Protein, coding gene thereof and application of protein in degradation of PET (Polyethylene Terephthalate)

Abstract

The invention discloses a protein, a coding gene thereof and application of the protein in degradation of PET (Polyethylene Terephthalate). The invention provides a protein which is any one of the following a1)-a4): a1) a protein as shown in SEQ ID NO.1; a2) protein which has more than 75% of homology with the amino acid sequence limited by a1) and has PET hydrolase activity; a3) a protein with an amino acid sequence defined by a1) or a2) in the sequence; and a4) a fusion protein obtained by connecting a tag to the N terminal and / or C terminal of any one of the proteins defined by a1)-a3). Protein is designed and screened from the beginning, new protein with the PET hydrolase function is obtained, and experiments prove that the prepared protein has the PET hydrolase function, can degrade PET into terephthalic acid and ethylene glycol, is a protein with a sequence different from that of the prior art and has the PET hydrolase function.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a protein and a coding gene thereof and application thereof in degrading PET. Background Art

[0002] In recent years, human demand for plastics has continued to increase, thanks to the advantages of plastics such as cheapness, transparency, lightness, strength, insulation, low thermal conductivity and corrosion resistance. However, since plastics are difficult to degrade naturally, whether it is plastic waste entering landfills or discarded plastics in soil and sludge and sewage, they often enter the ocean with the natural water cycle. Therefore, plastic pollution in the ocean is more serious than in the soil. Large pieces of plastic products that enter the ocean float on the sea surface because they usually contain air, or are washed ashore and become coastline garbage. Large pieces of plastic in the ocean gradually break down under ultraviolet rays and physical impact wear over time, becoming plastic fragments that sink to the seabed, or become microplastics that flow with ocean currents.

[0003] Among the many types of plastics, polyethylene terephthalate (PET) plastic has attracted much attention due to its wide application and large-scale production. In order to address the problem of PET plastic pollution, the methods currently developed to degrade PET are mainly divided into two categories: chemical degradation and biodegradation. Chemical degradation methods rely on chemical reagents or catalysts to decompose PET plastics into smaller compounds through chemical reactions. However, these methods usually require conditions such as high temperature, high pressure, and strong acids and alkalis, which not only have high energy consumption, but may also cause problems such as environmental pollution. In contrast, biodegradation methods have many advantages, including mild reaction conditions, high efficiency, and selective degradation capabilities.

[0004] In the field of research on biodegradable PET plastics, cutinases and PETase enzymes are two enzymes that have received widespread attention. Cutinases are a class of enzymes that can degrade natural polymers, esters of esters of terephthalic acid. In recent years, researchers have found that some cutinases have certain PET degradation capabilities. Among them, cutinases such as ThcCut1 and LCC are widely studied representatives. ThcCut1 is a cutinase isolated from the Thermobifida cellulosilytica strain. Studies have shown that ThcCut1 can catalyze the degradation of PET under relatively mild conditions. The enzyme breaks down PET polymers into smaller compounds such as terephthalic acid and ethylene glycol by binding and cutting them. LCC isolated from leaf branch compost is another cutinase with PET degradation capabilities. PETase enzyme is a highly specific PET hydrolase. The PETase enzyme originally isolated from the Ideonellasakaiensis strain can efficiently break down PET into terephthalic acid and ethylene glycol. In addition, the modification of these enzymes can also obtain enzymes with PET degradation ability. For example, LCC-G obtained by glycosylation of LCC has higher thermal stability and wider specificity. Mutation of ThermoPETase, a skeleton of PETase, can obtain FAST-PETase, which has dozens of times more activity than before mutation.

[0005] Existing PET hydrolases such as PETase and LCC are enzymes extracted from microorganisms with PET degradation capabilities in nature. Obtaining new enzymes through this method requires high costs and a long time, which means that this method is not suitable for rapid iterative development of enzymes. For example, enzymes such as LCC-G and Fast-PETase are new enzymes with improved functions obtained by mutation and modification of existing enzymes. The basis for performing this operation is the existence of enzymes that can be further redesigned. However, the number of such enzymes is currently limited, which means that even if mutation is used to improve enzyme performance, the diversity of the results is also limited.

[0006] Therefore, finding more new proteins with PET hydrolase function has become a research direction. Summary of the invention

[0007] The purpose of the present invention is to provide a protein and a coding gene thereof and application thereof in degrading PET.

[0008] In a first aspect, the present invention provides a protein, which is any one of the following a1)-a4):

[0009] a1) the protein shown in SEQ ID NO.1;

[0010] a2) a protein having PET hydrolase activity and having 99% or more, 95% or more, 90% or more, 85% or more, 80% or more homology with the amino acid sequence defined in a1);

[0011] a3) a protein whose sequence contains the amino acid sequence defined in a1) or a2);

[0012] a4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in any one of a1) to a3).

[0013] In a second aspect, the present invention provides a nucleic acid molecule encoding the protein described in the first aspect.

[0014] The nucleic acid molecule described above is a DNA molecule of any one of the following b1) to b3):

[0015] b1) The coding region is a DNA molecule shown in SEQ ID NO.2;

[0016] b2) a DNA molecule that hybridizes with the DNA sequence defined in b1) under stringent conditions and encodes a protein having PET hydrolase activity;

[0017] b3) is a DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in b1) and encodes a protein with PET hydrolase activity.

[0018] In a third aspect, the present invention provides a recombinant vector, an expression cassette or a recombinant bacterium containing the nucleic acid molecule described in the second aspect.

[0019] In a fourth aspect, the present invention provides the use of the protein described in the first aspect in any of the following:

[0020] c1) as a PET hydrolase;

[0021] c2) Hydrolyze or degrade PET.

[0022] In a fifth aspect, the present invention provides the use of the nucleic acid molecule described in the second aspect or the recombinant vector, expression cassette or recombinant bacteria described in the third aspect in any of the following:

[0023] d1) preparing a product having PET hydrolase activity;

[0024] d2) Hydrolyze or degrade PET.

[0025] In a sixth aspect, the present invention provides a method for hydrolyzing or degrading PET, using the protein described in the first aspect to degrade PET, thereby achieving hydrolysis or degradation of PET.

[0026] The main products after the above-mentioned hydrolysis or degradation of PET are TPA and MHET.

[0027] The present invention designs and screens proteins from scratch to obtain a new protein with PET hydrolase function, expresses the gene sequence of the new protein in prokaryotes to obtain the protein, and performs functional verification on the protein. Experiments have shown that the prepared protein has the function of PET hydrolase and can degrade PET into terephthalic acid and ethylene glycol. The protein is a non-natural protein with a sequence different from that of the past and having the function of PET hydrolase. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the plasmid map of TJJ4-pET-22b.

[0029] Figure 2 This is the SDS-PAGE electrophoresis of purified TJJ4.

[0030] Figure 3 The results of high performance liquid chromatography (HPLC) of PET nanoparticles degraded by the protein expressed by SEQ ID NO.1.

[0031] Figure 4 This is the HPLC test result of TPA standard.

[0032] Figure 5 The results of HPLC detection of MHET standard.

[0033] Figure 6 This is the HPLC test result of the blank control group for the degradation of PET nanoparticles.

[0034] Figure 7 The results are the product determination results of the degradation of PET nanoparticles by the protein expressed by SEQ ID NO.1 and the blank control group. DETAILED DESCRIPTION

[0035] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the accompanying drawings and examples. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0036] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0037] Terminology: As used herein, "PET" refers to polyethylene terephthalate, "TPA" refers to terephthalic acid, "MHET" refers to mono(hydroxyethyl) terephthalate, and "IPTG" refers to isopropyl-β-D-thiogalactoside.

[0038] In the present invention, the terms "vector" and "plasmid" can be used interchangeably.

[0039] In the present invention, the terms "protein" and "protein" can be used interchangeably.

[0040] In the present invention, the terms "hydrolysis" and "degradation" can be used interchangeably.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] The experimental methods described below are all routine laboratory methods unless otherwise specified. The experimental materials described below can be obtained online or from commercial channels unless otherwise specified. The present invention is described in detail below in conjunction with specific embodiments to better understand the present invention, but the scope of protection of the present invention is not limited to the following description.

[0044] In the following examples, Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd. and used as host cells to express the PET hydrolase protein of the present invention.

[0045] The culture medium formula involved in the following examples is as follows:

[0046] LB medium: peptone 1%, yeast extract 0.5%, NaCl 1%; the unit in the culture medium is % (W / V, g:mL).

[0047] Buffer solution A refers to a buffer solution composed of 100mM K2HPO4-HCl, 100mM NaCl and water, and buffer solution B refers to a buffer solution composed of 500mM imidazole, 100mM K2HPO4-HCl, 100mM NaCl and water. The solution formed by mixing buffer solutions A and B is called a mixed buffer solution, and the concentration of the mixed buffer solution refers to the proportion of buffer solution B. If the pH value of the buffer solution is not specified, it defaults to 7.0.

[0048] Example 1. Preparation of protein TJJ4

[0049] 1. Obtaining the protein TJJ4 sequence

[0050] Protein TJJ4 was obtained through screening, and its sequence is shown in SEQ ID NO.1. The nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO.2.

[0051] 2. Construction of expression vector

[0052] Artificially synthesize the gene sequence of protein TJJ4 shown in SEQ ID NO.2 ( Figure 1 The vector (TJJ4) was inserted between the NdeI and XhoI restriction sites of the pET-22b vector. Figure 1 ), named TJJ4-pET-22b, the vector expresses the protein TJJ4 shown in SEQ ID NO.1, and the recombinant plasmid is provided by Beijing Liuhe BGI Technology Co., Ltd.

[0053] 3. Expression of target protein TJJ4

[0054] 1. Obtaining recombinant bacteria

[0055] The vector TJJ4-pET-22b prepared in the above two was transformed into the competent cells of Escherichia coli BL21 (DE3) to obtain recombinant bacteria. Positive clones were screened using ampicillin resistance plates (containing 100 μg / mL Ampicillin) and cultured overnight at 37°C. Plasmids were extracted from the grown positive clones and sent for sequencing, and the result was vector TJJ4-pET-22b.

[0056] Therefore, the positive clone was named BL21(DE3) / TJJ4-pET-22b.

[0057] Select a single E. coli colony of the positive clone BL21 (DE3) / TJJ4-pET-22b from the above plate and transfer it to a resistance tube. The tube is cultured overnight at 37 ° C and 200 rpm. The cultured E. coli suspension is mixed with 50% glycerol that has been sterilized in advance at a volume ratio of 7:3. An appropriate amount of the mixture is placed in a sterilized centrifuge tube. The centrifuge tube is sealed and stored in an ultra-low temperature freezer at -80 ° C for long-term storage.

[0058] The recombinant bacteria BL21(DE3) / pET-22b obtained by transferring the pET-22b vector (Beijing Huayueyang Biological, VECT5200) into BL21(DE3) was used as the control strain.

[0059] 2. Induce protein expression

[0060] To obtain a crude enzyme solution, add 10 μL of ampicillin (100 μg / mL) and 100 μL of BL21 (DE3) / TJJ4-pET-22b glycerol culture to 10 mL of sterile LB medium, and culture at 37°C, 200 rpm for 12-14 hours to obtain a bacterial solution. Add 150 μL of ampicillin and 3 mL of the cultured bacterial solution to 150 mL of sterile LB medium, and culture at 37°C, 200 rpm until the optical density (OD 600 ) reached 0.6-0.8, added enough IPTG to the shake flask to make its final concentration reach 0.6mM, adjusted the temperature to 16°C, and continued to culture at a speed of 200rpm for 18 hours, and collected the culture products.

[0061] 3. Obtaining crude enzyme solution

[0062] After centrifuging the culture product obtained in 2 above at 8000rpm for 10 minutes, 30mL of buffer solution A was added to resuspend the bacteria, and then the bacteria were subjected to high-pressure homogenization under high pressure (1500bar). Finally, centrifuge at 12000rpm for 1 hour, collect the supernatant and precipitate (the precipitate was resuspended with 1mL of buffer solution A and collected, recorded as TJJ4 precipitate); filter the supernatant with a 0.45μm filter membrane, add 1.2mL of buffer solution B to the filtered supernatant; resuspend the precipitate with buffer solution A and collect it to obtain BL21(DE3) / TJJ4-pET-22b crude enzyme solution.

[0063] The same method was used to induce expression of the control strain and obtain a control protein solution.

[0064] 4. Protein Purification

[0065] The crude enzyme solution obtained in step 3 above was purified using a nickel column (Nanakai Biotechnology, PANF001-005C).

[0066] (1) In a chromatography cabinet at 4°C, equilibrate the purification column with 50 mL of 4% mixed buffer solution (10 times the sample volume).

[0067] (2) Add the BL21(DE3) / TJJ4-pET-22b crude enzyme solution extracted in step 3 and load the sample at a natural flow rate.

[0068] (3) Add 50 mL of 4% mixed buffer solution to rinse the column and collect the effluent, which is named TJJ4-4%.

[0069] (4) 10 mL of 10% mixed buffer solution and 10 mL of 15% mixed buffer solution were added successively to wash the impurities, and the effluent was collected and named TJJ4-10% and TJJ4-15%, respectively.

[0070] (5) Add 20 mL of 30% mixed buffer solution for elution and collect the effluent, which is named TJJ4-30%.

[0071] (6) Add 50 mL of buffer solution B to rinse the purification column.

[0072] (7) Add 100 mL of water to rinse the purification column.

[0073] (8) Seal the column with 20% ethanol.

[0074] Protein concentration and concentration determination: Each effluent and TJJ4 precipitate were used for SDS-PAGE protein gel electrophoresis ( Figure 2 ), the molecular weight of the target protein TJJ4 was 57 kDa, and the group with higher purity (TJJ4-30%) was selected, and the imidazole was removed by ultrafiltration using a 10 kDa ultrafiltration tube and concentrated to obtain a concentrated protein TJJ4 solution.

[0075] The concentration of the concentrated protein TJJ4 solution was measured using a BCA kit and the result was 0.44 mg / mL.

[0076] The control protein solution was purified using the same method to obtain a purified control protein.

[0077] Example 2, activity determination of PET hydrolase

[0078] 1. Determination of the activity of target enzyme in degrading PET nanoparticles

[0079] Weigh 50mg amorphous PET film (Goodfellow, ES301445) and add 5mL hexafluoroisopropanol, use glass rod to stir and mix at room temperature until the PET film is completely dissolved to obtain a polymer solution. Subsequently, the polymer solution (10mg / mL) is slowly injected into the distilled water of 200mL ice bath under ultrasound. The dropwise addition process should be maintained within 5 minutes to avoid excessive aggregation of the polymer. This step obtains a PET nanoparticle dispersion.

[0080] The PET nanoparticle dispersion was subjected to rotary distillation to remove excess liquid to concentrate the PET nanoparticles. The concentrated PET nanoparticle dispersion was filtered to remove impurities and obtain a pure PET nanoparticle solution. Finally, the concentration of the PET nanoparticles was diluted to 0.25 mg / mL using distilled water to obtain a PET nanoparticle solution of the desired concentration.

[0081] Take a 2 mL centrifuge tube and add 300 μL of PET nanoparticle solution (0.25 mg / mL) thereto, then add a certain amount of the concentrated protein TJJ4 solution obtained in Example 1, and use reaction buffer (100 mM K2HPO4-KH2PO4, the balance is water, pH 7.0) to supplement the total volume of the reaction system to 600 μL, and the concentration of the concentrated protein TJJ4 in the reaction system reaches 1 μM.

[0082] The centrifuge tube was placed in a thermostatic oscillator at 37°C and 1200 rpm and shaken for 2 hours to allow the enzyme to catalyze the degradation of PET nanoparticles. After the reaction was completed, 600 μL of acetonitrile was added to stop the hydrolysis reaction.

[0083] 2. Product analysis using high performance liquid chromatography

[0084] The reaction product after the termination of the above reaction 1 was centrifuged (13000rpm, 10min) to obtain the supernatant, which was filtered using a 0.22μm filter membrane. The analysis was performed using a high performance liquid chromatography system (Shimadzu LC-20AD) and a C18 chromatographic column (SilGreen, 4.6×250mm, 5μm). The mobile phase was 0.1% formic acid aqueous solution / acetonitrile, the flow rate was 0.8mL / min, the detection wavelength was 254nm, the elution condition was 0-36min, and the acetonitrile linear gradient was 5%-70%.

[0085] High performance liquid chromatography (HPLC) test results are as follows Figure 3 As shown, it can be seen that there are two peaks at retention times of 19.3 min and 20.4 min.

[0086] The peak at retention time of 19.3 min is consistent with the peak time of standard TPA (TCI, CAS: 100-21-0) ( Figure 4 ), so the substance with retention time 19.3min is TPA.

[0087] The peak at retention time of 20.4 min is consistent with the peak time of the standard MHET (Bidec Pharmaceuticals, CAS: 1137-99-1) ( Figure 5 ), so the substance with retention time 20.4min is MHET.

[0088] The above results show that the protein TJJ4 obtained in the present invention can degrade PET into TPA and MHET and has the activity of PET hydrolase.

[0089] The purified control protein was tested by HPLC using the same method. The results are as follows Figure 6 As shown, the peak appeared at the retention time of 19.3 min, indicating that the control protein could not degrade PET into MHET.

[0090] 3. Determination of TPA and MHET concentrations

[0091] The prepared TPA standard curve is y=5103777.16667x (x is the standard concentration, in mM; y is the peak area), and the MHET standard curve is y=6382022.45455x (x is the standard concentration, in mM; y is the peak area).

[0092] The concentrations of TPA and MHET in the reaction products of hydrolyzing PET material by purified control protein (denoted as blank control in the figure) and concentrated protein TJJ4 solution (denoted as SEQ ID NO.1 in the figure) were calculated by standard curve.

[0093] The results of the test are as follows Figure 7 As shown, it can be seen that the protein TJJ4 of the present invention has the ability to hydrolyze PET materials into TPA and / or MHET and can be used as a PET hydrolase.

[0094] It should be noted that the embodiments described above are only preferred embodiments of the present invention, which are used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A protein, which is any one of the following a1)-a4): a1) the protein shown in SEQ ID NO.1; a2) a protein having PET hydrolase activity and having 99% or more, 95% or more, 90% or more, 85% or more, 80% or more homology with the amino acid sequence defined in a1); a3) a protein whose sequence contains the amino acid sequence defined in a1) or a2); a4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in any one of a1) to a3).

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule of any one of the following b1) to b3): b1) The coding region is a DNA molecule shown in SEQ ID NO.2; b2) a DNA molecule that hybridizes with the DNA sequence defined in b1) under stringent conditions and encodes a protein having PET hydrolase activity; b3) is a DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in b1) and encodes a protein with PET hydrolase activity.

4. A recombinant vector, expression cassette or recombinant bacterium containing the nucleic acid molecule according to claim 2 or 3.

5. Use of the protein according to claim 1 in any of the following: c1) as a PET hydrolase; c2) Hydrolyze or degrade PET.

6. Use of the nucleic acid molecule according to claim 2 or 3 or the recombinant vector, expression cassette or recombinant bacterium according to claim 4 in any of the following: d1) preparing a product having PET hydrolase activity; d2) Hydrolyze or degrade PET.

7. A method for hydrolyzing or degrading PET, using the protein of claim 1 to degrade PET, thereby achieving hydrolysis or degradation of PET.

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