Degradation enzyme pufh for efficiently decomposing polyurethane sponge and application thereof

Abstract

The application discloses a degradation enzyme PufH for efficiently decomposing polyurethane sponge and application thereof. The amino acid sequence of the degradation enzyme is shown as SEQ ID NO. 1, and the coded gene sequence is shown as SEQ ID NO. 2. The degradation enzyme PufH used in the application can reach a degradation rate of 82.4% on commercial polyurethane sponge within 48 hours. The degradation effect of the degradation enzyme PufH on commercial polyurethane sponge is superior to that of the currently reported PU degradation enzyme, and the degradation enzyme PufH can be applied to the recycling or pollution repair of polyurethane sponge waste.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a degradation enzyme PufH for efficiently decomposing polyester polyurethane sponge and application thereof. BACKGROUND

[0002] Polyurethane (PU) is a common plastic, widely used in coatings, elastomers and foams. Polyurethane foams (PUF) is a thermoset plastic, more than 50% of the total production of PU [Magnin A, Entzmann L, Bazin A, et al. Green Recycling Process for Polyurethane Foams by a Chem-Biotech Approach. ChemSusChem. 2021 Oct 5;14(19):4234-4241.]. According to its soft and hard degree, PUF can be divided into soft, hard and semi-hard, among which soft polyurethane foam is commonly known as polyurethane sponge. Polyurethane sponge is widely used in furniture, seats and bedding. With the large use of polyurethane sponge, the problem of its waste disposal is increasingly prominent. Thermoset plastics are difficult to be recycled by heating. At present, polyurethane waste is mainly treated by landfill and incineration, which not only increases the emission of carbon dioxide, but also releases a large amount of microplastics and other toxic chemicals, polluting the environment and even endangering human health [MacLeod, M., Arp, H. P. H., Tekman, M. B., Jahnke, A., 2021. The global threat from plastic pollution. Science 373(6550), 61-65.]. At present, two problems are mainly concerned, one is the recycling of waste polyurethane sponge (before being released into the environment), the other is the efficient degradation of polyurethane sponge that has been released into the environment.For recycling, there are reports of physical and chemical methods [Cregut M, Bedas M, Durand M J, et al. New insights into polyurethane biodegradation and realistic prospects for the development of a sustainable waste recycling process [J]. Biotechnology advances, 2013, 31(8): 1634-1647.], but they are still in the experimental stage, and there are still problems such as low recycling rate [Zia K M, Bhatti H N, Bhatti I A. Methods for polyurethane and polyurethane composites, recycling and recovery: A review [J]. Reactive and functional polymers, 2007, 67(8): 675-692.] and poor economic benefits [Simón D, Borreguero A M, De Lucas A, et al. Recycling of polyurethanes from laboratory to industry, a journey towards the sustainability [J]. Waste Management, 2018, 76: 147-171.]. In recent years, biological methods using microorganisms or enzymes to recycle or degrade waste polyurethane sponge have been valued.

[0003] From the structure, polyurethane sponge is mainly composed of diisocyanate (such as

[0004] Polyurethane sponges are synthesized by the polymerization of diisocyanates (e.g. toluene-2,4-diisocyanate, TDI or diphenylmethane-4,4'-diisocyanate, MDI) and polyols [Hoyle C E, Kim K J. Effect of crystallinity and flexibility on the photodegradation of polyurethanes [J]. Journal of Polymer Science Part A: Polymer Chemistry, 1987, 25(10): 2631-2642.]. According to the difference in the configuration of the polyol raw materials used, polyurethane sponges can be divided into two categories: polyether type and polyester type. The essence of biological method is to use enzymes to open the chemical bonds of polyurethane sponges, achieving the recycling or complete degradation of monomers. The key to the biological treatment of waste polyurethane sponges is the need for efficient microbial or enzyme resources. Currently, the reported polyurethane sponge degrading enzymes include cutinases (LCC, HiC, TfCut2, Tcur1278, Tcur0390, CpCut1, BaCut1), esterases (PudA, PulA) and lipases (PueA, PueB, CALB) [Liu J, Xin K, Zhang T, et al. Identification and characterization of a fungal cutinase-like enzyme CpCut1 from Cladosporium sp. P7 for polyurethane degradation [J]. Applied and Environmental Microbiology, 2024, 90(4): e01477-23.][Jiang Z, Chen X, Xue H, et al. Novel polyurethane-degrading cutinase BaCut1 from Blastobotrys sp. G-9 with potential role in plastic bio-recycling [J]. Journal of Hazardous Materials, 2024, 472: 134493.].In terms of degradation efficiency, the degradation rate of cutinase CpCut1 on polyurethane sponge within 12h was 20.6% [Liu J, Xin K, Zhang T, et al. Identification and characterization of a fungal cutinase-like enzyme CpCut1 from Cladosporium sp. P7 for polyurethane degradation [J]. Applied and Environmental Microbiology, 2024, 90(4): e01477-23.]; the degradation rate of BaCut1 on polyurethane sponge within 48h was 50% [Jiang Z, Chen X, Xue H, et al. Novel polyurethane-degrading cutinase BaCut1 from Blastobotrys sp. G-9 with potential role in plastic bio-recycling [J]. Journal of Hazardous Materials, 2024, 472: 134493.]; and the degradation rate of CALB on polyurethane sponge within 24h was 25% [Magnin A, Entzmann L, Bazin A, et al. Green Recycling Process for Polyurethane Foams by a Chem-Biotech Approach. ChemSusChem. 2021 Oct 5; 14(19): 4234-4241.]. Overall, although there are many reports of polyurethane sponge degrading enzymes, the efficiency of these enzymes cannot meet the needs of biological methods, and more efficient polyurethane sponge degrading enzymes still need to be discovered.

[0005] The present application provides a degradation enzyme PufH for efficiently decomposing polyurethane sponge, which has a degradation rate of more than 80% within 48h, and has application potential in the recycling or pollution repair of waste polyurethane sponge pollution. SUMMARY

[0006] The present application solves the problem of low efficiency of polyurethane sponge degrading enzymes, and provides a degradation enzyme for efficiently decomposing polyurethane sponge and its application.

[0007] To solve the above technical problems, the present application discloses the following technical solutions:

[0008] A degradation enzyme PufH for efficiently decomposing polyurethane sponge, a sequence of which is shown in SEQ ID NO. 1.

[0009] A gene encoding the degradation enzyme PufH.

[0010] The nucleotide sequence of the gene is preferably shown in SEQ ID NO. 2.

[0011] A recombinant expression vector containing the gene.

[0012] Application of the degradation enzyme PufH in degrading polyurethane sponge.

[0013] The mass ratio of the degradation enzyme PufH to the polyurethane sponge is 1:50.

[0014] The degradation temperature is 30 DEG C, and the degradation pH is 7.0.

[0015] In summary, the present application provides a bacterial-derived degradation enzyme PufH which can efficiently degrade polyurethane sponge.

[0016] Advantages:

[0017] The degradation enzyme PufH used in the present application can efficiently degrade polyurethane sponge, and the degradation rate of PUF can reach 82.4% in 48h. The degradation effect is better than that of the reported degradation enzyme, and has application value in the recycling of waste polyurethane sponge and pollution remediation. BRIEF DESCRIPTION OF DRAWINGS

[0018] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings.

[0019] Figure 1 SDS-PAGE electrophoretogram and zymogram analysis of the crude enzyme solution of strain YX66, wherein M: marker (kDa); 1: crude enzyme solution staining; 2: PBA-PU plate color development.

[0020] Figure 2 SDS-PAGE electrophoretogram, enzyme activity verification diagram, amino acid sequence alignment and phylogenetic tree analysis of the degradation enzyme PufH, wherein, Figure 2 A is the SDS-PAGE electrophoretogram of the degradation enzyme PufH, Figure 2 B is the verification diagram of the degradation enzyme PufH degrading commercial PUF, Figure 2 C is the amino acid sequence alignment diagram of the degradation enzyme PufH, Figure 2 D is the phylogenetic tree analysis diagram of the degradation enzyme PufH.

[0021] Figure 3Enzymatic properties of the degrading enzyme PufH. Among them, Figure 3 A is the optimal degradation pH of the degrading enzyme PufH, Figure 3 B is the pH tolerance of the degrading enzyme PufH, Figure 3 C is the optimal degradation temperature of the degrading enzyme PufH, Figure 3 D is the temperature tolerance of the degrading enzyme PufH.

[0022] Figure 4 Product identification and hydrolysis mechanism speculation of the degrading enzyme PufH after degrading PBA-PU. Among them, Figure 4 A is the HPLC spectrum of the product of the degrading enzyme PufH after degrading PBA-PU, Figure 4 B is the LC-MS spectrum of the product of the degrading enzyme PufH after degrading PBA-PU, Figure 4 C is the hydrolysis mechanism speculation of the degrading enzyme PufH after degrading PBA-PU.

[0023] Figure 5 Degrading ability analysis of the degrading enzyme PufH on polyester commercial PUF. Among them, Figure 5 A is the weight loss rate of the degrading enzyme PufH degrading polyester commercial PUF, Figure 5 B is the actual picture of the degrading enzyme PufH degrading polyester commercial PUF.

[0024] Figure 6 Product detection and identification of the degrading enzyme PufH after degrading commercial PUF. Among them, Figure 6 A is the HPLC spectrum of the product of the degrading enzyme PufH after degrading commercial PUF, Figure 6 B is the LC-MS spectrum of the product of the degrading enzyme PufH after degrading commercial PUF. DETAILED DESCRIPTION

[0025] Example 1

[0026] First, the structural analog PBA-PU of polyurethane sponge (synthesis method reference Liu J, Zeng Q, Lei H, et al. Biodegradation of polyester polyurethane by Cladosporium sp. P7: Evaluating its degradation capacity and metabolic pathways. J Hazard Mater. 2023 Apr 15; 448: 130776.) was used as a substrate to screen for degrading bacteria Pueribacillus sp. YX66, and it was determined that the bacteria could decompose polyurethane sponge. Then, the degrading enzyme PufH was obtained through bioinformatics prediction combined with experimental verification, and its enzymatic properties and polyurethane sponge degradation properties were identified. The specific implementation method is as follows:

[0027] (1) In order to obtain the degrading enzyme, the extracellular crude enzyme solution of strain YX66 was collected and zymogram analysis was performed. Strain YX66 was cultured on LB solid plate at 50 degrees for 2-3 days, and the plate was washed with appropriate amount of 50 mM Tris-HCl buffer (pH 7.0). The collected supernatant was precipitated with saturated ammonium sulfate, then resuspended and dialyzed to obtain YX66 extracellular crude enzyme for zymogram analysis. As shown in Figure 1 , after renaturation of the enzyme solution in SDS-PAGE gel, a transparent band can be produced on the PBA-PU plate, and the corresponding protein size is about 25 kDa, suggesting that strain YX66 can secrete a polyurethane sponge degrading enzyme with a molecular weight of about 25 kDa. Combined with the annotation of the genome sequence of strain YX66, 21 degrading enzyme genes were retrieved by searching with cutinase, esterase and lipase as keywords. A potential lipase PufH was obtained by screening the 21 degrading enzymes based on molecular weight (about 25 kDa) and potential for secretion and expression, and the gene sequence is shown in SEQ ID NO. 2.

[0028] (2) In order to verify the function of PufH, the nucleotide sequence of the pufH gene was synthesized by a biological company, as shown in SEQ ID NO. 2 in the sequence listing, which encodes the lipase PufH, as shown in SEQ ID NO. 1 in the sequence listing. The synthesized pufH was cloned into pET28a(+) vector, and the recombinant vector was named pET28a-pufH, then transformed into E. coli BL21(DE3). Strain BL21(DE3)(pET28a-pufH) was grown in LB medium to OD 600= 0.5, and IPTG was added to a final concentration of 1 mM to induce the expression of PufH. After expression, PufH was purified by Ni-NTA column. SDS-PAGE protein electrophoresis showed that the protein band after purification was single, and the size was near 25 kDa, which was consistent with the expected size, indicating that the expression and purification of the degradation enzyme PufH were successful Figure 2 A).

[0029] (3) In order to verify the degradation ability of PufH to polyurethane sponge, 0.5 mg of purified degradation enzyme was mixed with 25 mg of polyurethane sponge (purchased from Nantong Dage Co., Ltd.) in 4 mL of Tris-HCl buffer. Sampling was taken at 0 h, 12 h, 24 h and 48 h, and dried for recovery. Compared with the control group without enzyme, the degradation effect of PufH on polyurethane sponge was obvious Figure 2 B).

[0030] (4) Similarity analysis of the PufH amino acid sequence with the protein sequences in the Protein Data Bank (PDB) database found that the similarity with the reported lipase of Bacillus subtilis (1T2N_A) was the highest, 47.83%, followed by the lipase derived from Bacillus pumilus (7R1K_A), 46.38%. As shown in Figure 2 C, there is a characteristic Ala-X-Ser-X-Gly motif centered on the lipase active serine residue in PufH. In addition, PufH has the typical catalytic triad site Ser-Asp-His (Ser97, Asp180, His203) of the α / β hydrolase superfamily protein. The above results show that PufH belongs to the lipase of the α / β hydrolase superfamily.

[0031] (5) Using evolutionary analysis, PufH was compared with the currently reported polyurethane sponge degrading enzymes Figure 2 D), the results showed that PufH had generally low similarity (less than 30%) with the reported polyurethane sponge degrading enzymes, such as PufH and the lipase CALB derived from Candida Antarctica, with a similarity of 25.49%, which indicated that PufH was a new polyurethane sponge degrading enzyme.

[0032] In the present application, the enzyme activity determination method is: using ultraviolet spectrophotometry to determine the amount of p-nitrophenol (pNP) produced by pNPB substrate at 410 nm to determine the activity of esterase. The reaction mixture is incubated at 37℃ for 10 min, and the reaction mixture is incubated at 37℃ for 10 min. 10 μL of 10 mM pNPB, 10 μL of enzyme solution, 980 μL of 50 mM Tris-HCl buffer (pH 7.0). One unit of esterase activity is defined as the amount of enzyme required to release 1 μmol of pNP per minute.

[0033] Wherein, the protease activity formula is: protease activity = A / (B*C), wherein A is the amount of p-nitrophenol produced (μmol), B is the reaction time (min), and C is the amount of enzyme added (mL).

[0034] The degradation rate of the polyurethane sponge evaluated in the examples of the present application is calculated as the weight loss rate of the polyurethane sponge. The product after enzymatic hydrolysis is filtered and washed multiple times with distilled water through double-layer filter paper, placed in a 60°C oven until constant weight, and then removed and weighed. The calculation formula is: = (initial weight - final weight) / initial weight x 100%.

[0035] The conditions for detecting the degradation product using HPLC are as follows: C18 reverse phase chromatographic column (4.6 x 250 nm, 3 μm), detection wavelength 240 nm, column temperature 40°C, flow rate 0.6 mL / min, mobile phase acetonitrile: water (80:20, V / V), sample size 5 μL, sample injection time 8 min. The instrument used for LC-MS is the above HPLC instrument equipped with an electrospray ionization probe connected to a Thermo LTQ Orbitrap XL hybrid mass spectrometer (USA, Massachusetts). The measurement conditions are the same as those of HPLC.

[0036] Example 2: Determination of the enzymatic properties of the degradation enzyme PufH

[0037] To determine the optimum pH and pH tolerance of the degradation enzyme PufH, the degradation enzyme PufH was placed in an activity measurement system of 50 mM citrate buffer (pH 4.0-6.0), 50 mM Tris-HCL buffer (pH 6.0-8.0), and 50 mM glycine-NaOH buffer (pH 8.0-10.0), and the enzyme activity was measured after 10 min at 30°C. The initial enzyme activity of the enzyme solution at the optimum pH was set to 100%, and the relative enzyme activity at each pH was calculated. As shown in Figure 3 A, the optimum pH of the degradation enzyme PufH is 7.0, and it is easily inactivated in an acidic environment, with an enzyme activity of less than 30% of the initial enzyme activity at pH < 5.5. The degradation enzyme PufH was placed in the above buffers at different pH, and the enzyme activity was measured after 24 h at 4°C, with the initial enzyme activity of the enzyme solution at the optimum pH set to 100%. As shown in Figure 3 B, the degradation enzyme PufH maintained 60% of the initial activity after incubation for 24 h at pH 6.0-8.0.

[0038] To determine the optimum temperature and temperature tolerance of the degradation enzyme PufH, the enzyme activity reaction system was placed in a water bath at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 10 min, and the enzyme activity of the enzyme solution at the optimum temperature was set to 100%, and the relative enzyme activity at each temperature was calculated. As shown inFigure 3 C It can be seen that the degradation enzyme PufH has activity at 20-90℃, and the highest activity at 30℃. The degradation enzyme PufH was placed in a 30℃, 37℃, and 50℃ water bath for gradient time sampling to determine the enzyme activity. The initial enzyme activity of the enzyme solution at the optimum reaction temperature was 100%, and the relative enzyme activity of the enzyme solution at each temperature was calculated. It can be seen from Figure 3 D that the degradation enzyme PufH has good stability at 30℃, and almost no enzyme activity loss after 24h of storage; the enzyme activity is reduced to less than 40% of the initial enzyme activity after 0.5h of treatment at 50℃.

[0039] Example 3: Decomposition of PBA-PU by degradation enzyme PufH and product identification

[0040] Since the structure of the commercial polyurethane sponge is not clear, the PBA-PU with a clear structure is synthesized in the application, which is used to study the way of PufH decomposing the polyurethane sponge. 200μg of the degradation enzyme PufH was incubated with Tris-HCl buffer (pH 7.0) containing 6mg of PBA-PU at 30℃ and 180rpm for 12h, and the supernatant was obtained by centrifugation at 12000rpm for 10min. The supernatant was acidified to pH 2.0, and then extracted with two volumes of ethyl acetate. The upper solution was dried, 500μL of methanol was added to resuspend the solution, and finally the solution was filtered through a 0.22μm organic phase filter membrane, and then detected by HPLC and LC-MS. It can be seen from Figure 4 A that some product absorption peaks appear near the characteristic peak of MDA by HPLC detection, which is speculated to be the derivative of MDA. Further identification by LC / MS mass spectrum. As shown in Figure 4 B, three PBA-PU hydrolysis products were detected in anion mode. The ion peak of metabolite C corresponds to 145.0508(M-H) - , which is identified as the PBA-PU soft segment rupture product adipic acid; the ion peaks of metabolites B and A correspond to 429.2030(M-H) - and 757.3542(M-H) - , respectively, which are identified as MDA derivatives connected by PBA-PU hard segment and dihydric alcohol or dihydric acid. In view of the above results, it can be inferred that the mode of PufH hydrolyzing PBA-PU is mainly to catalyze the depolymerization of the ester bond of the polyol soft segment to release long-chain oligomers. Then, these oligomers are further hydrolyzed into MDA derivatives (metabolite C) by PufH catalyzing other ester bonds. Finally, PufH hydrolyzes the ester bond in metabolite C to generate AA (metabolite A) and MDA derivative (metabolite B) Figure 4 C).

[0041] Example 4: Decomposition of commercial polyester sponge by degradation enzyme PufH and product identification

[0042] To further evaluate the degradation ability of PufH on commercial polyurethane sponge, PufH (0.1 mg, 0.3 mg and 0.5 mg) was mixed with polyurethane sponge (purchased from Nantong Dawu Co., Ltd.) (25 mg) in 4 mL 50 mM Tris-HCl buffer (pH 7.0) at 30 °C, 180 rpm for 48 h, and the pH change was monitored and adjusted during the reaction. As shown in Figure 5 As shown in FIG. 5A and 5B, the degradation enzyme PufH can degrade 82.4% of the commercial polyurethane sponge in 48 h. The product of enzymolysis for 48 h was detected and identified by HPLC and LC-MS (the method is the same as that in Example 3). As shown in Figure 6 FIG. 5A, the HPLC result shows that some product absorption peaks appear near the TDA characteristic peak, which is speculated to be the derivative of TDA. Further identified by LC / MS mass spectrum, Figure 6 As shown in FIG. 5B, the ion peak corresponding to metabolite A was detected as 145.0508 (M-H) - in anion mode, which was identified as adipic acid; the ion peak corresponding to metabolite B was detected as 683.3391 (M+H) + in cation mode, which was identified as the derivative of TDA. The results show that the polyurethane sponge used contains at least TDA, butanediol and adipic acid. PufH can break the ester bond between butanediol and adipic acid, thereby depolymerizing the polyurethane sponge, and the depolymerization rate reaches 82.4% in 48 h.

[0043] The above-described examples only express several embodiments of the present application, which are described in more detail and in more detail, but should not be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A degradation enzyme PufH for decomposing polyurethane sponge, wherein the sequence of the degradation enzyme PufH is shown as SEQ ID NO.

1. 2.A gene encoding the degradation enzyme PufH according to claim 1.

3. The gene of claim 2, wherein The nucleotide sequence is shown as SEQ ID NO.

2. 4.A recombinant expression vector containing the gene according to claim 2 or 3. 5.The degradation enzyme PufH according to claim 1 is used for degrading polyurethane sponge.

6. Use according to claim 5, characterized in that, The mass ratio of the degradation enzyme PufH to the polyurethane sponge is 1:

50.

7. Use according to claim 5, characterized in that, The degradation temperature is 30℃, and the degradation pH is 7.0.

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