Pet-degrading enzyme and use thereof in degradation of polyethylene terephthalate

Abstract

Provided are a PET-degrading enzyme and the use thereof in the degradation of PET plastics. LfPETase, a hydrolase capable of degrading PET plastics, is screened from the metagenome of a landfill soil sample, and the amino acid sequence thereof is as shown in SEQ ID NO. 2, which has the highest sequence similarity to the 10 currently known common PET-degrading enzymes of only 39.42%, indicating the novelty of the sequence scaffold thereof. An engineered Pichia pastoris strain capable of efficiently expressing a gene encoding the PET-degrading enzyme is constructed. The purified recombinant degrading enzyme LfPETase exhibits a specific activity of up to 250 U / mg when p-nitrophenyl octanoate (pNPO) is used as the substrate, and has a depolymerization rate of up to 92% after degradation at 70°C for 12 h when post-consumer waste PET (PCW-PET) bottle flakes are used as the substrate, thereby providing a new enzyme resource for the enzymatic depolymerization and recycling of plastics.

Description

A PET-degrading enzyme and its application in the degradation of polyethylene terephthalate. Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a PET degrading enzyme and its application in the degradation of polyethylene terephthalate. Background Technology

[0002] Polyethylene terephthalate (PET) is a linear polymer formed by the repeated ester bonds connecting terephthalic acid (TPA) and ethylene glycol (EG) monomers. Its high-density ester bonds and crystalline structure in the molecular chain endow it with excellent mechanical strength and chemical inertness, but also result in a degradation cycle of hundreds of years in the natural environment. Traditional PET recycling technologies (such as thermal cracking and methanol hydrolysis) rely on high temperature and high pressure conditions, resulting in high energy consumption, low product purity (containing tar byproducts), and highly corrosive equipment, making it difficult to achieve a closed-loop circular economy.

[0003] Currently, enzymatic degradation of PET is considered a core pathway for green recycling due to its environmental friendliness and sustainability. In recent years, with the increasing severity of global plastic pollution, researchers have accelerated the development and optimization of PET-degrading enzymes. Among them, IsPETase, secreted by *Ideonella sakaiensis*, has attracted attention due to its unique substrate recognition ability. This enzyme can catalyze the stepwise depolymerization of PET at room temperature, but its poor thermal stability and low catalytic efficiency limit its industrial application. On the other hand, the cutinase LCC variant LCC, screened through metagenomic analysis of leaf and branch compost, has also gained attention. ICCG These enzymes exhibit superior thermal stability and depolymerization efficiency. However, the number of reported PET-degrading enzymes remains limited, with only a few microbial enzymes in existing enzyme libraries possessing PET-degrading activity. The large-scale application of enzymatic depolymerization of PET plastics faces another major challenge: achieving industrial-scale enzyme expression levels. In recent years, with the rapid development of synthetic biology and protein engineering, researchers have significantly improved the expression levels of PET-degrading enzymes through multi-dimensional strategies such as rational design, host adaptability modification, and fermentation process optimization. Secondly, most enzymes exhibit low degradation efficiency for highly crystalline PET (>15%). Therefore, a high-yield bioenzyme with strong degradation performance is needed to address these issues. Summary of the Invention

[0004] Objective of the Invention: The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a PET-degrading enzyme and its application in the degradation of polyethylene terephthalate (PET). This invention discloses a novel PET-degrading enzyme screened through metagenomics, constructing a recombinant expression strain of this gene, and achieving a high level of enzyme expression through fermentation optimization. The resulting recombinant enzyme can efficiently degrade PET plastic under mild conditions, achieving a degradation rate of 92% for post-consumer PET bottle-grade plastic within 12 hours, indicating that this enzyme has significant application value in the fields of plastic biorecycling and environmental pollution control.

[0005] To address the aforementioned technical problems, this invention discloses a PET-degrading enzyme and its application in PET degradation. The specific technical solution is as follows:

[0006] In a first aspect, the present invention provides a PET degrading enzyme, LfPETase, the amino acid sequence of which is shown in SEQ ID NO.2. The highest sequence similarity with 10 currently known common PET degrading enzymes is only 39.42%.

[0007] Secondly, the present invention provides a gene encoding the PET degrading enzyme described in the first aspect. Preferably, its nucleotide sequence is shown in SEQ ID NO.1. The full-length (from start codon to stop codon) LfPETase gene of the PET degrading enzyme is 744 bp, encoding 247 amino acids, and the theoretical molecular weight of the protein is 26.2 kDa.

[0008] Thirdly, the present invention provides an expression cassette or recombinant expression vector containing the gene described in the second aspect. Preferably, the starting vector of the recombinant expression vector is plasmid pPIC9K. More preferably, the recombinant expression vector is obtained by cloning the gene encoding the PET degrading enzyme described in the second aspect into pPIC9K.

[0009] Fourthly, the present invention provides a recombinant bacterial strain containing the expression cassette or recombinant expression vector described in the third aspect. Preferably, the originating strain of the recombinant bacterial strain is Pichia pastoris GS115.

[0010] Fifthly, the present invention provides the application of the PET-degrading enzyme described in the first aspect or the recombinant bacteria described in the fourth aspect in the degradation of polyethylene terephthalate (PET).

[0011] The crystallinity of the PET is 5% to 25%. Preferably, the crystallinity is 5% to 15%, and more preferably 15%.

[0012] The PET degrading enzyme described in this invention is used for the enzymatic depolymerization and recycling of waste PET bottle flakes.

[0013] The method for degrading polyethylene terephthalate (PET) using the PET degrading enzyme described in the first aspect includes the following steps: Each g of PET is mixed with 400-550 U of PET degrading enzyme, and the degradation reaction is carried out for 10-14 hours at a temperature of 70-75°C and a pH of 7.5-8.5. Preferably, each g of PET is mixed with 500 U of PET degrading enzyme, and the degradation reaction is carried out for 12 hours at a temperature of 70°C and a pH of 8. The enzyme activity U of the PET degrading enzyme is defined as the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute using p-nitrophenyl octyl ester (pNPO) as a substrate at 37°C. More preferably, the mixing is carried out in a PB buffer solution. Beneficial effects

[0014] 1. The PET degrading enzyme was ligated into the Pichia pastoris GS115 high expression vector pPIC9K, transformed into the expression host strain P. pastoris GS115, and fermented at high density. The enzyme activity was 425 U / mL, which is equivalent to a protein expression level of 1.7 mg / mL.

[0015] 2. The present invention describes the enzyme activity of PET degrading enzyme by expressing the gene of PET degrading enzyme using p-nitrophenyl octanoate pNPO as a substrate. The PET degrading enzyme can effectively act on the ester bond of p-nitrophenyl octanoate and degrade it into p-nitrophenol, with a specific activity as high as 250 U / mg.

[0016] 3. The engineered strain constructed using this gene can efficiently express PET degrading enzyme. When using pretreated post-consumer bottle flake PET plastic (PCW-PET) as substrate, the depolymerization rate is as high as 92% at an initial substrate concentration of 200 g / L and a reaction temperature of 70℃ for 12 h. Attached Figure Description

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0018] Figure 1 shows the multiple sequence alignment diagram of LfPETase.

[0019] Figure 2 shows the phylogenetic tree of LfPETase and common PET-degrading enzymes.

[0020] Figure 3 shows the high-density fermentation parameters of LfPETase.

[0021] Figure 4 shows the SDS-PAGE of PET-degrading enzymes. The first column in Figure 4 is the protein marker, and the second column is the purified protein.

[0022] Figure 5 shows the depolymerization rate of PCW-PET. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more detailed description is provided below with reference to the accompanying drawings and specific embodiments. However, the present invention is not limited to the embodiments described in this specification. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0024] To identify as many potential PET degrading enzymes as possible accurately, 10 reported and experimentally validated PET degrading enzymes were selected as starting sequences for the search. Orthologous proteins were searched in the metagenomics of landfill soil samples. All retrieved results were merged and redundancy removed to construct a PET degrading enzyme database. These sequences were mapped to the constructed PET degrading enzyme database using a Hidden Markov Model (HMM), and Hits with a Bit Score > 180 were selected as candidate sequences for PET degrading enzymes. Using the above method, hydrolytic enzyme genes capable of degrading PET plastic were screened from the metagenomics of landfill soil samples. This invention further investigated the PET degrading enzyme LfPETase, whose amino acid sequence is shown in SEQ ID NO. 2.

[0025] By performing multiple sequence alignment (Figure 1) on the PET degrading enzyme LfPETase (whose amino acid sequence is shown in SEQ ID NO.2) obtained in Example 1 with the amino acid sequences of commonly used PET degrading enzymes, it was found that there are many differences between its sequence and those of other enzymes. The sequence similarity of LfPETase with common PET degrading enzymes such as Hic, CaPETase, LCC, BhrPETase, TfH, Cut190, IsPETase, PET12, PET5 and PET6 is 19.19%, 36.36%, 35.8%, 35.8%, 38.78%, 39.42%, 37.04%, 35.66%, 35.12% and 35.12%, respectively, showing that its sequence is unique.

[0026] Further, a phylogenetic tree was constructed using MEGA (Figure 2), and the evolutionary history of taxa was inferred using the neighbor-joining method. The optimal tree with a total branch length of 2.82835600 was shown. The percentage of repeated trees in which related taxa clustered in the bootstrap test (1000 repetitions) was labeled next to each branch. The tree was drawn to scale, and the unit of branch length was consistent with the unit of evolutionary distance (evolutionary distance was calculated using the p-distance method, with the unit being the number of amino acid differences per site). The analysis involved 11 amino acid sequences. After removing sites with less than 50% site coverage, the final dataset contained 295 sites. From the tree structure, the PET-degrading enzyme LfPETase screened in this invention formed an independent branch.

[0027] The results of comprehensive phylogenetic analysis and multiple sequence alignment show that the enzyme LfPETase screened in this invention is significantly different from known PET degrading enzymes in both evolutionary relationship and sequence similarity, and is a novel PET degrading enzyme.

[0028] 1. Determination of protein concentration and enzyme activity

[0029] Protein concentration determination method: The total volume of the reaction system was 220 μL, including 200 μL of Coomassie brilliant blue staining solution and 20 μL of protein solution. The absorbance was detected at 595 nm using an ELISA reader, and the protein concentration was measured in mg / mL.

[0030] Enzyme activity assay: The reaction system was 1 mL, specifically: 10 μL 10 mM p-nitrophenol octanoate (pNPO), 10 μL enzyme solution, and 980 μL PB buffer (pH 8.0). The mixture was heated at 37 ℃ for 3 min, and the absorbance was measured at 410 nm using a microplate reader. Enzyme activity definition: The amount of enzyme required to generate 1 μmol of p-nitrophenol per minute at 37 ℃ is defined as one unit of enzyme activity.

[0031] The protein expression levels mentioned in the following examples are all concentrations of active protein in protein solutions. The specific enzyme activity is known to be 250 U / mg, and the calculation formula is as follows:

[0032] 2. Fermentation medium

[0033] The fermentation medium used in this embodiment has the following formulation, where the percentages (%) shown in the following formulations represent w / v (g / mL) unless otherwise specified.

[0034] LLB medium: 0.5% yeast extract, 1% peptone, 0.5% NaCl (2% agar powder added to solid plates).

[0035] YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose. YPD plates are prepared by adding 2% agar powder to YPD liquid medium.

[0036] BSM liquid medium: 2.67% H3PO4, 0.093% CaSO4·2H2O, 1.82% K2SO4, 1.49% MgSO4·2H2O, 0.413% KOH, 4% v / v glycerol, 0.4% PMT1.

[0037] The PMT1 mentioned is a PMT1 salt solution: 0.6% CuSO4·5H2O, 8.8×10 -4 % KI, 0.3% MnSO4·H2O, 0.02% Na2MoO4·2H2O, 2×10 -3 % H3BO3, 0.05% CoCl2·6H2O, 2% ZnCl2, 6.5% FeSO4·7H2O, 0.02% Biotin, 0.5% Concentrated Sulfuric Acid.

[0038] 3. Construction of recombinant bacteria

[0039] Using the LfPETase gene (nucleotide sequence shown in SEQ ID NO.1) as a template, amplification primers LfPETase-F and LfPETase-R were designed, and Xho I and Pst I restriction sites were added to the 5' and 3' ends of the primers, respectively. The plasmid vector pPIC9K (purchased from Noven) was digested with Xho I and Pst I to obtain a linearized plasmid. The PCR-amplified gene fragment LfPETase and the linearized plasmid pPIC9K were transformed into E. coli DH5α through a one-step cloning reaction. Single clones were screened using LLB solid plates (containing antibiotics: 50 μg / mL kanamycin and 50 μg / mL bleomycin), and positive clones were obtained by sequencing verification, namely E. coli DH5α / pPIC9K-LfPETase. The pPIC9K-LfPETase plasmid was linearized using the restriction endonuclease Sac I, and then introduced into *Pichia pastoris* GS115 (purchased from Invitrogen) via electrotransduction. Single clones were screened using YPD plates (containing 100 μg / mL bleomycin) and identified by colony PCR and sequencing to obtain the positive expression strain *P. pastoris* GS115 / pPIC9K-LfPETase. The nucleotide sequences of the amplification primers LfPETase-F and LfPETase-R are as follows:

[0040] LfPETase-F: 5'-GGAGATATACATATGAAAAGATCCAACCCATAC-3' (SEQ ID NO. 3);

[0041] LfPETase-R: 5'-GTGGTGGTGCTCGAGCTGGCAGTGTCTGTTGTTAG-3' (SEQ ID NO. 4).

[0042] 4. High-density fermentation

[0043] Select healthy single colonies from YPD plates and inoculate them into shake flasks containing YPD liquid medium. Incubate at 30 °C and 250 rpm for 24 h to activate the bacterial culture and obtain the activated bacterial solution. Inoculate the activated bacterial solution into fresh shake flasks containing YPD liquid medium at a ratio of 4% v / v and incubate at 30 °C and 250 rpm until OD reaches 0.5%. 600 >10.

[0044] The cultured bacterial suspension was inoculated into a 5 L fermenter containing BSM liquid medium (10% v / v) at an inoculation rate of 8% v / v for fermentation, as follows:

[0045] The conditions for the batch fermentation stage of glycerol were as follows: pH was maintained at 6.0 using 25% v / v ammonia, temperature was controlled at 30 ℃, and aeration rate was 1-2 vvm. Dissolved oxygen was maintained at 30-60% by controlling the aeration rate and rotation speed. Rapid changes in dissolved oxygen were used to determine if glycerol was depleted. When glycerol was depleted, a glycerol feeding stage was initiated (25% v / v glycerol solution was added via a feedstock at a rate of 30 mL / L / h). The conditions for this stage were: pH maintained at 6.0 using 25% v / v ammonia, temperature controlled at 30 ℃, and aeration rate of 2 vvm. Dissolved oxygen was maintained at 20-30% by controlling the aeration rate and rotation speed. After reaching the required biomass of 250 g / L, glycerol feeding was stopped and the mixture was starved for 1 h to ensure complete glycerol depletion. Then, methanol-induced fed-batch fermentation (methanol was added via a feed-batch method at a flow rate of 10 L / h) was initiated. The conditions for this stage were: pH maintained at 5.5 using 25% v / v ammonia, temperature controlled at 28℃, aeration rate of 2 vvm, and dissolved oxygen maintained between 20-30% through a combined dissolved oxygen feeding and aeration system. The fermentation results are shown in Figure 3. The final supernatant protein concentration was measured to be 2.48 mg / mL, and the enzyme activity was 425 U / mL. Using the formula for calculating protein expression, the target protein concentration was calculated to be 1.7 mg / mL.

[0046] The fermentation supernatant from Example 3 was used to purify the protein using an AKTA protein purifier. An affinity chromatography pre-packed column (HisPrep FF) was pre-equilibrated with three column volumes of water and three column volumes of 50 mM PBS (pH 8.0). The sample was then loaded at a flow rate of 1 mL / min, and impurities were washed away with 50 mM PBS (pH 8.0). The target protein was then eluted with 200 mM imidazole (pH 8.0), with elution buffer collected in 5 mL tubes. The purified recombinant protein was analyzed by SDS-PAGE. The results are shown in Figure 4, showing a single target band in the gel image, similar to the predicted molecular weight of 26.2 kDa. The specific activity of the enzyme was determined to be 250 U / mg using the pNPO method and Coomassie Brilliant Blue method.

[0047] The formulations of the buffers used in the above protein purification are as follows:

[0048] 50 mM PBS (1 L): 0.34 g KH2PO4, 6.74 g Na2HPO4, 17.53 g NaCl, pH 8.0;

[0049] 500 mM imidazole (1 L): 6.46 g KH2PO4, 0.36 g Na2HPO4, 17.53 g NaCl, 34.04 g imidazole, pH 8.0.

[0050] 1. Preparation of degradation substrates

[0051] Preparation of post-consumer recycled PET powder (PCW-PET): After high-temperature washing to remove surface oil, PET sheets are amorphized using a twin-screw extruder. The extruded plastic is then rapidly recrystallized in water at 20°C. The resulting recrystallized plastic is micronized at room temperature using a disc crusher, followed by sieving to obtain PET powder with a particle size less than 380 μm. DSC analysis showed a crystallinity of 15%.

[0052] 2. Ability to degrade PCW-PET

[0053] Using the PCW-PET prepared in this example as the reaction substrate, 10 g of substrate and 20 mg of the pure enzyme prepared in Example 4 were added. The volume was increased to 50 mL with 50 mM PB buffer (pH=8.0), and the reaction was carried out at 70 °C for 12 h. During the reaction, the pH was maintained at 8 with 2 M sodium hydroxide solution (the alkaline solution was automatically added by a combination of acidity controller and peristaltic pump). Samples were taken at regular intervals, and the depolymerization rate was calculated by detecting the concentrations of terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) in the product using liquid chromatography. The formula for calculating the depolymerization rate is as follows:

[0054] The results are shown in Figure 5. The reaction tended to stabilize after 12 hours, at which point the depolymerization rate was 92%.

[0055] Analysis was performed using an Agilent 1260 Infinity II high-performance liquid chromatography system with a C18 column (Agilent 5 HC-C18 (2) 150 × 4.6 mm). The mobile phase consisted of 20% acetonitrile, 1% formic acid, and 79% water. The temperature was 30°C, the injection volume was 10 μL, the detection time was 12 min, the flow rate was 0.8 mL / min, and the detection wavelength was 240 nm. The concentrations of TPA and MHET were calculated based on a standard curve prepared from standard samples. PET degradation enzyme hydrolysates were filtered using a 0.22 μm filter and diluted appropriately according to the product concentration.

[0056] This invention provides a PET-degrading enzyme and its application in the degradation of polyethylene terephthalate (PET). Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A PET-degrading enzyme, characterized in that, The amino acid sequence of the PET-degrading enzyme is shown as SEQ ID NO.

2.

2. A gene encoding the PET-degrading enzyme of claim 1.

3. The gene of claim 2, wherein, The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

4. An expression cassette or a recombinant expression vector containing the gene of claim 2 or 3.

5. The expression cassette or recombinant expression vector of claim 4, wherein, The starting vector of the recombinant expression vector is plasmid pPIC9K.

6. A recombinant bacterium containing the expression cassette or the recombinant expression vector of claim 4.

7. The recombinant bacteria of claim 6, wherein The starting bacterium of the recombinant bacterium is Pichia pastoris GS115.

8. Use of the PET-degrading enzyme of claim 1 or the recombinant bacterium of claim 6 or 7 in degrading polyethylene terephthalate.

9. Use according to claim 8, characterized in that, The crystallinity of the polyethylene terephthalate is 5% to 25%.

10. Use according to claim 8, characterized in that, The method for degrading polyethylene terephthalate by using the PET-degrading enzyme of claim 1 comprises the following steps: mixing 400-550 U of the PET-degrading enzyme with per gram of polyethylene terephthalate, and then performing a degradation reaction, the degradation time is 10-14 h, the degradation temperature is 70-75℃, and the degradation pH is 7.5-8.5.

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