CN107056913A - A kind of method for preparing melittin - Google Patents

Abstract

The invention discloses a method for separating and purifying melittin, which belongs to the field of biotechnology. The melittin is a polypeptide extracted from bee venom, and the melittin is obtained by separation methods of Sephadex gel chromatography, freeze-drying, centrifugation and ethanol precipitation. The melittin of the present invention has high yield, short production time, simple process, electrophoretic purity, maintains the original structure and highest physiological activity of the melittin, and can be applied to the fields of medicine and the like.

Description

A kind of method for preparing melittin

technical field

The invention relates to a method for preparing melittin, which belongs to the field of biotechnology.

Background technique

Bee venom is a transparent liquid with aromatic odor secreted by the venom glands of worker bees. It is mainly composed of polypeptides, active enzymes and non-peptide substances. Protein polypeptide substances are the main components of bee venom, accounting for about 70% of bee venom. -80%. Melittin is a polypeptide composed of 26 amino acid residues, with a molecular weight of 2840Da, accounting for about 50% of the dry weight of bee venom, and its primary structure amino acid residue sequence is NH ₂ -Gly-Ile-Gly-Ala-Val- Leu-Lys-Val-Leu-Thr-Thr-Glu-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Glu-Gln-COOH. Melittin has anti-inflammatory, antibacterial, anti-cancer, anti-radiation, and inhibition of platelet aggregation effects, and can effectively treat many diseases such as frozen shoulder, rheumatoid arthritis, and rheumatoid arthritis. Applications.

Melittin is isolated and purified from bee venom. It has the characteristics of high biological activity and no pollution to the environment. It can develop products such as immune regulators and has great market development prospects, which has attracted widespread attention in the academic circles. s concern.

The method of preparing melittin by solvent extraction, three-column chromatography, and the combination of solvent extraction and three-column chromatography has a cumbersome preparation process, a long production cycle, and a large amount of organic solvents. Kreil (Kreil, G. Structure of melittin isolated from two species of honey bees [J]. Febs Letters, 1973,33(2): 241-244.) reported the method for preparing melittin by solvent extraction, and the yield of melittin It is about 20% (mass fraction), and the purity is 60.3% (mass fraction). The preparation method is simple. Extracting 1g of melittin requires 200mL of n-butanol. The demand for n-butanol is large, causing serious environmental pollution. Liu Ling and Yang Wenchao (1. Liu Ling, Li Changquan, Huang Xueqiang. Purification method of melittin and its anti-tumor effect in vitro[J]. China Biochemical Drug Impurities. 2003,24(4):16-17 2. Yang Wenchao, melittin Peptide separation and purification and its anti-radiation mechanism [D], Fujian Agriculture and Forestry University, 2007.) reported the preparation of melittin by three-column chromatography, the yield of melittin was about 48.67% (mass fraction), and the purity was 97.32% (mass fraction), high purity, which solves the problem of large amount of organic solvent, but the extraction process is complicated and the extraction time is long. Zhang Wenli and Xu Peng (1. Zhang Wenli, Sun Jingyuan. Separation and purification method of melittin [P], China, 101089017A.2007-12-19. 2. Xu Peng, Ouyang Yongwei, Huang Jingyao, Zhang Guiying, Xiao Cheng, Liu Bo . Experimental study on the separation and purification of melittin from bee venom. Chinese herbal medicine [J]. 2000, 31(12): 892-894.) reported the preparation of melittin by combining solvent extraction and chromatography, and the yield of melittin About 47% (mass fraction), the purity is electrophoretic pure, which shortens the time required for three-column chromatography, and Kreil (Kreil, G. Structure of melittin isolated from two species of honey bees [J]. Febs Letters, 1973, 33(2): 241-244.) Compared with the preparation method, the usage of n-butanol is reduced, but a large amount of n-butanol is still needed in the extraction process, which is not conducive to environmental protection.

The above separation and purification method has complex production process, long production cycle, low yield, low purity and serious pollution.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the existing separation and purification technology of melittin, and provide a method for rapidly preparing melittin. The melittin prepared by this method has high yield, high purity, short cycle and low energy consumption. A method for preparing melittin with low pollution and little pollution.

The mass fraction of the extraction rate of the melittin obtained by the present invention is 49%, and the mass fraction of the purity is 98%.

A kind of method for preparing melittin of the present invention, the steps are as follows:

①Removal of bee venom

Add the crude bee venom to a buffer solution with a pH of 4.0-5.5 to make the concentration 0.5-1.5mg/mL, centrifuge at 2500-3500r/min for 3-7min to remove insoluble impurities, and obtain a supernatant;

The crude bee venom is obtained by dissolving the venom discharged from bee stings with distilled water and freeze-drying;

The buffer is acetic acid-sodium acetate buffer solution;

The impurity removal is the removal of insoluble substances in the buffer solution.

② Separation of melittin

Pass the supernatant obtained in ① through a Sephadex G-25 column, elute with distilled water, control the flow rate at 25-30mL/h, measure the molecular weight of each separated component, and collect the molecular weight under the Sephadex column The 2840Da chromatographic component was freeze-dried to obtain a solid sample;

The Sephadex G-25 is represented by the English letter G for dextran, and G reflects the crosslinking degree, swelling degree and distribution range of the gel, and 25 is 2.5 grams of water absorption when every gram of gel expands;

The elution is to use distilled water as the mobile phase to continuously pass through the stationary phase of the chromatographic column, the force between the mobile phase and the stationary phase is weaker than that of the sample, and the components of the sample are washed out from the stationary phase in sequence;

The determination of the molecular weight is based on the standard curve regression equation of the elution retention time T (min) of the standard substance and its relative molecular mass logarithmic value (lgMr), and the standard substance is an antimicrobial peptide standard substance (Mr = 1422Da), oxidized glutathione Standard (Mr =612Da), reduced glutathione standard (Mr =307.32Da).

Described Da is the unit of melittin molecular weight;

The freeze-drying is a drying method in which the obtained components are frozen below the freezing point of water, placed in a high vacuum (10-40Pa) container, and the moisture in the material is directly sublimated from solid ice to water vapor by heating .

③Purification of melittin

Dissolve the solid obtained by freeze-drying in ② with 2-4 times the volume of absolute ethanol, centrifuge at 2500-3500r/min for 4-6min, and the precipitated substance is melittin.

The precipitate was a solid substance insoluble in absolute ethanol.

The production of melittin by this method simplifies the extraction process of melittin, reduces the production cycle by 1-2 days compared with the existing method, improves the yield by 1-2%, reduces the preparation cost, and the purity of melittin can reach Electrophoretic pure.

Compared with the prior art, the method for preparing melittin of the present invention has the following remarkable features:

(1) Use Sephadex G-25 column for separation;

(2) Compared with Liu Ling, Yang Wenchao, Zhang Wenli, Xu Peng (1. Liu Ling, Li Changquan, Huang Xueqiang. The purification method of melittin and its anti-tumor effect in vitro. Chinese biochemical drug impurities[J]. 2003,24(4):16-17 2. Yang Wenchao, Separation and purification of melittin and its anti-radiation mechanism [D], Fujian Agriculture and Forestry University, 2007. 3. Zhang Wenli, Sun Jingyuan. Separation and purification of melittin Method: China, 101089017A [P]. 2007-12-19. 4. Xu Peng, Ouyang Yongwei, Huang Jingyao, Zhang Guiying, Xiao Cheng, Liu Bo. Experimental study on the separation and purification of melittin from bee venom. Chinese herbal medicine [J] . 2000,31(12): 892-894.), the yield increased to 1-2%, and the preparation time was shortened by 2-3 days.

(3) The purity of melittin obtained by the present invention has reached electrophoretic purity.

Example 1:

①Removal of bee venom

Add crude bee venom to a buffer solution with a pH of 4.75 to make the concentration 1.0 mg/mL, and centrifuge at 3000 r/min for 5 min to remove insoluble impurities to obtain a supernatant;

The crude bee venom is obtained by dissolving the venom discharged from bee stings with distilled water and freeze-drying;

The buffer is acetic acid-sodium acetate buffer solution;

The impurity removal is the removal of insoluble substances in the buffer solution.

② Separation of melittin

Pass the supernatant obtained in ① through a Sephadex G-25 column, elute with distilled water, control the flow rate at 28mL/h, measure the molecular weight of each separated component, the molecular weight collected under the Sephadex column is 2840Da The chromatographic components were freeze-dried to obtain solid samples;

The Sephadex G-25 is represented by the English letter G for dextran, and G reflects the crosslinking degree, swelling degree and distribution range of the gel, and 25 is 2.5 grams of water absorption when every gram of gel expands;

The elution is to use distilled water as the mobile phase to continuously pass through the stationary phase of the chromatographic column, the force between the mobile phase and the stationary phase is weaker than that of the sample, and the components of the sample are washed out from the stationary phase in sequence;

The determination of the molecular weight is based on the standard curve regression equation of the elution retention time T (min) of the standard substance and its relative molecular mass logarithmic value (lgMr), and the standard substance is an antimicrobial peptide standard substance (Mr = 1422Da), oxidized glutathione Standard (Mr =612Da), reduced glutathione standard (Mr =307.32Da).

Described Da is the unit of melittin molecular weight;

The freeze-drying is a drying method in which the obtained components are frozen below the freezing point of water, placed in a high vacuum (10-40Pa) container, and the moisture in the material is directly sublimated from solid ice to water vapor by heating .

③Purification of melittin

Dissolve the solid obtained by freeze-drying in ② with 3 times the volume of absolute ethanol, centrifuge at 3000r/min for 5min, and the precipitated substance is melittin.

The precipitate was a solid substance insoluble in absolute ethanol.

The mass fraction of the extraction rate of melittin obtained in the current invention is 49%, and the mass fraction of the purity is 98%.

Example 2:

①Removal of bee venom

Add the crude bee venom to a buffer solution with a pH of 5.25, 1.5mg/mL, and centrifuge at 3200r/min for 6min to remove insoluble impurities to obtain a supernatant;

The crude bee venom is obtained by dissolving the venom discharged from bee stings with distilled water and freeze-drying;

The buffer is acetic acid-sodium acetate buffer solution;

The impurity removal is the removal of insoluble substances in the buffer solution.

②Separation of melittin

Pass the supernatant obtained in ① through a Sephadex G-25 column, elute with distilled water, and control the flow rate at 30mL/h, measure the molecular weight of each separated component, and the molecular weight collected under the Sephadex column is 2840Da The chromatographic components were freeze-dried to obtain solid samples;

The Sephadex G-25 is represented by the English letter G for dextran, and G reflects the crosslinking degree, swelling degree and distribution range of the gel, and 25 is 2.5 grams of water absorption when every gram of gel expands;

The elution is to use distilled water as the mobile phase to continuously pass through the stationary phase of the chromatographic column, the force between the mobile phase and the stationary phase is weaker than that of the sample, and the components of the sample are washed out from the stationary phase in sequence;

The determination of the molecular weight is based on the standard curve regression equation of the elution retention time T (min) of the standard substance and its relative molecular mass logarithmic value (lgMr), and the standard substance is an antimicrobial peptide standard substance (Mr = 1422Da), oxidized glutathione Standard (Mr =612Da), reduced glutathione standard (Mr =307.32Da).

Described Da is the unit of melittin molecular weight;

The freeze-drying is a drying method in which the obtained components are frozen below the freezing point of water, placed in a high vacuum (10-40Pa) container, and the moisture in the material is directly sublimated from solid ice to water vapor by heating .

③Purification of melittin

Dissolve the solid obtained by freeze-drying in ② with 4 times the volume of absolute ethanol, centrifuge at 3200r/min for 6min, and the precipitated substance is melittin.

The precipitate was a solid substance insoluble in absolute ethanol.

The mass fraction of the extraction rate of melittin obtained in the current invention is 48.65%, and the mass fraction of the purity is 97.21%.

Example 3:

①Removal of bee venom

Add the crude bee venom to a buffer solution with a pH of 4.5, 0.5mg/mL, and centrifuge at 2800r/min for 4min to remove insoluble impurities to obtain a supernatant;

The crude bee venom is obtained by dissolving the venom discharged from bee stings with distilled water and freeze-drying;

The buffer is acetic acid-sodium acetate buffer solution;

The impurity removal is the removal of insoluble substances in the buffer solution.

② Separation of melittin

Pass the supernatant obtained in ① through a Sephadex G-25 column, elute with distilled water, and control the flow rate at 26mL/h, measure the molecular weight of each separated component, and the molecular weight collected under the Sephadex column is 2840Da The chromatographic components were freeze-dried to obtain solid samples;

The Sephadex G-25 is represented by the English letter G for dextran, and G reflects the crosslinking degree, swelling degree and distribution range of the gel, and 25 is 2.5 grams of water absorption when every gram of gel expands;

The elution is to use distilled water as the mobile phase to continuously pass through the stationary phase of the chromatographic column, the force between the mobile phase and the stationary phase is weaker than that of the sample, and the components of the sample are washed out from the stationary phase in sequence;

The determination of the molecular weight is based on the standard curve regression equation of the elution retention time T (min) of the standard substance and its relative molecular mass logarithmic value (lgMr), and the standard substance is an antimicrobial peptide standard substance (Mr = 1422Da), oxidized glutathione Standard (Mr =612Da), reduced glutathione standard (Mr =307.32Da).

Described Da is the unit of melittin molecular weight;

The freeze-drying is a drying method in which the obtained components are frozen below the freezing point of water, placed in a high vacuum (10-40Pa) container, and the moisture in the material is directly sublimated from solid ice to water vapor by heating .

③Purification of melittin

Dissolve the solid obtained by freeze-drying in ② with 2 times the volume of absolute ethanol, centrifuge at 2800r/min for 4min, and the precipitated substance is melittin.

The precipitate was a solid substance insoluble in absolute ethanol.

The mass fraction of the extraction rate of melittin obtained in the current invention is 48.76%, and the mass fraction of the purity is 97.64%.

Claims (10)

1. A method for preparing melittin, the steps are as follows: ①Removal of bee venom Add the crude bee venom to a buffer solution with a pH of 4.0-5.5 to make the concentration 0.5-1.5mg/mL, centrifuge at 2500-3500r/min for 3-7min to remove insoluble impurities, and obtain a supernatant; ②Separation of melittin Pass the supernatant obtained in ① through a Sephadex G-25 column, elute with distilled water, control the flow rate at 25-30mL/h, measure the molecular weight of each separated component, and collect the molecular weight under the Sephadex column The 2840Da chromatographic component was freeze-dried to obtain a solid sample; ③Purification of melittin Dissolve the solid obtained by freeze-drying in ② with 2-4 times the volume of absolute ethanol, centrifuge at 2500-3500r/min for 4-6min, and the precipitated substance is melittin. 2. the method for preparing melittin as claimed in claim 1, thick bee venom is that the venom discharged by honeybee stings is dissolved in distilled water, and gained through freeze-drying. 3. the method for preparing melittin as claimed in claim 1, buffer solution is acetic acid-sodium acetate buffer solution. 4. the method for preparing melittin as claimed in claim 1, impurity removal is to remove the material insoluble in buffer solution. 5. the method for preparing melittin as claimed in claim 1, glucan gel G-25 is that dextran is represented by English letter G, and G reflects the degree of cross-linking of gel, degree of expansion and range of distribution, 25 It absorbs 2.5 grams of water per gram of gel swelling. 6. the method for preparing melittin as claimed in claim 1, elution is to pass through chromatographic column stationary phase continuously as mobile phase with distilled water, and mobile phase and stationary phase force are weaker than sample, and each component of sample is sequentially from The stationary phase is washed out. 7. the method for preparing melittin as claimed in claim 1, the mensuration of molecular weight is according to standard substance elution retention time T (min) and its relative molecular mass logarithmic value (lgMr) standard curve regression equation, and standard substance is antimicrobial peptide Standard (Mr =1422Da), oxidized glutathione standard (Mr =612Da), reduced glutathione standard (Mr =307.32Da). 8. the method for preparing melittin as claimed in claim 1, Da is the unit of melittin molecular weight. 9. the method for preparing melittin as claimed in claim 1, freeze-drying is that gained component is frozen to below the freezing point of water, and is placed in the container of high vacuum (10-40Pa), makes material in material by heat supply A drying method in which the moisture in the ice is directly sublimated from solid ice to water vapor. 10. The method for preparing melittin as claimed in claim 1, the precipitation is a solid substance insoluble in absolute ethanol.