阅读说明：本技术 一种酶催化制备赖氨酸寡肽及其单甲氧基聚乙二醇修饰的方法 (Method for preparing lysine oligopeptide through enzyme catalysis and modifying lysine oligopeptide by monomethoxypolyethylene glycol ) 是由 蒋晓晓 廖小凤 尹韦蔚 高鹏 杨长林 王峰 于 2019-10-17 设计创作，主要内容包括：本发明涉及一种酶催化制备赖氨酸寡肽及其单甲氧基聚乙二醇修饰的方法,属于酶催化反应技术领域。其采用蛋白酶为催化剂催化合成酪氨酸寡肽,然后在寡聚酪氨酸寡肽的氨基末端接枝单甲氧基聚乙二醇,制备得到单甲氧基聚乙二醇修饰赖氨酸寡肽。本发明方法涉及的聚肽合成步骤的催化剂为酶多相催化剂,具有反应条件温和、环境更友好、副反应少等优点,而且避免了繁琐的保护和去保护步骤,提供了一种应用前景良好的制备单甲氧基聚乙二醇修饰赖氨酸寡肽的方法,其制备所得的赖氨酸寡肽条件温和、无污染,产率最高达35%。(The invention relates to a method for preparing lysine oligopeptide by enzyme catalysis and a method for modifying monomethoxy polyethylene glycol thereof, belonging to the technical field of enzyme catalysis reaction. The method comprises the steps of catalytically synthesizing tyrosine oligopeptide by using protease as a catalyst, and grafting monomethoxypolyethylene glycol to the amino terminal of the oligomeric tyrosine oligopeptide to prepare monomethoxypolyethylene glycol modified lysine oligopeptide. The catalyst in the polypeptide synthesis step related by the method is an enzyme heterogeneous catalyst, has the advantages of mild reaction conditions, more environment-friendly property, less side reaction and the like, avoids fussy protection and deprotection steps, and provides the method for preparing the monomethoxy polyethylene glycol modified lysine oligopeptide with good application prospect, the prepared lysine oligopeptide has mild conditions and no pollution, and the yield can reach as high as 35%.)

1. A method for modifying lysine oligopeptide by monomethoxypolyethylene glycol is characterized in that: adding lysine oligopeptide, monomethoxy polyethylene glycol succinimide carbonate and triethylamine into anhydrous methanol, fully mixing, and reacting under the protection of nitrogen; standing overnight, vacuum filtering to obtain filtrate, vacuum drying, soaking in anhydrous ethanol, centrifuging, removing supernatant, and adding acetone until all is dissolved; standing the obtained solution overnight, and carrying out vacuum filtration to obtain a precipitate, namely the product of the monomethoxy polyethylene glycol modified lysine oligopeptide.

2. The method for modifying lysine oligopeptide by monomethoxy polyethylene glycol according to claim 1, which is characterized by comprising the following specific steps of adding 10-20 g of lysine oligopeptide, 12 ~ g of monomethoxy polyethylene glycol succinimide carbonate and 60 ~ g of triethylamine into 20mL of anhydrous methanol, reacting the reaction mixture at 30 ~ ℃ for 10 ~ hours under the protection of nitrogen, standing the reaction mixture at 0 ℃ overnight after the reaction is finished, carrying out vacuum filtration at 0-10 KPa, drying the filtrate at 30 ℃ for 0-10 KPa under vacuum for 2 ~ hours, adding 50 ~ mL of anhydrous ethanol, soaking for 7 ~ hours, centrifuging at 8000 ~ rpm for 2-4 minutes, removing the supernatant, adding acetone preheated to 30 ~ ℃ until the acetone is completely dissolved, standing the obtained solution at 0 ℃ for overnight, carrying out vacuum filtration at 0-10 KPa, and obtaining precipitate of monomethoxy polyethylene glycol lysine modified oligopeptide.

3. The method for modifying lysine oligopeptide by monomethoxypolyethylene glycol according to claim 1, which is characterized by comprising the following steps: the average molecular weight of the monomethoxypolyethylene glycol in the monomethoxypolyethylene glycol succinimide carbonate molecule is 200, 400 or 1000 Da.

4. A method for preparing lysine oligopeptide by enzyme catalysis, which is characterized by comprising the following steps: adding L-lysine methyl ester hydrochloride, tert-butyl alcohol and a protease heterogeneous catalyst into a disodium hydrogen phosphate-citric acid buffer solution, mixing, reacting, centrifuging, removing supernatant, extracting with ethanol by using glacial ethanol, centrifuging, taking precipitate, and freeze-drying to obtain the lysine oligopeptide.

5. The method for preparing lysine oligopeptide under enzyme catalysis according to claim 4, which is characterized by comprising the following steps of adding 2-6 g of L-lysine methyl ester hydrochloride and 10-20U of protease heterogeneous catalyst into 5.0-10.0 mL of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5 ~ 8.0.0, adding 30-45% by volume of tert-butyl alcohol into the buffer solution, fully mixing, placing the mixture in a constant temperature oscillator at the rotating speed of 300 ~ 600rpm and the temperature of 20-50 ℃ for reaction for 6-12 hours, centrifuging the reaction mixture at 8000 ~ 10000rpm for 2-4 minutes after the reaction is finished, adding 100mL of ice ethanol which is cooled to 0 ℃ in advance into supernate, centrifuging the mixture at 8000 ~ 10000rpm for 2-4 minutes, precipitating, and freeze-drying to obtain the lysine oligopeptide.

6. A preparation method of a protease heterogeneous catalyst is characterized by comprising the following steps: preparing protease into protein solution by using disodium hydrogen phosphate-citric acid buffer solution, adding ethylene glycol diglycidyl ether into the protein solution, stirring the mixture at room temperature for reaction, centrifugally collecting precipitates after the reaction is finished, and freeze-drying the precipitates to obtain the protease heterogeneous catalyst.

7. The preparation method of the heterogeneous catalyst according to claim 6 is characterized by comprising the steps of preparing a protein solution with 5% ~ 20% by mass of protease with a phosphate buffer solution, adding ethylene glycol diglycidyl ether into the protein solution, stirring and reacting for 4-8 hours at room temperature, centrifuging the reaction solution at 300 ~ 5000rpm for 5 ~ 10 minutes after the reaction is finished, collecting precipitates, and freeze-drying the precipitates to obtain the protease heterogeneous catalyst.

8. The method for preparing a heterogeneous catalyst according to claim 6, wherein: the protease is any one of bromelain, papain, trypsin, neutral protease and alkaline protease.

9. The method for preparing a heterogeneous catalyst according to claim 7, wherein the phosphate buffer is 0.1 ~ 0.2.2M disodium phosphate-citric acid buffer, and the pH of the phosphate buffer is 7.0 ~ 8.5.5.

Technical Field

The invention relates to a method for preparing lysine oligopeptide by enzyme catalysis and a method for modifying monomethoxy polyethylene glycol thereof, belonging to the technical field of enzyme catalysis reaction.

Background

The artificial polypeptide is a polyamino compound formed by taking natural amino acid as a monomer to carry out polymerization reaction through amido bond, and the properties of the artificial polypeptide are similar to those of natural peptide, polyamino acid and protein. The material formed by the artificial polypeptide has good biocompatibility and degradability. The polypeptide or the modified oligomer thereof shows unique structural properties such as self-assembly, liquid crystal behavior and the like, has stronger plasticity, and has great application potential in the fields of biology, medicine and natural high polymer materials.

The research on lysine polypeptide is relatively mature in foreign countries, especially in Japan, and is just started in China. It is a natural biological metabolite. Has good bactericidal capacity and thermal stability, and is a biological preservative with excellent preservative property and huge commercial potential. In Japan, lysine polypeptide has been approved as a preservative to be added to foods, and is widely used for preservation of instant rice, wet-boiled noodles, boiled dishes, seafood, sauces, soy sauce, fish fillet, and cookies. Meanwhile, the lysine polypeptide is matched with other natural bacteriostatic agents for use, so that the lysine polypeptide has an obvious synergistic effect and can improve the bacteriostatic ability.

Examples of the polymerization method of amino acids include a solid phase method, an NCA method and an enzyme catalysis method. Compared with other polymerization methods, the enzyme catalysis method has the advantages of mild reaction conditions, more environment-friendly property, less side reactions and the like, and avoids fussy protection and deprotection steps. The invention aims to adopt protease as a catalyst to catalyze and synthesize the lysine oligopeptide, and perform monomethoxy polyethylene glycol modification on the lysine oligopeptide to improve the hydrophilicity of the lysine oligopeptide.

Disclosure of Invention

The invention aims to overcome the defects and provide a method for preparing lysine oligopeptide and modifying the lysine oligopeptide by methoxy polyethylene glycol through enzyme catalysis, which has mild reaction conditions and is more environment-friendly.

The technical scheme of the invention is that the method for preparing lysine oligopeptide and single methoxyl polyethylene glycol modification thereof by enzyme catalysis specifically comprises (1) preparation of a protease heterogeneous catalyst; (2) synthesizing lysine oligopeptide; (3) modifying methoxy polyethylene glycol to prepare a lysine oligopeptide-monomethoxy polyethylene glycol copolymer.

(1) Preparation of protease heterogeneous catalyst: preparing protease into protein solution by using disodium hydrogen phosphate-citric acid buffer solution, adding ethylene glycol diglycidyl ether into the protein solution, stirring the mixture at room temperature for reaction, centrifugally collecting precipitates after the reaction is finished, and freeze-drying the precipitates to obtain the protease heterogeneous catalyst.

The method comprises the following specific steps of preparing a protein solution with the protease phosphate buffer solution in a mass percentage of 5 ~ 20%, adding ethylene glycol diglycidyl ether into the protein solution, stirring at room temperature, reacting for 4-8 hours, wherein the mass ratio of the protein to the ethylene glycol diglycidyl ether in the buffer solution is 1: 20-1: 50, centrifuging the reaction solution at 300 ~ 5000rpm for 5 ~ 10 minutes after the reaction is finished, collecting precipitates, and freeze-drying to obtain the protease heterogeneous catalyst.

The protease is any one of bromelain, papain, trypsin, neutral protease and alkaline protease.

The phosphate buffer is 0.1 ~ 0.2.2M disodium hydrogen phosphate-citric acid buffer, and the pH value of the phosphate buffer is 7.0 ~ 8.5.5.

(2) Synthesizing lysine oligopeptide by enzyme catalysis: adding L-lysine methyl ester hydrochloride, tert-butyl alcohol and a protease heterogeneous catalyst into a disodium hydrogen phosphate-citric acid buffer solution, mixing, reacting, centrifuging, removing supernatant, extracting with ethanol by using glacial ethanol, centrifuging, taking precipitate, and freeze-drying to obtain the lysine oligopeptide.

The method comprises the following specific steps of adding 2-6 g of L-lysine methyl ester hydrochloride and 10-20U of protease heterogeneous catalyst into 5.0-10.0 mL of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5 ~ 8.0.0, adding tert-butyl alcohol with the volume fraction of 30-45% of the buffer solution, fully mixing, placing in a constant temperature oscillator to react for 6-12 hours at 20-50 ℃ at the rotating speed of 300 ~ 600rpm, centrifuging the reaction mixture for 2-4 minutes at 8000 ~ 10000rpm after the reaction is finished, adding 100mL of ice ethanol which is pre-cooled to 0 ℃ into the supernatant, centrifuging for 2-4 minutes at 8000 ~ 10000rpm, precipitating, and freeze-drying to obtain the lysine oligopeptide.

The phosphate buffer is 0.1 ~ 0.2.2M disodium hydrogen phosphate-citric acid buffer, and the pH value of the phosphate buffer is 5.5 ~ 8.0.0.

The volume fraction of the tertiary butanol is 30-45% of phosphate buffer.

(3) The preparation of the monomethoxy polyethylene glycol modified lysine oligopeptide adopts the following method: adding lysine oligopeptide, monomethoxy polyethylene glycol succinimide carbonate and triethylamine into anhydrous methanol, fully mixing, and reacting under the protection of nitrogen; standing overnight, vacuum filtering to obtain filtrate, vacuum drying, soaking in anhydrous ethanol, centrifuging, removing supernatant, and adding acetone until all is dissolved; standing the obtained solution overnight, and carrying out vacuum filtration to obtain a precipitate, namely the product of the monomethoxy polyethylene glycol modified lysine oligopeptide.

The method comprises the following specific steps of adding 10-20 g of lysine oligopeptide, 12 ~ 40g of monomethoxy polyethylene glycol succinimide carbonate and 60 ~ 240g of triethylamine into 20mL of anhydrous methanol, reacting the reaction mixture at 30 ~ 50 ℃ for 10 ~ 24 hours under the protection of nitrogen, standing the reaction mixture at 0 ℃ overnight after the reaction is finished, carrying out vacuum filtration at 0-10 KPa, drying the filtrate at 30 ℃ for 0-10 KPa in vacuum for 2 ~ 6 hours, adding 50 ~ 100mL of anhydrous ethanol, soaking for 7 ~ 12 hours, centrifuging for 2-4 minutes at 8000 ~ 10000rpm, removing the supernatant, adding acetone preheated to 30 ~ 40 ℃ until all the acetone is dissolved, standing the obtained solution at 0 ℃ overnight, carrying out vacuum filtration at 0-10 KPa, and obtaining the precipitate as monomethoxy polyethylene glycol modified lysine oligopeptide.

The weight ratio of the lysine oligopeptide, the monomethoxy polyethylene glycol succinimide carbonate and the ethylamine is 1:1.2: 6-1: 2: 12.

The average molecular weight of the monomethoxy polyethylene glycol in the monomethoxy polyethylene glycol succinimide carbonate is 200Da, 400 Da and 1000 Da.

Detecting the monomethoxy polyethylene glycol modified lysine oligopeptide by using 1 HNMR: the 1HNMR spectra of the copolymers were determined at room temperature using an AVANCE III HD-400 MHz NMR spectrometer with CF3COOD as solvent and Tetramethylsilane (TMS) as internal standard.

The invention has the beneficial effects that: the catalyst in the polypeptide synthesis step related by the method is an enzyme heterogeneous catalyst, has the advantages of mild reaction conditions, more environment-friendly property, less side reaction and the like, avoids fussy protection and deprotection steps, and provides the method for preparing the monomethoxy polyethylene glycol modified lysine oligopeptide with good application prospect, the prepared lysine oligopeptide has mild conditions and no pollution, and the yield can reach as high as 35%.

Drawings

FIG. 1 is a HNMR detection chart of the product 1 obtained in example 1.

Detailed Description

In the following examples, the protease was obtained from Wakay biotech Inc. of Shanghai, the buffer was obtained from Nanchang rain and dew laboratory facilities Inc., and the other reagents were obtained from chemical reagents of the national pharmaceutical group, Inc. or Huateng pharmaceutical company, Hunan.