Degradation enzyme PufH for efficiently decomposing polyurethane sponge and application of degradation enzyme PufH

Abstract

The invention discloses a degrading enzyme PufH for efficiently decomposing polyurethane sponge and application of the degrading enzyme PufH. The amino acid sequence of the degrading enzyme is as shown in SEQ ID NO. 1, and the sequence of the coded gene is as shown in SEQ ID NO. 2. The degradation rate of the used degrading enzyme PufH on commercial polyurethane sponge within 48 hours can reach 82.4%. The degrading effect of the degrading enzyme PufH on commercialized polyurethane sponge is better than that of the PU degrading enzyme reported at present, and the degrading enzyme PufH can be applied to cyclic utilization or pollution remediation of polyurethane sponge waste.

Description

Technical Field

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

[0002] Polyurethane (PU) is a common plastic widely used in coatings, elastomers and foams. Polyurethane foams (PUF) are thermosetting plastics, accounting for more than 50% of the total PU production [MagninA, Entzmann L, Bazin A, et al. Green Recycling Process for Polyurethane Foams by a Chem-Biotech Approach. ChemSusChem. 2021Oct 5; 14(19): 4234-4241.]. According to its hardness, 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 extensive use of polyurethane sponge, the problem of its waste disposal has become increasingly prominent. Thermosetting plastics are difficult to reuse by heating. Currently, polyurethane waste is mainly disposed of by landfill and incineration, which not only increases carbon dioxide emissions, but also releases a large amount of microplastics and other toxic chemicals, polluting the environment and even endangering human health [MacLeod, M., Arp, HPH, Tekman, MB, Jahnke, A., 2021. The global threat from plastic pollution. Science 373 (6550), 61–65.]. Currently, there are two main issues of concern: one is the recycling of waste polyurethane sponges (before releasing them into the environment), and the other is the efficient degradation of polyurethane sponges that have been released into the environment.For recycling, physical and chemical methods have been reported [Cregut M, Bedas M, Durand MJ, 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 still have problems such as low reuse rate [Zia KM, Bhatti HN, 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, ajourney towards the sustainability [J]. Waste Management, 2018, 76: 147-171.]. In recent years, biological methods that use microorganisms or enzymes to recycle or degrade waste polyurethane sponges have received attention.

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

[0004] It is made of toluene-2,4-diisocyanate, TDI or diphenylmethane-4,4'-diisocyanate, MDI) and polyols [Hoyle CE, 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 the biological method is to use enzymes to open the chemical bonds of polyurethane sponges to achieve the recycling or complete degradation of monomers. The key to biological treatment of waste polyurethane sponges is the need for efficient microbial or enzyme resources. At present, 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 polyurethane sponge by cutinase CpCut1 within 12 hours was 20.6% [Liu J, Xin K, Zhang T, et al. Identification and characterization of afungal 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 polyurethane sponge by BaCut1 within 48 hours 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 polyurethane sponge by CALB within 24 hours was 25% [Magnin A, Entzmann L, Bazin A, et al. Green Recycling Process for Polyurethane Foams by a Chem-Biotech Approach. Chem Sus Chem. 2021 Oct 5; 14 (19): 4234-4241.]. Overall, although there are many reports on 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 invention provides a degradation enzyme PufH for efficiently decomposing polyurethane sponge, with a degradation rate of more than 80% within 48 hours, and has application potential in the recycling or pollution remediation of waste polyurethane sponge pollution. Summary of the invention

[0006] The present invention aims to solve the problem of low efficiency of polyurethane sponge degrading enzyme. The present invention provides a degrading enzyme capable of efficiently decomposing polyurethane sponge and application thereof.

[0007] In order to solve the above technical problems, the present invention discloses the following technical solutions:

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

[0009] The gene encoding the degradation enzyme PufH.

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

[0011] A recombinant expression vector containing the gene.

[0012] The application of the degrading enzyme PufH in degrading polyurethane sponge.

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

[0014] Wherein, the degradation temperature is 30°C; and the degradation pH is 7.0.

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

[0016] Beneficial effects:

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

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0019] Figure 1 The SDS-PAGE electrophoresis and enzyme spectrum analysis of the extracellular crude enzyme solution of strain YX66. Among them, M: marker (kDa); 1: crude enzyme solution staining; 2: PBA-PU plate color development.

[0020] Figure 2 The following are the SDS-PAGE electrophoresis diagram, enzyme activity verification diagram, amino acid sequence alignment and phylogenetic tree analysis of the degradation enzyme PufH. Figure 2 A is the SDS-PAGE electrophoresis of the degradation enzyme PufH. Figure 2 B is the verification diagram of the degradation of commercial PUF by the degradation enzyme PufH. Figure 2 C is the amino acid sequence alignment of the degradation enzyme PufH, Figure 2 D is the evolutionary tree analysis diagram of the degradation enzyme PufH.

[0021] Figure 3To study the enzymatic properties of the degradation enzyme PufH. Figure 3 A is the optimal degradation pH for the degradation enzyme PufH, Figure 3 B is the pH tolerance of the degradation enzyme PufH, Figure 3 C is the optimal degradation temperature of the degradation enzyme PufH, Figure 3 D is the temperature tolerance of the degradation enzyme PufH.

[0022] Figure 4 To identify the products after the degradation enzyme PufH degrades PBA-PU and speculate on the hydrolysis mechanism. Figure 4 A is the HPLC spectrum of the product after degradation of PBA-PU by the degradation enzyme PufH. Figure 4 B is the LC-MS spectrum of the product after degradation of PBA-PU by the degradation enzyme PufH. Figure 4 C is the speculated hydrolysis mechanism of PBA-PU degradation by the degradation enzyme PufH.

[0023] Figure 5 This is an analysis of the degradation ability of the degradation enzyme PufH on polyester-based commercial PUF. Figure 5 A is the weight loss rate of polyester commercial PUF degraded by the degradation enzyme PufH, Figure 5 A is a picture of the degradation enzyme PufH degrading polyester-type commercial PUF.

[0024] Figure 6 It is to detect and identify the products after the degradation enzyme PufH degrades the commercial PUF. Figure 6 A is the HPLC spectrum of the product after the degradation enzyme PufH degrades the commercial PUF. Figure 6 B is the LC-MS spectrum of the product after degradation of commercial PUF by the degradation enzyme PufH. DETAILED DESCRIPTION

[0025] Example 1

[0026] First, the structural analogue of polyurethane sponge PBA-PU (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 April 15; 448: 130776.) was used as a substrate for screening to obtain the degradation bacteria Pueribacillus sp. YX66, and it was determined that the bacteria can decompose polyurethane sponge. Then, the degradation 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 degradation enzyme, the extracellular crude enzyme solution of strain YX66 was collected and analyzed by zymogram. The strain YX66 was cultured on a LB solid plate at 50 degrees for 2-3 days, and the plate was washed with an appropriate amount of 50mM Tris-HCl buffer (pH 7.0). The collected supernatant was precipitated with saturated ammonium sulfate and then redissolved and dialyzed. The YX66 extracellular crude enzyme was analyzed by zymogram. Figure 1 As shown, the enzyme solution can produce a transparent band on the PBA-PU plate after renaturation in SDS-PAGE gel, and the corresponding protein size is about 25kDa, suggesting that strain YX66 can secrete a polyurethane sponge degrading enzyme with a molecular weight of about 25kDa. Combined with the genome sequence annotation of strain YX66, cutinase, esterase and lipase were used as keywords for retrieval, and a total of 21 degradation enzyme genes were retrieved (Appendix Table 1). The 21 degradation enzymes were screened based on the molecular weight (about 25kDa) and the potential for secretion expression as the standard, and a potential lipase PufH was obtained, and its gene sequence is shown in SEQ ID NO.2.

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

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

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

[0031] (5) PufH was compared with the previously reported polyurethane sponge-degrading enzymes using evolutionary analysis ( Figure 2 D), the results showed that PufH had a generally low similarity with reported polyurethane sponge-degrading enzymes (less than 30%). For example, the similarity between PufH and the lipase CALB from Candida Antarctica was 25.49%, indicating that PufH is a relatively new polyurethane sponge-degrading enzyme.

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

[0033] The protease activity formula 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).

[0034] The polyurethane sponge degradation rate evaluated in the present example is to calculate the weight loss rate of the polyurethane sponge. The enzymatic hydrolysis product is filtered and washed with distilled water through a double-layer filter paper for multiple times, placed in a 60°C oven to a constant weight, and then taken out and weighed. The calculation formula is: = (initial weight - final weight) / initial weight × 100%.

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

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

[0037] In order to determine the optimal pH and pH tolerance of the degradation enzyme PufH, the degradation enzyme PufH was placed in 50mM citrate buffer (pH4.0-6.0), 50mM Tris-HCL buffer (pH6.0-8.0) and 50mM glycine-NaOH buffer (pH8.0-10.0) respectively, and the enzyme activity was measured after 10 minutes at 30°C. The enzyme activity of the initial enzyme solution at the optimal pH was set as 100%, and the relative enzyme activity at each pH was calculated. Figure 3 A shows that the optimal pH value of the degradation enzyme PufH is 7.0. It is easy to be inactivated in an acidic environment. When pH < 5.5, the enzyme activity is less than 30% of the initial enzyme activity. The degradation enzyme PufH was placed in the above buffer solutions of different pH values ​​at 4°C for 24 hours, and the enzyme activity was measured. The initial enzyme activity of the enzyme solution at the optimal pH was 100%. Figure 3 B shows that after incubation at pH 6.0-8.0 for 24 hours, the degradation enzyme PufH retained 60% of its initial activity.

[0038] In order 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 as 100%, and the relative enzyme activity at each temperature was calculated. Figure 3 C shows that the degradation enzyme PufH is active at 20-90℃, and has the highest activity at 30℃. The degradation enzyme PufH was placed in a water bath at 30℃, 37℃, and 50℃, and the enzyme activity was measured at gradient time. 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. Figure 3 D shows that the degradation enzyme PufH has good stability at 30℃, and there is almost no loss of enzyme activity after storage for 24h; after treatment at 50℃ for 0.5h, the enzyme activity is reduced to less than 40% of the initial enzyme activity.

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

[0040] Since the structure of commercial polyurethane sponge is unclear, the present invention synthesizes PBA-PU with a clear structure to study the way PufH decomposes polyurethane sponge. 200μg of degradation enzyme PufH and Tris-HCl buffer (pH7.0) containing 6mg PBA-PU were incubated at 30℃ and 180rpm for 12h, centrifuged at 12000rpm for 10min, the supernatant was acidified to pH 2.0 and extracted with twice the volume of ethyl acetate. The upper solution was air-dried and resuspended in 500μL methanol. Finally, it was filtered through a 0.22μm organic phase filter membrane and detected by HPLC and LC-MS. Figure 4 A shows that, after HPLC detection, some product absorption peaks appeared near the characteristic peak of MDA, which may be the derivatives of MDA. Further LC / MS mass spectrometry identification. Figure 4 As shown in Figure B, a total of three PBA-PU hydrolysis products were detected in the negative ion mode. The ion peak corresponding to metabolite C was 145.0508 (MH) - , it was identified as adipic acid, a product of the cleavage of the soft segment of PBA-PU; the ion peaks corresponding to metabolites B and A were 429.2030 (MH) - and 757.3542 (MH) - , which were identified as MDA derivatives in which the PBA-PU hard segment was connected to a diol or a dibasic acid. In view of the above results, it can be inferred that the mode of hydrolysis of PBA-PU by the degradation enzyme PufH is mainly through catalyzing the depolymerization of the ester bonds 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 bonds in metabolite C to generate AA (metabolite A) and MDA derivatives (metabolite B) ( Figure 4 C).

[0041] Example 4: Decomposition of commercial polyester sponge by degradative 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 Dagong Co., Ltd.) (25 mg) in 4 mL of 50 mM Tris-HCl buffer (pH 7.0) and reacted at 30 °C and 180 rpm for 48 h. The pH change was monitored and adjusted during the reaction. Figure 5 A and 5B show that the degradation enzyme PufH can degrade 82.4% of the commercial polyurethane sponge in 48 hours. The product of 48 hours of enzymatic hydrolysis was detected and identified by HPLC and LC-MS (the method is the same as that in Example 3). Figure 6 As shown in A, the HPLC results showed that some product absorption peaks appeared near the characteristic peak of TDA, which was speculated to be a derivative of TDA. Figure 6 B shows that the ion peak corresponding to metabolite A detected in negative ion mode is 145.0508 (MH) - , it was identified as adipic acid; in the positive ion mode, the ion peak corresponding to metabolite B was detected at 683.3391 (M+H) + , which was identified as a TDA derivative. The results show that the polyurethane sponge 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 48h.

[0043] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A degradative enzyme PufH for efficiently decomposing polyurethane sponge, the sequence of which is shown in SEQ ID NO.

1.

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

3. The gene according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

2.

4. A recombinant expression vector containing the gene according to claim 2 or 3.

5. Use of the degradative enzyme PufH according to claim 1 in degrading polyurethane sponge.

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

50.

7. The use according to claim 5, characterized in that: The degradation temperature is 30°C; the degradation pH is 7.0.

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