A pet-degrading enzyme immobilized enzyme for insoluble plastic substrates and a preparation method and application thereof

Abstract

The application belongs to the technical field of immobilized enzyme and relates to a PET degrading enzyme immobilized enzyme for insoluble plastic substrates and a preparation method and application thereof. A pH responsive immobilized enzyme carrier is covalently connected with SpyCatcher, a degrading enzyme for insoluble substrates is connected with SpyTag, and the immobilized enzyme is formed through specific covalent combination of SpyCatcher-SpyTag. The pH value is selected so that the carrier is liquid, there is no steric hindrance with the insoluble substrate, the insoluble substrate can be efficiently degraded, the pH is adjusted after the reaction is completed, the immobilized enzyme is precipitated for easy recovery; the two-step connection in the immobilization process is covalent combination, which significantly improves the application and storage stability of the immobilized enzyme; the specific covalent combination of SpyCatcher-SpyTag can realize one-step purification of PETase-SpyTag in a fermentation crude enzyme solution, improve the activity of the immobilized enzyme, reduce the preparation cost, and still retain 50% activity after 10 cycles of catalysis.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme technology, specifically relating to an immobilized enzyme for degrading insoluble substrates, its preparation method, and its application. Background Technology

[0002] PET (polyethylene terephthalate) plastic, as an important petroleum-based synthetic polyester, is a polyester chain polymer formed by the polymerization of two monomer units—terephthalic acid (TPA) and ethylene glycol (EG). It is widely used in plastic packaging and textiles. Waste plastic pollution, due to its significant impact on the environment, health, and socio-economic conditions, has become a serious global concern. Enzymatic hydrolysis and recycling technology for waste PET plastic has significant application development potential and is expected to become a green approach to alleviate global plastic pollution and waste of plastic resources. However, to date, PET degrading enzymes still face bottlenecks in practical applications, such as high enzyme cost, low catalytic efficiency, and poor stability, which limit their industrial-scale promotion. Therefore, developing a PET degrading enzyme immobilization technology that can significantly improve enzyme stability, achieve efficient recycling, and reduce enzyme costs is of great practical significance and application value for promoting the industrialization of plastic biodegradation.

[0003] However, in existing immobilized enzyme technologies, many commonly used solid porous carrier materials immobilize enzyme molecules through physical adsorption and encapsulation, which can improve enzyme stability and reusability to some extent. However, the interaction between the carrier and enzyme molecules is weak, and the immobilized enzyme is easily detached due to mechanical shearing or substrate impact, resulting in poor stability during recycling. Furthermore, for insoluble solid substrates like PET plastic, even if chemical bonds are added to enhance the interaction between the enzyme and the carrier, improving the stability of the immobilized enzyme to some extent, a contact barrier still exists between the solid carrier and the solid substrate, such as steric hindrance and low mass transfer efficiency, preventing most of the enzyme from contacting the substrate and severely affecting catalytic efficiency. To overcome these problems, it is urgent to develop a novel method for preparing immobilized PET degrading enzymes to achieve efficient degradation and stable recycling of PET plastic solid substrates, thereby promoting the industrialization of PET enzymatic hydrolysis and utilization, improving degradation efficiency, and reducing enzyme costs. Summary of the Invention

[0004] In view of the current industrialization bottlenecks of low catalytic activity, poor stability, and high enzyme cost of degrading enzymes for insoluble substrates, this invention combines a pH-responsive methacrylate-ethyl acrylate copolymer carrier material with a SpyCatcher / SpyTag covalently bound protein pair to develop a high-purity, high-activity, and high-stability immobilized enzyme for degrading insoluble substrates, as well as its preparation method and application.

[0005] Since there are currently no immobilized enzyme materials and stable, mature immobilization methods for insoluble substrates, the stable immobilized enzyme preparation method based on pH-responsive polymer materials and double covalent bonding provided by this invention is theoretically applicable to all biological enzymes that can efficiently catalyze reactions of insoluble substrates under the pH conditions of carrier dissolution. This invention uses PET-degrading enzymes as an example to describe this invention in detail, but this does not limit the scope of protection of this invention.

[0006] The immobilized enzyme preparation method of the present invention includes: selecting a pH-responsive carrier, wherein the pH-responsive carrier of the present invention refers to a carrier whose dissolution state is related to different pH values; adjusting the pH value so that the carrier is in a dissolved state when reacting with an insoluble substrate; and after the reaction, adjusting the pH value to make the carrier precipitate for easy recovery. Preferably, this invention selects methacrylic acid-ethyl acrylate copolymer (MAEAP) as the immobilized enzyme carrier. This carrier has pH-responsive characteristics and is in a dissolved state at pH > 5.5. The optimal pH range for PET degrading enzymes is generally between pH 8.0 and 9.0. At this pH, the immobilized enzyme carrier is in a liquid dissolved state, making it easier for the enzyme to access the insoluble PET plastic substrate in the degradation reaction system compared to a solid carrier. It also has no steric hindrance and higher catalytic efficiency. After the reaction, adjusting the pH of the reaction solution to pH 4.5 allows the immobilized enzyme to precipitate, facilitating centrifugal recovery. Furthermore, this invention innovatively introduces a Spy covalent protein linkage system onto the MAEAP carrier, covalently linking SpyCatcher to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N... On a polyester carrier with carboxyl-activated hydroxysuccinimide (NHS), a PET degrading enzyme with a SpyTag linked to its C-terminus is then specifically covalently bound to SpyCatcher-SpyTag in a suitable system. Based on the specific covalent binding of SpyCatcher-SpyTag, one-step purification of the PET degrading enzyme can be achieved simultaneously, eliminating other impurities in fermentation and obtaining a high-purity immobilized PET degrading enzyme. Furthermore, since both steps of immobilization are covalent reactions, the stability of the immobilized enzyme is ensured during solid-liquid transitions, catalytic reactions, and enzyme preparation preservation, with almost no risk of detachment. The immobilized enzyme can be recycled, and 50% of the residual enzyme activity can be retained after 10 degradations of PET film, greatly reducing the cost of enzymes for PET plastic degradation, reducing interference from impurities in the enzymatic reaction system, and reducing the difficulty of product separation and purification.

[0007] Specifically, the present invention provides the following technical solution.

[0008] In a first aspect, the present invention provides an immobilized enzyme for degrading insoluble substrates, wherein the immobilization carrier of the immobilized enzyme for degrading insoluble substrates is a pH-responsive carrier, the pH-responsive carrier is covalently bound to SpyCatcher, the enzyme for degrading insoluble substrates is bound to SpyTag, and SpyTag is specifically covalently bound to SpyCatcher.

[0009] Preferably, the pH-responsive carrier is an anionic acrylic polymer, more preferably a methacrylate-ethyl acrylate copolymer; and / or preferably,

[0010] The degrading enzyme targeting insoluble substrates is a PET degrading enzyme or a PU degrading enzyme; and / or preferably,

[0011] The PET-degrading enzyme is HiT101, LCC, IsPETase, and / or TurboPETase;

[0012] The PU-degrading enzyme is PURase, AbPURase, and / or Aes72; and / or preferably,

[0013] The enzyme that degrades insoluble substrates binds to SpyTag at its C-terminus.

[0014] Secondly, the present invention provides a method for preparing an immobilized enzyme that degrades insoluble substrates, the method comprising the following steps:

[0015] (1) Pretreatment of the immobilization carrier for the enzyme immobilization of the insoluble substrate degradation enzyme, wherein the immobilization carrier is a pH-responsive carrier;

[0016] (2) SpyCatcher protein was immobilized onto the pH-responsive vector;

[0017] (3) The degradative enzyme for insoluble substrates bound to SpyTag is immobilized onto the pH-responsive carrier;

[0018] Preferably, the method further includes step (4) purifying the immobilized enzyme that degrades insoluble substrates according to the pH response characteristics of the pH-responsive carrier.

[0019] Furthermore, the method for preparing immobilized enzymes that degrade insoluble substrates as described above,

[0020] The pH-responsive carrier mentioned in step (1) is MAEAP, and the pretreatment includes activating the immobilized enzyme carrier using EDC and NHS activating reagents;

[0021] In step (3), the degrading enzyme targeting the insoluble substrate binds to SpyTag at its C-terminus, and the target degrading enzyme targeting the insoluble substrate is immobilized onto the pH-responsive carrier by the specific covalent binding of SpyTag and SpyCatcher; preferably, the degrading enzyme targeting the insoluble substrate is HiT101, LCC, IsPETase, and / or TurboPETase;

[0022] In step (4), the pH of the immobilized enzyme system is adjusted to acidity so that the immobilized enzyme precipitates for further purification; preferably, the pH is adjusted to pH < 5.5, more preferably the pH is adjusted to 4.5.

[0023] Furthermore, as described above, the method for preparing immobilized enzymes that degrade insoluble substrates...

[0024] Step (1) further includes dispersing the immobilized carrier MAEAP in a buffer solution and stirring until a uniform dispersion system is formed; adding EDC and NHS activation reagents in sequence, stirring to activate and remove excess activation reagents, and obtaining the activated pH-responsive carrier;

[0025] Step (2) further includes dissolving SpyCatcher protein in buffer to prepare a protein solution, mixing it with the activated carrier, reacting it to immobilize SpyCatcher on the surface of the activated carrier. Preferably, after the reaction, the unbound free SpyCatcher protein is washed with buffer to remove it, thereby obtaining the MAEMP-SpyCatcher complex.

[0026] Step (4) further includes mixing the C-terminal fused SpyTag degrading enzyme for insoluble substrates with the MAEMP-SpyCatcher complex, adjusting the pH of the system to alkaline, reacting to degrade the insoluble substrates, adjusting the pH to acidic after the reaction, washing to remove unbound impurities, and obtaining a high-purity immobilized degrading enzyme for insoluble substrates.

[0027] Furthermore, the method for preparing immobilized enzymes that degrade insoluble substrates, as described above,

[0028] In step (1), the ratio of the carrier to the buffer solution is 2-50 mg:1 ml, preferably 5-20 mg:1 ml, more preferably 10 mg:1 mL; the buffer solution is preferably phosphate buffer, more preferably with pH 6.0 and a concentration of 0.1 mol / L; the final concentration of EDC is 5-80 mmol / L, preferably 10-40 mmol / L, more preferably 20 mmol / L; the final concentration of NHS is 2.5-40 mmol / L, preferably 5-20 mmol / L, more preferably 10 mmol / L; and the activation is carried out at room temperature in the dark with stirring for 15-100 min, preferably 60 min.

[0029] In step (2), the buffer solution is pH 7.0 Tris-HCl buffer, and the concentration of the prepared protein solution is 0.25-4 mg / mL, preferably 0.5-2 mg / mL, and more preferably 1 mg / mL; the volume ratio of SpyCatcher protein solution to the activated carrier is 1:0.5-2, preferably 1:1.25.

[0030] In step (4), adjusting the pH of the system to alkaline means adjusting the pH value to 7.5-10.5, preferably 8-9, and more preferably pH 8. Adjusting the pH to acidic means adjusting the pH value to pH < 5.5, preferably 4.5.

[0031] Thirdly, the present invention provides a kit for preparing an immobilized enzyme that degrades an insoluble substrate, the kit comprising a pH-responsive carrier, a SpyCatcher, a SpyTag, and an enzyme that degrades an insoluble substrate, wherein the pH-responsive carrier, SpyCatcher, SpyTag, and the enzyme that degrades an insoluble substrate are spatially separated; preferably, the pH-responsive carrier is MAEAP.

[0032] The kit described above also includes an activator for MAEAP, preferably EDC and / or NHS; preferably, the kit also includes a pH adjuster.

[0033] Fourthly, the present invention provides the application of the described enzyme immobilization for degrading insoluble substrates, the method for immobilizing the described enzyme for degrading insoluble substrates, or the kit described therein in the degradation of insoluble substrates.

[0034] Compared with existing technologies, the high-purity, high-activity pH-responsive immobilized PET degrading enzyme prepared by this invention breaks through the technical bottleneck of traditional immobilized enzymes, and combines multiple advantages such as high catalytic activity, high purity, high stability, easy recovery and reuse, and low cost. Specific beneficial effects are as follows:

[0035] 1. Eliminating substrate steric hindrance and significantly improving the catalytic degradation efficiency of insoluble PET substrates: This invention uses methacrylic acid-ethyl acrylate copolymer as a pH-responsive carrier, abandoning the traditional rigid solid carrier. In the optimal catalytic pH system of 8.0-9.0 for PET degradation enzymes, the carrier completely dissolves, and the immobilized enzyme is dispersed in the reaction system in a homogeneous liquid form, which can freely and fully contact insoluble PET plastic films, particles, and other solid substrates. This completely solves the problems of steric hindrance, low mass transfer efficiency, and low probability of active center contact with substrates existing in traditional solid carriers. Compared with traditional immobilized enzymes, the catalytic efficiency of PET degradation is significantly improved, and it can be adapted to the degradation treatment of various solid PET waste plastics.

[0036] 2. Achieving simultaneous enzyme immobilization and one-step purification to obtain high-purity immobilized enzymes and reduce product separation difficulty: This invention employs the SpyCatcher-SpyTag specific covalent binding system, targeting only the PET-degrading enzyme with SpyTag fusion at its terminal end. This effectively retains the target enzyme protein and completely removes non-target components such as impurities, residual culture medium, and metabolic impurities from the fermentation broth. It eliminates the need for complex purification processes such as chromatography, ultrafiltration, and centrifugation, achieving enzyme immobilization and purification in one step, significantly simplifying the preparation process. Simultaneously, the reaction system free from impurities effectively improves the purity of the reaction system, reducing the difficulty of separating and purifying degradation products such as terephthalic acid and ethylene glycol, and improving product recovery purity.

[0037] 3. Dual covalent immobilization, extremely strong structural and catalytic stability, and reusable multiple times: This invention employs a two-step covalent bonding immobilization mechanism. First, the carboxyl groups of the carrier are activated using EDC / NHS activating reagents, achieving a stable covalent connection between the SpyCatcher and the carrier. Then, specific covalent binding achieves a strong coupling between the C-terminal SpyTag-fused PET degrading enzyme and the MAEAP-SpyCatcher. This dual covalent structure completely eliminates the problem of enzyme molecule detachment and leakage. Compared to physically adsorbed and encapsulated immobilized enzymes, the immobilized enzyme of this invention exhibits significantly improved resistance to mechanical shearing, pH fluctuations, and temperature changes, excellent preservation stability, and retains more than 50% of residual enzyme activity after 10 cycles of use. This significantly reduces enzyme preparation consumption and substantially lowers the industrial application cost of PET biodegradation.

[0038] 4. pH reversible solid-liquid conversion, simple and efficient recovery method with low energy consumption: The carrier of this invention has precise pH response and reversible phase change characteristics. Dissolution under alkaline catalytic conditions ensures high catalytic activity. After the reaction, adjusting the pH of the system to 4.5 can quickly cause agglomeration and precipitation. The immobilized enzyme can be completely recovered by conventional low-speed centrifugation. The recovery process is simple, energy-efficient, and has minimal enzyme loss, making it more suitable for large-scale continuous degradation production scenarios in industrial applications. Attached Figure Description

[0039] Figure 1 SDS-PAGE image of proteins purified by SpyCatcher.

[0040] Figure 2 Electrophoresis image of extracellular expression of the PET-degrading enzyme HiT101. Lane 1 is HiT101-SpyTag, and lane 2 is HiT101.

[0041] Figure 3Fourier transform infrared (FTIR) spectroscopy results of immobilized enzymes. Where: A: Immobilized enzyme in solid state; B: FTIR results of solid-state immobilized enzymes; C: Immobilized enzyme in liquid state; D: FTIR results of liquid-state immobilized enzymes.

[0042] Figure 4 Results of the recycling test of immobilized enzymes. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0044] Example 1: Preparation of high-purity, high-activity PET-degrading enzyme immobilized enzyme

[0045] 1.1 pH-responsive carrier pretreatment and activation

[0046] 100 mg of the methacrylate-ethyl acrylate copolymer (MAEAP) was selected as the immobilization carrier. 100 mg of the carrier was dispersed in 10 mL of phosphate buffer (pH 6.0, 0.1 mol / L) and stirred until a uniform dispersion was formed. EDC and NHS activating agents were added sequentially, and the final concentrations of EDC and NHS were controlled to be 20 mmol / L and 10 mmol / L, respectively. The carrier was activated by stirring at room temperature in the dark for 60 min to activate the carboxyl sites on the surface of the carrier. After activation, the excess activating agent was removed by low-speed centrifugation to obtain the activated pH-responsive carrier.

[0047] 1.2 SpyCatcher protein fixation

[0048] The SpyCatcher encoding gene was heterologously expressed using the E. coli BL21(DE3) and PET28a expression systems. The SpyCatcher protein obtained by IPTG-induced expression was purified by affinity chromatography. Figure 1 Dissolve the SpyCatcher protein in pH 7.0 Tris-HCl buffer to prepare a 1 mg / mL protein solution; take 8 mL of SpyCatcher protein solution and mix it with the activated carrier from step 1.1 above, and stir at room temperature for 2 h to immobilize SpyCatcher on the carrier surface through carboxyl covalent binding. After the reaction, dialyze against pH 7.0 Tris-HCl buffer for 4 h to remove unbound free SpyCatcher protein and obtain the MAEMP-SpyCatcher complex.

[0049] 1.3 Immobilization and simultaneous purification of the target PET-degrading enzyme

[0050] Take the fermented PET-degrading enzyme HiT101-SpyTag containing C-terminal fusion expression of SpyTag ( Figure 2 The fermentation broth (or other reported PET degrading enzymes such as LCC, IsPETase, TurboPETase, etc.) was centrifuged at low temperature and high speed to remove cell precipitate, and the supernatant was collected. The supernatant was mixed with the MAEMP-SpyCatcher complex at a volume ratio of 4:1, and the pH of the system was adjusted to 8.0 with 2M NaOH. The reaction was carried out at 30℃ with gentle stirring for 90 min. Only the target PET degrading enzyme with SpyTag in the system can specifically covalently bind to SpyCatcher on the carrier surface. Impurities such as proteins, polysaccharides, and metabolic impurities in the fermentation broth are retained in the liquid phase, achieving enzyme immobilization and one-step purification. Subsequently, the solution was precipitated with 3M acetic acid to pH=4.5, and centrifuged at 8000-10000 rpm at 4℃ for 10 min to obtain solid-state immobilized enzyme. At the same time, unbound impurities in the liquid were removed to obtain high-purity immobilized PET degrading enzyme HiT101ST-SC@MAEAP.

[0051] Example 2 Performance testing of immobilized enzymes

[0052] 2.1 Fourier Transform Infrared Spectroscopy (FTIR) Detection of Immobilized Enzymes

[0053] To verify the chemical structural stability and functional group changes of the pH-responsive immobilized enzyme in different phases, Fourier transform infrared (FTIR) spectroscopy was performed on the high-purity immobilized PET degrading enzyme in both liquid (pH 8.0, catalytic working state) and solid (pH 4.5, recovery and settling state) states. Combined with changes in the infrared absorption peaks of the carrier methacrylate-ethyl acrylate copolymer, the SpyCatcher-SpyTag covalent binding system, and the characteristic functional groups of the enzyme protein, structural change analysis was completed. Figure 3 The FTIR characteristic peaks of the immobilized enzyme in this invention mainly cover the core groups of the three major systems: polymer carrier, covalent linking structure, and enzyme protein: 3200-3600 cm⁻¹. -1 Broad peaks for OH and NH stretching vibrations (carrier hydrophilic groups, enzyme protein peptide bonds, bound water); 2920-2980 cm⁻¹ -1 The stretching vibration is alkyl CH (alkyl structure of copolymer main chain); 1725-1740 cm⁻¹ -1 Characteristic peaks of C=O groups in copolymer ester groups; 1630-1650 cm⁻¹ -1 The protein amide I band (peptide bond C=O stretching); 1520-1540 cm⁻¹ -1 It is a proteinamide II band (NH bending + CN stretching); 1400-1450cm -1 It is a carboxyl group symmetric stretching vibration; 1150-1250 cm⁻¹ -1 It is the stretching vibration of the ester group COC.

[0054] In an alkaline catalytic system at pH 8.0, the immobilized enzyme completely dissolves and forms a homogeneous liquid state. Figure 3 The FTIR spectral characteristics are as follows (C). Figure 3 D):

[0055] 1. Amide I band / support carbonyl group (1633 cm⁻¹) -1 / 1636cm -1 (C=O), the carbonyl peak of both the blank vector MAEAP and the free enzyme was stable at 1633 cm⁻¹ in liquid chromatography. -1 The immobilized enzyme HiT101ST-SC@MAEAP shifted slightly to 1636 cm⁻¹. -1 The enhanced hydration hydrogen bonding in the liquid environment and the slight peak shift indicate that the enzyme protein and the carrier have formed a covalent cross-link, and the microenvironment around the carbonyl group has changed. The three sets of peaks are all concentrated and sharp, without any split peaks, proving that the peptide bond structure of the enzyme protein is intact in the liquid homogeneous system, the SpyCatcher-SpyTag covalent bond is stable, the enzyme secondary structure has not been denatured or unfolded, and the carbonyl backbone of the carrier is also intact.

[0056] 2. Characteristic peaks of ester bonds on the carrier (1075 / 1078 cm⁻¹) -1 The peak positions of the COC, MAEAP carrier, and liquid-phase immobilized enzyme were all 1075 cm⁻¹. -1 The free enzyme concentration is 1078 cm⁻¹. -1 1075cm -1 This is a polymer-specific ester bond signal. The peak positions of the immobilized enzyme and the blank carrier coincide, directly proving that the ester COC backbone of the carrier and the cross-linking chemical bonds between the carrier and the enzyme are not broken in the liquid dissolved state; the free enzyme has a peak position of 1078 cm⁻¹. -1 The peak difference between the oxygen-containing groups of the protein itself can distinguish between the carrier and enzyme signals, providing direct evidence that the enzyme has been successfully immobilized on the carrier.

[0057] 3. Wide absorption region (3000-4000cm) -1 All three curves show a wide absorption dip in this region, a typical characteristic of hydrophilic (OH / NH) molecules in a liquid phase forming hydrated hydrogen bonds with a large number of water molecules. The absorption intensity of the immobilized enzyme in this region is similar to that of the carrier, indicating that the carboxyl groups of the carrier ionize and expand, and are fully hydrated and dissolved in the alkaline liquid phase. This allows the immobilized enzyme to be uniformly dispersed in the liquid phase without aggregation or precipitation. This provides a spectral explanation for the efficient contact between the homogeneous liquid phase and the substrate. In summary, under homogeneous liquid conditions: the carrier chemical framework is stable, the enzyme protein secondary structure is intact, and the covalent bond between the enzyme and the carrier is strong; the carrier is fully hydrated and dissolved, and the immobilized enzyme exists in a dispersed homogeneous form, with a structure adapted to the catalytic reaction.

[0058] After adjusting the pH of the system to 4.5, the immobilized enzyme rapidly aggregated and precipitated, forming a solid precipitate. Figure 3 (A), its FTIR spectrum shows a characteristic reversible change compared to the liquid state ( Figure 3 (Medium B): 1.3200-3600cm -1 (OH, NH stretching regions, Figure 3 The peak position of the free sample (blue, labeled 3409 and 3482) is 3409 cm⁻¹. -1 The immobilized enzyme HiT101ST-SC@MAEAP (red) and the blank vector MAEAP (light blue) were both shifted to 3482 cm⁻¹. -1 The shift of peak positions to higher wavenumbers and the broadening and dispersion of peak shapes indicate weakening of hydrogen bonds and an increase in hydrated hydrogen bonds in hydrophilic groups. After immobilization, the carboxyl groups on the support ionize, and the hydrophilic groups bind a large number of water molecules to form hydrated hydrogen bonds, providing direct spectral evidence of the support's dissolution and dispersion in alkaline systems. 2. Characteristic peaks of the C=O carbonyl group (1708, 1711 cm⁻¹). -1 The blank MAEAP is 1708cm. -1 The immobilized enzyme was 1711 cm⁻¹ -1 The free sample did not show a significant strong peak in this region. The 1708 / 1711 peak is attributed to the C=O stretching of the ester group / incompletely ionized carboxyl group in the carrier: This strong absorption is absent in the free state under alkaline conditions, while the immobilized sample retains this peak, indicating that the ester group structure of the carrier backbone and the covalent crosslinking bond between the carrier and SpyCatcher are not broken; simultaneously, the absence of this peak in the free sample confirms that the free enzyme does not possess the carbonyl structure of the polymer carrier. 3. Amide II band region (1578, 1547, 1544 cm⁻¹) -1 (Protein characteristics) Free: 1578 cm⁻¹ -1 Immobilized enzyme: 1547 cm -1 Blank vector MAEAP: 1544 cm⁻¹ -1 The blank carrier is only 1544cm. -1 Weak absorption, 1547cm -1 The amide II bands (NH bending + CN stretching) of the immobilized enzyme demonstrate successful loading of the enzyme protein structure; the free enzyme is 1578 cm⁻¹. -1 With immobilized enzyme 1547cm -1 The peaks are clearly distinguishable, sharp, and without splitting, indicating stable covalent binding between SpyCatcher and SpyTag, and that the secondary structure of the PET degradative enzyme is intact and undenatured / unfolded; the vector is 1544 cm⁻¹. -1 Background peaks do not interfere with the identification of protein characteristic peaks, and can clearly distinguish between carrier and enzyme protein signals.

[0059] In summary, the FTIR spectral changes of the immobilized enzyme of this invention exhibit high reversibility and structural stability: at pH 8.0, the carboxyl groups ionize and the molecules expand to become hydrophilic, ensuring homogeneous reaction in the catalytic system and eliminating substrate contact steric hindrance; at pH 4.5, the carboxyl groups protonate and the molecules aggregate to become hydrophobic, enabling rapid precipitation and recovery. Throughout the process, only the physical conformation of the carrier and the hydrogen bond network undergo reversible changes. The Spy covalent linkage system, the secondary structure of the enzyme protein, and the covalent cross-linked structure of the carrier remain stable. This microscopic chemical structure verifies the high activity, high stability, reversible cycling, and enzyme-free characteristics of the immobilized enzyme of this invention, providing a solid structural theoretical basis for its industrial-scale cyclic degradation application.

[0060] 2.2 Catalytic Activity Detection

[0061] Free enzyme detection group: Prepare a 50 mL pH 8.0 Tris-HCl buffer solution, add 0.008 mg of unimmobilized, terminally fused SpyTag purified free PET degrading enzyme, and use 8.0 mg (0.6 cm diameter) spherical PET films as substrate. Incubate at 70 °C and 150 rpm for 10 h with constant temperature shaking. After the reaction, remove the remaining film, dry and weigh it, and calculate the initial catalytic specific enzyme activity.

[0062] Immobilized enzyme assay group: High-purity immobilized PET-degrading enzyme prepared in Example 1 of this invention, containing 0.008 mg of enzyme protein, was used to detect enzyme activity in a standard system with temperature, pH, substrate amount, shaker speed, and reaction time completely identical to those of the free enzyme. The initial specific enzyme activity of the immobilized enzyme was calculated.

[0063] Definition of PET degrading enzyme activity: In this invention, the PET degrading enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the degradation of 1 mg of PET film substrate per hour under standard reaction conditions of 70°C and pH 8.0, which is one enzyme activity unit U. Specific enzyme activity is defined as U / mg, representing the catalytic activity of one milligram of enzyme protein.

[0064] Initial enzyme activity assays showed that, compared to the free PET degrading enzyme, the initial specific enzyme activity was 28.6 U / mg, obtained from three parallel replicate experiments. The covalently immobilized PET degrading enzyme prepared in Example 1 of this invention achieved an initial specific enzyme activity of 45.8 U / mg. Compared to the free enzyme, the immobilized enzyme exhibited a 60.1% increase in initial catalytic activity, significantly overcoming the technical shortcomings of traditional immobilized enzymes, such as decreased enzyme activity and masking of active sites after immobilization. This is attributed to the fact that the pH-responsive carrier of this invention completely dissolves in the catalytic system, thoroughly eliminating steric hindrance from the solid carrier and fully exposing the enzyme's active sites. Simultaneously, the Spy-specific covalent immobilization method precisely anchors the enzyme protein without disrupting its secondary spatial structure, and provides fixation and stability protection for the enzyme's spatial structure during long-term catalytic reactions.

[0065] Example 3: Application Test of Immobilized Enzyme Recovery and Recycling

[0066] Take the reaction solution after the immobilized enzyme degradation in Example 2, slowly adjust the pH of the system to 4.5 with dilute hydrochloric acid, let it stand at room temperature for 5 minutes, the immobilized enzyme rapidly aggregates to form a white precipitate, centrifuge at 8000 rpm for 5 minutes, and collect the precipitate completely to obtain the recovered immobilized enzyme. The immobilized enzyme mass recovery rate can reach 96.8%.

[0067] The recovered immobilized enzyme was redispersed in pH 8.0 buffer, and the PET film degradation experiment was repeated. The single degradation reaction conditions were exactly the same as in Example 2, and the cycle was repeated 10 times. The residual enzyme activity was measured after each cycle. With the initial immobilized enzyme activity as 100% baseline, the activity remaining was approximately 68% after the 8th cycle and approximately 50% after the 10th cycle. Figure 4 This fully demonstrates that the dual covalent fixation structure of the present invention can effectively prevent enzyme shedding and inactivation, exhibits excellent cycling stability, and can significantly reduce enzyme costs.

Claims

1. An immobilized enzyme for degrading insoluble substrates, characterized in that, The immobilization carrier for the enzyme that degrades insoluble substrates is a pH-responsive carrier. The pH-responsive carrier is covalently bound to SpyCatcher. The enzyme that degrades insoluble substrates is bound to SpyTag. SpyTag is specifically covalently bound to SpyCatcher. The pH-responsive carrier is an anionic acrylic polymer material, methacrylate-ethyl acrylate copolymer; The degrading enzyme targeting insoluble substrates is HiT101, and the degrading enzyme targeting insoluble substrates binds to SpyTag at its C-terminus.

2. A method for preparing an immobilized enzyme for degrading insoluble substrates as described in claim 1, characterized in that, The method Includes the following steps, (1) Pretreatment of the immobilization carrier for the enzyme immobilization of the insoluble substrate degradation enzyme, wherein the immobilization carrier is a pH-responsive carrier; (2) SpyCatcher protein was immobilized onto the pH-responsive vector; (3) The degradative enzyme for insoluble substrates bound to SpyTag is immobilized onto the pH-responsive carrier; The degradative enzyme targeting insoluble substrates is HiT101.

3. The method for preparing an immobilized enzyme for degrading insoluble substrates according to claim 2, characterized in that, The pH-responsive carrier mentioned in step (1) is MAEAP, and the pretreatment includes activating the immobilized enzyme carrier using EDC and NHS activating reagents; In step (3), the degrading enzyme targeting insoluble substrates binds to SpyTag at its C-terminus, and the degrading enzyme targeting insoluble substrates is immobilized onto the pH-responsive carrier by the specific covalent binding of SpyTag and SpyCatcher. It also includes step (4) purifying the immobilized enzyme that degrades insoluble substrates according to the pH response characteristics of the pH-responsive carrier.

4. The method for preparing an immobilized enzyme for degrading insoluble substrates according to claim 3, characterized in that, Step (1) further includes dispersing the immobilized carrier MAEAP in a buffer solution and stirring until a uniform dispersion system is formed; adding EDC and NHS activation reagents in sequence, stirring to activate and remove excess activation reagents, and obtaining the activated pH-responsive carrier; Step (2) further includes dissolving SpyCatcher protein in buffer to prepare a protein solution, mixing it with the activated carrier, reacting to immobilize SpyCatcher on the surface of the activated carrier, and washing with buffer after the reaction to remove unbound free SpyCatcher protein to obtain the MAEMP-SpyCatcher complex; In step (4), the pH of the immobilized enzyme system is adjusted to acidity so that the immobilized enzyme precipitates for further purification; the pH value is adjusted to pH < 5.

5.

5. The method for preparing an immobilized enzyme for degrading insoluble substrates according to claim 4, characterized in that, In step (1), the ratio of the carrier to the buffer is 2-50 mg: 1 ml; the buffer is phosphate buffer; the final concentration of EDC is 5-80 mmol / L; the final concentration of NHS is 2.5-40 mmol / L; and the activation is carried out at room temperature in the dark with stirring for 15-100 min. In step (2), the buffer solution is pH 7.0 Tris-HCl buffer, and the concentration of the prepared protein solution is 0.25-4 mg / mL; the volume ratio of SpyCatcher protein solution to the activated carrier is 1:0.5-2. Step (4) further includes mixing the C-terminal fused SpyTag degrading enzyme for insoluble substrates with the MAEMP-SpyCatcher complex, adjusting the pH of the system to alkaline, reacting to degrade the insoluble substrates, adjusting the pH to acidic after the reaction, washing to remove unbound impurities, and obtaining a high-purity immobilized degrading enzyme for insoluble substrates.

6. The method for preparing an immobilized enzyme for degrading insoluble substrates according to claim 4, characterized in that, The PET-degrading enzyme is HiT101, LCC, IsPETase, and / or TurboPETase; In step (1), the ratio of the carrier to the buffer is 5-20 mg: 1 ml; the buffer is phosphate buffer with pH 6.0 and a concentration of 0.1 mol / L; the final concentration of EDC is 10-40 mmol / L; the final concentration of NHS is 2.5-40 mmol / L; and the mixture is activated at room temperature in the dark with stirring for 60 min. In step (2), the concentration of the prepared protein solution is 0.5-2 mg / mL; the volume ratio of the SpyCatcher protein solution to the activated carrier is 1:1.

25. In step (4), adjusting the pH of the system to alkaline means adjusting the pH value to 8-9, and adjusting the pH to acidic means adjusting the pH value to 4.

5.

7. A kit for preparing immobilized enzymes that degrade insoluble substrates, characterized in that, The kit includes a pH-responsive carrier, SpyCatcher, SpyTag, and a degrading enzyme for insoluble substrates, wherein the pH-responsive carrier, SpyCatcher, SpyTag, and the degrading enzyme for insoluble substrates are spatially separated; the degrading enzyme for insoluble substrates is HiT101.

8. The kit according to claim 7, characterized in that the pH-responsive carrier is MAEAP; the kit further includes an activator of MAEAP.

9. The kit according to claim 8, characterized in that the activator is EDC and / or NHS; the kit further comprises a pH adjuster.

10. The use of the enzyme immobilized for degrading insoluble substrates according to claim 1, the method for immobilizing enzymes for degrading insoluble substrates according to any one of claims 2-6, or the kit according to any one of claims 7-9 in the degradation of insoluble substrates.

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