PET-degrading strains and PET-degrading enzymes derived from deep sea and their uses

Abstract

The present invention belongs to the field of microbiology and enzyme engineering technology, and relates to a PET-degrading strain from deep sea, a PET-degrading enzyme and its use. The PET-degrading strain from deep sea is a halophilic Pseudomonas Halopseudomonas sp .EEDS01, deposited in China Center for Type Culture Collection on November 28, 2024, with the deposit number CCTCC NO: M20242659. The present invention also provides PET degrading enzymes PnPETase1, PnPETase2, PnPETase3, and PnPETas4 derived from the strain, and their amino acid sequences are shown in the sequence table SEQ ID NO.2‑5, respectively. The PET degrading strains and four PET degrading enzymes provided by the present invention have strong PET hydrolysis ability under high salt concentration, which are of great significance in the fields of environmental protection and catalysis.

Description

Technical Field

[0001] The invention belongs to the technical field of microbiology and enzyme engineering, and relates to a PET-degrading strain from deep sea, a PET-degrading enzyme and uses thereof. Background Art

[0002] Polyethylene terephthalate (PET) is a plastic widely used in textiles, packaging, and beverage bottles. Due to its high strength, excellent thermal stability, and chemical inertness, it has become one of the most commonly used polymers in the world. However, the large-scale production and improper handling of PET have led to serious plastic pollution problems, posing a huge threat to the ecological environment and human health. Although traditional physical and chemical methods can be used to treat PET waste, they are costly and may cause secondary pollution. Therefore, green degradation pathways using microorganisms and enzymes have become a research hotspot for solving the PET pollution problem.

[0003] In recent years, studies have shown that certain microorganisms and related enzymes can catalyze the decomposition of PET into small molecular monomers (such as terephthalic acid and ethylene glycol), providing the possibility for efficient degradation and resource utilization of plastic waste. However, the types and efficiency of existing PET degrading enzymes are still limited, especially enzymes that can efficiently degrade PET under extreme environments are even more scarce. The deep-sea environment, with its unique extreme conditions such as high pressure and low temperature, contains rich microbial resources, which may provide important clues for the discovery of new and efficient PET degrading enzymes. The present invention mines enzymes with PET degradation ability from bacteria from deep sea sources, providing new methods and technical support for the biological treatment and recycling of plastic pollution. In recent years, bioenzymatic degradation based on PET hydrolases has made great progress, such as IsPETase from PET degrading bacteria Ideonella sakaiensis 201-F6, which demonstrates the great potential of biodegradation technology and provides a scientific basis for the development of more efficient and economical solutions in the future. As well as the cutinase LCC from the metagenome of leaf compost, the engineered LCC was used to hydrolyze PET, recover monomers and resynthesize PET, realizing the leap from theory to practice of PET bio-circular economy.

[0004] However, there are relatively few studies on PET-degrading bacteria. Although Ideonella sakaiensis 201-F6 isolated from a plastic recycling plant has the ability to degrade PET, and IsPETase was discovered from it, its thermal instability and low activity limit its application in the industrial treatment of PET waste.

[0005] Microbial communities in deep-sea sediments usually evolve enzyme tools that are highly efficient in degrading difficult-to-degrade organic matter because they are in an environment with extremely scarce materials and energy. However, research on PET-degrading bacteria in deep-sea environments is still in a blank stage. Exploring deep-sea microbial resources and discovering new PET-degrading bacteria and their enzymes is not only expected to break through the existing technical bottlenecks, but also provides new directions and possibilities for the biological treatment of plastic pollution. Summary of the invention

[0006] The purpose of the present invention is to solve the problem of insufficient research on deep-sea PET-degrading bacteria, and to provide a PET-degrading strain isolated and identified from 5890-meter deep-sea sediments in the western Pacific Ocean. Halopseudomonas sp EEDS01, which can degrade 15.3% of PET plastics within 20 days. It also provides four PET-degrading enzymes from the strain: HpPETase1, HpPETase2, HpPETase3, and HpPETase4, providing a new application tool for PET degradation.

[0007] The technical solution adopted by the present invention to solve the technical problem is: the PET degradation strain from deep sea is halophilic Pseudomonas Halopseudomonas sp. EEDS01 was deposited in the China Center for Type Culture Collection on November 28, 2024, and the deposit number is CCTCC NO: M20242659.

[0008] The present invention also provides a PET degrading enzyme derived from the strain, and the amino acid sequence thereof is shown in any one of SEQ ID NO. 2-5 in the sequence table.

[0009] The present invention also provides a gene encoding the PET degrading enzyme, and the nucleotide sequence thereof is shown in any one of SEQ ID NO.6-9 in the sequence table.

[0010] Preferably, the present invention also provides a vector comprising the gene encoding the PET degrading enzyme.

[0011] Preferably, the vector is a eukaryotic vector, a prokaryotic vector, a plasmid vector or a viral vector.

[0012] Preferably, the present invention also provides a host cell comprising the vector.

[0013] Preferably, the host cell is a bacterium.

[0014] Preferably, the present invention further provides the use of the strain, the PET degrading enzyme, the gene, the vector, and the host cell in degrading PET under high-salt conditions.

[0015] Preferably, the present invention further provides a PET degrading agent, which contains at least one of the strain, the PET degrading enzyme, the vector, and the host cell.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention screened and obtained a strain with PET degradation ability from deep-sea sediments. Halopseudomonas sp EEDS01, for the first time, fills the gap in the study of PET-degrading bacteria in deep-sea environments. The new enzymes contained in these bacteria have unique structural and functional characteristics, providing new resources for plastic degradation technology. The PET-degrading enzymes HpPETase1, HpPETase2, HpPETase3, and HpPETase4 provided by the present invention can show efficient PET hydrolysis ability under various high salt concentration conditions. Compared with existing PET-degrading enzymes such as IsPETase, the enzymes discovered by the present invention have higher thermal stability and catalytic activity, and can maintain good degradation performance under extreme conditions, which makes them more suitable for industrial large-scale PET waste treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 For strains Halopseudomonas sp . Electron microscope photo of EEDS01;

[0018] Figure 2 Schematic diagram of the general process of PET hydrolysis;

[0019] Figure 3 is the plasmid map of pDSP01 expressing HpPETase1;

[0020] Figure 4 is the plasmid map of pDSP02 expressing HpPETase2;

[0021] Figure 5 is the plasmid map of pDSP03 expressing HpPETase3;

[0022] Figure 6 is the plasmid map of pDSP04 expressing HpPETase4;

[0023] Figure 7 SDS-PAGE analysis of purified HpPETase1, HpPETase2, HpPETase3, and HpPETase4 proteins;

[0024] Figure 8 Visual comparison of HpPETase3 and IsPETase degradation using nano-PET as substrate;

[0025] Fig. 9 For strains Halopseudomonas sp EEDS01 degraded PET in 2216E medium containing 1 g / L nano-PET to produce TPA and MHET hydrolysis products;

[0026] Fig.10 The concentration of products produced by PET hydrolysis catalyzed by HpPETase1 at different salt concentrations for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0027] Fig.11 The concentration of products produced by PET hydrolysis catalyzed by HpPETase2 at different salt concentrations for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0028] Fig.12 The concentration of products produced by PET hydrolysis catalyzed by HpPETase3 at different salt concentrations for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0029] Fig.13 The concentration of products produced by PET hydrolysis catalyzed by HpPETase4 at different salt concentrations for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0030] Fig.14 The concentration of products produced by PET hydrolysis catalyzed by HpPETase1 at different temperatures for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0031] Fig.15 The concentration of products produced by PET hydrolysis catalyzed by HpPETase2 at different temperatures for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0032] Fig.16 The concentration of products of PET hydrolysis catalyzed by HpPETase3 at different temperatures for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0033] Fig.17The concentration of products produced by PET hydrolysis catalyzed by HpPETase4 at different temperatures for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0034] Fig.18 The product concentration of PET hydrolysis catalyzed by HpPETase1 at different pH for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0035] Fig.19 The concentration of products produced by PET hydrolysis catalyzed by HpPETase2 at different pH values ​​for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0036] Fig. 20 The concentration of products of PET hydrolysis catalyzed by HpPETase3 at different pH values ​​for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;

[0037] Fig.21 It is the product concentration of PET hydrolysis catalyzed by HpPETase4 at different pH for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity. DETAILED DESCRIPTION

[0038] In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific embodiments. However, the present invention is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0039] The present invention isolated a PET-degrading strain from deep-sea sediments and discovered four PET-degrading enzymes from it. The strain was named Pseudomonas halophilus Halopseudomonas sp .EEDS01, deposited in China Center for Type Culture Collection on November 28, 2024, with the deposit number CCTCC NO: M20242659. The deposit address is: Wuhan University, Hubei Province.

[0040] The four PET degrading enzymes obtained from the strain were named PnPETase1, PnPETase2, PnPETase3, and PnPETase4. The functional characteristics and catalytic activity of the enzymes were tested and verified by experiments as follows.

[0041] 1. Preparation of Degradation Substrate

[0042] GfPET (ES301445, Goodfellow, Huntingdon, England) was used to verify the function of PnPETase1-4, and a GfPET membrane with a diameter of 6 mm was prepared by a hole puncher as a substrate for the reaction.

[0043] Preparation of Nano-PET: Preparation of PET nanoparticles by dissolution-precipitation method. Nano-PET was prepared by self-made nano-PET to compare the hydrolysis activity of PET. The method for preparing nano-PET in this example is as follows: 1g GfPET was dissolved in 60ml 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), the PET solution was added to 240ml pure water at a rate of 6ml / min, rotary evaporated at 40℃ for 30min, freeze-dried for 4 days, and nano-PET was collected.

[0044] 2. Strains Halopseudomonas sp EEDS01 16S rRNA gene sequence analysis

[0045] strains Halopseudomonas sp EEDS01 is a microorganism that can efficiently degrade PET plastics and has unique enzymatic properties. The colony morphology is yellow, round and convex, and short rod-shaped under an electron microscope. The strains are connected by hyphae and can secrete vesicles outside the cell. It can secrete specific enzymes (such as PETase) to decompose polyester into small molecular monomers (such as terephthalic acid and ethylene glycol) through hydrolysis. The strain has good activity in a neutral to weakly alkaline environment and is suitable for growth at 10-40°C (optimal growth at 27°C). In order to determine the strain Halopseudomonas sp . The novelty of EEDS01, the present invention searches for its 16S rRNA gene in the EzBioCloud 16S-based ID database through the online EzBioCloud search tool (https: / / www.ezbiocloud.net) and arranges it by sequence similarity. As shown in Table 1, the 16S rRNA genes in the EZBioCloud 16S-based ID database that are similar to the strain Halopseudomonas sp EEDS01 16S rRNA gene sequence of the 30 strains with the highest consistency, among which Halopseudomonas sp EEDS01 and the most evolutionarily similar strain ( Pseudomonas nanhaiensis.SCS2-3) is 98.96%, indicating that the strain (SEQ ID NO.1 in the sequence list) has not been publicly disclosed and is novel.

[0046] Table 1. EZBioCloud 16S-based ID and strains Halopseudomonas sp EEDS01 16S rRNA gene sequences of the 30 strains with the highest consistency

[0047] name strains Accession Number Similarity(%) SCS 2-3 GCA_020025155.1_1 98.9648 NRRL B-51270 LT629736 98.89579 KBL-4-9 KC609749 97.51553 JCM 14963 LT629763 97.44651 Y22 MG589919 97.23948 CP033116_s Kongs-67 CP033116 97.17046 CL-AP6 AROI01000066 96.89441 XCD-X85 KC762324 96.8254 S-6-2 CP020100 96.8254 16W4-4-3 MN423266 96.71429 MT5 MF962536 96.58363 CCUG 46540 MUBC01000081 96.54934 CRS1 LN881559 96.3951 M5 KY988340 96.34231 C10-2 DQ088664 96.20428 R-20821 AM114527 96.13527 A31 / 70 QLAE01000067 96.13527 SSM26 KU716040 96.08819 COW40 GCA_019139895.1_1 96.06625 COR54 GCA_019139835.1_1 96.06625 RW9S1A JABWRH010000080 96.06625 RYU5 MH517510 95.99724 RW4S2 JABWRP010000055 95.99724 RW8P3 JABWRC010000038 95.99724 BCRC 17751 EU103629 95.98893 CIP 105259 AF072688 95.92823 DSM 17875 LT629785 95.92823 BML3 MK680061 95.92823 JV551A3 OPYN01000008 95.92823 CGMCC 1.9095 jgi.1058068 95.92823 .

[0048] 3. HpPETase1 gene and protein sequence analysis

[0049] In order to determine the novelty of the amino acid sequence of HpPETase1, the present invention searched for its homologous proteins in the NCBI non-redundant database through online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and arranged them according to sequence consistency. As shown in Table 2, the 30 sequences with the highest consistency with HpPETase1 in the NCBI non-redundant database, among which the consistency with the protein closest to HpPETase1 in evolution is 92.05%, indicating that the full-length sequence of the protein (sequence list SEQ ID NO.2) has not been publicly disclosed and is novel.

[0050] Table 2. The 30 protein sequences with the highest consistency with HpPETase1 in the NCBI non-redundant database

[0051] Accession Number Sequence length consistency WP_223655417.1 302 92.05% WP_172829808.1 303 91.78% PKM05449.1 309 80.46% WP_223655416.1 300 78.55% WP_169407362.1 300 78.48% WP_150299537.1 300 77.89% WP_150303136.1 300 76.90% WP_238869820.1 305 76.87% WP_169407363.1 300 76.57% WP_335605500.1 295 76.40% RGP52753.1 285 75.93% WP_169406150.1 303 75.91% WP_150277318.1 308 75.65% 7VMD_A 293 75.29% WP_339843916.1 314 75.28% WP_083724990.1 304 74.92% MEH6688921.1 305 74.83% WP_150300936.1 303 74.50% WP_044499735.1 302 74.34% 8AIS_A 310 74.34% MEL0168864.1 303 74.09% 6SCD_A 312 73.91% WP_228247074.1 304 73.91% WP_088276085.1 304 73.91% MAY07254.1 304 73.91% 6SBN_A 312 73.91% WP_338212404.1 304 73.91% WP_238869819.1 304 73.68% WP_285677831.1 304 73.58% .

[0052] 4. HpPETase2 gene and protein sequence analysis

[0053] In order to determine the novelty of the amino acid sequence of HpPETase2, the present invention searched for its homologous proteins in the NCBI non-redundant database through online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and arranged them according to sequence consistency. Table 3 shows the 30 sequences with the highest consistency with HpPETase2 in the NCBI non-redundant database, among which the consistency with the protein that is most evolutionarily similar to HpPETase2 is 90.33%, indicating that the full-length sequence of the protein (SEQ ID NO.3 in the sequence listing) has not been publicly disclosed and is novel.

[0054] Table 3. The 30 protein sequences with the highest consistency with HpPETase2 in the NCBI non-redundant database

[0055] Accession Number Sequence length consistency WP_223655416.1 300 90.33% WP_169407362.1 300 86.96% WP_150299537.1 300 86.67% WP_169407363.1 300 86.00% WP_150303136.1 300 84.67% WP_172829808.1 303 79.80% WP_223655417.1 302 79.40% WP_206668533.1 250 77.11% PKM05449.1 309 76.83% WP_339842536.1 309 75.68% 7VMD_A 293 75.57% MEH6800204.1 275 75.55% WP_238869820.1 305 75.19% WP_044499735.1 302 74.92% 8AIS_A 310 74.92% WP_036989706.1 302 74.92% 8AIT_A 310 74.92% RGP52753.1 285 74.81% WP_285259726.1 303 74.17% HEC53620.1 300 73.58% WP_105645204.1 302 73.42% MEH6688922.1 300 73.24% MEL0168864.1 303 73.15% WP_373185656.1 306 73.11% WP_185266504.1 302 73.09% MBL4610876.1 308 72.64% MEH6688921.1 305 72.48% WP_083724990.1 304 72.33% WP_335605360.1 308 72.31% WP_150277318.1 308 72.22% .

[0056] 5. HpPETase3 gene and protein sequence analysis

[0057] In order to determine the novelty of the amino acid sequence of HpPETase3, the present invention searched for its homologous proteins in the NCBI non-redundant database through online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and arranged them according to sequence consistency. As shown in Table 4, the 30 sequences with the highest consistency with HpPETase3 in the NCBI non-redundant database database, among which the consistency with the protein that is most similar in evolution to HpPETase1 is 89.29%, indicating that the full-length sequence of the protein (SEQ ID NO.4 in the sequence listing) has not been publicly disclosed and is novel.

[0058] Table 4. The 30 protein sequences with the highest consistency with HpPETase3 in the NCBI non-redundant database

[0059] Accession Number Sequence length consistency UAX00048.1 281 89.29% WP_223651295.1 286 88.81% WP_093392995.1 285 84.97% WP_223651294.1 287 84.34% WP_150301040.1 269 84.34% WP_371923585.1 271 84.34% WP_235818246.1 266 83.21% WP_372240216.1 270 83.13% WP_235833933.1 266 82.82% WP_239419990.1 268 82.61% WP_285259882.1 266 82.44% WP_150301039.1 271 80.56% WP_093392992.1 287 80.49% WP_149331932.1 267 80.49% WP_231703353.1 267 80.49% MBL4833285.1 278 80.32% WP_335606142.1 293 80.24% MBL4612017.1 278 80.24% QJD60744.1 271 80.16% WP_226944829.1 266 80.08% TKA91035.1 275 80.00% SER63710.1 287 79.79% EZQ14005.1 287 79.79% MFA5677944.1 268 79.67% WP_370920463.1 287 79.44% HIZ49548.1 287 79.44% WP_185266304.1 271 78.68% HDZ57343.1 272 78.68% WP_150304204.1 291 78.35% QIB53241.1 285 78.25% .

[0060] 6. HpPETase4 gene and protein sequence analysis

[0061] In order to determine the novelty of the amino acid sequence of HpPETase4, the present invention searched for its homologous proteins in the NCBI non-redundant database through online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and arranged them according to sequence consistency. As shown in Table 5, the 30 sequences with the highest consistency with HpPETase4 in the NCBI non-redundant database database, among which the consistency with the protein closest to HpPETase4 in evolution is 90.28%, indicating that the full-length sequence of the protein (sequence list SEQ ID NO.5) has not been publicly disclosed and is novel.

[0062] Table 5. The 30 protein sequences with the highest consistency with HpPETase4 in the NCBI non-redundant database

[0063] Accession Number Sequence length consistency WP_093392992.1 287 90.28% WP_223651294.1 287 89.93% WP_372240216.1 270 85.14% MBL4833285.1 278 84.74% WP_150301039.1 271 84.13% WP_335606142.1 293 83.73% MBL4612017.1 278 83.73% QJD60744.1 271 83.33% WP_231703353.1 267 82.93% WP_150301040.1 269 82.73% WP_371923585.1 271 82.73% WP_149331932.1 267 82.11% WP_339842624.1 293 80.89% HDZ57344.1 289 80.62% WP_223825416.1 293 80.55% WP_335606269.1 289 79.65% MBL4612019.1 289 79.30% MBL4833286.1 289 79.30% MFA5677944.1 268 79.27% PKM05044.1 288 79.17% WP_239419990.1 268 78.97% QJD57870.1 291 78.69% WP_235906743.1 292 78.50% WP_373288213.1 286 78.20% WP_226944829.1 266 78.05% WP_373286418.1 289 77.54% PKM05045.1 289 77.51% WP_372240192.1 292 77.47% WP_093392995.1 285 77.43% .

[0064] 7. Cloning of HpPETase1, HpPETase2, HpPETase3, and HpPETase4 genes

[0065] HpPETase1, HpPETase2, HpPETase3, HpPETase4 coding sequences were obtained from Halopseudomonas sp. EEDS01 The colonies were obtained by PCR amplification of HpPETase1, HpPETase2, HpPETase3, and HpPETase4 gene fragments using Pn1-F and Pn1-R, Pn2-F and Pn2-R, Pn3-F and Pn3-R, Pn4-F and Pn4-R as primers, as shown in the sequence table SEQ ID NO.6-9. Using pET28a plasmid as a template, 28a-F and 28a-R were used as primers to linearize pET28a. The primer sequences are shown in the sequence table SEQ ID NO.10-19. The PCR amplification conditions are: 98°C for 10 minutes; 98°C for 10 seconds, 55°C for 30 seconds, 72°C for 1 minute, 35 cycles; 72°C for 5 minutes. The obtained HpPETase1, HpPETase2, HpPETase3, and HpPETase4 fragments were ligated with the linearized pET28a vector fragment using a seamless cloning kit (ClonExpress Ultra OneStep Cloning Kit, Novogene, Nanjing) to form expression vectors pDSP01, pDSP02, pDSP03, and pDSP04, as shown in the following figure. Figure 3-6As shown, the Escherichia coli expression strain Escherichia coli BL21 (DE3) was transformed to obtain expression strains BL21DS1, BL21DS2, BL21DS3, and BL21DS4. The strains BL21DS1, BL21DS2, BL21DS3, and BL21DS4 all expressed fusion proteins with Trx-His6 tags at the C-terminus. HpPETase1, HpPETase2, HpPETase3, and HpPETase4 with protein tags were obtained by Ni-NTA affinity chromatography for activity detection.

[0066] 8. Expression and purification of HpPETase1-4

[0067] The BL21DS1-4 clone was selected and inoculated into the culture medium (containing 50 μg / mL ampicillin monoclonal antibody) for seed culture, and cultured at 37°C 220 rpm for 12 h; the expansion culture ratio was 1:100, and cultured at 37°C 220 rpm for about 4 to 6 hours. 600 When the concentration of IPTG reaches 0.6-0.8, IPTG is added to start induction, and the final concentration of IPTG is 0.5 mM. The induction condition is 18 ℃ for 18-24 h. After the induction, the bacterial solution is centrifuged at 4 ℃ 3000-6000 g for 10 minutes to collect the bacteria.

[0068] The collected bacteria were resuspended in 50 mL of pre-cooled lysis buffer and broken by ultrasonic disruptor in an ice bath. The breaking program was 290 W, ultrasonic disruption for 4 s, and stop for 8 s. The total ultrasonic duration was 15 min. The supernatant was centrifuged at 4 °C 13000 g for 60 min to obtain the supernatant. The supernatant was incubated with 3 mL Ni-NTA resin at 4 °C for 1 hour to allow the target protein to fully bind to the resin. The supernatant was rinsed with 15 mL lysis buffer and 15 mL wash buffer, and the target protein was eluted with 5 mLelution buffer. The eluate was concentrated to 2.5 mL using Ultracel-10K ultrafiltration tube. The desalting column PD-10 was balanced with 25 mL desalting buffer, 2.5 mL of concentrated protein solution was added to the desalting column PD-10, and 3.5 mL of desalting buffer was added to collect the protein. The protein was concentrated to 1 mL using an Ultracel-10K ultrafiltration tube, and the absorbance at 280 nm was measured using a micro-spectrophotometer to determine the concentration of the purified protein. 5 μL of protein was used for SDS-PAGE detection. The test results were as follows: Figure 7 The remaining protein was quickly frozen with liquid nitrogen and stored at -80 °C.

[0069] Protein purification - buffer recipes:

[0070] Lysis buffer (1 L): 3.03 g Tris, 8.77 g NaCl, 100 g glycerol, 1.36 g imidazole, pH 7.5.

[0071] Wash buffer (1 L): 3.03 g Tris, 8.77 g NaCl, 100 g glycerol, 2.72 g imidazole, pH 7.5.

[0072] Elution buffer (1 L): 3.03 g Tris, 8.77 g NaCl, 100 g glycerol, 10.2 g imidazole, pH 7.5.

[0073] Desalting buffer (1 L): 3.03 g Tris, 8.77 g NaCl, 100 g glycerol, pH 7.5.

[0074] 9. Comparison of PET degradation ability between HpPETase3 and IsPETase

[0075] Nano-PET prepared by the dissolution-precipitation method was used as the reaction substrate and added to 2 mL of 5.3 M NaCl pH 9.0 Tris-HCl buffer at a concentration of 1 g / L. 500 nM HpPETase3 and IsPETase were added to the test tube respectively. The reaction was carried out at 55°C for two days, and photos were taken every day to observe the degradation of nano-PET.

[0076] The instrument model used for HPLC detection was Agilent Technologies 1260 infinity LC, the analytical column model was ZORBAX SB-C18, and the column temperature was 30°C. Mobile phase A was deionized water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% TFA. The flow rate was fixed at 1 mL / min. The PET hydrolysis products TPA and MHET can be separated by the following gradient mobile phase: 0~5 minutes, 15% B; 5~20 minutes, 15%~100% B gradient; 20~25 minutes, 100%~15% B gradient; 25~30 minutes, 15% B. The detection wavelength was 254 nm. The concentrations of TPA and MHET produced were calculated based on the standard curve prepared from standard samples.

[0077] like Figure 8As shown, only a small amount of HpPETase3 remained after one day of reaction, and the reaction solution became clear on the second day. IsPETase was degraded to a certain extent within the first day, and then there was no obvious degradation effect. From the results, the degradation ability and stability of HpPETase3 were significantly higher than those of IsPETase.

[0078] 10. Strains Halopseudomonas sp. Degradation of PET by EEDS01

[0079] Nano-PET prepared by the dissolution-precipitation method was used as the reaction substrate and added to 2216E medium at a concentration of 1 g / L. 20 mL of 2216E medium mixed with nano-PET was added to a 50 mL conical flask and inoculated with Halopseudomonas sp. EEDS01 at a ratio of 1:100. The culture was incubated at 220 rpm in a shaker at 27°C for 20 days. 100 μl of the culture medium was taken every day in the first week, and then samples were taken once a week. The culture medium was treated with 3 times methanol, centrifuged at 12000 rpm for 3 min, and the supernatant was taken for HPLC detection.

[0080] The instrument model used for HPLC detection was Agilent Technologies 1260 infinity LC, the analytical column model was ZORBAX SB-C18, and the column temperature was 30°C. Mobile phase A was deionized water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% TCA. The flow rate was fixed at 1 mL / min. The PET hydrolysis products TPA and MHET can be separated by the following gradient mobile phase: 0~5 minutes, 15% B; 5~20 minutes, 15%~100% B gradient; 20~25 minutes, 100%~15% B gradient; 25~30 minutes, 15% B. The detection wavelength was 254 nm. The concentrations of TPA and MHET produced were calculated based on the standard curve prepared from standard samples.

[0081] from Fig. 9 It can be seen that the strain Halopseudomonas sp EEDS01 began to secrete PET hydrolase into the extracellular space on the third day, and a gradual increase in PET degradation products was observed. By the 20th day, the product concentration reached 0.73 mM, and PET had been degraded by 15.3%.

[0082] 11. HpPETase1-4 catalyzes the hydrolysis of GfPET at different salt concentrations

[0083] GfPET membrane with a diameter of 6 mm was used as the reaction substrate. Different concentrations of NaCl were added to 50 mM Tris-HCl as the reaction buffer, and the pH of each buffer was adjusted to 9.0. The concentrations of NaCl were: 0 M, 0.6 M, 1.2 M, 2.4 M, 3 M, 3.6 M, 4.2 M, 4.8 M, 5.3 M, the total reaction volume was 200 μL, the final enzyme concentration was 200 nM, and the reaction temperature was 55°C. Three replicates were set for each reaction. After 48 h of reaction, 100 μL was taken and 10 μL of 40% hydrochloric acid was added for freeze-drying to terminate the reaction. 400 μL of methanol was added, and the supernatant was centrifuged for HPLC detection.

[0084] The instrument model used for HPLC detection was Agilent Technologies 1260 infinity LC, the analytical column model was ZORBAX SB-C18, and the column temperature was 30°C. Mobile phase A was deionized water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% TFA. The flow rate was fixed at 1 mL / min. The PET hydrolysis products TPA and MHET can be separated by the following gradient mobile phase: 0~5 minutes, 15% B; 5~20 minutes, 15%~100% B gradient; 20~25 minutes, 100%~15% B gradient; 25~30 minutes, 15% B. The detection wavelength was 254 nm. The concentrations of TPA and MHET produced were calculated based on the standard curve prepared from standard samples.

[0085] The reaction results are as follows Figure 10-13 As shown in the results, the activity of HpPETase1-4 increased with the increase of NaCl concentration, and the highest activity was shown when it reached 5.3 M.

[0086] 12. HpPETase1-4 catalyzes the hydrolysis of GfPET at different temperatures

[0087] GfPET membrane with a diameter of 6 mm was used as the reaction substrate, 5.3 M NaCl was added to 50 mM Tris-HCl as the reaction buffer, and the pH of each buffer was adjusted to 9.0. The temperature gradient was: 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, the total reaction volume was 200 μL, and the final enzyme concentration was 200 nM. Three replicates were set for each reaction. After 48 h of reaction, 100 μL was taken and 10 μL of 40% hydrochloric acid was added to freeze-dry to terminate the reaction, 400 μL of methanol was added, and the supernatant was centrifuged for HPLC detection.

[0088] The instrument model used for HPLC detection was Agilent Technologies 1260 infinity LC, the analytical column model was ZORBAX SB-C18, and the column temperature was 30°C. Mobile phase A was deionized water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% TCA. The flow rate was fixed at 1 mL / min. The PET hydrolysis products TPA and MHET can be separated by the following gradient mobile phase: 0~5 minutes, 15% B; 5~20 minutes, 15%~100% B gradient; 20~25 minutes, 100%~15% B gradient; 25~30 minutes, 15% B. The detection wavelength was 254 nm. The concentrations of TPA and MHET produced were calculated based on the standard curve prepared from standard samples.

[0089] 48 hours reaction results Fig.14 As shown, the enzyme activity of HpPETase1 increased with increasing temperature from 30°C to 60°C and reached the highest at 60°C, and then decreased with increasing temperature from 60°C to 70°C.

[0090] 48 hours reaction results Fig.15 As shown, the enzyme activity of HpPETase2 increased with increasing temperature from 30°C to 55°C and reached the highest at 55°C, and then decreased with increasing temperature from 55°C to 70°C.

[0091] 48 hours reaction results Fig.16 As shown, the enzyme activity of HpPETase3 increased with increasing temperature from 30°C to 55°C and reached the highest at 55°C, and then decreased with increasing temperature from 55°C to 70°C.

[0092] 48 hours reaction results Fig.17 As shown, the enzyme activity of HpPETase4 increased with increasing temperature from 30°C to 55°C and reached the maximum at 55°C, and then decreased with increasing temperature from 55°C to 70°C.

[0093] 13. HpPETase1-4 catalyzes the hydrolysis of GfPET at different pH levels

[0094] The GfPET membrane with a diameter of 6 mm was used as the reaction substrate, and 50 mM KH 2 PO 4 (6.0-8.0), 50 mM Tris-HCl (8.0-9.0), 50 mM Gly-NaOH (9.0-10.0), 5.3 M NaCl was added. The total reaction volume was 200 μL, the final enzyme concentration was 200 nM, and the reaction pH was KH 2 PO4 6.0, 6.5, 7.0, 7.5, 8.0, Tris-HCl 8.0, 8.5, 9.0, Gly-NaOH 9.0, 9.5, 10.0, each reaction was repeated three times. After 48 h of reaction, 100 μL was added to 10 μL 40% hydrochloric acid for freeze-drying to terminate the reaction, 400 μL methanol was added, and the supernatant was centrifuged for HPLC detection.

[0095] The instrument model used for HPLC detection was Agilent Technologies 1260 infinity LC, the analytical column model was ZORBAX SB-C18, and the column temperature was 30°C. Mobile phase A was deionized water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid. The flow rate was fixed at 1 mL / min. The PET hydrolysis products TPA and MHET can be separated by the following gradient mobile phase: 0 ~ 5 minutes, 15% B; 5 ~ 20 minutes, 15% ~ 100% B gradient; 20 ~ 25 minutes, 100% ~ 15% B gradient; 25 ~ 30 minutes, 15% B. The detection wavelength was 254 nm. The concentrations of TPA and MHET produced were calculated based on the standard curve prepared from standard samples.

[0096] 48 hours reaction results Figure 18-21 As shown, the optimum pH for HpPETase1 is 8.0, HpPETase2 is 8.5, HpPETase3 is 8.5, and HpPETase4 is 9.0. Among them, HpPETase3 has the highest degradation activity. In Tris-HCl buffer at pH 8.5, the amount of reaction product is 5 mM after 48 h.

Claims

1. A PET-degrading strain from deep sea, characterized by: This strain is a halophilic Pseudomonas Halopseudomonas sp .) EEDS01, deposited in China Center for Type Culture Collection on November 28, 2024, with the deposit number being CCTCC NO: M20242659.

2. The PET degrading enzyme derived from the strain according to claim 1, characterized in that: The amino acid sequences thereof are shown in any one of SEQ ID NO. 2-5 in the sequence listing.

3. The gene encoding the PET degrading enzyme according to claim 2, characterized in that: The nucleotide sequences thereof are shown in any one of SEQ ID NO.6-9 in the sequence listing.

4. A carrier, characterized in that: Comprising the gene encoding the PET degrading enzyme according to claim 3.

5. The carrier according to claim 4, characterized in that: The vector is a eukaryotic vector, a prokaryotic vector, a plasmid vector or a viral vector.

6. A host cell, characterized in that: Comprising the vector according to claim 4 or 5.

7. The host cell according to claim 6, characterized in that: The host cell is a bacterium.

8. Use of any one of the strain according to claim 1, the PET degrading enzyme according to claim 2, the gene according to claim 3, the vector according to claim 4 or 5, and the host cell according to claim 6 or 7 in degrading PET under high salt conditions.

9. PET degradation agent, characterized in that: The PET degrading agent comprises any one of the strain according to claim 1, the PET degrading enzyme according to claim 2, the vector according to claim 4 or 5, and the host cell according to claim 6 or 7.

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