Mining and application of novel pvc-degrading enzymes

Abstract

The application discloses mining and application of a new PVC degradation enzyme and belongs to the field of environmental pollutant plastic degradation. The application uses the macroprotein technology, screens a new PVC plastic degradation enzyme based on different responses of a PVC degradation bacterial community to artificial polymers and natural polymers, and further characterizes catalytic activity of the enzyme. The application provides a new and effective strategy for screening the PVC degradation enzyme from a microbial community in an alpine meadow soil in the Qinghai-Tibet Plateau, so as to expand a resource library of the plastic degradation enzyme and be used for resource recovery and biological remediation.

Description

Technical Field

[0001] This invention relates to the discovery and application of novel PVC-degrading enzymes, belonging to the field of environmental pollutant plastic degradation. Background Technology

[0002] The use of plastics in daily life, agricultural production, and the chemical industry continues to rise. There is an urgent need to find efficient and environmentally friendly solutions to address the problem of plastic pollution. Polyvinyl chloride (PVC) accounts for 10% of global plastic production, second only to polyethylene (PE, 29.7%) and polypropylene (PP, 19.3%). Its high molecular weight, stable covalent bond structure, and hydrophobic properties make it difficult to degrade in the natural environment. Furthermore, the cutting process of PVC pipes can release millions of microplastic particles and additives such as phthalates and bisphenol A, posing a potential threat to ecosystems and human health. Compared with traditional treatment methods such as incineration and landfill, microbial degradation of plastic waste is considered an environmentally friendly alternative.

[0003] Currently, research has confirmed that over 430 microorganisms from more than 20 bacterial genera possess the ability to degrade various plastics, including PE, polystyrene (PS), polyethylene terephthalate (PET), and polyurethane (PUR). These include species such as *Gordonia*, *Novosphingobium*, and *Bacillus thuringiensis*. However, only a few microorganisms, such as *Klebsiella pneumoniae* sp. EMBL-1 and the degrading bacterial community EF1, can use PVC as their sole carbon source for growth and metabolism. While existing research confirms that some microorganisms can degrade PVC, the elucidation of their degradation pathways still focuses on multi-omics predictions, and the discovery and mechanism of action of PVC-degrading enzyme genes have not been thoroughly explored. Therefore, developing novel PVC-degrading enzymes has become a crucial issue urgently needing to be addressed in the field of plastic degradation, and is of great significance for enriching the existing gene pool related to plastic degradation and promoting the development of plastic biodegradation technology. Summary of the Invention

[0004] This invention provides a novel PVC-degrading enzyme, SerB.

[0005] The present invention also provides a gene encoding the PVC degrading enzyme SerB.

[0006] The present invention also provides recombinant microorganisms expressing the PVC-degrading enzyme SerB.

[0007] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli.

[0008] In one embodiment, the recombinant microorganism uses Escherichia coli BL21(DE3) as the host and pET-28a(+) as the expression vector.

[0009] This invention also provides a method for screening PVC plastic degrading enzymes from differentially expressed proteins of synthetic and natural polymers in PVC-degrading microbial communities (DCs), comprising:

[0010] (1) Metaproteome sequencing of PVC-DC and lignin-DC; (2) Construction of a gene library related to plastic degradation; (3) Screening of candidate enzymes by gene biology information and phylogenetic tree; (4) Construction and purification of target protein; (5) Determination of protein function by detection of epimorphology and degradation intermediates.

[0011] In one embodiment, the method involves culturing recombinant Escherichia coli expressing PVC-degrading enzymes in LB medium until OD... 600 If the value is ≥0.6, add IPTG to a final concentration of 0.4mM and incubate overnight at 18°C.

[0012] In one embodiment, the culture is carried out at 37°C and 180-200 rpm.

[0013] This invention also provides the application of the PVC-degrading enzymes obtained through screening in plastic degradation.

[0014] In one embodiment, the phosphoserine phosphatase SerB shown in SEQ ID No. 2 is added to a system containing PVC, and the reaction is carried out for at least 7 days.

[0015] In one embodiment, the system contains at least 4 mg·mL -1 PVC and at least 50 μg·mL -1 SerB.

[0016] In one embodiment, the system further contains 20–50 mmol / L HEPES and 5–10 mmol / L MgSO4.

[0017] In one embodiment, the reaction pH is 7–8.

[0018] In one implementation, the reaction pH is 7.5.

[0019] In one embodiment, the reaction conditions are 25–45°C.

[0020] In one embodiment, the PVC has a weight-average molecular weight (Mw) of 50–70 kDa, a number-average molecular weight (Mn) of 100–120 kDa, and a Z-average molecular weight (Mz) of 150–170 kDa.

[0021] In one embodiment, the PVC includes PVC film or PVC granules.

[0022] In one embodiment, PVC is administered at a concentration of 5 mg / mL. -1 The final concentration of HEPES, MgSO4, and MgSO4 were added to the reaction system and reacted at 25°C for at least 7 days. The reaction system contained a final concentration of 50 mmol / L HEPES, 5 mmol / L MgSO4, and 50 μg·mL⁻¹. -1 SerB.

[0023] The present invention also provides a method for increasing the surface roughness of a PVC film by adding the PVC film to a solution containing phosphoserine phosphatase SerB as shown in SEQ ID No. 2 and reacting for at least 7 days.

[0024] Beneficial effects:

[0025] (1) This invention provides a method for screening PVC degrading enzymes, which screens enzymes with PVC plastic degradation ability from differentially expressed proteins of lignocellulose and PVC by PVC degrading bacteria.

[0026] (2) This invention provides the application of PVC degrading enzyme in plastic waste treatment, which can make obvious pores appear on the PVC surface after continuous reaction at 25°C for 7 days. Attached Figure Description

[0027] Figure 1 This invention demonstrates that the PVC-degrading microbial communities (DCs) exhibit different response characteristics to synthetic and natural polymers. A shows the Venn diagram analysis of PVC-DCs and lignin-DCs; B compares the ACE indices of PVC-DCs and lignin-DCs; C shows the Shannon diversity index of PVC-DCs and lignin-DCs; D shows the differences in community structure between PVC-DCs and lignin-DCs based on nonmetric multidimensional scaling (NMDS); E shows the community composition characteristics of PVC-DCs and lignin-DCs at the phylum level; F shows the species abundance heatmap of PVC-DCs and lignin-DCs at the genus level; and G shows the microbial co-occurrence network analysis of PVC-DCs and lignin-DCs.

[0028] Figure 2This is a screening process for PVC plastic degrading enzymes according to one embodiment of the present invention; wherein A is based on the results of Bray-Curtis principal component analysis (PCA); B is the statistical analysis of the number of differentially expressed proteins (DEPs) between the PVC group and the lignin group; CD is the cluster analysis of protein expression patterns between the PVC and lignin groups; E is the relative abundance and heatmap of potential PVC degrading proteins in the macroproteome; F is the comparison of the relative abundance of carbohydrate-active enzymes (CAZymes) between the PVC group and the lignin group; and G is the protein interaction network analysis diagram in the macroproteome detection.

[0029] Figure 3 This is a potential degradation pathway for PVC plastic proposed in this invention.

[0030] Figure 4 This invention serves as a functional verification of the PVC plastic degrading enzyme. A shows a photograph of SerB and PVC film co-incubated in a test tube; B shows the detection of intermediate degradation products of the PVC film after 7 days of SerB treatment; C shows a scanning electron microscope (SEM) image of the SerB-treated PVC film; D shows an atomic force microscope (AFM) image of the SerB-treated PVC film; and E shows an analysis of the roughness changes in the SerB-treated PVC film.

[0031] Figure 5 This invention verifies the degradation efficiency of the PVC plastic degrading enzyme; where A is a photograph of SerB and PVC particles co-incubated in a test tube; B is an analysis of the change in molecular weight of PVC plastic after SerB treatment; and C is an ion chromatography detection of chloride ion release from PVC plastic. Detailed Implementation

[0032] The present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Any technical solution that can be conceived by those skilled in the art based on the present invention and in combination with common knowledge in the art shall fall within the scope of protection of the present invention.

[0033] The enzymes used in the following embodiments of the present invention are from the following sources:

[0034] PVC plastic degrading enzyme: Metaproteome sequencing of PVC-degrading bacteria was performed, and the protein was screened from genomes related to plastic degradation in the KEGG gene annotation. The protein was named SerB, and its amino acid sequence is shown in SEQ ID No. 2.

[0035] The PVC-degrading microbial community (DC) involved in the following examples was obtained by enrichment and culture from soil samples collected in the laboratory from the Haibei Research Station on the Qinghai-Tibet Plateau (37°36′38.53″N, 101°18′49.31″E).

[0036] Example 1: PVC-degrading microbial communities (DCs) exhibit different reactivity characteristics to synthetic and natural polymers.

[0037] The PVC used in the following examples has a number-average molecular weight (Mn) of 69 kDa, a weight-average molecular weight (Mw) of 128 kDa, and a Z-average molecular weight (Mz) of 196 kDa, and was purchased from Formosa Plastics Corporation, Taiwan.

[0038] Previous studies have confirmed that the gut microbiome exhibits similar responses to synthetic polymers (PS) and natural polymers (corn stalks). To further evaluate the microbial response strategies to PVC and lignin biodegradation, PVC-degrading bacteria (DCs) were inoculated into liquid carbon-free basal medium (LCBFM, 1% v / v) containing lignin or PVC, labeled as PVC-DC and lignin-DC, respectively. After one month of culture, the cells were collected by centrifugation and used... Total genomic DNA was extracted from the soil using a soil DNA kit, with in-situ soil at an altitude of 3600 meters serving as a control. Paired-end sequencing of the V3-V4 region of the bacterial 16S rRNA gene was performed using the Illumina PE300 / PE250 platform. The raw FASTQ files were quality filtered, and sequences were assembled using FLASH. The optimized sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity level using UPARSE, with the most abundant sequence in each OTU used as the representative sequence. Alpha diversity indices such as the ACE index and Shannon index were calculated using Mothur. Nonmetric multidimensional scaling (NMDS) based on the Bray-Curtis distance matrix was used in Vegan software to assess microbial community similarity. Visualization of the microbial community structure was performed using Python 2, and functional prediction was performed using PICRUSt2. The criteria for constructing the microbial co-occurrence network were set as follows: Spearman correlation coefficient absolute value greater than 0.6 and significance level P < 0.01.

[0039] The results show that ( Figure 1Compared to the control group (CK), both PVC-DC and lignin-DC showed a significant decreasing trend in ACE and Shannon diversity indices. Nonmetric multidimensional scaling (NMDS) analysis revealed a significant difference in bacterial community structure between PVC-DC and CK (P<0.05), and a clear separation of bacterial community structure between PVC-DC and lignin-DC. Species composition analysis further revealed that the relative abundance of Proteobacteria in PVC-DC and lignin-DC increased by 3.3-fold and 3.8-fold, respectively, compared to CK, accounting for 86.1% in PVC-DC and a high 99.1% in lignin-DC. At the genus level, the dominant genera in PVC-DC included Burkholderia (80.0%), Rhodococcus (13.9%), and Dyella (4.8%), while in lignin-DC, the proportions of these genera changed to 81.9%, 0.5%, and 14.6%, respectively. The relative abundance of Burkholderia did not change significantly between PVC-DC and lignin-DC; however, the relative abundance of Rhodococcus in PVC-DC increased by 96.4% compared to lignin-DC; and the relative abundance of Dyella in PVC-DC decreased by a factor of 3. Furthermore, microbial co-occurrence network analysis showed that the number of network nodes in PVC-DC and lignin-DC (both 56) was significantly reduced compared to the control (422). Figure 1 ).

[0040] Example 2: Screening of PVC-degrading enzymes based on differential protein responses of PVC-degrading microbial communities (DCs) to synthetic and natural polymers.

[0041] PVC-DC and lignin-DC bacterial samples were lysed with protein lysis buffer (8 mol / L urea, 1% SDS), processed using a high-throughput tissue homogenizer, and the supernatant was collected by centrifugation for subsequent analysis. After trypsin digestion, the enzymatically digested peptides were reconstituted with 0.1% trifluoroacetic acid (TFA), desalted using a hydrophilic-lipophilic balance (HLB) column, and dried using a vacuum concentrator. Peptide concentrations were quantified using the Peptide Quantification Kit, and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a Vanquish Neo liquid chromatography system coupled with an Orbitrap Astral mass spectrometer. For quantitative analysis, six characteristic peptides were selected for each protein, and three daughter ions were screened for each peptide. The analytical parameters were set as follows: protein false detection rate (FDR) ≤ 0.01, peptide confidence ≥ 99%, and extracted ion current (XIC) mass deviation ≤ 75 ppm. Bioinformatics analysis of the proteomics data was performed on the MajorBio cloud platform (https: / / cloud.majorbio.com). The screening criteria for differentially expressed proteins (DEPs) were: fold change (FC) > 2 or < 0.5 and p-value < 0.05. Proteins were functionally annotated using the Kyoto Encyclopedia of Genetics and Genomes (KEGG) database, and their sequences were compared with annotated full-length sequences in the BlastP and CAZy databases. A protein-protein interaction network was constructed using STRING, and network visualization was achieved using the NetworkX library in Python. Key nodes were selected based on topological characteristics of the protein-protein interaction network (PPI), including node degree, betweenness centrality, proximity centrality, and clustering coefficient.

[0042] Principal component analysis (PCA) results showed that the proteins of PVC-DC and lignin-DC formed independent clusters. Figure 2 A) In PVC-DC, the expression levels of 262 proteins were significantly upregulated (log2[FC]>0.5), while the expression levels of 105 proteins were significantly downregulated (log2[FC]<-0.5). Figure 2 B). Annotations based on the Carbohydrate Active Enzyme Database (CAZy) show ( Figure 2 F), the glycosidic hydrolases (GHs) family accounted for the largest proportion of annotated enzymes, reaching 34.2%, followed by carbohydrate esterases (CEs, 21.5%), glycosyltransferases (GTs, 15.4%), and cofactor enzymes (AAs, 13.4%). Among these, the GH3 family had the highest relative abundance, while the relative abundance of the AA2 family increased by 36.8% in PVC-DC compared to lignin-DC, while the CE3 family showed a significant decreasing trend. A protein-protein interaction network was constructed, containing 45 nodes and 400 edges, revealing the synergistic mechanism of the multi-enzyme system during PVC degradation. Figure 2G). Through annotation analysis of the KEGG database, this invention identified 48 potential PVC-degrading enzymes from metaproteomic data, including dehalogenases, dehydrogenases, peroxidases, esterases, oxidoreductases, and oxygenases. Figure 2 E). Therefore, the following degradation pathway is proposed: the dechlorination reaction mediated by the degrading enzyme SerB initiates the depolymerization of PVC, while the catalase-peroxidase (KatG) catalyzes a synergistic oxidation reaction, and the resulting low molecular weight polymer is further cleaved by monooxygenase (MO), dioxygenase (DOs), esterase (EST), and dehydrogenase (ADH / ALDH). Figure 3 Given that SerB expression was significantly upregulated 4.3-fold in the PVC-treated group and exhibited significant evolutionary differences from the halate dehalogenase (HAD) family, it was identified as a key candidate PVC-degrading enzyme. This enzyme shares high homology with the phosphoserine phosphatase (PSP) family, suggesting a potential novel catalytic mechanism.

[0043] Example 3: Activity Verification of Candidate Enzymes

[0044] 1. Expression and purification of candidate enzymes

[0045] The SerB degradation enzyme encoding gene (shown in SEQ ID No. 1) was cloned into the NdeI and NotI restriction sites of the pET-28a plasmid. The resulting recombinant plasmid was transformed into E. coli BL21(DE3) competent cells for protein expression induction. The specific steps are as follows: The engineered bacteria BL21-SerB containing the SerB recombinant plasmid was cultured in LB liquid medium (containing 50 mg / mL) at 37℃. -1 Cultured in kanamycin until OD 600 The concentration reached 0.6, and expression was subsequently induced for 18 h at 18°C ​​with 0.4 mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG). The collected cells were then lysed with lysis buffer (50 mmol / L Tris-HCl pH 7.4, 300 mmol / L NaCl, 1 mg / mL). -1 After resuspending the lysozyme, the mixture was sonicated for 30 min, and the supernatant was collected by centrifugation. The supernatant was loaded into a 5 mL HisTrap Ni-NTA affinity chromatography column (GE Healthcare) using an AKTA Pure chromatography system. Non-specifically bound proteins were eluted with buffer A (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 30 mmol / L imidazole), and the target protein was eluted with buffer B (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 150 mmol / L imidazole). The purified product was concentrated and desalted using a Centrifuge Biomax-5 ultrafiltration tube and stored at -80°C for later use.

[0046] 2. Verification of the PVC plastic degradation ability of candidate enzymes

[0047] The PVC used in the following examples has a number-average molecular weight (Mn) of 59 kDa, a weight-average molecular weight (Mw) of 106 kDa, and a Z-average molecular weight (Mz) of 154 kDa, and was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0048] PVC degradation enzyme activity test:

[0049] To evaluate the depolymerization activity of SerB on PVC, PVC films and particles were respectively injected at 5 mg·mL⁻¹. -1 The final concentrations were added to 3 mL of the reaction system (final concentrations of each substance were: 50 mmol / L HEPES, pH 7.5; 5 mmol / L MgSO4; 50 μg·mL). - 1 SerB was added and the mixture was incubated in a water bath at 25°C for 7 days. The control group did not contain SerB, and all other reaction conditions were the same. Figure 4 ).

[0050] Identification of PVC degradation products: Gas chromatography-mass spectrometry (GC-MS) was used to detect degradation intermediates. 2 mL of the supernatant was centrifuged at 12,000 g for 10 min and then lyophilized. The lyophilized product was extracted with HPLC-grade dichloromethane and filtered through a 0.22 μm nylon membrane. Metabolite separation was performed using an HP-5MS capillary column (0.25 mm × 0.25 μm), and mass spectrometry detection was performed using an electron impact ionization (EI) source with a scan range of 33-750 m / z. The column temperature program was set as follows: initial temperature 50 °C, held for 4 min, then increased at 20 °C / min. -1 The temperature was increased to 300℃ and maintained for 15 minutes. Using MS Workstation software, the sample spectra were compared with the NIST spectral library (match rate >85%), thereby achieving qualitative analysis of potential degradation products. Figure 4 ).

[0051] Characterization and analysis of PVC films after enzyme treatment: The morphology of SerB-treated and untreated PVC films was analyzed. PVC films were rinsed three times with 2% SDS and 75% ethanol to thoroughly remove extracellular secretions and biofilms, and then dried under natural conditions. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the SerB-treated and untreated PVC films, and roughness analysis was performed on the PVC films. Figure 4 ).

[0052] Degradation efficiency analysis of PVC particles by enzyme SerB: The treated PVC particles were washed sequentially with 30% hydrogen peroxide and ultrapure water, dissolved in tetrahydrofuran, and filtered through a 0.22 μm filter membrane. Gel permeation chromatography (GPC) was used to determine the changes in Mw, Mn, and Mz of the PVC after enzyme treatment. The solution after co-incubation of SerB and PVC particles was collected and centrifuged at 7000g for 4 minutes at room temperature. The supernatant obtained after centrifugation was then filtered through a 0.22 μm filter membrane, and the chloride ion concentration in the filtered culture medium was determined by ion chromatography (IC). Figure 5 ).

[0053] The results showed that the PVC film treated with SerB sank to the bottom of the test tube after 7 days, indicating a significant change in hydrophobicity. Figure 4 A), and the surface shows micro-pits, while the untreated film remains smooth and intact. Figure 4 C). Gas chromatography-mass spectrometry (GC-MS) identified several degradation intermediates of PVC film, including 2,6,10,15-tetramethylheptadecane (C). 21 H 44 ), hexadecyl trichloroacetate (C 18 H 33 Cl3O2), 3-(octadecyloxy)propyl stearate (C 39 H 78 O3), eicosyl oleate (C 38 H 74 O2) and isopropyl 11,12-methylene octadecanoate (C 20 H 38 O2) etc. Figure 4 B). The addition or emergence of new intermediates indicates that PVC undergoes depolymerization under SerB treatment. Furthermore, the roughness changes of the SerB-treated PVC plastic were observed at the nanoscale using atomic force microscopy. Figure 4 As shown in Figure D, the fiber surface of the PVC film treated with SerB becomes rough, exhibiting depressions and protrusions of different particle sizes. Surface roughness analysis results show that the arithmetic mean roughness (Ra) of the control group PVC film is approximately 26.5 nm, while the Ra of the PVC film treated with SerB increases to approximately 52.5 nm, an increase of nearly 2 times compared to the control group. Figure 4 E). Ion chromatography results showed that, compared with the control group (CK), the chloride ion release concentration in the SerB-treated group increased from 0.5 mg / L to 9.7 mg / L (E). Figure 5 C); meanwhile, the number-average molecular weight (Mn), weight-average molecular weight (Mw), and Z-average molecular weight (Mz) of PVC powder increased by 6.7%, 6.3%, and 4.4%, respectively. Figure 5 B) According to the literature "Identification of plastic-degrading bacteria in the human gut," this result further indicates that SerB can induce a limited degree of depolymerization in PVC. Previous studies have shown that the biodegradation process of PVC involves at least three key reactions: (a) depolymerization or breakage of polymer chains; (b) formation of oxidative intermediates accompanied by chloride ion release; and (c) final mineralization of intermediates into CO2 and H2O. Based on the observation of PVC films and the GC-MS identification of degradation intermediates in this study, it is inferred that the release of chloride ions likely occurs with the formation of intermediates after the breakage of PVC polymer chains, further demonstrating that the protein SerB has the function of degrading PVC plastics.

[0054] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of phosphoserine phosphatase (SerB) in the degradation of plastics, characterized in that, The plastic is PVC; the amino acid sequence of the phosphoserine phosphatase SerB is shown in SEQ ID No.

2.

2. The application as described in claim 1, characterized in that, Add the phosphoserine phosphatase SerB, as shown in SEQ ID No. 2, to a system containing PVC and react for at least 7 days.

3. The application as described in claim 2, characterized in that, The system contains at least 4 mg·mL -1 PVC and at least 50 μg·mL -1 SerB.

4. The application as described in claim 3, characterized in that, The system also contains 20-50 mmol / L HEPES and 5-10 mmol / L MgSO4.

5. The application as described in claim 4, characterized in that, The pH of the system is 7-8.

6. The application as described in claim 5, characterized in that, The reaction conditions are 25~45℃.

7. The application as described in claim 6, characterized in that, The PVC has a number-average molecular weight Mn of 50-70 kDa, a weight-average molecular weight Mw of 100-120 kDa, and a Z-average molecular weight Mz of 150-170 kDa.

8. The application as described in claim 7, characterized in that, The PVC includes PVC film or PVC granules.

9. The application as described in any one of claims 1 to 8, characterized in that, PVC was administered at 5 mg·mL -1 The final concentration of HEPES, MgSO4, and 50 μg·mL⁻¹ were added to the reaction system, and the reaction was carried out at 25°C for at least 7 days. The reaction system contained a final concentration of 50 mmol / L HEPES, 5 mmol / L MgSO4, and 50 μg·mL⁻¹. -1 SerB.

10. A method for increasing the surface roughness of a PVC film, characterized in that, The PVC film was added to a solution containing phosphoserine phosphatase SerB as shown in SEQ ID No. 2, and the reaction was carried out for at least 7 days.

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