Alkali-resistant degradable enzyme preparation for plastic and preparation method thereof

Abstract

The invention discloses an enzyme preparation for alkali-resistant degradable plastic and a preparation method of the enzyme preparation, and belongs to the technical field of plastic degrading enzyme preparations. The preparation process of the enzyme preparation comprises the following steps: synthesizing a norbornene intermediate and all-phosphate calix [6] arene, grafting tetrazine functionalized four-arm polyethylene glycol through a DA reaction to form a three-dimensional cross-linked carrier, and then loading a mixed enzyme composed of a plurality of enzymes. A rigid cavity of calix [6] arene can form a specific inclusion effect on enzyme molecules, a phosphate group enhances the enzyme-carrier binding force, and a cross-linked network improves the carrier stability and provides multiple loading sites. A multi-enzyme synergistic system can destroy the plastic surface structure, degrade the composite filler and cut off ester bonds. The enzyme activity retention rate of the enzyme preparation in an alkaline environment exceeds 85%, the half-life period at 50 DEG C exceeds 110 h, the weight loss rates of the enzyme preparation to polylactic acid films and composite materials containing 30% of starch filler both exceed 92%, and the degradation efficiency of degradable plastics in the alkaline environment is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plastic-degrading enzyme preparations, and in particular relates to an enzyme preparation for alkali-resistant degradable plastics and a preparation method thereof. Background Art

[0002] Polylactic acid (PLA), a typical biodegradable polyester material, is typically made from starch from crops such as corn, wheat, and sugarcane. It is produced through microbial fermentation to produce lactic acid, which is then chemically polycondensed or through ring-opening polymerization of lactide. PLA completely decomposes into carbon dioxide and water within 3-6 months under composting conditions, leaving no microplastic residue. The degradation products are harmless to the environment and humans, and the calorific value released upon combustion is only half that of traditional plastics, without producing toxic gases such as nitrogen and sulfides. Due to its excellent biocompatibility, degradability, and renewable raw material availability, PLA holds great promise for applications in packaging, healthcare, agriculture, and other fields. PLA degrades primarily through ester bond hydrolysis. Studies have shown that its degradation rate in alkaline conditions is significantly higher than in acidic or neutral environments. Alkaline environments (such as industrial wastewater treatment systems and alkaline composting) can accelerate the degradation of PLA by promoting ester bond hydrolysis.

[0003] Traditional enzyme preparation carrier materials are prone to structural destruction or changes in surface charge properties in alkaline environments, resulting in a decrease in the binding force between the enzyme molecules and the carrier, and the occurrence of enzyme molecule shedding or denaturation and inactivation. At the same time, the direct attack of hydroxide ions on the active center of the enzyme under alkaline conditions will destroy the spatial conformation of the enzyme protein and significantly reduce the catalytic efficiency of the enzyme. In addition, for degradable plastic mixtures containing composite fillers such as starch and cellulose, a single enzyme preparation is difficult to achieve synergistic degradation of multiple components, resulting in low overall degradation efficiency. Therefore, it is necessary to develop an enzyme preparation that can maintain high activity and stability in an alkaline environment and has multi-enzyme synergistic degradation function. Summary of the Invention

[0004] In response to the above situation, and to overcome the shortcomings of conventional enzyme preparations in the prior art, such as their easy inactivation in alkaline environments, poor carrier stability, and low degradation efficiency for degradable plastics containing composite fillers, the present invention provides an alkali-resistant enzyme preparation for degradable plastics and a method for preparing the same. The enzyme preparation achieves efficient degradation of degradable plastics in an alkaline environment by constructing an alkali-resistant functionalized carrier containing a calix[6]arene structure and loading it with a mixed enzyme composed of multiple functional enzymes.

[0005] In order to achieve the above object, the following technical solution is adopted: The present invention provides a method for preparing an enzyme preparation for alkali-resistant degradable plastics, comprising the following steps: S1. Sodium hydride is added to tetrahydrofuran, and nitrogen is introduced for protection. 5-bromomethyl-2-norbornene is slowly added to the tetrahydrofuran under continuous stirring. Hexylene glycol is then slowly added dropwise to the reaction system over 30-40 minutes using a constant pressure dropping funnel. After the addition is complete, the reaction temperature is raised to 60-70°C, and the reaction is continued with stirring for 6-8 hours. After the reaction is complete, the system is cooled to room temperature, and the tetrahydrofuran is removed by vacuum distillation to obtain a norbornene intermediate. S2. Add perbromocalix[6]arene to toluene, then add triethylamine and triethyl phosphite. Under nitrogen protection, raise the reaction temperature to 110-120°C, stir and react for 24-36 hours. After the reaction is completed, cool the system to room temperature, and remove toluene and excess triethyl phosphite by distillation under reduced pressure to obtain perphosphate calix[6]arene. S3. Add the norbornene intermediate prepared in step S1 and the perphosphate ester calix[6]arene prepared in step S2 to N,N-dimethylformamide, add alkali metal carbonate, and heat to 80-90° C. under nitrogen protection. Stir and react for 18-24 hours. After the reaction is completed, cool the system to room temperature, filter, and distill the filtrate under reduced pressure to remove DMF to obtain a norbornene-calix[6]arene intermediate; S4, adding four-arm polyethylene glycol succinimidyl glutarate to dimethyl sulfoxide, stirring thoroughly to dissolve, then adding tetrazine-amino hydrochloride and triethylamine, stirring at room temperature for 12-16 hours. After the reaction is completed, pouring the reaction solution into ice water, filtering and collecting the precipitated solid, and vacuum drying to obtain tetrazine-functionalized four-arm polyethylene glycol; S5. Add the norbornene-calix[6]arene intermediate obtained in step S3 and the tetrazine-functionalized four-arm polyethylene glycol obtained in step S2 to dichloromethane, stir and react at room temperature for 24-36 hours, extract with deionized water, and distill the organic phase under reduced pressure to remove dichloromethane to obtain an alkali-resistant functionalized carrier; S6. Add the alkali-resistant functionalized carrier prepared in step S5 to a phosphate buffer solution with a pH of 7.0, stir to fully disperse it, add the mixed enzyme to the system, and slowly stir at 4°C for 12-16 hours to allow the mixed enzyme to be fully loaded on the carrier. After the loading is completed, collect the solid by centrifugation at 8000 rpm for 10-30 minutes, wash it three times with a phosphate buffer solution with a pH of 7.0, and finally vacuum dry it to obtain the enzyme preparation for alkali-resistant degradable plastics.

[0006] Furthermore, the perbrominated calix[6]arene in step S2 is prepared by the following steps: adding calix[6]arene to dichloromethane, stirring thoroughly to dissolve it, adding liquid bromine dropwise in an ice bath, controlling the temperature not to exceed 5° C. during the addition, heating the reaction system to room temperature after the addition is completed, and continuing to stir and react for 12-16 hours. After the reaction is completed, adding a saturated sodium sulfite solution to the system, and then washing the organic phase with deionized water and a saturated sodium bicarbonate solution in sequence, and removing the dichloromethane solvent by distillation under reduced pressure to obtain perbrominated calix[6]arene.

[0007] Furthermore, the feeding ratio of the calix[6]arene, liquid bromine and dichloromethane is 20-30 g: 60-80 mL: 200-400 mL.

[0008] Furthermore, in step S1, the feeding ratio of sodium hydride, 5-bromomethyl-2-norbornene, hexylene glycol and tetrahydrofuran is 10-20 g: 15-40 g: 15-30 mL: 150-200 mL.

[0009] Furthermore, in step S2, the feeding ratio of perbromocalix[6]arene, triethylamine, triethyl phosphite and toluene is 5-15 g: 18-30 g: 8-12 g: 150-200 mL.

[0010] Furthermore, in step S3, the feeding ratio of norbornene intermediate, perphosphate calix[6]arene, alkali metal carbonate and N,N-dimethylformamide is 8-15 g: 10-25 g: 12-25 g: 120-150 mL; the alkali metal carbonate is one of sodium carbonate, potassium carbonate or cesium carbonate.

[0011] Furthermore, in step S4, the feeding ratio of four-arm polyethylene glycol succinimidyl glutarate, tetrazine-amino hydrochloride, triethylamine and dimethyl sulfoxide is 8-20 g: 3-5 g: 10-20 g: 100-150 mL.

[0012] Furthermore, in step S5, the feeding ratio of norbornene-calix[6]arene intermediate, tetrazine functionalized four-arm polyethylene glycol and dichloromethane is 5-12 g: 8-20 g: 120-150 mL.

[0013] Furthermore, in step S6, the feeding ratio of the alkali-resistant functionalized carrier, the mixed enzyme, and the phosphate buffer solution is 10-20 g: 5-12 g: 100-200 mL; the mixed enzyme contains the following components in parts by weight: 6-15 parts of sucrose fatty acid ester, 4-8 parts of alkaline protease, 4-8 parts of α-amylase, 2-5 parts of cutinase, and 2-5 parts of cellulase.

[0014] Among them, alkaline protease can specifically hydrolyze protein additives or pollutants on the surface of degradable plastics, destroying the dense surface structure of the material; α-amylase and cellulase can degrade starch-based or cellulose-based fillers that may remain in the plastic, forming a porous structure; cutinase, as a specific esterase, can recognize and cut the ester bonds of polyester materials such as polylactic acid; sucrose fatty acid esters, as surfactants, can reduce the surface tension of plastics and promote the adsorption and diffusion of enzyme molecules. This multi-enzyme synergistic system can not only efficiently degrade pure polylactic acid materials, but also has a good degradation effect on degradable plastic mixtures containing starch and cellulose composite fillers.

[0015] The beneficial effects of the present invention are: (1) The enzyme preparation prepared by the present invention has excellent alkali resistance and high-efficiency plastic degradation performance. By introducing the cup[6]arene structure in the carrier construction process, its rigid electron-rich cavity can form a specific inclusion complex on the enzyme molecule, effectively protecting the enzyme active center from direct attack by hydroxide ions in an alkaline environment, maintaining the stability of the enzyme protein spatial conformation, and at the same time, the phosphate groups on the cup[6]arene skeleton can form a hydrogen bond network and electrostatic interaction with polar groups such as amino groups and hydroxyl groups on the surface of the enzyme molecule, thereby enhancing the binding force between the enzyme and the carrier and avoiding the shedding or denaturation of the enzyme molecule under alkaline conditions. This special molecular recognition structure enables the enzyme preparation to maintain more than 85% of the enzyme activity in an alkaline environment of pH 8.0-10.0, and can efficiently degrade various degradable plastics such as polylactic acid, polyadipate, and polybutylene terephthalate; (2) The present invention introduces norbornene groups into calix[6]arene molecules to form norbornene-calix[6]arene intermediates with double bond active sites. Subsequently, the anti-electron demand Diels-Alder reaction of the tetrazine functional group and the norbornene group is used to achieve efficient grafting of calixarene and four-arm polyethylene glycol under mild conditions to form a pyrazine-norbornene bicyclic system and a three-dimensional cross-linked network structure. This cross-linked structure not only gives the carrier excellent mechanical strength and chemical stability, but also provides more loading sites for enzyme molecules by increasing the specific surface area. At the same time, the steric hindrance effect of the cross-linked network can effectively inhibit the aggregation and inactivation of enzyme molecules, thereby extending the storage half-life of the enzyme preparation. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0018] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and the experimental materials used in the following examples, unless otherwise specified, are all purchased from commercial channels.

[0019] Example 1: A method for preparing an enzyme preparation for alkali-resistant degradable plastics, comprising the following steps: S1. Add 10 g of sodium hydride to 150 mL of tetrahydrofuran, purge with nitrogen for protection, and slowly add 15 g of 5-bromomethyl-2-norbornene to the tetrahydrofuran while stirring continuously. Then, slowly add 15 mL of hexylene glycol to the reaction system over 30 minutes using a constant pressure dropping funnel. After the addition is complete, raise the reaction temperature to 60°C and continue stirring for 6 hours. After the reaction is complete, cool the system to room temperature and remove the tetrahydrofuran by vacuum distillation to obtain the norbornene intermediate. S2. Add 5 g of perbromocalix[6]arene to 150 mL of toluene, then add 18 g of triethylamine and 8 g of triethyl phosphite. Under nitrogen protection, raise the reaction temperature to 110°C and stir for 24 hours. After the reaction is completed, cool the system to room temperature and remove toluene and excess triethyl phosphite by distillation under reduced pressure to obtain perphosphate calix[6]arene. S3. Add 8 g of the norbornene intermediate prepared in step S1 and 10 g of the perphosphate ester calix[6]arene prepared in step S2 to 120 mL of N,N-dimethylformamide, add 12 g of sodium carbonate, and heat to 80° C. under nitrogen protection. Stir and react for 18 h. After the reaction is completed, cool the system to room temperature, filter, and distill the filtrate under reduced pressure to remove DMF to obtain a norbornene-calix[6]arene intermediate. S4. Add 8 g of four-arm polyethylene glycol succinimidyl glutarate to 100 mL of dimethyl sulfoxide and stir thoroughly to dissolve it. Then, add 3 g of tetrazine-amino hydrochloride and 10 g of triethylamine. Stir and react at room temperature for 12 h. After the reaction is completed, pour the reaction solution into ice water, collect the precipitated solid by filtration, and vacuum dry to obtain tetrazine-functionalized four-arm polyethylene glycol; S5. Add 5 g of the norbornene-calix[6]arene intermediate prepared in step S3 and 8 g of the tetrazine-functionalized four-arm polyethylene glycol prepared in step S2 to 120 mL of dichloromethane, stir and react at room temperature for 24 h, extract with deionized water, and distill the organic phase under reduced pressure to remove dichloromethane to obtain an alkali-resistant functionalized carrier; S6. Add 10 g of the alkali-resistant functionalized carrier prepared in step S5 to 100 mL of a phosphate buffer solution having a pH of 7.0, stir to fully disperse it, add 5 g of a mixed enzyme (comprising 6 parts of sucrose fatty acid ester, 4 parts of alkaline protease, 4 parts of α-amylase, 2 parts of cutinase, and 2 parts of cellulase, by mass) to the system, and slowly stir for 12 hours at 4°C to allow the mixed enzyme to be fully loaded onto the carrier. After loading, collect the solid by centrifugation at 8000 rpm for 10 minutes, wash it three times with a phosphate buffer solution having a pH of 7.0, and finally vacuum dry it to obtain the enzyme preparation for alkali-resistant degradable plastics.

[0020] In step S2, perbrominated calix[6]arene is prepared by the following steps: adding 20 g of calix[6]arene to 200 mL of dichloromethane, stirring thoroughly to dissolve the calix[6]arene, dropping 60 mL of liquid bromine in an ice bath, controlling the temperature not to exceed 5° C. during the addition, heating the reaction system to room temperature, and continuing to stir and react for 12 hours. After the reaction is completed, adding saturated sodium sulfite solution to the system, and then washing the organic phase with deionized water and saturated sodium bicarbonate solution in sequence, and removing the dichloromethane solvent by distillation under reduced pressure to obtain perbrominated calix[6]arene.

[0021] Example 2: A method for preparing an enzyme preparation for alkali-resistant degradable plastics, comprising the following steps: S1. Add 20g of sodium hydride to 200mL of tetrahydrofuran, purge with nitrogen for protection, and slowly add 40g of 5-bromomethyl-2-norbornene to the tetrahydrofuran under continuous stirring. Then, slowly add 30mL of hexylene glycol to the reaction system dropwise over 40 minutes using a constant pressure dropping funnel. After the addition is complete, raise the reaction temperature to 70°C and continue stirring for 8 hours. After the reaction is complete, cool the system to room temperature and remove the tetrahydrofuran by vacuum distillation to obtain the norbornene intermediate. S2. Add 15 g of perbromocalix[6]arene to 200 mL of toluene, then add 30 g of triethylamine and 12 g of triethyl phosphite. Under nitrogen protection, raise the reaction temperature to 120° C. and stir to react for 36 hours. After the reaction is completed, cool the system to room temperature and remove toluene and excess triethyl phosphite by vacuum distillation to obtain perphosphate ester calix[6]arene. S3. Add 15 g of the norbornene intermediate prepared in step S1 and 25 g of the perphosphate ester calix[6]arene prepared in step S2 to 150 mL of N,N-dimethylformamide, add 25 g of potassium carbonate, and heat to 90° C. under nitrogen protection. Stir and react for 24 h. After the reaction is completed, cool the system to room temperature, filter, and distill the filtrate under reduced pressure to remove DMF to obtain a norbornene-calix[6]arene intermediate. S4, adding 20 g of four-arm polyethylene glycol succinimidyl glutarate to 150 mL of dimethyl sulfoxide, stirring thoroughly to dissolve, then adding 5 g of tetrazine-amino hydrochloride and 20 g of triethylamine, stirring at room temperature for 16 h. After the reaction is completed, the reaction solution is poured into ice water, the precipitated solid is collected by filtration, and vacuum dried to obtain tetrazine-functionalized four-arm polyethylene glycol; S5. Add 12 g of the norbornene-calix[6]arene intermediate obtained in step S3 and 20 g of the tetrazine-functionalized four-arm polyethylene glycol obtained in step S2 to 150 mL of dichloromethane, stir and react at room temperature for 36 h, extract with deionized water, and distill the organic phase under reduced pressure to remove dichloromethane to obtain an alkali-resistant functionalized carrier; S6. Add 20 g of the alkali-resistant functionalized carrier prepared in step S5 to 200 mL of a phosphate buffer solution with a pH of 7.0, stir to fully disperse it, add 12 g of a mixed enzyme (containing 15 parts of sucrose fatty acid ester, 8 parts of alkaline protease, 8 parts of α-amylase, 5 parts of cutinase, and 5 parts of cellulase, by mass) to the system, and slowly stir for 16 hours at 4°C to allow the mixed enzyme to be fully loaded onto the carrier. After loading, collect the solid by centrifugation at 8000 rpm for 30 minutes, wash it three times with a phosphate buffer solution with a pH of 7.0, and finally vacuum dry it to obtain the enzyme preparation for alkali-resistant degradable plastics.

[0022] In step S2, perbrominated calix[6]arene is prepared by the following steps: adding 30 g of calix[6]arene to 400 mL of dichloromethane, stirring thoroughly to dissolve the calix[6]arene, adding dropwise 80 mL of liquid bromine in an ice bath, controlling the temperature not to exceed 5° C. during the addition, heating the reaction system to room temperature after the addition is completed, and continuing to stir and react for 16 hours. After the reaction is completed, adding saturated sodium sulfite solution to the system, and then washing the organic phase with deionized water and saturated sodium bicarbonate solution in sequence, and removing the dichloromethane solvent by distillation under reduced pressure to obtain perbrominated calix[6]arene.

[0023] Example 3: A method for preparing an enzyme preparation for alkali-resistant degradable plastics, comprising the following steps: S1. Add 15g of sodium hydride to 175mL of tetrahydrofuran, purge with nitrogen for protection, and slowly add 27g of 5-bromomethyl-2-norbornene to the tetrahydrofuran with continuous stirring. Then, slowly add 22mL of hexylene glycol to the reaction system dropwise over 35 minutes using a constant pressure dropping funnel. After the addition is complete, raise the reaction temperature to 65°C and continue stirring for 7 hours. After the reaction is complete, cool the system to room temperature and remove the tetrahydrofuran by vacuum distillation to obtain the norbornene intermediate. S2. Add 10 g of perbromocalix[6]arene to 175 mL of toluene, then add 24 g of triethylamine and 10 g of triethyl phosphite. Under nitrogen protection, raise the reaction temperature to 115°C and stir to react for 30 hours. After the reaction is completed, cool the system to room temperature and remove toluene and excess triethyl phosphite by distillation under reduced pressure to obtain perphosphate calix[6]arene. S3. Add 11 g of the norbornene intermediate prepared in step S1 and 17 g of the perphosphate ester calix[6]arene prepared in step S2 to 135 mL of N,N-dimethylformamide, add 18 g of cesium carbonate, and heat to 85° C. under nitrogen protection. Stir and react for 21 h. After the reaction is completed, cool the system to room temperature, filter, and distill the filtrate under reduced pressure to remove DMF to obtain a norbornene-calix[6]arene intermediate. S4, adding 14 g of four-arm polyethylene glycol succinimidyl glutarate to 125 mL of dimethyl sulfoxide, stirring thoroughly to dissolve, then adding 4 g of tetrazine-amino hydrochloride and 15 g of triethylamine, stirring at room temperature for 14 h. After the reaction is completed, the reaction solution is poured into ice water, the precipitated solid is collected by filtration, and vacuum dried to obtain tetrazine-functionalized four-arm polyethylene glycol; S5. Add 8 g of the norbornene-calix[6]arene intermediate prepared in step S3 and 14 g of the tetrazine-functionalized four-arm polyethylene glycol prepared in step S2 to 135 mL of dichloromethane, stir and react at room temperature for 30 h, extract with deionized water, and distill the organic phase under reduced pressure to remove dichloromethane to obtain an alkali-resistant functionalized carrier; S6. Add 15 g of the alkali-resistant functionalized carrier prepared in step S5 to 150 mL of a phosphate buffer solution having a pH of 7.0, stir to fully disperse it, add 8 g of a mixed enzyme (comprising 10 parts of sucrose fatty acid ester, 6 parts of alkaline protease, 6 parts of α-amylase, 3 parts of cutinase, and 3 parts of cellulase, by mass) to the system, and slowly stir for 14 hours at 4°C to allow the mixed enzyme to be fully loaded onto the carrier. After loading, collect the solid by centrifugation at 8000 rpm for 20 minutes, wash it three times with a phosphate buffer solution having a pH of 7.0, and finally vacuum dry it to obtain the enzyme preparation for alkali-resistant degradable plastics.

[0024] In step S2, perbrominated calix[6]arene is prepared by the following steps: adding 25 g of calix[6]arene to 300 mL of dichloromethane, stirring thoroughly to dissolve the calix[6]arene, adding dropwise 70 mL of liquid bromine in an ice bath, controlling the temperature not to exceed 5° C. during the addition, heating the reaction system to room temperature after the addition is completed, and continuing to stir and react for 14 hours. After the reaction is completed, adding saturated sodium sulfite solution to the system, and then washing the organic phase with deionized water and saturated sodium bicarbonate solution in sequence, and removing the dichloromethane solvent by distillation under reduced pressure to obtain perbrominated calix[6]arene.

[0025] Comparative Example 1: In this comparative example, conventional diatomaceous earth is used instead of the alkali-resistant functionalized carrier to carry out the loading in step S6, and the rest is the same as in Example 3.

[0026] Comparative Example 2: In this comparative example, a norbornene intermediate is used instead of a norbornene-calix[6]arene intermediate to carry out the reaction of step S5 with tetrazine-functionalized four-arm polyethylene glycol, and the rest is the same as in Example 3.

[0027] Comparative Example 3: In this comparative example, four-arm polyethylene glycol is used instead of the alkali-resistant functionalized carrier to carry out the loading in step S6, and the rest is the same as in Example 3.

[0028] The enzyme preparations prepared in each group of Examples and Comparative Examples of the present invention were tested and analyzed. The results are shown in Table 1: The enzyme preparations prepared in each group of Examples and Comparative Examples were dissolved in a sodium carbonate-sodium bicarbonate buffer solution at pH 10.0 and incubated at 45°C with constant shaking for 72 hours. The residual enzyme activity was measured and the activity retention rate was calculated. Alkaline protease activity (unit: U / mL) was determined using the Folin-phenol method with casein as the substrate. Other enzyme activities were determined spectrophotometrically using the corresponding substrates. Total enzyme activity was the sum of the activities of each enzyme.

[0029] The enzyme preparations prepared in each group of Examples and Comparative Examples were placed in a 50°C water bath, and samples were taken at regular intervals to measure the enzyme activity. The time required for the activity to drop to 50% of the initial value was calculated, which was the 50°C half-life of each group of enzyme preparations.

[0030] The degradation efficiency of the enzyme preparations prepared in each of the examples and comparative examples was tested on PLA films (50 μm thick, molecular weight 100,000) and PLA composites containing 30% starch filler in an alkaline composting environment. The conditions were: pH 9.0, temperature 55°C, humidity 80%, enzyme concentration 2 g / L, oscillation rate 150 rpm, and reaction time 14 days. After 14 days, the samples were removed, dried, and weighed. The weight loss was calculated, and the number-average molecular weight (Mn) of the PLA films was determined by GPC.

[0031] Table 1: Comparison of enzyme preparation test results

[0032] As can be seen from the results in the table above, the enzyme activity retention rate of each example in an alkaline environment of pH=10 exceeds 91%. This is because the rigid electron-rich cavity of calix[6]arene encapsulates the active center of the enzyme through molecular recognition and resists the attack of hydroxide ions; the phosphate group forms hydrogen bonds and electrostatic interactions with the enzyme molecules, enhancing the binding force and preventing the enzyme from falling off or denaturing. The half-life at 50°C of each example exceeds 110h, while that of comparative example 1 is only 45h, and that of comparative examples 2 and 3 is 68h and 75h respectively. The reason is that norbornene-calix[6]arene and four-arm polyethylene glycol form a three-dimensional cross-linked network through the Diels-Alder reaction, which increases the specific surface area and inhibits the aggregation of enzyme molecules, thereby improving mechanical strength and chemical stability. The cross-linked structure reduces the inactivation caused by the interaction between enzyme molecules and prolongs the storage period in practical applications. The weight loss rate of the PLA film of each example exceeds 93%, and the weight loss rate of the composite material exceeds 92%. In addition, the number average molecular weight of the PLA film is reduced from 100,000 to about 20,000, indicating that the ester bond is efficiently cut and the degradation efficiency of the enzyme preparation prepared by the present invention is relatively high.

[0033] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

[0034] The present invention and its embodiments are described above. Such description is not restrictive. The embodiment shown in the embodiment is only one embodiment of the present invention, and actual application is not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the invention, without creatively designing methods and embodiments similar to the technical solution, they shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing an enzyme preparation for alkali-resistant degradable plastics, characterized in that: The following steps are involved: S1. Sodium hydride is added to tetrahydrofuran, and nitrogen is introduced for protection. 5-bromomethyl-2-norbornene is slowly added to the tetrahydrofuran under continuous stirring. Then, hexylene glycol is slowly added dropwise to the reaction system over 30-40 minutes through a constant pressure dropping funnel; after the addition is complete, the reaction temperature is raised to 60-70° C., and the stirring reaction is continued for 6-8 hours. After the reaction is completed, the system is cooled to room temperature, and tetrahydrofuran is removed by distillation under reduced pressure to obtain a norbornene intermediate; S2. Add the norbornene intermediate and the perphosphate ester calix[6]arene to N,N-dimethylformamide, add an alkali metal carbonate, and heat to 80-90° C. under nitrogen protection. Stir and react for 18-24 hours. After the reaction is completed, cool the system to room temperature, filter, and remove DMF from the filtrate by vacuum distillation to obtain the norbornene-calix[6]arene intermediate; S3. Activate four-arm polyethylene glycol succinimidyl glutarate using tetrazine-amino hydrochloride to obtain tetrazine-functionalized four-arm polyethylene glycol, add the norbornene-calix[6]arene intermediate and the tetrazine-functionalized four-arm polyethylene glycol to dichloromethane, stir and react at room temperature for 24-36 hours, extract with deionized water, and remove dichloromethane from the organic phase under reduced pressure to obtain an alkali-resistant functionalized carrier; S4. Loading the functionalized carrier with mixed enzymes to obtain an enzyme preparation for alkali-resistant degradable plastics.

2. The method for preparing an enzyme preparation for alkali-resistant degradable plastics according to claim 1, wherein: The full phosphate calix[6]arene is prepared by the following steps: Add calix[6]arene to dichloromethane and stir thoroughly to dissolve it. Add liquid bromine dropwise in an ice bath, controlling the temperature not to exceed 5°C during the addition. After the addition is complete, warm the reaction system to room temperature and continue stirring to react for 12-16 hours. After the reaction is complete, add saturated sodium sulfite solution to the system, then wash the organic phase with deionized water and saturated sodium bicarbonate solution in sequence, and remove the dichloromethane solvent by vacuum distillation to obtain perbromocalix[6]arene. Perbromocalix[6]arene is added to toluene, and then triethylamine and triethyl phosphite are added. Under nitrogen protection, the reaction temperature is raised to 110-120°C, and the reaction is stirred for 24-36 hours. After the reaction is completed, the system is cooled to room temperature, and toluene and excess triethyl phosphite are removed by vacuum distillation to obtain perphosphate ester calix[6]arene.

3. The method for preparing an enzyme preparation for alkali-resistant degradable plastics according to claim 2, wherein: The tetrazine-functionalized four-arm polyethylene glycol is prepared by the following steps: four-arm polyethylene glycol succinimidyl glutarate is added to dimethyl sulfoxide, and the mixture is fully stirred to dissolve, and then tetrazine-amino hydrochloride and triethylamine are added. The mixture is stirred at room temperature for 12-16 hours. After the reaction is completed, the reaction solution is poured into ice water, and the precipitated solid is collected by filtration and vacuum dried to obtain the tetrazine-functionalized four-arm polyethylene glycol.

4. The method for preparing an enzyme preparation for alkali-resistant degradable plastics according to claim 3, wherein: The step S4 specifically includes the following steps: adding the alkali-resistant functionalized carrier to a phosphate buffer solution with a pH of 7.0, stirring to fully disperse it, adding a mixed enzyme to the system, slowly stirring at 4°C for 12-16 hours to fully load the mixed enzyme onto the carrier, and after loading, collecting the solid by centrifugation at 8000 rpm for 10-30 minutes, washing it three times with a phosphate buffer solution with a pH of 7.0, and finally vacuum drying to obtain the enzyme preparation for alkali-resistant degradable plastics.

5. The method for preparing an enzyme preparation for alkali-resistant degradable plastics according to claim 4, wherein: In the step S1, the feeding ratio of sodium hydride, 5-bromomethyl-2-norbornene, hexanediol and tetrahydrofuran is 10-20g: 15-40g: 15-30mL: 150-200mL; in the step S2, the feeding ratio of perbromocalix[6]arene, triethylamine, triethyl phosphite and toluene is 5-15g: 18-30g: 8-12g: 150-200mL; in the step S3, the norbornene intermediate, perphosphoric acid The feeding ratio of ester calix[6]arene, alkali metal carbonate and N,N-dimethylformamide is 8-15g:10-25g:12-25g:120-150mL; the alkali metal carbonate is one of sodium carbonate, potassium carbonate or cesium carbonate; the feeding ratio of norbornene-calix[6]arene intermediate, tetrazine functionalized four-arm polyethylene glycol and dichloromethane in step S3 is 5-12g:8-20g:120-150mL.

6. The method for preparing an enzyme preparation for alkali-resistant degradable plastics according to claim 5, wherein: The feeding ratio of the four-arm polyethylene glycol succinimidyl glutarate, tetrazine-amino hydrochloride, triethylamine and dimethyl sulfoxide is 8-20 g: 3-5 g: 10-20 g: 100-150 mL.

7. The method for preparing an enzyme preparation for alkali-resistant degradable plastics according to claim 6, wherein: The feeding ratio of the alkali-resistant functionalized carrier, the mixed enzyme, and the phosphate buffer solution is 10-20 g: 5-12 g: 100-200 mL.

8. The method for preparing an enzyme preparation for alkali-resistant degradable plastics according to claim 7, wherein: The enzyme mixture comprises the following components in parts by weight: 6-15 parts of sucrose fatty acid ester, 4-8 parts of alkaline protease, 4-8 parts of α-amylase, 2-5 parts of cutinase, and 2-5 parts of cellulase.

9. The method for preparing an enzyme preparation for alkali-resistant degradable plastics according to claim 8, wherein: The feeding ratio of the calix[6]arene, liquid bromine and dichloromethane is 20-30 g: 60-80 mL: 200-400 mL.

10. An enzyme preparation for alkali-resistant degradable plastics, characterized by: The invention is prepared by the preparation method according to any one of claims 1 to 9.

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