High-efficiency expression engineering strain of polyethylene glycol terephthalate degrading enzyme and application of high-efficiency expression engineering strain

Abstract

The invention relates to the field of gene engineering, and discloses a high-efficiency expression engineering strain of a polyethylene glycol terephthalate degrading enzyme and an application of the high-efficiency expression engineering strain. According to the invention, PET (Polyethylene Terephthalate) degrading enzymes LCCICCG and Turbo PETase are introduced into bacillus amyloliquefaciens, and a molecular element, a carrier and a host strain which are subjected to efficient secretory expression are further screened. According to the genetic engineering strain constructed by using the whole set of secretory expression system provided by the invention, a fermentation experiment shows that the extracellular expression quantity is the highest yield of the currently reported recombinant PET degrading enzyme, and meanwhile, the invention also discloses the efficient secretory expression of the PET degrading enzyme in bacillus amyloliquefaciens for the first time. The extracellular crude enzyme liquid produced by fermenting the engineering strain provided by the invention can be directly used for degrading PET (Polyethylene Terephthalate) plastics. Therefore, low-cost preparation and high-efficiency utilization of the key enzyme preparation in PET plastic recycling can be realized based on the method, and huge industrial application value is shown.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and specifically relates to a method for constructing an engineered Bacillus amyloliquefaciens strain that efficiently secretes and expresses PET plastic degrading enzymes, and directly degrading PET plastics by fermenting extracellular crude enzyme liquid. Background Art

[0002] Enzymatic recycling of waste PET plastics offers numerous advantages and promises to be a green approach to alleviating global plastic pollution and waste. However, the industrialization of this technology currently faces several challenges, particularly the low-cost preparation and application of key PET-degrading enzymes, which are considered key factors influencing its commercialization. Currently, over ten PET-degrading enzymes have been successfully expressed in various expression systems, including Escherichia coli, Bacillus subtilis, Pichia pastoris, and Yarrowia lipolytica. Bacillus and Pichia pastoris hold the greatest potential for heterologous expression. These systems are currently the mainstream strains for industrial enzyme production and possess efficient protein secretion and expression capabilities, offering significant advantages for the cost-effective preparation of novel enzymes such as PET plastic depolymerases. However, the yield of heterologously expressed PET-degrading enzymes, both domestically and internationally, remains significantly unattainable, hindering the economic viability of industrialized PET enzymatic degradation technology. To overcome this challenge, it is necessary to develop efficient, stable, and controllable expression systems to reduce enzyme production costs and ensure the economic viability of this technology. Summary of the Invention

[0003] In order to solve the problem of lack of commercial enzyme preparations for PET enzymatic degradation, the present invention explored the heterologous expression effects of different PET degradation enzyme genes in Bacillus amyloliquefaciens chassis cells and found that LCC ICCG The two PET-degrading enzymes, Turbo PETase and LCC, were efficiently secreted and expressed in Bacillus amyloliquefaciens 205. After systematic screening of expression molecular elements, it was determined that LCC could be expressed in this expression system. ICCG The optimal promoter and signal peptide elements of Turbo PETase were used to finally construct the engineered strain.

[0004] The present invention provides an efficient expression engineering strain of polyethylene terephthalate degrading enzyme, characterized in that the expression of PET degrading enzyme LCC is introduced into the starting strain of Bacillus amyloliquefaciens ICCG and Turbo PETase expression elements to enable the strain to express polyethylene terephthalate degrading enzyme.

[0005] Specifically, the promoter of the expression element is P43, PHapII and Pamy205, preferably Pamy205; The expression element also includes a signal peptide, which is the signal peptide SPamyE, SPhr29, SPpp2 and SPamy205, preferably SPamy205.

[0006] Preferably, the Bacillus amyloliquefaciens starting strain is Bacillus amyloliquefaciens Bacillus amyloliquefaciens 205.

[0007] Specifically, the degradative enzyme LCC ICCG The amino acid sequences of PETase and Turbo PETase are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively.

[0008] Preferably, the degrading enzyme LCC ICCG The coding nucleotide sequences of PETase and Turbo PETase were codon-optimized based on the starting bacteria.

[0009] More preferably, the degrading enzyme LCC ICCG The nucleotide sequences encoding the PETase and Turbo PETase are shown in SEQ ID NO: 2 and SEQ ID NO: 4.

[0010] The present invention provides the application of the high-efficiency expression engineering strain in the preparation of polyethylene terephthalate degrading enzyme.

[0011] The present invention further provides a method for degrading plastic film, which comprises the following steps: fermenting the high-efficiency expression engineered strain to obtain a fermentation crude enzyme solution, and using the fermentation crude enzyme solution to degrade the plastic film.

[0012] Specifically, the supernatant obtained by centrifugation of the crude enzyme solution after fermentation is added to the reaction system for degrading plastic film at a volume ratio of 4%; The preparation method of the fermentation crude enzyme liquid is as follows: after fermentation, the fermentation liquid is centrifuged at 12,000 xg for 30 minutes to obtain the plastic degrading enzyme crude enzyme liquid, and then filtered and sterilized using a 0.45 μm membrane.

[0013] More specifically, the plastic film is a 6 mm diameter film having a crystallinity of 7%; Fermentation conditions were 37°C, pH 7.0 (pH adjusted with ammonia and lactic acid), aeration of 400 L / h, and an initial rotational speed of 400 rpm (maximum 850 rpm, adjusted by feedback from DO 30%). Feeding was initiated when dissolved oxygen began to rise during fermentation. Samples were collected every 6 hours to measure the wet weight of the cell and the expression of the target enzyme in the fermentation broth. Specifically, the fermentation medium components are: sucrose 10 g / L, yeast extract 30 g / L, NaCl 8 g / L, K2HPO4 3 g / L, MgCl2 2 g / L, CaCl2·2H2O 0.3 g / L, MnSO4·2H2O 0.2 g / L, FeSO4·7H2O 0.02 g / L, ZnSO4·7H2O 0.02 g / L; Feed medium: 300 g sucrose, 10 g starch dissolved in 600 mL H2O, and 100 g yeast extract powder dissolved in 400 mL H2O.

[0014] The fermentation results showed that the engineered strain of Bacillus amyloliquefaciens provided by the present invention expressed the plastic degrading enzyme LCC. ICCG At 48h of fermentation, the enzyme production reached a maximum of 3.2g / L ( Figure 4 ), while the enzyme production of the plastic degrading enzyme Turbo PETase reached a maximum of 1.6 g / L at 36 h of fermentation ( Figure 5 ), it can be seen that the Bacillus amyloliquefaciens engineered strain provided by the present invention shows great application potential for industrial low-cost fermentation production of plastic degrading enzymes.

[0015] For the extracellular plastic degrading enzyme produced by fermentation in a 5 L fermenter of the present invention, the post-fermentation treatment is performed by a single step of centrifugation at 12,000 x g for 30 min to obtain a crude enzyme solution of the plastic degrading enzyme, which is then sterilized by filtration using a 0.45 μm membrane before the next step of the plastic film degradation experiment.

[0016] The results of experiments showed that the engineered strain constructed by the present invention was cultured in a 3L fermenter for 48 hours, and the extracellular recombinase LCC ICCGThe maximum yields of FAST-PETase and Turbo PETase reached 3.2 g / L and 1.6 g / L, respectively. The extracellular expression level of 3.2 g / L is the highest yield of recombinant PET degrading enzymes reported so far in prokaryotic expression systems. Although a 2024 report on the plastic degrading enzyme FAST-PETase also achieved high levels of extracellular secretion expression in Pichia pastoris, with a maximum yield of 3 g / L in a 30 L fermentor, the fermentation time required was as long as 212 hours (Complete decomposition of poly(ethylene terephthalate) by crude PET hydrolytic enzyme produced in Pichia pastoris, Chemical Engineering Journal, Chemical Engineering Journal Feb. 2024: DOI: 10.1016 / j.cej.2023.148418), the Bacillus amyloliquefaciens engineered strain constructed in the present invention has significantly excellent results in both fermentation yield and efficiency, and is of great significance for the low-cost preparation of plastic degrading enzymes. In addition, the present invention also provides a case of using crude enzyme liquid fermented by Bacillus amyloliquefaciens to degrade plastic film. The supernatant obtained by centrifugation after fermentation was added to the reaction system at a volume ratio of 4%, and a 6 mm diameter film with a crystallinity of 7% could be completely degraded within 24 hours. It can be seen that the crude enzyme liquid of PET degrading enzyme prepared by the engineered bacteria of Bacillus amyloliquefaciens provided by the present invention has good prospects for industrial production and application.

[0017] Strain deposit information: Bacillus amyloliquefaciens Bacillus amyloliquefaciens 205 was deposited in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC for short) on June 16, 2020, with the accession number: CGMCC No. 20093. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 . Screening of promoters for expressing PET-degrading enzyme in Bacillus amyloliquefaciens.

[0019] Figure 2 . Screening of signal peptides for PET-degrading enzyme secreted and expressed by Bacillus amyloliquefaciens.

[0020] Figure 3 Electrophoresis of extracellular proteins from shake flask fermentation of engineered Bacillus amyloliquefaciens strains; M: Marker, molecular weight (kDa) from top to bottom are 180, 130, 100, 70, 55, 40, 35, 25, 15, 1-2: LCC ICCG Extracellular protein expression in engineered strains, 3-4: Extracellular protein expression in Turbo PETase engineered strains.

[0021] Figure 4 . LCC production by Bacillus amyloliquefaciens engineered bacteria in a 5L fermenter ICCG Process diagram.

[0022] Figure 5 . Diagram of the fermentation process for producing Turbo PETase in a 5L fermenter using engineered Bacillus amyloliquefaciens bacteria.

[0023] Figure 6 . Image of Turbo PETase crude enzyme produced by fermentation of Bacillus amyloliquefaciens to degrade plastic film. DETAILED DESCRIPTION

[0024] The present invention is described below through specific embodiments in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0025] Example 1. Construction of an engineered Bacillus amyloliquefaciens strain that efficiently secretes and expresses PET-degrading enzyme 1.1 Codon optimization of PET degradation enzyme gene and construction of expression vector This embodiment is based on LCC ICCG The amino acid and nucleotide sequences of the genes for Turbo PETase are as follows: LCC ICCG Protein sequence (SEQ ID NO: 1): LVWASPSVEAQSNPYQRGPNPTRSALTADGPFSVASNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSP SAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ.

[0026] LCC ICCG Gene sequence (SEQ ID NO: 2): CTGGTTTGGGCTTCTCCGTCTGTTGAAGCTCAGTCTAACCCGTACCAGCGTGGTCCGAACCCGACCCGTTCTGCTCTGACCGCTGACGGTCCGTTCTCTGTTGCTAGTAACCCGTATCAGCGTGGCCCGAATCCGACCCGTAGTGCCCTGACCGCCGATGGTCCGTTTAGTGTTGCAACCTATACCGTTAGCCGCCTGAGTGTTAGTGGCTTTGGTGGTGGTGTTATTTATTATCCGACCGGTACCAGCCTGACCTTTGGCGGCATTGCAATGAGCCCGGGCTATACCGCCGATGCAAGTAGTCTGGCCTGGCTGGGTCGTCGTCTGGCCAGTCATGGCTTTGTGGTTCTGGTGATTAATACCAATAGCCGCTTTGATGGTCCGGATAGCCGCGCCAGTCAGCTGAGTGCCGCCCTGAATTATCTGCGCACCAGCAGCCCGAGTGCCGTGCGTGCTCGTCTGGATGCAAATCGCCTGGCAGTGGCAGGTCATAGCATGGGCGGCGGCGGTACCCTGCGTATTGCAGAACAGAATCCGAGTCTGAAAGCAGCCGTGCCGCTGACCCCGTGGCATACCGATAAAACCTTTAATACCAGCGTGCCGGTGCTGATTGTTGGCGCAGAAGCAGATACCGTTGCACCGGTGAGTCAGCATGCAATTCCGTTTTATCAGAATCTGCCGAGCACCACCCCGAAAGTGTATGTGGAACTGTGCAATGCAAGCCATATTGCCCCGAATAGCAATAATGCCGCAATTAGTGTTTATACCATTAGTTGGATGAAGCTGTGGGTGGATAATGATACCCGCTATCGTCAGTTTCTGTGCAATGTTAATGATCCGGCACTGTGCGATTTTCGTACCAATAATCGCCATTGCCAGTAA。

[0027] Turbo PETase protein sequence (SEQ ID NO: 3): QSNPYQRGPNPTRSALTTDGPFSVATYSVSRLSVSGFGGGVIYYPTGTTLTFGGIAMSPGYTADASSLALLGRRLASHGFVVIVINTNSRLDFPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGATLRISEQIPTLKAGVPLTPWHTDKTFNTPVPQLIVGAERDTVAPVSQSAIPIYQNLPSTTPKVYVELKNATHTAPNSPNACISVYTISWMKLWVDNDTRYRQFLCNVNDPCLSDFRSNNRHCQ。

[0028] Turbo PETase gene sequence (SEQ ID NO: 4): .

[0029] The ExpOptimizer codon optimization tool was used to optimize the codons for the Bacillus host. The optimized gene sequence was inserted into the multiple cloning site of the Bacillus expression vector pMA05, and the recombinant plasmid carrying the target gene was transformed into the expression host bacteria. Bacillus amyloliquefaciens 205 (Deposit Number: CGMCC No. 20093), the positive transformants were picked to extract the plasmids for sequencing verification, and the successfully constructed Bacillus amyloliquefaciens engineered strain was obtained.

[0030] The transformation method of Bacillus amyloliquefaciens is as follows: 1) Pick a single colony from the LB streak plate and transfer it to 2 mL of LB. Incubate at 37°C, 220 rpm for 12-16 hours.

[0031] 2) Transfer the bacterial solution to 40 mL of growth medium LBS at a 2% inoculum volume and culture in a baffled conical flask to increase ventilation. Incubate at 37°C and 220 rpm. Measure the OD value of the bacterial solution at regular intervals. 600nm .

[0032] 3) Stop the culture when the bacterial solution grows to an OD600nm between 0.85-0.95. Collect the bacterial solution into a 50mL centrifuge tube, place on ice for 20 minutes, and centrifuge at 12,000g at 4℃ for 10 minutes. Discard the supernatant and retain the bacterial cells.

[0033] 4) Add 0.1 mL of HEPES stock solution (1 M) to 100 mL of wash medium (SHMP). Wash the 40 mL of centrifuged bacteria with 25 mL of SHMP (prepare the cells in 1 mL of wash medium and then add the remaining wash medium). Gently centrifuge at 12,000 g at 4°C for 10 min. Discard the supernatant and retain the cells. If flocs are still present in the supernatant, continue centrifugation for 10 min.

[0034] 5) Repeat step 4 twice.

[0035] 6) Resuspend the cells in 1 mL of SHMP, mix thoroughly, and aliquot into 1.5 mL EP tubes at 200 μL / tube. Store at -80°C. Take one tube of competent culture and spread it on a common resistance plate to verify infection.

[0036] 7) Take one tube of competent cells, add DNA (or DNA methylation product), mix gently, and incubate on ice for 20 minutes. Add the mixture to a pre-chilled electroporation cuvette and electroporate at 1500-2500 V. Immediately add 600 μL of recovery medium (LBSPG) to the cuvette, mix thoroughly, and pipette the mixture into a 1.5 mL EP tube. Incubate at 37°C, 220 rpm for 7-8 hours.

[0037] 8) Take out the bacterial solution, shake it well, and place it in a 46℃ water bath for 8 minutes. Centrifuge the bacterial solution instantly and spread it on the resistance screening plate. Incubate it in a 37℃ incubator for 48 hours.

[0038] 1.2 Screening of protein expression elements and strain construction of Bacillus amyloliquefaciens Different protein expression elements were replaced in the recombinant expression vectors pMA05-ICCG and pMA05-turbo. First, expression strains containing different promoters were constructed: pMA05-p43-ICCG, pMA05-pHapII-ICCG, pMA05-pamy205-ICCG, pMA05-p43-turbo, pMA05-pHapII-turbo, and pMA05-pamy205-turbo. These plasmids were transformed into Bacillus amyloliquefaciens 205 according to the method in 1.1. The different promoter elements P43, pHapII, and Pamy205 were compared, and the optimal promoter Pamy205 was obtained ( Figure 1 ).

[0039] The promoter sequence is as follows: P43 (SEQ ID NO: 5): tgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgccgccgccggggctgtttgcgttcttgccgtgattt cgtgtaccattggtttatacttatttttttgccaaggctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagcggtaccattatag.

[0040] pHapII (SEQ ID NO: 6): gagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcg.

[0041] pamy205 (SEQ ID NO: 7): CAGGGTATTTTTTATGCTGTCCAGACTGTCCGCTGTGTAAAAAATAGGAATAAAGGGGGGTCGTTATTATTTTATTGATATGTAAAATATAATTTGTATAAGAAAATGAGAGGGAGAGGAAAC.

[0042] On the basis of the optimal promoter, the effects of different signal peptides on the extracellular secretion of the target enzyme were compared, including signal peptides SPamyE, SPhr29, SPpp2 and SPamy205. Expression strains containing different signal peptides were constructed, namely pMA05-SPamyE-ICCG, pMA05-SPhr29-ICCG, pMA05-SPpp2-ICCG, pMA05-SPamy205-ICCG, pMA05-SPamyE-turbo, pMA05-SPhr29-turbo, pMA05-SPpp2-turbo and pMA05-SPamy205-turbo. The above plasmids were transformed into Bacillus amyloliquefaciens 205 according to the method in 1.1. The optimal secretion signal peptide was SPamy205 ( Figure 2 ).

[0043] The signal peptide sequence used in the present invention is as follows: SPamyE (SEQ ID NO: 8): MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA; SPhr (SEQ ID NO: 9):MQVVLGRVRSAGLLAALLALAAWALVWASPSAEAQ; SPpp2 (SEQ ID NO: 10): MDGVLWRVRTAALMAALLALAAWALVWASPSVEAQ; SPamy205 (SEQ ID NO: 11): MIQKRKRTVSFRLVLMCTLLLFVSLPITKTSA.

[0044] Using the best expression element combination provided by the present invention, Bacillus amyloliquefaciens 205 were constructed to obtain efficient secretory expression of plastic degradation enzyme LCC ICCG and Turbo PETase engineered strains of Bacillus amyloliquefaciens.

[0045] Example 2: Fermentation of enzymes by engineered strains of Bacillus amyloliquefaciens and preparation of crude enzyme solution 2.1 Enzyme production by Bacillus amyloliquefaciens in shake flask fermentation The engineered strain of Bacillus amyloliquefaciens provided by the present invention was subjected to a shake flask fermentation experiment. An appropriately diluted glycerol tube bacterial solution was applied to a kanamycin-resistant LB plate. After overnight culture, a single colony was picked and placed in an LB liquid culture medium test tube at 37 o After culturing for 12 h at 220 rpm, the culture medium was transferred to a shake flask and the mixture was stirred for 37 oC and 220 rpm shake flask fermentation culture, after 48 h of fermentation, centrifuge to obtain the supernatant, and perform SDS-PAGE to detect the expression of target enzymes, such as Figure 3 As shown, LCC ICCG The extracellular expression level of PETase was slightly higher than that of Turbo PETase.

[0046] The formula of shake flask fermentation medium (SR+inducer) is as follows: peptone 10.0 g / L, beef extract powder 5.0 g / L, sodium chloride 5.0 g / L, inducer is 1% soluble starch, and the pH is adjusted to 7.5±0.2.

[0047] 2.2 Enzyme production in a 5 L fermenter of Bacillus amyloliquefaciens and preparation of crude enzyme solution Enzyme production experiments were also conducted in 5L fermenters using the two engineered strains provided by the present invention. The cultured seed solution was inoculated into a 5L fermenter containing 2.5L of fermentation medium. Fermentation conditions were a constant temperature of 37°C, pH 7.0 (pH adjusted with aqueous ammonia and lactic acid), aeration of 400 L / h, and an initial rotational speed of 400 rpm (with a maximum speed of 850 rpm adjusted by feedback control using a DO of 30%). Feeding was initiated when dissolved oxygen began to rise during fermentation. Samples were collected every 6 hours to measure the wet weight of the cells and the expression of the target enzyme in the fermentation broth.

[0048] Fermentation medium components: sucrose 10 g / L, yeast extract powder 30 g / L, NaCl 8 g / L, K2HPO4 3 g / L, MgCl2 2 g / L, CaCl2·2H2O 0.3 g / L, MnSO4·2H2O 0.2 g / L, FeSO4·7H2O 0.02 g / L, ZnSO4·7H2O 0.02 g / L.

[0049] Feed medium: 300 g sucrose, 10 g starch dissolved in 600 mL H2O, and 100 g yeast extract powder dissolved in 400 mL H2O.

[0050] The fermentation results showed that the engineered strain of Bacillus amyloliquefaciens provided by the present invention expressed the plastic degrading enzyme LCC. ICCG At 48h of fermentation, the enzyme production reached a maximum of 3.2g / L ( Figure 4 ), while the enzyme production of the plastic degrading enzyme Turbo PETase reached a maximum of 1.6 g / L at 36 h of fermentation ( Figure 5 ), it can be seen that the Bacillus amyloliquefaciens engineered strain provided by the present invention shows great application potential for industrial low-cost fermentation production of plastic degrading enzymes.

[0051] For the extracellular plastic degrading enzyme produced by fermentation in a 5 L fermenter of the present invention, the post-fermentation treatment is performed by a single step of centrifugation at 12,000 x g for 30 min to obtain a crude enzyme solution of the plastic degrading enzyme, which is then sterilized by filtration using a 0.45 μm membrane before the next step of the plastic film degradation experiment.

[0052] Example 3: Verification of the effect of Bacillus amyloliquefaciens fermentation on the degradation of PET plastics Application of the LCC prepared above ICCG and Turbo PETase crude enzyme solution, respectively at 72 o C and 65 o The plastic film with a diameter of 8 mm and a crystallinity of 7% was degraded at a temperature of 100 °C. The reaction system was 1 mL of PBS buffer with a pH of 8.0. After different amounts of crude enzyme solution added and catalytic reaction time, the results are shown in Table 1. Under the condition of not adjusting the pH value of the reaction system, it was found that LCC ICCG As the amount of crude enzyme solution added increases, the effect of the enzyme solution on the pH of the reaction system increases, resulting in a gradual weakening of the degradation effect on PET film. However, for the degradation enzyme Turbo PETase, although the amount of crude enzyme solution added has the same effect on the reaction system, it can still completely degrade the PET film substrate within the reaction time of 26 hours, and the enzyme protein of its crude enzyme solution is only LCC. ICCG It can be seen that, in comparison, the heterologously expressed PET plastic degrading enzyme Turbo PETase of the present invention shows applicability and enzyme activity in a wider reaction pH range and has higher industrial application value.

[0053] Table 1. LCC ICCG Comparison of PET film degradation performance with Turbo PETase crude enzyme solution

[0054] On this basis, the present invention further reduces the amount of enzyme added to the Turbo PETase crude enzyme solution, and the results are as follows Figure 6 As shown in the results, under the condition of the lowest enzyme addition amount of 4% (v / v) in the reaction system, the crude enzyme solution can still completely degrade PET film with a crystallinity of 7% and a diameter of 6 mm within 24 hours, showing great potential for low-cost industrial applications.

Claims

1. An engineered strain for highly efficient expression of polyethylene terephthalate degrading enzyme, characterized in that: Expression of PET-degrading enzyme LCC in Bacillus amyloliquefaciens starting strain ICCG and Turbo PETase expression elements to enable the strain to express polyethylene terephthalate degrading enzyme.

2. The high-efficiency expression engineered strain according to claim 1, characterized in that The promoter of the expression element is P43, PHapII and Pamy205, preferably Pamy205; The expression element also includes a signal peptide, which is the signal peptide SPamyE, SPhr29, SPpp2 and SPamy205, preferably SPamy205.

3. The high-efficiency expression engineered strain according to claim 1, characterized in that The Bacillus amyloliquefaciens starting bacteria is Bacillus amyloliquefaciens Bacillus amyloliquefaciens 205.

4. The high-efficiency expression engineered strain according to claim 1, characterized in that Degradation enzyme LCC ICCG The amino acid sequences of PETase and Turbo PETase are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively.

5. The high-efficiency expression engineered strain according to claim 4, characterized in that: Degradation enzyme LCC ICCG The coding nucleotide sequences of PETase and Turbo PETase were codon-optimized based on the starting bacteria.

6. The high-efficiency expression engineered strain according to claim 5, characterized in that: Degradation enzyme LCC ICCG The nucleotide sequences encoding the PETase and Turbo PETase are shown in SEQ ID NO: 2 and SEQ ID NO:

4.

7. Use of the high-efficiency expression engineered strain according to any one of claims 1 to 6 in the preparation of polyethylene terephthalate degrading enzyme.

8. A method for degrading plastic film, characterized in that: The method comprises the following steps: fermenting the high-efficiency expression engineered strain according to any one of claims 1 to 6 to obtain a crude fermentation enzyme solution, and using the solution for degrading plastic film.

9. The method according to claim 8, wherein The supernatant obtained by centrifugation of the crude enzyme solution after fermentation is added to the reaction system for degrading plastic film at a volume ratio of 4%; The preparation method of the fermentation crude enzyme liquid is as follows: after fermentation, the fermentation liquid is centrifuged at 12,000 xg for 30 minutes to obtain the plastic degrading enzyme crude enzyme liquid, and then filtered and sterilized using a 0.45 μm membrane.

10. The method according to claim 9, wherein The plastic film is a 6 mm diameter film with a crystallinity of 7%; Fermentation conditions were 37°C, pH 7.0 (pH adjusted with ammonia and lactic acid), aeration of 400 L / h, and an initial rotational speed of 400 rpm (maximum 850 rpm, adjusted by feedback from DO 30%). Feeding was initiated when dissolved oxygen began to rise during fermentation. Samples were collected every 6 hours to measure the wet weight of the cell and the expression of the target enzyme in the fermentation broth. Specifically, the fermentation medium components are: sucrose 10 g / L, yeast extract 30 g / L, NaCl 8 g / L, K2HPO4 3 g / L, MgCl2 2 g / L, CaCl2·2H2O 0.3 g / L, MnSO4·2H2O 0.2 g / L, FeSO4·7H2O 0.02 g / L, ZnSO4·7H2O 0.02 g / L; Feed medium: 300 g sucrose, 10 g starch dissolved in 600 mL H2O, and 100 g yeast extract powder dissolved in 400 mL H2O.

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