A method for complete depolymerization of thermoplastic polyester polyurethane plastics using two enzymes

Abstract

This invention belongs to the field of environmental science and relates to a method for the complete depolymerization of thermoplastic polyester polyurethane (PBA-PU) using a dual-enzyme approach. Specifically, PBA-PU is used as a substrate, and esterases PETase and Aes72 are added to initiate the depolymerization reaction. PETase hydrolyzes the ester bonds, while Aes72 primarily hydrolyzes the carbamate bonds. The two enzymes synergistically promote the complete depolymerization of PBA-PU. This invention utilizes a dual-enzyme approach to degrade PBA-PU, improving enzymatic hydrolysis efficiency while completely depolymerizing PBA-PU into the monomer MDA. The monomers generated after depolymerization can then be repolymerized or utilized for other high-value applications, thereby improving product utilization and enhancing the competitive advantage of enzymatic degradation of PBA-PU.

Description

Technical Field

[0001] This invention belongs to the field of environmental science and relates to a method for completely depolymerizing thermoplastic polyester polyurethane plastics using two enzymes. Background Technology

[0002] Polyurethane (PU) plastic is one of the five largest plastics produced and consumed globally, with applications spanning textiles, construction, building materials, automobiles, and defense. Statistics show that global plastic production reached 368 million tons in 2019, with PU plastic accounting for 6% to 7% of total production, making it the second largest polyester-type plastic produced globally. In my country alone, production reached 14.7 million tons in 2020, with consumption around 11.75 million tons. Most plastic products are disposable or for short-term use, and this massive consumption inevitably leads to a huge amount of post-consumer waste. As of 2015, the world had generated 6.3 billion tons of plastic waste, of which 91% was incinerated, landfilled, or dumped into soil and oceans. This not only wastes the Earth's carbon resources but also raises environmental concerns, such as microplastic pollution, which has drawn attention from all countries. Of the 9% of waste plastics recycled, most are waste plastics with simple structures, clean products, and clear components, such as scraps from plastic factories and food-grade polyethylene terephthalate (PET) packaging. However, PU plastics have complex structures and diverse types, making them difficult to effectively reuse through existing physical and chemical recycling methods.

[0003] With the establishment of enzymatic recycling technology for PET plastics, enzymatic recycling of waste plastics has become a focus of research both domestically and internationally. Carbios, a French company, utilizes cutinase LCC derived from plant compost to decompose 97% of PET plastics within 16 hours. The decomposition products are then re-reacted to generate new plastics. This recycling method is currently patented and represents the most efficient biological plastic recycling practice to date, effectively promoting a circular economy and reducing the use of virgin plastics. While enzymatic recycling of PU plastics is currently in the stage of exploring highly efficient depolymerization enzyme elements, the success of enzymatic recycling technology for PET plastics provides important feasibility references and technical lessons.

[0004] Compared to microorganisms, enzymatic degradation has the advantage of producing valuable degradation products after depolymerization, and it is easier to control and more reproducible than microbial degradation. Due to the complex structure of PU plastic, single-function enzymes cannot completely depolymerize it into monomeric substances, thus failing to yield valuable degradation products. Therefore, the application of a dual-enzyme system for the biorecycling of PU has significant practical implications. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for completely depolymerizing thermoplastic polyester polyurethane plastics by two enzymes, which addresses the shortcomings of the prior art.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] This invention discloses a method for the complete depolymerization of thermoplastic polyester polyurethane plastics using two enzymes, comprising the following steps:

[0008] (1) Dissolve polyurethane plastic in an organic solvent and sonicate to obtain a first mixture; pour the first mixture into a glass plate and let it air dry to obtain a polyurethane plastic film;

[0009] (2) Dissolve the esterase and the polyurethane plastic film obtained in step (1) in phosphate buffer to carry out the depolymerization reaction.

[0010] Specifically, in step (1), the polyurethane plastic is a polyester-type polyurethane.

[0011] Preferably, in step (1), the polyurethane plastic is PBA-PU.

[0012] Specifically, in step (1), the organic solvent is dichloromethane; the mass-to-volume ratio of the polyurethane plastic to the organic solvent is 1.5-2 g: 20 mL, preferably 1.5 g: 20 mL.

[0013] Specifically, in step (1), the ultrasonic dissolution is performed at an ultrasonic frequency of 40 Hz and an ultrasonic temperature of 25-30°C, preferably 25°C.

[0014] In step (1), the ultrasonic dissolution is performed for a duration of time until the solid in the first mixture is completely dissolved.

[0015] In step (1), the glass plate has a diameter of 9cm.

[0016] In step (1), after air drying, the polyurethane plastic film is cut into 1cm×1cm squares as a degradation substrate.

[0017] Specifically, in step (2), the esterase is PETase and Aes72; the mass ratio of PETase to Aes72 in the esterase is 1:37 to 1:40, preferably 1:37.

[0018] Specifically, in step (2), the PETase and Aes72 used p-NPB to determine enzyme activity. Enzyme activity is defined as: the production of 1 μmol of p-nitrophenol per minute by hydrolyzing p-NPB at 37°C is defined as one unit of protease activity.

[0019] The formula for protease activity is: Protease activity = A / (B*C), where A is the amount of p-nitrophenol produced (μmol), B is the reaction time (min), and C is the amount of enzyme added to the reaction (mL).

[0020] Wherein, specific enzyme activity = protease activity (U / mL) / protein concentration (mg / mL).

[0021] Specifically, in step (2), the specific enzyme activity of PETase is 33-40 U / mg; and the specific enzyme activity of Aes72 is 67-75 U / mg.

[0022] The preparation method of the PETase mentioned herein is described in A bacterium that degrades and aspirinates poly(ethylene terephthalate), SCIENCE, 351(6278), 1196-1199.

[0023] The preparation method of Aes72 is described in reference to patent EP 3587570A1.

[0024] Specifically, in step (2), the mass-to-volume ratio of the esterase to the phosphate buffer is 3.8–4.1 mg:5 mL, preferably 3.8 mg:5 mL.

[0025] Specifically, in step (2), the mass-to-volume ratio of the polyurethane plastic film to the phosphate buffer is 3-6 mg:1 mL, preferably 4-5 mg:1 mL.

[0026] Specifically, in step (2), the depolymerization reaction is carried out at a temperature of 37–40°C for 24–48 hours.

[0027] In step (2), the phosphate buffer (PB buffer) is a 50mM phosphate buffer with a pH of 7.74.

[0028] In step (2), the depolymerization reaction is preferably carried out in a shaker at a speed of 200 rpm.

[0029] In step (2), after the depolymerization reaction is completed, the polyurethane plastic film after the reaction is taken out, washed with pure water, dried in a 30℃ incubator, weighed to calculate the mass loss before and after the reaction, and Fourier transform infrared spectroscopy (FTIR) analysis is performed. After centrifuging the supernatant of the reaction solution at 12000 rpm for 2 min, the supernatant is taken for high performance liquid chromatography (HPLC) analysis.

[0030] The chromatographic conditions for the high-performance liquid chromatography (HPLC) analysis are as follows:

[0031] Liquid chromatography column: Agilent 5HC-C18(2) 150×4.6mm;

[0032] Detection wavelength: 240nm;

[0033] Column temperature: 30℃;

[0034] Flow rate: 1 mL / min;

[0035] Injection volume: 10 μL;

[0036] The mobile phases are water and acetonitrile, respectively;

[0037] Gradient elution: 0–5 min, water volume fraction 90%, acetonitrile volume fraction 10%; 5–14 min, water volume fraction changes from 90% to 35%, acetonitrile volume fraction changes from 10% to 65%.

[0038] Beneficial effects:

[0039] (1) The dual-enzyme system used in this invention improves the enzymatic hydrolysis effect while completely depolymerizing thermoplastic polyester polyurethane plastics, thereby improving product utilization. In the dual-enzyme system, the esterase PETase can hydrolyze the ester bonds in PBA-PU, but cannot hydrolyze the carbamate bonds. The esterase Aes72, however, has carbamate activity and can completely depolymerize the oligomers with carbamate bonds produced by PETase hydrolysis into monomers, thus relieving the product inhibition of PETase by the oligomers and improving degradation efficiency. Simultaneously, it increases the yield of monomers and improves product utilization.

[0040] (2) The dual-enzyme system used in this invention can improve the biodegradation efficiency of thermoplastic polyurethane plastics and completely depolymerize them to improve product utilization, which is of great significance for the bio-recycling and remanufacturing of PU plastics.

[0041] (3) The plastic bio-enzymatic depolymerization and high-value utilization technology of the present invention has mild conditions, few by-products, and is green and environmentally friendly. It is an ideal means for the same-level and upgraded utilization of waste plastics and is also a research hotspot at home and abroad.

[0042] (4) Plastics are polymer materials made from monomer raw materials through addition polymerization or condensation polymerization. If waste can be depolymerized to obtain monomers for reprocessing, making the entire industrial chain a closed loop, it is the most ideal way to treat waste resources. The dual-enzyme system used in this invention can completely depolymerize thermoplastic polyester polyurethane plastics into monomers 4,4-methylenediphenylamine (MDA). The monomers generated after depolymerization can be used for repolymerization or other high-value utilization, realizing the harmless recycling of polyurethane waste plastics. Attached Figure Description

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0044] Figure 1 A schematic diagram illustrating the degradation of PBA-PU by the PETase and Aes72 dual enzymes.

[0045] Figure 2 The mass loss of the PBA-PU film before and after degradation.

[0046] Figure 3 FTIR analysis of the degraded PBA-PU film.

[0047] Figure 4 HPLC analysis of the supernatant after degradation.

[0048] Figure 5 This is the standard curve for MDA concentration.

[0049] Figure 6 The concentration of MDA in the supernatant after degradation. Detailed Implementation

[0050] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0051] The esterases used in this invention are PETase and Aes72; wherein, the enzyme activity of PETase and Aes72 is determined by p-NPB, and the enzyme activity is defined as: at 37°C, the production of 1 μmol of p-nitrophenol per minute by hydrolyzing p-NPB is defined as one unit of protease activity.

[0052] The formula for protease activity is: Protease activity = A / (B*C), where A is the amount of p-nitrophenol produced (μmol), B is the reaction time (min), and C is the amount of enzyme added to the reaction (mL).

[0053] Wherein, specific enzyme activity = protease activity (U / mL) / protein concentration (mg / mL).

[0054] The specific enzyme activity of PETase was 33 U / mg, and the specific enzyme activity of Aes72 was 67 U / mg.

[0055] The preparation method of PETase is described in A bacterium that degrades and assimilates poly(ethylene terephthalate), SCIENCE, 351(6278), 1196-1199.

[0056] The preparation method of Aes72 is referenced in patent EP 3587570A1.

[0057] The phosphate buffer (PB buffer) used in this embodiment of the invention is a 50mM phosphate buffer with a pH of 7.74.

[0058] The high-performance liquid chromatography (HPLC) analysis in this embodiment of the invention uses a high-performance liquid chromatograph (Agilent Technologies), and the specific detection method is as follows:

[0059] Liquid chromatography column: Agilent 5HC-C18(2) 150×4.6mm;

[0060] Detection wavelength: 240nm;

[0061] Column temperature: 30℃;

[0062] Flow rate: 1 mL / min;

[0063] Injection volume: 10 μL;

[0064] The mobile phases are water and acetonitrile, respectively;

[0065] Gradient elution: 0–5 min, water volume fraction 90%, acetonitrile volume fraction 10%; 5–14 min, water volume fraction changes from 90% to 35%, acetonitrile volume fraction changes from 10% to 65%.

[0066] Example 1: Determination of mass loss of depolymerized polyurethane plastic film

[0067] Weigh 1.5g of PBA-PU powder and dissolve it in 20mL of dichloromethane solution. Sonicate the solution at 25℃ for 1h at a frequency of 40Hz. After sonication, pour the mixture into a glass plate with a diameter of 9cm and place it in a fume hood to air dry naturally until it forms a film. Cut the film into 1cm×1cm films as degradation substrates.

[0068] Four experimental groups were designed: a control group (CK), an Aes72 group, a PETase group, and a PETase+Aes72 group. In each group, a weighed PBA-PU membrane (1cm × 1cm, 20–25mg) was added to 5mL of 50mM PB buffer (pH = 7.74). The CK group received no enzyme, the Aes72 group received 3.7mg of Aes72, the PETase group received 0.1mg of PETase, and the PETase+Aes72 group received both 0.1mg of PETase and 3.7mg of Aes72.

[0069] Then, the above four reaction solutions were placed in a shaker at 37℃ and 200 rpm for 48 hours. The membrane was then removed, washed with pure water, dried at 30℃, and weighed. The mass loss before and after the reaction was calculated. The results are as follows. Figure 2 As shown. Figure 1 A schematic diagram illustrating the degradation of PBA-PU by the PETase and Aes72 dual enzymes.

[0070] After the reaction was completed, the supernatant of the reaction solution was centrifuged at 12,000 rpm for 2 minutes, and the supernatant was stored at 4°C for later use.

[0071] from Figure 2 The mass loss results show that the Aes72 group had a relatively low mass loss, indicating that this enzyme has weak exonuclease activity against PBA-PU and low activity against the polymer. PETase, on the other hand, showed a higher mass loss, indicating that this enzyme has strong exonuclease activity against PBA-PU and can effectively hydrolyze the polymer. The PETase+Aes72 group had a higher mass loss than both the PETase and Aes72 groups alone, indicating a synergistic effect between the two enzymes in the degradation of PBA-PU, thus improving the degradation rate.

[0072] Example 2: FTIR analysis of polyurethane plastic film after reaction

[0073] FTIR analysis was performed on the four groups of PBA-PU films after the reaction in Example 1. The FTIR was measured using a Nicolet iN10 infrared spectrometer, with a wavenumber range of 4000–500 cm⁻¹. -1 The scan was performed four times, equipped with a horizontal attenuation total reflection attachment. The results are as follows: Figure 3 As shown.

[0074] from Figure 3 It can be seen that the Aes72 group is at 1725cm. -1 The characteristic peak signal of the ester bond (C=O) and 1260 cm⁻¹ -1 The characteristic peak signal of the amide bond (CN) was weakened, but the degree of weakening was low, indicating that the enzyme has some activity against ester and carbamate bonds, but the activity is low; the PETase group showed a peak signal at 1725 cm⁻¹. -1 The characteristic peak signal of the ester bond (C=O) is significantly weakened, but at 1260 cm⁻¹... -1 The characteristic peak signal of the amide bond (CN) showed almost no attenuation, indicating that PETase can only hydrolyze ester bonds and has a strong hydrolytic ability, but cannot hydrolyze carbamate bonds; while the PETase+Aes72 group showed a peak signal at 1725 cm⁻¹. -1 The characteristic peaks of the ester bond (C=O) and 1260 cm⁻¹ -1The characteristic peak signal of the amide bond (CN) was weaker than that of the PETase group and the Aes72 group, indicating that the dual enzymes hydrolyzed the ester bond and carbamate bond on PBA-PU more completely, further proving that the dual enzymes have a synergistic effect on the degradation of PBA-PU.

[0075] Example 3: HPLC analysis of the supernatant after the reaction

[0076] The supernatants from the four reaction groups in Example 1 were analyzed by HPLC using an Agilent Technologies 1260 Infinity II high-performance liquid chromatograph. The specific detection method was as follows:

[0077] Liquid chromatography column: Agilent 5HC-C18(2) 150×4.6mm;

[0078] Detection wavelength: 240nm;

[0079] Column temperature: 30℃;

[0080] Flow rate: 1 mL / min;

[0081] Injection volume: 10 μL;

[0082] The mobile phases are water and acetonitrile, respectively;

[0083] Gradient elution: 0–5 min, water volume fraction 90%, acetonitrile volume fraction 10%; 5–14 min, water volume fraction changes from 90% to 35%, acetonitrile volume fraction changes from 10% to 65%. Specific detection results are as follows: Figure 4 As shown.

[0084] The MDA content in the supernatant represents the degree of complete depolymerization, because MDA is only produced when both ester and carbamate bonds are hydrolyzed simultaneously. Figure 4 The experimental results show that the PETase group did not produce MDA because PETase can only hydrolyze ester bonds and not urethane bonds. The Aes72 group produced trace amounts of MDA because Aes72 can hydrolyze both urethane and ester bonds, but its efficiency is low, resulting in very low yields. The MDA yield of the dual-enzyme system (PETase + Aes72) was significantly higher than that of the single-enzyme system, indicating that the dual-enzyme system can more completely depolymerize PBA-PU.

[0085] MDA Standard Curve Preparation: Weigh 19.8 mg of MDA and dissolve it in 10 mL of ethanol to prepare a 10 mM stock solution. Dilute this stock solution to prepare standards of 1 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, 0.1 mM, and 0.05 mM, respectively. Analyze the peak areas corresponding to different concentrations using HPLC to prepare an MDA concentration standard curve. The standard curve is shown below. Figure 5 As shown.

[0086] MDA content determination: The peak areas from the four experiments, determined by HPLC, were substituted into the standard curve to calculate the corresponding MDA concentrations. The results are as follows: Figure 6 As shown.

[0087] from Figure 4 , Figure 5 , Figure 6 HPLC analysis of the supernatants from the four experimental groups revealed that the Aes72 group produced 0.025 mM MDA, indicating that Aes72 possesses some hydrolytic ability towards PBA-PU, capable of hydrolyzing ester and carbamate bonds, although this ability is relatively weak. The PETase group did not produce MDA, indicating that PETase has no ability to degrade carbamate bonds and cannot further hydrolyze them to produce MDA. However, the PETase+Aes72 group produced a high amount of MDA, reaching 0.1875 mM, demonstrating that the dual-enzyme system PETase+Aes72 can simultaneously and efficiently hydrolyze both ester and carbamate bonds, thereby generating a large amount of monomeric MDA. This proves that the dual-enzyme system achieves more complete depolymerization of thermoplastic polyurethane.

[0088] This invention provides a method for the complete depolymerization of thermoplastic polyester polyurethane plastics using a dual-enzyme approach. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for the complete depolymerization of thermoplastic polyester- urethane plastics by two enzymes, characterized in that, The method comprises the following steps: (1) dissolving polyurethane plastics in an organic solvent to obtain a first mixed solution by ultrasonic dissolution; pouring the first mixed solution into a glass flat plate and naturally drying to obtain a polyurethane plastic film; (2) dissolving esterase and the polyurethane plastic film obtained in step (1) in a phosphate buffer to perform depolymerization reaction; The polyurethane plastics are PBA-PU; The esterase is PETase and Aes72; The organic solvent is dichloromethane.

2. The method of claim 1, wherein, In step (1), the mass-volume ratio of the polyurethane plastics to the organic solvent is 1.5-2 g:20 mL.

3. The method of claim 1, wherein, In step (1), the ultrasonic dissolution is performed at an ultrasonic frequency of 40 Hz and an ultrasonic temperature of 25-30 DEG C.

4. The method of claim 1, wherein, In step (2), the mass ratio of PETase to Aes72 in the esterase is 1:37-1:

40.

5. The method of claim 1, wherein, In step (2), the specific enzyme activity of the PETase is 33-40 U / mg; and the specific enzyme activity of the Aes72 is 67-75 U / mg.

6. The method of claim 1, wherein, In step (2), the mass-volume ratio of the esterase to the phosphate buffer is 3.8-4.1 mg:5 mL.

7. The method of claim 1, wherein, In step (2), the mass-volume ratio of the polyurethane plastic film to the phosphate buffer is 3-6 mg:1 mL.

8. The method of claim 1, wherein, In step (2), the depolymerization reaction is performed at a reaction temperature of 37-40 DEG C for 24-48 h.

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