A fusion protein with dual pet hydrolytic enzyme activity and a method for degrading pet

Abstract

The present application belongs to the technical field of enzyme engineering, and particularly relates to a fusion protein with double PET hydrolytic enzyme activity and a method for degrading PET. The technical scheme of the present application is a fusion protein with double PET hydrolytic enzyme activity, which fuses cutinase TfH and PETase (FAST-PETase) to obtain double PET hydrolytic enzyme fusion protein TfH-FPE or FPE-TfH. The present application also provides a method for synergistically promoting PET degradation by using the double PET hydrolytic enzyme activity of the fusion protein, which converts an engineering bacterium expressing the fusion protein, collects the expressed protein after fermentation, and reacts the protein with a PET-containing sample. The present application fuses TfH with PET hydrolytic enzyme, introduces molecular chaperone GroEL / ES for co-expression under the condition of obtaining high secretion amount of the fusion protein; takes into account the double advantages of efficient secretion and synergistic catalysis of double PET hydrolytic enzyme; avoids the cumbersome steps of protein purification; has the advantages of convenience, easy control, low cost, easy scale-up, etc., and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a fusion protein with dual PET hydrolase activity and a method for degrading PET. Background Technology

[0002] Plastics are long-chain man-made polymer molecules. Due to their durability, flexibility, non-toxicity, and low cost, they are increasingly used in people's daily lives, with production rising from 1.5 million tons in 1950 to 350 million tons in 2018. However, secondary recycling of plastics has been minimal, leading to a growing influx of waste plastics into the environment. For a long time, people considered it merely an unsightly addition to the environment. Until recently, new discoveries have raised concerns about the potential detrimental effects of plastics on biodiversity and human health. Chemicals released by plastics are known to cause cancer and reproductive abnormalities in humans, rodents, invertebrates, and even marine life. More seriously, potential health risks and societal concerns about virus-contaminated products have increased plastic consumption, resulting in even lower recycling rates. PET is one of the most commonly used plastics, with an annual production of approximately 50 million metric tons; however, the recycling rate is only 20-30%, with the remainder flowing into the environment. The use of these plastics is growing at an alarming rate every year, and despite ongoing efforts to find more effective recycling methods, the recycling situation remains unsatisfactory. It is reported that currently only one-third of post-consumer plastic waste in Europe is collected for recycling, with the majority (43%) incinerated in waste-to-energy plants and the remainder ending up in landfills. Chemical recycling involves a range of advanced recycling technologies that transform plastic waste into valuable raw materials for the chemical industry, such as monomers, oligomers, and hydrocarbons. Unfortunately, both physical and chemical recycling are energy-intensive and can cause secondary pollution; therefore, there is an urgent need to develop a green and inexpensive method for processing plastics.

[0003] Biodegradation has been one of the hottest research areas in the last two decades. In the past two decades, several enzymes capable of catalyzing the hydrolysis of PET have been discovered, including several lipases, carboxylesterases, and keratinases. Among them, the mutant PETase FAST-PETase, LCC, and TfH are currently the enzymes with high PET degradation activity at 40–70℃. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a new option for the biodegradation of PET.

[0005] The technical solution of the present invention is a fusion protein with dual PET hydrolase activity, which is obtained by fusing keratinase TfH and PET enzyme (FAST-PETase).

[0006] The structure of the dual PET hydrolase is TfH-FPE or FPE-TfH, where FPE represents FAST-PETase.

[0007] Specifically, the amino acid sequence of the keratinase TfH is shown in SEQ ID No. 1 or SEQ ID No. 3.

[0008] Specifically, the amino acid sequence of the PET enzyme is shown in SEQ ID No. 2 or SEQ ID No. 4.

[0009] In particular, there is a linker between keratinase TfH and PETase (FAST-PETase).

[0010] Furthermore, the amino acid sequence of the linker is GGGGSGGGG or GGGGSEAAAKGGGGS (SEQ ID No. 15).

[0011] Furthermore, a signal peptide was constructed at the N-terminus of the fusion protein.

[0012] Furthermore, a histidine tag is constructed at the C-terminus of the fusion protein.

[0013] Specifically, the amino acid sequence of the fusion protein is shown in SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9.

[0014] The present invention also provides a gene encoding the fusion protein.

[0015] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID No. 10.

[0016] The present invention also provides a vector loaded with the said gene.

[0017] The carrier is an expression carrier.

[0018] Specifically, the backbone of the expression vector is pET30.

[0019] The present invention also provides the use of the fusion protein or carrier in promoting the degradation of PET plastics or in the preparation of PET plastic degrading agents.

[0020] Furthermore, the PET plastic is PET plastic waste.

[0021] The present invention also provides a method for promoting PET degradation. It includes the following steps: treating the fusion protein with PET plastic at 40–60°C to promote PET plastic degradation.

[0022] Furthermore, the PET plastic is PET plastic waste.

[0023] Furthermore, the processing conditions involve using a liquid containing dissolved fusion proteins.

[0024] Specifically, the liquid used to dissolve the fusion protein is a glycine, Tris, or phosphate buffer solution with a pH of 8–10.

[0025] Furthermore, the processing time is 1 to 7 days.

[0026] Preferably, the method further includes the step of preparing the fusion protein: preparing the vector, transforming the obtained vector into engineered bacteria, and collecting the expressed fusion protein after fermentation.

[0027] Preferably, the method of the present invention further includes the following steps: constructing a vector for expressing a molecular chaperone, co-transforming engineered bacteria with a vector for expressing a fusion protein, collecting the expressed protein after fermentation, and using it to treat PET plastic waste.

[0028] Specifically, the molecular chaperones are GroEL and GroES.

[0029] The nucleotide sequence of the GroEL gene is shown in SEQ ID No. 5.

[0030] Specifically, the nucleotide sequence of the GroES gene is shown in SEQ ID No. 6.

[0031] Specifically, the backbone vector for expressing the molecular chaperone is pBAD.

[0032] The engineered bacteria is Escherichia coli.

[0033] Specifically, during the fermentation process, the addition of 0.5–1 mM IPTG or 2.5–5 g / L lactose induces TfH-FPE expression, and the addition of 0.5–1 mg / mL arabinose induces the expression of the molecular chaperone GroEL / ES.

[0034] Furthermore, after adding 0.5 mM IPTG, fermentation was carried out for 24–72 hours.

[0035] Preferably, fermentation is carried out for 24 hours after adding 0.5mM IPTG.

[0036] Specifically, after adding 5g / L of lactose, fermentation should be carried out for 24–72 hours.

[0037] Preferably, fermentation is carried out for 72 hours after adding 5g / L lactose.

[0038] Specifically, the fermentation temperature is 16–25°C.

[0039] Preferably, the fermentation temperature is 25℃.

[0040] Specifically, the collected expressed proteins are treated with PET plastic waste at 40–60°C for 1–7 days.

[0041] Preferably, the collected expressed protein is treated with PET plastic waste at 50°C for 3 days.

[0042] The beneficial effects of this invention are as follows: This invention utilizes the properties of keratinase TfH, which, in addition to degrading PET, also degrades cell membranes and alters cell membrane permeability. TfH is fused with another dominant PET hydrolase (FAST-PETase) to obtain an enzyme with dual activity. Surprisingly, the fusion protein without a signal peptide showed a 32.5-fold increase in secretion compared to the fusion protein with a signal peptide. The fusion protein of this invention relieved the product inhibition of MHET, resulting in the release of 1.5 times the product of FAST-PETase. Furthermore, the product released by the fusion protein of this invention is twice that of the two mixed enzymes, and its effect in degrading PET is superior to that of either enzyme alone or in combination, achieving unexpected results. This invention also provides a method for preparing the fusion protein, in which the molecular chaperone GroEL / ES is introduced for co-expression. After optimization of induction temperature, induction time, and inducer conditions, 104±5.2 mg / L Tf-FPE was obtained. Kinetic and PET-catalyzed degradation analyses of the secreted Tf-FPE with FAST-PETase showed that Tf-FPE has excellent degradation ability. Compared with cytoplasmic expression, this method utilizes the characteristic of efficient expression of the fusion protein without a signal peptide, and the enhanced solubility of THf-FPE under the action of the molecular chaperone GroEL / ES, resulting in efficient extracellular secretion. It combines the advantages of efficient secretion and synergistic catalysis by two PET hydrolases; it avoids the cumbersome steps of protein purification; and it has the advantages of being convenient, easy to control, inexpensive, and easy to scale up, showing broad application prospects. Attached Figure Description

[0043] Figure 1The images show SDS-PAGE images of pelB-TfH-FPE and TfH-FPE expression at different temperatures in Example 1. A, B, and C represent SDS-PAGE images of the *E. coli* control, pelB-TfH-FPE, and TfH-FPE induced by 0.5 mM IPTG at 16°C, 20°C, and 25°C, respectively. The corresponding band for TfH-FPE is marked with a solid triangle. The loading order was: Marker, *E. coli* control, pelB-TfH-FPE and TfH-FPE precipitate after disruption, soluble supernatant, and ammonium sulfate precipitation medium. I represents cell disruption precipitate, SN represents the supernatant from disruption and centrifugation, and CS medium supernatant. The experimental results show that compared to pelB secretion, TfH-FPE without a signal peptide was secreted in greater quantities. At 16°C and 20°C, it was mainly expressed in the cytoplasm, while at 25°C, it was secreted into the culture medium.

[0044] Figure 2 The figures show the growth curves of *E. coli* under three conditions: control (square curve), co-expression of molecular chaperone GroEL / ES and TfH-FPE (IPTG induction, dotted curve), and co-expression of molecular chaperone GroEL / ES and TfH-FPE (lactose induction, triangular curve) in Example 2. The results indicate that TfH-FPE degradation of phospholipids and increased cell membrane permeability have a certain impact on the growth of *E. coli*, and the effect is greater with IPTG induction than with lactose induction.

[0045] Figure 3 The images show SDS-PAGE images of the molecular chaperone GroEL / ES and TfH-FPE co-expression in Example 2. The corresponding TfH-FPE bands are marked with solid triangles; where I represents cell lysis pellet, SN represents the supernatant from lysis and centrifugation, CS medium supernatant, and E represents affinity purification results. A shows SDS-PAGE images after 24h, 48h, and 72h of induction with 0.5mM IPTG; the results indicate that IPTG induction after 24h does not increase TfH-FPE secretion. B shows SDS-PAGE images after 24h, 48h, and 72h of induction with 5g / L lactose; the results show that the induction amount is low at 24h, induction becomes stronger between 24 and 48h, and the highest secretion amount is achieved after 72h.

[0046] Figure 4 The values ​​represent the expression levels of IPTG-induced and lactose-induced affinity chromatography-purified proteins in Example 2 at 24h, 48h, and 72h. A represents the expression level of IPTG-induced affinity chromatography-purified protein, which is 86.2±1.6 mg / L of purified TfH-FPE; B represents the expression level of lactose-induced affinity chromatography-purified protein, which is 104±5.2 mg / L of purified TfH-FPE.

[0047] Figure 5The results of kinetic analysis for FAST-PETase and TfH-FPE in Example 3 are shown. The results indicate that the optimal initial degradation temperature for both FAST-PETase and TfH-FPE is 50℃, with TfH-FPE exhibiting a higher initial rate. Figure A shows the relationship between the initial rate of FAST-PETase and enzyme concentration at 40℃ and 50℃, fitted using kinetic equation 1 from Example 3; Figure B shows the data from A fitted using kinetic equation 2 from Example 3; Figure C shows the relationship between the initial rate of TfH-FPE and enzyme concentration at 40℃ and 50℃, fitted using equation 1. Figure 5 D is based on Figure 5 The data for C were fitted to the graph using Formula 2.

[0048] Figure 6 The figures show the product release from the degradation of PET membranes by FAST-PETase and TfH-FPE at 40°C and 50°C in Example 3. A and B show the product release after one week of degradation at 40°C for FAST-PETase and TfH-FPE, respectively. This indicates that TfH-FPE releases 1.5 times more total products than FAST-PETase due to reduced MHET product inhibition. C and D show the product release after one week of degradation at 50°C for FAST-PETase and TfH-FPE, respectively. This indicates that TfH-FPE releases significantly more products from the degradation of the PET membrane than FAST-PETase.

[0049] Figure 7 A, B, and C represent the product release after 72 hours of degradation of 600 nM FAST-PETase, 600 nM TfH, 300 nM FAST-PETase + 300 nM TfH, and 300 nM TfH-FPE at 40, 50, and 60 °C, respectively, in Example 3. D shows the morphology of PET membranes after degradation by FAST-PETase, TfH, FAST-PETase + TfH, and TfH-FPE at 50 °C. The results indicate that due to the proximity effect, TfH-FPE is significantly more abundant than FAST-PETase, TfH, and their mixture. Detailed Implementation

[0050] To provide a more efficient and practical solution for the biodegradation of PET, one approach is to combine multiple enzymes with PET-degrading activity. However, there are numerous PET enzymes with PET-degrading activity, but their effectiveness varies greatly, and their degradation mechanisms differ. Based on extensive prior work, this invention selects from numerous combinations the fusion of the keratinase TfH from *Thermobifidafusca* (a PET hydrolase) and the PET enzyme (FAST-PETase) from *Ideonella sakaiensis* to prepare a fusion protein.

[0051] The amino acid sequence (SEQ ID No. 1) of the TfH protein is as follows:

[0052] MANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHS MGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPF.

[0053] The amino acid sequence (SEQ ID No. 2) of the FPE (FAST-PETase) protein is as follows:

[0054] MPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWS MGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCS.

[0055] One method for obtaining the fusion protein of the present invention is through genetic engineering fusion expression. The present invention synthesizes a dual PET hydrolase gene containing keratinase TfH (a PET hydrolase) from *Thermobifida fusca* and PET enzyme (FAST-PETase) from *Ideonella sakaiensis*. A linker is positioned between the two; common linkers that ensure the spatial structure and activity of both units can be used. In one embodiment of the present invention, the linker used is GGGGSGGGG.

[0056] When using the GGGGSGGGG linker, the amino acid sequence of the fusion protein TfH-FPE of this invention is as follows (SEQ ID No. 7):

[0057] .

[0058] In some cases, especially during protein expression and purification, auxiliary functional sequences, such as restriction enzyme sites, are added to the above sequence. These restriction enzyme sites include restriction endonuclease sites introduced during the construction of the expression vector. For example, in one embodiment of the present invention, an XhoI restriction enzyme site is introduced, which will eventually be expressed as the amino acid "LE". In this case, the amino acid sequence of the fusion protein TfH-FPE of the present invention can be (SEQ ID No. 8):

[0059] .

[0060] To facilitate cleavage by the tag sequence introduced for affinity purification, a protease cleavage site can be introduced at the C-terminus. Various tag sequences that can bind to and elute affinity materials can be introduced for affinity purification; the most commonly used is the histidine tag. For example, in one embodiment of this invention, a histidine tag HHHHHH was further introduced at the C-terminus, with the amino acid sequence shown in SEQ ID No. 9. It is generally believed that the introduction of the aforementioned sites and tags will not significantly affect the performance of the fusion protein.

[0061] During vector construction, two expression vectors were constructed, one with and one without the pelB signal peptide. Both vectors were transformed into *E. coli* to obtain recombinant *E. coli*. Generally, the presence of a signal peptide promotes protein secretion; however, unexpectedly, because the keratinase TfH, in addition to degrading pelB, also degrades the cell membrane and alters cell membrane permeability, the fusion protein obtained in this invention exhibited significantly higher secretion levels even without the signal peptide, thus contributing to increased secretion. In subsequent studies, the expression vector without the signal peptide encoding gene was selected for further experiments.

[0062] To ensure the activity of the secreted fusion protein, this invention adds the molecular chaperone GroEL / ES to the expression system to promote the correct folding of the fusion protein, and uses the Golden Gate recombination method to obtain the expression vector containing the molecular chaperone; the molecular chaperone GroEL / ES expression vector and the fusion protein expression vector are co-transformed to obtain co-expressed recombinant Escherichia coli.

[0063] This invention further investigated the effects of different inducers on the secretion of fusion proteins and their degradation effects on PET at different temperatures, providing favorable support for the mass production of fusion proteins. Based on this, to study the enzymatic characteristics of the fusion enzyme, this invention also conducted kinetic analysis on the degradation of single enzymes and fusion proteins, demonstrating that the fusion of TfH with FAST-PETase does not affect the enzyme's properties, and that TfH-FPE has a better initial degradation efficiency for PET than FAST-PETase.

[0064] The results of the control experiment testing the degradation efficiency of the fusion protein showed that the release of degradation products of the fusion protein of the present invention was much higher than that of single enzyme degradation, and more than twice that of mixed degradation of two single enzymes, which has an unexpectedly excellent effect.

[0065] The present invention will be further described below with reference to specific embodiments.

[0066] The following examples are provided to better understand the present invention, but do not limit the invention. All experimental methods in the following examples are conventional methods. Unless otherwise specified, all reagents and consumables in the following examples are commercially available.

[0067] The plasmid pET30a and pBAD vector mentioned in this invention were purchased from Beijing Solarbio Science & Technology Co., Ltd. and are stored in our laboratory. Escherichia coli Dh5α and Escherichia coli shuffle T7 were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0068] Reagents used:

[0069] DNA Mix polymerase, plasmid extraction kit, and gel extraction kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; agarose was purchased from Qingke Biotechnology Co., Ltd.; DNA endonuclease and T4 ligase were purchased from ThermoFisher Scientific; and other commonly used reagents were purchased from Chengdu Kelon Chemical Co., Ltd.

[0070] All instruments:

[0071] PCR amplification instrument (Eppendorf), centrifuge (Thermo Fisher Scientific), protein purification instrument (GE Healthcare), high performance liquid chromatograph (Waters).

[0072] Example 1: Verification of TfH-FPE expression with or without signal peptide pelB

[0073] (1) For verification and comparative studies, nucleotide sequences encoding TfH and FAST-PETase were chemically synthesized in this embodiment. To facilitate subsequent vector construction and protein collection, XhoI restriction sites (LE) and histidine tag sequences for easy purification were added to the 3' end of the chemically synthesized TfH and FAST-PETase sequences. After expression, the purified amino acid sequences of TfH and FAST-PETase are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0074] SEQ ID No. 3 TfH protein amino acid sequence:

[0075] MANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFA PNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFLEHHHHHH.

[0076] SEQ ID No. 4 FPE (FAST-PETase) protein amino acid sequence:

[0077] MPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGG GSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCSLEHHHHHH.

[0078] Generally, signal peptides promote protein secretion. Therefore, when constructing the vector expressing TfH-FPE, two expression vectors were constructed, one with and one without the pelB signal peptide.

[0079] The nucleotide sequences of FpelB-TfH-FPE (using the flexible linker "GGGGSGGGG" between TfH and FAST-PETase) and TfH-FPE (using the flexible linker "GGGGSGGGG" between TfH and FAST-PETase) were chemically synthesized. To facilitate subsequent vector construction and protein collection, XhoI restriction sites and histidine tag sequences for easy purification were added to the 3' end of the chemically synthesized pelB-TfH-FPE and TfH-FPE sequences.

[0080] The amino acid sequence of SEQ ID No. 9 TfH-FPE protein (containing XhoI restriction site and histidine tag):

[0081] MANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGG GTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFGGGGSGGG GMPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGG GGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCSLEHHHHHH

[0082] SEQ ID No. 10 TfH-FPE nucleotide sequence (including XhoI restriction site and histidine tag):

[0083]

[0084] SEQ ID No. 11 pelB-TfH-FPE nucleotide sequence, 1–66 bp is the pelB signal peptide sequence, 67–1683 bp is the TfH-FPE sequence:

[0085]

[0086] Primers pelB F and R for amplifying pelB-TfH-FPE and ΔpelB F and R for amplifying TfH-FPE were also synthesized. PCR products were subjected to 1% agarose gel electrophoresis, and the target fragment was purified into DNA using a gel extraction kit.

[0087] The pelB-TfH-FPE gene sequence (with an NdeI restriction site at the 5' end and an XhoI restriction site at the 3' end) was chemically synthesized and linked to FAST-PETase using a flexible linker (GGGGSGGGG). PCR amplification yielded pelB-TfH-FPE (SEQ ID No. 10, using pelB F and R primers) and TfH-FPE (SEQ ID No. 11, using [primer name missing] primers). The primers involved are as follows:

[0088] pelB F: CATATGAAATACCTGCTGCCGACCGCT (SEQ ID No. 12)

[0089] ΔpelB F: CATATGAACCCTTATGAACGTGGTCCGAATC (SEQ ID No. 13)

[0090] R:CTCGAGGCTGCAATTCGCGGTACGAAAATCG(SEQ ID No.14)

[0091] The PCR reaction system and conditions are as follows: 30 ng DNA template, 1 μL primer F, 1 μL primer R, 12.5 μL PCR Mix, and ddH2O added to a final volume of 25 μL. The PCR fragment amplification program is as follows: pre-denaturation at 95℃ for 2 min, each cycle consisting of 98℃ for 30 s, 65℃ for 30 s, and 72℃ for 1 min, for a total of 34 cycles, followed by a final extension at 72℃ for 5 min.

[0092] pET30a was transformed into E. coli Dh5a competent cells for amplification and plasmid extraction was performed using a plasmid extraction kit.

[0093] (2) The obtained pelB-TfH-FPE, TfH-FPE and pET30a were digested with NdeI and XhoI at 37℃ for 12 hours, respectively. Then, the DNA was recovered by gel electrophoresis and purified. The enzyme digestion system was as follows: 2 μg of target DNA, 2 μL of 10× enzyme digestion buffer, 1 μL of NdeI, 1 μL of XhoI, and ddH2O to 20 μL.

[0094] The ligation system is as follows: 10 μL (50 ng) plasmid DNA, 5.0 μL (100 ng) PCR product, 2 μL T4 ligase buffer, 1 μL T4 ligase, and 2 μL ddH2O.

[0095] (3) Preparation and transformation of Escherichia coli DH5α in chemically transformed state:

[0096] a. Streak Escherichia coli DH5α on non-antibiotic LB solid plates and incubate overnight at 37°C.

[0097] b. Select the above single clones and culture them in 20 mL of antibiotic-free liquid LB until the OD600 = 0.8; take 1 mL of bacterial culture, centrifuge, blow with pre-cooled 0.1 M CaCl2 and place on ice for 30 minutes.

[0098] c. After pre-cooling for 30 minutes, centrifuge at 12000 rpm for 1 min, discard the supernatant and gently pipette with 100 μL of 0.1 M CaCl2 to prepare competent cells.

[0099] e. Take 10 μL of the above ligation product, add it to E.coli Dh5α competent cells and incubate on ice for 30 min, heat shock at 42℃ for 1 min, and then quickly incubate on ice for 2 min.

[0100] f. Add 700 μL of LB medium and thaw at 37°C for 1 h. Centrifuge and spread onto 50 μg / mL solid LB medium.

[0101] g. Pick single clones for subsequent plasmid extraction and sequencing verification.

[0102] (4) The pET30-pelB-Tf-FPE and pET30-Tf-FPE obtained from the sequencing were transformed into E. colishuffle T7 expression bacteria, and the preparation and transformation of competent cells were as described above.

[0103] (5) Take the above-mentioned recombinant E. coli shuffle T7 expression bacteria and E. coli shuffle T7 control and shake them in 20 mL LB medium at 37℃ for 12 h. Inoculate 2% into 100 mL TB medium and amplify to OD600=1.5 at 37℃ and 220 rpm. Add 0.5 mM IPTG and induce for 24 h at 16℃, 20℃ and 25℃ respectively.

[0104] (6) After expression for 24 hours, centrifuge at 4°C and 6000 rpm for 15 minutes. Transfer the supernatant of the culture medium to a 500 mL beaker and slowly add ammonium sulfate to 70% (w / v) while stirring. Let it stand at the appropriate temperature for 3 hours to precipitate the protein.

[0105] (7) Centrifuge the culture medium containing the above ammonium sulfate precipitate at 4°C and 12,000 rpm for 30 min, discard the supernatant, and agitate the precipitate with buffer A (50 mM Na2HPO4 100 mM NaCl). Dialyze the precipitate overnight in 2 L of buffer A using a 10 kD dialysis bag.

[0106] (8) The dialyzed samples were centrifuged at 4°C and 12,000 rpm for 30 min, and the protein was purified by ultrafiltration affinity chromatography using a 0.22 μm filter membrane. The purified protein was quantified using the Bradford method. After affinity chromatography purification, the Bradford method showed that the secretion of TfH-FPE mediated by the pelB signal peptide was only 3 ± 2 mg / L, while the secretion of TfH-FPE without the signal peptide reached 18.4 ± 2.5 mg / L.

[0107] (9) The recombinant E. coli shuffle T7 and E. coli shuffle T7 control expressed above were disrupted and centrifuged; and the protein from the disrupted precipitate, supernatant, and ammonium sulfate precipitate was subjected to SDS-PAGE expression verification. Figure 1 This indicates that compared to TfH-FPE secretion mediated by the pelB signal peptide, the amount of TfH-FPE secreted without the signal peptide is greater. At 16℃ and 20℃, it is mainly expressed in the cytoplasm, while at 25℃ it is secreted into the culture medium.

[0108] The SDS-PAGE adhesive formula is as follows:

[0109] 12% Resolving Gels (10 mL): 1.5 mol / L Tris-HCl pH 7.5 2.53 mL, 30% acrylamide 4.00 mL, deionized water 3.26 mL, 10% SDS 0.10 mL, 10% ammonium persulfate (APS) 0.10 mL, tetramethylethylenediamine (TEMED) 0.004 mL; 5% Stacking Gels (4 mL): 1.0 mol / L Tris-HCl pH 6.8 0.50 mL, 30% acrylamide 0.67 mL, deionized water 2.73 mL, 10% SDS 0.04 mL, 10% ammonium persulfate (APS) 0.04 mL, tetramethylethylenediamine (TEMED) 0.004 mL.

[0110] Example 2: Validation of co-expression of molecular chaperone GroEL / ES and TfH-FPE

[0111] (1) GroEL and GroES were chemically synthesized, and the two sequences (SEQ ID No. 5 and SEQ ID No. 6) were recombined and integrated into the pBAD vector to obtain the pGroEL / ES expression vector. The above TfH-FPE expression recombinant E. coli shuffle T7 was prepared into competent cells and transformed into pGroEL / ES to obtain co-expression recombinant E. coli shuffle T7. The preparation of competent cells and transformation methods are as described in Example 1.

[0112] (2) The expression level of TfH-FPE co-expressed by the molecular chaperone was expressed and verified according to the method described in Example 1.

[0113] (3) TfH-FPE expression was induced with 0.5 mM IPTG and 5 g / L lactose, respectively, and the expression level was measured at 24, 48, and 72 h after induction. The expression of the molecular chaperone GroEL / ES was induced with 0.5 mg / mL arabinose. When expression was induced with 0.5 mM IPTG ( Figure 3 A) Induction at 24h, 48h, and 72h successfully secreted TfH-FPE into the culture medium, and after 24h, the secretion of TfH-FPE did not increase. After affinity chromatography purification, the concentration was quantified by the Bradford method to be 80.3±2.5 mg / L; when expression was induced with 5 g / L lactose ( Figure 3 (B) Due to the slow absorption of lactose by *E. coli*, the secretion amount was only 3.2 ± 0.6 mg / L after 24 hours of induction, reaching 86.2 ± 1.6 mg / L after 48 hours, and as high as 104 ± 5.3 mg / L after 72 hours. Figure 4 A, B).

[0114] The nucleotide sequence of the molecular chaperone GroEL is SEQ ID No. 5:

[0115]

[0116] The nucleotide sequence of the molecular chaperone GroES is SEQ ID No. 6:

[0117] Atgaatattcgtccattgcatgatcgcgtgatcgtcaagcgtaaagaagttgaaactaaatctgctggcggcatcgttctgaccggctctgcagcggctaaatccacccgcggcgaagtgctggctgtcggcaatggccgtatcctt gaaaatggcgaagtgaagccgctggatgtgaaagttggcgacatcgttattttcaacgatggctacggtgtgaaatctgagaagatcgacaatgaagaagtgttgatcatgtccgaaagcgacattctggcaattgttgaagcgtaa

[0118] Example 3 Kinetic Analysis of FAST-PETase and TfH-FPE

[0119] (1) The chemically synthesized SEQ ID No. 2 sequence (with Nde I at the N-terminus and Xho I restriction site at the C-terminus) was constructed in pET30 using the Golden Gate method, transformed into E. coli shuffle T7 as described in Example 1, and fermented at 25°C to produce FAST-PETase. The purified FAST-PETase and TfH-FPE were subjected to kinetic analysis using an amorphous PET membrane.

[0120] (2) The PET membrane was punched into discs with a diameter of 0.6 cm. Two discs were placed in 600 μL of 50 mM Gly-NaOH buffer, and 0-500 nM of purified enzyme was added to each disc. The discs were incubated at 40 and 50 °C for 2 h respectively. The enzyme was then inactivated at 85 °C for 15 min. The reaction mixture was centrifuged at 12000 rpm for 15 min, ultrafiltered through a 0.22 μm filter membrane, and analyzed by HPLC with an Eclipse Plus-C18 column (5 μm, 4.6 × 250 mm). The degradation products were identified by UV at 260 nm. The HPLC reaction flow was as follows: from 0 to 18 min, the flow direction was changed from 95% 0.1% formic acid water and 5% acetonitrile to 70% 0.1% formic acid water and 30% acetonitrile.

[0121] (3) Degradation data were obtained using heterogeneous kinetic equation 1 The maximum initial rate was obtained by fitting. Figure 5 ); using dynamic equation 2 The hydrolysis constant k2 (Table 1) and adsorption constant K (Table 2) were obtained by fitting. Figure 5 This indicates that both FAST-PETase and TfH-FPE have optimal initial rates at 50 °C, with TfH-FPE having a slightly higher initial rate than FAST-PETase, at 93.2 ± 0.7 μmol·L⁻¹. -1 ·h -1 and 76.6±2.1 μmol·L -1 ·h -1 The hydrolysis constant of TfH-FPE at 50℃ is 79.05±6.32 nmol·cm⁻¹. -2 ·h -1 It is 1.3 times that of FAST-PETase (Table 1); however, the adsorption constant of FAST-PETase is slightly higher than that of TfH-FPE (Table 2), indicating that adsorption capacity is not the rate-limiting step for TfH-FPE to hydrolyze PET.

[0122] Table 1 Hydrolysis constants of FAST-PETase and TfH-FPE

[0123]

[0124] Table 2 Adsorption constants of FAST-PETase and TfH-FPE

[0125]

[0126] Example 4: Degradation Analysis of Amorphous PET Membranes Using FAST-PETase and TfH-FPE

[0127] The PET membrane was placed in 5 mL of 50 mM Gly-NaOH buffer, and 300 nM purification enzyme was added. The membrane was degraded at 40℃ and 50℃ for one week. Samples were taken daily and analyzed by HPLC using the method described above. Figure 6 The results showed that at 40℃, the main degradation products of FAST-PETase were hydroxyethyl terephthalate (MHET) and terephthalic acid (TPA), while the main degradation products of TfH-FPE were TPA and a small amount of MHET. This indicates that TfH-FPE mitigated the inhibitory effect of MHET, leading to an enhanced ability to degrade PET films. Figure 6 A, B). At 50℃, the stability of both enzymes is affected; therefore, the degradation ability of FAST-PETase is significantly reduced compared to 40℃, and TfH-FPE no longer increases product release after 3 days. Figure 6 C, D).

[0128] To analyze the effect of the proximity effect of TfH and FAST-PETase on PET membrane degradation, samples were degraded in 800 μL 50 mM Gly-NaOH buffer for 72 h using 600 nM FAST-PETase, 600 nM TfH, 300 nM FAST-PETase + 300 nM TfH, and 300 nM TfH-FPE, respectively. The degraded PET membranes were then analyzed by HPLC using the above method. After washing with 1% SDS, 20% ethanol, and deionized water, the degraded PET membranes were analyzed by scanning electron microscopy. The results showed that at 40℃ and 50℃, TfH-FPE, due to the proximity effect, resulted in a greater release of PET membrane degradation products than either of the two individual enzymes or the mixed enzyme. Furthermore, at 50℃, the product release of TfH-FPE was twice that of the mixed enzymes. Figure 7 B); SEM morphology also proved that TfH-FPE has the strongest hydrolysis ability (B); Figure 7 D).

Claims

1. A fusion protein with dual PET hydrolase activity, characterized in that: A dual PET hydrolase fusion protein was obtained by fusing keratinase TfH and PET enzyme FAST-PETase; the fusion protein structure is TfH-FPE or FPE-TfH, where FPE represents FAST-PETase.

2. The fusion protein according to claim 1, characterized in that: The amino acid sequence of the keratinase TfH is shown in SEQ ID No. 1; or, the PET enzyme is FAST-PETase, and the amino acid sequence is shown in SEQ ID No.

2.

3. The fusion protein according to claim 1, characterized in that: A linker is constructed between the keratinase TfH and the PET enzyme; or, a signal peptide is constructed at the N-terminus of the fusion protein; or, a histidine tag is constructed at the C-terminus of the fusion protein.

4. The fusion protein according to any one of claims 1 to 3, characterized in that, Meet at least one of the following: 1) The amino acid sequence of the linker is GGGGSGGGG or GGGGSEAAAKGGGGS; 2) The amino acid sequence of the histidine tag is HHHHHH.

5. The fusion protein according to claim 4, characterized in that: Its amino acid sequence is shown in SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No.

9.

6. A carrier, characterized in that, The vector is loaded with the encoding gene of the fusion protein according to any one of claims 1 to 5; further, the vector is an expression vector; further, the backbone of the expression vector is pET30.

7. The use of the fusion protein according to any one of claims 1 to 5 or the carrier according to claim 6 in promoting the degradation of PET plastic or in preparing a PET plastic degradation agent; further, the PET plastic is PET plastic waste.

8. A method for promoting PET degradation, characterized in that... Includes the following steps: The fusion protein according to any one of claims 1 to 5 is treated with PET plastic at 40 to 60°C to promote the degradation of PET plastic.

9. The method according to claim 8, characterized in that: The PET plastic is PET plastic waste.

10. The method according to claim 8, characterized in that: The processing conditions involve using a liquid containing dissolved fusion proteins.

11. The method according to claim 10, characterized in that: The liquid used to dissolve the fusion protein is a glycine, Tris, or phosphate buffer solution with a pH of 8–10.

12. The method according to claim 8, characterized in that: The processing time is 1 to 7 days.

13. The method according to claim 8, characterized in that: Furthermore, it also includes the step of preparing the fusion protein according to any one of claims 1 to 5: preparing the vector according to claim 6, transforming the obtained vector into engineered bacteria, fermenting at 16 to 25°C, and collecting the expressed fusion protein.

14. The method according to claim 13, characterized in that... The method also includes the following steps: constructing a vector for expressing a molecular chaperone, co-transforming engineered bacteria with a vector capable of expressing the fusion protein of any one of claims 1 to 5, collecting the expressed protein after fermentation at 16 to 25°C, and treating it with PET plastic waste at 40 to 60°C for 1 to 7 days.

15. The method according to claim 14, characterized in that: The molecular chaperones are GroEL and GroES, and the backbone vector for expressing the molecular chaperones is pBAD.

16. The method according to claim 15, characterized in that: The nucleotide sequence of the molecular chaperone GroEL encoding gene is shown in SEQ ID No. 5, and the nucleotide sequence of the molecular chaperone GroES encoding gene is shown in SEQ ID No.

6.

17. The method according to claim 15, characterized in that: The engineered bacteria is Escherichia coli; during the fermentation process, 0.5-1 mM IPTG or 2.5-5 g / L lactose is added to induce TfH-FPE expression, and 0.5-1 mg / mL arabinose is added to induce the expression of the molecular chaperone GroEL / ES.

18. The method according to claim 17, characterized in that: Ferment for 24–72 hours after adding 0.5 mM IPTG or after adding 5 g / L lactose.

Images