Carrier for immobilized enzymes, process for its preparation and use thereof

Abstract

The present application provides a carrier for immobilized enzyme, a preparation method thereof and use thereof. The present application provides a carrier for immobilized enzyme, which is a bacteriophage M13 hydrogel, wherein the bacteriophage M13 hydrogel is formed by N-terminal cross-linking of pVIII protein, and the N-terminal of pIII protein of the bacteriophage M13 is combined with a magnetic nanoparticle. The present application provides a preparation method of the carrier for immobilized enzyme. The present application also provides a novel immobilized enzyme comprising the carrier and an enzyme immobilized on the carrier. The carrier constructed based on the bacteriophage M13 and the magnetic nanoparticle has great application value in the field of enzyme immobilization.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and relates to a carrier for immobilizing enzymes. More specifically, this invention relates to a carrier for immobilizing enzymes based on bacteriophage M13. This invention also provides a method for preparing the carrier and its use in immobilizing enzymes. Background Technology

[0002] Enzymes, as natural high-molecular-weight biocatalysts, possess numerous advantages such as extremely high selectivity, high catalytic activity, mild reaction conditions, and low pollution, making them highly promising for applications in industries such as food processing and pharmaceuticals. However, the application of free enzymes in large-scale industrial production is limited due to difficulties in recovery and poor stability. Therefore, research on immobilized enzymes has received increasing attention. After decades of development, immobilized enzymes have been widely used in various fields, including food production, biopharmaceuticals, biosensors, the pharmaceutical industry, environmental protection technologies, and biotechnology.

[0003] In recent years, scientists and engineers have made great progress in the design and modification of enzyme molecules through various approaches such as rational design and directed evolution, and the activity and stability of many enzymes have been significantly improved. With the development of enzyme engineering and artificial intelligence (AI) technologies, free enzyme catalysis will be a very promising development direction in the near future, especially the catalysis of macromolecular substrate systems (Rix G, et al., Nat Commun 2020, 11(1), 5644.), but the problem of efficient separation and recovery needs to be solved as soon as possible. At present, more and more industrial enzymes, such as lipases, have sufficient stability and activity to achieve industrialization, and free lipases have been applied in industrial production. However, the problem of low recovery efficiency limits their industrialization. It is of great significance to develop a new method for immobilized enzymes that can achieve efficient biocatalysis like free enzymes and can be easily and cost-effectively recovered and reused.

[0004] Magnetic nanoparticles (MNPs) are widely used in immobilized enzymes, enabling rapid separation and reuse of enzymes via magnetic fields. However, due to size effects and electrostatic attraction, magnetic nanoparticles often exhibit poor dispersibility, requiring surface modification to prevent particle aggregation and provide enzyme-interacting groups such as amino and epoxy groups (Cheng G, et al., Chinese Chem Lett 2019, 30(3), 656-659). The functionalization step of magnetic nanoparticles increases the reaction steps and carrier costs to some extent. To improve enzyme loading, various magnetic composite materials, such as magnetic graphene composites, also require time-consuming pre-modification of the materials to obtain composite materials for subsequent enzyme immobilization (Li X, et al., Carbon 2013, 60, 488-497). To simplify the preparation process and reduce carrier costs, the direct use of unmodified magnetic nanoparticles is a promising research direction.

[0005] Bacteriophage M13 is a filamentous bacterial virus, approximately 6-8 nm × 900 nm in size. The surface of Bacteriophage M13 is primarily composed of approximately 2700 copies of the pVIII capsid protein assembled into a cylindrical structure. The N-terminal residues of the pVIII protein are exposed on the surface of Bacteriophage M13, making it a natural amino-rich carrier widely used in materials synthesis, biomedicine, and biosensors (Nguyen AH, et al., Biosens Bioelectron 2016, 85, 522-528.). To meet the growing applications of M13 in various fields, large-scale cultivation and harvesting of Bacteriophage M13 have been reported (Torres Acosta M, et al., J Chem Technol Biotechnol 2020, 95, 2822-2833.).

[0006] As a component of immobilized enzymes, the structure and properties of the carrier material have a significant impact on the immobilized enzyme. Due to the importance of carrier materials, many scholars have been dedicated to their research since the emergence of immobilized enzyme technology. To date, carrier materials for immobilized enzymes have evolved from the initial natural polymers to synthetic polymers, inorganic materials, and composite materials.

[0007] However, there is currently no research on using M13 phage itself as an immobilized enzyme carrier. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by providing a phage M13-based carrier for immobilized enzymes. This invention also provides a method for preparing the immobilized enzyme carrier and its use for immobilizing enzymes. Compared with existing technologies, the phage M13-based carrier for immobilized enzymes provided by this invention has high enzyme loading capacity, low enzyme activity loss, and low magnetic nanoparticle usage.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] On one hand, the present invention provides a carrier for immobilizing enzymes, the carrier being a bacteriophage M13 hydrogel, wherein the bacteriophage M13 hydrogel is formed by cross-linking the N-terminus of the pVIII protein of bacteriophage M13, and magnetic nanoparticles are bound to the N-terminus of the pIII protein of bacteriophage M13.

[0011] According to the present invention, the carrier for immobilizing enzymes contains a linker peptide (FAP) at the N-terminus of the pIII protein of phage M13 that is capable of specifically binding magnetic nanoparticles.

[0012] According to the carrier for immobilizing enzymes of the present invention, the magnetic nanoparticles are Fe3O4 particles; preferably, the Fe3O4 particles are unmodified Fe3O4 particles.

[0013] According to the present invention, the carrier for immobilizing enzymes is wherein the linker peptide is LPLSTQH(FAP) capable of specifically binding to Fe3O4 particles.

[0014] According to the carrier for immobilized enzymes of the present invention, the ratio of bacteriophage M13 to magnetic nanoparticles is 10:1. 12 -10 16 PFU phage M13 / g magnetic nanoparticles; preferably 1-9×10 15 pfu phage M13 / g magnetic nanoparticles; more preferably 1-9×10 15 pfu phage M13 / g Fe3O4.

[0015] In a preferred embodiment, the present invention provides a carrier (FAP-M13)-Fe3O4 for immobilizing enzymes.

[0016] On the other hand, the present invention provides a method for preparing the carrier for immobilized enzymes, the method comprising the following steps:

[0017] 1) Phage M13, which can specifically bind magnetic nanoparticles, was obtained by panning and selectively cultured on a large scale;

[0018] 2) Place the magnetic nanoparticles and the phage M13 obtained in step 1) in a buffer solution to allow the magnetic nanoparticles to bind to the phage M13;

[0019] 3) Add a cross-linking agent to the conjugate obtained in step 2) to cross-link the phage into a phage hydrogel, thereby obtaining the carrier for immobilizing the enzyme.

[0020] According to the method of the present invention, in step 1), the magnetic nanoparticles are Fe3O4 particles, and the bacteriophage M13 that specifically binds to the Fe3O4 particles is a bacteriophage M13 whose N-terminus of the pIII protein contains LPLSTQH(FAP).

[0021] According to the method of the present invention, in step 2), the concentration of the Fe3O4 particles is 1-10 mg / mL, preferably 5-8 mg / mL. In this step, sufficient Fe3O4 needs to be added to achieve stable magnetic recovery of the magnetically immobilized enzyme.

[0022] According to the method of the present invention, in step 2), the concentration of the bacteriophage M13 is 1 × 10⁻⁶. 12 -9×10 13 pfu / mL, preferably 1-5×10 13 pfu / mL. A relatively high concentration of phage M13 is required in this step to achieve the binding of phage M13 with Fe3O4 particles.

[0023] According to the method of the present invention, in step 2), the bonding conditions are: shaking at 20-40°C for 0.5-2 hours;

[0024] According to the method of the present invention, before adding the cross-linking agent in step 3), the conjugate obtained in step 2) is diluted 1-6 times, preferably 3-6 times, using a buffer solution. In this step, if the concentration of phage M13 is too high, the pores of the cross-linked phage M13 gel network will be too small, which will affect subsequent enzyme loading.

[0025] Preferably, in step 3), the crosslinking agent is glutaraldehyde; more preferably, the final concentration of glutaraldehyde is 0.1-2.5 wt.%. The inventors have found that a sufficient concentration of glutaraldehyde is beneficial for obtaining a phage M13 crosslinking network coupled with magnetic Fe3O4.

[0026] Preferably, in step 3), the crosslinking conditions are: shaking at 20-40°C for 4-10 hours. The inventors have found that excessively long crosslinking times in this step can affect subsequent enzyme loading.

[0027] According to the method of the present invention, in step 3), the obtained phage hydrogel is separated by an external magnet, washed with buffer solution, and then dissolved in a buffer solution of the same volume as before washing.

[0028] According to the method of the present invention, in step 2) or step 3), the buffer solution is a phosphate buffer solution; preferably, the phosphate buffer solution is a KH2PO4 or K2HPO4 buffer solution with pH 6-9; more preferably, the concentration of the phosphate buffer solution is 0.05-0.2M.

[0029] In another aspect, the present invention provides the use of the carrier for immobilized enzymes in immobilized enzymes;

[0030] Preferably, the enzyme is a lipase.

[0031] The present invention also provides an immobilized enzyme, comprising the carrier described in the present invention and the enzyme immobilized on the carrier.

[0032] The present invention also provides a method for immobilizing an enzyme, the method comprising the following steps:

[0033] The carrier of the present invention is mixed with the enzyme in phosphate buffer and shaken at 20-40°C for 6-12 hours. The enzyme is flexibly cross-linked to the N-terminus of the pVIII peptide of phage M13 and dispersed on the phage surface. Then, the enzyme is separated by an external magnet, washed and resuspended to obtain the immobilized enzyme.

[0034] Preferably, the concentration of the enzyme is 1-4 mg / mL;

[0035] Preferably, the enzyme is a lipase;

[0036] Preferably, the buffer solution is a phosphate buffer solution; preferably, the phosphate buffer solution is a KH2PO4 or K2HPO4 buffer solution with a pH of 6-9; more preferably, the concentration of the phosphate buffer solution is 0.05-0.2M.

[0037] The technical solution of this invention is based on the following research idea: The inventors discovered that nanoscale amino-rich filamentous bacteriophage M13 can be developed as a multifunctional biological scaffold for the surface dispersion and immobilization of enzymes. Therefore, based on the structure and capsid protein of bacteriophage M13, the inventors designed a magnetic hydrogel carrier that can directly immobilize enzymes using unmodified Fe3O4. In the carrier, bacteriophages M13 are cross-linked at the N-terminus of a pVIII peptide to form a bacteriophage hydrogel. Simultaneously, a linker peptide that specifically binds to Fe3O4 and a bacteriophage FAP-M13 carrying this linker peptide at the pIII terminus were developed, ultimately forming a magnetic hydrogel carrier (FAP-M13)-Fe3O4.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The carrier provided by the present invention immobilizes enzymes by flexible cross-linking of the N-terminus of the pVIII peptide of phage M13, which can flexibly adjust the orientation between the enzyme and the substrate to obtain catalytic efficiency similar to that of free enzymes, thereby achieving high enzyme activity recovery, strong stability and stable magnetic field recovery.

[0040] 2. The vector provided by this invention binds to and disperses unmodified Fe3O4 via the pIII protein of phage M13, simplifying the preparation process of the magnetic coupling vector and reducing the amount of magnetic particles used.

[0041] 3. The carrier provided by this invention has a high specific surface area, which can significantly increase the enzyme loading capacity;

[0042] 4. The method for immobilizing enzymes on a carrier provided by this invention is simple and easy to operate;

[0043] In summary, the magnetic hydrogel carrier constructed based on bacteriophage M13 and magnetic nanoparticles has significant application value in the field of enzyme immobilization. Attached Figure Description

[0044] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 The flowchart shown is a process for selecting bacteriophage M13 (FAP-bacteriophage M13) that specifically binds to Fe3O4 particles, according to an embodiment of the present invention.

[0046] Figure 2 The titer of the eluent in a five-round panning process of FAP-phage M13 is shown as an embodiment of the present invention.

[0047] Figure 3 The following is an example of the effect of phage concentration and binding time on the amount of FAP-M13 bound to Fe3O4 according to an embodiment of the present invention;

[0048] Figure 4 Transmission electron microscopy image of a magnetic hydrogel carrier prepared according to an embodiment of the present invention;

[0049] Figure 5 This is a schematic diagram of the magnetic hydrogel carrier immobilized lipase prepared in this invention.

[0050] Figure 6 Another schematic diagram of the magnetic hydrogel carrier prepared for this invention, and the lipase immobilized thereon.

[0051] Figure 7 A transmission electron microscope image of an immobilized enzyme prepared according to an embodiment of the present invention;

[0052] Figure 8 The enzyme activity yield of the immobilized enzyme prepared according to an embodiment of the present invention;

[0053] Figure 9 The results of the immobilized enzyme prepared according to an embodiment of the present invention at different pH values;

[0054] Figure 10 The results of the immobilized enzyme prepared according to an embodiment of the present invention at different temperatures;

[0055] Figure 11 The results show the thermal stability of the immobilized enzyme prepared according to an embodiment of the present invention;

[0056] Figure 12 The storage stability results of the immobilized enzyme prepared according to an embodiment of the present invention;

[0057] Figure 13 A rubidium magnet magnetic field recovery diagram of an immobilized enzyme prepared according to an embodiment of the present invention;

[0058] Figure 14 The results show the magnetic field recovery stability of the immobilized enzyme prepared according to an embodiment of the present invention;

[0059] Figure 15 Thermogravimetric curves of FAP-M13, lipase, iron oxide nanoparticles, magnetic hydrogel carrier, and immobilized enzyme according to the present invention. Detailed Implementation

[0060] The features and exemplary embodiments of various aspects of the present invention will now be described in detail. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without requiring some of these specific details. The following description of embodiments is merely intended to provide a better understanding of the invention by illustrating examples of the invention.

[0061] Example 1: Selection and Identification of FAP-M13

[0062] PhD-7 TMThe phage display peptide library kit was purchased from New England Biolabs (NEB) and contained phage M13 with 7 random amino acid pIII ends. This kit was used to pan for phages that specifically bind to unmodified Fe3O4 at their pIII ends. One round of panning consisted of four steps.

[0063] 1. The kit contains approximately 1.5 × 10⁻⁶ units. 11 PFU phages were mixed with 0.5 mg of unmodified 600 nm diameter Fe3O4 particles in 200 μL Tris-HCl buffer (TBS, pH 7.5) and incubated in a 24-well plate at room temperature for 60 minutes.

[0064] 2. The M13-Fe3O4 complex was recovered using a rubidium magnet and washed 10 times with 1 mL of 0.1% Tween-20 TBS buffer to remove unbound phages.

[0065] 3. Add 200 μL of Gly-HCl solution (0.2 M, pH = 2.2) to the M13-Fe3O4 complex and incubate at room temperature for 10 minutes to elute the phages bound to the Fe3O4 surface.

[0066] 4. Add 30 μL of 1M Tris-HCl solution (pH = 9.1) to neutralize the elution buffer, and then amplify the target phage in host Escherichia coli ER2738.

[0067] After the first round of panning, specific phage M13 with high affinity for Fe3O4 was initially obtained. The amplified phage was then mixed with Fe3O4 again, and the above panning process was repeated.

[0068] After five rounds of selection, the phages in the fifth round of elution were sequenced to obtain FAP-M13, which binds to Fe3O4 with high specificity. The specific pIII-terminal SAP sequence is LPLSTQH. The optimal three-dimensional structure of the FAP peptide predicted by the Scarch Protein Predictor 3Dpro model (http: / / scratch.proteomics.ics.uci.edu / ) is hook-shaped.

[0069] Figure 1 The flowchart shown is a process for selecting bacteriophage M13 (FAP-bacteriophage M13) that specifically binds to Fe3O4 particles, according to an embodiment of the present invention.

[0070] Figure 2 The titer of the elution buffer in a five-round panning process of FAP-phage M13 is shown as an embodiment of the present invention.

[0071] Example 2: Effect of phage concentration and binding time on the binding amount of FAP-M13 and Fe3O4

[0072] 0.5 mg Fe3O4 and 2 × 10 12 PFU was mixed with FAP-phage M13, and PB buffer was added to total volumes of 50 μL, 100 μL, 150 μL, and 200 μL to obtain a final concentration of 4 × 10⁻⁶. 13 pfu / mL, 2×10 13 pfu / mL, 1.33×10 13 pfu / mL, 1×10 13 FAP-M13 at pfu / mL. The final concentration using the unmodified pIII-terminated FAP-M13 was 4 × 10⁻⁶ pfu / mL. 13 wild-type phage M13 (2×10 pfu / mL) 12 A mixture of 50 μL of pfu and 0.5 mg of Fe3O4 was used as a control. The mixture was placed in a shaker at 37°C and 200 rpm for 0, 10, 30, and 60 minutes, and the amount of FAP-M13 bound to Fe3O4 was measured. The amount of FAP-M13 bound to Fe3O4 increased with increasing phage concentration and binding time (0-30 minutes), and then remained relatively stable after a period of time (30-60 minutes). At an FAP-M13 concentration of 4 × 10⁻⁶ mg / L... 13 In a system with pfu / mL, the highest binding amount of FAP-M13 to Fe3O4 after mixing for 30 minutes was approximately 5.3 × 10⁻⁶ pfu / mL. 14 pfu FAP-M13 / gFe3O4. In contrast, wild M13 hardly binds to Fe3O4, demonstrating the binding specificity of FAP-M13 to Fe3O4.

[0073] Figure 3 The following is an example of the effect of phage concentration and binding time on the amount of FAP-M13 bound to Fe3O4 according to an embodiment of the present invention.

[0074] Example 3: Large-scale culture of bacteriophage M13

[0075] To obtain sufficient quantities of phages for vector preparation, the phage culture process was optimized. Traditionally, phage culture involves inoculating a small amount of phage and performing 2-3 transfers to expand the culture volume. This preparation method simplifies the process by optimizing the inoculum size and culture time. The following is an example of the phage culture and purification process for a 1L culture volume. First, a single colony of host bacterium ER2738 was inoculated into 20mL LB medium (10g / L tryptone, 5g / L yeast extract, 10g / L NaCl) and cultured at 37℃ and 200rpm for 12h to obtain a seed culture. Then, an optimized inoculum size of 3×10⁻⁶ was used. 12Pfu phage M13 and 1 mL of ER2738 seed culture were simultaneously inoculated into ten 300 mL shake flasks containing 100 mL LB each, and cultured at 37°C and 200 rpm for 12 hours. The culture medium was centrifuged at 8000 rpm for 10 minutes to remove ER2738 cells; the resulting supernatant contained phage. 1 / 6 volume of polyethylene glycol 8000 / NaCl solution (20% (w / v) polyethylene glycol 8000, 2.5 M NaCl) was added to the supernatant, and the mixture was refrigerated at 4°C for 8 hours to allow M13 to precipitate. The phage was then harvested by centrifugation at 4°C and 8000 rpm for 15 minutes. Different volumes of deionized water were added to resuspend the phage, resulting in phage solutions of varying concentrations. For a 1 L LB culture volume, the net yield of phage M13 in this protocol was approximately 4 × 10⁻⁶. 14 pfu.

[0076] Example 4: Preparation of Magnetic Hydrogel Carrier

[0077] 0.5 mg of unmodified Fe3O4 (600 nm in diameter) was mixed with 2 × 10 12 A mixture of PFU and FAP-M13 was added to phosphate buffer (PB, pH 7.5, 0.1M KH2PO4 / K2HPO4) to a total volume of 50 μL. The mixture was placed in a shaker at 37°C and 200 rpm for 30 minutes, allowing the FAP-phage M13 ends to bind to the Fe3O4 surface. Subsequently, 2 μL of glutaraldehyde (50 wt.%) was added, followed by PB buffer to a final volume of 200 μL. The mixture was then placed in a shaker at 37°C and 200 rpm for 4 hours to crosslink and form a phage hydrogel. Fe3O4 was dispersed and embedded in the hydrogel through the ends of SAP-M13. Finally, the product was separated using a rubidium magnet and washed three times with 1 mL of PB buffer, and finally dispersed in 200 μL of PB buffer to obtain the magnetic hydrogel carrier ((FAP-M13)-Fe3O4).

[0078] Figure 4 Transmission electron microscopy (TEM) image of a magnetic hydrogel carrier prepared according to an embodiment of the present invention.

[0079] Example 5: Preparation of Immobilized Lipase

[0080] 100 μL of lipase (2 mg / mL) was mixed with 100 μL of the magnetic hydrogel carrier prepared in Example 4 and immobilized by mixing in a shaker at 37 °C and 200 rpm for 8 hours. The lipase was flexibly cross-linked to the N-terminus of the pVIII peptide and dispersed on the phage surface. Finally, the product was separated with a rubidium magnet and washed three times with 1 mL of PB buffer, and finally dispersed in 200 μL of PB buffer to obtain immobilized lipase (lipase@(FAP-M13)-Fe3O4).

[0081] Figure 5 This is a schematic diagram of the magnetic hydrogel carrier immobilized lipase prepared in this invention.

[0082] Figure 6 Another schematic diagram of the magnetic hydrogel carrier prepared for this invention, and the lipase immobilized thereon.

[0083] Figure 7 Transmission electron microscopy image of an immobilized enzyme prepared according to an embodiment of the present invention.

[0084] Example 6: Determination of enzyme loading on magnetic hydrogels

[0085] The enzyme activity retained after immobilization of 0.2 mg lipase was measured. The obtained immobilized lipase loading reached 0.2 mg / 100 μL of carrier (4 × 10⁻⁶). 15 The enzyme activity recovery rate reached over 95% (PFU FAP-M13 / g Fe3O4). Using 1 mg NH2-Fe3O4 (600 nm) as a control, its loading was only 2.5% of that of the magnetic hydrogel prepared from FAP-M13, indicating that FAP-M13, as a hydrogel, has a high specific surface area and can significantly increase the enzyme loading.

[0086] Figure 8 The enzyme activity yield of the immobilized enzyme prepared according to an embodiment of the present invention is shown.

[0087] Example 7 Lipase Activity Assay

[0088] Lipase activity was determined using p-nitrophenyl palmitate (pNPP) as a substrate. The substrate solution was prepared by mixing solutions A and B at a volume ratio of 1:9. Solution A was prepared by dissolving 9 mg of pNPP in 10 mL of isopropanol, and solution B was prepared by mixing 2 g of Triton X-100 with 450 mL of Tris-HCl solution (50 mM, pH 7.5). 0.1 mL of immobilized enzyme was added to 1 mL of the substrate solution, and the reaction was carried out at 37 °C for 10 min. The absorbance was measured at 410 nm, and the amount of p-nitrophenol (pNP) released was calculated. Under optimal experimental conditions, the lipase activity defined as the generation of 1 μmol of pNP per 1 mL of system per minute was considered to be one enzyme activity unit (U).

[0089] Example 8: Stability determination of immobilized enzymes

[0090] Using free lipase as a control, a series of evaluations were conducted on the stability of lipase immobilized on a magnetic hydrogel support.

[0091] The effect of pH changes on enzyme activity was determined. Enzyme activity was measured in solutions ranging from pH 5 to pH 9 at 37°C for 10 minutes. Immobilized enzymes exhibited higher pH tolerance compared to free enzymes at extreme pH conditions.

[0092] The effect of temperature changes on enzyme activity was determined. Lipase activity was measured by performing enzyme activity assays at different temperatures from 20°C to 60°C for 10 minutes at pH 7.5. Immobilized enzymes exhibit a wider catalytic temperature adaptability compared to free enzymes.

[0093] The thermostability of the immobilized enzyme at 50 °C was determined. The thermostability of the immobilized and free lipases was observed by incubation at 50 °C in PB buffer (pH = 7.5) for 0–3 hours, and the remaining enzyme activity was measured at 37 °C and pH = 7.5 for 10 minutes. After incubation at 50 °C for 3 hours, the immobilized enzyme retained approximately 45% of its initial activity, while the free lipase was almost completely inactivated.

[0094] The storage stability of the immobilized enzyme at 4°C was determined. The enzyme was stored in PB buffer (pH 7.5) at 4°C for 0–30 days, and its activity was measured after magnetic recovery. After 30 days of storage at 4°C, the immobilized enzyme maintained good magnetic recovery ability, and the activity of the immobilized lipase after magnetic recovery was approximately 60% higher than that of the free lipase.

[0095] Stability of the immobilized enzyme under magnetic field recovery. The magnetic recovery process of recovering and resuspending the immobilized enzyme using a rubidium magnet was repeated 20 times, with lipase activity measured every two rounds. After 20 magnetic cycles, the activity of the immobilized enzyme remained at approximately 94%, indicating that the immobilized enzyme has strong stability under magnetic field recovery.

[0096] Figure 9 The results of the immobilized enzyme prepared according to an embodiment of the present invention at different pH values;

[0097] Figure 10 The results of the immobilized enzyme prepared according to an embodiment of the present invention at different temperatures;

[0098] Figure 11 The results show the thermal stability of the immobilized enzyme prepared according to an embodiment of the present invention;

[0099] Figure 12 The storage stability results of the immobilized enzyme prepared according to an embodiment of the present invention;

[0100] Figure 13 A rubidium magnet magnetic field recovery diagram of an immobilized enzyme prepared according to an embodiment of the present invention;

[0101] Figure 14The results show the magnetic field recovery stability of an immobilized enzyme prepared according to an embodiment of the present invention.

[0102] Example 9: Thermogravimetric Analysis of Magnetic Hydrogels and Immobilized Enzymes

[0103] Thermogravimetric analysis (TGA) was performed on the magnetic hydrogels and immobilized enzymes under a nitrogen atmosphere at a heating rate of 10 °C / min, within a temperature range of 25–800 °C. Fe3O4, FAP-M13, and lipase were used as controls. Within different temperature ranges, the immobilized enzyme exhibited a slower weight loss rate than the free lipase, indicating superior thermal stability.

[0104] Figure 15 Thermogravimetric curves of FAP-M13, lipase, iron oxide nanoparticles, magnetic hydrogel carrier, and immobilized enzyme according to the present invention.

[0105] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A carrier for immobilizing an enzyme, the carrier being a hydrogel of bacteriophage M13, wherein the hydrogel of bacteriophage M13 is formed by cross-linking the N-terminus of the pVIII protein of bacteriophage M13, and the N-terminus of the pIII protein of bacteriophage M13 is bound to magnetic nanoparticles, the magnetic nanoparticles being Fe3O4 particles, and the N-terminus of the pIII protein of bacteriophage M13 containing a linker peptide FAP, the linker peptide FAP being LPLSTQH capable of specifically binding to Fe3O4 particles.

2. The carrier for immobilizing enzymes according to claim 1, wherein, The Fe3O4 particles are unmodified Fe3O4 particles.

3. The carrier for immobilizing enzymes according to claim 1 or 2, wherein, The ratio of bacteriophage M13 to magnetic nanoparticles is 10:

1. 12 -10 16 PFU phage M13 / g magnetic nanoparticles.

4. The carrier for immobilizing enzymes according to claim 3, wherein, 1-9×10 15 pfu bacteriophage M13 / gFe3O4.

5. A method for preparing a carrier for immobilizing enzymes according to any one of claims 1 to 4, the method comprising the following steps: 1) Place magnetic nanoparticles and phage M13 in a buffer solution to allow the magnetic nanoparticles to bind to phage M13. The magnetic nanoparticles are Fe3O4 particles, and the phage M13 that specifically binds to Fe3O4 particles is phage M13 whose N-terminus of pIII protein contains LPLSTQH. 2) Add a cross-linking agent to the conjugate obtained in step 1) to cross-link the phage into a phage hydrogel, thereby obtaining the carrier for immobilizing the enzyme.

6. The method according to claim 5, wherein, In step 1), the concentration of the Fe3O4 particles is 1-10 mg / mL.

7. The method according to claim 5, wherein, The concentration of the Fe3O4 particles is 5-8 mg / mL.

8. The method according to claim 5, wherein, In step 1), the concentration of bacteriophage M13 is 1 × 10⁻⁶. 12 -9×10 13 pfu / mL.

9. The method according to claim 8, wherein, In step 1), the concentration of bacteriophage M13 is 1-5 × 10⁻⁵. 13 pfu / mL.

10. The method according to claim 5, wherein, In step 1), the bonding conditions are: shaking for 0.5-2 hours at 20-40°C.

11. The method according to claim 5, wherein, In step 2), before adding the crosslinking agent, the conjugate obtained in step 1) is diluted 1-6 times with a buffer solution.

12. The method according to claim 11, wherein, In step 2), before adding the crosslinking agent, the binding obtained in step 1) is diluted 3-6 times using a buffer solution.

13. The method according to claim 5, wherein, In step 2), the crosslinking agent is glutaraldehyde.

14. The method according to claim 13, wherein, The final concentration of glutaraldehyde is 0.1-2.5 wt.%.

15. The method according to claim 5, wherein, In step 2), the crosslinking conditions are: shaking at 20-40°C for 4-10 hours.

16. The method according to claim 5, wherein, In step 2), the obtained phage hydrogel is separated using an external magnet, washed with buffer, and then dissolved in a buffer solution of the same volume as before washing.

17. The method according to claim 5, wherein, In step 1) or step 2), the buffer solution is a phosphate buffer solution.

18. The method according to claim 17, wherein, The phosphate buffer solution is a KH2PO4 or K2HPO4 buffer solution with a pH of 6-9.

19. The method according to claim 18, wherein, The concentration of the phosphate buffer solution is 0.05-0.2M.

20. Use of the carrier for immobilized enzyme according to any one of claims 1 to 4 in immobilized enzyme.

21. The use according to claim 20, wherein, The enzyme in question is a lipase.

22. An immobilized enzyme comprising a carrier for immobilizing an enzyme according to any one of claims 1 to 4 and an enzyme immobilized on said carrier.

23. The immobilized enzyme according to claim 22, wherein, The enzyme in question is a lipase.

24. A method for immobilizing an enzyme, the method comprising the following steps: The carrier for immobilizing the enzyme according to any one of claims 1 to 4 is mixed with the enzyme buffer and shaken at 20-40°C for 6-12 hours. The enzyme is flexibly cross-linked to the N-terminus of the pVIII peptide of phage M13 and dispersed on the phage surface. Then, the immobilized enzyme is obtained by separating with an external magnet, washing and resuspending.

25. The method according to claim 24, wherein, The concentration of the enzyme is 1-4 mg / mL.

26. The method of claim 24, wherein, The enzyme in question is a lipase.

27. The method according to claim 24, wherein, The buffer solution is a phosphate buffer.

28. The method according to claim 27, wherein, The phosphate buffer solution is a KH2PO4 or K2HPO4 buffer solution with a pH of 6-9.

29. The method according to claim 28, wherein, The concentration of the phosphate buffer solution is 0.05-0.2M.

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