Deep sea-derived pet hydrolase dspetase05 and use thereof

Abstract

The present invention relates to a deep sea-derived PET hydrolase and the use thereof. Provided is a deep sea-derived PET hydrolase dsPETase05, which has an amino acid sequence as shown in SEQ ID NO. 1. Further provided is a gene encoding the PET hydrolytic enzyme dsPETase05, which gene has a nucleotide sequence as shown in SEQ ID NO. 2. The PET hydrolase dsPETase05 can exhibit a high hydrolytic activity against PET in a high-salt or high-temperature environment.

Description

A deep-sea PET hydrolase dsPETase05 and its application Technical Field

[0001] The invention belongs to the technical field of biochemistry and enzyme engineering, and particularly relates to a deep-sea PET hydrolase and application thereof. Background Art

[0002] Plastics, owing to their excellent properties such as durability, ductility, plasticity, stability, and light transmittance, have become ubiquitous in all aspects of human life and production. However, the chemical inertness of plastics at room temperature has led to an increasing amount of white pollution caused by large amounts of plastic waste, which poses a threat to the ecological environment and human health. PET, a polyester plastic made from the polymerization of terephthalic acid and ethylene glycol, is widely used in the textile industry, food and beverage packaging, and other fields. Traditional PET processing methods, such as mechanical crushing, thermoplastic reprocessing, and chemical decomposition, are costly and can cause secondary environmental pollution. In recent years, bioenzymatic degradation based on PET hydrolases has shown great application potential. For example, IsPETase from the PET-degrading bacteria Ideonella sakaiensis 201-F6 (Yoshida et al., 2016) and LCC from the metagenome of leaf compost (Sulaiman et al., 2012) both have good PET hydrolysis activity. The use of their mutants for PET hydrolysis, monomer recovery, and PET resynthesis has demonstrated the theoretical feasibility of a circular economy for PET (Lu et al., 2022; Tournier et al., 2020).

[0003] There are few studies on the halophilicity of PET hydrolases. PET6 from the halophilic bacterium Vibrio gazogenes has been shown to tolerate 0-2.5 M NaCl, but its activity is much lower than that of IsPETase (Weigert et al., 2022). Therefore, there is currently a gap in the search for enzymes that can exhibit high PET hydrolysis activity under high-salt conditions.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to solve the problem that existing enzymes with high PET hydrolysis activity cannot tolerate high-salt environments. A PET hydrolase dsPETase05 is provided, which is excavated and identified from the metagenome of a hot spring at 1,199 meters in the deep sea. The enzyme can exhibit high hydrolysis activity against PET in high-salt or high-temperature environments.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a deep-sea PET hydrolase dsPETase05, whose amino acid sequence is shown in SEQ ID No.1.

[0007] Preferably, the present invention also provides a gene encoding the PET hydrolase dsPETase05, the nucleotide sequence of which is shown in SEQ ID No.2.

[0008] Preferably, the present invention also provides a vector of the PET hydrolase dsPETase05, wherein the vector comprises the gene.

[0009] Preferably, the vector is a eukaryotic vector or a prokaryotic vector.

[0010] Preferably, the vector is a plasmid vector or a viral vector.

[0011] Preferably, the present invention also provides a host cell comprising the vector.

[0012] Preferably, the host cell is a bacterium.

[0013] Preferably, the present invention further provides the use of the PET hydrolase dsPETase05, the vector, and the host cell in degrading PET under high salt or high temperature conditions.

[0014] Preferably, the present invention further provides a PET degrading agent, which contains at least one of the PET hydrolase dsPETase05, the epitope carrier, and the host cell.

[0015] Compared with existing technologies, the present invention has the following advantages: the PET hydrolase dsPETase05 provided by the present invention can demonstrate efficient PET hydrolysis under various high-salt concentrations or high-temperature conditions, producing monohydroxyethyl terephthalic acid (MHET), terephthalic acid (TPA), and ethylene glycol (EG), thereby achieving enzymatic degradation of PET. The dsPETase05 of the present invention is halophilic and exhibits higher activity and stability under high-salt conditions. Furthermore, this halophilic nature makes it more suitable for treating high-salt industrial wastewater and sewage. Therefore, the present invention has important implications in the fields of environmental protection and catalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 shows the hydrolysis of PET catalyzed by dsPETase05 at different NaCl levels;

[0017] FIG2 is a plasmid map of pDSP05 expressing dsPETase05;

[0018] FIG3 is an SDS-PAGE analysis of purified dsPETase05;

[0019] Figure 4 shows the product concentrations of PET hydrolysis catalyzed by dsPETase05 at different salt concentrations for 48 hours. The hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the TPA and MHET concentrations in the reaction solution represents the magnitude of PET hydrolysis activity.

[0020] Figure 5 shows the product concentrations of PET hydrolysis catalyzed by dsPETase05 for 120 hours at different salt concentrations. The hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the TPA and MHET concentrations in the reaction solution represents the magnitude of PET hydrolysis activity.

[0021] Figure 6 shows the complete degradation of scPET membrane catalyzed by dsPETase05; the reaction system in the test tube was 3 mL, the scPET content was 3 mg, the reaction time is indicated by the arrow on the left, and the enzyme concentration is indicated below the enzyme name;

[0022] Figure 7 shows the product concentrations of PET hydrolysis catalyzed by dsPETase05 at different temperatures for 48 hours. The hydrolysis products detectable by HPLC are TPA and MHET. The sum of the TPA and MHET concentrations in the reaction solution represents the magnitude of PET hydrolysis activity.

[0023] Figure 8 shows the product concentrations of PET hydrolysis catalyzed by dsPETase05 at different temperatures for 120 hours. The hydrolysis products detectable by HPLC are TPA and MHET. The sum of the TPA and MHET concentrations in the reaction solution represents the PET hydrolysis activity.

[0024] Figure 9 compares the concentrations of PET hydrolysis products catalyzed by dsPETase05, HotPETase, and FAST-PETase: A, comparison of the activities of dsPETase05 and FAST-PETase; B, comparison of the activities of dsPETase05 and FAST-PETase; No NaCl was added to the reaction systems of FAST-PETase-0 and HotPET-0, while 5.3 M NaCl was added to the reaction systems of dsPETase05, FAST-PETase-6, and HotPET-6. DETAILED DESCRIPTION

[0025] To facilitate understanding of this research, the following description provides a more detailed explanation of this research, with reference to the accompanying figures and specific examples. However, this research can be implemented in many different forms and is not limited to the examples described in this specification. Rather, these examples are provided to provide a more thorough and comprehensive understanding of the disclosure of this research.

[0026] The present invention screened deep-sea metagenomes to obtain an enzyme, named dsPETase05. The functional properties and catalytic activity of the enzyme were tested and verified in the following experiments.

[0027] 1. Preparation of dsPETase05 Substrate

[0028] GfPET (ES301445, Goodfellow, Huntingdon, England) was used to verify the function of dsPETase05, and a GfPET film with a diameter of 6 mm was prepared by a hole puncher as the substrate for the reaction.

[0029] Preparation of scPET membranes: Reports on the preparation of homemade scPET membranes by solvent dissolution-evaporation to test PET hydrolysis activity are available in references (Liu et al., 2023; Cui et al., 2021). The scPET membrane prepared in this example was prepared by uniformly coating a 10-cm-diameter flat glass plate with 40 mg / mL of GfPET dissolved in 2 mL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The HFIP was evaporated overnight at room temperature, and the resulting scPET membrane was incubated in 75% ethanol for 2 hours. The resulting scPET membrane was then peeled from the glass plate.

[0030] 2. dsPETase05 gene and protein sequence analysis

[0031] To determine the novelty of the dsPETase05 amino acid sequence, we searched for homologous proteins in GenBank using online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and ranked them by sequence similarity. Table 1 lists the 30 sequences with the highest identity to dsPETase05 in the GenBank database. The protein most closely related to dsPETase05 in evolutionary terms exhibited a 99.31% identity, indicating that the full-length sequence of this protein (SEQ ID No. 1) has not been publicly disclosed and is therefore novel.

[0032] Table 1. The 30 protein sequences with the highest identity to dsPETase05 in GeneBank

[0033] 3. Cloning of dsPETase05 gene

[0034] A complete gene synthesis was performed based on the dsPETase amino acid sequence and codon-optimized for expression in E. coli. The synthesized sequence is shown in SEQ ID No. 2. The dsPETase05 gene fragment was amplified using primers DSP05-F and DSP05-R. Linear amplification was performed using pET32a-LIC as a template and primers 32a-F and 32a-R in pET32a. Primer sequences are shown in Table 2. PCR amplification conditions were: 98°C for 10 minutes, followed by 35 cycles of 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute, and 72°C for 5 minutes. The obtained dsPETase05 fragment was ligated with a linearized pET32a vector fragment using a seamless cloning kit (ClonExpress II One Step Cloning Kit, Novozymes, Nanjing) to form the expression vector pDSP05, as shown in Figure 2. The vector was then transformed into the Escherichia coli Rosseta-gami 2 (DE3) expression strain to generate the expression strain RgDSP05. Strain RgDSP05 expresses a fusion protein with an N-terminal Trx-His6 tag, which can be cleaved by TEV protease. Tag-free dsPETase05 was obtained by two Ni-NTA affinity chromatography cycles for activity analysis.

[0035] Table 2. Primers and sequences for cloning dsPETase05 into pET32a-LIC

[0036] 4. Expression and Purification of dsPETase05

[0037] Pick up RgDSP05 monoclonal clones and inoculate them into culture medium (containing 50 μg / mL ampicillin and 10 μg / ml chloramphenicol dual resistance) for seed culture, and culture them on a shaker at 37°C and 220 rpm for 8-12 h; expand the culture at a ratio of 1:500 to 1:100, and culture them on a shaker at 37°C and 220 rpm for about 4-6 hours. 600 When the RI reaches 0.6-0.8, add IPTG to initiate induction. The final IPTG concentration can be 0.2-1 mM. Induction conditions are 18-30°C for 18-24 hours. After induction, centrifuge the culture at 4°C at 3000-6000g for 10 minutes to collect the cells.

[0038] The collected cells were resuspended in 50 mL of pre-chilled lysis buffer and disrupted using an ultrasonic disruptor in an ice bath at 290 W for 4 seconds on and 8 seconds off, for a total of 15 minutes. The supernatant was centrifuged at 13,000 g for 60 minutes at 4°C and incubated with 3 mL of Ni-NTA resin at 4°C for 1 hour to allow the target protein to bind fully to the resin. The supernatant was then rinsed with 15 mL of lysis buffer and then 15 mL of wash buffer. The target protein was eluted with 5 mL of elution buffer and concentrated to 2.5 mL using an Ultracel-30K ultrafiltration tube. A PD-10 desalting column was equilibrated with 25 mL of desalting buffer. 2.5 mL of the concentrated protein solution was added to the PD-10 column and the protein was collected by adding 3.5 mL of desalting buffer. 10 μL of His6-tagged TEV protease was added to the collected protein solution and incubated at 4°C for 12 hours. Equilibrate Ni-NTA resin with 20 mL of desalting buffer, add 3 mL of Ni-NTA resin, and incubate at 4°C for 1 hour to allow the Trx-His6 tag and TEV enzyme to fully bind to the resin. Collect the flow-through, which is untagged dsPETase05. Concentrate dsPETase05 to 1 mL using an Ultracel-30K ultrafiltration tube. Determine the concentration of the purified protein by measuring absorbance at 280 nm using a microspectrophotometer. A 5 μL sample of protein was analyzed by SDS-PAGE. The results are shown in Figure 3. The remaining protein was snap-frozen in liquid nitrogen and stored at -80°C.

[0039] Protein purification - buffer recipes:

[0040] Lysis buffer (1 L): 3.03 g Tris, 8.77 g NaCl, 100 g glycerol, 1.36 g imidazole, pH 7.5.

[0041] Wash buffer (1 L): 3.03 g Tris, 8.77 g NaCl, 100 g glycerol, 2.72 g imidazole, pH 7.5.

[0042] Elution buffer (1 L): 3.03 g Tris, 8.77 g NaCl, 100 g glycerol, 10.2 g imidazole, pH 7.5.

[0043] Desalting buffer (1 L): 3.03 g Tris, 8.77 g NaCl, 100 g glycerol, pH 7.5.

[0044] 5. dsPETase05 catalyzes the hydrolysis of GfPET at different salt concentrations

[0045] A 6mm diameter GfPET membrane was used as the reaction substrate. Various concentrations of NaCl were added to 50mM Tris-HCl as the reaction buffer, with the pH of each buffer adjusted to 9.0. NaCl concentrations were 0M, 0.6M, 1.2M, 1.9M, 2.8M, 3.7M, 4.5M, and 5.3M, in a total reaction volume of 500μL, with a final enzyme concentration of 50nM and a reaction temperature of 37°C. IsPETase was reacted under the same conditions for activity comparison, with three replicates per reaction. 100μL samples were taken at 48 and 120 hours of reaction, terminated by the addition of 200μL of methanol, and then inactivated by treatment at 100°C for 10 minutes. The supernatant was centrifuged and analyzed by HPLC.

[0046] HPLC analysis was performed using an Agilent Technologies 1220 Infinity LC instrument with a ZORBAX SB-C18 analytical column at 30°C. Mobile phase A consisted of deionized water containing 0.1% trichloroacetic acid, and mobile phase B consisted of acetonitrile containing 0.1% TCA. The flow rate was fixed at 1 mL / min. The PET hydrolysis products, TPA and MHET, were separated using the following mobile phase gradient: 15% B (0-5 minutes); 15% to 100% B (5-20 minutes); 100% to 15% B (20-25 minutes); and 15% B (25-30 minutes). Detection was performed at a wavelength of 254 nm. The concentrations of TPA and MHET generated were calculated using a standard sample.

[0047] The reaction results are shown in Figures 4 and 5. As can be seen from the results, IsPETase exhibited the highest activity under the condition without the addition of NaCl, so the PET hydrolysis reaction catalyzed by IsPETase under this condition was used as a reference.

[0048] The results of the 48-hour reaction are shown in Figure 4. dsPETase05 exhibited PET hydrolysis activity under all NaCl conditions, and the activity increased with increasing NaCl concentration. It exhibited the highest activity when it reached 5.3 M. At this time point, the activity exhibited was 5.4 times that of IsPETase.

[0049] The results of the 120-hour reaction are shown in Figure 5. dsPETase05 exhibited PET hydrolysis activity under all NaCl conditions, and the activity increased with increasing NaCl concentration. It exhibited the highest activity when it reached 5.3 M. At this time point, the activity exhibited was 16 times that of IsPETase.

[0050] 6. dsPETase05 catalyzes the hydrolysis of scPET membranes at different salt concentrations

[0051] The PET hydrolysis activity of dsPETase05 was demonstrated by complete hydrolysis of a homemade scPET film. 3 mg of scPET film was weighed into a glass test tube, followed by the addition of 3 mL of the following reaction solutions: the IsPETase reaction solution contained 50 mM tris-HCl buffer (pH 9.0) and 500 nM enzyme; the dsPETase05 reaction solution contained 50 mM tris-HCl buffer (pH 9.0), 5.3 M NaCl, and 500 nM enzyme. The degradation of the scPET film was observed every two days over a total of 10 days. As shown in Figure 6, dsPETase05 at a concentration of 500 nM significantly degraded the scPET film after six days and maintained its activity throughout the entire 10-day reaction, catalyzing the degradation of most of the scPET. In contrast, the IsPETase control showed no significant degradation over the 10-day reaction.

[0052] The above results indicate that dsPETase05 has higher activity and higher stability under high salt conditions.

[0053] 7. dsPETase05 catalyzes the hydrolysis of GfPET at different temperatures

[0054] A 6mm diameter GfPET membrane was used as the reaction substrate. 5.3M NaCl was added to 50mM Tris-HCl, and the pH of each buffer was adjusted to 9.0. The total reaction volume was 500μL, the final enzyme concentration was 50nM, and the reaction temperatures were 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C. Each reaction was repeated three times. A 100μL sample was taken at 48 and 120 hours of reaction, and the reaction was terminated by adding 200μL of methanol. The sample was then inactivated by heating at 100°C for 10 minutes. The supernatant was centrifuged and analyzed by HPLC.

[0055] HPLC analysis was performed using an Agilent Technologies 1220 Infinity LC instrument with a ZORBAX SB-C18 analytical column at 30°C. Mobile phase A consisted of deionized water containing 0.1% trichloroacetic acid, and mobile phase B consisted of acetonitrile containing 0.1% TCA. The flow rate was fixed at 1 mL / min. The PET hydrolysis products, TPA and MHET, were separated using the following mobile phase gradient: 15% B (0-5 minutes); 15% to 100% B (5-20 minutes); 100% to 15% B (20-25 minutes); and 15% B (25-30 minutes). Detection was performed at a wavelength of 254 nm. The concentrations of TPA and MHET generated were calculated using a standard sample.

[0056] The results of the 48-hour reaction are shown in FIG7 . The amount of hydrolysis products catalyzed by dsPETase05 increases with increasing temperature from 25°C to 55°C and reaches a maximum at 55°C. It then decreases with increasing temperature from 55°C to 75°C.

[0057] The results of the 120-hour reaction are shown in FIG8 . The amount of hydrolysis products catalyzed by dsPETase05 increases with increasing temperature from 25°C to 55°C and reaches a maximum at 55°C. It then decreases with increasing temperature from 55°C to 75°C.

[0058] 8. Comparison of catalytic activity of dsPETase05 with the highly active mutants HotPETase and FAST-PETase

[0059] HotPETase (Bell et al., 2022) and FAST-PETase (Lu et al., 2022), two of the most representative mutants of isPETase, exhibit higher activity than the wild-type and are currently the best-performing PET hydrolase mutants reported. In this example, the activities of dsPETase05 and these two mutants were compared using a 6 mm diameter GfPET membrane as the reaction substrate. The dsPETase05 reaction system consisted of 50 mM Tris-HCl + 5.3 M NaCl and incubated at 55°C; the HotPETase reaction system consisted of either 50 mM Tris-HCl or 50 mM Tris-HCl + 5.3 M NaCl and incubated at 50°C; and the FAST-PETase reaction system consisted of either 50 mM Tris-HCl or 50 mM Tris-HCl + 5.3 M NaCl and incubated at 40°C. All enzymes were incubated at a final concentration of 50 nM. Each reaction was performed in triplicate, with sampling intervals of 8, 24, 48, 72, 96, and 120 hours. 20 μL of sample was taken respectively, 120 μL of methanol and 40 μL of deionized water were added to terminate the reaction, and then the sample was treated at 100° C. for 10 min to inactivate the enzyme. The supernatant was centrifuged and subjected to HPLC detection.

[0060] HPLC analysis was performed using an Agilent Technologies 1220 Infinity LC instrument with a ZORBAX SB-C18 analytical column at 30°C. Mobile phase A consisted of deionized water containing 0.1% trichloroacetic acid, and mobile phase B consisted of acetonitrile containing 0.1% TCA. The flow rate was fixed at 1 mL / min. The PET hydrolysis products, TPA and MHET, were separated using the following mobile phase gradient: 15% B (0-5 minutes); 15% to 100% B (5-20 minutes); 100% to 15% B (20-25 minutes); and 15% B (25-30 minutes). Detection was performed at a wavelength of 254 nm. The concentrations of TPA and MHET generated were calculated using a standard sample.

[0061] The reaction results are shown in Figure 9. In the presence of 5.3 M NaCl, HotPETase and FAST-PETase lost most of their activity and only displayed normal activity in a NaCl-free buffer. dsPETase05, on the other hand, exhibited higher activity than both HotPETase and FAST-PETase at all time points. At 120 hours, the amount of total product was 2.5 times that of HotPETase and 4.5 times that of FAST-PETase. This demonstrates the halophilic and thermophilic properties of dsPETase05, demonstrating superior catalytic activity compared to an engineered mutant of isPETase under high salt and high temperature conditions.

Claims

1. A deep-sea PET hydrolase dsPETase05, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The gene encoding the PET hydrolase dsPETase05 according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

2.

3. A vector comprising the gene according to claim 2.

4. The carrier according to claim 3, characterized in that: The vector is a eukaryotic vector or a prokaryotic vector.

5. The carrier according to claim 3, characterized in that: The vector is a plasmid vector or a viral vector.

6. A host cell comprising the expression vector according to any one of claims 3 to 5.

7. The host cell according to claim 6, characterized in that: The host cell is a bacterium 8. Use of the PET hydrolase dsPETase05 according to claim 1, the vector according to any one of claims 3 to 5, or the host cell according to any one of claims 6 to 7 in degrading PET.

9. The use according to claim 8, characterized in that: Degradation of PET under high salt or high temperature conditions.

10. PET degradation agent, characterized in that: Contains at least one of the PET hydrolase dsPETase05 according to claim 1, the vector according to any one of claims 3-5, and the host cell according to any one of claims 6-7.

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