A fungal cutinase for efficient hydrolysis of polyester polyurethane plastics and use thereof

Abstract

The application discloses a kind of high-efficiency hydrolysis of polyester polyurethane plastic fungal cutinase and application thereof, the nucleotide sequence of the coding gene cpcut1 of the cutinase is as shown in SEQ ID NO.1.The fungal cutinase CpCut1 used in the application can efficiently degrade different types of polyester PU substrates (DLN, PBA-PU and rigid PU foam) in a short time.When DLN is used as substrate, the degradation rate of CpCut1 to DLN reaches 100% after 30 min;The degradation rate of PBA-PU film after 12 h reaches 40.5%;When PU foam is used as substrate, the degradation rate of CpCut1 to PU foam is 20.6%.And it is found that the degradation effect of CpCut1 on the three PUs is better than that of the other three reported polyester cutinases.

Description

Technical Field

[0001] This invention belongs to the field of environmental science and relates to a fungal cutinase for the efficient hydrolysis of polyester polyurethane plastics and its application. Background Technology

[0002] Polyurethane (PU) plastics are a class of polymers widely used in medical, automotive, and industrial fields. PU accounts for nearly 8% of all plastics produced, making it the sixth largest polymer in the world. According to statistics, my country's PU consumption reached 11.75 million tons in 2020 and is projected to reach 18.28 million tons by 2025. The large-scale use of PU plastics inevitably generates PU waste. The continued use and accumulation of PU plastics, as well as their pollution of soil and water, pose a very serious threat to the environment. The degradation products of PU plastics in the natural environment are mainly small-molecule products such as alcohols, acids, and amines, including diamine compounds, such as 2,4-diaminotoluene (2,4-TDA) or 2,6-diaminotoluene (2,6-TDA) and 4,4-diaminodiphenylmethane (4,4-MDA), which are more toxic than the hydrolysis products of other plastics. Therefore, strengthening the effective disposal of PU and other plastic waste has become a major practical problem that urgently needs to be addressed by all of humanity.

[0003] Enzymatic depolymerization of PU plastics is a green way to solve the environmental problems caused by these waste plastics. The reaction occurs at suitable temperatures without the participation of chemical catalysts, making this gentle process more environmentally friendly than chemical processes. Furthermore, the monomers generated from enzymatic depolymerization of PU can be used to synthesize new macromolecules. A typical example is the recycling of polyethylene terephthalate (PET). The French company Carbios uses keratinase LCC derived from plant compost to decompose 97% of PET plastic within 16 hours, and then reacts the decomposition products to generate new plastics. This method is considered a promising biotechnology recycling method. However, very few types of PU-degrading enzymes have been discovered so far. Esterase PulA from Pseudomonas fluorescens, lipase PudA from Commonos acidovorans TB-35, and lipases PueA and PueB from Pseudomonas chlororaphis are effective for waterborne polyurethane. Both DLNs exhibited hydrolytic activity; the keratinase LCC derived from plant compost and the keratinase TfCut2 derived from Thermobifida fusca KW3 resulted in a mass loss of 3.2% and 1.9% respectively in polyester-type PU within 100 h; furthermore, the keratinase HiC derived from Humicola insolens depolymerized polyester-type polyurethane, achieving a number-average molecular weight (Mn) loss within 7 days. nThe concentration of PU decreased by 84%. Highly efficient depolymerizing enzymes for polyester-type PU are still under development. The highly efficient keratinase CpCut1 for hydrolyzing PU provided in this study will provide a highly efficient enzyme resource for the enzymatic depolymerization of PU plastics, which will be beneficial for the future upgrading or recycling of waste PU plastics, and realize the green recycling of waste plastic resources. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a fungal cutinase for hydrolyzing polyester polyurethane plastics and its application, which addresses the shortcomings of the prior art.

[0005] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0006] In a first aspect, the present invention discloses a keratinase-encoding gene cpcut1.

[0007] The nucleotide sequence of the keratinase encoding gene cpcut1 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0008] In a second aspect, the present invention discloses the keratinase CpCut1 encoded by the keratinase encoding gene cpcut1 described in the first aspect above.

[0009] Thirdly, this invention discloses a genetically engineered bacterium that produces the keratinase CpCut1.

[0010] The genetically engineered bacteria were obtained by cloning the cutinase-encoding gene cpcut1 using the pMCO-AOXα plasmid as a vector and Pichia pastoris as a host.

[0011] Fourthly, the present invention discloses the application of the genetically engineered bacteria described in the third aspect above in the production of keratinase CpCut1.

[0012] In this process, the seed culture of the genetically engineered bacteria was inoculated into YPD liquid medium containing bleomycin resistance at 4%–8% v / v to obtain the first bacterial culture. The first bacterial culture was then inoculated into BMGY liquid medium at 0.5%–2% v / v to obtain the second bacterial culture. The second bacterial culture was centrifuged to obtain the bacterial cells. The obtained bacterial cells were resuspended in BMMY liquid medium and cultured. Ethanol was added for fermentation to obtain the keratinase CpCut1.

[0013] Fifthly, the present invention discloses a keratinase CpCut1, which is expressed by the genetically engineered bacteria described in the third aspect above.

[0014] In this process, the seed culture of the genetically engineered bacteria was inoculated into YPD liquid medium containing bleomycin resistance at 4%–8% v / v to obtain the first bacterial culture. The first bacterial culture was then inoculated into BMGY liquid medium at 0.5%–2% v / v to obtain the second bacterial culture. The second bacterial culture was centrifuged to obtain the bacterial cells. The obtained bacterial cells were resuspended in BMMY liquid medium and cultured. Ethanol was added for fermentation to obtain the keratinase CpCut1.

[0015] In a sixth aspect, the present invention discloses the application of the above-mentioned keratinase CpCut1 in depolymerized polyester-type urethane plastics.

[0016] The polyester-type urethane plastics include water-based PU, PBA-PU film, and solid PU foam.

[0017] The ratio of the amount of keratinase CpCut1 to aqueous PU is 12 μg: 0.01-0.09 mL; the mass ratio of keratinase CpCut1 to PBA-PU film is 0.4: 25-35; the mass ratio of keratinase CpCut1 to solid PU foam is 0.4: 25-35; and the specific enzyme activity of keratinase CpCut1 is 450-460 U / mg, such as 455 U / mg.

[0018] The depolymerization temperature is 50-60℃; the depolymerization pH is 7.5-8.5.

[0019] In summary, this invention provides a fungal-derived keratinase CpCut1 that can efficiently depolymerize polyester polyurethane (PU) plastics and has the ability to efficiently catalyze the hydrolysis of ester bonds in PU plastics.

[0020] Beneficial effects:

[0021] (1) The fungal keratinase CpCut1 used in this invention can efficiently degrade different types of polyester PU substrates in a short time. DLN, PBA-PU, and solid PU foam). When DLN is used as a substrate, CpCut1 reacts to the substrate after 30 minutes. The degradation rate of DLN reached 100%; the degradation rate of PBA-PU film reached 40.5% after 12 hours; and when using PU foam as a substrate, CpCut1 achieved a degradation rate of 20.6% for PU foam. Furthermore, CpCut1 was found to be superior to three other previously reported polyester keratinases in its degradation effect on all three types of PU.

[0022] (2) This invention identified the metabolites of PU, speculated on the hydrolysis mechanism of fungal keratinase CpCut1 hydrolyzing polyester PU, and determined that the enzyme mainly hydrolyzes PU by acting on ester bonds in PU, providing efficient depolymerization elements and theoretical guidance for PU plastic enzymatic depolymerization technology. Attached Figure Description

[0023] 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.

[0024] Figure 1 SDS-PAGE electrophoresis image, amino acid sequence alignment, and phylogenetic analysis of keratinase CpCut1.

[0025] Figure 2 To study the enzymatic properties of keratinase CpCut1.

[0026] Figure 3 To verify the degradation function of keratinase on different PU substrates, and to compare its degradation ability with that of other polyester depolymerases.

[0027] Figure 4 SEM characterization of the degradation of PU plastic by the keratinase CpCut1.

[0028] Figure 5 FTIR, GPC and TGA characterization of the degradation of PU plastic by the keratinase CpCut1.

[0029] Figure 6 Mass spectrometry identification of metabolites and hypothesis of hydrolysis mechanism after keratinase CpCut1 degrades PBA-PU film. Detailed Implementation

[0030] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0031] In this invention, the protein expression strain of keratinase CpCut1, P. pastoris GS115 / pMCO-AOXα-cpcut1, was constructed in the laboratory in the early stage (NCBI: OR245267). The extracellular enzyme was produced by fermentation in the Pichia pastoris expression system. The culture media used were YPD medium, BMGY medium and BMMY medium. The antibiotic used was bleomycin.

[0032] In this invention, the cloning and expression of the fungal cutinase CpCut1 in the high-efficiency polyester PU plastic are as follows:

[0033] (1) The cpcutl gene derived from the fungus Cladosporium sp. P7, which was optimized by codons, was cloned into the plasmid vector pMCO-AOXα through two restriction enzyme sites, Xho I and Pst I. The recombinant plasmid was digested with Sal I and transformed into Pichia pastoris GS115 by electroporation. It was then plated on YPDZ selective medium containing 300 μg / mL Zecoin and cultured at 30℃.

[0034] (2) Inoculate the bacterial culture at a rate of 5% (v / v) into YPD liquid medium containing 100 μg / mL bleomycin resistance. After culturing at 30°C for 24 h, add the bacterial culture to BMGY liquid medium at a rate of 1% (v / v) and culture at 30°C for 24 h. Then, transfer the bacterial culture to a sterile centrifuge tube and centrifuge at 6000 rpm for 10 min. Discard the supernatant, resuspend the bacterial cells in an equal volume of BMMY liquid medium, and transfer the culture to a shake flask. Place the flask in a shaker at 30°C and culture. Add 1% (v / v) methanol every 24 h and continue fermenting for 5 days.

[0035] The YPD liquid culture medium formula is: 20 g / L peptone, 20 g / L glucose, and 10 g / L yeast extract.

[0036] The formula for BMGY liquid culture medium is: 20 g / L peptone, 10 g / L yeast extract, 13.4 g / L YNB (containing ammonium sulfate), 3.01 g / L K2HPO4·3H2O, 11.81 g / L KH2PO4, 0.4 mg / L biotin, and 1% glycerol.

[0037] The BMMY liquid culture medium formula is: 20 g / L peptone, 10 g / L yeast extract, 13.4 g / L YNB (containing ammonium sulfate), 3.01 g / L K2HPO4·3H2O, 11.81 g / L KH2PO4, 0.4 mg / L biotin, and 1% (v / v) methanol.

[0038] In this invention, the enzyme activity assay method is as follows: using p-NPB as a substrate, reacting at 30°C for 3 minutes, the amount of enzyme required to produce p-nitrophenol per unit time is one enzyme activity unit (U).

[0039] 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).

[0040] Wherein, specific enzyme activity = protease activity (U / mL) / protein concentration (mg / mL).

[0041] The specific enzyme activity of CpCut1 was detected to be 455 U / mg.

[0042] The phosphate buffer (PBS buffer) used in this embodiment of the invention is 50 mM PBS with pH = 8.0.

[0043] In this invention, the preparation method of PBA-PU film is as follows: 1.5g of PBA-PU film is dissolved in 20mL of dichloromethane, stirred on a magnetic stirrer until dissolved, sonicated for 30min, poured onto a clean glass petri dish, and placed in a fume hood to evaporate.

[0044] In the following examples, the depolymerization effects of different polyester hydrolysases on three types of polyester PU plastics were compared by measuring changes in turbidity and mass loss.

[0045] Specifically, the depolymerization efficiency of the four keratinases on aqueous PU was measured by OD. 400 The turbidity change measured at nm was compared; the depolymerized PBA-PU film and PU foam were dried in an oven at 60℃ to constant weight, and the mass loss before and after depolymerization was measured to compare the depolymerization effect of keratinase from different sources on PBA-PU film and PU foam.

[0046] After the keratinase CpCut1 depolymerized PBA-PU film and PU foam, the changes in the surface morphology of PBA-PU film and PU foam before and after depolymerization were observed by photographic images and scanning electron microscopy (SEM). The hydrolysis sites of keratinase CpCut1 on polyester PU were inferred by Fourier transform infrared spectroscopy (FTIR) of the depolymerized PBA-PU film and PU foam. The molecular weight change of PBA-PU after depolymerization was determined by gel permeation chromatography (GPC). The products of PBA-PU depolymerization supernatant were analyzed by gas chromatography-mass spectrometry (GC-MS). Thermogravimetric analysis (TGA) was performed on the depolymerized PU foam to study the changes in the thermodynamic properties of PU foam.

[0047] Example 1: Amino acid sequence analysis and phylogenetic analysis of keratinase CpCut1

[0048] According to genome annotation, the keratinase CpCut1 is expressed in secretory form by Pichia pastoris, producing extracellular proteins. After purification of the fermentation broth using an AKTA protein purifier and verification by SDS-PAGE electrophoresis, multiple bands were observed above the target band, suggesting that glycosylation modification occurred during yeast expression. Figure 1A). A phylogenetic tree was constructed by comparing the protein sequence of CpCut1 (without the signal peptide) with the protein sequences of keratinases from different sources. The comparison revealed that CpCut1 shared 45.1% to 58.3% sequence homology with fungal keratinases, while its similarity with bacterial keratinases was only 26.2% to 33.7%, indicating that CpCut1 is a PU depolymerase derived from fungi. Figure 1 As shown in Figure C. Furthermore, it exhibits high similarity (41.5% and 43.69%) to fungal keratinases HiC (Humicola insolens) and FsC (Fusarium solani) reported to possess PU hydrolytic activity. CpCut1 was also found to possess the typical triplet catalytic active site Ser113-Asp168-His181 of the α / β hydrolase superfamily proteins, as well as the characteristic sequence tag (GYSQG) of this family of proteins. (e.g.) Figure 1 (as shown in B).

[0049] Example 2: Determination of the enzymatic properties of the keratinase CpCut1 for depolymerizing polyurethane plastics

[0050] The enzymatic properties of keratinase CpCut1 include the optimal enzyme activity temperature and pH, temperature stability and pH stability, as well as the effects of metal ions and chemical reagents on CpCut1 enzyme activity.

[0051] Specifically, the optimal reaction pH of the enzyme was determined by placing the reaction system in an activity assay system containing 50 mM citrate buffer (pH 4.0–6.0), 50 mM PBS buffer (pH 6.0–8.0), and 50 mM glycine-NaOH buffer (pH 8.0–10.0), reacting at 55°C for 10 min, and then measuring the enzyme activity to determine the optimal reaction pH of the enzyme.

[0052] Specifically, the pH stability of the enzyme was determined by placing it in buffer solutions of different pH values, incubating it at 4°C for 24 hours, and measuring the enzyme activity. The initial enzyme activity at the optimal pH was taken as 100%, and the enzyme's tolerance to pH was calculated.

[0053] Specifically, the reaction system was placed in water baths at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃ and 90℃ for 10 min to determine the optimal reaction temperature of the enzyme.

[0054] Specifically, the keratinase was placed in water baths at 37℃, 55℃, and 80℃ for 12 hours, and samples were taken every hour to measure the enzyme activity. The initial enzyme activity of the enzyme solution at the optimal reaction temperature was taken as 100%, and the relative enzyme activity of the enzyme solution at each temperature was calculated to determine the enzyme's temperature tolerance.

[0055] Specifically, the keratinase CpCut1 was added to a solution containing 1 mM of different metal ions (Fe). 3+ Ca 2+ Mn 2+ Mg 2+ K + Co 2+ Al 3+ Cu 2+ Zn 2+ Ba 2+ Ni 2+ and Na + The enzyme activity was measured in an organic solvent (acetone, isopropanol, methanol, ethanol, DMSO, p-β-mercaptoethanol and DMF), chelating agent (EDTA), and surfactant (TrionX-100, Tween80, SDS and CTAB), and placed at 55°C for 10 min. The enzyme activity of the blank group was set as 100%, and the relative enzyme activity after the addition of metal ions and chemical reagents was calculated.

[0056] Depend on Figure 2 As shown in A and 2B, the keratinase CpCut1 has high activity in the pH range of 6.0 to 9.0, with the optimal pH value being 8.0. After storage for 24 hours in the pH range of 6.0 to 10.0, the residual activity of CpCut1 enzyme is above 50%. When the pH is less than 6.0 or higher than 9.0, the stability decreases significantly under different pH conditions.

[0057] Depend on Figure 2 As shown in D and 2E, the keratinase CpCut1 is active from 20 to 90°C, with the highest activity at 55°C. It has good stability at 37°C, and after 12 hours of storage, the residual enzyme activity is 95%. After 10 hours of treatment at 55°C, the enzyme activity is reduced by 50%. As the temperature continues to rise, the stability of the enzyme activity weakens. After 2 hours of treatment at 80°C, the enzyme completely loses its activity.

[0058] Depend on Figure 2 C indicates that metal ions have a certain influence on the enzyme activity of keratinase CpCut1, among which the metal ion Mn 2+ K + Al 3+ and Zn 2+ It has a certain activating effect on enzyme activity, with relative enzyme activities of 130.80%, 137.40%, 123.10%, and 145.50%, respectively; the metal ion Cu 2+ Ba 2+ Na + And has little effect on CpCut1; Fe 3+ Mg 2+ Ca 2+ and Co 2+It has an inhibitory effect on enzyme activity.

[0059] Depend on Figure 2 As shown in F, Triton X-100, SDS, CTAB, acetone, and other chemicals have a strong inhibitory effect on the activity of β-mercaptoethanol keratinase. Other chemical reagents have little effect on the enzyme activity of CpCut1. After treatment with 10 mM metal chelating agent EDTA, the relative enzyme activity of CpCut1 is above 70%, indicating that CpCut1 may not be a strictly metal ion-dependent enzyme. CpCut1 can resist various environmental stresses without significantly losing its activity.

[0060] Example 3: Degradation of different polyurethane plastic substrates by keratinase

[0061] 12 μg of keratinase CpCut1 was added to a solution containing 1.0% (v / v) of [amount missing]. DLN was reacted in 5 mL of PBS buffer at 55 °C and pH 8.0 for 48 h, and the OD was measured using a UV spectrophotometer. 400nm The absorbance value changes under different conditions, and the degradation rate is calculated.

[0062] Depend on Figure 3 As can be seen from A, the solution rapidly changes from an emulsion to a clear solution after 30 minutes of degradation. The degradation rate of DLN is close to 100%.

[0063] 0.4 mg of keratinase CpCut1 was added to 5 mL of PBS buffer containing 30 mg of PBA-PU membrane, and the reaction was carried out at 55 °C and pH 8.0 for 48 h. After the degradation of the PBA-PU membrane by keratinase CpCut1 was completed, the membrane was recovered, rinsed, dried in a 60 °C oven to constant weight, and the mass loss rate was calculated.

[0064] Depend on Figure 3 As shown in Figure B, after CpCut1 degraded the PBA-PU film for 12 hours, the film became transparent, and the mass loss rate reached 40.5%, as can be seen from the actual image.

[0065] 0.4 mg of keratinase CpCut1 was added to 5 mL of PBS buffer containing 30 mg of solid PU foam, and the mixture was reacted at 55 °C and pH 8.0 for 48 h. After the reaction, the foam was rinsed with pure water and dried in a 60 °C oven to constant weight. The net weight of the foam after degradation was calculated, and the foam mass loss rate was calculated.

[0066] Depend on Figure 3 As shown in C, after 12 hours of degradation, the obvious sharp edges of the PU foam surface were eroded and degraded by enzymes, the surface became irregular and turned yellow, and the mass loss rate of the PU foam reached 20.6%.

[0067] Four enzymes, CpCut1, LCC, TfCut2, and HiC, were added to PBS buffer containing the three PU substrates according to the aforementioned dosage ratio. The mixtures were then placed at 55°C and pH 8.0 to carry out depolymerization reactions, and the degradation rates of the three PU substrates after depolymerization by different enzymes were calculated.

[0068] Depend on Figure 3 D indicates that the keratinase CpCut1 can completely degrade a 1.0% (v / v) concentration of [a substance] after a reaction at 55℃ for 3 hours. DLN, LCC, TfCut2 and HiC pairs The degradation rates of DLN were 23.5%, 5.3%, and 10.7%, respectively, significantly lower than that of CpCut1 (100.0%). At 55℃, after 12 hours of treatment with three keratinases (LCC, TfCut2, and HiC), the weight loss rates of the PBA-PU film were 20.8%, 9.3%, and 13.7%, respectively, significantly lower than the degradation rate after CpCut1 treatment (40.5%). After 12 hours of degradation, the mass loss of PU foam caused by the three keratinases (LCC, TfCut2, and HiC) was 9.5%, 3.7%, and 5.8%, respectively, significantly lower than the degradation rate of PU foam caused by CpCut1 (20.6%). These results indicate that the keratinase CpCut1 has a significantly better depolymerization effect on polyester PU than the other three enzymes.

[0069] Example 4: Characterization of the degradation of PU plastic by keratinase CpCut1

[0070] After depolymerization of PBA-PU film and polyester PU foam by CpCut1, the changes in material properties of the degraded polyester PU were analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), and thermogravimetric analysis (TGA).

[0071] Depend on Figure 4 It can be seen that after SEM characterization of the degraded PBA-PU film and PU foam, at a magnification of 1000×, the surface of the PBA-PU film became rougher and had many pores compared with the control group; compared with the control group, the PU foam skeleton was obviously eroded, the skeleton became rough and cracked, indicating that both were severely attacked by enzymes.

[0072] like Figure 5 As shown in A and 5C, FTIR analysis revealed that the degraded polyester PU plastic (PBA-PU film): Figure 5 A. PU foam: Figure 5 B) Compared with the control group, the characteristic peak of PU showed ester bond C=O (1725cm). -1 The signal of the α bond was significantly weakened, while the urethane bond (CNH) signal was 1530 cm⁻¹. -1The near peaks showed almost no change, indicating that ester bonds, rather than carbamate bonds, were hydrolyzed during degradation, suggesting that the keratinase CpCut1 only acts on the ester bond portion of the polyester PU.

[0073] like Figure 5 As shown in Figure B, the GPC characterization results show that, compared with the control group, the Mw and Mn of the degraded PBA-PU film decreased by 13.3% and 14.5%, respectively, indicating that the PBA-PU structure breaks down and produces low molecular weight substances after degradation by CpCut1.

[0074] like Figure 5 As shown in D, compared with the untreated control sample (T) 50％ =382.9℃, T max1 =307.3℃, T max2 Compared to (402.0℃), the PBA-PU film treated with CpCut1 (T) 50％ =339.0℃, T max1 =296.3℃, T max2 =374.9℃), the temperature at which the maximum weight loss rate was lower than that of the control group, indicating that the PU foam after enzymatic polymerization is more easily pyrolyzed and the thermal stability of the foam is reduced.

[0075] Example 5: Mass spectrometry analysis of PBA-PU film metabolites by keratinase CpCut1

[0076] 0.4 mg of keratinase CpCut1 was added to 5 mL of PBS buffer containing 30 mg of PBA-PU and incubated at 55 °C and pH 8.0 for 48 h. The supernatant was collected by centrifugation, lyophilized to remove moisture, dissolved in methanol, and filtered through a 0.22 μm organic filter membrane for sterilization. The metabolites were analyzed by mass spectrometry (MS).

[0077] Specifically, MS analysis was performed in ESI mode using an Agilent 6500Q-TOF instrument. Detection conditions: sequential positive and negative ion mode; positive electrospray voltage 4.0 kV, negative electrospray voltage 3500 kV, CDL 200℃, heater temperature 200℃, nebulizing gas (N2) 5 L / min, and drying gas (N2) pressure 0.1 MPa.

[0078] Depend on Figure 6 It can be seen that PBA-PU metabolism produces four metabolites, namely 1,4-butanediol (BDO), Figure 6 C), adipic acid (AA, Figure 6 D), two MDA derivatives, bis(4-hydroxybutyl)(methylenebis(4,1-phenylene))dicarbamate (BMC, Figure 6E) and derivatives of MDA with alcohols or acids attached to both ends (MDA derivatives, Figure 6 F).

[0079] Specifically, after 48 hours of degradation, high-performance liquid chromatography (HPLC) and analysis based on the ion peak at 145.05 (MH) were performed. - The corresponding mass spectrometry (MS) analysis showed the production of AA (metabolite 2). Figure 6 A and 6D). Similarly, gas chromatography (GC) of the hydrolysis products and the ion peak corresponding to 89.07 (MH) were analyzed. - Mass spectrometry analysis also confirmed the release of BDO (metabolite 1). Figure 6 (B and 6C). These two monomers indicate that CpCut1 hydrolyzes the ester bond in the PU soft segment (polyol). Based on the ion peak 453.20 (M+Na). + Mass spectrometry analysis at the location ( Figure 6 E), it is speculated that metabolite 3 is BMC; based on ion peak 581.25 (M+Na). + Mass spectrometry analysis at the location ( Figure 6 F), it is speculated that metabolite 4 is an MDA derivative ( Figure 6 Based on these results, a possible degradation pathway is proposed (F). Figure 6 G): PBA-PU first undergoes depolymerization via CpCut1 catalysis, breaking the ester bonds in the soft segments of the polyol to release long-chain oligomers. These oligomers are then hydrolyzed by CpCut1 into BDO (metabolite 1), AA (metabolite 2), and an MDA derivative (metabolite 4). Further, CpCut1 hydrolyzes the ester bonds in metabolite 4 to generate AA and BMC (metabolite 3).

[0080] 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. Use of a cutinase CpCutl in depolymerization of polyester polyurethane plastic, characterized in that, The keratinase CpCut1 was developed using the pMCO-AOXα plasmid as a vector and Pichia pastoris as a host, by cloning the keratinase-encoding gene. CpCut1 The keratinase-encoding gene was obtained. CpCut1 The nucleotide sequence is shown in SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The seed liquid of the genetically engineered bacteria is inoculated into YPD liquid medium containing bleomycin resistance at 4%-8% v / v for first culture to obtain a first bacterial liquid, the obtained first bacterial liquid is inoculated into BMGY liquid medium at 0.5%-2% v / v for culture to obtain a second bacterial liquid, the obtained second bacterial liquid is centrifuged to obtain bacterial bodies, the obtained bacterial bodies are resuspended in BMMY liquid medium for culture, ethanol is added for fermentation, and the cutinase CpCut1 is obtained.

3. Use according to claim 1, characterized in that, The polyester polyurethane plastic is water-based PU, PBA-PU film or solid PU foam.

4. Use according to claim 3, characterized in that, The mass ratio of the cutinase CpCut1 to water-based PU is 12 μg: 0.01-0.09 mL, the mass ratio of the cutinase CpCut1 to PBA-PU film is 0.4: 25-35, and the mass ratio of the cutinase CpCut1 to solid PU foam is 0.4: 25-35.

5. Use according to any one of claims 1 to 4, characterized in that, The temperature of the depolymerization is 50-60 ℃, and the pH of the depolymerization is 7.5-8.

5. CpCut1 CpCut1 The seed liquid of the genetically engineered bacteria is inoculated into YPD liquid medium containing bleomycin resistance at 4%-8% v / v for first culture to obtain a first bacterial liquid, the obtained first bacterial liquid is inoculated into BMGY liquid medium at 0.5%-2% v / v for culture to obtain a second bacterial liquid, the obtained second bacterial liquid is centrifuged to obtain bacterial bodies, the obtained bacterial bodies are resuspended in BMMY liquid medium for culture, ethanol is added for fermentation, and the cutinase CpCut1 is obtained. The polyester polyurethane plastic is water-based PU, PBA-PU film or solid PU foam. The mass ratio of the cutinase CpCut1 to water-based PU is 12 μg: 0.01-0.09 mL, the mass ratio of the cutinase CpCut1 to PBA-PU film is 0.4: 25-35, and the mass ratio of the cutinase CpCut1 to solid PU foam is 0.4: 25-35. The temperature of the depolymerization is 50-60 ℃, and the pH of the depolymerization is 7.5-8.5.

Images