Application of laccase-mediated system in polyethylene degradation

Abstract

The present invention discloses the application of a laccase mediator system in polyethylene degradation, belonging to the field of enzyme engineering technology. The present invention adds laccases derived from Botrytis aclada (BaLac) and Bacillus subtilis (BsLac) to a 50mM citric acid buffer containing ultraviolet-treated polyethylene film (UVPE), pH 5 and 6, and adds 10mM mediator solution to initiate an enzymatic hydrolysis reaction. The reaction is allowed to proceed for 120 hours, which can partially degrade the UV-treated polyethylene film (UVPE) into small molecule compounds such as aldehydes, ketones, alcohols, and acids, causing significant etching on the film surface and a significant reduction in weight-average molecular weight. The method for degrading UV-treated polyethylene film provided by the present invention has mild reaction conditions and is environmentally friendly, and has extremely high application prospects in the field of plastic degradation.

Description

Technical Field

[0001] The invention relates to application of a laccase mediator system in polyethylene degradation, and belongs to the technical field of enzyme engineering. Background Art

[0002] The "white pollution" caused by plastics has become a "long-standing" problem for the global environment. Due to the inefficient recycling of discarded plastics, the vast majority of plastic waste is released into the environment, ubiquitous from land to the hydrosphere. On October 21, 2021, the United Nations Environment Programme (UNEP) released the report "From Pollution to Solutions: A Global Assessment of Marine Litter and Plastic Pollution". The report shows that 85% of marine debris is plastic. Polyethylene in petroleum-based plastics is widely used in various fields such as food, construction, and agriculture due to its good durability, and global production is increasing exponentially. Due to its strong chemical inertness, it is difficult to effectively degrade in the natural environment.

[0003] At present, non-biological treatment methods such as landfill incineration are commonly used at home and abroad, which easily lead to waste of resources and secondary pollution. Biodegradable plastic methods have great potential for ecological sustainable development and environmentally friendly protection.

[0004] According to literature reports on polyethylene biodegradation, lignin peroxidases are currently recognized as oxidoreductases that play a role in the initial oxidative degradation of polyolefin plastics. Laccase (EC 1.10.3.2), a member of the lignin peroxidase family, differs from other peroxidases and phenolic oxidases in that its electron acceptor is oxygen molecules rather than hydrogen peroxide, and its only byproduct is water. Furthermore, laccases can effectively oxidize other non-phenolic substrates with high redox potentials by oxidizing small molecule mediators with low redox potentials into highly catalytically active cationic free radicals. Therefore, laccases are the most environmentally friendly of the potential polyolefin plastic degrading enzymes. Summary of the Invention

[0005] The first object of the present invention is to provide a polyethylene degradation method, which uses laccase to degrade in a mediator system; the mediator includes but is not limited to 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), 4-acetyl-2,2,6,6-tetramethylpiperidone-1-oxyl free radical (TEMPO), and 1-hydroxybenzotriazole (HBT).

[0006] In one embodiment, the amino acid sequence of the laccase is as shown in Genbank Accession No. WP_003243170.1 or AFC76164.1.

[0007] In one embodiment, the polyethylene is further subjected to ultraviolet irradiation treatment before the enzymatic hydrolysis treatment.

[0008] In one embodiment, the laccase is produced by microbial fermentation.

[0009] In one embodiment, the laccase is prepared by expressing the laccase having an amino acid sequence such as Genbank accession number WP_003243170 in E. coli BL21 (DE3) using pET-24a(+) as a vector.

[0010] In one embodiment, the nucleotide sequence encoding the laccase is shown as SEQ ID NO.1.

[0011] In one embodiment, the laccase is prepared by expressing the laccase having an amino acid sequence as shown in Genbank accession number AFC76164 in P. pastoris KM71 using pPIC9K as a vector.

[0012] In one embodiment, the nucleotide sequence encoding the laccase is shown as SEQ ID NO.2.

[0013] In one embodiment, the amount of laccase added to the reaction system is ≥4 U / mL.

[0014] In one embodiment, the amount of laccase added to the reaction system is 0.8-1 U / mg polyethylene film.

[0015] In one embodiment, the reaction temperature is 30° C. and the pH is 5-6.

[0016] In one embodiment, the degradation temperature is 30° C. and the degradation time is 120 h.

[0017] In one embodiment, the degradation system further contains pH 5, 50 mM citric acid buffer.

[0018] The present invention also provides the use of the laccase shown in Genbank accession number WP_003243170.1 or AFC76164.1, the gene encoding the laccase, the recombinant plasmid carrying the gene, or the host cell expressing the laccase in degrading ultraviolet pretreated polyethylene film (UVPE).

[0019] In one embodiment, the vector of the recombinant plasmid is plasmid pET-24a(+) or pPIC9K.

[0020] In one embodiment, the gene encoding the laccase comprises the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0021] In one embodiment, the host cell is Escherichia coli BL21 (DE3) or Pichia pastoris KM71.

[0022] Beneficial effects:

[0023] (1) The present invention provides a laccase capable of degrading UV-pretreated polyethylene film (UVPE). Laccase is added to a 50 mM citric acid buffer solution containing UV-pretreated polyethylene film (UVPE), pH 5-6, and a 10 mM mediator solution is added to initiate an enzymatic hydrolysis reaction. The reaction is carried out at 30°C and 200 rpm for 120 h, and the UV-pretreated polyethylene film (UVPE) can be partially degraded into small molecular compounds such as aldehydes, ketones, alcohols, and acids. The surface of the film is obviously etched, the hydrophilicity is significantly enhanced, and the weight-average molecular weight of the UVPE is significantly reduced.

[0024] (2) The present invention provides a method for degrading ultraviolet-treated polyethylene film (UVPE). The method has mild reaction conditions and is environmentally friendly. Therefore, the laccase provided by the present invention has extremely high application prospects in degrading ultraviolet-treated polyethylene film (UVPE). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : SDS-PAGE analysis of laccase BaLac and BsLac; wherein, (a): BaLac; (b): BsLac;

[0026] Figure 2 : FTIR analysis of LDPE film after high-temperature UV irradiation treatment (A) and UVPE film after surface cleaning (B);

[0027] Figure 3 : GC-MS analysis of olefins and oxides in the buffer after UVPE washing; Control: UVPE unwashed control group; First wash: experimental group rinsed with ethyl acetate; Second wash: experimental group rinsed with n-hexane;

[0028] Figure 4 : SEM images of UVPE3 after oxidation by LMS (BaLac); AD: magnification 600 times; EH: magnification 2400 times; A, E: control UVPE3; B, F: HBT medium experimental group; C, G: ABTS medium experimental group; D, H: TEMPO medium experimental group;

[0029] Figure 5: SEM images of UVPE3 after oxidation by LMS (BsLac); AD: magnification 600 times; EH: magnification 2400 times; A, E: control UVPE3; B, F: HBT medium experimental group; C, G: ABTS medium experimental group; D, H: TEMPO medium experimental group;

[0030] Figure 6 : FTIR spectra and oxygen-containing functional group index of UVPE membranes pre-treated with BaLac and BsLac enzymatic hydrolysis; (A) UVPE-2 enzymatically pretreated with BaLac, (B) UVPE-3 enzymatically pretreated with BaLac, (C) UVPE oxygen functional group index after enzymatic treatment with BaLac, (D) UVPE-2 enzymatically treated with BsLac, (E) UVPE-3 enzymatically treated with BsLac, (F) UVPE oxygen functional group index after enzymatic treatment with BsLac.

[0031] Figure 7 : GC-MS detection of the carbon chain distribution of oxygen-containing products after UVPE-3 reaction of BaLac (AC) and BsLac (DF) laccase mediator systems; A, D: ABTS mediator experimental group; B, E: HBT mediator experimental group; C, F: TEMPO mediator experimental group. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific embodiments.

[0033] The LDPE films used in the following examples were purchased from Chengxin Plastics & Hardware Packaging on Taobao. They had a thickness of 20 μm. 1-Hydroxybenzotriazole (HBT), 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO), and 2,2-nitro-bis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich (China). Hexane, ethanol, and ethyl acetate were purchased from Sinopharm Reagent. All chemicals were of reagent grade.

[0034] The culture medium involved in the following examples is as follows:

[0035] LB medium: 10.00 g / L tryptone, 10.00 g / L NaCl, 5.00 g / L yeast extract, and 15.00 g / L agar powder for solid medium.

[0036] YPD medium: yeast extract 10.00 g / L, tryptone 20.00 g / L, glucose 20.00 g / L, solid medium supplemented with agar powder 14.00 g / L.

[0037] MD solid medium: glucose 20.00 g / L, biotin 4.00×10-4 g / L, YNB 13.40 g / L, agar powder 15.00 g / L.

[0038] BMMY medium: yeast extract 10.00 g / L, tryptone 20.00 g / L, YNB 13.40 g / L, biotin 4.00 × 10-4 g / L.

[0039] BMGY medium: glycerol 10.00 g / L, yeast extract 10.00 g / L, tryptone 20.00 g / L, YNB 13.40 g / L, biotin 4.00×10-4 g / L.

[0040] The detection methods involved in the following embodiments are as follows:

[0041] Laccase activity detection method:

[0042] Enzyme activity assay: Add 20 μL of 10 mM ABTS solution and 930 μL of citric acid-sodium citrate buffer (pH 5.0) to a cuvette and preheat in a 30°C water bath. Add 50 μL of enzyme solution and shake rapidly to mix. Measure the change in OD at 420 nm and calculate the enzyme activity. One unit (U) of enzyme activity is defined as the amount of enzyme required to oxidize 1 μmol of substrate in 1 minute.

[0043] Detection method of surface microstructure of polyethylene (PE) plastic film:

[0044] UV-pretreated polyethylene (UVPE) films treated with a laccase-mediated system were rinsed three to four times with deionized water. The films were then ultrasonically cleaned twice with 2% SDS (v / v), deionized water, and 50% ethanol for 30 minutes each to remove surface impurities such as proteins. After rinsing, the films were wrapped in weighing paper and dried in an oven at 60°C. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and high-temperature gel permeation chromatography (HT-GPC) were used to examine the surface microstructure and molecular weight of untreated UVPE films and those treated with the laccase-mediated system, respectively.

[0045] Detection of UVPE degradation products:

[0046] The types and proportions of small molecules in the reaction solution were determined using a gas chromatography-mass spectrometer (TSQ 8000, Thermo; USA). Gas chromatography detection conditions were: DB-5MS column, injection rate 1.0 ml / min, initial temperature 40°C, ramp rate 10°C / min to 250°C, and hold for 4 min. Mass spectrometry detection conditions were: molecular weight range 33–450, ion source temperature 70°C, and electron energy 70.0.

[0047] Solid-phase microextraction (SPME) was performed by inserting an extraction head (DVB, Sigma-Aldrich) into the headspace vial for target adsorption. After 7 min of adsorption, the injection needle was inserted into the GC injection port for injection under a constant helium flow rate (1.0 ml / min).

[0048] Example 1: Recombinant expression of laccase

[0049] (1) Construction of recombinant plasmid

[0050] Using a chemical method, the nucleotide sequences of genes encoding laccases as shown in SEQ ID NO.1 and SEQ ID NO.2 are respectively ligated between the EcoRI-XhoI restriction sites of the vector pET-24a(+) and the EcoRI-NotI restriction sites of the vector pPIC9K to obtain recombinant plasmids, and the recombinant plasmids are transformed into Escherichia coli JM109 to obtain transformation products; the transformation products are spread on LB solid medium (containing 40 μg / mL kanamycin) and incubated upside down in a 37°C constant temperature incubator for 8-12 hours to obtain transformants; the transformants are picked and inoculated into LB liquid medium, and shake-cultured at 37°C and 120-180 rpm for 8-12 hours, after which the plasmids are extracted and sequenced for verification. If the verification is correct, the recombinant plasmids pET-24a-bslac and pPIC9K-balac are obtained;

[0051] (2) Construction of recombinant Escherichia coli

[0052] Take out the E. coli BL21 (DE3) competent cells stored at -80℃ and place them in an ice box for 5 minutes. Then, add an appropriate amount of the recombinant plasmid pET-24a-bslac constructed in step (1) to the competent cells and gently tap the tube wall with your fingers to mix. After placing it in an ice box for 30 minutes, take out the EP tube containing the competent cells and place it in a 42℃ water bath. After heat shock for 90 seconds, immediately place it in an ice box to cool. After 5 minutes, pour in 1 mL of LB liquid culture medium and incubate at 37℃, 200 r·min -1Incubate for 40-50 minutes, spread the bacterial solution on a solid LB plate containing Kan resistance under sterile conditions, and incubate in a 37°C incubator for 10-12 hours. Select 4-6 single colonies and inoculate them into 10 mL of liquid LB medium (Kan resistance, 30 μg mL -1 ) and cultured with shaking at 37°C for 8-10 h. The correct plasmid of the double enzyme digestion band was verified by nucleic acid gel electrophoresis to obtain the recombinant E. coli BL21-PET24a-bslac and stored in a -80°C refrigerator for future use.

[0053] (3) Construction of recombinant Pichia pastoris

[0054] Linearization system (20 μL): plasmid pPIC9K-balac 14.0 μL, 10×L buffer 2.0 μL, Sac I 1.0 μL, ddH2O 3.0 μL. Incubate the reaction system at 37°C for 2 hours. Store the linearized plasmid at 4°C until use.

[0055] Add 10 μL of the linearized plasmid to a competent P. pastoris KM71 cell and gently tap the tube to mix. Then, add the competent cell along the wall of a pre-chilled electrode cuvette and gently tap the bottom of the cuvette to remove any bubbles. Place the cuvette in a 1500V electroporator and perform electroporation. Immediately remove the cuvette and quickly add 1 mL of pre-chilled 1M sorbitol, gently pipetting to mix. Transfer the cell suspension to an EP tube and incubate at 30°C on a shaker for 1–2 hours. Pipette 100 μL of the cell suspension from the EP tube and evenly spread it onto MD solid medium. Incubate at 30°C on a shaker for 1.5–2 days.

[0056] 96 single colonies were picked from the MD plate and spotted at equal intervals on a new MD plate. After incubation at 30°C for 1-2 days, all the colonies were transferred to a deep-well plate containing 1 mL of BMGY and cultured for 48 hours. The cells were then centrifuged, the supernatant discarded, and the cells were suspended in 0.5 mL of BMMY and 1% (v / v) BMMY was added. -1 ) were induced with methanol for 2 days, and the supernatant was obtained by centrifugation to measure the enzyme activity. Eight transformants with higher enzyme activity were selected and named as recombinant Pichia pastoris KM71-pPIC9K-balac and stored at -80℃.

[0057] Example 2: Enzyme production by shake flask fermentation of recombinant E. coli BL21-pET24a-bslac

[0058] Take the glycerol tube containing the recombinant E. coli BL21-PET24a-bslac from the -80℃ freezer and aspirate 20μL of the bacterial solution to inoculate 10mL of LB liquid medium (Kan resistance, 30μg·mL-1). Then, shake and culture in a 37℃ shaker for 8-10h. Then, aspirate 5mL of the bacterial solution and transfer it to 100mL of TB medium (Kan resistance, 30μg·mL-1) and culture at 37℃ for 2h. Then, add IPTG to a final concentration of 0.2mM and culture at 25℃ and 200rpm for 24h. Centrifuge the fermentation broth at 8000rpm for 20min, discard the supernatant, and resuspend it in 50mM citrate buffer, pH 5.0 to an OD of 0. 600 The cell wall was broken by high-pressure homogenizer, and then centrifuged at 10,000 rpm for 15 minutes. The supernatant was the crude enzyme solution.

[0059] Example 3 Shake flask fermentation of recombinant P. pastoris KM71

[0060] Remove the glycerol tube containing recombinant P. pastoris KM71-pPIC9K-balac from the -80°C freezer and inoculate 30 μL of the culture into 10 mL of YPD liquid medium. Incubate the tube in a shaker at 30°C for 24-36 hours. Transfer 2.5 mL of the culture to 50 mL of BMGY liquid medium and incubate at 30°C for 24 hours. Collect the cells after centrifugation at 4000 rpm for 10 minutes and resuspend the cells in 25 mL of BMMGY liquid medium containing 1% (v / v) methanol. Add 1% (v / v) methanol every 24 hours and incubate at 30°C for 120 hours. Centrifuge the resulting fermentation broth at 10,000 rpm for 15 minutes, and remove the supernatant as the crude enzyme solution. Replace the culture medium in the crude enzyme solution with 50 mM citrate buffer at pH 5.0 using an ultrafiltration tube, and assay the enzyme activity.

[0061] Table 1 Laccase enzyme activity

[0062] Laccase Enzyme activity (U / L) BaLac <![CDATA[3.2×10 3 ]]> BsLac <![CDATA[7.2×10 3 ]]>

[0063] Example 4: Polyethylene (PE) film UV pre-oxidation and cleaning

[0064] The polyethylene film was pre-oxidized using ultraviolet heat treatment: the PE plastic film was spread flat on a stainless steel rack and placed in an ultraviolet aging box (UVC, 275–200nm), and the aging temperature was set to 70°C. Starting from the second day, samples were taken every 24 hours for 3 consecutive days. The films treated for 24 hours, 48 ​​hours, and 72 hours were named UVPE-1day (UVPE-1), UVPE-2day (UVPE-2), and UVPE-3day (UVPE-3), respectively. The UVPE-2day and UVPE-3day films were cut into 4×4cm 2 The membranes were ultrasonically rinsed in ethyl acetate, n-hexane, and deionized water, respectively, with each solution rinsed twice for 30 minutes each time. After removing any residual organic reagents from the surface, the membranes were placed in a fume hood and air-dried for later use. To detect the substances released from the UVPE surface after rinsing, clean tweezers were used to clamp the rinsed membranes into a 50ml conical flask. 10mL of 50mM citric acid buffer (pH 5.0) was added and the product migration reaction was carried out in a constant temperature water bath shaker at 30°C and 150rpm for a total of 5 days. A blank control was performed using unwashed UVPE membranes, with three replicates per group. Changes in the components in the buffer were detected and identified by GCMS.

[0065] Figure 2 The ATR-FTIR spectra of PE films after UV aging for different days are shown. Compared with the control group without pretreatment, the experimental group has a higher concentration of 1712 cm-1 and 1720 cm-2 respectively due to UV high temperature pre-oxidation. -1 、1174cm -1 and 908cm -1 Nearby peak, 1712cm -1 The peak position of ketone carbonyl peak is shown in the figure. As the treatment time increases, the peak intensity also increases. At the same time, the appearance of carbonyl peak also indicates that oxidation has occurred on the long chain of polyethylene molecules. -1 It may be the CO stretching vibration and OH bending vibration peak areas of alcohols and carboxylic acids respectively. The appearance of these oxygen-containing functional groups can increase the hydrophilicity of UVPE and the attackability of carbon-carbon chains.

[0066] from Figure 3It can be seen that the GC-MS results of the experimental group that was soaked in UVPE membranes without surface cleaning contained a rich variety of alkanes and oxygen-containing compounds. After two rounds of cleaning, the content of normal alkanes has been significantly reduced and the types of oxygen-containing products are basically controlled within ten, and their peak intensity is also controlled within an acceptable range, thus effectively controlling the problem of product interference in the later stage. It can be seen that using ethyl acetate and n-hexane to extract the surface of UVPE membranes can reduce the interfering substances introduced into the enzymatic hydrolyzate by the UVPE membrane itself. Figure 2 B also shows that after being cleaned with organic solvents, the surface of the UVPE film still retains a certain amount of oxygen-containing groups.

[0067] Example 5: Effect of Laccase Mediator System on UVPE Film Degradation

[0068] The mediator is selected from the group consisting of: 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), 4-acetyl-2,2,6,6-tetramethylpiperidone-1-oxyl radical (TEMPO), and 1-hydroxybenzotriazole (HBT).

[0069] Weigh 50 mg of the UVPE-2 day to UVPE-3 day membranes prepared in Example 4 as reaction substrates, use clean tweezers to clamp the membranes into a 50 ml conical flask, add 10 mL of enzyme solution (4 U / mL) and 10 mM mediator (ABTS, TEMPO, or HBT) prepared in Examples 2 and 3, respectively, and react in a constant temperature water bath shaker at 30 ° C and 150 rpm. The total reaction time is 5 days, during which 10 mM of the corresponding mediator is added every 24 h. A blank control is added with inactivated enzyme solution and 50 mM citric acid buffer of equal pH, with three parallels per group.

[0070] After the reaction is complete, the membrane is filtered through a 600-mesh nylon filter cloth and the filtrate is recovered. The membrane is ultrasonically cleaned twice with 2% SDS (v / v), deionized water, and 50% ethanol solution, respectively, to remove surface impurities such as proteins. Each wash lasts 30 minutes. After rinsing, the membrane is wrapped in weighing paper and dried in a 60°C oven. The dried film is then analyzed by SEM, IR spectroscopy, and HT-GPC. Changes in the reaction solution composition are detected and identified by GC-MS.

[0071] The results of SEM observation of the microscopic morphology of PE film showed that the control group of UVPE-3day without enzyme treatment had no obvious etching aging phenomenon on the film surface at two magnifications ( Figure 4 、 5 ), while the experimental groups treated with laccase BaLac and BsLac ( Figure 4 BD, FH; Figure 5Obvious etching and fragment shedding can be seen on BD and FH).

[0072] After laccase mediator treatment, the effective experimental groups of BaLac and BsLac were both at 4000-3000 cm-1, compared with the UVPE control group. -1 , 1800-1500cm -1 ,1200-900cm -1 There are different degrees of peak changes and the emergence of new absorption peaks in the wave number band ( Figure 6 ).from Figure 6 From the graphs of carbonyl index and hydroxyl index of C and F, it can be seen that the carbonyl index and hydroxyl index of UVPE film increased after LMS treatment, which also indicates that the oxidation degree of the plastic film is further enhanced.

[0073] Currently, molecular weight changes measured by gel permeation chromatography (HT-GPC) are considered a reliable method for determining polyethylene biodegradation. The weight-average molecular weight (Mw) of UVPE treated with the laccase-mediated system decreased to some extent. The average decrease in the three BaLac mediator groups was approximately 40%, while the decrease in the BsLac group ranged from 31% to 52% (Table 2). These results demonstrate that both laccase-mediated systems are effective in biodegrading UVPE.

[0074] Table 2 Molecular weight of UVPE-3 after treatment with laccase mediator system

[0075]

[0076]

[0077] Note:

[0078] Many possible oxidative degradation products appeared in the GC-MS results, ranging from Figure 7The product distribution and product types show that the enzymatic hydrolysis products are primarily oxygenated products such as ketones, alcohols, and aldehyde esters, with carbon chain lengths primarily concentrated in the C3-C20 range. However, the types and distribution of UVPE oxidation products produced by the two laccases with different redox potentials when paired with different mediators differ. The products produced by the ABTS and HBT mediators were primarily alcohols, while those produced by the TEMOP mediator were primarily ketones. The similarity in product types between the two laccases and the same mediator also suggests that, although the oxidation process in the mediator system is random, there is a certain tendency for product accumulation. The carbon chain lengths of the BaLac system tended to be short, ranging from C4-C8, while those produced by the BsLac system tended to be C7-C15. The detection of short-chain oxygenated products not only demonstrates that the laccase-mediator system is capable of attacking the activated carbon chains of UVPE, oxidatively cleaving carbon-carbon bonds and thus depolymerizing UVPE, but also indirectly suggests that the mediator system is capable of chain oxidation reactions on the degradation products, resulting in a rich array of oxidation products.

[0079] Although the present invention has been disclosed above in terms of preferred embodiments, 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 film, characterized in that: Polyethylene is degraded using laccase in a mediator system; the mediator includes 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, 4-acetyl-2,2,6,6-tetramethylpiperidone-1-oxyl free radical, or 1-hydroxybenzotriazole; the amino acid sequence of the laccase is shown in Genbank accession number WP_003243170.1 or AFC76164.1, and the amount of the laccase added is 0.8-1 U / mg of polyethylene film; the enzymatic reaction temperature is 25-35°C, and the reaction system pH is 5-6; and the polyethylene is also subjected to ultraviolet irradiation treatment before the enzymatic hydrolysis treatment.

2. The method according to claim 1, characterized in that The laccase is prepared by microbial fermentation.

3. The method according to claim 2, characterized in that The laccase is prepared by expressing the laccase having an amino acid sequence such as Genbank accession number WP_003243170.1 in Escherichia coli using pET-24a(+) as a vector.

4. The method according to claim 2, characterized in that The laccase is prepared by expressing the laccase with an amino acid sequence as shown in Genbank accession number AFC76164.1 in Pichia pastoris using pPIC9K as a vector.

5. The method according to claim 1, wherein The degradation system further contains a citric acid buffer.

6. Use of laccase, a gene encoding the laccase, a recombinant plasmid carrying the gene, or a host cell expressing the laccase in the degradation of ultraviolet pretreated polyethylene film, characterized in that: The amino acid sequence of the laccase is shown in Genbank accession number WP_003243170.1 or AFC76164.1.

Images