Plastic degrading enzyme variant and application thereof in degrading polyester plastic

Abstract

The invention belongs to the technical field of synthetic biology and enzyme engineering, and particularly relates to a plastic degrading enzyme variant and application thereof in degrading polyester plastic. Existing enzymes are modified through a protein engineering technology, the global flexibility of the polyester hydrolase LCC-A2 protein is researched through molecular dynamics simulation, and a key loop region combined with a substrate is focused on. The flexibility of a loop region is improved by designing a mutant, so that the flexibility of a substrate binding pocket is enhanced, and the degradation capability of enzyme on polyester plastic substrates with diversified structures is realized. The novel mutant improves the hydrophobicity of the enzyme surface, enhances the capability of hydrolyzing an intermediate product, obtains higher monomer yield, finally achieves the purpose of improving the enzymatic hydrolysis efficiency of PET, PBAT and other polyester plastics, and enhances the hydrolytic activity at low pH so as to enhance the value of the polyester plastics in actual industrial application. The difficulty and the cost of recycling the mixed polyester type plastic garbage are reduced, so that the method has a good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical fields of synthetic biology and enzyme engineering, and particularly relates to a plastic-degrading enzyme variant and application thereof in degrading polyester plastics. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Polyethylene terephthalate (PET) is a polyester plastic made from the monomers terephthalic acid (TPA) and ethylene glycol (EG). It is one of the most widely used petroleum-based plastics worldwide. Its advantages include light weight and high transparency, making it suitable for applications such as disposable packaging and clothing. However, with its widespread use worldwide, post-consumer PET waste is mostly discarded into the environment. It is rarely recycled, resulting in significant environmental pollution. Currently, the main disposal methods are landfill and incineration, which can easily cause secondary pollution.

[0004] In recent years, the use of enzymatic degradation of PET waste has become a promising way to treat PET waste due to its green and environmentally friendly nature. Currently, a large number of PET-degrading enzymes have been discovered from the environment, including esterases, cutinases, lipases, etc. For example, the thermophilic leaf compost cutinase (LCC) identified from the leaf compost metagenome in 2013 can degrade PET at higher temperatures, and the hydrolysis products are TPA and ethylene glycol monomers. In 2016, Yoshida et al. Ideonella sakaigenesis Two PET hydrolases were found in 201-F69 Is PETase and MHETase, among which Is PETase preferentially recognizes PET as a substrate and exhibits strong specificity for PET, hydrolyzing it into MHET, TPA, and ethylene glycol at room temperature. MHETase, in turn, hydrolyzes MHET into TPA and ethylene glycol. These two enzymes are currently being extensively studied for their excellent PET degradation properties and are considered potential candidates for future treatment of PET waste pollution.

[0005] Polybutylene adipate-terephthalate (PBAT) is a polyester plastic made from terephthalic acid (TPA), butanediol, and adipic acid. It is not only biodegradable but also possesses mechanical and processing properties similar to those of low-density polyethylene (LDPE). It is currently used in packaging such as plastic bags, as well as in other applications such as agricultural mulch, and is gradually replacing LDPE. Under environmental conditions, PBAT, as a biodegradable plastic, is relatively more susceptible to degradation by esterases secreted by environmental microorganisms than traditional polyester plastics such as polyethylene terephthalate (PET). However, the widespread use of PBAT plastics still places significant pressure on the environment. Furthermore, as plastic waste remains exposed to the environment for extended periods, if biodegradable plastics are not degraded promptly, a large amount of microplastics will be released into the environment. Due to the extensive exposure of PBAT plastics, the TPA produced by their degradation may be environmentally toxic, potentially harmful to microorganisms and even some small and medium-sized animals and plants.

[0006] The reason why microorganisms can degrade PBAT is that the esterases and cutinases secreted by microorganisms act on the ester bonds of PBAT. For example, Fumihiro Muroi found that PBAT can be degraded by bacteria in an aerobic and mesophilic environment. Bacillus pumilus An enzyme that slowly degrades PBAT under mild conditions was discovered. Thermobifida fusca Thermoresistant cutinase Tf Cut1 and Tf Cut2 has been found to have PET degradation activity. Recently, researchers have found Tf Cut2 not only has the degradation activity of PET, but also has the degradation activity of PBAT. The researchers used the DM strategy to Tf Cut2 was transformed to obtain new mutants Tf Cut-DM improves the degradation efficiency of PBAT.

[0007] Plastic-degrading enzymes are key to improving enzymatic degradation and upcycling of plastics. Current modifications focus on enhancing enzyme thermal stability and substrate affinity. For example, LCC-A2 not only demonstrates significantly improved thermal stability but also significantly enhanced affinity for PET. LCC is not a specific PET-degrading enzyme, and therefore may have hydrolytic activity against other plastics, including PBAT. However, experiments have shown that while LCC-A2 has hydrolytic activity against PBAT, the activity is low. Furthermore, the hydrolysis products are primarily short aromatic chains and TPA, while the concentration of the aliphatic degradation product, adipic acid, is low. This suggests that LCC-A2 primarily hydrolyzes aromatic polyesters within PBAT molecules. PBAT contains two distinct ester bond structures: a sterically hindered aromatic ester bond formed by the polymerization of TPA and butanediol, and an elongated aliphatic ester bond formed by adipic acid and butanediol. These two ester bond structures differ significantly. In particular, since most plastic waste exists as a mixture, there is an urgent need for simultaneous enzymatic degradation of mixed plastic waste. Summary of the Invention

[0008] In response to the problems existing in the prior art, the present invention provides a plastic degrading enzyme variant and its application in degrading polyester plastics. Specifically, the present invention transforms the existing enzyme through protein engineering technology, studies the global flexibility of the polyester hydrolase LCC-A2 protein through molecular dynamics simulation, and focuses on the key loop region for substrate binding. By designing mutants to improve the flexibility of the loop region, the flexibility of the substrate binding pocket is enhanced to achieve the enzyme's ability to degrade polyester plastic substrates with diverse structures. In addition, the new mutant improves the hydrophobicity of the enzyme surface, enhances the ability to hydrolyze intermediates, and obtains a higher monomer yield, ultimately achieving the purpose of improving the enzymatic hydrolysis efficiency of polyester plastics such as PET and PBAT, while enhancing its hydrolysis activity at low pH to enhance its value in actual industrial applications and reduce the difficulty and cost of recycling mixed polyester plastic waste. Based on the above research results, the present invention was completed.

[0009] Specifically, the present invention relates to the following technical solutions: In a first aspect of the present invention, a plastic degrading enzyme variant is provided. The plastic degrading enzyme variant is based on polyester hydrolase LCC-A2 and has mutations in any one or more of the following sites: A178S, S206P, S206L, S206V, S206I, S206T, S206F, S212L, S212M, S212R, S212G; The amino acid sequence of the polyester hydrolase LCC-A2 is shown in SEQ ID NO.1.

[0010] In another specific embodiment of the present invention, the amino acid sequence of the plastic degrading enzyme variant has at least 80% homology compared with SEQ ID NO.1; more preferably, it has at least 90% homology; most preferably, it has at least 95% homology; such as at least 95%, 96%, 97%, 98%, 99% homology.

[0011] Furthermore, the plastic degrading enzyme variant is based on polyester hydrolase LCC-A2, and the cutinase variant is selected from any one or more mutants in the following groups: Single mutants: LCC-A2-S206P, LCC-A2-S206L, LCC-A2-S206V, LCC-A2-S206I, LCC-A2-S206T, LCC-A2-S206F, and LCC-A2-A178S.

[0012] Double mutants: LCC-A2-S206P / S212L, LCC-A2-S206P / S212M, LCC-A2-S206P / S212R, and LCC-A2-S206P / S212G.

[0013] Through research, the present invention found that the flexibility of the above-mentioned plastic degrading enzyme variant in the substrate binding pocket region is greatly improved compared with the polyester hydrolase LCC-A2, and the depolymerization efficiency of polyester plastics such as PET, PBAT, PBS is improved compared with LCC-A2, and it also has higher degradation activity at lower pH.

[0014] At the same time, in order to facilitate subsequent protein purification, the plastic degrading enzyme variant can be modified with a His tag (LEHHHHHH, SEQ ID NO. 2) at the carboxyl terminus.

[0015] The second aspect of the present invention provides a nucleic acid molecule encoding the plastic degrading enzyme variant described in the first aspect.

[0016] The third aspect of the present invention provides a recombinant expression vector, which contains the nucleic acid molecule described in the second aspect.

[0017] The recombinant expression vector is obtained by effectively linking the above-mentioned nucleic acid molecule to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid or an artificial chromosome; the viral vector may include an adenoviral vector, a retroviral vector or an adeno-associated viral vector, and the artificial chromosome includes a bacterial artificial chromosome, a phage P1-derived vector, a yeast artificial chromosome or a mammalian artificial chromosome; preferably, the expression vector is a plasmid.

[0018] The fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect or the chromosome integrated with the nucleic acid molecule described in the second aspect, or is capable of expressing the plastic degrading enzyme variant described in the first aspect.

[0019] The host cell can be a prokaryotic cell or a eukaryotic cell.

[0020] In another embodiment of the present invention, the host cell is any one or more of a bacterial cell and a fungal cell; wherein the bacterial cell is any species within the genus Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus; In another embodiment of the present invention, the bacterial cell is Escherichia coli, Agrobacterium tumefaciens, Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus or Pseudomonas fluorescens.

[0021] The fungal cells include yeast (such as Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica) and filamentous fungi.

[0022] The fifth aspect of the present invention provides a method for preparing the above-mentioned plastic degrading enzyme variant, comprising the steps of: culturing the host cell described in the fourth aspect to express the plastic degrading enzyme variant; and isolating and purifying to obtain the plastic degrading enzyme variant.

[0023] The sixth aspect of the present invention provides the use of the plastic degrading enzyme variant described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant expression vector described in the third aspect, and the host cell described in the fourth aspect in the fields of plastic hydrolysis, depolymerization, degradation and catalysis.

[0024] Furthermore, the plastic may be a polyester plastic, including but not limited to any one or more of polyethylene terephthalate (PET), polylactic acid (PLA), polybutylene succinate (PBS), and polybutylene adipate terephthalate (PBAT). Furthermore, the plastic may be a blend / mixture of the above polyester plastics, preferably polybutylene adipate terephthalate and its blend with polylactic acid.

[0025] A seventh aspect of the present invention provides a method for degrading polyester plastics, the method comprising: contacting the polyester plastics with the plastic degrading enzyme variant or host cells, thereby degrading the plastic product; Furthermore, the method further comprises recovering monomers and / or oligomers.

[0026] The eighth aspect of the present invention is an enzyme design and screening method for enhancing the degradation ability of mixed polyester plastic substrates with different molecular structures by improving the flexibility of the plastic hydrolyzate substrate binding pocket. Specifically, a screening method for plastic degrading enzyme variants is provided, the screening method comprising: molecular docking of a PBAT model substrate (TPA-BDO-AA), an aromatic polyester model substrate 3TPA-3BDO (3 TPAs ​​and 3 butanediols alternately connected), and an aliphatic polyester model substrate 3AA-3BDO (3 adipic acids and 3 butanediols alternately connected) with LCC-A2, and molecular dynamics simulation of LCC-A2 and LCC-A2 and two model substrates with different ester bonds. By comparing the global RMSF, key flexible loop regions are identified, conserved sites are removed, and candidate mutation sites are obtained by analyzing the affinity of the loop region with the two substrates and the RMSF of the loop region sites; PBAT degradation activity is verified by constructing a saturated mutation library.

[0027] Furthermore, the candidate mutation sites include: A178, S206, I208, and S212.

[0028] Beneficial technical effects of one or more of the above technical solutions: The above technical solution provides a plastic degrading enzyme variant and its application in degrading polyester plastics. Specifically, the present invention is directed to improving the flexibility of the substrate binding pocket of cutinase to enhance the binding of the enzyme to polyesters of different structures, and ultimately successfully obtains a series of plastic degrading enzyme variants. Experiments have shown that its ability to degrade polyester plastic substrates with diverse structures is significantly improved. At the same time, the enzyme variant improves the hydrophobicity of the enzyme surface, enhances the ability to hydrolyze intermediates, and obtains a higher monomer yield, ultimately achieving the purpose of improving the enzymatic hydrolysis efficiency of polyester plastics such as PET and PBAT, while enhancing its hydrolysis activity at low pH to enhance its value in actual industrial applications and reduce the difficulty and cost of recycling mixed polyester plastic waste, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1 is the root mean square fluctuation (RMSF) of the LCC-A2 of the present invention and the complex structures of LCC-A2 and three substrates.

[0031] Figure 2 is the root mean square fluctuation (RMSF) of LCC-A2-S206P and LCC-A2 of the present invention, and the dotted line is the RMSF value of the S206P site.

[0032] Figure 3 The relative activities of all high-activity mutants in the first round of transformation in degrading PBAT at pH 8.5 and 72° C. were calculated using LCC-A2 as the control in Example 2 of the present invention.

[0033] Figure 4 Taking LCC-A2-S206P in Example 2 of the present invention as the control, the total concentration of terephthalic acid (TPA) and the intermediate product (BTa) of TPA connected to butanediol produced by the degradation of PBAT by all high-activity mutants at the S212 site in the second round of transformation at pH 8.5 and 72°C.

[0034] Figure 5 Taking LCC-A2 in Example 2 of the present invention as a control, LCC-A2-S206P degraded PET at pH values ​​of 6.5, 7.5, and 8.5, releasing the total concentration of TPA, MHET, and BHET within 2 h.

[0035] Figure 6 Using LCC-A2 in Example 2 of the present invention as a control, the mutant LCC-A2-S206P degraded PBAT at pH 8.5 and 72° C. to release the total concentration of products within 12 h, including TPA, BTa, butanediol, and adipic acid.

[0036] Figure 7 The LCC-A2 in Example 2 of the present invention is used as a control. The mutant LCC-A2-S206P degrades PBS at pH 8.5 and 72°C for 2 h and 12 h, and releases butanediol and succinic acid.

[0037] Figure 8 This is the process of degrading a 1×1 cm commercial PBAT film under the conditions of pH 7 and 81° C. using the mutant LCC-A2-S206P in Example 2 of the present invention.

[0038] Figure 9 This is the process of degrading a 1×1 cm commercial PBAT / PLA film by the mutant LCC-A2-S206P in Example 2 of the present invention under the conditions of pH 7 and 81°C.

[0039] Figure 10 These are the concentrations of the products when the mutant LCC-A2-S206P in Example 2 of the present invention degrades the PBAT film and the PBAT / PLA film to clarity at pH 7 and 81°C.

[0040] Figure 11The degradation rate of post-consumer PET in a large-scale bioreactor at pH 8.0 and 78° C. was calculated using LCC-A2 as a control in Example 2 of the present invention.

[0041] Figure 12 The percentages of various products of large-scale degradation of post-consumer PET by the mutant LCC-A2-S206P in Example 2 of the present invention at pH 8.0 and 78°C in a bioreactor. DETAILED DESCRIPTION

[0042] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0043] It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. In the following specific embodiments, if the experimental methods of specific conditions are not specified, they are generally in accordance with the conventional methods and conditions of molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: A Laboratory Manual", or in accordance with the conditions recommended by the manufacturer.

[0044] Design, screening and validation of cutinase mutants The present invention aims to improve the flexibility of the substrate binding pocket of cutinase to enhance the enzyme's binding to polyesters of different structures. We used Autodock Vina1.1.2 to analyze the binding modes of PBAT model substrates (TPA-BDO-AA), aromatic polyester model substrates 3TPA-3BDO (3 TPAs ​​and 3 butanediols alternately connected), and aliphatic polyester model substrates 3AA-3BDO (3 adipic acids and 3 butanediols alternately connected) with LCC-A2. We also performed molecular dynamics simulations of LCC-A2 and LCC-A2 with two model substrates with different ester bonds using GROMACS-2024.2, and identified four key flexible loop regions by comparing the global RMSF. Figure 1), Loop 1 (P58-D63), Loop 2 (A172-V180), Loop 3 (C203-I208), and Loop 4 (S212-N214). We removed conserved sites and, by analyzing the affinity of the loop regions for two substrates and the RMSF of the loop regions, identified four candidate mutation sites: A178, S206, I208, and S212. A saturation mutation library was constructed and validated for PBAT degradation activity.

[0045] In the first round of modification, mutations at the S206 and A178 sites significantly impacted PBAT degradation activity, with S206P, S206L, S206V, S206I, S206T, S206F, and A178S increasing their activity by 90%, 68%, 27%, 15%, 10%, 4%, and 41%, respectively. In particular, the S206P mutant showed a significant improvement in activity under reaction conditions of pH 6.5-8.5.

[0046] The second round of transformation was carried out based on LCC-A2-S206P, and double mutants S206P / S212G, S206P / S212M, S206P / S212L, and S206P / S212R were obtained. After Autodock Vina 1.1.2 and molecular dynamics simulation, the binding of mutants to substrates was analyzed. Among them, mutant S206P significantly improved the flexibility of Loop 3 ( Figure 2 ), while Loop 3 not only facilitates substrate recruitment to the substrate binding pocket but is also crucial for substrate binding. This enhances enzyme-substrate interaction, thereby increasing activity in degrading PET, PBAT, and PBS. In the second round of modification, using LCC-A2-S206P as the starting protein, the activity-enhancing mutation site S212 was selected. S212G, S212M, S212L, and S212R were selected. The LCC-A2-S206P / S212G, S206P / S212M, S206P / S212L, and S206P / S212R double mutants further improved PBAT degradation efficiency.

[0047] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0048] Example 1: Engineering of cutinase mutant LCC-A2 as the starting enzyme to obtain a mutant with enhanced esterase activity 1. Selection of mutation sites In the first round of evolution, we used Autodock Vina 1.1.2 and GROMACS-2024.2 software to analyze the binding patterns of LCC-A2 with 3AA-3BDO, 3TPA-3BDO, and TPA-BDO-AA, as well as the global flexibility of LCC-A2. We identified a series of potential enzyme-substrate interaction sites: A178S, S206, I208, and S212. In the second round of evolution, we added mutations at the S212 site, potentially enhancing activity, to the highly active mutant LCC-A2-S206P obtained in the first round of evolution.

[0049] 2. Construction of expression vectors for cutinase mutants The LCC-A2 expression vector was maintained in the laboratory. Amino acid mutations were designed in primers and introduced into the cutinase LCC-A2 DNA sequence using PCR and Gibson seamless cloning techniques to generate mutant expression vectors. The plasmid vector expressing the cutinase mutant was transfected into competent Escherichia coli BL21 (DE3) cells and plated on LB agar plates containing kanamycin. Correct transformants were selected after DNA sequence analysis.

[0050] 3. Relative activity detection of cutinase mutants in hydrolyzing PET and PBAT nanoparticles E. coli containing LCC-A2 and its mutants were inoculated into 96-well plates containing 1 mL of LB medium. Protein secretion was induced by the addition of IPTG. Fifty microliters of fermentation supernatant containing the cutinase mutants was added to 950 microliters of phosphate buffered saline (PBS) containing 0.2 mg of PET or PBAT nanoparticles. After incubation at 72°C for 2 hours, 200 microliters of the supernatant was measured for absorbance at 240 nm. Terephthalic acid, the hydrolysis product of PET and PBAT, contains benzene rings and exhibits a characteristic absorption peak at 240 nm. Therefore, the catalytic activity of the mutants was assessed based on the absorbance at 240 nm. The relative hydrolysis activity of the mutants against PET and PBAT was calculated, assuming the absorbance of the starting enzyme, LCC-A2, was set as 1.

[0051] 4. Expression and Purification of Cutinase Mutants 1. Expression of cutinase mutants The above-mentioned E. coli expressing LCC-A2 or cutinase mutants were inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm. 1 mL of culture was transferred into 50 mL of fresh LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm until the OD reached 0. 600After adding IPTG to a final concentration of 0.5 mM, the strain was cultured for 16 h to allow protein expression and extracellular secretion.

[0052] 2. Purification of Cutinase Mutants The cutinase fermentation broth was centrifuged at 12,000 rpm for 10 minutes, and the collected supernatant was filtered through a 0.45 µm filter. The filtered supernatant was purified by nickel ion chromatography. Contaminants were washed away with wash buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole), and the target protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole). The purified protein was then filtered through a 10 kDa ultrafiltration tube, and the buffer was exchanged with storage buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl). The concentrated enzyme was concentrated to a concentration greater than 1 mg / mL using a protein concentration assay kit and stored at 4°C.

[0053] Example 2 Testing the Hydrolysis Activity of Cutinase Mutants on Polyester-Based Plastics 1. Hydrolysis of PET, PBAT and PBS powders using LCC-A2 and cutinase mutants 7.5 µg of LCC-A2 or a cutinase mutant and 10 mg of plastic powder were added to 0.5 mL of 0.1 M phosphate buffer (pH 8.5) and mixed thoroughly. PET (purchased from Goodfellow, Germany, Cat. No. ES301445) and PBAT (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., Cat. No. P909229, Mn ~120,000) were incubated at 72°C for 2 h and terminated by the addition of 0.5 mL of acetonitrile. PBS (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., Cat. No. P909170) was incubated at 72°C for 12 h and terminated by the addition of 0.5 mL of sulfuric acid to adjust the pH to 2-3. Each reaction was performed in triplicate.

[0054] 2. Detect the concentration of plastic hydrolysis products After the reaction was terminated, the reaction solution was filtered through a 0.22 µm filter membrane and the product concentration was determined by high-performance liquid chromatography (Shimadzu LA-20AT). The analytical column was a ZORBAX extend-c18 column (150 × 4.6 mm, 5 µm, Agilent). Mobile phase A consisted of diluted trifluoroacetic acid (0.1% v / v) and mobile phase B consisted of acetonitrile, with mobile phase A comprising 80% of the total volume. The flow rate was 0.8 mL / min, and the detection wavelength was 240 nm. A standard curve was prepared using commercially available PET hydrolysis products, bis(hydroxyethyl)terephthalate (BHET, Sigma), hydroxyethyl terephthalate (MHET, Aladdin), and terephthalic acid (TPA, Sigma). Butanediol, adipic acid, and succinic acid were separated using an organic acid column (300 × 7.8 mm, Aminex HPX-87H, Bio-Rad) and detected using a differential refractive index detector (DRD). Mobile phase A (5 mM sulfuric acid) was used at a flow rate of 0.6 mL / min. The concentrations of the hydrolysis products in the reaction solution were determined using a standard curve.

[0055] The first round of screening revealed that mutations at S206 and A178 increased the degradation activity of PBAT (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., Cat. No. P909229, Mn ~120,000). The activity of S206P, S206L, S206V, S206I, S206T, S206F, and A178S increased by 90%, 68%, 27%, 15%, 10%, 4%, and 41%, respectively. Figure 3 We then performed a new round of mutations based on the S206P mutant to obtain double mutants LCC-A2-S206P / S212L, S206P / S212M, S206P / S241R, and S206P / S212G, with activity increases of 35%, 30%, 25%, and 4%, respectively ( Figure 4 ).

[0056] The PET hydrolysis product TPA is acidic, and as the hydrolysis reaction proceeds, the pH value of the reaction system gradually decreases. To ensure catalytic activity, NaOH needs to be continuously added during the reaction to maintain a suitable reaction pH. This not only requires real-time monitoring of the pH value of the entire reaction process, which increases the complexity and cost of the biodepolymerization PET process, but also the hydrolysis product TPA needs to be crystallized and recovered in a strong acid environment. The addition of NaOH requires more sulfuric acid for TPA purification and produces a by-product of low economic value, Na2SO4, which is not conducive to the recovery of the final product and the treatment of wastewater. Therefore, improving the PET hydrolysis activity of the LCC-A2 mutant under acidic conditions has obvious industrial application value. We found that the activity of the most active single mutant S206P at a lower pH was also improved compared to the control LCC-A2 ( Figure 5 The activities of the two mutants were tested using reaction systems at pH 6.5, 7.5, and 8.5. The S206P mutant showed a 28% increase in activity compared to LCC-A2 at pH 6.5. While maintaining high catalytic activity at its optimal pH of 8.5, this mutant also exhibits high activity in weakly acidic environments, demonstrating unique advantages for industrial applications.

[0057] We reacted the most active mutant LCC-A2-S206P and the control LCC-A2 with PBAT powder (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number P909229, Mn ~ 120,000) for 12 h, and recorded the total product release during the 12 h period ( Figure 6 ), S206P released 30% more product than LCC-A2 at the end of the 12 h reaction, and the product release increased by 23.21 mM. We then measured the PBS degradation activity of LCC-A2 and S206P at 2 h and 12 h ( Figure 7 ), the amount of S206P degradation product released increased by 6.64 mM compared with LCC-A2 at 2 h, an increase of 30%, and the product release increased by 40.12 mM at 12 h, an increase of 33%.

[0058] We applied S206P to the degradation of PBAT commercial materials. At pH 7.0 and 81°C, it took only 4 h to completely degrade a 1×1 cm PBAT film (Mn: 120,000). Figure 8 ), a 1×1 cm PBAT / PLA blend film (Mn: 400,000) can be completely degraded in 3 h ( Figure 9 ), and the monomer yield reached 100% ( Figure 10 ).

[0059] We then tested the degradation activity of S206P on PET, selecting daily PET mineral water bottles as substrates and LCC-A2 as a control. We used LCC-A2-S206P as the experimental group to simulate large-scale industrial degradation in the laboratory.

[0060] The specific implementation plan is: PET mineral water bottles were removed from their labels and caps and cut into small pieces. The pieces were melted at 280°C and immersed in cold water to de-crystallize. The pieces were then frozen in liquid nitrogen and ground into a powder. First, 10 g of PET powder and 30 mg of PET hydrolase (LCC-A2 and LCC-A2-S206P) were added to a bioreactor containing phosphate buffer to a final volume of 50 ml. The reaction temperature was maintained at 78°C in a water bath, and the pH was maintained at 8.0 using 5 M aqueous sodium hydroxide. The reaction was stirred at 300 rpm for 8 hours, and the sodium hydroxide consumption was recorded. For every 1 M of the acidic product TPA produced, 2 M of sodium hydroxide was consumed. Therefore, the amount of TPA produced can be calculated from the sodium hydroxide consumption. The amount of PET degradation was then calculated based on the TPA production.

[0061] The hydrolysis curves of LCC-A2 and LCC-A2-S206P to PET were calculated based on the sodium hydroxide consumption curve. Figure 11 The mutant LCC-A2-S206P significantly improved the depolymerization rate of post-consumer PET waste. LCC-A2 required 3.3 h to degrade 90% of PET, while the mutant LCC-A2-S206P only needed 3.1 h, saving 0.2 h compared to LCC-A2, and the maximum degradation rate increased by 21%. LCC-A2-S206P only needed 5.5 h to degrade to 100%, while LCC-A2 took more than 6 h. Figure 11 After the reaction, more than 99% of the PET hydrolysis products of the single mutant LCC-A2-S206P were terminal hydrolysis products TPA and EG, with almost no intermediate degradation products MHET and BHET. Figure 12 This shows that the mutant we designed not only has a faster hydrolysis rate for PET substrates, but also a more thorough hydrolysis reaction, which is more conducive to meeting the practical application needs of depolymerization and reuse of PET waste. The mutant can also quickly degrade PET under conditions of lower enzyme concentrations, which is also of great help in reducing the cost of use in practical applications.

[0062] Amino acid / nucleotide sequence information involved in the present invention Polyester hydrolase LCC-A2 amino acid sequence SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSM GGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQYAIPFYQNLPSTTPKVYVELCNASHIAPNSDNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ (SEQ ID NO.1) The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A plastic degrading enzyme variant, characterized in that The plastic degrading enzyme variant is based on the polyester hydrolase LCC-A2, and is mutated at any one or more of the following sites: A178S, S206P, S206L, S206V, S206I, S206T, S206F, S212L, S212M, S212R, S212G; Wherein, the amino acid sequence of the polyester hydrolase LCC-A2 is shown in SEQ ID NO.

1.

2. The plastic degrading enzyme variant according to claim 1, characterized in that The plastic degrading enzyme variant is based on polyester hydrolase LCC-A2, and the cutinase variant is selected from any one or more mutants in the following groups: single mutants: LCC-A2-S206P, LCC-A2-S206L, LCC-A2-S206V, LCC-A2-S206I, LCC-A2-S206T, LCC-A2-S206F, and LCC-A2-A178S; Double mutants: LCC-A2-S206P / S212L, LCC-A2-S206P / S212M, LCC-A2-S206P / S212R, and LCC-A2-S206P / S212G.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the plastic degrading enzyme variant according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 3.

5. A host cell, characterized in that The host cell contains the recombinant expression vector according to claim 4 or has the nucleic acid molecule according to claim 3 integrated into its chromosome, or is capable of expressing the plastic degrading enzyme variant according to any one of claims 1-2.

6. The host cell according to claim 5, wherein The host cell is a prokaryotic cell or a eukaryotic cell.

7. Use of the plastic degrading enzyme variant according to claim 1 or 2, the nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4, or the host cell according to claim 5 or 6 in the fields of plastic hydrolysis, depolymerization, degradation, and catalysis; Furthermore, the plastic is a polyester plastic, including but not limited to any one or more of polyethylene terephthalate, polylactic acid, polybutylene succinate, and polybutylene adipate terephthalate; further, it is a blend / mixture of the above polyester plastics, preferably polybutylene adipate terephthalate and its blend with polylactic acid.

8. A method for degrading polyester plastics, characterized in that: The method comprises: contacting the polyester plastic with the plastic degrading enzyme variant according to any one of claims 1 to 2 or the host cell according to any one of claims 5 to 6, thereby degrading the plastic product; Furthermore, the method further comprises recovering monomers and / or oligomers.

9. A method for screening plastic degrading enzyme variants, characterized in that: The screening method includes: molecular docking of a PBAT model substrate (TPA-BDO-AA), an aromatic polyester model substrate 3TPA-3BDO (three TPAs ​​and three butanediols alternately linked), and an aliphatic polyester model substrate 3AA-3BDO (three adipic acids and three butanediols alternately linked) with LCC-A2, and molecular dynamics simulations of LCC-A2 and LCC-A2 and model substrates with two different ester bonds. Key flexible loop regions are identified by comparing global RMSFs, conserved sites are removed, and candidate mutation sites are obtained by analyzing the affinity of the loop regions with the two substrates and the RMSFs of the loop region sites. PBAT degradation activity is verified by constructing a saturated mutation library, that is, by increasing the flexibility of the plastic hydrolyzate substrate binding pocket, the degradation ability for mixed polyester plastic substrates with different molecular structures is enhanced.

10. The screening method according to claim 9, wherein The candidate mutation sites include: A178, S206, I208 and S212.

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