Sustainable enzyme-loaded hydrogel based on heterodimer alpha-helix induced self-assembly

Abstract

The invention discloses a sustainable enzyme-loaded hydrogel based on heterodimer alpha-helix induced self-assembly, and belongs to the technical field of immobilized enzyme and hydrogel. The method comprises the following steps: artificially synthesizing alpha-helical collagen hydrogel E3VBK3, modifying the N end of a mutant 6B of bacillus subtilis lipase A (BSLA) by using an E3 adhesion module to obtain a modified enzyme E36B, and immobilizing the enzyme in the hydrogel based on the salt bridge interaction between E3 in the E36B and K3 in the E3VBK3. The hydrogel-enzyme compound prepared by the method disclosed by the invention has relatively high immobilization rate, enzyme activity retention rate, repeatability and stability.

Description

Technical Field

[0001] The present invention relates to a sustainable enzyme-loaded hydrogel based on hetero-dimer α-helix induced self-assembly, belonging to the technical fields of immobilized enzymes and hydrogels. Background Art

[0002] In recent years, the field of biocatalysis has developed rapidly and is gradually becoming a major tool in the industrial field. Lipase (EC 3.1.1.3), also known as triacylglycerol hydrolase, is used in various biochemical reactions such as transesterification, esterolysis, and esterification. Due to its excellent catalytic synthesis ability and catalytic hydrolysis ability, it is widely used in the food industry, energy industry, and pharmaceutical industry. However, in an environment of high temperature or organic solvents, the activity of lipase usually drops sharply, and the utilization rate of free lipase is low and it is difficult to separate, which all limit the industrial application of lipase.

[0003] To overcome these difficulties, various methods have been adopted in domestic and foreign research, such as directed evolution, rational design, covalent modification of enzymes, immobilization of enzymes, etc. Among them, the enzyme immobilization technology has significant advantages in industrial applications compared with other methods. Immobilized enzymes can solve problems such as low reusability, poor stability, and difficult operation of enzymes by immobilizing enzymes on solid carriers or in polymer matrices. As an insoluble solid catalyst, immobilized enzymes can be easily recovered by methods such as filtration or centrifugation, thereby promoting their reuse and product separation and reducing production costs.

[0004] Currently, the methods for enzyme immobilization are mainly divided into two categories, physical methods and chemical methods. Physical methods include adsorption methods and embedding methods, and chemical methods usually include covalent methods, encapsulation methods, and cross-linking methods. Traditional inorganic carriers such as activated carbon and magnetic materials have the disadvantages of poor biocompatibility and complex immobilization processes. As a material for enzyme immobilization, hydrogels have advantages such as non-toxicity, good biocompatibility, and biodegradability compared with other immobilization materials, which makes hydrogel carriers have great potential in medical applications.

[0005] Patent CN116656664A discloses the steps of carboxylating polycyclodextrin hydrogel microspheres and using the carboxylated microspheres to immobilize lipase by physical adsorption to obtain an amphiphilic lipase / polycyclodextrin hydrogel microsphere catalytic material; Patent CN119193569A discloses an in-situ polymerization method to form a three-dimensional network structure to immobilize carbonyl reductase or lipase inside the hydrogel, improving the pore volume, specific surface area, average pore size, and pore width of the immobilized enzyme, thereby increasing the enzyme loading amount; however, the above methods cannot solve the problem of reduced enzyme activity of lipase in an organic solvent environment.

[0006] Therefore, developing an immobilization method that can improve the stability of lipase in an organic solvent environment while maintaining a high loading rate and enzyme activity has extremely high practical and economic value. Summary of the Invention

[0007] To solve the above problems, the present invention designs a new way of immobilizing enzymes. Based on the ionic bond interaction between E3 / K3, the enzyme is immobilized on a collagen-based hydrogel, and a mild, sustainable and biocompatible new enzyme immobilization technology is developed. The present invention artificially synthesizes an α-helical collagen hydrogel E3VBK3, uses the E3 adhesion module to modify the N-terminus of the mutant 6B of Bacillus subtilis lipase A (BSLA) to obtain the modified enzyme E36B, and realizes the immobilization of the enzyme in the hydrogel based on the salt bridge interaction between E3 in E36B and K3 in E3VBK3.

[0008] The first object of the present invention is to provide a method for loading lipase based on a collagen self-assembled hydrogel. An adhesion module is connected to the N-terminus of the lipase, and through the salt bridge interaction between the adhesion module and collagen, a collagen hydrogel loaded with lipase is obtained;

[0009] Among them, the amino acid sequence of collagen is shown in SEQ ID NO.1;

[0010] The amino acid sequence of the adhesion module (E3) is shown in SEQ ID NO.9.

[0011] In one embodiment, the collagen has an α-helical structure and biological activity.

[0012] In one embodiment, the amino acid sequence of the lipase (6B) is shown in SEQ ID NO.3.

[0013] In one embodiment, the amino acid sequence of the lipase (E36B) connected with the adhesion module is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0014] In one embodiment, the preparation method of the collagen hydrogel loaded with lipase is: mixing the hydrogel and the lipase solution, shaking, centrifuging, and standing to obtain a collagen hydrogel loaded with lipase;

[0015] Optionally, in the hydrogel loaded with lipase, the enzyme loading amount is 5 - 50 mg / g hydrogel;

[0016] Optionally, the shaking is at 4 - 35 °C, 100 - 300 rpm for 4 - 20 h;

[0017] Optionally, the standing is at 4 °C for 0 - 20 h;

[0018] Optionally, the buffer is a phosphate buffer with a pH of 5.0 to 10.0;

[0019] Optionally, the dosage ratio of the hydrogel to the lipase solution is 3 - 5 mg: 100 - 200 μL.

[0020] In one embodiment, the concentration of the lipase solution is 0.3 mg / L to 3 mg / L.

[0021] Preferably, the enzyme loading is 5 - 10 mg / g hydrogel;

[0022] Preferably, the shaking is at 4 - 20 °C and 200 rpm for 4 - 12 h;

[0023] Preferably, the standing is at 4 °C for 4 - 20 h; more preferably, the standing is at 4 °C for 4 - 12 h;

[0024] Preferably, the buffer is a phosphate buffer with a pH of 5.0 to 9.0.

[0025] The second object of the present invention is to provide a collagen hydrogel loaded with lipase prepared by any of the above - mentioned methods.

[0026] The third object of the present invention is to provide a product, which contains the above - mentioned collagen hydrogel loaded with lipase; the product includes food catalysts, medical products, decontamination products, cosmetic additives, and biodegradable materials.

[0027] In one embodiment, the medical products include drugs, drug carriers, wound dressings, diagnostic reagents, etc.

[0028] In one embodiment, the decontamination products include detergents.

[0029] The fourth object of the present invention is to provide the application of any of the above - mentioned methods or the above - mentioned collagen hydrogel loaded with lipase in the fields of food, medicine, and biocatalysis.

[0030] In one embodiment, the application in the food field includes synthesizing flavor substances using the collagen hydrogel loaded with lipase;

[0031] The application in the medical field includes preparing drug controlled - release systems and wound dressings using the collagen hydrogel loaded with lipase;

[0032] The application in the biocatalysis field includes preparing detergents using the collagen hydrogel loaded with lipase.

[0033] The fifth object of the present invention is to provide a method for simultaneously improving the enzyme activity of lipase in organic solvents, the recycling rate of the enzyme, and the storage stability, by using a collagen hydrogel to load lipase; an adhesion module is connected to the N-terminus of the lipase, and through the salt bridge interaction between the adhesion module and collagen, a collagen hydrogel loaded with lipase is obtained;

[0034] Among them, the amino acid sequence of collagen is shown in SEQ ID NO.1;

[0035] The amino acid sequence of the adhesion module (E3) is shown in SEQ ID NO.9.

[0036] In one embodiment, the preparation method of the collagen hydrogel loaded with lipase is: mixing the hydrogel and the lipase solution, shaking, centrifuging, and standing still to obtain a collagen hydrogel loaded with lipase;

[0037] Optionally, in the hydrogel loaded with lipase, the enzyme loading amount is 5-50 mg / g of the hydrogel;

[0038] Optionally, the shaking is carried out at 4-35°C and 100-300 rpm for 4-20 h;

[0039] Optionally, the standing still is carried out at 4°C for 0-20 h;

[0040] Optionally, the buffer solution is a phosphate buffer solution with a pH of 5.0-10.0;

[0041] Optionally, the dosage ratio of the hydrogel to the lipase solution is 3-5 mg: 100-200 μL.

[0042] Preferably, the enzyme loading amount is 5-10 mg / g of the hydrogel;

[0043] Preferably, the shaking is carried out at 4-20°C and 200 rpm for 4-12 h;

[0044] Preferably, the standing still is carried out at 4°C for 4-20 h; more preferably, the standing still is carried out at 4°C for 4-12 h;

[0045] Preferably, the buffer solution is a phosphate buffer solution with a pH of 5.0-9.0.

[0046] The sixth object of the present invention is to provide a biocatalyst, which contains the above-mentioned collagen hydrogel loaded with lipase.

[0047] Advantages of the present invention

[0048] (1) The lipase-loaded collagen hydrogel of the present invention has strong designability and controllable structure; the artificially synthesized α-helical collagen hydrogel E3VBK3 is a self-assembled hydrogel based on the ionic bond force between E3 / K3. It can be constructed by genetic engineering of protein motifs, thereby changing its macroscopic material properties to meet the application requirements of different scenarios;

[0049] From the perspective of application, its viscoelasticity can be adjusted. For example, by changing its protein motif, its elasticity can be weakened, viscosity increased, and mechanical strength reduced, bringing potential advantages for drug delivery or applications that require enhanced material flexibility. By adjusting the sequence or cross-linking degree of collagen, the pore size and degradation rate of the hydrogel can be precisely controlled, thereby realizing the controlled release of drugs; while the existing hydrogels may have lower regulation accuracy in degradation behavior or drug diffusion rate.

[0050] (2) The lipase-loaded collagen hydrogel of the present invention has good mechanical properties on the basis of excellent biocompatibility; immobilized enzymes based on natural hydrogels usually face the defect of low mechanical strength. By using them in combination with other materials (such as carbon nanotubes, graphene, silicon, and zeolites), improving their mechanical stability may reduce their biocompatibility and biodegradability.

[0051] At the same time, the assembly methods usually require chemical cross-linking (photo-cross-linking, chemical reagents) or physical cross-linking (freezing-thawing), involving catalysts, solvents, cross-linking agents (such as glutaraldehyde, isocyanate, and carbodiimide). The synthesis steps are complex and low-concentration residues may still cause cytotoxicity, calcification, or foreign body reactions. Compared with other synthetic self-assembled hydrogels, the lipase-loaded collagen hydrogel of the present invention has a decisive advantage in biocompatibility due to its biological origin. E3VBK3 self-assembles through salt bridge forces, and its high cross-linking degree makes its mechanical strength and viscoelasticity match its broad application prospects in the medical field.

[0052] (3) The lipase-loaded collagen hydrogel of the present invention shows excellent adaptability in a wide range of application scenarios such as tissue engineering, drug delivery, cosmetics, and wound healing compared with the existing hydrogels.

[0053] Specifically,

[0054] (1) The immobilization efficiency of the lipase-loaded collagen hydrogel prepared by the present invention for lipase reaches 61.5%, the enzyme activity is 29.5 U / mg, and the enzyme activity retention rate is 59.8%;

[0055] (2) After incubating the lipase-loaded collagen hydrogel prepared by the present invention in 90% (v / v) DMSO, acetonitrile, and ethanol for 36 h, the remaining enzyme activities are 63.2%, 52.7%, and 57.4% respectively;

[0056] (3) After 5 cycles, the residual enzyme activity of the lipase-loaded collagen hydrogel prepared by the present invention reaches 60%;

[0057] (4) The lipase-loaded collagen hydrogel prepared by the present invention still retains 89.87% of the residual enzyme activity after being stored at 25 °C for 30 days. Brief Description of the Drawings

[0058] Figure 1 is the immobilization principle of E36B enzyme; wherein, a is the structural schematic diagram of E3VBK3; b is the cross-linking principle diagram of E3VBK3-E36B;

[0059] Figure 2 is the influence of different immobilization conditions on the immobilization efficiency, enzyme activity and activity recovery rate; wherein, a is the enzyme loading amount, b is the mixing time, c is the standing time, d is the pH value, and e is the immobilization temperature;

[0060] Figure 3 is the change of enzyme activity of E36B and E3VBK3-E36B in the presence of 90% (v / v) organic solvent at 25 °C; wherein, a is DMSO, b is acetonitrile, c is ethanol, and d is isopropanol;

[0061] Figure 4 is the stability of E36B and E3VBK3-E36B in 90% (v / v) organic solvent at 25 °C; wherein, a is DMSO, b is acetonitrile, c is ethanol, and d is isopropanol;

[0062] Figure 5 is the detection result of the reusability and storage stability of immobilized E3VBK3-E36B (using ρNPC hydrolysis as the substrate at 37 °C and pH 8.0); wherein, a is the reusability of five cycles, and the activity of the first cycle is set to 100%; b is the image of ρNPC hydrolysis after the first cycle; c is the image of ρNPC hydrolysis after the fifth cycle; d is the storage stability at 4 °C; e is the storage stability at 25 °C;

[0063] Figure 6 is the detection result of the mechanical properties of the hydrogel. Detailed Embodiments

[0064] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0065] Raw materials used in the examples:

[0066] ρ-Nitrophenyl octanoate (ρNPC) was purchased from Sigma-Aldrich (USA);

[0067] Absolute ethanol, absolute methanol (water content ≤ 0.05%), acetonitrile and Triton X-100 were purchased from Sinopharm Chemical Reagent Co., Ltd. (China);

[0068] The coding genes of 6B and E36B mutants were synthesized by GENEWIZ (Suzhou, China) and expressed in Escherichia coli BL21(DE3); the amino acid sequence of 6B is shown in SEQ ID NO.3, and the amino acid sequence of E36B is shown in SEQ ID NO.5.

[0069] Testing methods:

[0070] 1. Enzyme activity assay

[0071] Using ρ-nitrophenyl octanoate (ρNPC) as the substrate, the hydrolysis activities of free enzyme and immobilized enzyme were measured at 37°C.

[0072] In 10 mM phosphate buffer at 37°C and pH 8.0, when 2.5 mM p-NPC was hydrolyzed, the enzyme activity was evaluated by measuring the increase in absorbance at 420 nm caused by the release of nitrophenol. The enzyme activity assay was carried out in a 1 mL system containing a free enzyme solution or an immobilized enzyme suspension. To obtain more accurate spectrophotometer readings, the amount of enzyme was adjusted appropriately. The enzyme activity was defined as the amount of enzyme required to release 1 μmol of nitrophenol per minute.

[0073] 2. Determination of immobilization parameters

[0074] Immobilization efficiency (%) = (mass of immobilized enzyme) / (total enzyme mass) × 100%;

[0075] Enzyme activity recovery rate (%) = (activity of immobilized enzyme) / (activity of free enzyme, i.e., the enzyme activity of lipase without immobilization) × 100%;

[0076] Residual activity (%) = (current enzyme activity) / (initial enzyme activity) × 100%;

[0077] Enzyme loading (mg / g) = (total enzyme mass) / (mass of carrier).

[0078] 3. Organic solvent stability

[0079] The free lipase and immobilized lipase were incubated in 90% (v / v) DMSO, methanol, acetonitrile, ethanol and isopropanol at 25°C for different times. Using ρNPC as the substrate, their residual activity and specific activity were measured at 37°C and pH 8 to evaluate the stability of free enzyme and immobilized enzyme in organic solvents.

[0080] 4. Reusability and storage stability

[0081] The reusability of the immobilized lipase was evaluated at 37 °C using ρNPC as the substrate.

[0082] After each round of reaction, the immobilized enzyme was recovered by centrifugation, washed at least three times with 10 mM phosphate buffer (pH 7.4), and stored at 4 °C for use in the next round. The enzyme activity of E3VBK3-E36B in the first round was defined as 100% relative activity. The residual enzyme activity after each round was calculated.

[0083] The immobilized enzyme and free enzyme were stored at 4 °C and 25 °C respectively, and measured every 5 days until the end of 30 days. The storage stability was expressed as the residual activity.

[0084] 5. Protein expression and purification methods

[0085] The recombinant plasmid was transformed into the strain E. coli BL21(DE3) to construct a mutant strain. The fermentation and purification process was as follows: 20 μL of the mutant strain expressing E36 (or 6BK3) was taken into 3 mL of LB medium, and kana was added dropwise to a final concentration of 100 μg / mL. It was cultured in a constant temperature incubator at 37 °C and 220 rpm for 12 h. It was inoculated into 100 mL of TB medium at an inoculation ratio of 1% v / v and cultured in a constant temperature incubator at 37 °C for about 4 h until OD was 0.6 - 0.8. IPTG was added to a final concentration of 1 mmol / L, and induced fermentation was carried out at 25 °C and 220 rpm for 20 h.

[0086] Purification was carried out using a Ni 2+ affinity chromatography column. All target proteins were inserted with 6 histidine tags at the N-terminus and purified using an AKTA instrument. All solutions used for purification were ultrafiltered through a 0.22 μm aqueous membrane. Solution A (8×PBS: disodium hydrogen phosphate dodecahydrate 28.57 g·L -1 , sodium dihydrogen phosphate dihydrate 12.53 g·L -1 , sodium chloride 233.8 g·L -1 , dissolved in deionized water, adjusted to pH = 7.4 with sodium hydroxide, and made up to 1 L; 2 M imidazole: imidazole 136.16 g, dissolved in deionized water, adjusted to pH = 7.4 with hydrochloric acid, and made up to 1 L; protein loading buffer (Solution A): 125 mL of 8×PBS (pH = 7.4), 10 mL of 2 M imidazole (pH = 7.4), made up to 1 L with deionized water, and ultrafiltered through a 0.22 μm filter membrane; protein elution buffer (Solution B): 125 mL of 8×PBS (pH = 7.4), 250 mL of 2 M imidazole (pH = 7.4), made up to 1 L with deionized water, and ultrafiltered through a 0.22 μm filter membrane) was used to resuspend and mix the above-mentioned fermented bacterial cells, and an ultrasonic crusher was used for crushing (ultrasonic for 3 s, stop for 2 s), and crushed at 4 °C for 30 min until the resuspended solution became clear.

[0087] The above clarification and resuspension solution was centrifuged at 4 °C and 10,000 rpm for 40 min, and the supernatant was collected. It was ultrafiltered through a 0.45 μm aqueous membrane and purified using an AKTA instrument. During the protein elution process, the supernatant of K36B was first gradient eluted with a 75 mM imidazole eluent to remove impurities, and then linearly eluted with an imidazole eluent from 75 mM to 500 mM (linear rising rate 0.5%·ml -1 ), and the protein sample at the target peak was collected at 150 mM - 250 mM.)

[0088] Example 1: Preparation of a sustainable enzyme-loaded hydrogel based on heterodimer α-helix-induced self-assembly

[0089] 1. Preparation of hydrogel E3VBK3

[0090] According to the method in the literature "Design Strategies to Tune the Structural and MechanicalProperties ofSynthetic Collagen Hydrogels; DOI: 10.1021 / acs.biomac.1c00520", the hydrogel E3VBK3 was prepared as follows:

[0091] The nucleotide of E3VBK3 (as shown in SEQ ID NO.2) was inserted between StyI and BanHI in the pCold-III plasmid, and expressed, induced, and purified in E. coli BL21(DE3) to obtain the protein E3VBK3.

[0092] The collected E3VBK3 protein (amino acid sequence as shown in SEQ ID NO.1) was dialyzed through a regenerated cellulose dialysis membrane with a molecular weight of 3.5 kDa and desalted in a 10 mM PB solution for 48 h, and then freeze-dried; the freeze-dried E3VBK3 was dissolved in a 10 mM PB solution to 3 mg / mL, incubated at 4 °C and 200 rpm for 20 h, centrifuged at 4 °C and 6,000 rpm for 10 min, and the bottom hydrogel was collected; the supernatant was separated, and the bottom hydrogel was allowed to stand at 4 °C for 20 h to promote the self-assembly of the E3VBK3 hydrogel.

[0093] 2. Preparation of lipase enzyme 6B and E36B

[0094] The nucleotides of 6B (amino acid sequence shown in SEQ ID NO.3, nucleotide sequence shown in SEQ ID NO.4) and E36B (amino acid sequence shown in SEQ ID NO.5, nucleotide sequence shown in SEQ ID NO.6) were respectively inserted between the T7-promoter and T7-terminator in the pET-28a plasmid, and expressed, induced, and purified using E. coli BL21(DE3) to prepare lipases 6B and E36B.

[0095] 3. Preparation of sustainable enzyme-loaded hydrogels based on heterodimer α-helix-induced self-assembly

[0096] Take the hydrogels E3VBK3, lipases 6B and E36B prepared in 1 and 2, and carry out immobilization. The steps are as follows:

[0097] Mix 3 mg of hydrogel E3VBK3 with 100 μL of purified E36B (enzyme activity 50.25 U / mg, concentration 0.3 mg / mL) and 6B (enzyme activity 70 U / mg, concentration 0.3 mg / mL) respectively to obtain a mixture (enzyme loading amount 10 mg / g). The mixture is shaken and mixed at 4 °C and 200 rpm in 10 mM phosphate buffer (pH 7.4) for 20 h; centrifuge at 4 °C and 6000 rpm for 10 min, and collect the hydrogel-enzyme complex and the supernatant, named E3VBK3-6B and E3VBK3-E36B respectively.

[0098] The hydrogel-enzyme complex is washed with 10 mM phosphate buffered saline at least 3 times, and the washing solution is collected; the concentration of unbound enzyme in the washing solution and the supernatant is measured to calculate the amount of immobilized enzyme (i.e., immobilization rate %).

[0099] 4. Detection of immobilization performance

[0100] Take the hydrogel-enzyme complexes E3VBK3-6B and E3VBK3-E36B prepared in 3, and detect their immobilization performance. The results are shown in Table 1.

[0101] Table 1 Comparison of immobilization parameters of E3VBK3-6B and E3VBK3-E36B

[0102] Immobilized enzyme Immobilization rate (%) Enzyme activity (U / mg) Enzyme activity retention rate (%) E3VBK3-6B 23.6±4.5 37.4±1.3 53.5±1.9 E3VBK3-E36B 52.5±4.1 24.5±0.8 49.9±1.6

[0103] The results showed that the immobilization efficiency of E3VBK3-E36B was 52.5%, more than twice that of E3VBK3-6B (23.6%). This significant improvement can be attributed to the salt-bridge-driven specific interaction between the E3 adhesion module at the N-terminus of E36B and the K3 sticky module of E3VBK3, which enhanced the selectivity and efficiency of the immobilization process, verifying the feasibility of the immobilization strategy designed in Example 1.

[0104] From the data in Table 1, it was also found that the enzyme activity of E3VBK3-E36B was 24.5 U / mg, which decreased by approximately 34.76% compared to 37.4 U / mg of E3VBK3-6B. To explore whether this decrease was due to differences in the immobilization method or a reduction in the specific activity of E36B compared to 6B caused by the modification of the E3 adhesion module, the enzyme activities of the free enzymes E36B and 6B were further measured.

[0105] The results showed that the enzyme activity of the free enzyme 6B was 70 U / mg, while that of E36B was 50.25 U / mg. The enzyme activity of E36B decreased by approximately 28.5% compared to 6B, and this trend was consistent with the change in the enzyme activity of the immobilized enzyme. It can be seen that the introduction of the E3 adhesion module did not significantly interfere with the overall structure of 6B, but may have reduced the enzyme activity of E36B by changing its site conformation (increasing the enzyme specificity). Even so, the enzyme activity retention rates of E3VBK3-6B and E3VBK3-E36B were very close, being 53.5% and 49.9% respectively.

[0106] In summary, the above results indicate that the E3VBK3-E36B enzyme loading strategy has high specificity, has a small impact on enzyme activity, and has an ideal immobilization rate.

[0107] Example 2: Optimization of the immobilization conditions of E3VBK3-E36B

[0108] Based on Example 1, the enzyme loading amount, mixing time, static incubation time, immobilization pH, and immobilization temperature were changed as follows:

[0109] (1) Enzyme loading amount

[0110] 3 mg of hydrogel E3VBK3 was respectively mixed with 100 μL of purified E36B to obtain mixtures (with enzyme loading amounts of 5 - 50 mg / g respectively). The mixtures were shaken and mixed at 4°C and 200 rpm in 10 mM phosphate buffer (pH 7.4) for 20 h.

[0111] The mixture was centrifuged at 6000 rpm for 10 min (4 °C) to collect the hydrogel-enzyme complex; the hydrogel-enzyme complex was allowed to stand at 4 °C for 20 h to promote the self-assembly and adsorption equilibrium of the hydrogel; and the complex was washed at least three times with 10 mM phosphate buffer (pH 7.4).

[0112] The immobilization efficiency, enzyme activity, and enzyme activity recovery rate of the hydrogel-enzyme complex with different enzyme loadings were detected. The results are as Figure 2 shown in a of []. The results indicate that when the enzyme loading exceeds 10 mg / g, the immobilization efficiency significantly decreases, that is, 10 mg / g is the optimal enzyme loading.

[0113] (2) Mixing time

[0114] 3 mg of hydrogel E3VBK3 was mixed with 100 μL of purified E36B respectively to obtain a mixture (to make the enzyme loading 10 mg / g). The mixture was shaken and mixed at 4 °C and 200 rpm for 4 - 20 h in 10 mM phosphate buffer (pH 7.4);

[0115] The mixture was centrifuged at 6000 rpm for 10 min (4 °C) to collect the hydrogel-enzyme complex; the hydrogel-enzyme complex was allowed to stand at 4 °C for 20 h to promote the self-assembly and adsorption equilibrium of the hydrogel; and the complex was washed at least three times with 10 mM phosphate buffer (pH 7.4).

[0116] The immobilization efficiency of the hydrogel-enzyme complex with different mixing times was detected. The results are as Figure 2 shown in b of []. The results indicate that as the mixing time prolongs, the immobilization efficiency increases, but after 12 h, the enzyme activity significantly decreases, that is, 12 h is the optimal mixing time.

[0117] (3) Standing time

[0118] 3 mg of hydrogel E3VBK3 was mixed with 100 μL of purified E36B respectively to obtain a mixture (to make the enzyme loading 10 mg / g). The mixture was shaken and mixed at 4 °C and 200 rpm for 12 h in 10 mM phosphate buffer (pH 7.4);

[0119] The mixture was centrifuged at 6000 rpm for 10 min (4 °C) to collect the hydrogel-enzyme complex; the hydrogel-enzyme complex was allowed to stand at 4 °C for 0 - 20 h to promote the self-assembly and adsorption equilibrium of the hydrogel; and the complex was washed at least three times with 10 mM phosphate buffer (pH 7.4).

[0120] The immobilization efficiency of the hydrogel-enzyme complex with different standing times was detected. The results are as Figure 2As shown in c, the results indicate that the static time significantly improves the immobilization efficiency. As the static time prolongs, the improvement of the immobilization efficiency gradually slows down. That is, 12 h is the optimal static time.

[0121] (4) pH

[0122] Mix 3 mg of hydrogel E3VBK3 with 100 μL of purified E36B respectively to obtain a mixture (to make the enzyme loading amount 10 mg / g). The mixture is shaken and mixed at 200 rpm for 12 h at 4 °C in 10 mM phosphate buffer (pH 5.0 - 10.0).

[0123] Centrifuge the mixture at 6000 rpm for 10 min (4 °C) to collect the hydrogel - enzyme complex. The hydrogel - enzyme complex is left standing at 4 °C for 12 h to promote the self - assembly of the hydrogel and the adsorption equilibrium. Then wash the complex with 10 mM phosphate buffer (pH 7.4) at least three times.

[0124] Detect the immobilization efficiency of the hydrogel - enzyme complex at different buffer pH values. The results are as Figure 2 shown in d. The activity and immobilization efficiency of the immobilized enzyme reach the highest at pH 9. That is, pH 9 is the optimal buffer pH.

[0125] (5) Immobilization temperature

[0126] Mix 3 mg of hydrogel E3VBK3 with 100 μL of purified E36B respectively to obtain a mixture (to make the enzyme loading amount 10 mg / g). The mixture is shaken and mixed at 200 rpm for 12 h at 4 - 35 °C in 10 mM phosphate buffer (pH 9.0).

[0127] Centrifuge the mixture at 6000 rpm for 10 min (4 °C) to collect the hydrogel - enzyme complex. The hydrogel - enzyme complex is left standing at 4 °C for 12 h to promote the self - assembly of the hydrogel and the adsorption equilibrium. Then wash the complex with 10 mM phosphate buffer (pH 7.4) at least three times.

[0128] Detect the immobilization efficiency of the hydrogel - enzyme complex at different buffer pH values. The results are as Figure 2 shown in e. When the temperature exceeds 20 °C, the immobilization efficiency significantly decreases. The immobilization efficiency and enzyme activity are the highest at 15 °C. That is, 15 °C is the optimal temperature.

[0129] In summary, the optimal conditions for preparing the hydrogel - enzyme complex are:

[0130] Mix 3 mg of hydrogel E3VBK3 with 100 μL of purified E36B separately to obtain a mixture (with an enzyme loading of 10 mg / g). The mixture is shaken at 200 rpm in 10 mM phosphate buffer (pH 9.0) at 15 °C for 12 h.

[0131] Centrifuge the mixture at 6000 rpm for 10 min (4 °C) to collect the hydrogel-enzyme complex. The hydrogel-enzyme complex is allowed to stand at 4 °C for 12 h to promote the self-assembly of the hydrogel and adsorption equilibrium. Then, wash the complex with 10 mM phosphate buffer (pH 7.4) at least three times.

[0132] The immobilization efficiency of the prepared hydrogel-enzyme complex reaches 61.5%, the enzyme activity is 29.5 U / mg, and the enzyme activity retention rate is 59.8%.

[0133] Example 3: Performance Detection

[0134] Take the hydrogel-enzyme complex prepared in Example 2 under the optimal conditions and detect its performance.

[0135] (1) Stability in high-concentration organic solvents

[0136] Evaluate the stability in high-concentration organic solvents by detecting the changes in enzyme activity and residual activity of the immobilized enzyme (i.e., hydrogel-enzyme complex) and free enzyme over time in 90% (v / v) organic solvents (DMSO, acetonitrile, ethanol, isopropanol).

[0137] The results are as Figure 3 、 Figure 4 shown. The results show that the enzyme activities of free enzyme E36B in 90% (v / v) DMSO, acetonitrile, ethanol, and isopropanol are 50.68 U / mg, 20.33 U / mg, 71.36 U / mg, and 85.75 U / mg, respectively, which are significantly lower than its enzyme activity in 10 mM PB (50.25 U / mg). The enzyme activities of the immobilized enzyme E3VBK3-E36B are consistent in all four solvent systems, approximately 31 U / mg, slightly higher than its enzyme activity in 10 mM PB (29.5 U / mg). After incubation for 36 h, the residual activities of E36B in 90% (v / v) DMSO, acetonitrile, and ethanol are 63.2%, 52.7%, and 57.4%, respectively, all lower than the residual activity of E3VBK3-E36B. Only in 90% (v / v) isopropanol, the residual enzyme activities of E36B and E3VBK3-E36B are similar, 62.69% and 59.86%, respectively.

[0138] It can be seen that the immobilization treatment slows down the rate of enzyme activity decline in these solvent systems. Especially in highly polar solvents, compared with free enzymes, the immobilized enzymes show obvious improvement.

[0139] (2) Reusability

[0140] The reusability of the hydrogel-enzyme complex (i.e., the immobilized enzyme) was detected and evaluated by detecting the residual enzyme activity after multiple hydrolysis reactions.

[0141] The results are as Figure 5 shown. The results show that after 5 cycles, the residual enzyme activity in the immobilized enzyme reaches 60%. Compared with free enzymes, the utilization rate of the enzyme is significantly improved (free enzymes can only be used once); at the same time, E3VBK3-E36B can be easily recovered by centrifugation, simplifying the enzyme recovery process, and its structural integrity is maintained after five cycles.

[0142] (3) Storage stability

[0143] The immobilized enzyme and the free enzyme were stored at 4 °C and 25 °C respectively, and the enzyme activity was measured every 5 days.

[0144] The results are as Figure 5 shown. The results show that at 4 °C, both enzymes maintained high residual activities; while at 25 °C, the residual enzyme activity of E36B decreased to 74.06% after 30 days, which was due to protein aggregation and slow denaturation. In contrast, E3VBK3-E36B showed excellent storage stability and still retained 89.87% of the residual enzyme activity after 30 days.

[0145] (4) Mechanical property characterization

[0146] The storage modulus (G’) and loss modulus (G”) of the E3VBK3 hydrogel at a concentration of 2% (w·v-1, i.e., 20 mg·mL-1) were measured by micro-rheological experiments. The results are as Figure 6 shown. The results show that when the frequency is below 7 rad·s-1, G’ is greater than G”, showing gel-like characteristics; when the frequency is higher than 7 rad·s-1, G’ is less than G”, turning into a viscous liquid state. The crossover point between the gel state and the viscous liquid state is 7 rad·s-1, generally showing the characteristics of a hard gel and showing good mechanical properties.

[0147] Comparative Example 1: Hydrogel-immobilized enzyme

[0148] Compared with the hydrogel-immobilized enzyme disclosed in the prior art, the hydrogel-immobilized enzyme of the present application has the advantages of milder processing conditions and high biocompatibility, which provides excellent potential in biomedical applications.

[0149] For example, in the literature "ATwo-Step Cross-Linked Hydrogel Immobilization Strategy for Diacetylchitobiose Deacetylase; doi.org / 10.3390 / catal12090932", a two-step cross-linking method was adopted, using gelatin as the immobilization carrier and genipin as the cross-linking agent. After 1 day of "soft cross-linking" and 5 days of "hard cross-linking", an immobilization efficiency of about 87% of diacetylchitobiose deacetylase (Dac) was achieved.

[0150] In contrast, the hydrogel-immobilized enzyme of the present application is more time-saving and does not introduce chemical cross-linking agents. The immobilization process is milder and has better biocompatibility. At the same time, maintaining the triple-helix structure of collagen can provide certain biological functions for future applications.

[0151] In the literature "Naturally-derived biopolymers: Potential platforms for enzyme immobilization; doi.org / 10.1016 / j.ijbiomac.2019.02.152", glucose oxidase was encapsulated in a PEGDA hydrogel with a relatively high molecular weight, a relatively high water content, and a relatively large mesh size, so the mass transfer of the substrate was better; the glucose oxidase maintained its activity without leakage within one week, but the hydrogel showed weak mechanical stability.

[0152] In contrast, the hydrogel-immobilized enzyme of the present application has more excellent mechanical properties on the basis of maintaining good biocompatibility.

[0153] Comparative Example 2: Using K36B connection

[0154] K3 (amino acid sequence shown in SEQ ID NO.10) was connected with 6B to obtain lipase 6BK3 (amino acid sequence shown in SEQ ID NO.7, nucleotide sequence shown in SEQ ID NO.8). The nucleotide of lipase 6BK3 was inserted between the T7-promoter and T7-terminator in the pET-28a plasmid, and E.coli BL21(DE3) was used for expression, induction, and purification to prepare lipase 6BK3.

[0155] During the purification process, it was found that the target band of 6BK3 was relatively light and accompanied by impurity bands. The reason is that the K3 adhesion module was fused to the C-terminus of lipase 6B, thus reducing the expression level.

[0156] It can be seen that there is a certain particularity between the adhesion module and the lipase, and it cannot be constructed simply by binding.

[0157] Sequences used in the present invention

[0158] Amino acid sequence of E3VBK3 SEQ ID NO.1

[0159] MNHKVPMGHHHHHHG EISALEKEISALEKEISALEK GGGGGGGADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDPGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGGGGGGGG KISALKEKISALKEKI SALKE

[0160] Nucleotide sequence of E3VBK3 SEQ ID NO.2

[0161] ATGAATCACAAAGTGCCCATGGGCCACCATCATCACCATCATGGCGAGATCAGCGCCCTGGAGAAGGAAATCAGCGCCCTGGAGAAAGAGATCAGCGCCCTGGAAAAAGGTGGTGGTGGTGGTGGCGGTGCCGATGAACAGGAAGAGAAAGCCAAAGTGCGCACCGAACTGATTCAGGAATTAGCCCAGGGCCTGGGCGGCATCGAGAAGAAAAATTTCCCGACCCTGGGCGACGAGGATCTGGATCATACCTACATGACCAAACTGCTGACCTATCTGCAGGAGCGCGAACAGGCCGAAAATAGCTGGCGCAAACGCCTGCTGAAAGGCATTCAGGATCATGCCTTAGATCCTGGTCCTCGTGGTGAGCAAGGTCCGCAGGGTCTGCCGGGTAAGGACGGTGAAGCCGGTGCCCAGGGTCCTGCAGGTCCTATGGGCCCGGCAGGTTTTCCGGGTGAACGCGGTGAAAAAGGTGAACCGGGCACCCAGGGCGCAAAAGGCGATCGTGGCGAAACCGGTCCGGTGGGTCCTCGTGGTGAACGTGGTGAGGCCGGCCCGGCAGGCAAAGATGGTGAGCGTGGTCCGGTTGGTCCGGCCGGTGGTGGTGGTGGTGGTGGCAAGATCAGCGCCCTGAAGGAGAAAATCAGCGCCCTGAAAGAAAAAATCAGCGCCCTGAAAGAA

[0162] 6B amino acid sequence SEQ ID NO.3

[0163] MAEHNPVVMVHGIGGSSSNFEGIKSYLVSQGWSRDKLYAVDFWDKTGTNYNNGPVLSRFVQKVLDETGAKKVDIVAHSMGGANTLYYIKYLDGGNKVANVVTLGGANRLTTDKAPPGTDPNQKILYTSIYSSDDEIVPNYLSRLDGARNVQIHGVGHMGLLYSPQVYSLIKEGLNGGGQNTNGGGSHHHHHH

[0164] 6B nucleotide sequence SEQ ID NO.4

[0165] ATGGCGGAGCATAACCCGGTGGTGATGGTGCATGGCATTGGCGGCAGCAGTAGCAACTTTGAAGGCATTAAAAGCTATCTGGTGAGCCAAGGCTGGAGCCGCGATAAACTGTATGCGGTGGATTTTTGGGATAAAACCGGCACCAACTATAACAACGGCCCGGTGCTGAGCCGCTTTGTGCAGAAAGTGCTGGATGAAACCGGCGCGAAAAAAGTGGATATTGTGGCGCATAGCATGGGTGGCGCGAACACCCTGTATTATATTAAATATCTGGATGGCGGCAACAAAGTGGCGAACGTGGTGACCTTAGGCGGCGCGAACCGTCTGACCACCGATAAAGCGCCGCCGGGCACCGATCCGAATCAGAAAATTCTGTATACGAGCATTTATAGCAGCGATGATGAAATTGTGCCGAACTATCTGAGCCGCCTGGATGGCGCGCGCAACGTGCAGATTCATGGCGTGGGCCATATGGGCCTGCTGTATAGCCCGCAAGTGTATAGCCTGATTAAAGAAGGCCTGAATGGTGGCGGTCAGAACACCAACGGTGGCGGCAGCCATCACCATCACCATCAT

[0166] The amino acid sequence of E36B SEQ ID NO.5

[0167] MEIAALEKEIAALEKEIAALEKGGGSGGGSGGGSAEHNPVVMVHGIGGSSSNFEGIKSYLVSQGWSRDKLYAVDFWDKTGTNYNNGPVLSRFVQKVLDETGAKKVDIVAHSMGGANTLYYIKYLDGGNKVANVVTLGGANRLTTDKAPPGTDPNQKILYTSIYSSDDEIVPNYLSRLDGARNVQIHGVGHMGLLYSPQVYSLIKEGLNGGGQNTNGGGSHHHHHH

[0168] The nucleotide sequence of E36B SEQ ID NO.6

[0169] ATGGAAATTGCCGCCCTGGAGAAAGAAATTGCGGCGCTGGAAAAAGAGATCGCGGCGTTAGAGAAGGGTGGTGGCAGCGGCGGTGGCAGCGGCGGTGGCAGTGCGGAGCATAACCCGGTGGTGATGGTGCATGGCATTGGCGGCAGCAGTAGCAACTTTGAAGGCATTAAAAGCTATCTGGTGAGCCAAGGCTGGAGCCGCGATAAACTGTATGCGGTGGATTTTTGGGATAAAACCGGCACCAACTATAACAACGGCCCGGTGCTGAGCCGCTTTGTGCAGAAAGTGCTGGATGAAACCGGCGCGAAAAAAGTGGATATTGTGGCGCATAGCATGGGTGGCGCGAACACCCTGTATTATATTAAATATCTGGATGGCGGCAACAAAGTGGCGAACGTGGTGACCTTAGGCGGCGCGAACCGTCTGACCACCGATAAAGCGCCGCCGGGCACCGATCCGAATCAGAAAATTCTGTATACGAGCATTTATAGCAGCGATGATGAAATTGTGCCGAACTATCTGAGCCGCCTGGATGGCGCGCGCAACGTGCAGATTCATGGCGTGGGCCATATGGGCCTGCTGTATAGCCCGCAAGTGTATAGCCTGATTAAAGAAGGCCTGAATGGTGGCGGTCAGAACACCAACGGTGGCGGCAGCCATCACCATCACCATCATTAA[[ID=***1***]] [[ID=***2***]]

[0170] [[ID=***3***]]6BK3 Amino acid sequence SEQ ID NO.7[[ID=***4***]] [[ID=***5***]]

[0171] [[ID=***6***]]MHHHHHHGGGSAEHNPVVMVHGIGGSSSNFEGIKSYLVSQGWSRDKLYAVDFWDKTGTNYNNGPVLSRFVQKVLDETGAKKVDIVAHSMGGANTLYYIKYLDGGNKVANVVTLGGANRLTTDKAPPGTDPNQKILYTSIYSSDDEIVPNYLSRLDGARNVQIHGVGHMGLLYSPQVYSLIKEGLNGGGQNTNGGGSGGGSGGGSKIAALKEKIAALKEKIAALKE[[ID=***7***]] [[ID=***8***]]

[0172] It should be noted that in the original text, the tags ,

[0170] , etc. are likely some kind of specific identifiers in a particular context. Since there is no clear indication of how they should be precisely translated in a general sense, they are kept as they are in the translation. If there is more specific information about these tags, a more accurate translation could be provided. Also, the amino acid sequence part is translated according to the common amino acid abbreviations and naming conventions.6BK3 nucleotide sequence SEQ ID NO.8

[0173] ATGCATCACCATCACCATCATGGCGGCGGTAGCGCGGAACATAACCCGGTGGTGATGGTGCATGGCATTGGCGGCAGCAGTAGCAACTTTGAAGGCATTAAAAGCTATCTGGTGAGCCAAGGCTGGAGCCGCGATAAACTGTATGCGGTGGATTTTTGGGATAAAACCGGCACCAACTATAACAACGGCCCGGTGCTGAGCCGCTTTGTGCAGAAAGTGCTGGATGAAACCGGCGCGAAAAAAGTGGATATTGTGGCGCATAGCATGGGCGGCGCGAACACCCTGTATTATATTAAATATCTGGATGGCGGCAACAAAGTGGCGAACGTGGTGACCCTGGGTGGTGCGAACCGTCTGACCACCGATAAAGCGCCGCCGGGCACCGATCCGAATCAGAAAATTCTGTATACGAGCATTTATAGCAGCGATGATGAAATTGTGCCGAACTATCTGAGCCGCCTGGATGGCGCGCGCAACGTGCAGATTCATGGCGTGGGCCATATGGGCCTGCTGTATAGCCCGCAAGTGTATAGCCTGATTAAAGAAGGCCTGAACGGCGGTGGTCAGAACACCAACGGTGGCGGTAGTGGCGGTGGCAGCGGCGGTGGCAGTAAAATTGCGGCCTTAAAGGAGAAGATTGCGGCCCTGAAAGAAAAAATTGCGGCGCTGAAAGAATAA

[0174] Amino acid sequence of E3 SEQ ID NO.9

[0175] EIAALEKEIAALEKEIAALEK

[0176] Amino acid sequence of K3 SEQ ID NO.10

[0177] KISALKEKISALKEKISALKE

[0178] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for loading lipase based on collagen self-assembled hydrogel, characterized in that An adhesion module is connected to the N-terminus of lipase, and through the salt bridge interaction between the adhesion module and collagen, a collagen hydrogel loaded with lipase is obtained; Among them, the amino acid sequence of collagen is shown in SEQ ID NO.1; The amino acid sequence of the adhesion module is shown in SEQ ID NO.

9.

2. The method according to claim 1, wherein The amino acid sequence of the lipase is shown in SEQ ID NO.

3.

3. The method according to any one of claims 1 to 2, characterized in that, The preparation method of the collagen hydrogel loaded with lipase is: mixing the hydrogel and lipase solution, shaking, centrifuging, and standing still to obtain a collagen hydrogel loaded with lipase; Optionally, in the hydrogel loaded with lipase, the enzyme loading amount is 5-50 mg / g; Optionally, the shaking is at 4-35 °C and 100-300 rpm for 4-20 h; Optionally, the standing still is at 4 °C for 0-20 h; Optionally, the buffer solution is a phosphate buffer solution with a pH of 5.0-10.0; Optionally, the dosage ratio of the hydrogel to the lipase solution is 3-5 mg: 100-200 μL.

4. The collagen hydrogel loaded with lipase prepared by the method according to any one of claims 1 to 3.

5. A product, characterized in that, Containing the collagen hydrogel loaded with lipase according to claim 4; the product includes food catalysts, medical products, decontamination products, cosmetic additives, and biodegradable materials.

6. The application of the method according to any one of claims 1 to 3 or the collagen hydrogel loaded with lipase according to claim 4 in the fields of food, medicine, and biocatalysis.

7. The application according to claim 6, wherein The application in the food field includes synthesizing flavor substances using the collagen hydrogel loaded with lipase; The application in the medical field includes preparing a drug controlled release system and a wound dressing using the collagen hydrogel loaded with lipase; The application in the biocatalysis field includes preparing detergents using the collagen hydrogel loaded with lipase.

8. A method for simultaneously improving the enzyme activity, the repeated utilization rate, and the storage stability of lipase in organic solvents, characterized in that, Use a collagen hydrogel to load lipase; connect an adhesion module to the N-terminus of lipase, and through the salt bridge interaction between the adhesion module and collagen, obtain a collagen hydrogel loaded with lipase; Among them, the amino acid sequence of collagen is shown in SEQ ID NO.1; The amino acid sequence of the adhesion module is shown in SEQ ID NO.

9.

9. The method according to claim 8, characterized in that The preparation method of the collagen hydrogel loaded with lipase is: mixing the hydrogel and lipase solution, shaking, centrifuging, and standing still to obtain a collagen hydrogel loaded with lipase; Optionally, in the hydrogel loaded with lipase, the enzyme loading amount is 5-50 mg / g; Optionally, the shaking is at 4-35 °C and 100-300 rpm for 4-20 h; Optionally, the standing still is at 4 °C for 0-20 h; Optionally, the buffer solution is a phosphate buffer solution with a pH of 5.0-10.0; Optionally, the dosage ratio of the hydrogel to the lipase solution is 3-5 mg: 100-200 μL.

10. A biocatalyst, characterized in that, The biocatalyst contains the collagen hydrogel loaded with lipase according to claim 4.

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