method of hairtail liver antibacterial peptide

technical field

The invention relates to a preparation method of fish visceral active polypeptide, in particular to a preparation method of hairtail liver antibacterial peptide.

Background technique

Antibacterial peptides (ABPs), also known as antimicrobial peptides and polypeptide antibiotics, refer to a class of small peptides with less than 100 amino acids and broad-spectrum resistance. Because antimicrobial peptides have a wide range of inhibitory effects on bacteria, fungi, parasites, etc., and with the emergence of more and more antibiotic-resistant microorganisms, antimicrobial peptides have better applications in the pharmaceutical industry and food additives. prospect. In a broad sense, antimicrobial peptides refer to a class of small-molecule cationic peptides that exist in the body to resist the invasion of external microorganisms and eliminate mutant cells in the body. Antimicrobial peptides are encoded by specific genes of biological cells and induced by specific external conditions. So far, there are more than 1,200 known antimicrobial peptides in the world, and there are thousands of peptidomimetic peptides that are artificially synthesized using natural cationic antimicrobial peptides as templates.

Based on this, the applicant uses hairtail liver as raw material and uses modern biotechnology to prepare antimicrobial peptides, which not only increases the utilization rate of hairtail resources, reduces the harm to the environment from scraps such as liver, but also provides candidate drugs for the development of marine antibacterial drugs .

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a preparation method of hairtail liver antibacterial peptide with scientific and reasonable process, easy operation, and good antibacterial effect of the extracted polypeptide.

The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: a preparation method of hairtail liver antibacterial peptide, which is characterized by comprising the following steps:

1) Preparation of hairtail liver enzymolysate: hairtail liver was taken out from frozen hairtail, thawed, homogenized with a high-speed tissue masher, and then added with phosphate buffer (0.02 mol/L) according to a solid-liquid ratio of 1 g: 8-10 mL. , pH 6.5-7.5), mix well, heat to 20-30°C, ultrasonically extract for 90-120 min in an ultrasonic generator with a power of 900W and a frequency of 30-50kHz, and add neutral protease (0.3-0.5% of the liver mass). Enzyme activity ≥ 2.0×10 ⁴ U/g), enzymatic hydrolysis at 55-65°C for 6-8 h, centrifugation at 10 000 r/min for 25-30 min, and the supernatant was collected using a 3 kDa ultrafiltration membrane for ultrafiltration Process, collect the fraction with molecular weight less than 3 kDa, freeze-dry to obtain ultrafiltration enzymatic hydrolysate;

2) Preparation of antibacterial peptides from hairtail liver: The prepared hairtail liver ultrafiltration enzymatic hydrolyzate was sequentially prepared by macroporous resin removal, gel filtration chromatography, cell membrane chromatography purification and reversed-phase high performance liquid chromatography (RP-HPLC), Get hairtail liver antibacterial peptides.

Preferably, the hairtail in the step 1) is hairtail ( Ttichiutus haumela ).

Preferably, the specific process of macroporous resin removal, gel filtration chromatography, cell membrane chromatography purification and reversed-phase high performance liquid chromatography (RP-HPLC) preparation in the step 2) is as follows:

Removal of impurities by macroporous resin: The obtained ultrafiltration enzymolysate was made into a solution with a concentration of 10-15 mg/mL with double distilled water, and was added to a D201 macroporous resin chromatography column at a flow rate of 2-3 mL/min. 2 to 3 times the column volume of double-distilled water to remove impurities, and then 3 to 5 times the column volume of 75% ethanol for elution, the eluent is rotated at a low pressure below 50 ℃ to remove the ethanol, and freeze-dried to obtain a macroporous resin Preparation of enzymatic hydrolysate.

Gel filtration chromatography: Dissolve the enzymatic hydrolyzate prepared from the macroporous resin above in triple-distilled water to make a solution with a concentration of 10-15 mg/mL, and separate it by Sephadex G-25 column chromatography, and use triple-distilled water for separation. After elution, the eluted fractions were collected according to the absorbance curve at 225 nm, among which, the fraction with the highest inhibition rate against Escherichia coli ( Escherichia coli ) and Bacillus megaterium ( Bacillus megaterium ) was gel filtration chromatography enzymatic hydrolysate.

Purification by Cell Membrane Chromatography: The above gel filtration chromatography enzymolysate was made into a solution of 80-100 μg/mL with triple distilled water, added to a cell membrane chromatography column for purification, and the eluted fractions were collected according to the absorbance curve at 225 nm. Among them, the components with the highest inhibition rate to Escherichia coli and Bacillus megaterium are the enzymatic hydrolysates purified by cell membrane chromatography.

RP-HPLC preparation: The above-mentioned cell membrane chromatography-purified enzymatic hydrolysate was prepared into a solution of 80-100 μg /mL with double distilled water, and purified by RP-HPLC. ) was a highly active antimicrobial peptide Lys-Val-Phe-Trp-Gly-Leu-His-Pro (KVFWGLHP).

Preferably, the cell membrane chromatography conditions are: the cell membrane is the cell membrane of Bacillus megaterium ; the injection volume is 15-20 μL; the cell membrane chromatography column (150 mm × 4.6 mm, 5 μm); the column temperature is 37 °C; The mobile phase was triple distilled water; the UV detection wavelength was 225 nm; the flow rate was 0.3-0.5 mL/min.

Preferably, the RP-HPLC conditions are: the injection volume is 15-20 μL; the chromatographic column is Zorbax C18 (250 mm × 4.6 mm, 5 μm); the mobile phase is 20% acetonitrile (containing 0.1% trifluoroacetic acid); The elution rate was 0.8-1.0 mL/min; the UV detection wavelength was 225 nm.

Compared with the prior art, the advantages of the present invention are:

(1) The present invention uses hairtail liver as raw material, which improves the utilization rate of hairtail resources and reduces environmental pollution.

(2) The antimicrobial peptide Lys-Val-Phe-Trp-Gly-Leu-His-Pro (KVFWGLHP) prepared by the present invention is effective against Gram ^- negative bacteria (G-) ( Escherichia coli ), Pseudomonas fluorescens ( Pseudomonas fluorescens ), Pseudomonas aeruginosa ) and Gram-positive bacteria (G ⁺ ) ( Staphylococcus aureus , Bacillus megaterium , Sarcina lutea ) ) have strong inhibitory effect and high safety, and can be used in medicine, cosmetics and food industries.

Description of drawings

Fig. 1 is the step flow schematic diagram of the present invention;

Fig. 2 is the Sephadex G-25 chromatogram of the macroporous resin of the present invention to prepare the enzymatic hydrolysate;

Fig. 3 is a cell membrane chromatogram of the enzymatic hydrolyzate (Fr.C) prepared by Sephadex G-25 of the present invention.

Fig. 4 is a reversed-phase high performance liquid chromatography (RP-HPLC) diagram of the enzymatic hydrolyzate (Fr.C-I) prepared by cell membrane chromatography of the present invention.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments.

A method for preparing antibacterial peptides from hairtail liver. The preparation process is as follows: hairtail liver → protein extraction, enzymatic hydrolysis → ultrafiltration → macroporous resin impurity removal → gel filtration chromatography → cell membrane chromatography purification → high performance liquid chromatography preparation → Antimicrobial Peptides.

Example:

1) Preparation of hairtail liver enzymolysate: hairtail ( Ttichiutus haumela ) liver was taken out from frozen hairtail, thawed, homogenized with a high-speed tissue masher, and then added with phosphate buffer (0.02 mol) according to the solid-to-liquid ratio of 1 g: 10 mL /L, pH 7.0), mix well, heat to 25°C, ultrasonically extract for 120min in an ultrasonic generator with a power of 900W and a frequency of 40kHz, and add neutral protease (enzyme activity ≥2.0×10 ⁴ U/L) according to 0.5% of the liver mass. g), enzymolyzed at 60°C for 8 hours, centrifuged at 10 000 r/min for 30 min, took the supernatant for ultrafiltration with a 3 kDa ultrafiltration membrane, collected the fraction with molecular weight less than 3 kDa, freeze-dried, and obtained ultrafiltration enzymatic hydrolysis thing;

2) Preparation of antibacterial peptides from hairtail liver: The prepared hairtail liver ultrafiltration enzymatic hydrolyzate was sequentially prepared by macroporous resin removal, gel filtration chromatography, cell membrane chromatography purification and reversed-phase high performance liquid chromatography (RP-HPLC), Get hairtail liver antibacterial peptides.

①Removal of impurities by macroporous resin: The obtained ultrafiltration enzymolysate was made into a solution with a concentration of 15 mg/mL with double distilled water, and added to a D201 macroporous resin chromatography column at a flow rate of 3 mL/min, using 2 times the column volume The impurities were removed by elution with double-distilled water, and then eluted with 5 times the column volume of 75% ethanol. The eluate was evaporated at a low pressure below 50 °C to remove the ethanol, and then freeze-dried to obtain a macroporous resin to prepare the enzymatic hydrolyzate.

②Gel filtration chromatography: The enzymatic hydrolyzate prepared from the macroporous resin was dissolved in triple-distilled water to make a solution with a concentration of 15 mg/mL, separated by Sephadex G-25 column chromatography, and washed with triple-distilled water. The eluted fractions were collected according to the absorbance curve at 225 nm, among which, the fraction with the highest inhibition rate to Escherichia coli and Bacillus megaterium was gel filtration chromatography enzymatic hydrolysate Fr.C ( figure 2).

③ Purification by Cell Membrane Chromatography: The above gel filtration chromatography enzymolysate was prepared into a solution of 100 μg/mL with three distilled water, and added to the cell membrane chromatography column for purification (condition: the cell membrane is the cell membrane of Bacillus megaterium ); The injection volume was 15 μL; the cell membrane column (150 mm × 4.6 mm, 5 μm); the column temperature was 37 °C; the mobile phase was triple distilled water; the UV detection wavelength was 225 nm; the flow rate was 0.3 mL/min), according to 225 The absorbance curve under nm was used to collect the eluted fractions. Among them, the fraction with the highest inhibition rate against Escherichia coli and Bacillus megaterium was the cell membrane chromatography-purified enzymatic hydrolysate Fr.CI (Figure 3).

④ RP-HPLC preparation: The above-mentioned cell membrane chromatography-purified enzymatic hydrolysate was made into a 100 μg/mL solution with double distilled water, and purified by RP-HPLC (condition: the injection volume was 20 μL; the chromatographic column was Zorbax C18 (250 mm) × 4.6mm, 5 μm); mobile phase is 20% acetonitrile (containing 0.1% trifluoroacetic acid); elution rate is 0.8 mL/min; UV detection wavelength is 225 nm), according to Escherichia coli and Megaspore Bacillus ( Bacillus megaterium ) inhibition rate was a highly active antimicrobial peptide (Figure 4).

⑤Structure determination: The highly active antimicrobial peptide was detected as a single peak, and the amino acid sequence determined by protein/peptide sequence analyzer was Lys-Val-Phe-Trp-Gly-Leu-His-Pro (KVFWGLHP), and the molecular weight detected by ESI-MS was 983.15Da.

⑥ Determination of antibacterial activity: as shown in Table 1: antimicrobial peptide Lys-Val-Phe-Trp-Gly-Leu-His-Pro (KVFWGLHP) against Gram ^- negative bacteria (G-) ( Escherichia coli ), Pseudomonas fluorescens , Pseudomonas aeruginosa ) and Gram-positive bacteria (G ⁺ ) ( Staphylococcus aureus ), Bacillus megaterium , Garcinia Coccus ( Sarcina lutea )) has a strong inhibitory effect, with broad-spectrum antibacterial activity, and the antibacterial effect on G ^- bacteria is generally stronger than that of G ⁺ bacteria.

Table 1 Antibacterial spectrum of antimicrobial peptide Lys-Val-Phe-Trp-Gly-Leu-His-Pro (KVFWGLHP) (n = 3)

Note: A sterilization hole punch (diameter = 4mm) was used to punch holes, and 20 μL of antibacterial solution was added to each hole; the data in the table are the mean ± standard deviation of the diameter of the inhibition zone (n = 3).

Finally, it should be noted that the above list is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All deformations that those of ordinary skill in the art can directly derive or associate from the disclosure of the present invention shall be considered as the protection scope of the present invention.

SEQUENCE LISTING

<110> Zhejiang Ocean University

<120> Preparation method of hairtail liver antibacterial peptide

<130> zjou-2015-wb0505

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Synthetic

<400> 1

Lys Val Phe Trp Gly Leu His Pro

1 5

Claims (1)

1. the preparation method of hairtail liver antimicrobial peptide, described hairtail liver antimicrobial peptide is Lys-Val-Phe-Trp-Gly-Leu-His-Pro, and its preparation method comprises the following steps: 1) Preparation of hairtail liver enzymolysate: The hairtail liver was taken out from the frozen hairtail, thawed, homogenized with a high-speed tissue masher, and then added with 0.02mol/L, pH 6.5-7.5 according to the solid-liquid ratio of 1g: 8-10mL. Phosphate buffer solution was mixed and heated to 20-30°C; ultrasonic extraction was performed in an ultrasonic generator with a power of 900W and a frequency of 30-50kHz for 90-120min, neutral protease was added according to 0.3-0.5% of the liver mass, and the temperature was Enzymolysis was carried out at 55-65°C for 6-8 hours, centrifuged at 10,000 r/min for 25-30 minutes, the supernatant was taken for ultrafiltration treatment with a 3kDa ultrafiltration membrane, the fraction with a molecular weight less than 3kDa was collected, and freeze-dried to obtain an ultrafiltration enzymatic hydrolyzate; 2) Preparation of antibacterial peptides from hairtail liver: The prepared hairtail liver ultrafiltration enzymatic hydrolysate was sequentially subjected to macroporous resin removal, gel filtration chromatography, cell membrane chromatography purification and reversed-phase high performance liquid chromatography RP-HPLC preparation to obtain hairtail. Liver antimicrobial peptides; The specific process of the macroporous resin impurity removal, gel filtration chromatography, cell membrane chromatography purification and reversed-phase high performance liquid chromatography RP-HPLC preparation in the step 2) is as follows: Macroporous resin impurity removal: The obtained ultrafiltration enzymolysate is made into a solution with a concentration of 10-15 mg/mL with double-distilled water, and is added to a D201 macroporous resin chromatography column at a flow rate of 2-3 mL/min. 3 times the column volume of double-distilled water to remove impurities, and then 3 to 5 times the column volume of 75% ethanol for elution, the eluate is rotated at a low pressure below 50 ° C to remove the ethanol, and freeze-dried to obtain the macroporous resin preparation enzyme solution; Gel filtration chromatography: Dissolve the enzymatic hydrolyzate prepared from the macroporous resin in triple-distilled water to prepare a solution with a concentration of 10-15 mg/mL, separate it by Sephadex G-25 column chromatography, and carry out Elution, collect elution fractions according to the absorbance curve at 225nm, among which, the fraction with the highest inhibition rate to Escherichia coli and Bacillus megaterium is gel filtration chromatography enzymolysate; Cell membrane chromatography purification: the gel filtration chromatography enzymolysate was made into a solution of 80-100 μg/mL with three-distilled water, added to a cell membrane chromatography column for purification, and the eluted fractions were collected according to the absorbance curve at 225 nm, wherein , the highest inhibition rate of Escherichia coli and Bacillus megaterium is the cell membrane chromatography purified enzymatic hydrolysate; the cell membrane chromatography conditions are: the cell membrane is the cell membrane of Bacillus megaterium ; the injection volume is 15-20 μL; the cell membrane chromatography column specifications are 150mm×4.6mm, 5μm; column temperature is 37℃; mobile phase is triple distilled water; UV detection wavelength is 225nm; flow rate is 0.3～0.5mL/min; RP-HPLC preparation: The cell membrane chromatography-purified enzymatic hydrolysate was prepared into a 100 μg/mL solution with double distilled water, and purified by RP-HPLC. According to the inhibition rate of Escherichia coli and Bacillus megaterium , 1 product was obtained. Highly active antimicrobial peptide, its amino acid sequence is Lys-Val-Phe-Trp-Gly-Leu-His-Pro; the RP-HPLC conditions are: the injection volume is 15-20 μL; the chromatographic column is 250mm×4.6mm, 5μm Zorbax C18; mobile phase is 20% acetonitrile containing 0.1% trifluoroacetic acid; elution rate is 0.8-1.0 mL/min; UV detection wavelength is 225 nm.

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