Application of an α / β hydrolase in polyethylene degradation

Abstract

The present invention relates to an application of α / β hydrolase in polyethylene degradation, and belongs to the field of enzyme engineering technology. The method for degrading polyethylene or plastic containing polyethylene comprises: (1) pre-treating polyethylene or plastic containing polyethylene by ultraviolet radiation; (2) contacting the pre-treated polyethylene or plastic containing polyethylene with α / β hydrolase in a reaction system, and performing an enzymatic hydrolysis reaction. The present invention provides an α / β hydrolase capable of degrading ultraviolet pre-treated polyethylene film, adding the ultraviolet treated polyethylene film to a HEPES buffer system containing α / β hydrolase for enzymatic hydrolysis reaction, reacting at 35-50° C. for 72-144 hours, and adding α / β hydrolase once every 24 hours during the intermediate reaction process, so that the ultraviolet treated polyethylene film can be partially degraded into small molecular compounds such as alcohols, and the film surface is obviously etched, and the hydrophilicity is significantly enhanced.

Description

Technical Field

[0001] The invention relates to application of alpha / beta hydrolase in polyethylene degradation, and belongs to the technical field of enzyme engineering. Background Art

[0002] Since the beginning of the 20th century, the production and application of plastics have developed rapidly, and the main types include polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), etc. Plastics have been widely used in various fields due to their high strength, good flexibility, durability, light weight and low cost, and have aging resistance of several years to decades.

[0003] Polyethylene (PE) is a widely used and inexpensive plastic that accounts for 36% of the global market share. It is widely used in daily necessities such as food packaging bags and garbage bags, and is applied to all aspects of the national economy and human life. Due to its long chain and hydrophobic macromolecular structure, it is generally believed that the biodegradation process of polyethylene in the natural environment is extremely slow. For a long time, researchers from various countries have been working hard to explore effective methods and ways to biodegrade polyethylene. Among them, the development of environmentally friendly biodegradable polyethylene has become a key direction to solve the problem of plastic waste.

[0004] It is reported that more than 100 microbial strains have been found to be able to degrade PE, among which bacteria, fungi and actinomycetes can significantly reduce the quality and mechanical properties of PE films under different conditions. However, the biodegradation of polyethylene includes multiple stages and is a complex process, so the research on PE degrading enzymes is still relatively limited. The exoenzymes currently disclosed to be involved in PE degradation mainly include peroxidase, laccase, alkane oxidase, esterase and lipase.

[0005] At present, there are no relevant reports on the role of α / β hydrolases in PE degradation, and their binding mode and degradation mechanism are still unclear. Therefore, it is urgent to develop new PE-degrading enzymes to enrich the existing PE-degrading enzyme library and propose new methods for degrading polyethylene. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides an application of α / β hydrolase in polyethylene degradation.

[0007] The technical solution of the present invention is as follows:

[0008] An alpha / beta hydrolase, the amino acid sequence of which is shown as SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO.1.

[0009] According to the present invention, the α / β hydrolase is derived from the strain Vreelandella venusta .

[0010] Application of the α / β hydrolase in the degradation of polyethylene or plastics containing polyethylene.

[0011] Preferably according to the present invention, the polyethylene-containing plastic includes but is not limited to polyethylene film.

[0012] Further preferably, the polyethylene-containing plastic includes but is not limited to rigid or flexible packaging (film, bottle, pallet), agricultural film, bag, disposable items, textiles, fabrics, non-woven fabrics, floor coverings, plastic waste or fiber waste, etc.

[0013] The present invention also provides a method for degrading polyethylene or plastics containing polyethylene, comprising the following steps:

[0014] (1) Pre-treating polyethylene or plastics containing polyethylene by ultraviolet radiation;

[0015] (2) The polyethylene or plastic containing polyethylene pretreated in step (1) is contacted with the above-mentioned α / β hydrolase in a reaction system to cause an enzymatic reaction.

[0016] Preferably according to the present invention, in step (1), the polyethylene or plastic containing polyethylene is a polyethylene film with a thickness of 25 μm.

[0017] Preferably, according to the present invention, in step (1), the ultraviolet radiation pretreatment is specifically:

[0018] Polyethylene or plastic containing polyethylene is cut into 0.5 cm × 0.5 cm and 1 mg in weight, fixed in a UV aging box, and irradiated with UV light at 50-80°C and a UV radiation intensity of 200V for 2-5 days.

[0019] Preferably, according to the present invention, in step (2), the mass ratio of polyethylene or plastic containing polyethylene to α / β hydrolase in the reaction system is 1:(0.1-0.4).

[0020] Preferably, according to the present invention, in step (2), the initial enzyme amount of the α / β hydrolase in the reaction system is 0.1-0.4 mg, and the α / β hydrolase solution is added once every 24 hours, so that the amount of the α / β hydrolase in the reaction system increases by 0.1-0.4 mg after the addition.

[0021] Preferably, according to the present invention, in step (2), the temperature of the enzymatic hydrolysis reaction is 35-50° C., and the degradation time is 72-144 h.

[0022] Preferably, in step (2), the enzymatic hydrolysis reaction is carried out in 4-hydroxyethylpiperazineethanesulfonic acid buffer (HEPES buffer); the mass volume ratio of the α / β hydrolase and the 4-hydroxyethylpiperazineethanesulfonic acid buffer is 1:2, unit: mg / mL; the concentration of the 4-hydroxyethylpiperazineethanesulfonic acid buffer is 50-100 mM, and the pH value is 7.0-9.0.

[0023] The above-mentioned α / β hydrolase is prepared by heterologous expression of microorganisms, and the specific method is as follows:

[0024] 1) Connecting the α / β hydrolase gene (HvH) shown in SEQ ID NO.1 to the plasmid vector pQE80-L to obtain a recombinant plasmid; transforming the recombinant plasmid into Escherichia coli to obtain recombinant Escherichia coli;

[0025] 2) Cultivating the recombinant Escherichia coli constructed in step 1), collecting the culture, separating and purifying, and obtaining α / β hydrolase.

[0026] The present invention also provides the use of the α / β hydrolase gene, a recombinant plasmid containing the α / β hydrolase gene, or a microbial cell carrying the α / β hydrolase gene in degrading polyethylene or plastics containing polyethylene.

[0027] Beneficial effects:

[0028] 1. The present invention provides an α / β hydrolase capable of degrading an ultraviolet pretreated polyethylene film (UV-PE). The ultraviolet pretreated polyethylene film (UV-PE) is added into a HEPES buffer system containing the α / β hydrolase for enzymatic hydrolysis, and the reaction is carried out at 35-50°C for 72-144 hours. During the intermediate reaction, the α / β hydrolase is added once every 24 hours, so that the ultraviolet pretreated polyethylene film (UV-PE) can be partially degraded into small molecular compounds such as alcohols, and the surface of the film is obviously etched, and the hydrophilicity is significantly enhanced.

[0029] 2. The present invention provides a method for degrading ultraviolet-treated polyethylene film (UV-PE). The method has mild reaction conditions and is environmentally friendly. Therefore, the α / β hydrolase provided by the present invention has extremely high application prospects in degrading ultraviolet-treated polyethylene film (UV-PE). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the gel electrophoresis diagram of α / β hydrolase;

[0031] In the figure, lane M is the maker and lane 1 is the α / β hydrolase.

[0032] Figure 2 The diagram shows the structure of the recombinant plasmid HvH-pQE-80L.

[0033] Figure 3 This is the SDS-PAGE electrophoretogram of α / β hydrolase after nickel column affinity chromatography purification;

[0034] In the figure, lane M is Maker, lane 1 is bacterial disruption solution, lane 2 is α / β hydrolase crude enzyme solution, lane 3 is lysis eluate, lane 4 is washing eluate, lane 5 is flow-through solution, and lanes 6 to 8 are target protein eluates.

[0035] Figure 4 The results of Fourier transform infrared spectroscopy (FTIR) of UV-pretreated polyethylene film (UV-PE) after α / β-hydrolase hydrolysis;

[0036] In the figure, the red line segment represents the UV-PE-experimental group, and the black line segment represents the UV-PE-control group.

[0037] Figure 5 Scanning electron microscopy (SEM) observation results of UV-pretreated polyethylene film (UV-PE) after enzymatic hydrolysis with α / β hydrolase.

[0038] Figure 6 The water contact angle test results of UV-PE pretreated polyethylene film after α / β hydrolase hydrolysis;

[0039] In the figure, A represents the UV-PE-control group, and B represents the UV-PE-experimental group.

[0040] Figure 7 The results of gas chromatography-mass spectrometry (GC-MS) analysis of the degradation products of UV-pretreated polyethylene film (UV-PE) after enzymatic hydrolysis with α / β hydrolase. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with the embodiments and the accompanying drawings, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art.

[0042] Materials and reagents involved in the examples are common commercially available products unless otherwise specified; experimental operations involved in the examples are performed according to routine operations in the art unless otherwise specified.

[0043] Example 1. Construction of a recombinant plasmid carrying an α / β hydrolase gene

[0044] 1. The α / β hydrolase gene (HvH) shown in SEQ ID NO.1 was artificially synthesized by Genewise, and then PCR amplification was performed using the α / β hydrolase gene as a template and HvH-F / R as primers to obtain the HvH sequence. The PCR amplification product was subjected to 1wt% agarose gel electrophoresis, and the results were as follows Figure 1 As shown, the amplified DNA fragments were then recovered using the Omega DNA recovery kit according to its instructions.

[0045] The PCR primer sequences are as follows:

[0046] HvH-F: 5'-CGCGGATCCATGGTGCCGACCACCGTGGA-3' (SEQ ID NO. 3);

[0047] HvH-R: 5'-CGGGGTACTTACGGGGTCAGGCGTTCAATAAAT-3' (SEQ ID NO. 4).

[0048] The PCR amplification program was as follows: pre-denaturation at 98°C for 30 sec; denaturation at 98°C for 10 sec, annealing at 61°C for 15 sec, extension at 72°C for 6 sec, 32 cycles; and extension at 72°C for 10 min.

[0049] The PCR amplification system (25 μL) was as follows: 9.5 μL of sterile distilled water, 12.5 μL of Primer star, 1 μL of primer HvH-F, 1 μL of primer HvH-R, and 1 μL of template DNA.

[0050] Depend on Figure 1 It can be seen that the gene HvH encoding α / β hydrolase was successfully amplified.

[0051] 2. Use restriction enzymes Bam HI and Kpn I. Double-digest the plasmid vector pQE-80L and the HvH sequence amplified in step 1, and then use In-Fusion (purchased from TaKaRa) to connect the double-digested HvH sequence to the linearized plasmid vector pQE-80L. The expression vector was transformed into E. coli BL21 (DE3) competent cells according to the heat shock transformation method in the Molecular Cloning Experiment Guide. The specific operation is as follows: add 5 μL of the ligation system to 100 μL of E. coli BL21 (DE3) competent cells (purchased from TransGen), and let it stand on ice for 30 min; heat shock at 42℃ for 60 sec; quickly transfer to ice and let it stand for 2 min; add 500 μL of LB liquid culture medium (no resistance) and culture at 37℃ for 1 h; spread the bacteria on LB solid culture medium containing kanamycin at a final concentration of 100 μg / mL and culture at 37℃ overnight.

[0052] The transformants were selected and inoculated into LB liquid medium. After shaking flask culture at 37°C and 120-180 rpm for 8-12 hours, the plasmids were extracted and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification. The sequencing results showed that the α / β hydrolase gene (HvH) was successfully inserted into the restriction site of the plasmid vector pQE-80L. Bam HI and Kpn I, the insertion direction is correct, and no base mutation, deletion or addition occurs. The recombinant plasmid is named HvH-pQE-80L. The specific structure is shown in the figure Figure 2 shown.

[0053] Example 2: Construction of recombinant Escherichia coli expressing α / β hydrolase gene

[0054] The recombinant plasmid HvH-pQE-80L constructed in Example 1 was transformed into Escherichia coli BL21 (DE3) competent cells to obtain a transformation product; the transformation product was spread on LB solid culture medium (no resistance) and cultured overnight at 37° C. to obtain a transformant; the transformant was picked and inoculated into LB liquid culture medium, and after shaking flask culture at 37° C. and 120-180 rpm for 8-12 h, the plasmid was extracted for enzyme digestion verification and sequencing verification. If the verification was correct, the recombinant Escherichia coli BL21 (DE3) / HvH-pQE-80L was obtained.

[0055] Example 3. Preparation and purification of α / β hydrolase gene

[0056] 1. The recombinant Escherichia coli BL21 (DE3) / HvH-pQE-80L constructed in Example 1 was inoculated into LB liquid culture medium containing 100 μg / mL kanamycin, and cultured at 37° C. overnight to obtain seed solution;

[0057] The seed solution was then transferred to LB liquid medium containing 100 μg / mL kanamycin at a 1% (v / v) inoculum and cultured at 37°C and 150 rpm until OD 600 When nm was about 0.6-0.8, IPTG (0.1 mM) was added and induced at 16°C and 150 rpm for 16 h to obtain fermentation broth; the fermentation broth was centrifuged at 4°C and 4000 rpm for 15 min to collect the bacteria.

[0058] 2. Resuspend the cells in lysis buffer (containing 50 mM Tris-HCl, 100 mM NaCl, 100 mL glycerol, pH = 8.0), use a high-pressure homogenizer to break the cells, centrifuge at 4°C, 12000 rpm for 60 min, and the resulting supernatant is the crude α / β hydrolase enzyme solution.

[0059] 3. After the nickel affinity column is pretreated with lysis buffer, the crude α / β hydrolase solution is loaded onto the nickel affinity column, and 1 column volume is eluted with lysis buffer and wash buffer in sequence. Finally, the target protein is eluted with elution buffer to obtain α / β hydrolase.

[0060] The α / β hydrolase purified by nickel column affinity chromatography was analyzed by SDS-PAGE. Figure 3 shown.

[0061] Depend on Figure 3 It can be seen that the target protein band appears near the 30 kDa band, which is α / β hydrolase, indicating that the heterologous expression of α / β hydrolase is successful.

[0062] Example 4

[0063] A method for degrading polyethylene film comprises the following steps:

[0064] (1) A PE film with a thickness of 25 μm was cut into a size of 0.5 cm × 0.5 cm (mass of 1 mg), fixed in a UV aging box, and irradiated with UV light at 50°C and a UV radiation intensity of 200 V; in order to ensure the uniformity of UV irradiation, the polyethylene film was turned over every 24 hours; a sample of the polyethylene film irradiated with UV light for 2 days was retained to obtain a pretreated polyethylene film (UV-PE);

[0065] (2) 1 mg of the polyethylene film (UV-PE) pretreated in step (1) and the α / β hydrolase prepared in Example 3 were added to 1 mL of HEPES buffer to construct a reaction system. The mass of the α / β hydrolase in the reaction system was 0.1 mg. The reaction was carried out at 35° C. for 72 h. During the reaction, the α / β hydrolase solution was added once every 24 h. After the addition, the mass of the α / β hydrolase in the reaction system increased by 0.1 mg.

[0066] The above method was for the UV-PE experimental group, and was repeated 3 times.

[0067] UV-PE-control group: The experimental steps were the same as above, except that the same volume of HEPES buffer was used instead of α / β hydrolase solution, and a total of 3 replicates were performed.

[0068] Example 5

[0069] A method for degrading polyethylene film comprises the following steps:

[0070] (1) A PE film with a thickness of 25 μm was cut into a size of 0.5 cm × 0.5 cm (mass of 1 mg), fixed in a UV aging box, and irradiated with UV light at 60°C and a UV radiation intensity of 200 V; in order to ensure the uniformity of UV irradiation, the polyethylene film was turned over every 24 hours; a sample of the polyethylene film irradiated with UV light for 3 days was retained to obtain a pretreated polyethylene film (UV-PE);

[0071] (2) 1 mg of the polyethylene film (UV-PE) pretreated in step (1) and the α / β hydrolase prepared in Example 3 were added to 1 mL of HEPES buffer to construct a reaction system. The mass of the α / β hydrolase in the reaction system was 0.2 mg. The reaction was carried out at 40° C. for 96 h. During the reaction, the α / β hydrolase solution was added once every 24 h. After the addition, the mass of the α / β hydrolase in the reaction system increased by 0.2 mg.

[0072] Example 6

[0073] A method for degrading polyethylene film comprises the following steps:

[0074] (1) A PE film with a thickness of 25 μm was cut into a size of 0.5 cm × 0.5 cm (mass of 1 mg), fixed in a UV aging box, and irradiated with UV light at 70°C and a UV radiation intensity of 200 V; in order to ensure the uniformity of UV irradiation, the polyethylene film was turned over every 24 hours; a sample of the polyethylene film irradiated with UV light for 4 days was retained to obtain a pretreated polyethylene film (UV-PE);

[0075] (2) 1 mg of the polyethylene film (UV-PE) pretreated in step (1) and the α / β hydrolase prepared in Example 3 were added to 1 mL of HEPES buffer to construct a reaction system. The mass of the α / β hydrolase in the reaction system was 0.3 mg. The reaction was carried out at 45° C. for 120 h. During the reaction, the α / β hydrolase solution was added once every 24 h. After the addition, the mass of the α / β hydrolase in the reaction system increased by 0.3 mg.

[0076] Example 7

[0077] A method for degrading polyethylene film comprises the following steps:

[0078] (1) A PE film with a thickness of 25 μm was cut into a size of 0.5 cm × 0.5 cm (mass of 1 mg), fixed in a UV aging box, and irradiated with UV light at 80°C and a UV radiation intensity of 200 V; in order to ensure the uniformity of UV irradiation, the polyethylene film was turned over every 24 hours; a sample of the polyethylene film irradiated with UV light for 5 days was retained to obtain a pretreated polyethylene film (UV-PE);

[0079] (2) 1 mg of the polyethylene film (UV-PE) pretreated in step (1) and the α / β hydrolase prepared in Example 3 were added to 1 mL of HEPES buffer to construct a reaction system. The mass of the α / β hydrolase in the reaction system was 0.4 mg. The reaction was carried out at 50° C. for 120 h. During the reaction, the α / β hydrolase solution was added once every 24 h. After the addition, the mass of the α / β hydrolase in the reaction system increased by 0.4 mg.

[0080] Test example

[0081] After the enzymatic hydrolysis reaction in Example 4 was completed, the films after the reaction of the UV-PE-experimental group and the UV-PE-control group were taken out, ultrasonically cleaned once with 2% SDS (v / v), ultrasonically cleaned once with 75% ethanol, and ultrasonically cleaned twice with ultrapure water to remove the attachments on the surface of the film, and each ultrasonic cleaning lasted for 20 minutes. After the cleaning, the films were placed in an oven and dried at 60°C.

[0082] 1. Perform Fourier transform infrared spectroscopy (FTIR) on the dried film. The results are as follows: Figure 4 shown.

[0083] Depend on Figure 4 It can be seen that under the enzymatic hydrolysis of α / β hydrolase, the infrared spectrum of the UV-PE-experimental group changed significantly compared with the UV-PE-control group. -1 A broad peak was observed at 1650 cm-1, which was due to the stretching of -OH, indicating the formation of hydroxyl groups. -1 A new characteristic peak was found in the region, and analysis showed that the peak corresponded to vinyl (C=C) stretching. -1 A new peak was also observed nearby, resulting in the generation of new functional groups (e.g., -ROOH). These changes in functional groups demonstrated, to some extent, the biodegradation effect of α / β hydrolases on UV-PE.

[0084] 2. The dried film was observed by scanning electron microscope (SEM). The results are as follows: Figure 5 shown.

[0085] Depend on Figure 5It can be seen that the formation of cracks, roughness and pits can be observed in the UV-PE-experimental group. At 600 times magnification, the effect of enzymatic hydrolysis on the PE film is relatively slight, while at 5000 times magnification, the degree of damage to the PE surface is significantly increased. In the UV-PE-control group, there is no obvious damage to the structure except for some physical scratches.

[0086] 3. Perform water contact angle test (WCA) on the dried film. The results are as follows: Figure 6 shown.

[0087] Depend on Figure 6 It can be seen that after degradation with α / β hydrolase, the water contact angle of the UV-PE experimental group decreased by 6.4°. The decrease in the water contact angle of the UV-PE experimental group indicates that α / β hydrolase changes the hydrophilicity of the UV-PE film surface, and the increase in hydrophilicity also indicates the increase in hydrophilic functional groups on the UV-PE film surface (such as -OH). There was no significant change in the UV-PE control group.

[0088] 4. Extract the dried film, and then analyze the extract by gas chromatography-mass spectrometry (GC-MS). The results are as follows: Figure 7 shown.

[0089] Depend on Figure 7 It can be seen that alcohol substances, especially 2,6-di-tert-butylbenzene-1,4-diphenol, were generated in the extract of the UV-PE experimental group, which fully demonstrates that the α / β hydrolase of the present invention has the ability to degrade UV-PE film.

[0090] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for degrading polyethylene or plastics containing polyethylene, characterized in that: The steps include: (1) Pre-treating polyethylene or plastics containing polyethylene by ultraviolet radiation; (2) contacting the polyethylene or plastic containing polyethylene pretreated in step (1) with α / β hydrolase in a reaction system to cause enzymatic hydrolysis; The amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; the mass ratio of polyethylene or polyethylene-containing plastic to α / β hydrolase in the reaction system is 1:(0.1-0.4); the temperature of the enzymatic hydrolysis reaction is 35-50°C, and the degradation time is 72-144h; the initial enzyme amount of the α / β hydrolase in the reaction system is 0.1-0.4mg, and the α / β hydrolase liquid is added once every 24h, so that the amount of α / β hydrolase in the reaction system increases by 0.1-0.4mg after the addition.

2. The method for degrading polyethylene or plastics containing polyethylene according to claim 1, characterized in that: In step (1), the polyethylene-containing plastic is a polyethylene film.

3. The method for degrading polyethylene or plastics containing polyethylene according to claim 1, characterized in that: In step (1), the ultraviolet radiation pretreatment is specifically as follows: polyethylene or plastic containing polyethylene is cut into 0.5 cm×0.5 cm and has a mass of 1 mg, fixed in an ultraviolet aging box, and subjected to ultraviolet radiation for 2 to 5 days at 50 to 80° C. and a UV radiation intensity of 200 V.

4. The method for degrading polyethylene or plastics containing polyethylene according to claim 1, characterized in that: The enzymatic hydrolysis reaction is carried out in 4-hydroxyethylpiperazineethanesulfonic acid buffer.

5. The method for degrading polyethylene or plastics containing polyethylene according to claim 4, characterized in that: The mass volume ratio of the α / β hydrolase and the 4-hydroxyethylpiperazineethanesulfonic acid buffer is 1:2, unit: mg / mL; the concentration of the 4-hydroxyethylpiperazineethanesulfonic acid buffer is 50-100 mM, and the pH value is 7.0-9.

0.

6. The method for degrading polyethylene or plastics containing polyethylene according to claim 1, characterized in that: The preparation method of the α / β hydrolase is as follows: 1) Connecting the α / β hydrolase gene shown in SEQ ID NO.1 to the plasmid vector pQE80-L to obtain a recombinant plasmid; transforming the recombinant plasmid into Escherichia coli to obtain recombinant Escherichia coli; 2) Cultivating the recombinant Escherichia coli constructed in step 1), collecting the culture, separating and purifying, and obtaining α / β hydrolase.

7. Use of the method according to any one of claims 1 to 6 in degrading polyethylene or plastics containing polyethylene.

8. Use of a gene of an α / β hydrolase having a nucleotide sequence as shown in SEQ ID NO.1, a recombinant plasmid containing the gene of the α / β hydrolase, or a microbial cell carrying the gene of the α / β hydrolase in degrading polyethylene or plastics containing polyethylene.

9. Use of the α / β hydrolase having an amino acid sequence as shown in SEQ ID NO.2 in the degradation of polyethylene or plastics containing polyethylene.

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