Use of degrading enzyme tfcut-b3 in depolymerization of waste plastic film

Abstract

The application belongs to the technical field of biotechnology, and discloses application of a degrading enzyme TfCut-B3 in depolymerization of waste plastic film.The amino acid sequence of the degrading enzyme is shown as SEQ ID NO.1, and the coding gene sequence is shown as SEQ ID NO.2.The degrading enzyme TfCut-B3 used in the application can efficiently depolymerize various common commercial PBAT films, and can completely depolymerize PBAT film in 48 h at the fastest speed, and the degrading enzyme TfCut-B3 has good degrading effect, and has application value in recycling of waste PBAT film and pollution repair.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of the degradation enzyme TfCut-B3 in the depolymerization of waste plastic films. Background Technology

[0002] Polybutylene adipate-co-terephthalate (PBAT) is one of the most commonly used biodegradable plastics in our daily lives. It is a copolyester composed of terephthalic acid (TPA), adipic acid (AA), and 1,4-butanediol (BDO). The PBAT molecule consists of two parts: an aliphatic segment (polybutyleneadipate, BA) composed of adipic acid and 1,4-butanediol, and an aromatic segment (polybutyleneterephthalate, BT) composed of terephthalic acid and 1,4-butanediol. Due to its excellent mechanical properties and special barrier properties, PBAT has been widely used in food packaging, agriculture, and other industries. Although PBAT degrades more readily in the environment than traditional plastics, its natural degradation rate is still relatively slow, and incomplete depolymerization during the degradation process can lead to the formation of microplastics. Therefore, the accumulation of PBAT exacerbates the environmental burden. Currently, the main methods for processing waste such as packaging bags are physical or chemical recycling, which are not only costly but also complex to operate. Compared to physical and chemical recycling methods, biodegradation is not only low-cost but also does not pollute the environment.

[0003] Biodegradation methods mainly include composting, microbial degradation, and enzymatic degradation. Among them, enzymatic depolymerization has high efficiency, directly using degrading enzymes to depolymerize PABT into monomers, omitting the step of strain secreting degrading enzymes, and the depolymerized monomers can be recycled and further used in PBAT production. Currently reported PBAT depolymerizing enzymes mainly include esterases (PpEst, EstB3, EstC7, Chath_Est1), keratinases (AaCut4, AaCut10, TfCut-DM(Q132Y), LCC) WCCGThe enzymes mentioned above include IsPETase, PbPL, BurPL, TcCut, TfCut-DM, and TfCut, as well as lipases (PfL1). Although these enzymes can completely depolymerize PBAT membranes, they still have shortcomings in terms of catalytic efficiency and stability, making it difficult to meet the needs of industrial applications. More efficient PBAT degrading enzymes are still needed. Among the enzymes mentioned above, TfCut-DM is one of the enzymes with the best known PBAT degradation effect. It is obtained by rationally designing and modifying the keratinase TfCut, by transforming the large binary body (H224-F228) of the catalytic center into a small binary body (S224-I228) [Complete bio-degradation of poly (butylene adipate-coterephthalate) via engineered cutinases. Nature Communications, 2023, 14(1):1645.]. However, TfCut-DM has a better degradation effect on pure PBAT membranes, but its depolymerization effect on commercially available PBAT plastic membranes is not ideal.

[0004] In practice, commercially available PBAT production involves adding substances such as polylactic acid, styrene, and dibutyl maleate to enhance product performance. It is also subjected to ultraviolet irradiation to increase the degree of cross-linking. Therefore, the depolymerization of waste plastic film requires enzymes with stronger substrate adaptability, anti-interference ability, and stability. Its depolymerization difficulty is much higher than that of the simple system of pure PBAT. These factors have increased the difficulty of depolymerization and recycling of waste PBAT film. At present, there is a lack of enzymes that can efficiently depolymerize commercial PBAT plastic film. Summary of the Invention

[0005] To address the shortcomings of existing PBAT degrading enzymes in terms of catalytic efficiency, stability, and depolymerization efficiency on commercially available PBAT membranes, this invention provides the application of the degrading enzyme TfCut-B3 in the depolymerization of waste plastic films. This invention represents a directed evolution of the enzyme TfCut-DM, providing a highly efficient PBAT degrading enzyme, TfCut-B3. Its degradation capacity significantly surpasses existing PBAT degrading enzymes and exhibits stronger depolymerization ability on commercially available PBAT membranes, showing potential applications in the recycling and remediation of waste PBAT plastics.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The application of the degrading enzyme TfCut-B3 in the depolymerization of waste plastic film, wherein the amino acid sequence of the degrading enzyme TfCut-B3 is shown in SEQ ID NO.1.

[0008] Furthermore, the waste plastic film includes polybutyl adipate.

[0009] Furthermore, the degradation temperature is 50~70℃.

[0010] Furthermore, the degradation pH is 8-10.

[0011] Furthermore, the mass ratio of the degrading enzyme TfCut-B3 to waste plastic film is 1~3:100.

[0012] The present invention also provides a method for depolymerizing waste plastic film, wherein the waste plastic film is mixed with a degrading enzyme TfCut-B3 and incubated, the amino acid sequence of which is shown in SEQ ID NO.1.

[0013] Furthermore, the incubation time is 30-60 hours.

[0014] Furthermore, the incubation speed is 180 rpm.

[0015] Furthermore, the incubation temperature is 60°C.

[0016] Furthermore, the incubation pH was 9.0.

[0017] Beneficial effects

[0018] The degradation enzyme TfCut-B3 provided by this invention can efficiently depolymerize waste plastic films, completely depolymerizing PBAT within 48 hours. Compared with the existing PBAT degradation enzyme TfCut-DM, TfCut-B3 exhibits 3.08 times higher activity and 6 times higher thermal stability; its depolymerization ability for commercially available PBAT films from different sources is significantly improved, especially for the three tested commercially available products PB-3, PB-6, and PM-4, where its depolymerization ability is increased by 2.78 times, 5.13 times, and 1.96 times, respectively. Therefore, TfCut-B3 has application value in the recycling of waste PBAT films and pollution remediation. Attached Figure Description

[0019] 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.

[0020] Figure 1 To screen TfCut-DM mutant libraries on PBAT plates;

[0021] Figure 2 To verify the degradation ability of the mutant TfCut-B3; among which, Figure 2 Figure A shows a schematic diagram of TfCut-DM and TfCut-B3 incubated with PBAT white plastic film (0.008 mm) for 48 hours. Figure 2 In Figure B, the liquid chromatography chromatograms of TfCut-DM and TfCut-B3 incubated with PBAT white mulch film (0.008 mm) for 48 h are obtained. Figure 2 The bar chart in section C represents the yield of terephthalic acid (TPA) after incubation of TfCut-DM and TfCut-B3 with PBAT white mulch film (0.008 mm) for 48 h. Figure 2 The bar chart in section D shows the enzyme activity of TfCut-DM and TfCut-B3 after 12 h of incubation with PBAT white mulch film (0.008 mm).

[0022] Figure 3 Thermal stability diagrams for TfCut-DM and TfCut-B3; where, Figure 3 In the figure, A represents the thermal stability of TfCut-DM at temperatures of 40℃, 50℃, 60℃, 70℃, and 80℃. Figure 3 In the figure, B represents the thermal stability of TfCut-B3 at temperatures of 40℃, 50℃, 60℃, 70℃, and 80℃.

[0023] Figure 4 The optimal reaction temperature and pH for the decomposition of PBAT films by TfCut-B3 were determined; among them, Figure 4 In the middle, A is a bar chart showing the yield of terephthalic acid (TPA) produced by the decomposition of PBAT by TfCut-B3 at different temperatures; Figure 4 B is a bar chart showing the TPA production from the decomposition of PBAT by TfCut-B3 at different pH levels;

[0024] Figure 5 Applications of TfCut-B3 depolymerized commercial PBAT-based plastic products; among which... Figure 5 China A Figure 5 The diagrams in the middle (B) illustrate the degradation of 10 PBAT products by TfCut-DM and TfCut-B3 at different times; the control, TfCut-DM, and TfCut-B3 represent no enzyme addition, TfCut-DM enzyme addition, and TfCut-B3 enzyme addition, respectively.

[0025] Figure 6 The liquid phase results of TfCut-DM and TfCut-B3 at 12 h, 48 h and 72 h are shown; where PB-1, PB-2, PB-3, PB-4, PB-5, PB-6, PM-1, PM-2, PM-3 and PM-4 represent the different PBAT products listed in Table 1.

[0026] Figure 7Bar charts showing the production of terephthalic acid (TPA) after incubation of TfCut-DM and TfCut-B3 with 10 PBAT plastic products for different times (significant differences are indicated by ***: P < 0.001; ****: P < 0.0001; ns: no significant difference). Detailed Implementation

[0027] Example 1

[0028] Directed evolution of PBAT degrading enzyme TfCut-DM

[0029] The purpose of this embodiment is to obtain mutants with improved catalytic ability and stability through directed evolution, using the PBAT degrading enzyme TfCut-DM [Complete bio-degradation of poly(butylene adipate-coterephthalate) via engineered cutinases. Nature Communications, 2023, 14(1):1645.] as the initial template. Specifically, based on the amino acid sequence of TfCut-DM (SEQ ID NO.3), the gene TfCut-DM (SEQ ID NO.4) was synthesized according to the codon preference of Bacillus subtilis. The TfCut-DM gene was amplified by error-prone PCR using the StarMut random mutation kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). The amplified product was ligated into the expression plasmid pHCMC05 [Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid. 2005 Nov;54(3):241-8.] and introduced into Bacillus subtilis SCK6 [Preparation and transformation conditions optimization of supercompetent cells of Bacillus subtilis SCK6 ([J]. Chinese Journal of Biotechnology, 2017,33(04):692-698.)] to obtain a random mutant library of the TfCut-DM gene. The library was spotted onto LB plates containing PBAT powder and incubated at 37°C. Since PBAT is insoluble in water, TfCut-DM expressed by Bacillus subtilis decomposes PBAT and forms a transparent hydrolysis zone. The size of the hydrolysis zone is positively correlated with the enzyme activity. From approximately 10,000 clones, one clone with the largest hydrolysis zone was selected through screening. Figure 1The mutant was named MT-B3. To confirm the mutation site, the coding region of the mutant MT-B3 was sequenced, and it was found that serine (Ser) at position 224 of TfCut-DM was mutated to phenylalanine (Phe). The mutant was named TfCut-B3, and its amino acid sequence is shown in SEQ ID NO.1. The gene sequence encoding TfCut-B3 is shown in SEQ ID NO.2.

[0030] SEQ ID NO.1 (Amino acid sequence of the degradative enzyme TfCut-B3)

[0031] MANPYERGPNPTDALLEARSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHS MGGGGSLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATFAKPIYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPF.

[0032] SEQ ID NO.2 (Gene sequence of the degradative enzyme TfCut-B3)

[0033] ATGGCTAACCCTTATGAAAGAGGACCTAACCCTACAGATGCATTACTGGAAGCTCGCAGCGGACCTTTTTCAGTTTCAGAAGAGAATGTGTCTAGACTTAGCGCTAGCGGATTTGGAGGCGGAACAATCTACTATCCTAGAGAAAACAATACGTACGGAGCAGTGGCAATTTCTCCTGGATATACGGGAACAGAAGCTAGCATTGCATGGCTTGGCGAACGCATTGCCTCTCACGGATTTGTTGTCATTACAATTGATACAATTACGACACTTGATCAACCGGACTCTAGAGCCGAACAGTTAAATGCTGCATTAAACCATATGATTAACAGAGCCTCATCTACAGTGAGATCAAGAATCGATTCATCTAGATTAGCAGTCATGGGACATAGCATGGGAGGAGGAGGCTCTCTTCGCTTAGCTTCACAAAGACCGGACCTGAAAGCAGCAATTCCTTTAACGCCGTGGCATCTGAACAAAAACTGGAGCTCAGTGACAGTTCCGACGTTAATCATTGGCGCTGATTTAGATACAATTGCCCCGGTCGCAACATTTGCCAAGCCTATCTATAACTCTTTACCTAGCAGCATTAGCAAGGCATATCTTGAATTAGATGGAGCCACACATTTTGCGCCGAACATTCCTAACAAGATCATTGGCAAATATTCAGTAGCTTGGCTTAAGAGATTCGTCGACAATGACACACGCTATACACAATTTCTTTGTCCGGGACCGAGAGATGGATTATTTGGCGAAGTTGAAGAATACAGATCAACATGTCCTTTTTAA。

[0034] SEQ ID NO.3 (Amino acid sequence of the degrading enzyme TfCut-DM)

[0035] MANPYERGPNPTDALLEARSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGGGSLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATSAKPIYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPF。

[0036] SEQ ID NO.4 (Gene sequence of the degrading enzyme TfCut-DM)

[0037] ATGGCTAACCCTTATGAAAGAGGACCTAACCCTACAGATGCATTACTGGAAGCTCGCAGCGGACCTTTTTCAGTTTCAGAAGAGAATGTGTCTAGACTTAGCGCTAGCGGATTTGGAGGCGGAACAATCTACTATCCTAGAGAAAACAATACGTACGGAGCAGTGGCAATTTCTCCTGGATATACGGGAACAGAAGCTAGCATTGCATGGCTTGGCGAACGCATTGCCTCTCACGGATTTGTTGTCATTACAATTGATACAATTACGACACTTGATCAACCGGACTCTAGAGCCGAACAGTTAAATGCTGCATTAAACCATATGATTAACAGAGCCTCATCTACAGTGAGATCAAGAATCGATTCATCTAGATTAGCAGTCATGGGACATAGCATGGGAGGAGGAGGCTCTCTTCGCTTAGCTTCACAAAGACCGGACCTGAAAGCAGCAATTCCTTTAACGCCGTGGCATCTGAACAAAAACTGGAGCTCAGTGACAGTTCCGACGTTAATCATTGGCGCTGATTTAGATACAATTGCCCCGGTCGCAACATCTGCCAAGCCTATCTATAACTCTTTACCTAGCAGCATTAGCAAGGCATATCTTGAATTAGATGGAGCCACACATTTTGCGCCGAACATTCCTAACAAGATCATTGGCAAATATTCAGTAGCTTGGCTTAAGAGATTCGTCGACAATGACACACGCTATACACAATTTCTTTGTCCGGGACCGAGAGATGGATTATTTGGCGAAGTTGAAGAATACAGATCAACATGTCCTTTTTAA。

[0038] Example 2

[0039] Identification of the ability of TfCut-B3 to degrade PBAT

[0040] To verify the degradation ability of the mutant TfCut-B3, TfCut-DM and TfCut-B3 were purified by affinity chromatography using a nickel column (Sangon Biotech Co., Ltd., Shanghai, China). The purified TfCut-DM and TfCut-B3 were then reacted with PBAT membranes (Shanghai Hongrui Biotechnology Co., Ltd., Shanghai, China, model 0.008 mm). The reaction conditions were: 40 μg enzyme, 3 mg PBAT membrane (Shanghai Hongrui Biotechnology Co., Ltd., Shanghai, China, model 0.008 mm), Gly-NaOH buffer (50 mM) at pH 9.0, incubation at 60℃ and 180 rpm for 48 h, with a total reaction volume of 4 mL. A reaction system without enzyme was used as a control group. Samples were taken at 12 h and 48 h, and the amount of TPA monomer generated was detected by high-performance liquid chromatography (HPLC). After 48 hours of reaction, the film in the control group showed no change, while a large number of fragments of the white PBAT film could still be observed in the TfCut-DM group. In the TfCut-B3 group, the film disappeared and the reaction system became clear. Figure 2 (A). HPLC results showed that only TPA product peaks were detected in the TfCut-B3 group, indicating that the PBAT mulch film had been completely depolymerized. Figure 2 (Middle B). After incubation for 48 h, the amounts of TPA generated by the depolymerization of PBAT in TfCut-DM and TfCut-B3 were 1.05 mg and 2.15 mg, respectively. Figure 2 (C). Furthermore, the specific enzyme activity of TfCut-B3 was 19.85 U / mg ( Figure 2 The efficiency of TfCut-B3 (3.08 times that of TfCut-DM) is 3.08 times that of TfCut-DM. These results indicate that TfCut-B3 has a more efficient PBAT decomposition efficiency than TfCut-DM.

[0041] The sample preparation method used in this invention is as follows: 500 μL of the enzyme reaction system is taken, boiled at high temperature for 10 min, centrifuged at 12000 rpm for 10 min, and the supernatant is collected. After filtration through a 0.22 μm filter membrane, the amount of monomer TPA generated is detected by HPLC. The HPLC detection conditions for degradation products are as follows: The instrument used for HPLC is a Thermo Dionex MLtiMate 3000 (Massachusetts, USA), a C18 reversed-phase column (4.6 × 250 mm, 3 µm), a detection wavelength of 241 nm, a column temperature of 25℃, a flow rate of 0.8 mL / min, an injection volume of 5 µL, and a mobile phase of acetonitrile: 0.1% formic acid (0-12.5 min, 15%-50% acetonitrile; 12.5-14 min, 50%-15% acetonitrile) (v / v).

[0042] The enzyme activity determination method in this invention is as follows: the activity of the degrading enzyme is determined based on the TPA produced by PBAT under enzymatic hydrolysis. The amount of enzyme that generates 1 μmol of TPA per hour is defined as one unit of enzyme activity. The specific enzyme activity formula is: Specific enzyme activity = A / (B*C), where A is the amount of terephthalic acid produced (μmol), B is the reaction time (h), and C is the amount of enzyme added to the reaction (mg).

[0043] Example 3

[0044] Thermal stability of TfCut-B3

[0045] To determine the thermostability of TfCut-DM and TfCut-B3, equal amounts of recombinant enzymes TfCut-DM and TfCut-B3 were incubated at 40℃, 50℃, 60℃, 70℃, and 80℃, with samples taken every 12 hours. The enzyme activities of TfCut-DM and TfCut-B3 were measured after reacting at 60℃ for 3 minutes to determine the enzyme thermostability. The enzyme activity of the untreated enzyme solution was set as 100%, and the retained enzyme activity after heat treatment was calculated. Figure 3 China A Figure 3 The results showed that the two enzymes exhibited significant differences in stability at 60℃: with prolonged incubation time, the enzyme activity of TfCut-DM gradually decreased, retaining less than 10% of its activity after 72 h; in contrast, TfCut-B3 showed excellent thermostability under the same conditions, maintaining approximately 60% of its enzyme activity after 72 h of incubation. In conclusion, TfCut-B3 demonstrates superior thermostability at 60℃.

[0046] The enzyme activity assay method used in this invention is based on that of Yan et al. [Influence of ammonium salts on the lipase / esterase activity assay using p-nitrophenyl esters as substrates. Biotechnology and Applied Biochemistry, 2013, 60:343-347.]. The enzyme reaction system consisted of 1 mL of 10 μL of 10 mM pNPA, 10 μL of enzyme solution, and 980 μL of 50 mM Tris-HCl (pH 8.0). The absorbance at 405 nm was measured using a spectrophotometer. A reaction system without enzyme was used as a control group.

[0047] Example 4

[0048] Optimal reaction temperature and pH for TfCut-B3 decomposition of PBAT membranes

[0049] (1) To investigate the optimal temperature for TfCut-B3 to decompose PBAT film, the method of Yang et al. [Complete bio-degradation of poly (butylene adipate-coterephthalate) via engineeredcutinases. Nature Communications, 2023, 14(1):1645.] was followed. TfCut-B3 (40 μg) and 3 mg of white PBAT film (Shanghai Hongrui Biotechnology Co., Ltd., Shanghai, China, model 0.008 mm) were incubated in water baths at 40℃, 50℃, 60℃, 70℃, and 80℃ for 12 h, respectively. The yield of TPA was detected by HPLC (sample processing and detection methods are as described in Example 2) to determine the optimal temperature for TPA production by TfCut-B3. A reaction system without enzyme was used as a control group, and three biological replicates were set up for each group. Figure 4 The results from the study A showed that the optimal operating temperature for TfCut-B3 to produce TPA after incubation in a water bath at different temperatures for 12 h was 60℃. At this temperature, the average amount of TPA monomer produced (product I) reached 1.42 mg.

[0050] (2) To investigate the optimal pH for TfCut-B3 to decompose PBAT films, TfCut-B3 (40 μg) and 3 mg of white PBAT film (Shanghai Hongrui Biotechnology Co., Ltd., Shanghai, China, model 0.008 mm) were incubated in a 60°C water bath for 12 h in Tris-HCl buffer (pH 8.0) and Gly-NaOH buffer (pH 8.0-10.0). The amount of TPA generated was then detected by HPLC (sample processing and detection methods are as described in Example 2). A reaction system without enzymes was used as a control group, and three biological replicates were set up for each group. Figure 4 The results from the study showed that TfCut-B3 produced the highest amount of TPA (product I) after 12 h of incubation at pH 9.0.

[0051] Example 5

[0052] Application of the degradative enzyme TfCut-B3 in depolymerized commercial PBAT plastic film products

[0053] To compare the ability of TfCut-DM and TfCut-B3 to degrade commercial PBAT-based plastic film products under the same conditions, ten commercial PBAT-based plastic products were selected (Table 1). Each PBAT-based plastic film was cut into small squares, and 3 mg was weighed into 10 mL centrifuge tubes. 40 μg of TfCut-DM and TfCut-B3 were incubated with 3 mg of each different PBAT-based plastic product at 60°C and 180 rpm. The enzyme reaction system was 4 mL (buffer Gly-NaOH, pH 9.0). The reaction system without enzyme was used as a control group. Samples were taken at 12 h, 48 h, and 72 h, and the TPA generation was detected by HPLC (sample processing and detection methods are as described in Example 2). Three biological replicates were set up for each group.

[0054] Table 1. Commercially available PBAT-based plastic film products used for testing.

[0055]

[0056] Note: PBAT: Polybutylene adipate; PLA: Polylactic acid; St: Styrene; MD: Dibutyl maleate.

[0057] Figure 5 Results from sections A and B show that variant TfCut-B3 exhibits stronger degradation capabilities. It effectively depolymerized nine PBAT-based plastic products within 12 hours, with only product PB-6 requiring 48 hours of incubation to depolymerize into fragments. In contrast, TfCut-DM showed lower degradation efficiency, depolymerizing only PM-3 and PB-4 products within 12 hours, while five other products (PB-1, PB-2, PB-5, PM-2, and PM-1) required an extended incubation period of up to 48 hours to depolymerize into fragments. Particularly noteworthy is (…). Figure 5 Even after 72 hours of incubation, the three products, PB-3, PB-6 and PM-4, maintained a relatively intact cubic morphology under TfCut-DM treatment, and no obvious depolymerization was observed.

[0058] HPLC analysis was used to quantitatively detect the monomer TPA generated by the depolymerization of TfCut-DM and TfCut-B3 during incubation. The results showed that TfCut-B3 could essentially depolymerize and convert six samples (PB-1, PB-2, PB-4, PM-1, PM-2, and PM-3) into TPA within 48 h. Product PB-5 could also essentially depolymerize into TPA after 72 h of incubation with TfCut-B3. Notably, for product PB-3, which could not be completely depolymerized by TfCut-DM… Figure 5In Figure 5 (A), TfCut-B3 can almost completely depolymerize it into monomers in just 48 hours. While products PB-6 and PM-4 show signs of depolymerization into fragments by TfCut-B3 (Figure 5 (A)), HPLC results show... Figure 6 The product still contains a large amount of derivatives that are not completely converted into TPA. In contrast, the amount of TPA produced by TfCut-DM catalyzing PBAT is significantly lower than that produced by TfCut-B3. Even after incubating these 10 products with TfCut-DM for 72 hours, it failed to completely depolymerize and convert them into TPA. Figure 6 ).

[0059] Through calculation, it was found that ( Figure 7 PB-1, PB-2, and PM-3, after incubation with TfCut-B3 for 12 h, generated a large amount of TPA, with average yields of 1.50 mg, 1.45 mg, and 2.17 mg, respectively. After incubation with TfCut-B3 for 48 h, the TPA produced by the depolymerization of PB-1, PB-2, PB-3, PB-4, PM-2, PM-2, and PM-3 accounted for 95% of the total TPA production (after complete degradation of PBAT). Specifically, the TPA produced by incubating TfCut-DM / TfCut-B3 with PB-3, PB-6, and PM-4 for 72 h was 0.89 mg / 2.49 mg, 0.37 mg / 1.89 mg, and 0.28 mg / 0.56 mg, respectively, while the TPA produced by TfCut-B3 was 2.78 times, 5.13 times, and 1.96 times that of TfCut-DM, respectively.

[0060] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of the degrading enzyme TfCut-B3 in the depolymerization of waste plastic film, characterized in that, The amino acid sequence of the degradation enzyme TfCut-B3 is shown in SEQ ID NO.

1. The pH of the depolymerization of waste plastic film is 8.0~10.0 and the temperature is 50~70℃. The waste plastic film includes poly(butyl adipate).

2. The application according to claim 1, characterized in that, The mass ratio of the degrading enzyme TfCut-B3 to waste plastic film is 1~3:

100.

3. A method for depolymerizing waste plastic film, characterized in that, Waste plastic film was mixed with degrading enzyme TfCut-B3 and incubated. The amino acid sequence of the degrading enzyme TfCut-B3 is shown in SEQ ID NO.

1. The incubation pH was 8.0~10.0 and the temperature was 50~70℃. The waste plastic film included polybutyl adipate.

4. The method for depolymerizing waste plastic film according to claim 3, characterized in that, The incubation time is 30-60 hours.

5. The method for depolymerizing waste plastic film according to claim 3, characterized in that, The incubation speed is 180 rpm.

6. The method for depolymerizing waste plastic film according to claim 3, characterized in that, The incubation temperature is 60℃.

7. The method for depolymerizing waste plastic film according to claim 3, characterized in that, The incubation pH was 9.0.

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