A method for constructing protein vesicle aggregates for improving enzyme utilization

Abstract

A method for constructing a protein vesicle aggregate that improves enzyme utilization, including: by introducing dextran into the protein vesicle building block, the glycosylation functionalization of the protein vesicle is completed; mesoporous silica nanoparticles are prepared by a classical sol-gel method, and amino and phenylboronic acid groups are modified sequentially by a silane coupling agent and 4-formylphenylboronic acid to obtain phenylboronic mesoporous silica nanoparticles; based on the bioorthogonal reaction of phenylboronic acid and polysaccharide, the mesoporous silica nanoparticles grafted with phenylboronic acid act as a linker to form protein vesicle aggregates; by this structure, the protein vesicle loaded with enzyme is prevented from passing through a small-aperture capillary, so that it remains in a gravity flow empty column and performs multiple enzyme catalytic reactions. The enzyme catalytic reaction of the present invention is efficient and specific, flexible and controllable, and the enzyme is loaded in a protein film-coated manner. Under the premise of greatly ensuring the reversible catalytic activity of the enzyme, the utilization rate of the enzyme is increased by up to 3 times.

Description

Technical Field

[0001] The invention belongs to the technical field of cell model application, and particularly relates to a method for constructing protein vesicle aggregates for improving enzyme utilization. Background Art

[0002] In recent years, based on the research methods of synthetic biology, the bionic assembly construction of the structure and function of organisms has received widespread attention. From the spontaneous assembly of building blocks to the generation of microcompartments, and then to the formation of higher-level hierarchical structures, its gradual development process is similar to the formation process of early life on Earth. The research process of bionic assembly not only helps to reveal the mysteries of organisms from the perspective of chemical self-assembly, deepen human understanding of nature and life, and further transform and utilize them in order to build a bridge between materials science and life science, but also provides a theoretical basis for the design of bionic materials. Cells, as the smallest structural and functional units of organisms on Earth, have become the research basis of bionic assemblies. So far, various types of functional microcompartment structures that can simulate the basic characteristics of living cells have been developed, but there are relatively few studies on this basis to show the characteristics of bionic materials.

[0003] Collective behavior is a unique social interaction behavior and a phenomenon that exists widely in nature and social situations. Its basic characteristic is self-organization, which is manifested as a large number of unorganized individuals acting together under the promotion and influence of the same event, and this leaderless self-organizing behavior has the ability to integrate physical and biological systems. This intelligent feature exists in both the microscopic and macroscopic worlds, such as the aggregation of microbial quorum sensing cells to form biological tissues with advanced structures, and the resistance of fish schools to natural enemies. Compared with scattered individuals, this new property generated by group aggregation shows great advantages. Therefore, based on a single microcompartment bionic cell, by constructing a multi-compartment aggregate model, the new properties of bionic cells can be further developed, and new ideas can be provided for the research in the field of bionic cells. Summary of the invention

[0004] The present invention provides ideas for the field of bionic cells as bionic materials, aims to achieve the simulation of life, and provides a method for constructing protein vesicle aggregates based on the bioorthogonal reaction of phenylboronic acid and polysaccharides. On the basis of maintaining the reversible catalytic activity of natural enzymes, the enzyme utilization rate is increased by up to 3 times.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A method for constructing protein vesicle aggregates for improving enzyme utilization, the method comprising:

[0007] Step 1: Glycosylation functionalization of protein vesicles

[0008] (1) Preparation of dextran-polymer motif: Aminated dextran and polyisopropylacrylamide were mixed in a mass ratio of 1:1, incubated overnight in a phosphate buffer solution with a pH of 6 to 8, dialyzed and freeze-dried for use;

[0009] (2) preparing a functional protein-polymer motif: mixing an amino functional protein with polyisopropylacrylamide at a mass ratio of 1:1, incubating overnight in a phosphate buffer solution at a pH of 6 to 8, and lyophilizing after dialysis for use; the functional protein is one of bovine serum albumin, amylase, glucose oxidase or catalase;

[0010] (3) Preparation of glycosylated protein vesicles: The above two moieties were prepared into 5-10 mg / mL aqueous solutions, mixed, added to the isooctyl alcohol oil phase, shaken vigorously, and then transferred to the aqueous phase for later use;

[0011] Step 2: Synthesis of mesoporous silica nanoparticles by phenylboration

[0012] (1) Synthesis of mesoporous silica nanoparticles: Hexadecyltrimethylammonium bromide was fully dissolved in ultrapure water, and then ethyl orthosilicate was added and stirred for hydrolysis under alkaline conditions of 60 to 90°C to obtain a white powder; the mass ratio of hexadecyltrimethylammonium bromide to ethyl orthosilicate was 0.4:100;

[0013] (2) Modification of amino groups of mesoporous silica nanoparticles: mesoporous silica nanoparticles were suspended in toluene, aminopropyltriethoxysilane was added, refluxed overnight under inert conditions, washed and dried in vacuum;

[0014] (3) Mesoporous silica nanoparticles modified with phenylboronic acid: Amination-modified mesoporous silica nanoparticles and 4-formylphenylboronic acid were ultrasonically dispersed in anhydrous toluene at a concentration of 0.01 g mL -1 , add a few drops of glacial acetic acid, and reflux overnight at 50-80°C;

[0015] Step 3: Preparation of protein vesicle aggregates

[0016] The glycosyl functionalized protein vesicles obtained in step 1 and the phenylborated mesoporous silica nanoparticles obtained in step 2 are mixed in a phosphate buffer solution with a pH of 6 to 8, and the mixture is gently shaken at room temperature for 10 to 30 minutes, and the upper solution is removed to obtain protein vesicle aggregates.

[0017] Furthermore, an appropriate amount of enzyme is added during the process of preparing the vesicles in step 1 (3), and the method further comprises step 4: transferring the preparation process of the protein vesicle aggregates in step 3 to a gravity flow column (bottom pore size of 50 to 300 μm), forming protein vesicle aggregates at the bottom, adding a substrate to cause an enzymatic reaction, releasing the reactants from the bottom, re-adding the substrate to continue the reaction, and repeating the operation.

[0018] Furthermore, in step 1 (3), the preparation of glycosylated functionalized protein vesicles is specifically as follows: using a pipette to draw the building block solution into a centrifuge tube (the ratio of different blocks is the same, and the ratio can also be matched as required), then adding phosphate buffer, controlling the pH to 6-8, mixing evenly, adding isooctyl alcohol oil phase and shaking vigorously for 30-120s to obtain emulsion vesicles. In addition, if there is a requirement to transfer the aqueous phase, 0.5-2.5mg (final concentration is 0.02-0.1mg / μL) of cross-linking agent is added before preparation. After the cross-linking reaction is allowed to proceed overnight, the solution is transferred to the aqueous phase by gradient dialysis using ethanol of different concentrations.

[0019] Furthermore, in step 1 (3), the ratio of the two building blocks is 1:1-5, and the ratio of water to oil is 1:20-40.

[0020] Furthermore, in step 2 (1), the alkalinity is regulated by adding 3-5 mL of 2M sodium hydroxide into 250 mL of the system.

[0021] Furthermore, in step 2 (2), the ratio of the mesoporous silica nanoparticles, toluene and aminopropyltriethoxysilane is 1 g: 50-200 mL: 1-3 mL.

[0022] Furthermore, in step 2 (3), the mass ratio of the amino-modified mesoporous silica nanoparticles to 4-formylphenylboronic acid is 2:1-5.

[0023] Furthermore, in step three, the concentration of the protein vesicles in the solution is 0.1-2.0 mg / mL.

[0024] Furthermore, in step three, the concentration of the phenylborated mesoporous silica nanoparticles in the solution is 2-7 mg / mL.

[0025] Furthermore, in step 4, the assembly time of the protein vesicle aggregates in the gravity flow column is 10 to 30 minutes, and the enzymatic reaction time is 5 to 30 minutes.

[0026] Compared with the prior art, the protein vesicle aggregates of the present invention have the following beneficial effects:

[0027] (1) The unique advantage of the protein vesicle-based system is that it uses various functional proteins as building blocks, and its flexible membrane can display various functions through simple design. Since there are millions of proteins with clear primary sequences and various three-dimensional structures and functions in nature, their use as building blocks provides a broad space for subsequent applications with different functions;

[0028] (2) The protein vesicles have a flexible membrane structure on the outside and a hollow structure on the inside, which can provide a stable guarantee for the substances loaded inside;

[0029] (3) A method for constructing protein vesicle aggregates based on the bioorthogonal reaction of phenylboronic acid and polysaccharides. This non-covalent bond method brings a reversible effect to the formation and disaggregation of aggregates;

[0030] (4) This overall non-covalent bond construction method ensures the activity of the enzyme loaded inside, and this protein vesicle aggregate model has the new function of improving enzyme utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the H NMR spectrum of dextran carboxylation;

[0032] Figure 2 It is a microscope photo of glycosylated protein vesicles and a size statistical chart;

[0033] Figure 3 This is a scanning electron micrograph of glycosylated protein vesicles in a dry state;

[0034] Figure 4 This is a test diagram of the flexibility of glycosylated protein vesicles in water phase;

[0035] Figure 5 Scanning electron micrograph of phenylborated mesoporous silica nanoparticles.

[0036] Figure 6 Confocal microscopy photograph of protein vesicle aggregates.

[0037] Figure 7 Schematic diagram of multiple enzyme-catalyzed reactions;

[0038] Figure 8 This is a test chart for improving enzyme utilization in protein vesicle aggregates. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention that does not depart from the spirit and scope of the technical solution of the present invention should be included in the protection scope of the present invention.

[0040] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention can also be used to realize the present invention.

[0041] The present invention completes the glycosylation functionalization of protein vesicles by introducing dextran (70kDa) into the protein vesicle building block; mesoporous silica nanoparticles are prepared by the classic sol-gel method, and amino and phenylboronic acid groups are modified sequentially by silane coupling agent and 4-formylphenylboronic acid to obtain phenylboronic mesoporous silica nanoparticles; based on the bioorthogonal reaction of phenylboronic acid and polysaccharide, the mesoporous silica nanoparticles grafted with phenylboronic acid act as a linker to form protein vesicle aggregates; the protein vesicles loaded with enzymes are hindered from passing through small-aperture capillaries by this "hand-in-hand" structure, so that they remain in the gravity flow empty column and perform multiple enzyme catalytic reactions. The construction method of the present invention is green and pollution-free, the enzyme catalytic reaction is efficient and specific, flexible and controllable, and the enzyme is loaded in a protein film-coated manner, and the utilization rate of the enzyme is increased by up to 3 times under the premise of greatly ensuring the reversible catalytic activity of the enzyme.

[0042] The method of the present invention is applicable to the formation of protein vesicles and protein vesicle aggregates by a variety of different proteins, such as amylase, glucose oxidase, catalase, etc.; and when different proteins are introduced, different effects will be introduced, such as the introduction of catalase, the increase of the same enzyme on the membrane, so that the substrate conversion rate is increased by 5-10%; or the introduction of glucose oxidase, through the enzyme cascade reaction, increases the substrate selection space (glucose) while the final product remains unchanged. In short, the introduction of different functional proteins will increase the function of the protein vesicle aggregate and bring more advantages.

[0043] Embodiment 1:

[0044] 1. Glycosylation functionalization of protein vesicles

[0045] (1) Preparation of dextran-polymer moiety: Dextran is carboxylated and amino-modified. Figure 1 The carboxylation of dextran was confirmed by H NMR spectrum, which proved the success of the carboxyl modification process. Then, 10 mg of amino dextran was dissolved in 10 mL of ultrapure water and slowly added to the polyisopropylacrylamide solution (10 mL, 1 mg mL -1), react at room temperature overnight, dialyze the solution in ultrapure water (dialysis bag molecular weight cutoff is 10000), filter the dialysate and freeze-dry to obtain a white solid, which is stored at -20°C.

[0046] (2) Preparation of bovine serum albumin-polymer motif: 10 mg of aminated bovine serum albumin was dissolved in 10 mL of ultrapure water and slowly added dropwise to a polyisopropylacrylamide solution (10 mL, 1 mg / mL). -1 ) was reacted at room temperature overnight, the solution was dialyzed in ultrapure water (dialysis bag molecular weight cutoff 35000), the dialyzate was filtered and freeze-dried to obtain a white solid, which was stored at -20°C.

[0047] (3) Preparation of glycosylated protein vesicles: The building block solution was prepared at 10 mg / mL -1 , use a pipette to draw 20 μL of the building block solution into a 1.5 mL centrifuge tube, then add 8 μL of phosphate buffer (pH 8.0, 50 mM), mix well, add 400 μL of isooctyl alcohol and shake vigorously for 30 seconds to obtain emulsion vesicles ( Figure 2 ), and the membrane structure after drying can be observed by scanning electron microscopy. In addition, if there is a requirement to transfer the aqueous phase, 0.5 mg of cross-linking agent (NHS-PEG 16 -NHS), and after the cross-linking reaction overnight, the mixture was transferred to the aqueous phase by gradient dialysis with 75%, 50%, and 25% ethanol, respectively. Figure 4 The throughput of the micropipette can be observed under an optical microscope, indicating the flexibility of the membrane.

[0048] 2. Synthesis of Benzene-borated Mesoporous Silica Nanoparticles

[0049] (1) Synthesis of mesoporous silica nanoparticles: First, weigh hexadecyltrimethylammonium bromide (1.0 g) and fully dissolve it in 240 mL of ultrapure water, then add 3.5 mL of sodium hydroxide solution (NaOH, 2 M), heat the system to 80°C and add 5 mL of ethyl orthosilicate, keep the constant temperature at 80°C and continue to stir for 2 h, remove the solution by vacuum filtration to obtain a white solid, then use vacuum filtration to wash with a large amount of ultrapure water and methanol, vacuum dry overnight to obtain a white powder. In order to remove the CTAB template, weigh 1.0 g of the product obtained in the previous step and suspend it in 100 mL of methanol, ultrasonicate it thoroughly, add 6 mL of concentrated HCl under heating conditions and reflux for 6 h, filter and wash with a large amount of water and methanol under reduced pressure, vacuum dry overnight to obtain a white powder.

[0050] (2) Modification of amino groups in mesoporous silica nanoparticles: This method mainly utilizes the hydrolysis process of hydroxyl groups on the surface of nanoparticles by amino-containing silane coupling agents. It is necessary to ensure an anhydrous and oxygen-free environment by suspending 1.0 g of mesoporous silica nanoparticles in 100 mL of anhydrous toluene, adding 2.4 mL of aminopropyltriethoxysilane, heating and reflux under inert gas protection for 12 h, filtering and washing under reduced pressure with a large amount of water and methanol, and vacuum drying overnight.

[0051] (3) Mesoporous silica nanoparticles modified with phenylboronic acid: This method utilizes the aldehyde group on 4-formylphenylboronic acid to react with the amino group modified with aminated mesoporous silica nanoparticles to modify the phenylboronic acid groups on the surface of the nanoparticles and in the mesoporous channels. First, 100 mg of aminated mesoporous silica nanoparticles and 100 mg of 4-formylphenylboronic acid were thoroughly dispersed in 100 mL of anhydrous toluene using an ultrasonic cleaner, and a few drops (about 20 to 50 μL) of glacial acetic acid were added. The suspension was heated to 70°C and refluxed overnight. It was then filtered and washed under reduced pressure with a large amount of water and methanol, and vacuum dried overnight to obtain a white powder. The final product can be observed by scanning electron microscopy ( Figure 5 ).

[0052] 3. Preparation of protein vesicle aggregates

[0053] After transferring the aqueous phase, the glycosylated protein vesicles were concentrated to a concentration of 0.4 mg mL -1 50 μL of protein vesicles were taken, and then 40 μL of phosphate buffer solution (pH = 7.4, 100 mM) and 10 μL of phenylboronic acid mesoporous silica nanoparticles (5 mg mL -1 ), neutral solution environment to ensure supramolecular interaction between phenylboronic acid and sugar, and slight shaking for 30 min to form protein vesicle aggregates ( Figure 6 ).

[0054] 4. Construction of enzyme utilization model and effect testing

[0055] The preparation process of protein vesicles (with 5-10 μg of enzyme loaded inside) was transferred to a gravity flow column (bottom pore size of 200 μm), as Figure 7 As shown, protein vesicle aggregates are formed at the bottom, substrate is added to cause enzymatic reaction, the reactant is released from the bottom, substrate is added again to continue the reaction, and the operation is repeated. The reaction is carried out for 5 to 30 minutes, and 5 cycles are experienced. Figure 8 It can be seen that the utilization rate of the enzyme has increased by 2 to 3 times. For example, when the reaction time is 5 minutes, after 5 cycles, the conversion rate of the substrate increased from 29% to 97%, and the utilization rate of the enzyme increased by about 3 times.

[0056] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0057] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for constructing protein vesicle aggregates for improving enzyme utilization, characterized in that: The method is: Step 1: Glycosylation functionalization of protein vesicles (1) Preparation of dextran-polymer motif: amino dextran and polyisopropylacrylamide were mixed in a mass ratio of 1:1, incubated overnight in a phosphate buffer solution at pH 6-8, dialyzed and freeze-dried for use; (2) Preparing a functional protein-polymer motif: mixing the amino functional protein with polyisopropylacrylamide at a mass ratio of 1:1, incubating overnight in a phosphate buffer solution at a pH of 6 to 8, and lyophilizing after dialyzing for use; the functional protein is one of bovine serum albumin, amylase, glucose oxidase or catalase; (3) Preparation of glycosylated protein vesicles: The above two moieties were prepared into 5-10 mg / mL aqueous solution, mixed, added to the isooctyl alcohol oil phase, shaken vigorously, and then transferred to the aqueous phase for later use; Step 2: Synthesis of mesoporous silica nanoparticles by phenylboration (1) Synthesis of mesoporous silica nanoparticles: Hexadecyltrimethylammonium bromide was fully dissolved in ultrapure water, and then ethyl orthosilicate was added and stirred for hydrolysis under alkaline conditions of 60-90°C to obtain a white powder; the mass ratio of hexadecyltrimethylammonium bromide to ethyl orthosilicate was 0.4:100; (2) Modification of mesoporous silica nanoparticles with amino groups: Mesoporous silica nanoparticles were suspended in toluene, aminopropyltriethoxysilane was added, and the mixture was refluxed overnight under inert conditions. The mixture was washed and dried in vacuum. (3) Mesoporous silica nanoparticles modified with phenylboronic acid: Amination of mesoporous silica nanoparticles and 4-formylphenylboronic acid were ultrasonically dispersed in anhydrous toluene at a concentration of 0.01 g mL -1 , add 20-50 μL of glacial acetic acid, and reflux overnight at 50-80°C; Step 3: Preparation of protein vesicle aggregates The glycosyl functionalized protein vesicles obtained in step 1 and the phenylborated mesoporous silica nanoparticles obtained in step 2 are mixed in a phosphate buffer solution with a pH of 6 to 8, and the mixture is gently shaken at room temperature for 10 to 30 minutes, and the upper solution is removed to obtain protein vesicle aggregates.

2. The method for constructing a protein vesicle aggregate for improving enzyme utilization according to claim 1, characterized in that: During the process of preparing vesicles in step 1 (3), 5-10 μg of enzyme is added. The method further comprises step 4: transferring the process of preparing protein vesicle aggregates in step 3 to a gravity flow column, forming protein vesicle aggregates at the bottom, adding substrate to cause enzymatic reaction, releasing the reactants from the bottom, re-adding substrate to continue the reaction, and repeating the operation.

3. A method for constructing a protein vesicle aggregate for improving enzyme utilization according to claim 1 or 2, characterized in that: In step 1 (3), the preparation of glycosylated functionalized protein vesicles is as follows: pipette the building block solution into a centrifuge tube, then add phosphate buffer, control the pH to 6-8, mix well, add isooctyl alcohol oil phase and shake vigorously for 30-120 s to obtain emulsion vesicles. In addition, if there is a requirement to transfer the aqueous phase, add 0.5-2.5 mg of cross-linking agent before preparation, and the final concentration of the cross-linking agent is 0.02-0.1 mg / μL. After the cross-linking reaction is allowed to proceed overnight, the solution is transferred to the aqueous phase by gradient dialysis using 75%, 50%, and 25% ethanol.

4. The method for constructing a protein vesicle aggregate for improving enzyme utilization according to claim 3, characterized in that: In step 1 (3), the ratio of the two building blocks is 1:1-5, and the ratio of water to oil is 1:20-40.

5. The method for constructing a protein vesicle aggregate for improving enzyme utilization according to claim 1, characterized in that: In step 2 (1), the alkaline condition used is to add 3-5 mL of 2 M sodium hydroxide into 250 mL of the system.

6. The method for constructing a protein vesicle aggregate for improving enzyme utilization according to claim 1, characterized in that: In step 2 (2), the ratio of the mesoporous silica nanoparticles, toluene and aminopropyltriethoxysilane is 1 g: 50-200 mL: 1-3 mL.

7. The method for constructing a protein vesicle aggregate for improving enzyme utilization according to claim 1, characterized in that: In step 2 (3), the mass ratio of the amino-modified mesoporous silica nanoparticles to 4-formylphenylboronic acid is 2:1-5.

8. The method for constructing a protein vesicle aggregate for improving enzyme utilization according to claim 1, characterized in that: In step 3, the concentration of the protein vesicles in the solution is 0.1-2.0 mg / mL.

9. The method for constructing a protein vesicle aggregate for improving enzyme utilization according to claim 1, characterized in that: In step 3, the concentration of the phenylborated mesoporous silica nanoparticles in the solution is 2-7 mg / mL.

10. The method for constructing a protein vesicle aggregate for improving enzyme utilization according to claim 2, characterized in that: In step 4, the assembly time of the protein vesicle aggregates in the gravity flow column is 10 to 30 min, and the enzymatic reaction time is 5 to 30 min.

Images