A PET degrading enzyme and its application in polyethylene terephthalate degradation

Abstract

The present invention discloses a PET degrading enzyme and its application in degrading PET plastics. The present invention screened a hydrolase, LfPETase, capable of degrading PET plastics from the metagenome of landfill soil samples. Its amino acid sequence, shown in SEQ ID NO. 2, shows a maximum sequence similarity of only 39.42% with 10 currently known common PET degrading enzymes, demonstrating the novelty of its sequence backbone. The present invention constructed an engineered Pichia pastoris strain capable of efficiently expressing the gene encoding the PET degrading enzyme. The purified recombinant LfPETase was then purified and quenched with p-nitrophenyl octyl ester ( p NPO) as a substrate, the specific activity is as high as 250 U / mg; using post-consumer PCW-PET bottle flakes as a substrate, the depolymerization rate can reach 92% after degradation at 70°C for 12 h, providing a new enzyme resource for the enzymatic depolymerization and recycling of plastics.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a PET degrading enzyme and application thereof in the degradation of polyethylene terephthalate. Background Art

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

[0003] At present, bioenzymatic degradation of PET is regarded as the core path of green recycling due to its environmental friendliness and sustainability. In recent years, with the intensification of global plastic pollution, researchers have accelerated the development and optimization of PET degradation enzymes. Ideonella sakaiensis secreted by bacteria Is PETase has attracted attention due to its unique substrate recognition ability. The enzyme can catalyze the gradual 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 was screened from the branch and leaf compost metagenome. ICCG It exhibits better thermal stability and depolymerization efficiency. However, the number of PET-degrading enzymes reported so far is still limited, and only a few enzymes of microbial origin in the existing enzyme library have PET degradation activity. The large-scale application of enzymatic depolymerization of PET plastics faces another major challenge, which is whether industrial-level enzyme expression can be achieved. In recent years, with the rapid development of synthetic biology and protein engineering, researchers have significantly improved the expression level of PET-degrading enzymes through multi-dimensional strategies such as rational design, host adaptability modification, and fermentation process optimization. Secondly, most enzymes have low degradation efficiency for high-crystallinity PET (>15%). Therefore, it is necessary to provide a biological enzyme with high yield and strong degradation performance to solve the above problems. Summary of the Invention

[0004] Purpose of the Invention: The present invention addresses the shortcomings of existing technologies by providing a PET-degrading enzyme and its application in the degradation of polyethylene terephthalate (PET). The present invention discloses a novel PET-degrading enzyme identified through metagenomic screening. A recombinant expression strain of this gene was constructed, and high levels of enzyme expression were achieved through fermentation optimization. The resulting recombinant enzyme efficiently degrades PET plastic under mild conditions, achieving a degradation rate of 92% for post-consumer PET bottle-grade plastic within 12 hours. This demonstrates the significant application value of this enzyme in the fields of plastic biorecycling and environmental pollution control.

[0005] In order to solve the above technical problems, the present 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, whose amino acid sequence is shown in SEQ ID NO. 2. The highest sequence similarity with the 10 currently known common PET degrading enzymes is only 39.42%.

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

[0008] In a third aspect, the present invention provides an expression cassette or recombinant expression vector containing the gene described in the second aspect. Preferably, the recombinant expression vector is derived from the plasmid pPIC9K. Further preferably, the recombinant expression vector is obtained by cloning the gene encoding the PET degrading enzyme described in the second aspect into pPIC9K.

[0009] In a fourth aspect, the present invention provides a recombinant bacterium containing the expression cassette or recombinant expression vector described in the third aspect. Preferably, the starting strain of the recombinant bacterium is Pichia pastoris. Pichia pastoris GS115.

[0010] In a fifth aspect, the present invention provides use of the PET degrading enzyme described in the first aspect or the recombinant bacteria described in the fourth aspect in degrading polyethylene terephthalate (PET).

[0011] The PET has a crystallinity of 5% to 25%, preferably 5% to 15%, and more preferably 15%.

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

[0013] The method for degrading polyethylene terephthalate using the PET degrading enzyme described in the first aspect comprises the following steps: mixing 400-550 units of PET degrading enzyme per gram of polyethylene terephthalate, followed by a degradation reaction, with a degradation time of 10-14 hours, a degradation temperature of 70-75°C, and a degradation pH of 7.5-8.5. Preferably, 500 units of PET degrading enzyme per gram of polyethylene terephthalate are mixed, followed by a degradation reaction, with a degradation time of 12 hours, a degradation temperature of 70°C, and a degradation pH of 8. The enzyme activity (U) of the PET degrading enzyme is defined as the amount of enzyme required to produce 1 μmol of p-nitrophenol per minute using p-nitrophenyl octyl ester (pNPO) as a substrate at 37°C. Furthermore, preferably, the mixing is performed in a solvent, PB buffer.

[0014] Beneficial effects:

[0015] 1. Linking the PET-degrading enzyme to Pichia pastoris P. pastoris GS115 high expression vector pPIC9K, transformed into expression host bacteria P. pastoris GS115 was used for high-density fermentation, and the enzyme activity was 425 U / mL, which was converted into a protein expression level of 1.7 mg / mL of the target protein.

[0016] 2. The present invention is to treat the product of PET degradation enzyme gene expression with p-nitrophenyl octanoate p The enzyme activity of PET degrading enzyme was determined using NPO as 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 of up to 250 U / mg.

[0017] 3. The engineered strain constructed using this gene can efficiently express PET degrading enzyme. When using pretreated post-consumer bottle flakes PET plastic (PCW-PET) as the substrate, the depolymerization rate reached 92% at an initial substrate concentration of 200 g / L and 70°C for 12 h. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0019] Figure 1 This is a multiple sequence alignment of LfPETase.

[0020] Figure 2 This is the phylogenetic tree of LfPETase and common PET-degrading enzymes.

[0021] Figure 3This is a diagram of the high-density fermentation parameters of LfPETase.

[0022] Figure 4 This is the SDS-PAGE diagram of PET degrading enzyme. Figure 4 The first column is the protein marker, and the second column is the purified protein.

[0023] Figure 5 is the depolymerization rate of PCW-PET. DETAILED DESCRIPTION

[0024] To facilitate understanding of the present invention, the present invention will be described in more detail below with reference to 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.

[0025] Example 1 New enzyme mining

[0026] To accurately identify as many potential PET-degrading enzymes as possible, 10 reported and experimentally validated PET-degrading enzyme sequences were selected as starting sequences. Orthologous proteins were searched for in the metagenome of landfill soil samples. All retrieved results were merged to remove redundancies and 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 PET-degrading enzyme sequences. Using this method, hydrolase genes capable of degrading PET plastic were screened in the metagenome of landfill soil samples. The present invention further investigated the PET-degrading enzyme LfPETase, whose amino acid sequence was shown in SEQ ID NO. 2.

[0027] Example 2 Sequence homology and phylogenetic tree analysis

[0028] The PET degrading enzyme LfPETase (whose amino acid sequence is shown in SEQ ID NO. 2) obtained by screening in Example 1 was compared with the amino acid sequences of the currently common PET degrading enzymes ( Figure 1 ), it was found that there were many differences between its sequence and other enzymes, LfPETase and Hic, Ca PETase, LCC, Bhr PETase, Tf H, Cut190, IsThe sequence similarities of common PET-degrading enzymes such as PETase, PET12, PET5 and PET6 were 19.19%, 36.36%, 35.8%, 35.8%, 38.78%, 39.42%, 37.04%, 35.66%, 35.12% and 35.12%, respectively, indicating that their sequences are unique.

[0029] The phylogenetic tree was further constructed using MEGA ( Figure 2 ), using the neighbor-joining method to infer the evolutionary history of taxa, presents an optimal tree with a total branch length of 2.82835600. Branches are annotated with the percentage of replicates in which related taxa clustered in a bootstrap test (1000 replicates). The tree is drawn to scale, and the units of branch length correspond to evolutionary distance (evolutionary distance is calculated using the p-distance method, in units of amino acid differences per site). The analysis involved 11 amino acid sequences, and after removing positions with less than 50% site coverage, the final dataset contained 295 positions. The tree structure indicates that the PET-degrading enzyme, LfPETase, identified by the present invention, forms a separate branch.

[0030] The results of comprehensive phylogenetic analysis and multiple sequence alignment showed that the enzyme LfPETase screened by the present invention is significantly different from known PET degrading enzymes in terms of evolutionary relationship and sequence similarity, and is a new type of PET degrading enzyme.

[0031] Example 3 Expression of PET-degrading enzyme

[0032] 1. Determination of protein concentration and enzyme activity

[0033] Protein concentration determination method: The total reaction volume was 220 μL, including 200 μL Coomassie Brilliant Blue dye and 20 μL protein solution. The absorbance was measured at 595 nm using a microplate reader, and the measured protein concentration was expressed in mg / mL.

[0034] Enzyme activity assay method: The reaction system is 1 mL, specifically: 10 μL 10 mM p-nitrophenol octanoate ( p NPO), 10 μL enzyme solution, 980 μL PB buffer (pH 8.0). Heat at 37°C for 3 min. Absorbance was measured at 410 nm using a microplate reader. Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute at 37°C.

[0035] The protein expression levels described in the following examples are all the concentrations of active proteins in the protein solution. The specific enzyme activity is known to be 250 U / mg, and the calculation formula is:

[0036]

[0037] 2. Fermentation medium

[0038] The formula of the fermentation medium used in this example is as follows, wherein the percentages shown in the following formula represent w / v (g / mL) unless otherwise specified.

[0039] LLB medium: 0.5% yeast powder, 1% peptone, 0.5% NaCl (for solid plates, add 2% agar powder).

[0040] YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose. YPD plates were prepared by adding 2% agar powder to YPD liquid medium.

[0041] 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.

[0042] The PMT1 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.

[0043] 3. Construction of recombinant bacteria

[0044] 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 Novegen) was double-digested with Xho I and Pst I to obtain a linearized plasmid. The gene fragment LfPETase amplified by PCR and the linearized plasmid pPIC9K were transformed into Escherichia coli through a one-step cloning reaction. E. coli DH5α was cultured and LLB solid plates (containing antibiotics: 50 μg / mL kanamycin and 50 μg / mL bleomycin) were used to screen single clones and sequence the positive clones. E. coli DH5α / pPIC9K-LfPETase. The pPIC9K-LfPETase plasmid was linearized using the restriction endonuclease Sac I and then introduced into Pichia pastoris by electroporation. P. pastorisGS115 (purchased from Invitrogen). Monoclonal strains were screened on YPD plates (containing 100 μg / mL bleomycin) and positive expression strains were identified by colony PCR and sequencing. P. pastoris GS115 / pPIC9K -LfPETase. The nucleotide sequences of the amplification primers LfPETase-F and LfPETase-R are:

[0045] LfPETase-F: 5'-GGAGATATACATATGAAAAGATCCAACCCATAC-3' (SEQ ID NO. 3);

[0046] LfPETase-R: 5'-GTGGTGGTGCTCGAGCTGGCAGTGTCTGTTGTTAG-3' (SEQ ID NO. 4).

[0047] 4. High-density fermentation

[0048] Select a single colony with good growth from the YPD plate and inoculate it into a shake flask containing YPD liquid medium. Incubate it at 30℃ and 250 rpm for 24 h to activate the bacteria and obtain an activated bacterial solution. Inoculate the activated bacterial solution into a new shake flask containing YPD liquid medium at a ratio of 4% v / v and incubate it at 30℃ and 250 rpm until the OD 600 >10.

[0049] The cultured bacterial liquid was inoculated into a 5 L fermenter filled with BSM liquid medium (10% v / v) at an inoculum volume of 8% v / v for fermentation as follows:

[0050] During the glycerol batch fermentation phase, the following conditions were used: pH maintained at 6.0 with 25% v / v ammonia, temperature controlled at 30°C, aeration at 1-2 vvm, and dissolved oxygen maintained at 30-60% by controlling the speed and aeration rate. Glycerol depletion was determined by a sudden change in dissolved oxygen. When glycerol was depleted, a glycerol feed phase was initiated (a 25% v / v glycerol solution was fed via a fed-batch method at a rate of 30 mL / L / h). During this phase, the following conditions were used: pH maintained at 6.0 with 25% v / v ammonia, temperature controlled at 30°C, aeration at 2 vvm, and dissolved oxygen maintained at 20-30% by controlling the speed and aeration rate. After the desired biomass reached 250 g / L, glycerol feeding was stopped and starvation treatment was carried out for 1 h to ensure complete depletion of glycerol. Then, methanol-induced feeding (methanol was added by feeding at a flow rate of 10 L / h) was carried out. The conditions in this stage were: pH was maintained at 5.5 using 25% v / v ammonia water, temperature was controlled at 28°C, ventilation was 2 vvm, and dissolved oxygen was maintained between 20-30% by feeding-linked dissolved oxygen. Figure 3 As shown in the figure, the final protein concentration of the fermentation supernatant was 2.48 mg / mL, and the enzyme activity was 425 U / mL. Using the above protein expression calculation formula, the target protein content was 1.7 mg / mL.

[0051] Example 4 Purification of PET Degrading Enzyme

[0052] The fermentation supernatant from Example 3 was taken and purified using an AKTA protein purifier. The affinity chromatography prepacked 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. Contaminants were washed away with 50 mM PBS pH 8.0, and the target protein was eluted with 200 mM imidazole (pH 8.0). The eluate was collected in a tube every 5 mL. The purified recombinant protein was subjected to SDS-PAGE analysis. The results are shown in Figure 2. Figure 4 As shown in the protein gel image, a single target band was shown, which was similar to the predicted molecular weight of 26.2 kDa. p The specific activity of the enzyme was 250 U / mg as determined by the NPO method and the Coomassie Brilliant Blue method.

[0053] The formulas of the buffers used in the above protein purification are as follows:

[0054] 50 mM PBS (1 L): 0.34 g KH2PO4, 6.74 g Na2HPO4, 17.53 g NaCl, pH 8.0;

[0055] 500 mM imidazole (1 L): 6.46 g KH2PO4, 0.36 g Na2HPO4, 17.53 g NaCl, 34.04 g imidazole, pH 8.0.

[0056] Example 5 Ability of PET-degrading enzyme to degrade PET

[0057] 1. Preparation of degradation substrate

[0058] Preparation of post-consumer recycled PET powder (PCW-PET): PET flakes were washed at high temperature to remove surface oil and then amorphized using a twin-screw extruder. The extruded plastic was then rapidly recrystallized in water at 20°C. The resulting recrystallized plastic was micronized using a disc crusher at room temperature and then sieved to obtain a PET powder with a particle size less than 380 μm. The crystallinity, as determined by DSC, was 15%.

[0059] 2. Ability to degrade PCW-PET

[0060] The PCW-PET prepared in this example was used as the reaction substrate, 10 g of substrate and 20 mg of the pure enzyme prepared in Example 4 were added, and 50 mM PB buffer (pH = 8.0) was added to 50 mL. The reaction was carried out at 70 ° C for 12 h. During the reaction, the pH = 8 was maintained with a 2 M sodium hydroxide solution (alkaline solution was automatically added by a combined acidity controller and a peristaltic pump). Samples were taken at regular intervals, and the depolymerization rate was calculated by liquid phase detection of the product terephthalic acid (TPA) and mono (2-hydroxyethyl) terephthalate (MHET) concentrations. The depolymerization rate calculation formula is:

[0061]

[0062] The results are as follows Figure 5 As shown in Figure 3, the reaction stabilized after 12 h, at which time the depolymerization rate was 92%.

[0063] An Agilent 1260 Infinity II HPLC system was used for analysis using 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 produced were calculated based on a standard curve prepared from standard samples. The PET degradation products were filtered using a 0.22 μm filter and diluted appropriately according to the product concentration.

[0064] The present invention provides a PET-degrading enzyme and its application in the degradation of polyethylene terephthalate (PET). Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of the present invention. Any components not specified in this embodiment may 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 in SEQ ID NO.

2.

2. A gene encoding the PET degrading enzyme according to claim 1.

3. The gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

4. An expression cassette or recombinant expression vector containing the gene according to claim 2 or 3.

5. The expression cassette or recombinant expression vector according to claim 4, characterized in that The starting vector of the recombinant expression vector is plasmid pPIC9K.

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

7. The recombinant bacterium according to claim 6, characterized in that The starting strain of the recombinant bacteria is Pichia pastoris Pichia pastoris GS115.

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

9. The use according to claim 8, characterized in that The crystallinity of the polyethylene terephthalate is 5% to 15%.

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

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