PET degrading enzyme and application thereof in degradation of polyethylene glycol terephthalate

Abstract

The invention discloses a PET (Polyethylene Terephthalate) degrading enzyme and an application thereof in the aspect of degrading PET plastics. The hydrolase LfPETase capable of degrading the PET plastic is screened from metagenomes of landfill soil samples of a garbage dump, the amino acid sequence of the hydrolase LfPETase is shown as SEQ ID NO.2, the similarity between the amino acid sequence of the hydrolase LfPETase and the highest sequence of 10 kinds of currently known common PET degrading enzymes is only 39.42%, and the novelty of a sequence skeleton of the hydrolase LfPETase is indicated. According to the invention, a pichia pastoris engineering strain capable of efficiently expressing and coding the PET degrading enzyme gene is constructed, and the specific activity of the recombinant degrading enzyme LfPETase obtained by purification is up to 250 U / mg when p-nitrophenyl octyl ester (pNPO) is taken as a substrate; after consumption, PCW-PET bottle flake plastic is taken as a substrate, degradation is carried out at 70 DEG C for 12 h, the depolymerization rate can reach 92%, and a new enzyme resource is provided for plastic enzymatic depolymerization recycling.

Description

Technical Field

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

[0002] Polyethylene terephthalate (PET) is a linear polymer formed by the repeated connection of terephthalic acid (TPA) and ethylene glycol (EG) monomers through ester bonds. The high-density ester bonds and crystalline region structure in its molecular chain endow it with excellent mechanical strength and chemical inertness, but also result in a degradation period of up to hundreds of years in the natural environment. Traditional PET recycling technologies (such as pyrolysis and methanol alcoholysis) rely on high temperature and high pressure conditions, and have problems such as high energy consumption, low product purity (containing tar by-products), and strong equipment corrosion, making it difficult to achieve a closed-loop circular economy.

[0003] Currently, the enzymatic degradation of PET is regarded as the core path of green recycling due to its environmental protection and sustainability. In recent years, with the exacerbation of the global plastic pollution problem, researchers have accelerated the development and optimization of PET degrading enzymes. Among them, Ideonella sakaiensis PETase secreted by Is bacteria 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 ICCG screened from leaf compost metagenome shows better thermal stability and depolymerization efficiency. However, the currently reported PET degrading enzymes are still limited, and only a few enzymes from microbial sources in the existing enzyme library have PET degrading activity. Another major challenge for the large-scale application of enzymatic depolymerization of PET plastic 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 highly crystalline PET (>15%). Therefore, there is a need to provide a biological enzyme with high yield and strong degradation performance to solve the above problems. Summary of the Invention

[0004] Objective of the Invention: The technical problem to be solved by the present invention is to provide a PET-degrading enzyme and its application in the degradation of polyethylene terephthalate (PET) in view of the deficiencies of the prior art. The present invention discloses a novel PET-degrading enzyme screened by metagenomics, constructs a recombinant expression strain of this gene, and realizes a high level of enzyme expression through fermentation optimization means. The corresponding recombinant enzyme obtained therefrom can efficiently degrade PET plastics under mild conditions, and the degradation rate of post-consumer PET bottle-grade plastics reaches 92% within 12 hours, indicating that this enzyme has great application value in the fields of plastic biorecycling and environmental pollution control.

[0005] To solve the above technical problems, the present invention discloses a PET-degrading enzyme and its application in PET degradation. The specific technical solutions are as follows: In the first aspect, the present invention provides a PET-degrading enzyme LfPETase, and the amino acid sequence of the PET-degrading enzyme is shown as SEQ ID NO.2. The highest sequence similarity with the currently known 10 common PET-degrading enzymes is only 39.42%.

[0006] In the second aspect, the present invention provides a gene encoding the PET-degrading enzyme described in the first aspect. Preferably, its nucleotide sequence is shown as SEQ ID NO.1. The full length of the gene LfPETase of the PET-degrading enzyme (from the start codon to the stop codon) is 744 bp, encoding 247 amino acids, and the theoretical molecular weight of the protein is 26.2 kDa.

[0007] In the third aspect, 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. Further preferably, the recombinant expression vector is obtained by cloning the gene encoding the PET-degrading enzyme described in the second aspect into pPIC9K.

[0008] In the 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 bacterium of the recombinant bacterium is Pichia pastoris Pichia pastoris GS115.

[0009] In the fifth aspect, the present invention provides the application of the PET-degrading enzyme described in the first aspect or the recombinant bacterium described in the fourth aspect in the degradation of polyethylene terephthalate (PET).

[0010] Among them, the crystallinity of the PET is 5% - 25%. Preferably, the crystallinity is 5% - 15%, and further preferably 15%.

[0011] The PET-degrading enzyme described in the present invention is used for the enzymatic depolymerization and cyclic recycling of PET waste bottle chips plastics.

[0012] Among them, the method for degrading polyethylene terephthalate using the PET degrading enzyme described in the first aspect includes the following steps: Mix 1 g of polyethylene terephthalate with 400 - 550 U of PET degrading enzyme and then carry out a degradation reaction. The degradation time is 10 - 14 h, the degradation temperature is 70 - 75 °C, and the degradation pH is 7.5 - 8.5. Preferably, 1 g of polyethylene terephthalate is mixed with 500 U of PET degrading enzyme and then the degradation reaction is carried out. The degradation time is 12 h, the degradation temperature is 70 °C, and the degradation pH is 8. The enzyme activity unit U of the described PET degrading enzyme is defined as: at 37 °C, the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute with p-nitrophenyl octanoate (pNPO) as the substrate is used as one enzyme activity unit U. Further preferably, the mixing is carried out in the solvent PB buffer.

[0013] Beneficial effects: 1. Connect the PET degrading enzyme to the Pichia pastoris P. pastoris GS115 high-expression vector pPIC9K, transform the expression host strain P. pastoris GS115, carry out high-density fermentation, the enzyme activity is 425 U / mL, and the protein expression level of the target protein is converted to 1.7 mg / mL.

[0014] 2. For the product of the PET degrading enzyme gene expression of the present invention, use p-nitrophenyl octanoate p NPO as the substrate to measure the enzyme activity of the PET degrading enzyme. This PET degrading enzyme can effectively act on the ester bond of p-nitrophenyl octanoate and degrade it into p-nitrophenol, and the specific activity is as high as 250 U / mg.

[0015] 3. The engineered strain constructed using this gene can highly express the PET degrading enzyme. When using pretreated post-consumer bottle flake PET plastic (PCW-PET) as the substrate, at an initial substrate concentration of 200 g / L, the depolymerization rate is as high as 92% after reacting at 70 °C for 12 h. Brief description of the drawings

[0016] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0017] Figure 1 It is a multiple sequence alignment map of LfPETase.

[0018] Figure 2 It is a phylogenetic tree of LfPETase and common PET degrading enzymes.

[0019] Figure 3 It is a high-density fermentation parameter map of LfPETase.

[0020] 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.

[0021] Figure 5 is the depolymerization rate of PCW-PET. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1 New enzyme mining In order to find as many potential PET degrading enzymes as possible and accurately, 10 PET degrading enzymes that have been reported and experimentally verified to be active were selected as the starting sequences for the search, and orthologous proteins were searched in the metagenome of landfill soil samples. All retrieved results were merged to remove redundancy and a PET degrading enzyme database was constructed. These sequences were mapped to the constructed PET degrading enzyme database through a hidden Markov model (HMM), and hits with Bit Score>180 were screened as candidate sequences for PET degrading enzymes. By the above method, hydrolase genes that can degrade PET plastics were screened in the metagenome of landfill soil samples. The present invention further studied the PET degrading enzyme LfPETase whose amino acid sequence was screened as shown in SEQ ID NO.2.

[0024] Example 2 Sequence homology and phylogenetic tree analysis 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 ), and found that there are many differences between its sequence and other enzymes, LfPETase and Hic, Ca PETase, LCC, Bhr PETase, Tf H.Cut190 Is The 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.

[0025] Furthermore, a phylogenetic tree was constructed using MEGA ( Figure 2 ), and the neighbor-joining method was used to infer the evolutionary history of taxa. The optimal tree with a total branch length of 2.82835600 was shown. The percentage of replicate trees in which the associated taxa clustered was labeled beside the branches in the bootstrap test (1000 replicates). The tree was drawn to scale, and the branch length unit was consistent with the evolutionary distance unit (the evolutionary distance was calculated by the p-distance method, and the unit was the number of amino acid differences per site). The analysis involved 11 amino acid sequences. After removing positions with a site coverage rate of less than 50%, the final dataset contained 295 positions. From the structure of the tree, the PET-degrading enzyme LfPETase screened in the present invention formed an independent branch.

[0026] Comprehensive phylogenetic analysis and multiple sequence alignment results showed that the enzyme LfPETase screened in the present invention was significantly different from known PET-degrading enzymes in terms of evolutionary relationship and sequence similarity, and it was a novel PET-degrading enzyme.

[0027] Example 3 Expression of PET-degrading enzyme 1. Determination of protein concentration and enzyme activity Method for determining protein concentration: The total volume of the reaction system was 220 μL, including 200 μL of Coomassie brilliant blue dye solution and 20 μL of protein solution. The absorbance was detected at 595 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the unit of the measured protein concentration was mg / mL.

[0028] Method for determining enzyme activity: The reaction system was 1 mL, specifically: 10 μL of 10 mM p-nitrophenyl octanoate ( p NPO), 10 μL of enzyme solution, and 980 μL of PB buffer (pH 8.0). It was heated at 37 °C for 3 min, and the absorbance was detected at 410 nm using an ELISA reader. Definition of enzyme activity: At 37 °C, the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute was defined as one enzyme activity unit.

[0029] In the following examples, the protein expression level refers to the concentration of active protein in the protein solution. The known specific enzyme activity is 250 U / mg, and the calculation formula is:

[0030] 2. Fermentation medium The formula of the fermentation medium used in this example is as follows. The percentages (%) shown in the following formula represent w / v (g / mL) unless otherwise specified.

[0031] LLB medium: 0.5% yeast powder, 1% peptone, 0.5% NaCl (2% agar powder was added to the solid plate).

[0032] YPD liquid medium: 1% yeast powder, 2% peptone, 2% glucose. YPD plate is obtained by adding 2% agar powder to YPD liquid medium.

[0033] 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.

[0034] The PMT1 mentioned above is 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.

[0035] 3. Construction of recombinant bacteria Using the LfPETase gene (nucleotide sequence as shown in SEQ ID NO.1) as a template, design amplification primers LfPETase-F and LfPETase-R, and add Xho I and Pst I restriction enzyme sites to the 5' end and 3' end of the primers respectively. Linearize the plasmid vector pPIC9K (purchased from Novegen) by double digestion with Xho I and Pst I. Transform the gene fragment LfPETase obtained by PCR amplification and the linearized plasmid pPIC9K into Escherichia coli E. coli DH5α using a one-step cloning reaction, and use an LLB solid plate (containing antibiotics: 50 μg / mL kanamycin and 50 μg / mL bleomycin) to screen monoclonal colonies for sequencing verification to obtain a positive clone strain E. coli DH5α / pPIC9K-LfPETase. Linearize the pPIC9K-LfPETase plasmid using the restriction enzyme Sac I, and then introduce it into Pichia pastoris P.pastoris GS115 (purchased from Invitrogen) by electroporation. Screen monoclonal strains through a YPD plate (containing 100 μg / mL bleomycin) and perform colony PCR and sequencing identification to obtain a positive expression strain P.pastoris GS115 / pPIC9K -LfPETase. The nucleotide sequences of the amplification primers LfPETase-F and LfPETase-R are: LfPETase-F: 5'-GGAGATATACATATGAAAAGATCCAACCCATAC-3' (SEQ ID NO.3); LfPETase-R: 5'-GTGGTGGTGCTCGAGCTGGCAGTGTCTGTTGTTAG-3' (SEQ ID NO.4).

[0036] 4. High-density fermentation Pick a single colony with good growth from the YPD plate and inoculate it into a shake flask containing YPD liquid medium. Incubate at 30 °C and 250 rpm for 24 h to activate the strain, obtaining the 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 at 30 °C and 250 rpm until OD 600 > 10.

[0037] Inoculate the cultured bacterial solution into a 5 L fermenter filled with BSM liquid medium (liquid filling volume 10% v / v) at an inoculation amount of 8% v / v for fermentation, as follows: The conditions for the glycerol batch fermentation stage are: the pH is maintained at 6.0 using 25% v / v ammonia water, the temperature is controlled at 30 °C, the aeration rate is 1 - 2 vvm, and the dissolved oxygen is maintained at 30 - 60% by controlling the rotation speed and aeration rate; judge whether the glycerol is exhausted by the sharp change of dissolved oxygen. When the glycerol is exhausted, carry out the glycerol feeding (a 25% v / v glycerol solution is added by feeding, and the feeding rate is 30 mL / L / h) stage culture. The conditions for this stage are: the pH is maintained at 6.0 using 25% v / v ammonia water, the temperature is controlled at 30 °C, the aeration rate is 2 vvm, and the dissolved oxygen is maintained at 20 - 30% by controlling the rotation speed and aeration rate. After culturing to the required biomass of 250 g / L, stop the glycerol feeding and carry out starvation treatment for 1 h to ensure that the glycerol is completely exhausted, and then carry out the methanol induction feeding (methanol is added by feeding, and the flow rate is 10 L / h) stage fermentation. The conditions for this stage are: the pH is maintained at 5.5 using 25% v / v ammonia water, the temperature is controlled at 28 °C, the aeration rate is 2 vvm, and the dissolved oxygen is maintained between 20 - 30% by the method of linking dissolved oxygen and feeding. The fermentation results are as Figure 3 shown. Finally, the protein concentration in the fermentation supernatant is measured to be 2.48 mg / mL, and the enzyme activity is 425 U / mL. Using the above protein expression calculation formula, it is converted to obtain 1.7 mg / mL of the target protein.

[0038] Example 4 Purification of PET degrading enzyme The fermentation supernatant in Example 3 was taken and the protein was purified by AKTA protein purifier. The affinity chromatography pre-packed column (HisPrep FF) was pre-equilibrated with three column volumes of water and three column volumes of 50 mM PBS pH8.0, and then the sample was loaded at a flow rate of 1 mL / min, and the impurities were washed away with 50 mM PBS pH8.0, and then the target protein was eluted with 200 mM imidazole (pH8.0). The eluate was collected in one tube every 5 mL. The purified recombinant protein was subjected to SDS-PAGE detection, and the results were as follows: Figure 4 As shown in the protein gel image, a single target band is shown, which is similar to the predicted molecular weight of 26.2 kDa. p The specific activity of the enzyme measured by NPO method and Coomassie Brilliant Blue method was 250 U / mg.

[0039] The formulas of the buffers used in the above protein purification are as follows: 50 mM PBS (1 L): 0.34 g KH2PO4, 6.74 g Na2HPO4, 17.53 g NaCl, pH 8.0; 500 mM Imidazole (1 L): 6.46 g KH2PO4, 0.36 g Na2HPO4, 17.53 g NaCl, 34.04 g Imidazole, pH 8.0.

[0040] Example 5 Ability of PET-degrading enzyme to degrade PET 1. Preparation of degradation substrate Preparation of post-consumer recycled PET powder (PCW-PET): After high-temperature washing to remove surface oil, the PET flakes were amorphized using a twin-screw extruder, and then the extruded plastic was quickly placed in 20°C water for recrystallization. The obtained recrystallized plastic was micronized using a disc crusher at room temperature and then sieved to obtain PET powder with a particle size of less than 380 μm, and the DSC detected a crystallinity of 15%.

[0041] 2. Ability to degrade PCW-PET The PCW-PET prepared in this example was used as the reaction substrate, 10 g of the substrate and 20 mg of the pure enzyme prepared in Example 4 were added, 50 mM PB buffer (pH = 8.0) was added to 50 mL, and the reaction was carried out at 70 ° C for 12 h. During the reaction, 2 M sodium hydroxide solution was used to maintain pH = 8 (alkali solution was automatically added by combining the acidity controller and the peristaltic pump), and samples were taken at regular intervals. 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:

[0042] The results are as Figure 5 shown. The reaction tended to reach equilibrium after 12 h, and the depolymerization rate was 92% at this time.

[0043] Analysis was carried out using an Agilent 1260 Infinity Ⅱ high-performance liquid chromatography system through a C18 chromatographic column (Agilent 5 HC-C18(2) 150×4.6 mm). A solvent composed of 20% acetonitrile, 1% formic acid and 79% water was used as the mobile phase, 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 the generated TPA and MHET were calculated according to the standard curve made from the standard samples. The enzymatic hydrolysis products of PET degradation were filtered using a 0.22 μm filter and reasonably diluted according to the product concentration.

[0044] The present invention provides an idea and method for a PET-degrading enzyme and its application in the degradation of polyethylene terephthalate. There are many methods and ways to specifically implement this technical solution. The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by the prior art.

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, wherein The starting strain of the recombinant strain 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 application according to claim 8, wherein The crystallinity of the polyethylene terephthalate is 5% to 25%.

10. The application according to claim 8, wherein, The method for degrading polyethylene terephthalate using the PET-degrading enzyme according to claim 1 comprises the following steps: 400 - 550 U of the PET-degrading enzyme is mixed with each gram of polyethylene terephthalate and then subjected to a degradation reaction, the degradation time is 10 - 14 h, the degradation temperature is 70 - 75 °C, and the degradation pH is 7.5 - 8.5.

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