A highly active immobilized plastic-degrading enzyme preparation and its application

Abstract

This invention discloses a highly active immobilized plastic-degrading enzyme preparation and its application, belonging to the field of plastic degradation technology. The enzyme preparation is prepared through the following steps: using Pinus tabuliformis pollen as raw material, acid treatment, carbonization, and activation are performed to form a multi-level porous active pollen carrier; lanthanum-doped magnetic nanoparticles and thiol-functionalized modification are used to endow the carrier with magnetic responsiveness and directional immobilization sites; a three-dimensional cross-linked structure is constructed using a rigid triazine ring conjugated monomer and a hyperbranched network, combined with UV click reaction to achieve spatial confinement immobilization of enzyme molecules; keratinase, lipase, cellulase, laccase, and peroxidase are synergistically loaded to form a multi-enzyme catalytic system. The multi-level pores of the pollen base combined with the rigid triazine ring network significantly improve the enzyme loading and distribution uniformity; the cross-linked network protects the enzyme preparation, maintaining over 90% enzyme activity retention after 10 cycles; and rapid magnetic separation is achieved through lanthanum-doped iron oxide nanoparticles.

Description

Technical Field

[0001] This invention belongs to the field of plastic degradation technology, and in particular relates to a highly active immobilized plastic degradation enzyme preparation and its application. Background Technology

[0002] Plastics, as an important synthetic polymer material, have been widely used in industrial production and daily life for decades due to their excellent durability and low cost. However, traditional petroleum-based plastics (such as polyethylene, polypropylene, and polystyrene) are difficult to degrade in the natural environment, with degradation cycles typically lasting hundreds of years. These plastic wastes entering soil and oceans cause plastic pollution problems that negatively impact ecosystems and human health. Although the development and application of biodegradable plastics (such as polylactic acid and polyhydroxyalkanoates) have been considered an important way to alleviate plastic pollution in recent years, biodegradable plastics, under specific natural conditions such as composting, are degraded by naturally occurring microorganisms, eventually degrading completely into carbon dioxide, methane, water, and mineralized inorganic salts of their constituent elements.

[0003] Microorganisms and enzymes are indispensable in the plastic degradation process. For example, keratinase and lipase can break down ester bonds in plastic molecular chains through hydrolysis, while laccase and peroxidase promote the disintegration of plastic structures through oxidation. However, free enzymes have significant drawbacks in practical applications: firstly, enzyme molecules are easily denatured and inactivated in high temperatures, extreme pH levels, or organic solvents, leading to a sharp drop in catalytic efficiency; secondly, free enzymes are difficult to recycle and reuse, significantly increasing usage costs; and thirdly, the low contact efficiency between enzymes and hydrophobic plastic substrates limits the degradation rate. To overcome these problems, immobilized enzyme technology has been introduced into the field of plastic degradation. In existing technologies, carrier materials immobilize enzyme molecules through physical adsorption or chemical bonding, which can improve enzyme stability and reusability to a certain extent. However, traditional immobilized carriers have a simple pore structure and limited specific surface area, resulting in low enzyme loading and uneven distribution. The interaction between the carrier and enzyme molecules is weak, and the immobilized enzyme is easily detached due to mechanical shearing or substrate impact. Therefore, there is an urgent need to develop an immobilized plastic degradation enzyme preparation that has high enzyme activity retention, strong structural stability, easy separation and recovery, and synergistic effects of multiple enzymes. Summary of the Invention

[0004] To address the above issues and overcome the shortcomings of existing technologies, this invention provides a highly active immobilized plastic-degrading enzyme formulation based on the synergistic effect of pollen hierarchical porous structure and three-dimensional cross-linked network. By constructing a composite carrier with magnetic response and directional enzyme immobilization sites, combined with a multi-enzyme synergistic catalytic system, the problems of low enzyme loading, poor enzyme activity retention, and insufficient reusability of traditional immobilized enzymes are solved. This formulation uses a pollen carbon-based carrier modified with lanthanum-doped magnetic nanoparticles, combined with a hyperbranched network formed by UV click reaction, achieving spatial confinement immobilization and conformational stabilization protection of enzyme molecules. At the same time, through the rigid conjugation effect of the triazine ring and the synergistic effect of the porous structure, the substrate mass transfer efficiency and catalytic activity of the enzyme formulation are significantly improved, enabling it to maintain high degradation performance under complex environmental conditions.

[0005] To achieve the above objectives, the following technical solution is adopted: On one hand, the present invention provides a highly active immobilized plastic-degrading enzyme preparation, which is prepared through the following steps:

[0006] S1. Preparation of active pollen: Add pine pollen to a 0.5-1.5 mol / L hydrochloric acid solution and stir in a water bath at 60-80℃ for 2-4 hours to remove impurities and waxes. Then filter, wash with deionized water until neutral, and dry with hot air at 80℃ to constant weight. Place the dried pollen in a tube furnace and introduce a mixture of nitrogen and carbon dioxide gas. Heat to 500-700℃ at 2-10℃ / min for 1-3 hours for carbonization. After cooling, immerse the carbonized pollen in a 10-30% (w / w) sodium lignosulfonate solution and activate at 700-800℃ for 0.5-2 hours. Wash the product with deionized water until no chloride ions remain to obtain active pollen.

[0007] S2. Magnetic composite modification: The active pollen obtained in step S1 is impregnated in a mixed solution of hydrated ferric chloride and hydrated ferrous sulfate, hydrated lanthanum nitrate is added, the pH is adjusted to 9-11, and the mixture is stirred at 60-80℃ for 2-4 hours to generate lanthanum-doped iron oxide nanoparticles. Subsequently, mercaptopropionic acid and polyethylene glycol diacrylate are added, and the mixture is reacted at 50-70℃ for 6-12 hours under ultrasonic assistance at a power of 200-500W and a frequency of 28-40kHz. After the reaction is completed, the product is collected by magnetic separation, washed three times with ethanol and deionized water, and dried under vacuum at 50℃ to obtain mercapto-functionalized magnetic pollen.

[0008] S3. Construction of cross-linked network: 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazine trione was dissolved in N,N-dimethylformamide, and dihydroxyethylglycine was dissolved in deionized water to prepare a first solution and a second solution, respectively. The second solution was added to the first solution, and then the magnetic pollen was added. The mixture was stirred at 40-60°C for 4-8 hours. The product was filtered, washed three times alternately with DMF and deionized water, and dried at room temperature. The product was placed in an ultraviolet light reactor for ultraviolet irradiation for 10-30 minutes to allow the thiol groups and epoxy groups to cross-link by click. The final product was washed with acetone to remove unreacted monomers and dried under vacuum at 40°C for 12 hours to obtain an immobilized carrier with a three-dimensional cross-linked network colonized.

[0009] S4. Enzyme Immobilization: Keratinase, lipase, cellulase, laccase, and peroxidase were mixed in proportion and dissolved in phosphate buffer at pH 7.5 to prepare a composite enzyme solution. The carrier was mixed with the composite enzyme solution and immersed in the solution by shaking at 25-37℃ for 2-4 hours. After magnetic separation, the immobilized enzyme was washed three times with phosphate buffer at pH 7.5 to remove free enzyme. The washed immobilized enzyme was then shaken in 0.1mol / L NaCl solution for 1 hour. After magnetic separation again, the enzyme was rinsed with deionized water, pre-frozen at -40℃ for 4 hours, and then dried in a freeze dryer for 12 hours to obtain the highly active immobilized plastic degradation enzyme preparation.

[0010] Furthermore, in step S1, the feeding ratio of Pinus tabuliformis pollen to hydrochloric acid solution is 1g:8-12mL, and the feeding ratio of carbonized pollen to sodium lignosulfonate solution in step S1 is 1g:3-5mL.

[0011] Furthermore, in step S1, the volume ratio of the nitrogen to carbon dioxide mixture is nitrogen:carbon dioxide = 1:0.2-0.5, and the flow rate of the mixture is 10-20 L / min.

[0012] Further, in step S2, the molar ratio of ferric ions to ferrous ions in the mixed solution is 1.5-2.5:1, and the total ferric ion concentration of the mixed solution is 0.5-1.5 mol / L.

[0013] Furthermore, in step S2, the ratio of active pollen to mixed solution is 1g:20-50mL.

[0014] Furthermore, in step S2, the amount of lanthanum nitrate hydrate added is 5-15% of the total mass of iron salts in the mixed solution, the amount of mercaptopropionic acid added in step S2 is 8-20% of the mass of active pollen, and the amount of polyethylene glycol diacrylate added is 5-15% of the mass of active pollen.

[0015] Furthermore, in step S3, the feeding ratio of 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazinetrione to N,N-dimethylformamide in the first solution is 1g:20-40mL.

[0016] Furthermore, in step S3, the ratio of dihydroxyethylglycine to deionized water in the second solution is 1g: 50-100mL.

[0017] Furthermore, in step S3, the feeding ratio of the first solution, the second solution, and the magnetic pollen is 10-20 mL: 5-15 mL: 1-3 g.

[0018] Furthermore, in step S3, the ultraviolet wavelength is 365nm, the power is 50-100W, the irradiation distance is maintained at 10-15cm, and nitrogen gas with a flow rate of 10-20L / min is introduced for protection during ultraviolet irradiation.

[0019] Further, in step S4, the mass ratio of keratinase, lipase, cellulase, laccase and peroxidase in the complex enzyme is 1-3:1-3:2-5:0.1-0.5:0.5-1, and the concentration of the complex enzyme in the complex enzyme solution in step S4 is 10-30 mg / mL.

[0020] Further, in step S4, the carrier and the complex enzyme solution are mixed at a carrier-to-enzyme mass ratio of 1:5-15.

[0021] On the other hand, the present invention also provides the application of the highly active immobilized plastic-degrading enzyme preparation, in which the enzyme preparation is added to biodegradable plastics to promote the degradation of biodegradable plastics.

[0022] The beneficial effects of this invention are:

[0023] (1) The immobilized plastic degrading enzyme preparation disclosed in this invention is based on the multi-level pore structure of the pollen-based carrier to form an immobilized carrier with a three-dimensional cross-linked network for spatial confinement loading of keratinase, lipase, cellulase, laccase and peroxidase. Compared with the free plastic degrading enzyme preparation, the immobilized plastic degrading enzyme preparation constructed in this invention has significantly improved thermal stability, pH stability, mechanical stability and storage resistance, and still has a high enzyme activity retention rate after 10 cycles.

[0024] (2) The carbonized pollen was modified by thiolization and magnetic composite treatment. A hyperbranched polyetheramine network was formed on the surface of the pollen carrier through the self-assembly reaction of 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazinetrione and dihydroxyethylglycine. This network uniformly covered the surface of the pollen carrier, etched the cellulose structure in the pollen carrier, reduced the crystallinity, improved the pore structure of the carrier, and made it have higher porosity, more diverse pore sizes and distributions. It also increased the oxygen-containing functional groups on the surface of the active pollen carrier. This was further enhanced by ultraviolet induction. The thiol-alkenyl click reaction implants the structure formed by the self-assembly reaction onto the surface of the pollen carrier, thereby preventing the random aggregation of enzyme molecules on the carrier surface and forming ordered enzyme-directed fixation sites. At the same time, the addition of polyethylene glycol diacrylate forms an interpenetrating polymer network with the pollen carrier, further improving the specific surface area and mechanical strength of the pollen carrier. The carboxyl groups introduced by dihydroxyethylglycine, the sulfur groups remaining from the click reaction, and polyethylene glycol diacrylate all contribute to the adsorption and fixation of enzymes by the carrier, improving the substrate affinity of the final product enzyme preparation.

[0025] (3) The triazine structure in 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazinetrione has planar rigid conjugation characteristics. The electron-deficient characteristics of the triazine ring enable it to bind tightly with enzyme molecules or carrier materials through π-π interactions to form a stable network structure, thereby effectively inhibiting the loss of enzyme activity caused by conformational unfolding, thus maintaining the catalytic activity of the enzyme. The conjugation characteristics of the triazine ring enhance the cross-linking density of the carrier, so that the immobilized carrier will not deform or break during the degradation of plastics, thus improving the stability of the enzyme preparation for recycling.

[0026] (4) Lanthanum doping optimizes the magnetic properties of iron oxide nanoparticles, reduces coercivity and increases magnetic saturation, making it easier for iron oxide nanoparticles to be rapidly separated under an external magnetic field for immobilized enzyme preparations. Lanthanum doping can also increase the number of hydroxyl groups and oxygen vacancies on the surface of nanoparticles, providing more anchoring sites for subsequent modification with mercaptopropionic acid and polyethylene glycol diacrylate, thereby enhancing the adsorption capacity of the carrier and enzyme. Attached Figure Description

[0027] Figure 1 The images show the microstructure of Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 after PLA film degradation for 200 hours.

[0028] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0032] Example 1: A highly active immobilized plastic-degrading enzyme preparation, wherein the enzyme preparation is prepared by the following steps:

[0033] S1. Preparation of active pollen: Add pine pollen to a 1.5 mol / L hydrochloric acid solution and stir in an 80°C water bath for 4 hours to remove impurities and waxes. Then filter, wash with deionized water until neutral, and dry with hot air at 80°C to constant weight. Place the dried pollen in a tube furnace and introduce a mixed gas of nitrogen and carbon dioxide (nitrogen:carbon dioxide volume ratio of 1:0.5) at a flow rate of 20 L / min. Carbonize at 700°C for 3 hours at a rate of 10°C / min. After cooling, immerse the carbonized pollen in a 30% (w / w) sodium lignin sulfonate solution (the ratio of carbonized pollen to sodium lignin sulfonate solution is 1 g:5 mL) and activate at 800°C for 2 hours. Wash the product with deionized water until no chloride ions remain to obtain active pollen.

[0034] S2. Magnetic Composite Modification: The active pollen obtained in step S1 was impregnated in a mixed solution of hydrated ferric chloride and hydrated ferrous sulfate (the molar ratio of ferric ions to ferrous ions in the mixed solution was 2.5:1, the total ferric ion concentration in the mixed solution was 1.5 mol / L, and the feed ratio of active pollen to mixed solution was 1 g: 50 mL). Hydrated lanthanum nitrate was added (the amount of hydrated lanthanum nitrate added was 15% of the total mass of iron salts in the mixed solution), the pH was adjusted to 11, and the reaction was stirred at 80 °C for 4 h to generate lanthanum-doped iron tetroxide nanoparticles. Subsequently, mercaptopropionic acid and polyethylene glycol diacrylate were added (the amount of mercaptopropionic acid added was 20% of the mass of active pollen, and the amount of polyethylene glycol diacrylate added was 15% of the mass of active pollen). Under the ultrasonic assistance of 500 W power and 40 kHz frequency, the reaction was carried out at 70 °C for 12 h. After the reaction was completed, the product was collected by magnetic separation, washed three times with ethanol and deionized water, and dried under vacuum at 50 °C to obtain mercaptofunctionalized magnetic pollen.

[0035] S3. Construction of the cross-linked network: 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazinone was dissolved in N,N-dimethylformamide (the ratio of 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazinone to N,N-dimethylformamide in the first solution was 1 g: 40 mL), and dihydroxyethylglycine was dissolved in deionized water (the ratio of dihydroxyethylglycine to deionized water in the second solution was 1 g: 100 mL). The first and second solutions were prepared separately. The second solution was added to the first solution (first solution, second solution, magnetic pollen). The feed ratio was 20mL:15mL:3g. Then, the magnetic pollen was added, and the mixture was stirred at 60℃ for 8 hours. The product was filtered, washed three times alternately with DMF and deionized water, and dried at room temperature. The product was then placed in an ultraviolet light reactor for ultraviolet irradiation (ultraviolet wavelength 365nm, power 100W, irradiation distance 15cm, and nitrogen gas at a flow rate of 20L / min was introduced for protection during ultraviolet irradiation). The reaction was carried out for 30 minutes to allow the thiol groups and epoxy groups to crosslink by click. The final product was washed with acetone to remove unreacted monomers and dried under vacuum at 40℃ for 12 hours to obtain an immobilized carrier with a three-dimensional crosslinked network.

[0036] S4. Enzyme Immobilization: Keratinase, lipase, cellulase, laccase, and peroxidase were mixed in a mass ratio of 3:3:5:0.5:1 and dissolved in phosphate buffer at pH 7.5 to prepare a composite enzyme solution (the concentration of the composite enzyme in the composite enzyme solution was 30 mg / mL). The carrier and the composite enzyme solution were mixed at a mass ratio of 1:15 and immersed at 37°C with shaking for 4 hours. After magnetic separation, the immobilized enzyme was washed three times with phosphate buffer at pH 7.5 to remove free enzyme. The washed immobilized enzyme was then shaken in 0.1 mol / L NaCl solution for 1 hour and magnetically separated again. After rinsing with deionized water, it was pre-frozen at -40°C for 4 hours and then dried in a freeze dryer for 12 hours to obtain the highly active immobilized plastic degradation enzyme preparation.

[0037] Example 2: A highly active immobilized plastic-degrading enzyme preparation, wherein the enzyme preparation is prepared by the following steps:

[0038] S1. Preparation of active pollen: Add pine pollen to a 0.5 mol / L hydrochloric acid solution, stir in a 60℃ water bath for 2 hours to remove impurities and waxes, then filter, wash with deionized water until neutral, and dry with hot air at 80℃ to constant weight. Place the dried pollen in a tube furnace, and introduce a mixed gas of nitrogen and carbon dioxide (nitrogen:carbon dioxide volume ratio of 1:0.2) at a flow rate of 10 L / min. Carbonize at 500℃ for 1 hour at a rate of 2℃ / min. After cooling, immerse the carbonized pollen in a 10% (w / w) sodium lignin sulfonate solution (the ratio of carbonized pollen to sodium lignin sulfonate solution is 1 g:3 mL), activate at 700℃ for 0.5 hours, and wash the product with deionized water until no chloride ions remain to obtain active pollen.

[0039] S2. Magnetic Composite Modification: The active pollen obtained in step S1 was impregnated in a mixed solution of hydrated ferric chloride and hydrated ferrous sulfate (the molar ratio of ferric ions to ferrous ions in the mixed solution was 1.5:1, the total ferric ion concentration in the mixed solution was 0.5 mol / L, and the feed ratio of active pollen to mixed solution was 1 g: 20 mL). Hydrated lanthanum nitrate was added (the amount of hydrated lanthanum nitrate added was 5% of the total mass of iron salts in the mixed solution), the pH was adjusted to 9, and the reaction was stirred at 60 °C for 2 h to generate lanthanum-doped iron tetroxide nanoparticles. Subsequently, mercaptopropionic acid and polyethylene glycol diacrylate were added (the amount of mercaptopropionic acid added was 8% of the mass of active pollen, and the amount of polyethylene glycol diacrylate added was 5% of the mass of active pollen). Under the ultrasonic assistance of 200 W power and 28 kHz frequency, the reaction was carried out at 50 °C for 6 h. After the reaction was completed, the product was collected by magnetic separation, washed three times with ethanol and deionized water, and dried under vacuum at 50 °C to obtain mercaptofunctionalized magnetic pollen.

[0040] S3. Construction of the cross-linked network: 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazintrione was dissolved in N,N-dimethylformamide (the ratio of 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazintrione to N,N-dimethylformamide in the first solution was 1 g: 20 mL), and dihydroxyethylglycine was dissolved in deionized water (the ratio of dihydroxyethylglycine to deionized water in the second solution was 1 g: 50 mL). The first and second solutions were prepared separately. The second solution was added to the first solution (first solution, second solution, magnetic flower). The powder was added in a ratio of 10mL:5mL:1g, and then the magnetic pollen was added. The mixture was stirred at 40℃ for 4 hours, the product was filtered, and washed three times alternately with DMF and deionized water. The product was dried at room temperature and placed in an ultraviolet light reactor for ultraviolet irradiation (ultraviolet wavelength 365nm, power 50W, irradiation distance 10cm, nitrogen gas with a flow rate of 10L / min was introduced for protection during ultraviolet irradiation). The reaction was carried out for 10 minutes to allow the thiol groups and epoxy groups to crosslink by click. The final product was washed with acetone to remove unreacted monomers and dried under vacuum at 40℃ for 12 hours to obtain an immobilized carrier with a three-dimensional crosslinked network.

[0041] S4. Enzyme Immobilization: Keratinase, lipase, cellulase, laccase, and peroxidase were mixed in a mass ratio of 1:1:2:0.1:0.5 and dissolved in phosphate buffer at pH 7.5 to prepare a composite enzyme solution (the concentration of the composite enzyme in the composite enzyme solution was 10 mg / mL). The carrier and the composite enzyme solution were mixed at a mass ratio of 1:5 and immersed in the mixture at 25°C with shaking for 2 hours. After magnetic separation, the immobilized enzyme was washed three times with phosphate buffer at pH 7.5 to remove free enzyme. The washed immobilized enzyme was then shaken in 0.1 mol / L NaCl solution for 1 hour and magnetically separated again. After rinsing with deionized water, the enzyme was pre-frozen at -40°C for 4 hours and then dried in a freeze dryer for 12 hours to obtain the highly active immobilized plastic degradation enzyme preparation.

[0042] Example 3: A highly active immobilized plastic degrading enzyme preparation, wherein the enzyme preparation is prepared by the following steps: S1, preparation of active pollen: Pinus tabuliformis pollen is added to a 1.0 mol / L hydrochloric acid solution, stirred in a water bath at 70°C for 3 hours to remove impurities and waxes, then filtered, washed with deionized water until neutral, and dried with hot air at 80°C to constant weight. The dried pollen is placed in a tube furnace, and a mixture of nitrogen and carbon dioxide gas (nitrogen:carbon dioxide volume ratio of 1:0.35) is introduced at a flow rate of 15 L / min. The temperature is increased to 600°C at 6°C / min for 2 hours for carbonization. After cooling, the carbonized pollen is immersed in a 20% mass fraction sodium lignosulfonate solution (the feeding ratio of carbonized pollen to sodium lignosulfonate solution is 1 g:4 mL), activated at 750°C for 1.25 hours, and the product is washed with deionized water until no chloride ions remain to obtain active pollen;

[0043] S2. Magnetic Composite Modification: The active pollen obtained in step S1 was impregnated in a mixed solution of hydrated ferric chloride and hydrated ferrous sulfate (the molar ratio of ferric ions to ferrous ions in the mixed solution was 2.0:1, the total ferric ion concentration in the mixed solution was 1.0 mol / L, and the feed ratio of active pollen to mixed solution was 1 g:35 mL). Hydrated lanthanum nitrate was added (the amount of hydrated lanthanum nitrate added was 10% of the total mass of iron salts in the mixed solution), the pH was adjusted to 10, and the reaction was stirred at 70℃ for 3 h to generate lanthanum-doped iron oxide nanoparticles. Subsequently, mercaptopropionic acid and polyethylene glycol diacrylate were added (the amount of mercaptopropionic acid added was 14% of the mass of active pollen, and the amount of polyethylene glycol diacrylate added was 10% of the mass of active pollen). Under the ultrasonic assistance of 350 W power and 34 kHz frequency, the reaction was carried out at 60℃ for 9 h. After the reaction was completed, the product was collected by magnetic separation, washed three times with ethanol and deionized water, and dried under vacuum at 50℃ to obtain mercapto-functionalized magnetic pollen.

[0044] S3. Construction of the cross-linked network: 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazinone was dissolved in N,N-dimethylformamide (the ratio of 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazinone to N,N-dimethylformamide in the first solution was 1 g:30 mL), and dihydroxyethylglycine was dissolved in deionized water (the ratio of dihydroxyethylglycine to deionized water in the second solution was 1 g:75 mL). The first and second solutions were prepared separately. The second solution was added to the first solution (first solution, second solution, magnetic pollen...). The feed ratio was 15mL:10mL:2g. Then, the magnetic pollen was added, and the mixture was stirred at 50℃ for 6 hours. The product was filtered, washed three times alternately with DMF and deionized water, and dried at room temperature. The product was then placed in an ultraviolet light reactor for ultraviolet irradiation (ultraviolet wavelength 365nm, power 75W, irradiation distance 12.5cm, and nitrogen gas at a flow rate of 15L / min was introduced for protection during ultraviolet irradiation). The reaction was carried out for 20 minutes to allow the thiol groups and epoxy groups to crosslink by click. The final product was washed with acetone to remove unreacted monomers and dried under vacuum at 40℃ for 12 hours to obtain an immobilized carrier with a three-dimensional crosslinked network.

[0045] S4. Enzyme Immobilization: Keratinase, lipase, cellulase, laccase, and peroxidase were mixed in a mass ratio of 2:2:3.5:0.3:0.75 and dissolved in phosphate buffer at pH 7.5 to prepare a composite enzyme solution (the concentration of the composite enzyme in the composite enzyme solution was 20 mg / mL). The carrier and the composite enzyme solution were mixed at a carrier-to-enzyme mass ratio of 1:10 and immersed at 31°C with shaking for 3 hours. After magnetic separation, the immobilized enzyme was washed three times with phosphate buffer at pH 7.5 to remove free enzyme. The washed immobilized enzyme was then shaken in 0.1 mol / L NaCl solution for 1 hour and magnetically separated again. After rinsing with deionized water, the enzyme was pre-frozen at -40°C for 4 hours and then dried in a freeze dryer for 12 hours to obtain the highly active immobilized plastic degradation enzyme preparation.

[0046] Comparative Example 1: This comparative example omits the sodium lignin sulfonate activation treatment in step S1 and directly uses carbonized pollen as a carrier. All other aspects are the same as in Example 3.

[0047] Comparative Example 2: In this comparative example, lanthanum doping with hydrated lanthanum nitrate was not added in step S2, and all other steps were the same as in Example 3.

[0048] Comparative Example 3: This comparative example omits the construction of the crosslinked network in step S3 and directly uses thiol-functionalized magnetic pollen as the carrier in step S4. The rest is the same as in Example 3.

[0049] Comparative Example 4: In this comparative example, polyethylene glycol diacrylate was not added in step S2, and all other steps were the same as in Example 3.

[0050] The enzyme preparations prepared in each group of embodiments and comparative examples of the present invention were tested and analyzed as follows, and the results are shown in Table 1:

[0051] PLA films with a thickness of 0.1 mm were mixed with the immobilized enzyme preparations of each example and comparative example at a mass ratio of 1:10, placed in PBS buffer at pH 7.4, and shaken at 37°C. After 30 days, the samples were removed, dried, weighed, and the mass loss rate of the PLA films was calculated.

[0052] The immobilized enzyme preparations of each example and comparative example were reacted with PLA films in PBS at pH 7.4 at 37°C for 2 hours. The carrier was recovered by magnetic separation, washed 3 times with PBS, and repeated 10 times. The enzyme activity after the reaction was recorded, and the enzyme activity retention rate after 10 cycles was calculated.

[0053] Record the initial total volume (V0) and enzyme concentration (C0) of the enzyme solution, and calculate the initial total enzyme amount (Q0=C0×V0). Mix each group of carriers with the enzyme solution and react. After immobilization, separate the carriers and measure the remaining enzyme concentration (C1) and volume (V1) in the supernatant. Calculate the remaining enzyme amount (Q1=C1×V1). The immobilized enzyme amount is the difference between the initial enzyme amount and the remaining enzyme amount: Qimmobilized=Q0−Q1. Then, the enzyme loading of the immobilized carrier = (Q0−Q1) / carrier mass.

[0054] The immobilized enzyme suspensions of each example and comparative example were placed in a round-bottom 96-well plate, and magnetic beads were adsorbed using a magnetic separator. After separation, the supernatant was taken and the residual enzyme activity was detected. The magnetic separation efficiency E=(A0−Ar) / A0×100%, where A0 is the initial enzyme activity and Ar is the residual enzyme activity in the supernatant.

[0055] The immobilized enzymes of each example and comparative example were dispersed in PBS buffer at pH 7.5 and incubated in a water bath at 60°C. Samples were taken at 0, 5, 10, 20, and 30 minutes, and the reaction was quickly terminated by placing the sample on ice. The residual enzyme activity was measured, and the time to 50% enzyme activity was calculated by fitting a curve using an exponential decay model.

[0056] Table 1: Results of enzyme preparation testing and analysis

[0057] project Quality loss rate (%) Enzyme activity retention rate (%) Enzyme loading (mg / g) Magnetic separation efficiency (%) Half-life at 60℃ (min) Example 1 82 92.6 145.2 99.5 150 Example 2 79 92.0 138.5 97.2 152 Example 3 85 93.2 130.3 98.8 143 Comparative Example 1 50 28.6 75.0 89.3 46 Comparative Example 2 62 72.5 105.2 85.6 80 Comparative Example 3 45 35.8 72.1 76.4 40 Comparative Example 4 58 68.2 95.5 92.1 55

[0058] Figure 1 The microstructure of Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 after PLA film degradation for 200 h is shown.

[0059] The test and observation results above show that the PLA film mass loss rate, enzyme activity retention rate after 10 cycles, enzyme loading, and magnetic separation efficiency of each embodiment are significantly higher than those of the comparative example, and the half-life at 60°C is significantly longer than that of the comparative example. Figure 1As shown, the film surface treated in Example 3 produced a large number of wrinkles, cracks and cavities, indicating that enzyme molecules efficiently contact the substrate through the carrier pores and utilize enzymes such as keratinase and lipase to synergistically cleave the ester bonds in the PLA molecular chain.

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0061] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A highly active immobilized plastic-degrading enzyme preparation, characterized in that: Prepared by the following steps: S1. Preparation of active pollen: Add pine pollen to a 0.5-1.5 mol / L hydrochloric acid solution and stir in a water bath at 60-80℃ for 2-4 hours to remove impurities and waxes. Then filter, wash with deionized water until neutral, and dry with hot air at 80℃ to constant weight. Place the dried pollen in a tube furnace and introduce a mixture of nitrogen and carbon dioxide gas. Heat to 500-700℃ at 2-10℃ / min for 1-3 hours for carbonization. After cooling, immerse the carbonized pollen in a 10-30% (w / w) sodium lignosulfonate solution and activate at 700-800℃ for 0.5-2 hours. Wash the product with deionized water until no chloride ions remain to obtain active pollen. S2. Magnetic composite modification: The active pollen obtained in step S1 is impregnated in a mixed solution of hydrated ferric chloride and hydrated ferrous sulfate, hydrated lanthanum nitrate is added, the pH is adjusted to 9-11, and the mixture is stirred at 60-80℃ for 2-4 hours to generate lanthanum-doped iron oxide nanoparticles. Subsequently, mercaptopropionic acid and polyethylene glycol diacrylate are added, and the mixture is reacted at 50-70℃ for 6-12 hours under ultrasonic assistance at a power of 200-500W and a frequency of 28-40kHz. After the reaction is completed, the product is collected by magnetic separation, washed three times with ethanol and deionized water, and dried under vacuum at 50℃ to obtain mercapto-functionalized magnetic pollen. S3. Construction of cross-linked network: 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazine trione was dissolved in N,N-dimethylformamide, and dihydroxyethylglycine was dissolved in deionized water to prepare a first solution and a second solution, respectively. The second solution was added to the first solution, and then the magnetic pollen was added. The mixture was stirred at 40-60°C for 4-8 hours. The product was filtered, washed three times alternately with DMF and deionized water, and dried at room temperature. The product was placed in an ultraviolet light reactor for ultraviolet irradiation for 10-30 minutes to allow the thiol groups and epoxy groups to cross-link by click. The final product was washed with acetone to remove unreacted monomers and dried under vacuum at 40°C for 12 hours to obtain an immobilized carrier with a three-dimensional cross-linked network colonized. S4. Enzyme Immobilization: Keratinase, lipase, cellulase, laccase, and peroxidase were mixed in proportion and dissolved in phosphate buffer at pH 7.5 to prepare a composite enzyme solution. The carrier was mixed with the composite enzyme solution and immersed in the solution by shaking at 25-37℃ for 2-4 hours. After magnetic separation, the immobilized enzyme was washed three times with phosphate buffer at pH 7.5 to remove free enzyme. The washed immobilized enzyme was then shaken in 0.1mol / L NaCl solution for 1 hour. After magnetic separation again, the enzyme was rinsed with deionized water, pre-frozen at -40℃ for 4 hours, and then dried in a freeze dryer for 12 hours to obtain the highly active immobilized plastic degradation enzyme preparation.

2. The highly active immobilized plastic-degrading enzyme preparation according to claim 1, characterized in that: In step S1, the ratio of pine pollen to hydrochloric acid solution is 1g:8-12mL, and the ratio of carbonized pollen to sodium lignosulfonate solution is 1g:3-5mL.

3. The highly active immobilized plastic-degrading enzyme preparation according to claim 1, characterized in that: In step S1, the volume ratio of the nitrogen to carbon dioxide mixture is nitrogen:carbon dioxide = 1:0.2-0.5, and the flow rate of the mixture is 10-20 L / min.

4. The highly active immobilized plastic-degrading enzyme preparation according to claim 1, characterized in that: In step S2, the molar ratio of ferric ions to ferrous ions in the mixed solution is 1.5-2.5:1, and the total ferric ion concentration in the mixed solution is 0.5-1.5 mol / L.

5. The highly active immobilized plastic-degrading enzyme preparation according to claim 1, characterized in that: In step S2, the ratio of active pollen to mixed solution is 1g: 20-50mL; the amount of lanthanum nitrate hydrate added in step S2 is 5-15% of the total mass of iron salts in the mixed solution; the amount of mercaptopropionic acid added in step S2 is 8-20% of the mass of active pollen; and the amount of polyethylene glycol diacrylate added is 5-15% of the mass of active pollen.

6. The highly active immobilized plastic-degrading enzyme preparation according to claim 1, characterized in that: In step S3, the ratio of 1,3-bis(epoxyethylenemethyl)-5-(2-propenyl)-triazinetrione to N,N-dimethylformamide in the first solution is 1g:20-40mL; and the ratio of dihydroxyethylglycine to deionized water in the second solution in step S3 is 1g:50-100mL.

7. The highly active immobilized plastic-degrading enzyme preparation according to claim 1, characterized in that: In step S3, the feeding ratio of the first solution, the second solution, and the magnetic pollen is 10-20 mL: 5-15 mL: 1-3 g; in step S3, the ultraviolet wavelength is 365 nm, the power is 50-100 W, the irradiation distance is maintained at 10-15 cm, and nitrogen gas with a flow rate of 10-20 L / min is introduced for protection during ultraviolet irradiation.

8. The highly active immobilized plastic-degrading enzyme preparation according to claim 1, characterized in that: In step S4, the mass ratio of keratinase, lipase, cellulase, laccase, and peroxidase in the complex enzyme is 1-3:1-3:2-5:0.1-0.5:0.5-1, and the concentration of the complex enzyme in the complex enzyme solution in step S4 is 10-30 mg / mL. In step S4, the carrier and the complex enzyme solution are mixed at a carrier-to-enzyme mass ratio of 1:5-15.

9. The application of the highly active immobilized plastic-degrading enzyme preparation according to any one of claims 1-8, characterized in that: The enzyme preparation is added to the biodegradable plastic to promote its degradation.

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