Degradation enzyme PA5 as well as coding gene, recombinant vector, recombinant strain, enzyme preparation and application thereof

Abstract

The invention relates to the technical field of enzyme engineering and biology, and discloses a degrading enzyme PA5 as well as a coding gene, a recombinant vector, a recombinant strain, an enzyme preparation and application thereof. The amino acid sequence of the degrading enzyme PA5 is as shown in SEQ ID NO. 1. The specific amino acid sequence endows the enzyme with efficient degradation activity on thermoplastic polyurethane, characteristic chemical bonds in thermoplastic polyurethane molecules can be specifically hydrolyzed, the problem that an existing degrading enzyme is insufficient in thermoplastic polyurethane degradation capacity is effectively solved, the enzyme can keep stable catalytic performance under mild conditions, and the degradation efficiency is improved. The degradation bottleneck of high-crystallinity and high-molecular-weight thermoplastic polyurethane is broken through, a special enzyme preparation is provided for biodegradation treatment of thermoplastic polyurethane waste, and a key technical support is also provided for resource utilization of waste polyurethane and bioremediation of environmental plastic pollution.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and biotechnology, specifically to a degradation enzyme PA5 and its encoding gene, recombinant vector, recombinant strain, enzyme preparation, and applications. Background Technology

[0002] Polyurethane (PU) is a class of polymeric materials produced by the reaction of polyols and polyisocyanates. Due to its excellent mechanical properties, abrasion resistance, and chemical corrosion resistance, it is widely used in foam materials, elastomers, coatings, adhesives, footwear materials, medical devices, and electronic products. Among them, thermoplastic polyurethane (TPU) combines the elasticity of rubber with the processing properties of thermoplastics, and its ability to be repeatedly melted and processed makes it one of the fastest-growing polyurethane materials.

[0003] However, the TPU molecule contains multiple chemical bonds, including urethane bonds (–NHCOO–), ester bonds, and ether bonds, making it structurally stable and highly resistant to aging, which hinders its degradation in the natural environment. With the continuous increase in the use of TPU products, a large amount of waste TPU material enters the environment. Traditional landfill and incineration methods not only occupy land resources but may also generate toxic and harmful gases and secondary pollution, becoming an environmental problem that urgently needs to be addressed.

[0004] Currently, the recycling and degradation of polyurethane mainly rely on physical recycling and chemical degradation methods. Physical recycling typically requires crushing and reprocessing of the material, resulting in a decline in the performance of the product and limiting its applications. Chemical degradation methods, such as alcoholysis, hydrolysis, and ammonolysis, often require high temperature, high pressure, or strong acid / alkali conditions, resulting in high energy consumption, complex processes, and potential equipment corrosion and environmental safety hazards. Therefore, developing mild, efficient, and environmentally friendly polyurethane degradation technologies is of great significance.

[0005] In recent years, biodegradation methods have attracted attention due to their mild reaction conditions and environmental friendliness. Studies have found that certain microorganisms and their secreted enzymes, such as lipases, esterases, ureases, and polyurethane enzymes, have a certain ability to catalyze the hydrolysis of some chemical bonds in polyurethane. However, existing polyurethane-degrading enzymes generally suffer from problems such as low degradation efficiency, narrow substrate applicability, and limited ability to degrade thermoplastic polyurethanes, making it difficult to meet practical application needs. In particular, there are still significant technical bottlenecks in the efficient degradation of highly crystalline, high-molecular-weight TPU materials.

[0006] Therefore, there is an urgent need to develop an enzyme preparation and its application method that has a high efficiency in degrading thermoplastic polyurethane, so as to achieve efficient and green degradation of TPU materials and provide a new technical approach for the resource utilization and environmentally friendly treatment of polyurethane waste. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of low degradation efficiency, narrow substrate applicability, and limited degradation ability of thermoplastic polyurethane (TPU) in existing polyurethane degrading enzymes, and to provide a degrading enzyme PA5, its encoding gene, recombinant vector, recombinant strain, enzyme preparation, and applications. This degrading enzyme PA5 exhibits highly efficient and specific degradation ability for thermoplastic polyurethane, achieving efficient degradation of TPU materials under mild conditions, and providing a new technical approach for the resource utilization and environmentally friendly treatment of polyurethane waste.

[0008] To achieve the above objectives, the present invention provides a degradation enzyme PA5, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0009] A second aspect of the present invention provides an enzyme preparation comprising the previously described degradation enzyme PA5.

[0010] A third aspect of the present invention provides a gene for a degrading enzyme PA5, wherein the gene for the degrading enzyme PA5 is a nucleotide sequence encoding the degrading enzyme PA5 as described above.

[0011] A fourth aspect of the present invention provides a recombinant vector containing the gene as described above.

[0012] A fifth aspect of the present invention provides a recombinant strain containing the gene as described above or the recombinant vector as described above.

[0013] The sixth aspect of the present invention provides the use of at least one of the aforementioned degrading enzyme PA5, the aforementioned enzyme preparation, the aforementioned gene, the aforementioned recombinant vector, and the aforementioned recombinant strain in the degradation of polyurethane.

[0014] Through the above technical solution, the present invention provides a degradation enzyme PA5 with an amino acid sequence as shown in SEQ ID NO. 1. This specific amino acid sequence endows it with highly efficient degradation activity against thermoplastic polyurethane, and can specifically hydrolyze the characteristic chemical bonds in thermoplastic polyurethane molecules. This effectively solves the problem of insufficient degradation capacity of existing degradation enzymes against thermoplastic polyurethane. Moreover, this enzyme can maintain stable catalytic performance under mild conditions, breaking through the degradation bottleneck of high crystallinity and high molecular weight thermoplastic polyurethane. It provides a dedicated enzyme preparation for the biodegradation treatment of thermoplastic polyurethane waste, and also provides key technical support for the resource utilization of waste polyurethane and the bioremediation of environmental plastic pollution. Attached Figure Description

[0015] Figure 1 This is an SDS-PAGE electrophoresis analysis of the purified degrading enzyme PA5 in Example 2; Figure 2 This is a graph showing the enzyme activity detection results of the degrading enzyme PA5 in Example 3. Detailed Implementation

[0016] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0017] In one aspect, the present invention provides a degrading enzyme PA5, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0018] The present invention provides a degrading enzyme PA5 with an amino acid sequence as shown in SEQ ID NO. 1. This specific amino acid sequence endows it with highly efficient degradation activity against thermoplastic polyurethane, and it can specifically hydrolyze the characteristic chemical bonds in thermoplastic polyurethane molecules, effectively solving the problem of insufficient degradation ability of existing degrading enzymes against thermoplastic polyurethane. Moreover, this enzyme can maintain stable catalytic performance under mild conditions, breaking through the degradation bottleneck of highly crystalline and high molecular weight thermoplastic polyurethane. It provides a dedicated enzyme preparation for the biodegradation treatment of thermoplastic polyurethane waste, and also provides key technical support for the resource utilization of waste polyurethane and the bioremediation of environmental plastic pollution.

[0019] A second aspect of the present invention provides an enzyme preparation comprising the previously described degradation enzyme PA5.

[0020] In this invention, the degrading enzyme PA5 can be prepared into a corresponding enzyme preparation, specifically, the enzyme preparation can exist in solid, semi-solid or liquid form.

[0021] A third aspect of the present invention provides a gene for a degrading enzyme PA5, wherein the gene for the degrading enzyme PA5 is a nucleotide sequence encoding the degrading enzyme PA5 as described above.

[0022] The nucleotide sequences provided by this invention can generally be obtained using polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities using recombination. Typically, the obtained nucleotide sequence is cloned into a vector, then transformed into genetically engineered bacteria, and then the relevant nucleotide sequence is isolated from the proliferated host cells using conventional methods. Alternatively, the relevant nucleotide sequence can also be synthesized using known artificial chemical synthesis methods.

[0023] According to the present invention, preferably, the nucleotide sequence of the gene encoding the degradative enzyme PA5 is shown in SEQ ID NO. 2. The inventors have discovered that this nucleotide sequence can efficiently encode the degradative enzyme PA5 with complete biological activity. The protein it encodes can stably perform the function of degrading thermoplastic polyurethane, laying the core genetic foundation for the in vitro heterologous expression and large-scale preparation of this degradative enzyme, and ensuring the efficient production and practical application of the degradative enzyme PA5.

[0024] A fourth aspect of this invention provides a recombinant vector containing the gene as described above. In this invention, the "vector" used in the recombinant vector can be any of the vectors known in the art, such as commercially available plasmids, granules, bacteriophages, and retroviruses. The preferred expression vector of this invention is selected from any one of pET22b, pET28a, and pET32a. Exemplarily, the expression vector and the target fragment are ligated using seamless cloning to obtain the recombinant vector.

[0025] A fifth aspect of the present invention provides a recombinant strain containing the gene as described above or the recombinant vector as described above.

[0026] In this invention, the recombinant vector can be transformed, transduced, or transfected into host cells (strains) using conventional methods in the art, such as chemical transformation by calcium chloride method or high-voltage electroporation transformation. The host cell can be a prokaryotic cell or a eukaryotic cell; preferably, the host cell is Escherichia coli, such as Escherichia coli BL21(DE3).

[0027] The sixth aspect of this invention provides the application of at least one of the aforementioned degrading enzyme PA5, the aforementioned enzyme preparation, the aforementioned gene, the aforementioned recombinant vector, and the aforementioned recombinant strain in the degradation of polyurethane. The inventors have discovered that the application of the above substances in polyurethane degradation can achieve mild biodegradation of polyurethane by leveraging the highly efficient degrading activity of PA5, thus avoiding many shortcomings of traditional physical and chemical methods. Various functional material forms can be adapted to different practical treatment scenarios, providing a practical and feasible technical approach for the resource utilization of polyurethane waste and the bioremediation of environmental plastic pollution.

[0028] According to the present invention, preferably, the polyurethane is selected from at least one of TPU, PU Fiber, and CPU. TPU is thermoplastic polyurethane, PU Fiber is polyurethane fiber (spandex), and CPU is cast polyurethane elastomer. All three are subcategories of polyurethane containing urethane bonds, differing only in polymerization process and molding method, resulting in differentiated product forms. TPU is further preferred. Applying it to the degradation of the above-mentioned preferred polyurethane materials can specifically improve the degradation efficiency of thermoplastic polyurethane, adapting to the treatment needs of mainstream polyurethane waste in actual production, and significantly enhancing the practical application value of this technology in the field of plastic pollution control.

[0029] According to the present invention, preferably, the application conditions include: pH 7-9 and temperature 30-50°C. The inventors have discovered that these reaction conditions provide a suitable catalytic environment for the degrading enzyme PA5, allowing it to fully exert its degradation activity. Furthermore, these conditions are mild and easily controllable, requiring no special equipment, and are suitable for practical industrial applications.

[0030] According to the present invention, preferably, the amount of the degrading enzyme is 0.02-0.1 g relative to 1 g of the polyurethane. The inventors have discovered that this enzyme dosage achieves efficient degradation of polyurethane, controlling the cost of enzyme use while ensuring degradation effectiveness, thus balancing degradation efficiency with the economics of practical application.

[0031] The present invention will be described in detail below through examples. In the following examples, Escherichia coli BL21(DE3) was purchased from Takara Bio Engineering (Dalian) Co., Ltd., product number 9126; pET28a was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B540183; the seamless cloning kit was purchased from Beyotime Biotechnology Co., Ltd.; MDA, AA, and 1,4-BDO standards were purchased from Maclean's Reagent Company; and the remaining raw materials and reagents were all commercially available products.

[0032] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH adjusted to 6.5, autoclaved for 21 min, ready for use; LB agar plate medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 12 g / L, pH adjusted to 7, autoclaved for 21 min, ready for use; Detection of MDA, AA, 1,4-BDO: First, perform HPLC analysis with standard solutions of gradient concentrations to obtain a standard curve of peak area versus standard concentration; then, take 200 μL of sample supernatant and stop the reaction in 200 μL of methanol, filter through a 0.22 μm filter membrane, and perform HPLC analysis. The HPLC test conditions were as follows: column: C18 reversed-phase column; column temperature: 30℃; detection wavelength: 240 nm; flow rate: 0.7 mL / min. -1 Injection volume: 10 μL; Mobile phase: 40 wt% methanol (chromatographic grade), 60 wt% water (1 / 1000 trifluoroacetic acid).

[0033] Example 1 Construction of a recombinant strain expressing the degrading enzyme PA5 To obtain candidate enzyme molecules with target functions, this invention employs a bioinformatics mining strategy based on Sequence Similarity Network (SSN) to systematically screen and predict the functions of protein sequences in public databases.

[0034] First, using reported reference enzyme sequences with the target catalytic activity as seed sequences, homology sequence searches were conducted in NCBI, UniProt, or other public protein databases to obtain an initial set of candidate sequences. Subsequently, redundancy removal and length screening were performed on the obtained sequences to ensure sequence integrity and comparability. Based on this, a sequence similarity network (SSN) was constructed. Further location analysis was performed by combining the network clusters of known functional enzymes, screening unknown or uncharacterized sequences in the same or adjacent clusters as key candidates. Simultaneously, combining phylogenetic analysis, conserved site analysis, domain annotation, and key catalytic residue alignment results, the candidate enzyme PA5 was obtained.

[0035] Design blunt-end primer pairs (including primer R and primer F) using SnapGene software, to Pseudomonas aeruginosa The gene encoding TPU hydrolase (amino acid sequence as shown in SEQ ID NO. 1, nucleotide sequence as shown in SEQ ID NO. 2) was used as a template, and then PCR amplification was performed. The PCR reaction system is as follows: PrimeSTARMix high-fidelity enzyme 25 μL, upstream primer (10 pmol / μL) 2 μL, downstream primer (10 pmol / μL) 2 μL, template 2 μL, ddH2O 19 μL; PCR cycling process: Preheating: 95℃, 10 min; Denaturation: 95℃, 30 s; Annealing: 60℃, 30 s; Extension: 72℃, 4 min; 2-4 cycles, 25 times; Extension: 72℃, 10 min; Cool to 4℃ and store. The PCR product was recovered, and agarose gel electrophoresis was used to detect whether the amplification was complete. Then, DpnI (1 μL) was added to 50 μL of the reaction mixture, and the mixture was kept at 37 °C for 3 h to digest the template. The mixture was then cooled to 4 °C and stored.

[0036] Construction of recombinant vector: pET28a was selected as the expression vector. The linearized pET28a vector digested with restriction endonuclease was mixed with the target fragment of the PA5 encoding gene digested above at a molar ratio of 3:1. 10 μL of 2X Seamless Cloning Mix was added to the mixture, and ddH2O was added to make up to 20 μL. The mixture was incubated at 50℃ for 15 min. After the reaction was completed, the system was quickly placed on ice for 5 min to obtain the recombinant vector ligation solution.

[0037] Construction and screening of recombinant strains: 5 μL of the above recombinant vector ligation solution was transformed into 100 μL of E. coli DH5α competent cells. After resuscitation and culture, the bacterial culture was plated on LB agar plates containing kanamycin and incubated at 37℃ for 12 h. Single colonies on the plates were picked for colony PCR verification. Colonies with positive PCR verification were sequenced for DNA. Colonies with correct sequencing results were the recombinant vector engineered bacteria containing the PA5 encoding gene. The recombinant plasmid of the above positive engineered bacteria was extracted and transformed into E. coli BL21 (DE3) competent cells. The cells were also plated on LB agar plates containing kanamycin and incubated at 37℃ for 12 h. Single colonies were picked and verified by colony PCR and sequencing to obtain the recombinant strain E. coli BL21 (DE3) / pET28a-PA5 expressing the degradation enzyme PA5.

[0038] Example 2: Induction, Expression, Isolation, and Purification of Degradative Enzyme PA5 Inducible expression of the degradative enzyme PA5 (1) Seed culture: single colonies of the recombinant strain E. coli BL21 (DE3) / pET28a-PA5 obtained in Example 1 were picked and inoculated into LB liquid medium containing 50 mg / L kanamycin. The culture was carried out at 37°C and 200 rpm for 12 h to obtain seed culture. (2) Fermentation induction culture: The above seed culture was inoculated at a rate of 2% by volume into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium containing 50 mg / L kanamycin. The culture was then incubated at 37°C with shaking at 180 rpm until the cell concentration in the culture reached OD500. 600When the concentration reaches 0.6, add isopropyl-β-D-thiogalactoside (IPTG) to make the final concentration 0.1mM, and continue to induce culture at 16℃ and 180 rpm for 24 h to obtain fermentation broth; (3) Preparation of crude enzyme solution: Centrifuge the fermentation broth at 4℃ and 8000 rpm for 10 min, collect the cell pellet, wash the cell pellet twice with physiological saline to obtain resting cells; suspend the resting cells in 10mL of pH7.5 phosphate buffer, and sonicate them under ice bath conditions. After disruption, centrifuge at 4℃ and 12000 rpm for 20 min, collect the supernatant, which is the crude enzyme solution of degrading enzyme PA5.

[0039] The isolation and purification of the degradative enzyme PA5 was carried out using Ni 2+ The crude enzyme solution was purified using an affinity chromatography column. The specific steps were as follows: the PA5 crude enzyme solution was filtered through a 0.22 μm filter membrane and then loaded onto a Nisepharose 6 Fast Flow (FF) Ni filter. 2+ Affinity chromatography packing material; first, elute contaminating proteins with 10 column volumes of buffer A (10 mM Tris-HCl pH 7, 250 mM NaCl, 20 mM imidazole), then elute the target protein with 5 column volumes of buffer B (10 mM Tris-HCl pH 7, 250 mM NaCl, 250 mM imidazole). Collect the elution buffer corresponding to the elution peak, which is the purified PA5 degradative enzyme solution. Analyze the purified PA5 enzyme solution by SDS-PAGE electrophoresis. The results are as follows: Figure 1 As shown, the electrophoretic bands are single, indicating that the purity of the obtained degradative enzyme PA5 meets the experimental requirements, and its amino acid sequence is shown in SEQ ID NO. 1.

[0040] Example 3: Enzyme activity assay of degrading enzyme PA5 Enzyme activity assay system establishment: An enzyme activity assay system based on the substrate 7-carboxyethoxyamino-4-methylcoumarin (EMACC) was established. A sterile flat-bottomed 96-well plate was used as the reaction carrier. 40 μL of the prepared buffer solution (25 mM Tris-HCl, 150 mM NaCl, pH 7.5) and the purified PA5 enzyme solution obtained in Example 2 were added to each well. 5 μL of 10 mM EMACC substrate solution was quickly added and mixed rapidly.

[0041] Enzyme activity assay: Place the 96-well plate with the added samples in a microplate reader, set the detection wavelength to 365 nm, and the detection time to 30 min. Monitor the absorbance changes in real time throughout the process, and calculate the enzyme activity based on the absorbance change trend. The results are as follows: Figure 2 As shown, the obtained degradative enzyme PA5 has stable and efficient catalytic activity.

[0042] Example 4: Application of degradative enzyme PA5 in the hydrolysis of TPU The TPU degradation reaction system was constructed using PBS buffer at pH 7.5 as the reaction medium. A total volume of 2 mL of degradation reaction system was constructed, in which 60 mg of TPU powder was added as substrate, and the purified PA5 enzyme solution obtained in Example 2 was added to make the enzyme concentration 20 μg / mL, that is, the mass ratio of degrading enzyme PA5 to TPU is 1:30, which is within the dosage range of 0.02-0.1 g enzyme / g TPU.

[0043] The degradation reaction conditions were as follows: the above reaction system was placed in a constant temperature shaking incubator, the temperature was set at 30℃ and the rotation speed at 900 rpm, and the reaction was carried out under constant temperature shaking for 24 h.

[0044] After 24 h of reaction, samples were taken and mixed with an equal volume of methanol-water mixture (1:1 volume ratio). The mixture was diluted 1-fold and filtered through a 0.22 μm filter. High-performance liquid chromatography (HPLC) was used to detect the content of terminal degradation products AA (adipic acid), BDO (1,4-butanediol), and MDA (4,4'-diphenylmethanediamine). HPLC detection conditions: C18 reversed-phase column; column temperature 30℃; detection wavelength 240 nm; flow rate 0.7 mL. min -1 The injection volume was 10 μL; the mobile phase was 40 wt% chromatographic grade methanol and 60 wt% ultrapure water containing 1 / 1000 trifluoroacetic acid; HPLC analysis was first performed with AA, BDO, and MDA standard solutions of varying concentrations, and a standard curve was plotted between the peak area and the standard concentration. The product content was then calculated based on the peak area of ​​the sample. The results showed that the AA content in the system was 12.6 mg / L, the BDO content was 88.3 mg / L, and the MDA content was 1.1 mg / L, indicating that the degrading enzyme PA5 can efficiently hydrolyze TPU, break the characteristic chemical bonds in its molecule, and generate small molecule terminal products.

[0045] Comparative Example 1 The degradation products were detected according to the method used in Example 4, except that the amount of TPU substrate added was 100 mg, the degradation reaction temperature was 40°C, and the rest of the reaction system and conditions were the same as in Example 4. The results are shown in Table 1. The results show that the AA content in the system was 11.9 mg / L, the BDO content was 79.4 mg / L, and the MDA content was 0.8 mg / L. This indicates that after changing the amount of substrate and the reaction temperature, the degrading enzyme PA5 can still effectively degrade TPU, with only the product generation slightly decreasing with the reaction conditions.

[0046] Comparative Example 2 The degradation products were detected according to the method used in Example 4, except that the PA5 enzyme solution was added to achieve an enzyme concentration of 50 μg / mL, and the degradation reaction temperature was 40℃. The remaining reaction system and conditions were the same as in Example 4. The results are shown in Table 1. The results show that the AA content in the system was 14.8 mg / L, the BDO content was 93.6 mg / L, and the MDA content was 1.9 mg / L, indicating that appropriately increasing the enzyme dosage can further improve the degradation efficiency of TPU and significantly increase the amount of end products generated.

[0047] Table 1

[0048] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A degradative enzyme PA5, characterized in that, The amino acid sequence of the degradative enzyme PA5 is shown in SEQ ID NO.

1.

2. An enzyme preparation, characterized in that, The enzyme preparation comprises the degrading enzyme PA5 as described in claim 1.

3. A gene for a degradation enzyme PA5, characterized in that, The gene for the degrading enzyme PA5 is the nucleotide sequence encoding the degrading enzyme PA5 as described in claim 1.

4. The gene for the degrading enzyme PA5 according to claim 3, characterized in that, The nucleotide sequence of the gene encoding the degradation enzyme PA5 is shown in SEQ ID NO.

2.

5. A recombinant vector, characterized in that, The recombinant vector contains the gene as described in claim 3 or 4.

6. The recombinant vector according to claim 5, characterized in that, The expression vector of the recombinant vector is selected from any one of pET22b, pET28a and pET32a.

7. A recombinant bacterial strain, characterized in that, The recombinant strain contains the gene as described in claim 3 or 4, or the recombinant vector as described in claim 5 or 6.

8. The use of at least one of the following in the degradation of polyurethane: the degrading enzyme PA5 of claim 1, the enzyme preparation of claim 2, the gene of claim 3 or 4, the recombinant vector of claim 5 or 6, and the recombinant strain of claim 7.

9. The application according to claim 8, characterized in that, The polyurethane is selected from at least one of TPU, PU Fiber, and CPU; Preferably, the polyurethane is TPU.

10. The application according to claim 8, characterized in that, The conditions for the application include: pH 7-9 and temperature 30-50℃; And / or, relative to 1 g of the polyurethane, the amount of the degrading enzyme is 0.02-0.1 g.

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