Alkali-resistant degradable plastic enzyme preparation and preparation method thereof

Abstract

The application discloses an enzyme preparation for alkali-resistant degradable plastics and a preparation method thereof, and belongs to the technical field of plastic degradation enzyme preparations. The preparation process of the enzyme preparation comprises the following steps: synthesizing a norbornene intermediate and a full phosphate cup [6] arene, grafting a 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 multiple enzymes. The rigid cavity of the cup [6] arene can form specific inclusion with enzyme molecules, the phosphate group can enhance the enzyme-carrier binding force, and the cross-linked network can improve the stability of the carrier and provide multiple loading sites. The multi-enzyme synergistic system can destroy the surface structure of plastics, degrade the composite filler and cut the ester bond. The enzyme activity retention rate of the enzyme preparation in an alkaline environment is more than 85%, the half-life at 50 DEG C is more than 110h, the weight loss rates of polylactic acid film and composite materials containing 30% starch filler are both more than 92%, and the degradation efficiency of degradable plastics in an alkaline environment is significantly improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plastic-degrading enzyme preparation, and particularly relates to an alkali-resistant enzyme preparation for degradable plastics and a preparation method thereof. BACKGROUND

[0002] As a typical degradable polyester material, polylactic acid (PLA) is usually prepared by using starch in crops such as corn, wheat and sugarcane as raw material, through microbial fermentation to generate lactic acid, and then through chemical polycondensation or ring-opening polymerization of lactide. PLA can be completely decomposed into carbon dioxide and water in 3-6 months under composting conditions, without microplastic residues. The degradation products are harmless to the environment and human body, and the heat value released during combustion is only half of that of traditional plastics, and no toxic gases such as nitrogen compounds and sulfides are generated. Due to the good biocompatibility, degradability and renewable raw materials of polylactic acid, it has broad application prospects in the fields of packaging, medical treatment and agriculture. The degradation of polylactic acid is mainly realized through ester bond hydrolysis. Studies have shown that its degradation rate in alkaline conditions is significantly higher than that in acidic or neutral environments. Alkaline environments (such as industrial wastewater treatment systems and alkaline composting environments) can accelerate the degradation process of polylactic acid materials by promoting ester bond hydrolysis.

[0003] Traditional enzyme preparation carrier materials are prone to structural damage or surface charge property changes in alkaline environments, resulting in a decrease in the binding force between enzyme molecules and the carrier, and phenomena such as enzyme molecule shedding or denaturation and inactivation. At the same time, the direct attack of hydroxyl ions on the active center of the enzyme under alkaline conditions can destroy the spatial conformation of the enzyme protein, significantly reducing 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 the 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 alkaline environments and has the function of multiple enzyme synergistic degradation. SUMMARY

[0004] In view of the above situation, in order to overcome the defects of traditional enzyme preparations in the prior art, such as easy inactivation in alkaline environments, poor carrier stability and low degradation efficiency for degradable plastics containing composite fillers, the application provides an alkali-resistant enzyme preparation for degradable plastics and a preparation method thereof. The enzyme preparation realizes efficient degradation of degradable plastics in alkaline environments by constructing an alkali-resistant functional carrier containing a calix[6]arene structure and loading a mixed enzyme composed of multiple functional enzymes.

[0005] In order to achieve the above purpose, the following technical scheme is adopted: the application provides a preparation method of an alkali-resistant enzyme preparation for degradable plastics, comprising the following steps:

[0006] S1, sodium hydride was added to tetrahydrofuran, nitrogen was introduced for protection, 5-bromomethyl-2-norbornene was slowly added to tetrahydrofuran under continuous stirring, then hexanediol was slowly added to the reaction system through a constant pressure dropping funnel within 30-40 min. After the addition was completed, the reaction temperature was raised to 60-70℃, and the stirring reaction was continued for 6-8h. After the reaction was completed, the system was cooled to room temperature, and tetrahydrofuran was removed by reduced pressure distillation to obtain a norbornene intermediate;

[0007] S2, the total bromine calix[6]arene was added to toluene, then triethylamine and triethyl phosphite were added, the reaction temperature was raised to 110-120℃ under nitrogen protection, and the stirring reaction was carried out for 24-36h. After the reaction was completed, the system was cooled to room temperature, and toluene and excess triethyl phosphite were removed by reduced pressure distillation to obtain a total phosphonate calix[6]arene;

[0008] S3, the norbornene intermediate prepared in step S1 and the total phosphonate calix[6]arene prepared in step S2 were added to N,N-dimethylformamide, an alkali metal carbonate was added, and the system was heated to 80-90℃ under nitrogen protection. The stirring reaction was carried out for 18-24h. After the reaction was completed, the system was cooled to room temperature, filtered, and the filtrate was distilled under reduced pressure to remove DMF to obtain a norbornene-calix[6]arene intermediate;

[0009] S4, the four-arm polyethylene glycol succinimidyl glutarate was added to dimethyl sulfoxide, and was fully stirred to dissolve. Then, tetrazine-amino hydrochloride and triethylamine were added, and the stirring reaction was carried out at room temperature for 12-16h. After the reaction was completed, the reaction liquid was poured into ice water, and the precipitated solid was collected by filtration and vacuum dried to obtain a tetrazine-functionalized four-arm polyethylene glycol;

[0010] S5, the norbornene-calix[6]arene intermediate prepared in step S3 and the tetrazine-functionalized four-arm polyethylene glycol prepared in step S2 were added to dichloromethane, and the stirring reaction was carried out at room temperature for 24-36h. Then, the organic phase was extracted with deionized water, and the dichloromethane was removed by reduced pressure distillation to obtain an alkali-resistant functional carrier;

[0011] S6, the alkali-resistant functional carrier prepared in step S5 was added to a pH=7.0 phosphate buffer solution, and was fully dispersed by stirring. Then, a mixed enzyme was added to the system, and the stirring was slowly carried out at 4℃ for 12-16h to fully load the mixed enzyme onto the carrier. After the loading was completed, the solid was collected by centrifugation at 8000rpm for 10-30min, and was washed with a pH=7.0 phosphate buffer solution for 3 times. Finally, the vacuum drying was carried out to obtain an enzyme preparation for the alkali-resistant degradable plastic.

[0012] Further, the perbromo-calix[6]arene in step S2 is prepared by adding calix[6]arene into dichloromethane, stirring to dissolve, adding liquid bromine dropwise in ice bath, controlling the temperature not to exceed 5℃ during the dropwise addition, warming the reaction system to room temperature after the dropwise addition is completed, continuing to stir for 12-16h, adding saturated sodium sulfite solution into the system after the reaction is completed, washing the organic phase with deionized water and saturated sodium bicarbonate solution in turn, removing the dichloromethane solvent by reduced pressure distillation, and obtaining the perbromo-calix[6]arene.

[0013] Further, the feeding ratio of the calix[6]arene, liquid bromine and dichloromethane is 20-30g: 60-80mL: 200-400mL.

[0014] Further, the feeding ratio of sodium hydride, 5-bromomethyl-2-norbornene, hexanediol and tetrahydrofuran in step S1 is 10-20g: 15-40g: 15-30mL: 150-200mL.

[0015] Further, the feeding ratio of perbromo-calix[6]arene, triethylamine, triethyl phosphite and toluene in step S2 is 5-15g: 18-30g: 8-12g: 150-200mL.

[0016] Further, the feeding ratio of norbornene intermediate, perphosphonate calix[6]arene, alkali metal carbonate and N,N-dimethylformamide in step S3 is 8-15g: 10-25g: 12-25g: 120-150mL; the alkali metal carbonate is one of sodium carbonate, potassium carbonate or cesium carbonate.

[0017] Further, the feeding ratio of four-arm polyethylene glycol succinimidyl glutarate, tetrazine-amino hydrochloride, triethylamine and dimethyl sulfoxide in step S4 is 8-20g: 3-5g: 10-20g: 100-150mL.

[0018] Further, the feeding ratio of norbornene-calix[6]arene intermediate, tetrazine-functionalized four-arm polyethylene glycol and dichloromethane in step S5 is 5-12g: 8-20g: 120-150mL.

[0019] Further, the feeding ratio of alkali-resistant functionalized carrier, mixed enzyme, phosphate buffer solution in step S6 is 10-20g: 5-12g: 100-200mL; the mixed enzyme contains the following components by weight: sucrose fatty acid ester 6-15 parts, alkaline protease 4-8 parts, α-amylase 4-8 parts, cutinase 2-5 parts, cellulase 2-5 parts.

[0020] The alkaline protease can specifically hydrolyze protein additives or pollutants on the surface of degradable plastics, and destroy the dense structure of the material surface; the alpha-amylase and cellulase can degrade starch-based or cellulose-based fillers possibly remaining in the plastics to form a porous structure; the cutinase as a specific esterase can recognize and cut the ester bond of polyester materials such as polylactic acid; and the sucrose fatty acid ester as a surfactant can reduce the surface tension of the plastics, promote the adsorption and diffusion of the enzyme molecules. The multi-enzyme synergistic system can not only efficiently degrade pure polylactic acid materials, but also has good degradation effect on degradable plastic mixtures containing starch and cellulose composite fillers.

[0021] The beneficial effects of the present application are:

[0022] (1) The enzyme preparation prepared by the present application has excellent alkali resistance and high plastic degradation performance. By introducing calix[6]arene structure in the carrier construction process, the rigid electron-rich cavity thereof can form specific packaging with enzyme molecules, effectively protecting the enzyme active center from direct attack by hydroxyl ions in an alkaline environment, maintaining the stability of the spatial conformation of the enzyme protein. Meanwhile, the phosphate groups on the calixarene skeleton can form a hydrogen bond network and electrostatic interaction with the polar groups such as amino and hydroxyl groups on the surface of the enzyme molecules, enhancing the binding force between the enzyme and the carrier, avoiding the shedding or denaturation of the enzyme molecules 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 with a pH of 8.0-10.0, and can efficiently degrade various degradable plastics such as polylactic acid, polyadipic acid, and polybutylene terephthalate.

[0023] (2) The present application introduces norbornene groups into calix[6]arene molecules to form a norbornene-calix[6]arene intermediate with double bond active sites, and then realizes efficient grafting of calixarene and four-armed polyethylene glycol under mild conditions by using the Diels-Alder reaction of the tetrazine functional group with the norbornene group, forming a pyrazine-norbornene double ring system and a three-dimensional crosslinked network structure. This crosslinked 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. Meanwhile, the steric hindrance effect of the crosslinked network can effectively inhibit the aggregation and inactivation of enzyme molecules, prolonging the storage half-life of the enzyme preparation. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor belong to the scope of protection of the present application.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present application. The preferred materials and methods are described herein, although any method and material similar or equivalent to those described herein can be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0026] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0027] Example 1: A method for preparing an enzyme preparation for alkali-resistant degradable plastics, comprising the following steps:

[0028] S1, 10g of sodium hydride was added to 150mL of tetrahydrofuran, and nitrogen was introduced for protection. 15g of 5-bromomethyl-2-norbornene was slowly added to the tetrahydrofuran under continuous stirring. Then, 15mL of hexanediol was slowly added to the reaction system through a constant pressure dropping funnel within 30min. After the addition was completed, the reaction temperature was raised to 60℃, and the stirring reaction was continued for 6h. After the reaction was completed, the system was cooled to room temperature, and the tetrahydrofuran was removed by reduced pressure distillation to obtain a norbornene intermediate;

[0029] S2, 5g of perbrominated calix[6]arene was added to 150mL of toluene, and then 18g of triethylamine and 8g of triethyl phosphite were added. The reaction temperature was raised to 110℃ under nitrogen protection, and the stirring reaction was continued for 24h. After the reaction was completed, the system was cooled to room temperature, and the toluene and excess triethyl phosphite were removed by reduced pressure distillation to obtain a perphosphoric acid ester calix[6]arene;

[0030] S3, 8g of the norbornene intermediate prepared in step S1 and 10g of the perphosphoric acid ester calix[6]arene prepared in step S2 were added to 120mL of N,N-dimethylformamide, 12g of sodium carbonate was added, and the temperature was raised to 80℃ under nitrogen protection. The stirring reaction was continued for 18h. After the reaction was completed, the system was cooled to room temperature, filtered, and the filtrate was distilled under reduced pressure to remove DMF to obtain a norbornene-calix[6]arene intermediate;

[0031] S4, 8g of four-arm polyethylene glycol succinimidyl glutarate was added to 100mL of dimethyl sulfoxide, and was fully stirred to dissolve. Then, 3g of tetrazine-amino hydrochloride and 10g of triethylamine were added, and the stirring reaction was continued at room temperature for 12h. After the reaction was completed, the reaction liquid was poured into ice water, and the precipitated solid was collected by filtration and vacuum dried to obtain a tetrazine-functionalized four-arm polyethylene glycol;

[0032] S5, 5 g of the norbornene-calix[6]arene intermediate prepared in step S3 and 8 g of the tetrazine-functionalized four-armed polyethylene glycol prepared in step S2 were added to 120 mL of dichloromethane, and the reaction was stirred at room temperature for 24 h. The organic phase was extracted with deionized water, and dichloromethane was removed from the organic phase by distillation under reduced pressure to obtain an alkali-resistant functionalized carrier;

[0033] S6, 10 g of the alkali-resistant functionalized carrier prepared in step S5 was added to 100 mL of a phosphate buffer solution with pH = 7.0, and the mixture was stirred to fully disperse. 5 g of mixed enzymes (containing sucrose fatty acid ester 6 parts, alkaline protease 4 parts, alpha-amylase 4 parts, cutinase 2 parts, and cellulase 2 parts by mass fraction) were added to the system, and the mixed enzymes were fully loaded onto the carrier by slowly stirring at 4°C for 12 h. After the loading was completed, the solid was collected by centrifugation at 8000 rpm for 10 min, washed three times with a phosphate buffer solution with pH = 7.0, and finally dried under vacuum to obtain the enzyme preparation for degradable plastics resistant to alkali.

[0034] The perbromo-calix[6]arene in step S2 was prepared by the following steps: 20 g of calix[6]arene was added to 200 mL of dichloromethane and stirred to dissolve. In an ice bath, 60 mL of liquid bromine was added dropwise, and the temperature was controlled to be no more than 5°C during the addition. After the addition was completed, the reaction system was warmed to room temperature, and the stirring was continued for 12 h. After the reaction was completed, saturated sodium sulfite solution was added to the system, and the organic phase was successively washed with deionized water and saturated sodium bicarbonate solution. The dichloromethane solvent was removed by distillation under reduced pressure to obtain the perbromo-calix[6]arene.

[0035] Example 2: A method for preparing an enzyme preparation for degradable plastics resistant to alkali, comprising the following steps:

[0036] S1, 20 g of sodium hydride was added to 200 mL of tetrahydrofuran, and nitrogen was introduced for protection. 40 g of 5-bromomethyl-2-norbornene was slowly added to the tetrahydrofuran under continuous stirring. Then, 30 mL of hexanediol was slowly added to the reaction system through a constant pressure dropping funnel within 40 min. After the addition was completed, the reaction temperature was raised to 70°C, and the stirring was continued for 8 h. After the reaction was completed, the system was cooled to room temperature, and the tetrahydrofuran was removed by distillation under reduced pressure to obtain the norbornene intermediate.

[0037] S2, 15 g of perbromo-calix[6]arene was added to 200 mL of toluene, and then 30 g of triethylamine and 12 g of triethyl phosphite were added. The reaction temperature was raised to 120°C under nitrogen protection, and the stirring was continued for 36 h. After the reaction was completed, the system was cooled to room temperature, and the toluene and excess triethyl phosphite were removed by distillation under reduced pressure to obtain the perphosphonate calix[6]arene.

[0038] S3, 15 g of the norbornene intermediate prepared in step S1 and 25 g of the perphosphonate calix[6]arene prepared in step S2 were added to 150 mL of N,N-dimethylformamide, 25 g of potassium carbonate was added, and the reaction was stirred at 90℃ for 24 h under nitrogen protection. After the reaction was completed, the system was cooled to room temperature, filtered, and the filtrate was distilled under reduced pressure to remove DMF to obtain a norbornene-calix[6]arene intermediate;

[0039] S4, 20 g of the four-arm polyethylene glycol succinimidyl glutarate was added to 150 mL of dimethyl sulfoxide and stirred until dissolved. Then 5 g of tetrazine-amino hydrochloride and 20 g of triethylamine were added, and the reaction was stirred at room temperature for 16 h. After the reaction was completed, the reaction solution was poured into ice water, and the precipitated solid was collected by filtration and dried under vacuum to obtain the tetrazine-functionalized four-arm polyethylene glycol.

[0040] S5, 12 g of the norbornene-calix[6]arene intermediate prepared in step S3 and 20 g of the tetrazine-functionalized four-arm polyethylene glycol prepared in step S2 were added to 150 mL of dichloromethane and stirred at room temperature for 36 h. The organic phase was extracted with deionized water, and the dichloromethane was removed by distillation under reduced pressure to obtain an alkali-resistant functional carrier.

[0041] S6, 20 g of the alkali-resistant functional carrier prepared in step S5 was added to 200 mL of a pH = 7.0 phosphate buffer solution and stirred until fully dispersed. Then 12 g of mixed enzymes (containing sucrose fatty acid ester 15 parts, alkaline protease 8 parts, alpha-amylase 8 parts, cutinase 5 parts, and cellulase 5 parts by mass) was added to the system, and the mixed enzymes were slowly loaded onto the carrier by stirring at 4℃ for 16 h. After the loading was completed, the solid was collected by centrifugation at 8000 rpm for 30 min, washed three times with a pH = 7.0 phosphate buffer solution, and finally dried under vacuum to obtain the enzyme preparation for alkali-resistant degradable plastics.

[0042] The perbromo calix[6]arene in step S2 was prepared by the following steps: 30 g of calix[6]arene was added to 400 mL of dichloromethane and stirred until dissolved. Then 80 mL of liquid bromine was added dropwise in an ice bath, and the temperature was controlled not to exceed 5℃ during the addition. After the addition was completed, the reaction system was warmed to room temperature and stirred for 16 h. After the reaction was completed, saturated sodium sulfite solution was added to the system, and then the organic phase was washed with deionized water and saturated sodium bicarbonate solution in sequence. The dichloromethane solvent was removed by distillation under reduced pressure to obtain the perbromo calix[6]arene.

[0043] Example 3: A method for preparing an enzyme preparation for alkali-resistant degradable plastics, comprising the following steps:

[0044] S1, 15 g of sodium hydride was added to 175 mL of tetrahydrofuran, and nitrogen was introduced for protection. 27 g of 5-bromomethyl-2-norbornene was slowly added to the tetrahydrofuran under continuous stirring, and then 22 mL of hexanediol was slowly added to the reaction system through a constant pressure dropping funnel within 35 min. After the addition was completed, the reaction temperature was raised to 65 °C, and the stirring reaction was continued for 7 h. After the reaction was completed, the system was cooled to room temperature, and tetrahydrofuran was removed by reduced pressure distillation to obtain a norbornene intermediate;

[0045] S2, 10 g of perbrominated calix[6]arene was added to 175 mL of toluene, and then 24 g of triethylamine and 10 g of triethyl phosphite were added. Under nitrogen protection, the reaction temperature was raised to 115 °C, and the stirring reaction was continued for 30 h. After the reaction was completed, the system was cooled to room temperature, and toluene and excess triethyl phosphite were removed by reduced pressure distillation to obtain a perphosphonate calix[6]arene.

[0046] S3, 11 g of the norbornene intermediate prepared in step S1 and 17 g of the perphosphonate calix[6]arene prepared in step S2 were added to 135 mL of N,N-dimethylformamide, and 18 g of cesium carbonate was added. Under nitrogen protection, the temperature was raised to 85 °C, and the stirring reaction was continued for 21 h. After the reaction was completed, the system was cooled to room temperature, and the filtrate was subjected to reduced pressure distillation to remove DMF to obtain a norbornene-calix[6]arene intermediate.

[0047] S4, 14 g of four-arm polyethylene glycol succinimidyl glutarate was added to 125 mL of dimethyl sulfoxide, and was fully stirred to dissolve. Then, 4 g of tetrazine-amino hydrochloride and 15 g of triethylamine were added, and the stirring reaction was continued at room temperature for 14 h. After the reaction was completed, the reaction solution was poured into ice water, and the precipitated solid was collected by filtration and vacuum dried to obtain a tetrazine-functionalized four-arm polyethylene glycol.

[0048] S5, 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 were added to 135 mL of dichloromethane, and the stirring reaction was continued at room temperature for 30 h. The organic phase was extracted with deionized water, and dichloromethane was removed by reduced pressure distillation to obtain an alkali-resistant functional carrier.

[0049] S6, 15 g of the alkali-resistant functional carrier prepared in step S5 was added to 150 mL of a phosphate buffer solution with pH = 7.0, and was fully dispersed by stirring. 8 g of a mixed enzyme (containing sucrose fatty acid ester 10 parts, alkaline protease 6 parts, α-amylase 6 parts, cutinase 3 parts, and cellulase 3 parts by mass fraction) was added to the system, and the mixed enzyme was fully loaded onto the carrier by slowly stirring at 4 °C for 14 h. After the loading was completed, the solid was collected by centrifugation at 8000 rpm for 20 min, and was washed with a phosphate buffer solution with pH = 7.0 for 3 times. Finally, vacuum drying was performed to obtain the enzyme preparation for the alkali-resistant degradable plastic.

[0050] The perbromo-calix[6]arene in step S2 is prepared by the following steps: 25 g of calix[6]arene is added to 300 mL of dichloromethane, and dissolved by stirring, 70 mL of liquid bromine is added dropwise in an ice bath, the temperature is controlled not to exceed 5 ℃ during the dropwise addition, after the dropwise addition is completed, the reaction system is warmed to room temperature, and stirring is continued for 14 h, after the reaction is completed, saturated sodium sulfite solution is added to the system, and then the organic phase is washed with deionized water and saturated sodium bicarbonate solution in sequence, and the dichloromethane solvent is removed by reduced pressure distillation to obtain the perbromo-calix[6]arene.

[0051] Comparative Example 1: In this comparative example, conventional diatomite is used instead of the alkali-resistant functionalized carrier to perform the loading in step S6, and the rest is the same as Example 3.

[0052] Comparative Example 2: In this comparative example, a norbornene intermediate is used instead of the norbornene-calix[6]arene intermediate to perform the reaction in step S5 with the tetrazine functionalized four-armed polyethylene glycol, and the rest is the same as Example 3.

[0053] Comparative Example 3: In this comparative example, a four-armed polyethylene glycol is used instead of the alkali-resistant functionalized carrier to perform the loading in step S6, and the rest is the same as Example 3.

[0054] The enzyme preparations prepared in each group of examples and comparative examples are tested and analyzed, and the results are shown in Table 1.

[0055] The enzyme preparations prepared in each group of examples and comparative examples are dissolved in a sodium carbonate-sodium bicarbonate buffer solution with pH = 10.0, and are oscillated at 45 ℃ for 72 h, the residual enzyme activity is measured, and the activity retention rate is calculated. Casein is used as the substrate, and the alkaline protease activity is measured by the Folin-phenol method (unit: U / mL), the activities of other enzymes are measured by spectrophotometry using the corresponding substrates, and the total enzyme activity is the sum of the activities of each enzyme.

[0056] The enzyme preparations prepared in each group of examples and comparative examples are placed in a 50 ℃ water bath, and the enzyme activity is measured at intervals to calculate the time required for the activity to decrease to 50% of the initial value, which is the 50 ℃ half-life of each group of enzyme preparations.

[0057] PLA film (thickness 50 μm, molecular weight 100,000) and PLA composite material containing 30% starch filler are taken respectively, and the degradation efficiency of the enzyme preparations prepared in each group of examples and comparative examples is tested in an alkaline compost simulation environment, with the conditions being: pH = 9.0, temperature 55 ℃, humidity 80%, enzyme preparation concentration 2 g / L, oscillation rate 150 rpm, and reaction time 14 days. After 14 days, the samples are taken out, dried and weighed, the weight loss ratio is calculated, and the number average molecular weight Mn of the PLA film is measured by GPC.

[0058] Table 1: Comparison table of enzyme preparation test results

[0059]

[0060] From the results of the above table, it can be seen that the enzyme activity retention rates of the various embodiments are all over 91% in an alkaline environment with pH = 10. Since the rigid electron-rich cavity of calix[6]arene can encapsulate the enzyme active center through molecular recognition, it can resist the attack of hydroxyl ions. The phosphonate groups form hydrogen bonds and electrostatic interactions with the enzyme molecules, thereby enhancing the binding force and avoiding enzyme shedding or denaturation. The half-life of each embodiment at 50℃ is over 110h, while the half-life of Comparative Example 1 is only 45h, and the half-lives of Comparative Examples 2 and 3 are 68h and 75h, respectively. The reason is that the Diels-Alder reaction between norbornene-calix[6]arene and four-arm polyethylene glycol forms a three-dimensional cross-linked network, which increases the specific surface area and inhibits the aggregation of enzyme molecules, thereby improving the mechanical strength and chemical stability. The cross-linked structure reduces the inactivation of enzyme molecules caused by the interaction between the enzyme molecules, thereby prolonging the storage period in actual application. The weight loss rate of the PLA film of each embodiment is over 93%, and the weight loss rate of the composite material is over 92%. In addition, the number average molecular weight of the PLA film decreases from 100,000 to about 20,000, indicating that the ester bond is efficiently cleaved, and the degradation efficiency of the enzyme preparation prepared by the present application is high.

[0061] Although embodiments of the present application have been shown and described, it is to be understood that for the purpose of the present application, the embodiments shown and described can be modified, substituted, replaced and changed in many ways without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

[0062] The above describes the present application and its embodiments, which are not restrictive, and the embodiments shown in the embodiments are only one of the embodiments of the present application, and the actual application is not limited thereto. In summary, if a person skilled in the art is inspired by it, without departing from the purpose of the present application, without creative design, similar ways and embodiments of the technical solutions should belong to the protection scope of the present application.

Claims

1. A process for the preparation of an enzyme formulation for alkali resistant degradable plastics, characterized by: Comprise the following steps: S1, sodium hydride is added to tetrahydrofuran, nitrogen is blown in for protection, 5-bromomethyl-2-norbornene is slowly added to tetrahydrofuran under continuous stirring, then hexanediol is slowly dropped into the reaction system through a constant pressure dropping funnel within 30-40 min; after dropping is completed, the reaction temperature is raised to 60-70 DEG C, and the stirring reaction is continued for 6-8 h, the system is cooled to room temperature after the reaction is completed, and tetrahydrofuran is removed by reduced pressure distillation, to obtain a norbornene intermediate; S2, the norbornene intermediate and the full phosphonate calix[6] arene are added to N, N-dimethylformamide, an alkali metal carbonate is added, and the temperature is raised to 80-90 DEG C under nitrogen protection, and the stirring reaction is carried out for 18-24 h, the system is cooled to room temperature after the reaction is completed, filtration is carried out, and the filtrate is distilled under reduced pressure to remove DMF, to obtain a norbornene-calix[6] arene intermediate; S3, the four-arm polyethylene glycol succinimidyl glutarate is activated by using a tetrazine-amino hydrochloride, to obtain a tetrazine functionalized four-arm polyethylene glycol, the norbornene-calix[6] arene intermediate and the tetrazine functionalized four-arm polyethylene glycol are added to dichloromethane, and the stirring reaction is carried out at room temperature for 24-36 h, the organic phase is extracted with deionized water, dichloromethane is removed from the organic phase by reduced pressure distillation, to obtain an alkali-resistant functionalized carrier; S4, the functionalized carrier is loaded with mixed enzymes to obtain an enzyme preparation for alkali-resistant degradable plastics.

2. The process for the preparation of enzyme formulation for alkali resistant degradable plastics as claimed in claim 1, wherein: The full phosphonate calix[6] arene is prepared by the following steps: The calix[6] arene is added to dichloromethane, and is dissolved by stirring; liquid bromine is added dropwise in an ice bath, the temperature is controlled to be not more than 5 DEG C during the dropping process, the reaction system is warmed to room temperature after dropping is completed, the stirring reaction is continued for 12-16 h, and after the reaction is completed, saturated sodium sulfite solution is added to the system, and then the organic phase is washed with deionized water and saturated sodium bicarbonate solution in sequence, and dichloromethane solvent is removed by reduced pressure distillation, to obtain a full bromine calix[6] arene; The full bromine calix[6] arene is added to toluene, triethylamine and triethyl phosphite are added, the reaction temperature is raised to 110-120 DEG C under nitrogen protection, the stirring reaction is carried out for 24-36 h, the system is cooled to room temperature after the reaction is completed, and toluene and excess triethyl phosphite are removed by reduced pressure distillation, to obtain the full phosphonate calix[6] arene.

3. The process for the preparation of enzyme formulation for alkali resistant degradable plastics as claimed in claim 2, wherein: The tetrazine functionalized four-arm polyethylene glycol is prepared by the following steps: the four-arm polyethylene glycol succinimidyl glutarate is added to dimethyl sulfoxide, and is dissolved by stirring, then tetrazine-amino hydrochloride and triethylamine are added, and the stirring reaction is carried out at room temperature for 12-16 h, and after the reaction is completed, the reaction liquid is poured into ice water, the precipitated solid is collected by filtration, and vacuum drying is carried out, to obtain the tetrazine functionalized four-arm polyethylene glycol.

4. The process for the preparation of enzyme formulation for alkali resistant degradable plastics as claimed in claim 3, wherein: The step S4 specifically comprises the following steps: adding the alkali-resistant functional carrier into a phosphate buffer solution with pH=7.0, stirring to fully disperse, adding mixed enzymes into the system, slowly stirring at 4℃ for 12-16h to fully load the mixed enzymes onto the carrier, after the loading is completed, collecting the solid by centrifugation at 8000rpm for 10-30min, washing 3 times with a phosphate buffer solution with pH=7.0, and finally vacuum drying to obtain the enzyme preparation for alkali-resistant degradable plastics.

5. The process for the preparation of enzyme formulation for alkali resistant degradable plastics as claimed in claim 4, wherein: The feeding ratio of sodium hydride, 5-bromomethyl-2-norbornene, hexanediol and tetrahydrofuran in the step S1 is 10-20g:15-40g:15-30mL:150-200mL; the feeding ratio of perbromide calix[6]arene, triethylamine, triethyl phosphite and toluene in the step S2 is 5-15g:18-30g:8-12g:150-200mL; the feeding ratio of norbornene intermediate, perphosphate calix[6]arene, alkali metal carbonate and N,N-dimethylformamide in the step S3 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-armed polyethylene glycol and dichloromethane in the step S3 is 5-12g:8-20g:120-150mL.

6. The process for the preparation of enzyme formulation for alkali resistant degradable plastics as claimed in claim 5, wherein: The feeding ratio of the four-armed polyethylene glycol succinimidyl glutarate, tetrazine-amino hydrochloride, triethylamine and dimethyl sulfoxide is 8-20g:3-5g:10-20g:100-150mL.

7. The process for the preparation of enzyme formulation for alkali resistant degradable plastics as claimed in claim 6, wherein: The feeding ratio of the alkali-resistant functional carrier, mixed enzymes and phosphate buffer solution is 10-20g:5-12g:100-200mL.

8. The process for the preparation of enzyme formulation for alkali resistant degradable plastics as claimed in claim 7, wherein: The mixed enzymes comprise the following components by weight: sucrose fatty acid ester 6-15 parts, alkaline protease 4-8 parts, alpha-amylase 4-8 parts, cutinase 2-5 parts, cellulase 2-5 parts.

9. The method of claim 8, wherein the method is characterized by: The feeding ratio of calix[6]arene, liquid bromine and dichloromethane is 20-30g:60-80mL:200-400mL.

10. An enzyme preparation for alkali resistant degradable plastics, characterized by: Prepared by the preparation method of any one of claims 1-9. Prepared by the preparation method of any one of claims 1-9.

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