A pet hydrolytic enzyme dsPETase06 from deep sea and application thereof

Abstract

The application belongs to the technical field of biochemistry and enzyme engineering, and particularly relates to a PET hydrolase from deep sea and application thereof. The PET hydrolase from deep sea is dsPETase06, and the amino acid sequence of the PET hydrolase is shown as SEQ ID No. 1. The application further provides a gene for coding the PET hydrolase dsPETase06, and the nucleotide sequence of the gene is shown as SEQ ID No. 2. The application provides a PET hydrolase dsPETase06 which is mined and identified from a metagenome from deep sea, and the enzyme can exhibit high hydrolysis activity on PET in a high-salt or high-temperature environment, and has important significance in the fields of environmental protection and catalysis.

Description

Technical Field

[0001] This invention belongs to the fields of biochemistry and enzyme engineering technology, specifically relating to a deep-sea-derived PET hydrolase and its applications. Background Technology

[0002] Plastics, due to their excellent properties such as durability, ductility, plasticity, stability, and light transmittance, are ubiquitous in all aspects of human production and life. However, the chemical inertness of plastics at room temperature has led to increasingly serious white pollution caused by large amounts of plastic waste, harming the ecological environment and human health. PET is a type of polyester plastic polymerized from terephthalic acid and ethylene glycol monomers, widely used in textiles, food and beverage packaging, and many other fields. Traditional PET processing methods, such as mechanical crushing, thermoplastic reprocessing, and chemical decomposition, are costly and cause secondary pollution to the environment. In recent years, bioenzymatic degradation based on PET hydrolases has shown great application potential. For example, IsPETase from the PET-degrading bacterium Ideonella sakaiensis201-F6 (Yoshida et al., 2016) and LCC from the metagenomics of leaf and branch 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] Studies on the halophilicity of PET hydrolases are limited. PET6 from the halophilic Vibrio gazogenes has been shown to tolerate 0–2.5 M NaCl, but its activity is far lower than that of IsPETase (Weigert et al., 2022). Therefore, enzymes that can exhibit high PET hydrolytic activity under high salt conditions are currently a blank. Summary of the Invention

[0004] The purpose of this invention is to solve the problem that existing enzymes with high PET hydrolytic activity cannot tolerate high-salt environments, and to provide a PET hydrolase dsPETase06, which was mined and identified from a metagenomic sample at a depth of 10,400 meters in the Mariana Trench. This enzyme can exhibit high hydrolytic activity on PET under high-salt or high-temperature environments.

[0005] The technical solution adopted by the present invention to solve its technical problem is: a deep-sea PET hydrolase dsPETase06, the amino acid sequence of which is shown in SEQ ID No.1.

[0006] Preferably, the present invention also provides a gene encoding the PET hydrolase dsPETase06, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0007] Preferably, the present invention also provides a vector for the PET hydrolase dsPETase06, wherein the vector contains the gene.

[0008] Preferably, the carrier is a eukaryotic carrier or a prokaryotic carrier.

[0009] Preferably, the vector is a plasmid vector or a viral vector.

[0010] Preferably, the present invention also provides a host cell comprising the aforementioned vector.

[0011] Preferably, the host cell is a bacterium.

[0012] Preferably, the present invention further provides the application of the PET hydrolase dsPETase06, the carrier, and the host cell in the degradation of PET under high salt or high temperature conditions.

[0013] Preferably, the present invention further provides a PET degrading agent containing at least one of the PET hydrolase dsPETase06, the surface carrier, and the host cell.

[0014] Compared with the prior art, the present invention has the following beneficial effects: the PET hydrolase dsPETase06 provided by the present invention can catalyze the hydrolysis reaction of PET in a high-salt or high-temperature environment to generate MHET and TPA. Subsequently, MHET and TPA can be recycled and repolymerized to generate new PET to achieve the purpose of recycling. At the same time, the halophilic nature of this enzyme makes it more suitable for the treatment of high-salt industrial wastewater and sewage. Therefore, the present invention has important significance in the fields of environmental protection and catalysis. Attached Figure Description

[0015] Figure 1 dsPETase06 catalyzes the hydrolysis of PET under different NaCl concentrations;

[0016] Figure 2 The pDSP06 plasmid map expressing dsPETase06;

[0017] Figure 3 SDS-PAGE analysis of purified dsPETase06; M, protein marker; 1. Purified dsPETase06;

[0018] Figure 4The concentrations of dsPETase06 products catalyzing PET hydrolysis for 48 hours at different salt concentrations are given. 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.

[0019] Figure 5 The concentrations of dsPETase06 products after 120 hours of PET hydrolysis catalyzed by different salt concentrations are given. 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.

[0020] Figure 6 This experiment demonstrates the complete degradation of scPET membranes catalyzed by dsPETase06. The reaction volume in the test tube is 3 mL, the scPET concentration is 3 mg, the reaction time is indicated by the arrow on the left, and the enzyme concentration is indicated below the enzyme name.

[0021] Figure 7 The concentrations of dsPETase06 products catalyzing PET hydrolysis for 48 hours at different temperatures are given. 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.

[0022] Figure 8 The product concentrations of dsPETase06 catalyzing PET hydrolysis for 120 hours at different temperatures were determined. The hydrolysis products detectable by HPLC were TPA and MHET. The sum of the concentrations of TPA and MHET in the reaction solution was used to represent the PET hydrolysis activity. Detailed Implementation

[0023] To facilitate understanding of this research, a more detailed description is provided below with reference to the accompanying drawings and specific embodiments. However, this research can be implemented in many different forms and is not limited to the embodiments described in this specification. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of this research.

[0024] This invention involves screening deep-sea metagenomics to obtain an enzyme, named dsPETase06. The functional characteristics and catalytic activity of this enzyme are then tested and verified experimentally.

[0025] 1. Preparation of substrates for dsPETase06

[0026] As a PET for verifying the function of DsPETase06, we purchased GfPET (ES301445, Goodfellow, Huntingdon, England) and prepared a PET film with a diameter of 6 mm using a punch as the substrate for the reaction.

[0027] Preparation of scPET membrane: Reports on the preparation of scPET membranes using a solvent dissolution-evaporation method for detecting PET hydrolysis activity can be found in the references (Liu et al., 2023; Cui et al., 2021). The scPET membrane prepared in this example was prepared as follows: 2 mL of GfPET (40 mg / mL) dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was uniformly coated onto a 10 cm diameter flat glass plate. The HFIP was evaporated overnight at room temperature, followed by incubation in 75% ethanol for 2 hours. The resulting scPET membrane was then peeled off the glass plate.

[0028] 2. dsPETase06 gene and protein sequence analysis

[0029] To determine the novelty of the DsPETase06 amino acid sequence, we searched for its homologous proteins in GenBank using online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and sorted them by sequence similarity. Table 1 lists the 30 sequences in the GeneBank database that are most identical to dsPETase06. Among them, the protein that is most evolutionarily similar to DsPETase06 has an identity of 86.76%, indicating that the full-length sequence of this protein (SEQ ID No. 1) has not been publicly disclosed before and is novel.

[0030] Table 1. 30 protein sequences in GeneBank with the highest identity to dsPETase06

[0031]

[0032]

[0033] 3. Cloning of the dsPETase06 gene

[0034] The entire dsPETase gene was synthesized using the amino acid sequence of dsPETase, and codon optimization was performed for expression in *E. coli*. The synthesized sequence is shown in SEQ ID No. 2. The dsPETase06 gene fragment was amplified using primers 5076-F and 5076-R. pET32a was linearized and amplified using pET32a-LIC as a template and primers 32a-F and 32a-R. Primer sequences are shown in Table 2. PCR amplification conditions were: 98℃ for 10 minutes; 98℃ for 10 seconds, 55℃ for 30 seconds, 72℃ for 1 minute, 35 cycles; 72℃ for 5 minutes. The obtained dsPETase06 fragment and the linearized pET32a vector fragment were ligated using a seamless cloning kit (ClonExpressII One Step Cloning Kit, Novizan, Nanjing) to form the expression vector pDSP06. The chromatogram is shown below. Figure 2 As shown, the strain was transformed into *Escherichia coli* Rosseta-gami 2(DE3) to obtain strain RgDSP06. Strain RgDSP06 expresses a fusion protein with an N-terminal Trx-His6 tag, which can be cleaved by TEV protease. Tag-free dsPETase06 was obtained through two Ni-NTA affinity chromatography analyses for activity assay.

[0035] Table 2. Primers and their sequences for cloning dsPETase06 into pET32a-LIC

[0036]

[0037] 4. Expression and purification of DsPETase06

[0038] RgDSP06 single clones were picked and inoculated into a culture medium (containing 50 μg / mL ampicillin and 10 μg / mL chloramphenicol dual resistance) for seed culture, and cultured at 37℃ and 220 rpm for 8-12 h. The scale-up culture ratio was 1:500 to 1:100, and cultured at 37℃ and 220 rpm for approximately 4-6 hours. When OD... 600 When the concentration reaches 0.6–0.8, add IPTG to begin induction. The final IPTG concentration can be 0.2–1 mM. Induction conditions are 18–30℃ for 18–24 h. After induction, centrifuge the bacterial culture at 4℃ and 3000–6000 g for 10 minutes to collect the bacterial cells.

[0039] The collected bacterial cells were resuspended in 50 mL of pre-chilled lysis buffer and sonicated in an ice bath using an ultrasonic homogenizer. The homogenization program was 290 W, 4 s of sonication followed by an 8 s pause, for a total sonication time of 15 min. The cells were centrifuged at 13000 g for 60 min at 4 °C, and the supernatant was collected. The supernatant was incubated with 3 mL of Ni-NTA resin at 4 °C for 1 h to allow the target protein to fully bind to the resin. The cells were then washed with 15 mL of lysis buffer and 15 mL of wash buffer, followed by elution with 5 mL of elution buffer to collect the target protein. The eluent was concentrated to 2.5 mL using an Ultracel-30K ultrafiltration tube. The 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 desalting column, followed by 3.5 mL of desalting buffer to collect the protein. 10 μL of TEV protease with a His6 tag was added to the collected protein solution, and the cells were incubated at 4 °C for 12 h. Ni-NTA resin was equilibrated with 20 mL desalting buffer. 3 mL of Ni-NTA resin was added and incubated at 4°C for 1 hour. The Trx-His6 tag and TEV enzyme were fully bound to the resin. The effluent was collected as unlabeled dsPETase06. dsPETase06 was concentrated to 1 mL using an Ultracel-30K ultrafiltration tube. The absorbance at 280 nm was measured using a micro-spectrophotometer to determine the concentration of the purified protein. 10 μL of protein was used for SDS-PAGE analysis. The results are shown below. Figure 3 As shown. The remaining protein was flash-frozen in liquid nitrogen and stored at -80°C.

[0040] The above protein purification buffer formulations are as follows:

[0041] Lysis buffer (1L): 3.03g Tris, 8.77g NaCl, 100g glycerol, 1.36g imidazole, pH 7.5.

[0042] Wash buffer (1L): 3.03g Tris, 8.77g NaCl, 100g glycerol, 2.72g imidazole, pH 7.5.

[0043] Elution buffer (1L): 3.03g Tris, 8.77g NaCl, 100g glycerol, 10.2g imidazole, pH 7.5.

[0044] Desalting buffer (1L): 3.03g Tris, 8.77g NaCl, 100g glycerol, pH 7.5.

[0045] 5. dsPETase06 catalyzes the hydrolysis of GfPET at different salt concentrations

[0046] A 6 mm diameter GfPET membrane was used as the reaction substrate. Different concentrations of NaCl were added to 50 mM Tris-HCl as reaction buffers, and the pH of each buffer was adjusted to 9.0. The NaCl concentrations were: 0 M, 0.6 M, 1.2 M, 1.9 M, 2.8 M, 3.7 M, 4.5 M, and 5.3 M. The total reaction volume was 500 μL, the final enzyme concentration was 50 nM, and the reaction temperature was 37 °C. IsPETase was reacted under the same conditions for activity comparison, with three replicates for each reaction. At 48 and 120 hours of reaction, 100 μL of sample was taken, and the reaction was terminated with 200 μL of methanol. The enzyme was then inactivated by treatment at 100 °C for 10 min, centrifuged, and the supernatant was collected for HPLC analysis.

[0047] The HPLC instrument used was an Agilent Technologies 1220infinity LC, with a ZORBAX SB-C18 column and a column temperature of 30℃. Mobile phase A was deionized water containing 0.1% trichloroacetic acid, and mobile phase B was acetonitrile containing 0.1% TCA, with a fixed flow rate of 1 mL / min. PET hydrolysis products TPA and MHET were separated using the following gradient mobile phases: 0–5 min, 15% B; 5–20 min, 15%–100% B gradient; 20–25 min, 100%–15% B gradient; 25–30 min, 15% B. The detection wavelength was 254 nm. The concentrations of TPA and MHET were calculated based on standard samples.

[0048] The reaction results are as follows Figure 4 , Figure 5 As shown in the figure, IsPETase exhibits the highest activity under conditions without NaCl addition; therefore, the IsPETase-catalyzed PET hydrolysis reaction under these conditions is used as a reference.

[0049] 48-hour reaction results as follows Figure 4 As shown, dsPETase06 exhibited PET hydrolysis activity under all NaCl conditions, and the activity increased with increasing NaCl concentration. It showed the highest activity at 4.5M, which was twice that of IsPETase.

[0050] The results of the 120-hour reaction are as follows Figure 5As shown, dsPETase06 exhibited PET hydrolysis activity under all NaCl conditions, and the activity increased with increasing NaCl concentration. It showed the highest activity at 4.5M, which was 5.5 times that of IsPETase.

[0051] 6. dsPETase06 catalyzes the hydrolysis of scPET membranes under different salt concentrations.

[0052] The PET hydrolytic activity of dsPETase06 was demonstrated by the complete hydrolysis of a self-made scPET membrane. 3 mg of scPET membrane was weighed into a glass test tube, followed by 3 mL of reaction solution: the IsPETase reaction solution contained 50 mM tris-HCl buffer (pH 9.0) and 500 nM enzyme; the dsPETase reaction solution contained 50 mM tris-HCl buffer (pH 9.0), 4.5 M NaCl, and either 300 nM or 500 nM enzyme. The degradation of the scPET membrane was observed by photographing every two days over a total reaction period of 10 days. Results are as follows: Figure 6 As shown, dsPETase06 at a concentration of 500 nM catalyzed the degradation of most scPET membranes after 4 days, with almost no scPET remaining after 6 days; while at a concentration of 300 nM, significant degradation of scPET membranes was observed after 4 days, with only a small portion of scPET membrane remaining on days 8 and 10; and as a control, IsPETase did not show significant degradation during the 10-day reaction process.

[0053] The above results indicate that dsPETase06 exhibits both higher activity and high stability under high salt conditions.

[0054] 7. dsPETase06 catalyzes the hydrolysis of GfPET at different temperatures

[0055] A 6 mm diameter GfPET membrane was used as the reaction substrate. Different concentrations of NaCl were added to 50 mM Tris-HCl as reaction buffers, and the pH of each buffer was adjusted to 9.0. The NaCl concentration was 4.5 M, the total reaction volume was 500 μL, the final enzyme concentration was 50 nM, and the reaction temperatures were 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, and 75℃, with three replicates for each reaction. At 48 and 120 hours of reaction, 100 μL of sample was taken, and the reaction was terminated with 200 μL of methanol. The enzyme was then inactivated by treatment at 100℃ for 10 min, centrifuged, and the supernatant was collected for HPLC analysis.

[0056] The HPLC instrument used was an Agilent Technologies 1220infinity LC, with a ZORBAX SB-C18 column and a column temperature of 30℃. Mobile phase A was deionized water containing 0.1% trichloroacetic acid, and mobile phase B was acetonitrile containing 0.1% TCA, with a fixed flow rate of 1 mL / min. PET hydrolysis products TPA and MHET were separated using the following gradient mobile phases: 0–5 min, 15% B; 5–20 min, 15%–100% B gradient; 20–25 min, 100%–15% B gradient; 25–30 min, 15% B. The detection wavelength was 254 nm. The concentrations of TPA and MHET were calculated based on standard samples.

[0057] 48-hour reaction results as follows Figure 7 As shown, the amount of hydrolysis products catalyzed by dsPETase06 increases with increasing temperature from 25°C to 45°C, reaching a maximum at 45°C, and then decreases with increasing temperature from 45°C to 75°C.

[0058] The results of the 120-hour reaction are as follows Figure 8 As shown, the amount of hydrolysis products catalyzed by dsPETase06 increases with increasing temperature from 25°C to 45°C, reaching a maximum at 45°C, and then decreases with increasing temperature from 45°C to 75°C.

[0059] This result indicates that dsPETase06 can exhibit better performance than [other products] in the 37–45℃ range. Figure 7 and Figure 8 Higher activity.

Claims

1. A deep-sea-derived PET hydrolase, dsPETase06, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The gene encoding the PET hydrolase dsPETase06 as described in claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

2.

3. A vector containing the gene as described in claim 2.

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

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 vector as described in any one of claims 3-5.

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

8. The use of the PET hydrolase dsPETase06 as described in claim 1, the carrier as described in any one of claims 3-5, or the host cell as described in any one of claims 6-7 in the degradation of PET.

9. The application according to claim 8, characterized in that: PET is degraded under high salt or high temperature conditions; the high salt conditions refer to a NaCl concentration of 4.5 M; the high temperature conditions refer to a temperature of 40℃, 45℃ or 50℃.

10. A PET degrading agent, characterized in that: It contains at least one of the following: the PET hydrolase dsPETase06 as described in claim 1, the vector as described in any one of claims 3-5, or the host cell as described in any one of claims 6-7.

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