CN117143847B - Preparation process and application of polypeptide hydrogel supported biological enzyme - Google Patents
Preparation process and application of polypeptide hydrogel supported biological enzyme Download PDFInfo
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- CN117143847B CN117143847B CN202311105986.4A CN202311105986A CN117143847B CN 117143847 B CN117143847 B CN 117143847B CN 202311105986 A CN202311105986 A CN 202311105986A CN 117143847 B CN117143847 B CN 117143847B
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- lysozyme
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- modified polypeptide
- carrying
- polypeptide
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
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- Biomedical Technology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Immunology (AREA)
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- Inorganic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation process and application of a polypeptide hydrogel supported biological enzyme, and relates to the technical field of polypeptide hydrogels. The invention is used for solving the technical problems of carrying biological enzymes by utilizing the modified polypeptide polymer to control the release of the biological enzymes, achieving long-acting antibacterial and bacteriostatic properties and maintaining effective degradation. The technology comprises the steps of preparing a modified polypeptide polymer, loading lysozyme and preparing polypeptide hydrogel, wherein the lysozyme is well loaded by the loaded microsphere with good water absorption swelling performance, pH and ion concentration responsiveness, so that the activity of the lysozyme is kept, the long-acting release and sterilization of the lysozyme are promoted, the loaded microsphere is convenient to reuse, and the cost is reduced; the modified polypeptide polymer is added into a buffer solution and then is covalently crosslinked with lysozyme loaded microspheres, and the polypeptide hydrogel carrying biological enzymes is obtained after incubation, so that the long-acting antibacterial and bacteriostatic effects are exerted, the modified polypeptide polymer can be effectively degraded, and the biological safety is high.
Description
Technical Field
The invention belongs to the technical field of polypeptide hydrogels, and particularly relates to a preparation process and application of a polypeptide hydrogel supported biological enzyme.
Background
Hydrogels are a class of hydrophilic three-dimensional network structure gels that swell rapidly in water and in this swollen state can hold a large volume of water without dissolution. Due to the presence of the crosslinked network, hydrogels can swell and hold large amounts of water, the amount of water absorbed being closely related to the degree of crosslinking. The higher the degree of crosslinking, the lower the water absorption. The polypeptide hydrogel has good biocompatibility due to the addition of natural hydrophilic high molecular polypeptides (collagen, poly-L-lysine, poly-L-glutamic acid and the like), and can be applied to biological medicine materials and food additives.
The prior art one (CN 105669832B) discloses a polypeptide for preparing hydrogel and the hydrogel prepared by the polypeptide, which are catalyzed by the enzyme of organisms to form the hydrogel, so that the damage of external chemical agents, ultraviolet light and the like to tissues is avoided; and the gel can be degraded by biological enzyme, so that the gel is degraded by the endogenous substances of organisms, and the potential toxicity and immunogenicity risks caused by introducing exogenous chemical agents are avoided. The prior art two (CN 105693823B) discloses a polypeptide for preparing hydrogel, which is formed by using plasma ammonia oxidase to catalyze the gel, and is degraded by using matrix metalloproteinase-II biological enzyme; the prepared hydrogel takes polypeptide self-assembled fibers as a bracket, and has stable properties. The prepared stent can be effectively degraded after being implanted, so that the recovered tissue is further integrated into the body. Although the preparation of bioactive materials by combining polypeptide hydrogels with biological enzymes has been reported, how to carry the biological enzymes by using modified polypeptide polymers to control the release of the biological enzymes, so as to achieve long-acting antibacterial and bacteriostatic properties and maintain effective degradation, and further research is still needed.
Disclosure of Invention
The invention aims to provide a preparation process and application of a polypeptide hydrogel supported biological enzyme, which are used for solving the technical problems of how to carry the biological enzyme by utilizing a modified polypeptide polymer to control the release of the biological enzyme, achieving long-acting antibacterial and bacteriostatic properties and maintaining effective degradation in the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a preparation process of polypeptide hydrogel supported biological enzyme, which comprises the following steps:
s1, preparing a modified polypeptide polymer: the L-serine is subjected to benzyl alcohol side hydroxyl protection, triphosgene cyclization, ring opening polymerization initiation and palladium hydrocarbon reduction in sequence to obtain a modified polypeptide polymer;
s2, lysozyme loading: dissolving lysozyme in PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out centrifugal separation for 8-10 min at the rotating speed of 4600-5000 rpm after uniform ultrasonic oscillation, filtering to remove lower solid impurities, retaining an enzyme liquid phase, placing dried load microspheres in the enzyme liquid phase, measuring the enzyme concentration in the enzyme liquid phase every half an hour until the enzyme concentration of two adjacent times is the same and both are positioned at 2-3 mg/mL, stopping loading, and carrying out vacuum freezing at the temperature of-20 to-10 ℃ to remove internal moisture to obtain lysozyme load microspheres;
s3, preparing polypeptide hydrogel: adding the modified polypeptide polymer into PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out ultrasonic oscillation uniformly, heating in a water bath at 80-90 ℃ for 2 hours, naturally cooling to room temperature, adding lysozyme-loaded microspheres, and incubating at 37 ℃ for 15 days to obtain the polypeptide hydrogel carrying the biological enzyme.
As a further improved scheme of the invention, the preparation method of the modified polypeptide polymer comprises the following steps:
adding L-serine, toluene and p-toluenesulfonic acid into a three-neck flask provided with a mechanical stirrer and a water separator, dropwise adding benzyl alcohol through a constant-pressure dropping funnel, heating to 120-130 ℃ in an oil bath, mechanically stirring for 24-26 hours, naturally cooling to room temperature, freezing in an ice-ethanol bath at-10 ℃ for 2 hours, adding ethyl acetate for dilution, and dropwise adding 1mol/L sodium carbonate solution to adjust the pH to 9; layering by a separating funnel, taking an upper organic phase, drying by using anhydrous magnesium sulfate, filtering, adding the upper organic phase into 0.3mol/L oxalic acid methanol solution, uniformly oscillating, and freezing at-10 ℃ for 12 hours to separate out light yellow solid matters; heating and dissolving by using ethanol, recrystallizing for 24 hours at the temperature of minus 20 ℃, carrying out vacuum filtration under reduced pressure, and carrying out vacuum drying to obtain a white crystal crude product, and refining the crude product to obtain an intermediate 1;
the synthetic chemical reaction formula of the first step is as follows:
molecular weight, m/z, was measured on intermediate 1 using ESI-MS: 195.09 (100%), 196.09 (11.3%);
step two, adding the intermediate 1 into a three-neck flask equipped with a mechanical stirrer, adding tetrahydrofuran and triphosgene, stirring at room temperature for reaction for 2-3 hours, naturally standing for 12 hours, adding petroleum ether, precipitating white needle-like crystals at 5 ℃, carrying out vacuum filtration, and vacuum drying a filter cake until the weight is constant to obtain an intermediate 2;
the synthetic chemical reaction formula of the second step is as follows:
molecular weight, m/z, was measured on intermediate 2 using ESI-MS: 205.07 (100%), 206.08 (12.1%), 207.08 (1.3%);
adding isobutanol amine into a three-neck flask equipped with a mechanical stirrer, adding a mixed solution of N, N-dimethylformamide and chloroform under the protection of nitrogen, stirring for 5min, adding an intermediate 2, stirring at room temperature for 24 hours, adding frozen diethyl ether for precipitation, performing vacuum filtration, and performing vacuum drying on a filter cake to obtain a solid powdery intermediate 3;
the synthetic chemical reaction formula of the third step is as follows:
and step four, adding the intermediate 3 into a three-neck flask equipped with a mechanical stirrer and a constant-pressure dropping funnel, adding trifluoroacetic acid in a hydrogen environment, adding palladium carbon after the intermediate 3 is completely dissolved, continuously introducing hydrogen, stirring at room temperature for reaction for 70-72 hours, filtering by a thin-layer silica gel column, precipitating by diethyl ether, centrifuging to remove supernatant, drying in vacuum until the constant weight is achieved to obtain a viscous crude product, and carrying out post-treatment on the viscous crude product to obtain the modified polypeptide polymer.
The synthetic chemical reaction formula of the fourth step is as follows:
as a further improved scheme of the invention, the dosage ratio of the L-serine to toluene, p-toluenesulfonic acid, benzyl alcohol and ethyl acetate is 0.2mol: 300-350 mL:0.2mol: 180-200 mL: 180-200 mL; the dosage ratio of the intermediate 1 to tetrahydrofuran, triphosgene and petroleum ether is 0.02mol: 200-250 mL:0.02mol: 160-180 mL; the dosage ratio of the isobutolamine to the N, N-dimethylformamide, the chloroform, the intermediate 2 and the frozen diethyl ether is 0.12g:5mL:10mL:1.8g:30mL.
As a further improved scheme of the invention, the dosage ratio of the intermediate 3 to trifluoroacetic acid and palladium-carbon is 1g:20mL:0.5g; the specific method for post-treatment is as follows: adding deionized water into the viscous crude product, removing low-boiling impurities by rotary evaporation at 80 ℃, concentrating under reduced pressure at 120 ℃, and freeze-drying at-10 ℃ to obtain white powdery modified polypeptide polymer.
As a further improved scheme of the invention, the preparation method of the supported microsphere comprises the following steps: dissolving tween-80 in dodecane, stirring uniformly, and placing in a three-neck flask for later use; mixing hyperbranched polyglycidyl methacrylate, acrylamide, dimethyl diallyl ammonium chloride and deionized water, uniformly stirring, and adding sodium persulfate to obtain a mixed solution a; and (3) dropwise adding the mixed solution a into a three-neck flask through a constant-pressure dropping funnel, heating to 60 ℃ under a nitrogen atmosphere, carrying out heat preservation reaction for 2 hours, adding absolute ethyl alcohol, cooling and precipitating at-10 ℃ to obtain white crystals, carrying out vacuum filtration, eluting three times by using petroleum ether and ethanol successively, and drying at 60 ℃ to constant weight to obtain the load microsphere.
As a further improved scheme of the invention, the dosage ratio of the tween-80, the dodecane, the hyperbranched polyglycidyl methacrylate, the acrylamide, the dimethyldiallylammonium chloride, the deionized water, the sodium persulfate and the absolute ethyl alcohol is 0.08g:60g:0.2g:1.2g:2g:1.6g:0.01g:200mL.
As a further improved scheme of the invention, the addition amount of the lysozyme is 5-10U/L, and the addition amount of the load microsphere is 35-50 mg/g; the lysozyme is selected from one or more of cholinesterase-like lysozyme, glucosamine-like lysozyme, xylanase-like lysozyme and DNAzyme-like lysozyme.
Lysozyme is also called muramidase or N-acetylmuramidase hydrolase, and is an alkaline enzyme capable of hydrolyzing mucopolysaccharide in bacteria; the bacterial lysis is mainly achieved by breaking the beta-1, 4 glycosidic bond between the N-acetylmuramic acid and N-acetylglucosamine in the cell wall, which breaks down the insoluble mucopolysaccharide of the cell wall into soluble glycopeptides, resulting in the escape of the broken cell wall contents. Lysozyme can also bind directly to negatively charged viral proteins, forming complexes with DNA, RNA, apoproteins, inactivating the virus.
As a further improved scheme of the invention, the addition amount of the modified polypeptide polymer is 12-18 mM, and the addition amount of the lysozyme-loaded microsphere is 8-15U/L.
The invention also provides an application method of the polypeptide hydrogel loaded biological enzyme, and the polypeptide hydrogel loaded biological enzyme is applied to antibiosis and bacteriostasis.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
1. the preparation process of the polypeptide hydrogel comprises the steps of preparing a modified polypeptide polymer, loading lysozyme and preparing the polypeptide hydrogel, taking chiral amino acid L-serine as a raw material, carrying out ring opening polymerization and hydrogenation reduction to obtain the modified polypeptide polymer with terminal hydroxyl groups and a plurality of amino hydrophilic groups, improving hydrophilicity and water solubility, and displaying temperature-sensitive performance of hydration and dehydration along with temperature rise in a buffer solution to trigger the secondary structure of the polymer to change from random curling to folding; the lysozyme is well loaded by the loading microsphere with good water absorption swelling performance and pH and ion concentration responsiveness, so that the activity of the lysozyme is kept, the long-acting release sterilization of the lysozyme is promoted, the repeated use of the loading microsphere is facilitated, and the cost is reduced; the modified polypeptide polymer is added into a buffer solution and then is covalently crosslinked with lysozyme loaded microspheres, and the polypeptide hydrogel carrying biological enzymes is obtained after incubation, so that the long-acting antibacterial and bacteriostatic effects are exerted, the modified polypeptide polymer can be effectively degraded, and the biological safety is high.
2. According to the modified polypeptide polymer, benzyl alcohol is adopted to protect active side hydroxyl, then triphosoyl is used to synthesize an intermediate 2 with an oxazolidinedione structure, the intermediate 2 is subjected to ring-opening polymerization under the initiation of an isobutolamine as an initiator to synthesize an intermediate 3, and the intermediate 3 is subjected to homopolymerization under the condition of palladium hydrocarbon reduction to obtain a product; the synthesis method reasonably protects the lateral hydroxyl, and can obtain the polymer with the polypeptide chain segment in high yield.
3. The load microsphere is synthesized by a polymerization method by taking hyperbranched polyglycidyl methacrylate, acrylamide and dimethyl diallyl ammonium chloride as monomers, sodium persulfate as an initiator, tween-80 as an emulsifier and dodecane as a dispersing agent; the amphiphilicity of hyperbranched polyglycidyl methacrylate can reduce the surface tension of an oil-water interface, so that the monomer is uniformly and stably dispersed, the particle size consistency of the microspheres is higher, the crosslinked network structure of the hyperbranched polyglycidyl methacrylate has good load capacity on biological enzymes, and the slow release of the biological enzymes in the use process is promoted.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows an electron micrograph of a polypeptide hydrogel prepared in example 1 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a preparation process of polypeptide hydrogel supported biological enzyme, which comprises the following steps:
s1, preparing a modified polypeptide polymer: the L-serine is subjected to benzyl alcohol side hydroxyl protection, triphosgene cyclization, ring opening polymerization initiation and palladium hydrocarbon reduction in sequence to obtain a modified polypeptide polymer;
s2, lysozyme loading: dissolving lysozyme in PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out centrifugal separation for 8min at the rotation speed of 4800rpm after ultrasonic oscillation is uniform, filtering to remove lower solid impurities, retaining an enzyme liquid phase, placing dried load microspheres in the enzyme liquid phase, measuring the enzyme concentration in the enzyme liquid phase every half an hour until the enzyme concentration is the same for two adjacent times and the enzyme concentration is 2-3 mg/mL, stopping loading, and carrying out vacuum freezing at the temperature of-16 ℃ to remove internal moisture to obtain lysozyme load microspheres; the addition amount of lysozyme is 8U/L, and the addition amount of the load microsphere is 45mg/g; lysozyme is prepared from cholinesterase-like lysozyme, glucosamine-like lysozyme, xylanase-like lysozyme and DNAzyme-like lysozyme according to a mass ratio of 1:1:1:1, a mixture of two or more of the above-mentioned materials;
s3, preparing polypeptide hydrogel: adding the modified polypeptide polymer into PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out ultrasonic oscillation uniformly, heating in water bath at 85 ℃ for 2 hours, naturally cooling to room temperature, adding lysozyme-loaded microspheres, and incubating at 37 ℃ for 15 days to obtain the polypeptide hydrogel carrying the biological enzyme. The addition amount of the modified polypeptide polymer is 16mM, and the addition amount of the lysozyme-loaded microsphere is 12U/L.
The preparation method of the modified polypeptide polymer comprises the following steps:
adding 21.02g L-serine, 330mL of toluene and 34.44g of p-toluenesulfonic acid into a three-neck flask equipped with a mechanical stirrer and a water separator, dropwise adding 190mL of benzyl alcohol through a constant pressure dropping funnel, heating an oil bath to 125 ℃, mechanically stirring for 25 hours, naturally cooling to room temperature, freezing in an ice-ethanol bath at-10 ℃ for 2 hours, adding 190mL of ethyl acetate for dilution, and dropwise adding 1mol/L of sodium carbonate solution to adjust the pH to 9; layering by a separating funnel, taking an upper organic phase, drying by using anhydrous magnesium sulfate, filtering, adding the upper organic phase into 0.3mol/L oxalic acid methanol solution, uniformly oscillating, and freezing at-10 ℃ for 12 hours to separate out light yellow solid matters; heating and dissolving by using ethanol, recrystallizing for 24 hours at the temperature of minus 20 ℃, carrying out vacuum filtration under reduced pressure, and carrying out vacuum drying to obtain a white crystal crude product, and refining the crude product to obtain an intermediate 1;
the specific method for refining the crude product is as follows: adding ethyl acetate with the mass of 8 times of that of the crude product into the crude product, dropwise adding 1mol/L sodium carbonate solution to adjust the pH value to 9, layering by a separating funnel, taking an organic phase, drying anhydrous magnesium sulfate, carrying out vacuum suction filtration, removing the ethyl acetate by rotary evaporation, diluting methanol, adding 1mol/L sodium hydroxide aqueous solution, uniformly stirring, removing the methanol by rotary evaporation, separating and purifying the concentrated solution by using strong base type anion exchange resin, eluting 1mol/L acetic acid solution, concentrating eluent, separating out white crystals by using ice water bath at the temperature of 10 ℃, filtering, and carrying out vacuum drying to constant weight to obtain an intermediate 1;
adding 3.9g of the intermediate 1 into a three-neck flask with a mechanical stirrer, adding 230mL of tetrahydrofuran and 5.9g of triphosgene, stirring at room temperature for reaction for 2.5 hours, naturally standing for 12 hours, adding 170mL of petroleum ether, precipitating white needle-like crystals at 5 ℃, carrying out vacuum filtration, and vacuum drying a filter cake until the weight is constant to obtain an intermediate 2;
adding 0.12g of isobutanol amine into a three-neck flask equipped with a mechanical stirrer, adding a mixed solution of 5mLN, N-dimethylformamide and 10mL of chloroform under the protection of nitrogen, stirring for 5min, adding 1.8g of intermediate 2, stirring at room temperature for 24 h, adding 30mL of frozen diethyl ether for precipitation, performing vacuum filtration, and performing vacuum drying on a filter cake to obtain a solid powdery intermediate 3;
adding 1g of intermediate 3 into a three-neck flask equipped with a mechanical stirrer and a constant-pressure dropping funnel, adding 20mL of trifluoroacetic acid under a hydrogen environment, adding 0.5g of palladium carbon after the intermediate 3 is completely dissolved, continuously introducing hydrogen, stirring at room temperature for reaction for 71 hours, filtering by a thin-layer silica gel column, precipitating by diethyl ether, centrifugally separating to remove supernatant, vacuum drying to constant weight to obtain a viscous crude product, and performing post-treatment on the viscous crude product to obtain a modified polypeptide polymer; the specific method for post-treatment is as follows: adding deionized water into the viscous crude product, removing low-boiling impurities by rotary evaporation at 80 ℃, concentrating under reduced pressure at 120 ℃, and freeze-drying at-10 ℃ to obtain white powdery modified polypeptide polymer.
The preparation method of the loaded microsphere comprises the following steps: dissolving 0.08g of Tween-80 in 60g of dodecane, uniformly stirring, and placing in a three-neck flask for later use; mixing 0.2g of hyperbranched polyglycidyl methacrylate, 1.2g of acrylamide, 2g of dimethyl diallyl ammonium chloride and 1.6g of deionized water, uniformly stirring, and adding 0.01g of sodium persulfate to obtain a mixed solution a; and (3) dropwise adding the mixed solution a into a three-neck flask through a constant-pressure dropping funnel, heating to 60 ℃ under the nitrogen atmosphere, preserving heat and reacting for 2 hours, adding 200mL of absolute ethyl alcohol, cooling and precipitating at-10 ℃ to obtain white crystals, carrying out vacuum filtration, eluting three times by using petroleum ether and ethanol successively, and drying at 60 ℃ to constant weight to obtain the loaded microspheres.
Referring to fig. 1, it can be seen that the polypeptide hydrogel prepared in this embodiment has a porous structure connected with each other, which indicates that the cross-linking density is high, the structure is controllable, and the hydrogel is suitable for controlled release of biological enzymes.
Example 2
The embodiment provides a preparation process of polypeptide hydrogel supported biological enzyme, which comprises the following steps:
s1, preparing a modified polypeptide polymer: the L-serine is subjected to benzyl alcohol side hydroxyl protection, triphosgene cyclization, ring opening polymerization initiation and palladium hydrocarbon reduction in sequence to obtain a modified polypeptide polymer;
s2, lysozyme loading: dissolving lysozyme in PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out centrifugal separation for 10min at the rotation speed of 5000rpm after ultrasonic oscillation is uniform, filtering to remove lower solid impurities, retaining an enzyme liquid phase, placing dried load microspheres in the enzyme liquid phase, measuring the enzyme concentration in the enzyme liquid phase every half an hour until the enzyme concentration is the same for two adjacent times and the enzyme concentration is 2-3 mg/mL, stopping loading, and carrying out vacuum freezing at-18 ℃ to remove internal moisture to obtain lysozyme load microspheres; the adding amount of lysozyme is 9U/L, and the adding amount of the load microsphere is 40mg/g; lysozyme is prepared from cholinesterase-like lysozyme and glucosamine-like lysozyme according to a mass ratio of 1:1, mixing;
s3, preparing polypeptide hydrogel: adding the modified polypeptide polymer into PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out ultrasonic oscillation uniformly, heating in a water bath at the temperature of 90 ℃ for 2 hours, naturally cooling to the room temperature, adding lysozyme-loaded microspheres, and incubating at the temperature of 37 ℃ for 15 days to obtain the polypeptide hydrogel carrying the biological enzyme. The addition amount of the modified polypeptide polymer is 18mM, and the addition amount of the lysozyme-loaded microsphere is 15U/L.
The preparation method of the modified polypeptide polymer comprises the following steps:
adding 21.02g L-serine, 350mL of toluene and 34.44g of p-toluenesulfonic acid into a three-neck flask equipped with a mechanical stirrer and a water separator, dropwise adding 200mL of benzyl alcohol through a constant pressure dropping funnel, heating an oil bath to 130 ℃, mechanically stirring for 26 hours, naturally cooling to room temperature, freezing in an ice-ethanol bath at-10 ℃ for 2 hours, adding 200mL of ethyl acetate for dilution, and dropwise adding 1mol/L of sodium carbonate solution to adjust the pH to 9; layering by a separating funnel, taking an upper organic phase, drying by using anhydrous magnesium sulfate, filtering, adding the upper organic phase into 0.3mol/L oxalic acid methanol solution, uniformly oscillating, and freezing at-10 ℃ for 12 hours to separate out light yellow solid matters; heating and dissolving by using ethanol, recrystallizing for 24 hours at the temperature of minus 20 ℃, carrying out vacuum filtration under reduced pressure, and carrying out vacuum drying to obtain a white crystal crude product, and refining the crude product to obtain an intermediate 1;
the specific method for refining the crude product is as follows: adding ethyl acetate with the mass of 9 times of that of the crude product, dropwise adding 1mol/L sodium carbonate solution to adjust the pH value to 9, layering by a separating funnel, taking an organic phase, drying anhydrous magnesium sulfate, carrying out vacuum suction filtration, removing the ethyl acetate by rotary evaporation, diluting methanol, adding 1mol/L sodium hydroxide aqueous solution, uniformly stirring, removing the methanol by rotary evaporation, separating and purifying the concentrated solution by using strong base type anion exchange resin, eluting 1mol/L acetic acid solution, concentrating eluent, separating out white crystals by using ice water bath at the temperature of 10 ℃, filtering, and carrying out vacuum drying to constant weight to obtain an intermediate 1;
step two, adding 3.9g of the intermediate 1 into a three-neck flask with a mechanical stirrer, adding 250mL of tetrahydrofuran and 5.9g of triphosgene, stirring at room temperature for reaction for 3 hours, naturally standing for 12 hours, adding 180mL of petroleum ether, precipitating white needle-like crystals at 5 ℃, carrying out vacuum filtration, and carrying out vacuum drying on a filter cake until the weight is constant to obtain an intermediate 2;
adding 0.12g of isobutanol amine into a three-neck flask equipped with a mechanical stirrer, adding a mixed solution of 5mLN, N-dimethylformamide and 10mL of chloroform under the protection of nitrogen, stirring for 5min, adding 1.8g of intermediate 2, stirring at room temperature for 24 h, adding 30mL of frozen diethyl ether for precipitation, performing vacuum filtration, and performing vacuum drying on a filter cake to obtain a solid powdery intermediate 3;
adding 1g of intermediate 3 into a three-neck flask equipped with a mechanical stirrer and a constant-pressure dropping funnel, adding 20mL of trifluoroacetic acid under a hydrogen environment, adding 0.5g of palladium carbon after the intermediate 3 is completely dissolved, continuously introducing hydrogen, stirring at room temperature for reaction for 71 hours, filtering by a thin-layer silica gel column, precipitating by diethyl ether, centrifugally separating to remove supernatant, vacuum drying to constant weight to obtain a viscous crude product, and performing post-treatment on the viscous crude product to obtain a modified polypeptide polymer; the specific method for post-treatment is as follows: adding deionized water into the viscous crude product, removing low-boiling impurities by rotary evaporation at 80 ℃, concentrating under reduced pressure at 120 ℃, and freeze-drying at-10 ℃ to obtain white powdery modified polypeptide polymer.
The preparation method of the supported microspheres is the same as in example 1.
Example 3
The embodiment provides a preparation process of polypeptide hydrogel supported biological enzyme, which comprises the following steps:
s1, preparing a modified polypeptide polymer: the procedure is the same as in example 1;
s2, lysozyme loading: dissolving lysozyme in PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out centrifugal separation for 9min at the rotation speed of 5000rpm after ultrasonic oscillation is uniform, filtering to remove lower solid impurities, retaining an enzyme liquid phase, placing dried load microspheres in the enzyme liquid phase, measuring the enzyme concentration in the enzyme liquid phase every half an hour until the enzyme concentration is the same for two adjacent times and the enzyme concentration is 2-3 mg/mL, stopping loading, and carrying out vacuum freezing at-18 ℃ to remove internal moisture to obtain lysozyme load microspheres; the addition amount of lysozyme is 10U/L, and the addition amount of the load microsphere is 48mg/g; lysozyme is prepared from glucosamine enzyme-like lysozyme and DNase-like lysozyme according to a mass ratio of 1:1, mixing; the preparation method of the supported microspheres is the same as that of example 1;
s3, preparing polypeptide hydrogel: adding the modified polypeptide polymer into PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out ultrasonic oscillation uniformly, heating in a water bath at 86 ℃ for 2 hours, naturally cooling to room temperature, adding lysozyme-loaded microspheres, and incubating at 37 ℃ for 15 days to obtain the polypeptide hydrogel carrying the biological enzyme. The addition amount of the modified polypeptide polymer is 18mM, and the addition amount of the lysozyme-loaded microsphere is 12U/L.
Comparative example 1
In the preparation process of the polypeptide hydrogel supported biological enzyme of the comparative example, compared with the preparation process of the modified polypeptide polymer in example 1, the isobutanol amine is replaced by the ethanolamine in the step three.
Comparative example 2
In the preparation process of the polypeptide hydrogel supported biological enzyme of the comparative example, compared with the embodiment 1, the lysozyme loading step is omitted, and the lysozyme is directly added in the step S3.
Comparative example 3
In the preparation process of the polypeptide hydrogel supported biological enzyme of the present comparative example, compared with example 1, the polypeptide hydrogel preparation step adds the modified polypeptide polymer into Tris-acetate buffer.
Antibacterial property experiment
The sterilization performance of the biological enzyme-carrying polypeptide hydrogels prepared in examples 1-3 and comparative examples 1-3 was measured according to the national standard GB/T39101-2020 "method for measuring bacteriostasis circle of polypeptide antibacterial activity", the diameter of the bacteriostasis circle was measured by using a conventional method for measuring biological activity of a bactericide-the bacteriostasis circle method, and a blank group was set, and according to the bacteriostasis ratio= (diameter of bacteriostasis circle of experimental group-diameter of bacteriostasis circle of blank group)/diameter of bacteriostasis circle of treatment group multiplied by 100%, the bacteriostasis ratio of staphylococcus aureus, escherichia coli and candida albicans on days 1, 7 and 14 was measured, and the specific experimental results are shown in the following table:
from the experimental results of the above table, the observation analysis gave: 1) The antibacterial rates of the polypeptide hydrogel prepared by the embodiment of the invention on staphylococcus aureus, escherichia coli and candida albicans on the 1 st day are basically no difference, and the antibacterial rates on the 7 th day and the 14 th day are obviously higher than those of the comparative example, which shows that the polypeptide hydrogel of the embodiment can keep the sustained release of lysozyme due to the lysozyme loaded microsphere, so that the cell wall rupture content escapes to dissolve and kill bacteria, and has long-acting sterilizing performance; 2) In comparative example 1, the modified polypeptide polymer is prepared by replacing isobutanol amine with ethanolamine, so that the long-acting antibacterial performance on staphylococcus aureus, escherichia coli and candida albicans is reduced, and the possible reason is that the non-covalent bond effect deteriorates the water solubility, so that the temperature sensitivity is reduced, a good gel network cannot be formed, and the crosslinking effect with lysozyme is deteriorated; 3) In comparative example 2, as lysozyme is directly added in the step S3, no effective load is carried out on the lysozyme, so that the lysozyme is released in a short period of time and has no long-acting sterilization effect; 4) Comparative example 3 has reduced long-acting antibacterial performance against staphylococcus aureus, escherichia coli and candida albicans due to the change of the buffer type, probably due to the poor gel network formation effect, resulting in a slight increase in the release rate of lysozyme.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (4)
1. The preparation process of the polypeptide hydrogel supported biological enzyme is characterized by comprising the following steps of:
s1, preparing a modified polypeptide polymer: the L-serine is subjected to benzyl alcohol side hydroxyl protection, triphosgene cyclization, ring opening polymerization initiation and palladium hydrocarbon reduction in sequence to obtain a modified polypeptide polymer;
s2, lysozyme loading: dissolving lysozyme in PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out centrifugal separation for 8-10 min at the rotating speed of 4600-5000 rpm after uniform ultrasonic oscillation, filtering to remove lower solid impurities, retaining an enzyme liquid phase, placing dried load microspheres in the enzyme liquid phase, measuring the enzyme concentration in the enzyme liquid phase every half an hour until the enzyme concentration is the same for two adjacent times and the enzyme concentration is 2-3 mg/mL, and carrying out vacuum freezing at the temperature of-20 to-10 ℃ to remove internal moisture to obtain lysozyme load microspheres, wherein the adding amount of lysozyme is 5-10U/L, and the adding amount of load microspheres is 35-50 mg/g; the lysozyme is selected from one or more of cholinesterase-like lysozyme, glucosamine-like lysozyme, xylanase-like lysozyme and DNAzyme-like lysozyme;
the preparation method of the load microsphere comprises the following steps: dissolving tween-80 in dodecane, stirring uniformly, and placing in a three-neck flask for later use; mixing hyperbranched polyglycidyl methacrylate, acrylamide, dimethyl diallyl ammonium chloride and deionized water, uniformly stirring, and adding sodium persulfate to obtain a mixed solution a; dropwise adding the mixed solution a into a three-neck flask through a constant-pressure dropping funnel, heating to 60 ℃ under nitrogen atmosphere, carrying out heat preservation reaction for 2 hours, adding absolute ethyl alcohol, cooling and precipitating at minus 10 ℃ to obtain white crystals, carrying out vacuum filtration, eluting three times by using petroleum ether and ethanol successively, and drying at 60 ℃ to constant weight to obtain the load microsphere;
s3, preparing polypeptide hydrogel: adding the modified polypeptide polymer into PBS buffer solution with the concentration of 0.01mol/L, pH =7, carrying out ultrasonic oscillation uniformly, heating in a water bath at 80-90 ℃ for 2 hours, naturally cooling to room temperature, adding lysozyme-loaded microspheres, and incubating at 37 ℃ for 15 days to obtain the polypeptide hydrogel carrying biological enzymes, wherein the addition amount of the modified polypeptide polymer is 12-18 mM, and the addition amount of the lysozyme-loaded microspheres is 8-15U/L;
the preparation method of the modified polypeptide polymer comprises the following steps:
adding L-serine, toluene and p-toluenesulfonic acid into a three-neck flask provided with a mechanical stirrer and a water separator, dropwise adding benzyl alcohol through a constant-pressure dropping funnel, heating to 120-130 ℃ in an oil bath, mechanically stirring for 24-26 hours, naturally cooling to room temperature, freezing in an ice-ethanol bath at-10 ℃ for 2 hours, adding ethyl acetate for dilution, and dropwise adding 1mol/L sodium carbonate solution to adjust the pH to 9; layering by a separating funnel, taking an upper organic phase, drying by using anhydrous magnesium sulfate, filtering, adding the upper organic phase into 0.3mol/L oxalic acid methanol solution, uniformly oscillating, and freezing at-10 ℃ for 12 hours to separate out light yellow solid matters; dissolving with ethanol under heating, recrystallizing at-20deg.C for 24 hr, vacuum filtering, vacuum drying to obtain white crystal crude product, and refining to obtain intermediate 1, wherein the intermediate 1 has structural formula of；
Step two, adding the intermediate 1 into a three-neck flask with a mechanical stirrer, adding tetrahydrofuran and triphosgene, stirring at room temperature for reaction for 2-3 hours, naturally standing for 12 hours, adding petroleum ether, precipitating white needle-like crystals at 5 ℃, carrying out vacuum filtration, and vacuum drying a filter cake until the weight is constant to obtain an intermediate 2, wherein the structural formula of the intermediate 2 is as follows；
Adding isobutanol amine into a three-neck flask equipped with a mechanical stirrer, adding a mixed solution of N, N-dimethylformamide and chloroform under the protection of nitrogen, stirring for 5min, adding an intermediate 2, stirring at room temperature for 24 hours, adding frozen diethyl ether for precipitation, performing vacuum filtration, and vacuum drying a filter cakeObtaining a solid powdery intermediate 3, wherein the structural formula of the intermediate 3 is as follows；
Adding the intermediate 3 into a three-neck flask equipped with a mechanical stirrer and a constant-pressure dropping funnel, adding trifluoroacetic acid in a hydrogen environment, adding palladium carbon after the intermediate 3 is completely dissolved, continuously introducing hydrogen, stirring at room temperature for reaction for 70-72 hours, filtering by a thin-layer silica gel column, precipitating by diethyl ether, centrifuging to remove supernatant, vacuum drying to constant weight to obtain a viscous crude product, and performing post-treatment on the viscous crude product to obtain a modified polypeptide polymer, wherein the structural formula of the modified polypeptide polymer is as follows。
2. The process for preparing the polypeptide hydrogel supported biological enzyme according to claim 1, wherein the dosage ratio of the L-serine to toluene, p-toluenesulfonic acid, benzyl alcohol and ethyl acetate is 0.2mol: 300-350 mL:0.2mol: 180-200 mL: 180-200 mL; the dosage ratio of the intermediate 1 to tetrahydrofuran, triphosgene and petroleum ether is 0.02mol: 200-250 mL:0.02mol: 160-180 mL; the dosage ratio of the isobutolamine to the N, N-dimethylformamide, the chloroform, the intermediate 2 and the frozen diethyl ether is 0.12g:5mL:10mL:1.8g:30mL.
3. The preparation process of the polypeptide hydrogel supported biological enzyme according to claim 1, wherein the dosage ratio of the intermediate 3 to trifluoroacetic acid to palladium-carbon is 1g:20mL:0.5g; the specific method for post-treatment is as follows: adding deionized water into the viscous crude product, removing low-boiling impurities by rotary evaporation at 80 ℃, concentrating under reduced pressure at 120 ℃, and freeze-drying at-10 ℃ to obtain white powdery modified polypeptide polymer.
4. The process for preparing the polypeptide hydrogel supported biological enzyme according to claim 1, wherein the dosage ratio of tween-80, dodecane, hyperbranched polyglycidyl methacrylate, acrylamide, dimethyldiallylammonium chloride, deionized water, sodium persulfate and absolute ethanol is 0.08g:60g:0.2g:1.2g:2g:1.6g:0.01g:200mL.
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