An enzyme and its use in degrading PET plastic

Abstract

This invention relates to the field of protein engineering technology, specifically to an enzyme and its use in degrading PET plastic. The enzyme provided by this invention comprises any one of the following: (1) an amino acid sequence as shown in SEQ ID NO.1; (2) an amino acid sequence having at least 50% identity with the amino acid sequence shown in SEQ ID NO.1 and possessing enzymatic activity; the above enzyme can be used as a degrading enzyme to degrade plastics, especially as a PET degrading enzyme to degrade PET plastics.

Description

Technical Field

[0001] This invention relates to the field of protein engineering technology, specifically to an enzyme and its use in degrading PET plastic. Background Technology

[0002] Polyethylene terephthalate (PET) is widely used in food and pharmaceutical packaging due to its excellent physical properties and low cost. However, PET's chemical inertness and extremely low natural degradation rate make it a persistent environmental pollutant, and traditional recycling methods are limited by high energy consumption, high cost, and potential secondary pollution problems. PET-degrading enzymes can depolymerize PET, breaking the ester bonds of long-chain polymers and converting them into monomers or small molecules, such as terephthalic acid (TPA) and ethylene glycol (EG). However, the degradation efficiency and stability of existing wild-type enzymes are insufficient to meet industrial requirements, and their ability to degrade highly crystalline PET is poor, especially at low and medium temperatures, where the degradation efficiency is particularly low. Currently, most PET hydrolase mutants that have some ability to degrade highly crystalline PET at low and medium temperatures are modified from IsPETase (Yoshida et al., 2016) found in Ideonella sakaiensis 201-F6, but their wild-types have poor reactivity in high-crystalline PET at low and medium temperatures and poor ability to completely hydrolyze PET into monomers. Therefore, there is an urgent need for a PET hydrolase that can exhibit high reactivity with highly crystalline PET at medium and low temperatures. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide an enzyme and its use in degrading PET plastic.

[0004] Therefore, the present invention provides the following technical solution:

[0005] This invention discloses an enzyme comprising any one of the following:

[0006] (1) Contains the amino acid sequence shown in SEQ ID NO.1;

[0007] (2) An amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO.1 and has enzyme activity.

[0008] In some embodiments, in the amino acid sequence having at least 50% identity with the amino acid sequence shown in SEQ ID NO. 1 and having enzymatic activity, the identity with the amino acid sequence shown in SEQ ID NO. 1 can be at least 50%, 60%, 70%, 80%, or 90% or more. In a preferred embodiment, it is the sequence shown in SEQ ID NO. 1 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, or even more preferably at least 99% or more sequence identity with it.

[0009] The enzyme of the present invention can be obtained by substituting, deleting, and / or adding at least one amino acid based on the specific amino acid sequences listed above, and the resulting enzyme has enzymatic activity. The number of amino acids substituted, deleted, or added can be any value, such as 1, 5, 10, 15, or more, such that the sequence identity between the changed amino acid sequence and its corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0010] As used in this article, the term “sequence identity” refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences.

[0011] In some embodiments, the enzyme is derived from the prokaryotic bacterium Asanoahainanensis.

[0012] This invention discloses a nucleic acid molecule comprising a nucleotide sequence encoding the enzyme described above. In a preferred embodiment, the nucleic acid molecule is shown in SEQ ID NO.2.

[0013] As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably, including DNA molecules or RNA molecules. DNA molecules can be single-stranded or double-stranded.

[0014] Due to the degeneracy of the genetic code, a large number of polynucleotides that can be used to encode the enzymes of this invention can be obtained. Therefore, given the identification of a specific amino acid sequence, those skilled in the art can easily prepare any number of different nucleic acids by modifying the sequence of one or more codons without altering the amino acid sequence encoding the protein. More preferred polynucleotides can be selected through codon optimization based on the preferences of the host cells used in the actual preparation process.

[0015] The polynucleotides can be obtained using conventional methods, such as PCR amplification or artificial synthesis. Currently, the polynucleotide sequences can be obtained entirely through chemical synthesis.

[0016] In some preferred, but not limiting, embodiments, the nucleotide sequence is represented by SEQ ID NO.2.

[0017] This invention discloses a carrier comprising the aforementioned nucleic acid molecule.

[0018] In some embodiments, the vector may be a recombinant vector or an expression vector, which can be transformed into host cells to express the nucleic acid molecule or the enzyme.

[0019] As used herein, "vector" refers to a construct capable of delivering, preferably expressing in, a host cell, one or more target genes or sequences. Vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules, or phage vectors, DNA or RNA expression vectors bound to cationic condensers, and DNA or RNA expression vectors encapsulated in liposomes.

[0020] This invention discloses a host cell, the host cell comprising:

[0021] Express the PET-degrading enzyme described above;

[0022] And / or, comprising the aforementioned nucleic acid molecules;

[0023] And / or, includes the carrier described above.

[0024] The host cell of this invention can be a prokaryotic cell, a lower eukaryotic cell, or a higher eukaryotic cell. Prokaryotic cells include bacterial cells, lower eukaryotic cells include yeast cells, and higher eukaryotic cells include mammalian cells. Representative examples include Escherichia coli and Bacillus subtilis.

[0025] In some preferred, but not limiting, embodiments, the host cell includes *Asanoa hainanensis*.

[0026] Transforming the vector into host cells can be performed using conventional methods well known to those skilled in the art. Examples include the CaCl2 method, electroporation, calcium phosphate co-precipitation, and conventional mechanical methods such as microinjection, electroporation, and liposome packaging. The resulting transformants can be cultured using conventional methods well known to those skilled in the art, and the culture medium can be a standard culture medium. The nanobodies generated from the transformants can be separated and purified using physical and chemical methods, such as salting out, centrifugation, cell disruption, and chromatography.

[0027] This invention discloses a fusion protein containing the aforementioned enzyme. The enzyme can be fused with other functional polypeptides.

[0028] The embodiments of the present invention disclose the use of the enzyme as a degrading enzyme or for preparing a degrading enzyme.

[0029] In some embodiments, the degrading enzyme is used in the degradation of plastics.

[0030] In some preferred embodiments, the plastic includes PET plastic.

[0031] In some embodiments, the enzyme is included for use in the preparation of heat-resistant, organic solvent-resistant, and highly active PET plastic degrading agents.

[0032] In some preferred embodiments, the PET plastic is a PET plastic with a crystallinity greater than 50%.

[0033] In some embodiments, the products of degradation of the PET plastic include terephthalic acid and / or 2-hydroxyethyl methyl terephthalate.

[0034] In some preferred embodiments, the molar percentage of terephthalic acid in the product of degrading the PET plastic is ≥90%.

[0035] In some embodiments, the enzyme is used in the degradation of PET plastic containing plastic contaminants. Optionally, the plastic contaminant is 2-hydroxyethyl methyl terephthalate (MHET).

[0036] In some embodiments, the enzyme is included for use in the preparation of MHET degrading agents that are heat-resistant, organic solvent-resistant, highly active, or catalyst-free.

[0037] The present invention discloses a PET plastic degrading agent comprising the enzyme, the nucleic acid molecule, the carrier, the host cell, or the polypeptide molecule.

[0038] In some embodiments, the PET plastic degrader may or may not contain process-permitted additives.

[0039] This invention discloses a method for degrading PET plastic, which includes using the aforementioned PET plastic degrading agent.

[0040] In some embodiments, the degradation conditions are: a degradation temperature of 37–72°C. In some embodiments, the degradation temperature can be any one of 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 72°C, or a range between any two of these values.

[0041] In some embodiments, the degradation reaction solvent includes a buffer solution, such as phosphate buffer, Tris buffer, or other conventional buffer solutions. Exemplary examples include, but are not limited to, KH₂PO₄-NaOH buffer. In some embodiments, the pH of the buffer solution is 7.5–9, such as any one of 7.5, 8, 8.5, or 9, or a range between any two values.

[0042] As used herein, the term "high crystallinity" refers to semi-crystalline PET, and "medium-low temperature" refers to 37°C. The chemical recycling of PET requires the addition of a catalyst or high-temperature, high-pressure conditions. The PET hydrolase in this invention can digest the target substrate at medium-low temperature and normal pressure without the need for an additional catalyst.

[0043] The technical solution of this invention has the following advantages:

[0044] 1. An enzyme provided by the present invention comprises any one of the following: (1) containing an amino acid sequence as shown in SEQ ID NO.1; (2) having an amino acid sequence having at least 50% identity with the amino acid sequence shown in SEQ ID NO.1 and having enzymatic activity; the above enzyme can be used as a degrading enzyme to degrade plastics, especially as a PET degrading enzyme to degrade PET plastics.

[0045] Furthermore, the enzyme can degrade highly crystalline PET plastic, with the degradation product mainly being the monomer TPA, which can greatly promote the recycling of PET.

[0046] Furthermore, the enzyme can catalyze the hydrolysis of PET under medium and low temperature conditions, and its catalytic ability is superior to that of known wild-type PET hydrolases, giving it great potential for industrial applications. Therefore, this invention is of great significance in the fields of environmental protection and catalysis.

[0047] Furthermore, the enzyme preparation method of the present invention uses readily available carriers and the preparation method is simple. Attached Figure Description

[0048] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0049] Figure 1 This is a plasmid map of the expression plasmid pET21b-AhPETase in Example 1 of this invention;

[0050] Figure 2 This is the SDS-PAGE analysis of AhPETase hydrolase in Example 1 of the present invention; M, protein marker; Lane 1, AhPETase;

[0051] Figure 3 This is a comparison of the degradation ability of AhPETase, a PET hydrolase, with other known wild-type PET hydrolases in Example 2 of this invention on semi-crystalline PET.

[0052] Figure 4 This is the result of AhPETase degrading semi-crystalline PET within 20 days in Example 2 of the present invention. Detailed Implementation

[0053] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0054] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0055] The genes involved in the following examples were synthesized by GenScript.

[0056] The pET21b carrier was purchased from Genscript.

[0057] BhrPETase enzyme: See Xi

[0058] Cut190 enzyme: Kawai F, Oda M, Tamashiro T, et al. A novel Ca2+-activated, thermostabilized polyesterase capable of hydrolyzing polyethylene terephthalate from Saccharomonospora viridis AHK190[J]. Applied microbiology and biotechnology, 2014, 98: 10053-10064.

[0059] IsPETase enzyme: Yoshida S, Hiraga K, Takehana T, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate)[J]. Science, 2016, 351(6278): 1196-1199.

[0060] LCC enzyme: Sulaiman S, Yamato S, Kanaya E, et al. Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach[J]. Applied and Environmental Microbiology, 2012, 78(5): 1556-1562.

[0061] TfCut2 enzyme: Chen S, Tong X, Woodard R W, et al. Identification and characterization of bacterial cutinase[J]. Journal of biological chemistry, 2008, 283(38): 25854-25862.

[0062] Preparation method of AhPETase enzyme in Example 1

[0063] (1) Construction of recombinant plasmid:

[0064] The gene sequence shown in SEQ ID NO.2 was synthesized (the gene sequence corresponds to the amino acid sequence shown in SEQ ID NO.1; during the construction of the recombinant plasmid, a gene encoding six consecutive histidine residues was added to the 3' end of this gene). The gene sequence product was then inserted into the Nde I and Xho I restriction sites of the vector pET21b. The specific method is as follows:

[0065] The enzyme digestion system was as follows: 10 μg plasmid; 2 μl each of enzymes (Nde I enzyme (commercially available) and Xho I enzyme (commercially available)), and the total volume was made up to 50 μl with sterile, enzyme-free water. Digestion conditions: 37℃ for 30 min.

[0066] Ligation system: 50 ng gene fragment, 200 ng digested plasmid vector, 1 μl T4 ligase, and sterile enzyme-free water to a total volume of 20 μl. Ligation conditions: T4 ligase reaction at room temperature for 1 h to complete plasmid ligation, yielding the expression plasmid pET21b-AhPETase. The plasmid map is shown below. Figure 1 As shown, the sequence of the recombinant plasmid is shown in SEQ ID NO.3.

[0067] (2) Construction of engineered bacteria for heterologous expression of PET hydrolase:

[0068] The expression plasmid pET21b-AhPETase was transformed into Escherichia coli C41(DE3) under the following conditions: 5 μl of recombinant plasmid was incubated with 100 μl of E. coli C41(DE3) competent cells on ice for 30 min, followed by heat shock at 42°C for 90 s. Then, 1 ml of LB liquid medium (LB medium: 10.0 g peptone, 5.0 g yeast extract, 10 g NaCl, 1000 ml distilled water, pH 7.3; solid medium supplemented with 20.0 g agar) was added, and the mixture was incubated at 37°C for 1 h. The culture was then plated on LB ampicillin-resistant plates. Genetically engineered bacteria containing the corresponding PET hydrolase gene recombinant plasmid were obtained.

[0069] (3) Select a single clone of the genetically engineered *E. coli* obtained in step (2) and inoculate it into LB liquid medium containing 50 μg / ml ampicillin. Incubate overnight at 37°C and 250 rpm. Inoculate a 1 v / v% bacterial culture into LB liquid medium containing 50 μg / ml ampicillin. Incubate in a shaker at 37°C and 250 rpm until OD... 600nmWhen the concentration reached 0.8, IPTG was added to a final concentration of 0.5 mM to induce protein expression, and the mixture was cultured at 16°C and 150 rpm for 20 h. After fermentation was terminated, the bacterial cells were collected by centrifugation, resuspended in Tris-HCl buffer, and sonicated to disrupt the cells. The cells were then centrifuged at 12000 rpm for 30 min to remove the precipitate, obtaining the supernatant of the *E. coli* lysate. The supernatant was purified using a Ni-NTA affinity chromatography column, and imidazole was removed by ultrafiltration to obtain the PET hydrolase AhPETase described in this invention (the amino acid sequence of the above-mentioned PET hydrolase was detected and was consistent with SEQ ID NO.1), with a concentration of approximately 39 mg / ml. The obtained AhPETase was used for SDS-PAGE detection, and the detection results are as follows. Figure 2 As shown. The remaining AhPETase was flash-frozen in liquid nitrogen and stored at -80°C for subsequent analysis.

[0070] Example 2. Hydrolysis of semi-crystalline PET catalyzed by AhPETase and other known wild-type PET hydrolases.

[0071] 2.1 Comparison of AhPETase efficacy with other known wild-type PET hydrolases

[0072] 10 mg of semi-crystalline PET powder (purchased from GoodFellow, semi-crystalline PET powder with a crystallinity greater than 50%) was used as the reaction substrate. A 100 mM, pH 8.5 KH₂PO₄-NaOH buffer solution was used as the reaction solvent. A final concentration of 10 μM of PET-degrading enzyme was added, and the reaction was carried out at 37 °C. The total reaction volume was 500 μL. The PET-degrading enzymes used were AhPETase, other wild-type BhrPETase enzymes, Cut190 enzyme, IsPETase enzyme, LCC enzyme, and TfCut2 enzyme. Each PET-degrading enzyme was reacted under the same conditions, and enzyme activity was compared. Each reaction was performed in triplicate. The enzymatic digestion process lasted 96 hours. 100 μL of the reaction solution was sampled from each reaction, and an equal volume of methanol was added to terminate the reaction. The supernatant was collected by centrifugation and analyzed by UPLC.

[0073] 2.2 AhPET degradation PET reaction activity cycle test

[0074] 5 mg of semi-crystalline PET powder (purchased from GoodFellow, semi-crystalline PET powder with a crystallinity greater than 50%) was used as the reaction substrate. 100 mM, pH 8.5 KH₂PO₄-NaOH buffer was used as the reaction solvent. A final concentration of 5 μM PET-degrading enzyme (AhPETase) was added, and the reaction was carried out at 37 °C. The total reaction volume was 500 μL, and each reaction was repeated in triplicate. The reaction was terminated by adding an equal volume of methanol to the reaction solution at 6, 12, 24, 48, 96 hours, 6 days, 10 days, 14 days, and 20 days. The supernatant was collected by centrifugation and analyzed by UPLC.

[0075] The BhrPETase, Cut190, IsPETase, LCC, and TfCut2 enzymes showed 45.38%, 47.66%, 47.04%, and 48.03% identity with AhPETase, respectively. The amino acid sequences of the BhrPETase, Cut190, IsPETase, LCC, and TfCut2 enzymes are SEQ ID NO. 4–8, and their encoding genes are SEQ ID NO. 9–13. These five wild-type enzymes were prepared according to the method described in Example 1.

[0076] The UPLC instrument used was a Waters ACQUITY Premier, and the analytical column was an ACQUITY Premier BEH C18 Column (1.7 μm, 2.1 x 50 mm). The column oven temperature was 40℃. Mobile phase A was deionized water containing 0.1 v / v% formic acid, and mobile phase B was acetonitrile. The flow rate was fixed at 0.4 mL / min; the detection wavelength was 260 nm. PET hydrolysis products can be separated using the following mobile phase gradient program:

[0077] Table 1. Elution Procedure

[0078] Time (min) Flow rate (mL / min) A％ B％ 0.00 0.4 80.0 20.0 1.00 0.4 50.0 50.0 1.20 0.4 30.0 70.0 3.20 0.4 30.0 70.0 3.30 0.4 80.0 20.0 5.00 0.4 80.0 20.0

[0079] The concentrations of the products TPA and MHET were calculated based on the standard samples.

[0080] The reaction results of each enzyme degradation after 96 hours are as follows: Figure 3The following enzymes are listed (μM represents concentration in μmol / L): BhrPETase (117.42 μM of degraded TPA and 4.66 μM of MHET), Cut190 (54.65 μM of degraded TPA and 0.04 μM of MHET), IsPETase (98.02 μM of degraded TPA and 19.31 μM of MHET), LCC (51.67 μM of degraded TPA and 0.17 μM of MHET), TfCut2 (67.55 μM of degraded TPA and 0.25 μM of MHET), and AhPETase (295.32 μM of degraded TPA and 12.04 μM of MHET). Therefore, it can be concluded that AhPETase enzyme has a greater ability to catalyze the hydrolysis of semi-crystalline PET powder than known wild-type PET hydrolases, and the molar proportion of TPA monomer in the hydrolysis product is more than 90%.

[0081] like Figure 4 As shown, during the 20-day reaction period, the AhPETase activity was relatively stable within 14 days, and the concentration of the monomer product TPA accumulated over time, reaching its highest concentration on 14 days, until the hydrolysis reaction gradually terminated after 14 days.

[0082] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An enzyme characterized in that, Including any of the following: (1) Contains the amino acid sequence shown in SEQ ID NO.1; (2) An amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO.1 and has enzyme activity.

2. The enzyme according to claim 1, characterized in that, The enzyme is derived from the prokaryotic bacterium Asanoa hainanensis.

3. A nucleic acid molecule, characterized in that, Includes the nucleotide sequence encoding the enzyme of claim 1 or 2.

4. A carrier, characterized in that, Includes the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, The host cells include: Express the enzyme according to claim 1 or 2; And / or, comprising the nucleic acid molecule of claim 3; And / or, comprising the carrier as described in claim 4.

6. A fusion protein, characterized in that, Includes the enzyme described in claim 1 or 2.

7. Use of the enzyme according to claim 1 or 2 as a degrading enzyme or for the preparation of a degrading enzyme.

8. The use according to claim 7, characterized in that, The use of the degrading enzyme in degrading plastics; Optionally, the plastic includes PET plastic; Optionally, the PET plastic is a PET plastic with a crystallinity greater than 50%; Optionally, the products of degradation of the PET plastic include terephthalic acid and / or 2-hydroxyethyl methyl terephthalate; Optionally, the molar percentage of terephthalic acid in the product of degrading the PET plastic is ≥90%.

9. A PET plastic degrading agent, characterized in that, It comprises the enzyme of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4, the host cell of claim 5, or the polypeptide molecule of claim 6.

10. A method for degrading PET plastic, characterized in that, It includes the PET plastic degrading agent as described in claim 9.

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