A cutinase variant, preparation method thereof, and application in plastic degradation

Abstract

The present invention belongs to the fields of enzyme engineering and plastic degradation technology, and specifically relates to a cutinase variant, a preparation method thereof, and its application in plastic degradation. The present invention uses cutinase ICCG as a starting enzyme and utilizes bioinformatics and protein engineering techniques to engineer a series of mutants with enhanced affinity for the substrate PET. Experimental verification shows that the obtained cutinase variants have a higher affinity for PET and higher hydrolytic activity for PET, hydrolyzing 90% of the PET substrate in just 3.7 hours or less. Furthermore, over 99% of the hydrolysis products are terephthalic acid and ethylene glycol monomers, with virtually no incompletely degraded BHET and MHET. This more thorough hydrolysis process is therefore more conducive to meeting the practical application needs of depolymerization and reuse of PET waste, and therefore has excellent practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering and plastic degradation, and particularly relates to a cutinase variant, a preparation method thereof, and application in plastic degradation. 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 formed by the ester polymerization of terephthalic acid and ethylene glycol. It is one of the most abundant petroleum-based synthetic polymers and boasts excellent properties such as light weight, good insulation, high strength, and transparency. It is widely used in disposable beverage bottles, packaging, clothing, and electrical accessories, greatly facilitating people's lives. However, over 300 million tons of PET are consumed globally each year. Only a small portion of this post-consumer PET is recycled, with the majority being discarded, causing significant environmental pollution and resource waste. Currently, the primary disposal methods are landfill and incineration, which can lead to resource waste and secondary pollution.

[0004] In recent years, the use of enzymatic catalytic depolymerization of PET waste has provided a green and environmentally friendly approach to addressing PET pollution. A variety of cutinases, lipases, and carboxylesterases have been discovered that can hydrolyze the nonspecific substrate PET into oligomers (BHET, MHET) and monomers (terephthalic acid and ethylene glycol). For example, in 2016, Yoshida et al. discovered two PET hydrolases, IsPETase and MHETase, from the bacterium Ideonella sakaigenesis 201-F69. These enzymes preferentially recognize PET as a substrate over aliphatic esters and are capable of degrading PET at room temperature. However, their low catalytic activity and thermal stability currently hinder their practical application in the recycling of PET waste. Furthermore, a thermophilic leaf and branch compost cutinase (LCC), identified in 2013 from a leaf and branch compost metagenome, although not specifically targeting PET, exhibits higher thermal stability and can nonspecifically bind to PET at higher temperatures, hydrolyzing the ester bonds to release terephthalic acid and ethylene glycol monomers. Its depolymerization efficiency is significantly higher than that of IsPETase and MHETase.

[0005] Protein engineering of these hydrolases is key to improving the enzymatic depolymerization and upcycling of PET. Current research focuses on enhancing the enzyme's thermal stability. Because PET is a semicrystalline polymer, the enzyme struggles to interact with the highly crystalline regions of the substrate. However, when the temperature rises to around 70°C, PET undergoes a glass transition, transforming from a hard solid into a soft, elastic form. Simultaneously, molecular thermal motion intensifies, interchain interactions weaken, and mobility increases, resulting in an active form that is more susceptible to biodegradation. Therefore, improving the enzyme's thermal stability is more beneficial for PET hydrolysis. Examples include the rational design of ThermoPETase mutants based on protein structure, the DuraPETase mutants derived through GRAPE computational evolution, the FAST-PETase mutants derived through machine learning-assisted evolution, and the addition of additional disulfide bonds to enhance the thermal stability of LCC. Currently, the most thermally stable PET hydrolase is the LCC-derived mutant ICCG, with a Tm of 94°C. It can degrade 90% of PET within 10 hours at 72°C, making it the most catalytically active PET hydrolase mutant reported to date. However, when the reaction temperature exceeds 72°C, the recrystallization rate of PET increases significantly. However, the catalytic rate of existing enzymes is insufficient, causing some PET to recrystallize before it is degraded, making it unavailable for degradation. ICCG's PET degradation rate at 75°C is only 60%. Therefore, in addition to thermal stability, increasing the enzyme's catalytic rate is key to further improving the efficiency of PET enzymatic degradation.

[0006] Unlike IsPETase, which is a specific PET-degrading enzyme (EC3.1.1.101), ICCG belongs to the class of cutinases (EC3.1.1.74). Its natural substrates are long- and short-chain fatty acid esters, not ethylene terephthalate (PET), which contains a benzene ring. Therefore, the substrate binding pocket of ICCG is not fully adapted to PET, and its affinity for PET could be further improved. Summary of the Invention

[0007] To address the challenges of the prior art, the present invention provides a cutinase variant, its preparation method, and its application in plastic degradation. The present invention utilizes bioinformatics and protein engineering techniques to modify cutinase ICCG, thereby increasing its affinity for the PET substrate, thereby improving the efficiency of enzymatic hydrolysis of PET and enhancing its value in practical industrial applications such as plastic degradation. Based on the above research, the present invention was completed.

[0008] Specifically, the present invention relates to the following technical solutions:

[0009] In a first aspect, the present invention provides a cutinase variant, wherein any one or more sites selected from the group consisting of:

[0010] T176A, T176N, H183W, A62D, T176Q, H183F, T176K, H183Y, S212E, N213Q, S65K, S65I, N213D, S65H, W69C, Q182F, D63E, S212A, I208Q, W69Y, S212R, N211Y, Q182D, Q182G, S206A, S212H, I208V, Q182W, D91S, Y60W, D91C, I208P, I208S, Q182T, Q182V, S206R, W69V, I208N, I208R; wherein, the amino acid residues are numbered using SEQ ID Numbering shown in NO.1 (amino acid sequence of Cutinase ICCG, SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ).

[0011] The Cutinase variant is mutated based on the Cutinase ICCG shown above, and the Cutinase variant is selected from the following groups of mutants:

[0012] LCC-A1:H183Y;

[0013] LCC-A2:H183Y / N213D;

[0014] LCC-A3:H183Y / N213D / S212A.

[0015] To facilitate subsequent protein purification, the cutinase variant may be modified with a His tag (LEHHHHHH, SEQ ID NO. 2) at the carboxyl terminus.

[0016] The second aspect of the present invention provides a polynucleotide encoding the Cutinase variant described in the first aspect.

[0017] The third aspect of the present invention provides a recombinant expression vector, which contains the polynucleotide described in the second aspect.

[0018] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect or the chromosome has the polynucleotide described in the second aspect integrated therein or expresses the cutinase variant described in the first aspect.

[0019] In a fifth aspect, the present invention provides a method for preparing the aforementioned cutinase variant, comprising the steps of: culturing the host cell described in the fourth aspect to express the cutinase variant; and isolating and purifying the cutinase variant.

[0020] The sixth aspect of the present invention provides the use of the cutinase variant described in the first aspect, the polynucleotide 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 hydrolysis, depolymerization, degradation and catalysis of plastic products.

[0021] Wherein, the plastic product is a product containing polyester plastic, and the polyester plastic is specifically polyethylene terephthalate.

[0022] A seventh aspect of the present invention provides a method for degrading polyester, comprising: applying the cutinase variant described in the first aspect or the host cell described in the fourth aspect to the polyester for reaction.

[0023] An eighth aspect of the present invention provides a method for screening cutinase variants described in the first aspect, the screening method comprising using the crystal structure of the starting enzyme to perform molecular docking with a 3PET small molecule substrate to obtain a three-dimensional structure of the enzyme-substrate complex; analyzing the dynamic protein conformation of the ICCG cutinase and the 3PET model substrate over time through molecular dynamics simulation, performing computer virtual saturation mutations on key amino acid sites, and obtaining the three-dimensional structure of the mutants through homology modeling; using molecular docking technology to analyze the affinity of mutants at different sites for the PET substrate, and selecting mutants with improved affinity for experimental verification.

[0024] Beneficial technical effects of one or more of the above technical solutions:

[0025] The above technical solution provides a cutinase variant, a preparation method thereof, and its application in plastic degradation. Specifically, the above technical solution uses the cutinase mutant ICCG as a starting enzyme and utilizes bioinformatics and protein engineering techniques to engineer a series of mutants with improved affinity for the substrate PET. Experimental verification shows that the obtained cutinase variants have a higher affinity for PET and higher hydrolytic activity for PET, as demonstrated by: 1. Faster hydrolysis of the PET substrate, requiring only 3.7 hours or less to hydrolyze 90% of the PET substrate; 2. Over 99% of the hydrolysis products are terephthalic acid and ethylene glycol monomers, with virtually no incompletely degraded BHET and MHET, resulting in more complete hydrolysis, which is more conducive to meeting the practical application needs of depolymerization and reuse of PET waste, and therefore has excellent practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 This is the concentration of the PET hydrolyzate of the mutant with enhanced activity in the first round of protein engineering in Example 1 of the present invention.

[0028] Figure 2 This is the concentration of the PET hydrolyzate of the mutant with enhanced activity in the second round of protein engineering in Example 1 of the present invention.

[0029] Figure 3 This is the concentration of the PET hydrolyzate of the mutant with enhanced activity in the third round of protein engineering in Example 1 of the present invention.

[0030] Figure 4 The concentrations of PET hydrolysis products of the starting enzyme ICCG, the single-site mutants H183Y, N213D, and S212A, and the site combinations LCC-A2 and LCC-A3.

[0031] Figure 5 These are the hydrolysis curves of the starting enzyme ICCG, double mutant LCC-A2, and triple mutant LCC-A3 in Example 2 of the present invention for high-concentration (200 g / L) post-consumer PET waste.

[0032] Figure 6 It is composed of the PET hydrolysis products after 6 hours of reaction of the starting enzyme ICCG, the double mutant LCC-A2 and the triple mutant LCC-A3 in Example 2 of the present invention.

[0033] Figure 7The hydrolysis curves of the double mutant LCC-A2 in Example 2 of the present invention for high concentration (200 g / L) post-consumer PET waste at different reaction temperatures. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] In a typical embodiment of the present invention, a cutinase variant is provided, wherein the cutinase variant is mutated at any one or more sites selected from the group consisting of:

[0037] T176A, T176N, H183W, A62D, T176Q, H183F, T176K, H183Y, S212E, N213Q, S65K, S65I, N213D, S65H, W69C, Q182F, D63E, S212A, I208Q, W69Y, S212R, N211Y, Q182D, Q182G, S206A, S212H, I208V, Q182W, D91S, Y60W, D91C, I208P, I208S, Q182T, Q182V, S206R, W69V, I208N, I208R; wherein, the amino acid residues are numbered using SEQ ID Numbering shown in NO.1 (amino acid sequence of Cutinase ICCG, SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ).

[0038] In another embodiment of the present invention, the amino acid sequence of the Cutinase variant has at least 80% homology to SEQ ID NO. 1; more preferably, at least 90% homology; most preferably, at least 95% homology; such as at least 95%, 96%, 97%, 98%, or 99% homology.

[0039] In another embodiment of the present invention, the number of mutation sites in the Cutinase variant is 1-5, more preferably 1-3, such as 1, 2 or 3.

[0040] In another embodiment of the present invention, the cutinase variant is mutated based on the cutinase ICCG shown in SEQ ID NO. 1, and the cutinase variant is selected from the following group of mutants:

[0041] LCC-A1:H183Y;

[0042] LCC-A2:H183Y / N213D;

[0043] LCC-A3:H183Y / N213D / S212A.

[0044] In another embodiment of the present invention, to facilitate subsequent protein purification, the cutinase variant may be modified with a His tag (LEHHHHHH, SEQ ID NO. 2) at the carboxyl terminus.

[0045] The present invention demonstrates through experiments that, compared to the existing cutinase ICCG, the activities of the mutants LCC-A1, LCC-A2, and LCC-A3 were increased by 27%, 56%, and 61%, respectively. To characterize the effects of each of the three mutation sites on the increased enzyme activity, the single-site mutants H183Y, N213D, and S212A, as well as the site combination mutants LCC-A2 and LCC-A3, were compared to the enzyme activity of ICCG. The activities of H183Y, N213D, and S212A were increased by 35.7%, 13.7%, and 8.1%, respectively, compared to ICCG. The double mutant LCC-A2 (H183Y / N213D) showed a 57.2% increase in activity compared to ICCG, while the triple mutant LCC-A3 (H183Y / N213D / S212A) showed a 71.7% increase in activity compared to ICCG. That is, the enzyme activity of double-site mutations and triple-site mutations is significantly higher than the sum of the enzyme activities of the corresponding single-site mutations. Considering that different mutants have different sequences and therefore different structures, it is still difficult to expect that the enzyme activity of double-site mutations and triple-site mutations is significantly higher than the sum of the enzyme activities of the corresponding single-site mutations.

[0046] At a reaction temperature of 72°C, the double mutant H183Y / N213D degraded 90% of PET waste within 5.8 hours, 3.5 hours less than the most active ICCG mutant reported so far. The triple mutant catalyzed the degradation of 80% of post-consumer PET waste in just 4.7 hours, while ICCG required 6 hours. At the same time, the affinity of the two mutants for the PET substrate was significantly improved. The enzyme kinetic results showed that the K of LCC-A2 was 1.34. m The values ​​for LCC-A2 and LCC-A3 were 2.13 nM and 1.96 nM, respectively, significantly lower than ICCG's 3.99 nM, indicating a significantly improved affinity for the PET substrate. Due to this increased affinity, over 99% of the degradation products of mutants LCC-A2 and LCC-A3 were terminal degradation products, terephthalic acid and ethylene glycol, with virtually no intermediate degradation products, BHET and MHET. In contrast, ICCG's degradation products also contained 18% of the intermediate hydroxyethyl terephthalate.

[0047] In addition, after further optimization of the reaction temperature, the catalytic efficiency of the double mutant H183Y / N213D was further improved. At the optimal reaction temperature, it only took 3.7 hours to degrade 90% of PET waste, which was 5.6 hours less than the most active ICCG mutant reported so far. It also broke through the limitation of substrate recrystallization and finally achieved a PET waste degradation rate of 100%.

[0048] In summary, the aforementioned cutinase variants exhibit a higher affinity for PET and exhibit enhanced PET hydrolysis activity. Specifically, they demonstrate: 1. PET substrate hydrolysis is faster, requiring only 3.7 hours or less to hydrolyze 90% of the PET substrate. 2. Over 99% of the hydrolysis products are terephthalic acid and ethylene glycol monomers, with virtually no incompletely degraded BHET and MHET. This more thorough hydrolysis is therefore more conducive to meeting the practical application needs of depolymerization and recycling of PET waste. In particular, as previously mentioned, the inventors unexpectedly discovered in experimental studies that the enzyme activities of both double-site and triple-site mutations were significantly higher than the sum of the activities of the corresponding single-site mutations. Considering that different mutants have different sequences and, therefore, different structures, the enzyme activities of both double-site and triple-site mutations were significantly higher than the sum of the activities of the corresponding single-site mutations, demonstrating a "synergistic" effect and achieving unexpected results.

[0049] In another embodiment of the present invention, a polynucleotide is provided, which encodes the above-mentioned cutinase variant.

[0050] In another specific embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector contains the above-mentioned polynucleotide of the present invention.

[0051] In another specific embodiment of the present invention, the recombinant expression vector is obtained by effectively linking the above-mentioned polynucleotide to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid, a phage, 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, and in a specific embodiment of the present invention, the plasmid is PET-26b.

[0052] In another specific embodiment of the present invention, a host cell is provided, wherein the host cell contains the vector described in the third aspect of the present invention or the chromosome is integrated with the above-mentioned polynucleotide.

[0053] The host cell can be a prokaryotic cell or a eukaryotic cell.

[0054] In another embodiment of the present invention, the host cell is any one or more of a bacterial cell and a fungal cell;

[0055] wherein the bacterial cell is any species within the genus Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus;

[0056] In another embodiment of the present invention, the bacterial cell is Escherichia coli (such as Escherichia coli BL21 (DE3)), Agrobacterium tumefaciens (such as GV3101), Agrobacterium rhizogenes, Bacillus subtilis, Bacillus cereus or Pseudomonas sp.

[0057] The fungal cells include yeast (such as Pichia pastoris) and the like.

[0058] In another embodiment of the present invention, a method for preparing the aforementioned cutinase variant is provided, comprising the steps of: culturing the aforementioned host cell of the present invention to express the aforementioned cutinase variant; and isolating and purifying the cutinase variant.

[0059] In another embodiment of the present invention, there is provided the use of the above-mentioned cutinase variants, polynucleotides, recombinant expression vectors, and host cells in the fields of hydrolysis, depolymerization, degradation, and catalysis of plastic products.

[0060] The plastic product may be a plastic product comprising polyester, wherein the polyester is selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyisosorbate terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends / mixtures of these materials, preferably polyethylene terephthalate.

[0061] In the application, the reaction temperature is controlled to be not less than 70°C, and further not less than 72°C. The inventors have found that within a certain temperature range, as the reaction temperature increases, the catalytic degradation efficiency of the cutinase on plastic products is further improved. Therefore, the reaction temperature can be 72-84°C, such as 72°C, 75°C, 78°C, 81°C and 84°C, and the catalytic degradation efficiency is optimal at 84°C.

[0062] In another embodiment of the present invention, a method for degrading polyester is provided, comprising: applying the above-mentioned cutinase variant or host cell to the polyester for reaction.

[0063] The polyester is selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyisosorbate terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends / mixtures of these materials, preferably polyethylene terephthalate.

[0064] The reaction temperature is controlled to be not less than 70°C, and further not less than 72°C. The inventors have found that within a certain temperature range, as the reaction temperature increases, the catalytic degradation efficiency of the cutinase on plastic products is further improved. Therefore, the reaction temperature can be 72-84°C, such as 72°C, 75°C, 78°C, 81°C and 84°C, and the catalytic degradation efficiency is optimal at 84°C.

[0065] In another specific embodiment of the present invention, a screening method for the above-mentioned cutinase variants is provided, which includes using the crystal structure of the starting enzyme to perform molecular docking with the 3PET small molecule substrate to obtain the three-dimensional structure of the enzyme-substrate complex; analyzing the dynamic protein conformation of cutinase ICCG and the 3PET model substrate over time through molecular dynamics simulation, performing computer virtual saturation mutations on key amino acid sites, and obtaining the three-dimensional structure of the mutants through homology modeling; using molecular docking technology to analyze the affinity of mutants at different sites for the PET substrate, and selecting mutants with improved affinity for experimental verification.

[0066] Among them, the key amino acid site judgment standard is that the site that interacts with the 3PET substrate for more than 20% of the time is considered to be a key amino acid site;

[0067] In the screening method, the starting enzyme used in the first round is cutinase ICCG (its amino acid sequence is shown in SEQ ID NO.1);

[0068] After experimental verification, the mutant starting enzyme with better or best hydrolysis efficiency can be used as the starting enzyme for the second round to repeat the above screening method, and the cycle can be repeated to the third round, fourth round, and so on.

[0069] In one embodiment of the present invention, in the first round of transformation, 33 mutants with improved affinity for PET substrates were verified, of which 8 mutants had increased PET hydrolysis efficiency. The most efficient mutant, H183Y, was used as the starting enzyme for the second round of protein engineering. In the second round of transformation, 44 mutants with improved affinity for PET substrates were verified, of which 4 mutants had a hydrolysis efficiency higher than that of ICCG. The third round of mutation was carried out using the most efficient H183Y / N213D mutant as the starting enzyme. Among the 72 mutants with improved affinity for PET substrates, 17 had a hydrolysis efficiency higher than that of ICCG. After three rounds of protein transformation, the two mutants with the highest degradation rates were screened out, namely the double mutant H183Y / N213D (LCC-A2) and the triple mutant H183Y / N213D / S212A (LCC-A3).

[0070] 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.

[0071] Example 1: Cutinase ICCG was used as the starting enzyme for engineering to obtain a series of mutants with improved affinity for the substrate PET, and their hydrolysis activity towards PET was tested.

[0072] 1. Prediction of cutinase mutants with improved affinity for PET substrates using bioinformatics techniques

[0073] To identify cutinase mutants with enhanced affinity for the PET substrate, the present invention developed a bioinformatics strategy for predicting the affinity of protein mutants for PET substrates. Molecular docking was performed using Autodock vina 1.1.2 with the ICCG crystal structure retrieved from the PDB and a 3PET molecule drawn in ChemOffice 2019 to obtain the three-dimensional structure of the enzyme-substrate complex. Molecular dynamics analysis of the complex was performed using the OPLS-AA / M force field in NAMD 2.12. Based on the dynamic protein conformational information from the molecular dynamics trajectory, key amino acid residues that play an important role in substrate binding were identified. The three-dimensional structures of all mutants were obtained through in silico virtual saturation mutagenesis and homology modeling. Molecular docking was used to evaluate the affinity of mutants at different sites for the PET substrate. Mutants with improved affinity were selected for experimental validation and their ability to degrade PET nanoparticles was tested. The mutant with the best hydrolysis efficiency was used as the starting enzyme for the next round of protein engineering.

[0074] Three rounds of protein engineering were performed. In the first round, cutinase ICCG was used as the starting enzyme. 33 mutants with increased affinity for the PET substrate were predicted by computation. Their affinity for 3PET is shown in Table 1.

[0075] Table 1: Mutants predicted to have increased affinity for PET substrates in the first round of modification and their relative activities in catalyzing the hydrolysis of PET nanoparticles

[0076]

[0077]

[0078] In the second round, using mutant H183Y as the starting enzyme, 44 mutants with improved affinity for PET substrates were predicted through calculation. Their affinities with 3PET substrates are shown in Table 2.

[0079] Table 2: Mutants predicted to have increased affinity for PET substrates in the second round of modification and their relative activities in catalyzing the hydrolysis of PET nanoparticles

[0080]

[0081] In the third round, using the mutant H183Y / N213D as the starting enzyme, 72 mutants with improved affinity for the PET substrate were predicted through calculation. Their affinities with the 3PET substrate are shown in Table 3.

[0082] Table 3: Mutants predicted to have increased affinity for PET substrates in the third round of modification and their relative activities in catalyzing the hydrolysis of PET nanoparticles

[0083]

[0084]

[0085] 2. Construction of expression vectors for cutinase mutants

[0086] 1. Construction of ICCG expression vector

[0087] The amino acid sequence of ICCG was first described by Tournier et al. in the April 2020 issue of Nature. The cutinase nucleotide sequence, minus the signal peptide, was codon-optimized and commercially synthesized, then ligated into the expression vector pET26b(+) between the NdeI (5' end) and XhoI (3' end) restriction sites. A His tag consisting of six histidine residues was included downstream of the XhoI restriction site to facilitate protein purification using affinity chromatography. The amino acid sequence of the cutinase after insertion into the vector is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 4.

[0088] 2. Construction of expression vector for cutinase mutant

[0089] Mutations were introduced into the wild-type cutinase DNA sequence using PCR and Gibson seamless cloning techniques to generate expression vectors for the mutants. The plasmid vectors expressing the cutinase mutants were transfected into Escherichia coli BL21(DE3) cells and plated on LB agar plates containing kanamycin. Correct transformants were selected after DNA sequence analysis.

[0090] 3. Relative activity detection of cutinase mutants in hydrolyzing PET nanoparticles

[0091] Escherichia coli containing ICCG and a cutinase mutant were inoculated into a 96-well plate containing 1 ml of LB medium. The inducer IPTG was added to induce protein secretion. 50 μl of fermentation supernatant containing the cutinase mutant was added to 950 μl of phosphate buffered saline containing 0.2 mg of PET nanoparticles. After incubation at 72°C for 4 hours, 200 μl of the supernatant was measured for absorbance at 240 nm. Terephthalic acid, the hydrolysis product of PET, contains benzene rings and has a characteristic absorption peak at 240 nm. Therefore, the catalytic activity of the mutant was assessed based on the absorbance at 240 nm. The relative PET hydrolysis activity of the mutant was calculated, with the absorbance of the starting enzyme ICCG as 1. The relative PET hydrolysis activities of the mutants from each round of protein engineering are shown in Tables 1, 2, and 3, respectively.

[0092] It is worth noting that the mutations at sites A62, S65, R89, D91, P93, M131, T153, H156, T157, T176, A178, P179, and N211 reported in patent CN 109642221 A may all help to improve the polyester degradation activity of cutinase. However, the present invention has experimentally demonstrated that mutations at these sites significantly reduce the hydrolysis activity of PET.

[0093] 4. Expression and Purification of Cutinase Mutants

[0094] 1. Expression of cutinase mutants

[0095] The above-mentioned E. coli expressing ICCG 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 the 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 600 After adding IPTG to a final concentration of 0.5 mM, the strain was cultured for 16 h to allow protein expression and extracellular secretion.

[0096] 2. Purification of Cutinase Mutants

[0097] The cultured cutinase fermentation broth was centrifuged at 12000rpm for 10min, and the collected supernatant was filtered with a 0.45μm filter membrane. The filtered supernatant was purified by nickel ion chromatography column. After washing away impurities with wash buffer (20mM Tris-HCl, pH8.0, 300mM NaCl, 20mM imidazole), the target protein was eluted using elution buffer (20mM Tris-HCl, pH8.0, 300mM NaCl, 250mM imidazole). The collected purified protein was replaced with storage buffer (20mM Tris-HCl, pH8.0, 100mM NaCl) using a 10KDA ultrafiltration tube and concentrated to a concentration greater than 1mg / ml. The concentration of the concentrated enzyme was determined using a protein concentration test kit and stored at 4°C.

[0098] 5. Testing the PET hydrolysis activity of cutinase mutants

[0099] 1. Hydrolysis of PET powder using ICCG and cutinase mutants

[0100] Add 50 μg of ICCG or cutinase mutant and 10 mg of PET powder (Goodfellow, ES301445) to 1 ml of phosphate buffer and mix thoroughly. Incubate at 72°C for 5 h and terminate the reaction by adding 1 ml of acetonitrile. Perform each reaction in triplicate.

[0101] 2. Detection of PET hydrolysis product concentration

[0102] After the reaction was terminated, the reaction solution was filtered through a 0.22 μm filter membrane and the product concentration was determined using 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 0.6 ml / min, and the detection wavelength was 240 nm. A calibration curve was generated using commercially available PET hydrolysis products: bis(hydroxyethyl)terephthalate (BHET, Sigma), hydroxyethyl terephthalate (MHET, Aladdin), and terephthalic acid (TPA, Sigma). The concentration of the hydrolysis product in the reaction solution was determined based on the calibration curve.

[0103] The results of the PET degradation product concentration test of the cutinase mutants with enhanced activity in the first round of protein engineering are shown in Figure 1 The most active mutant, H183Y, showed a 27% increase in activity compared to ICCG.

[0104] The results of the PET degradation product concentration test of the cutinase mutant with improved activity in the second round of protein modification are shown in Figure 2 The most active cutinase mutant, H183Y / N213D, showed a 56% increase in activity compared to ICCG.

[0105] The results of the PET degradation product concentration test of the cutinase mutant with improved activity in the third round of protein modification are shown in Figure 3 The most active cutinase mutant, H183Y / N213D / S212A, showed a 61% increase in activity compared to ICCG.

[0106] To characterize the effects of each of the three mutation sites on the improvement of enzyme activity, single-site mutants N213D and S212A were constructed. The degree of improvement in enzyme activity of the single-site mutants H183Y, N213D, and S212A, and the site combination mutants LCC-A2 and LCC-A3 relative to ICCG was compared. The concentrations of the products produced by the degradation of PET by each mutant at 72°C for 2 hours are shown in Table 1. Figure 4 The activities of H183Y, N213D, and S212A increased by 35.7%, 13.7%, and 8.1%, respectively, compared to ICCG. The double mutant LCC-A2 (H183Y / N213D) increased its activity by 57.2%, and the triple mutant LCC-A3 (H183Y / N213D / S212A) increased its activity by 71.7%. This indicates that the enzyme activities of double- and triple-site mutations are significantly higher than the sum of the activities of the corresponding single-site mutations.

[0107] Example 2: Hydrolysis of high concentrations of post-consumer PET waste by cutinase mutants

[0108] To validate the potential of the cutinase mutants described in this invention for recycling post-consumer PET waste, we conducted large-scale PET depolymerization experiments using common PET mineral water bottles as substrates. The amino acid sequence of the double mutant LCC-A2 is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO. 6. The amino acid sequence of the triple mutant LCC-A3 is shown in SEQ ID NO. 7, and the nucleotide sequence is shown in SEQ ID NO. 8.

[0109] The specific implementation plan is:

[0110] Remove the label and cap from a PET mineral water bottle and cut it into small pieces. Melt it at 280°C and immerse it in cold water to remove crystallization. Use liquid nitrogen to freeze and grind it into powder. Add 10g of PET powder and 30mg of PET hydrolase (ICCG, LCC-A2 or LCC-A3) to a bioreactor containing phosphate buffer to a final volume of 50ml. Maintain the reaction temperature by heating in a water bath and use a 5M sodium hydroxide aqueous solution to maintain the pH at 8.5. Maintain stirring at 300rpm for 12h and record the consumption of sodium hydroxide. 2M sodium hydroxide will be consumed for every 1M acidic product TPA generated. Therefore, the amount of product TPA generated can be calculated by the consumption of sodium hydroxide. The amount of PET degradation is then calculated based on the amount of TPA generated.

[0111] The hydrolysis curve of PET was calculated based on the consumption curve of sodium hydroxide. At 72℃, the depolymerization rate of post-consumer PET waste by mutants LCC-A2 and LCC-A3 was significantly improved, see Figure 5 . It takes 6 hours for ICCG to degrade 80% of PET, while the double mutant LCC-A2 only takes 3.8 hours, saving 2.2 hours compared to ICCG. LCC-A2 degraded 90% of PET waste within 5.8 hours, which is 3.5 hours less than the currently reported most active PET hydrolase ICCG. It takes only 4.6 hours for the triple mutant LCC-A3 to degrade 80% of PET waste, saving 1.4 hours compared to ICCG. More importantly, due to the increase in substrate affinity, after 6 hours of reaction, more than 99% of the PET hydrolysis products of LCC-A2 and LCC-A3 are terminal hydrolysis products TPA and EG, with almost no intermediate degradation products MHET and BHET, while 18% of the hydrolysis products of the starting enzyme ICCG are intermediate degradation products MHET, see Figure 6 .

[0112] In addition, after further optimization of the reaction temperature, the catalytic efficiency of the double mutant H183Y / N213D was further improved. Figure 7 At the optimal reaction temperature (84°C), it only takes 3.7 hours to degrade 90% of PET waste, which is 5.6 hours less than the most active ICCG mutant reported so far. It also breaks through the limitation of substrate recrystallization and ultimately achieves a 100% degradation rate of PET waste.

[0113] This shows that the mutants we designed not only have 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.

[0114] SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGI

[0115] AMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNY

[0116] LRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTD

[0117] KTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ(SEQ ID NO.1)

[0118] LEHHHHHH(SEQ ID NO.2)

[0119] SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGI

[0120] AMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNY

[0121] LRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTD

[0122] KTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNS

[0123] NNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQLEHHHHHH(SEQ ID NO.3)

[0124] AGCAACCCGTACCAGCGTGGCCCGAATCCGACCCGCAGCGCACTGACCGC

[0125] AGATGGCCCGTTTAGCGTGGCAACCTACACCGTCTCACGCCTGTCAGTCTC

[0126] GGGTTTTGGCGGTGGCGTGATTTATTACCCGACCGGCACGTCTCTGACGTT

[0127] CGGTGGCATCGCGATGAGTCCGGGTTATACCGCAGATGCTAGCTCTCTGGC

[0128] ATGGCTGGGTCGTCGCCTGGCTTCCCATGGCTTTGTGGTTCTGGTGATTAAC

[0129] ACGAATTCACGTTTCGATGGTCCGGACAGCCGCGCCTCTCAGCTGAGTGCC

[0130] GCCCTGAACTACCTGCGTACCAGTTCCCCGAGCGCCGTTCGCGCACGTCTG

[0131] GATGCAAATCGTCTGGCGGTTGCCGGTCATTCTATGGGTGGCGGTGGCACC

[0132] CTGCGTATTGCAGAACAAAACCCGAGCCTGAAAGCGGCTGTCCCGCTGAC

[0133] CCCGTGGCACACCGATAAAACGTTTAATACCAGTGTCCCGGTGCTGATTGT

[0134] TGGCGCAGAAGCTGACACCGTGGCGCCGGTTTCGCAGCATGCCATCCCGTT

[0135] TTATCAAAACCTGCCGAGCACCACGCCGAAAGTTTACGTCGAACTGTGCA

[0136] ACGCATCGCACATTGCTCCGAATAGCAACAATGCGGCCATTTCCGTTTATAC

[0137] GATCTCATGGATGAAACTGTGGGTCGATAATGACACCCGTTACCGCCAGTT

[0138] CCTGTGTAATGTGAACGACCCGGCTCTGTGCGACTTCCGCACCAATAATCG

[0139] CCACTGCCAActcgagcaccaccaccaccaccac(SEQ ID NO.4)

[0140] MSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGG

[0141] IAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNY

[0142] LRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTD

[0143] KTFNTSVPVLIVGAEADTVAPVSQYAIPFYQNLPSTTPKVYVELCNASHIAPNS

[0144] DNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQLEHHH

[0145] HHH(SEQ ID NO.5)

[0146] atgAGCAACCCGTACCAGCGTGGCCCGAATCCGACCCGCAGCGCACTGACC

[0147] GCAGATGGCCCGTTTAGCGTGGCAACCTACACCGTCTCACGCCTGTCAGTC

[0148] TCGGGTTTTGGCGGTGGCGTGATTTATTACCCGACCGGCACGTCTCTGACG

[0149] TTCGGTGGCATCGCGATGAGTCCGGGTTATACCGCAGATGCTAGCTCTCTGG

[0150] CATGGCTGGGTCGTCGCCTGGCTTCCCATGGCTTTGTGGTTCTGGTGATTAA

[0151] CACGAATTCACGTTTCGATGGTCCGGACAGCCGCGCCTCTCAGCTGAGTGC

[0152] CGCCCTGAACTACCTGCGTACCAGTTCCCCGAGCGCCGTTCGCGCACGTCT

[0153] GGATGCAAATCGTCTGGCGGTTGCCGGTCATTCTATGGGTGGCGGTGGCAC

[0154] CCTGCGTATTGCAGAACAAAACCCGAGCCTGAAAGCGGCTGTCCCGCTGA

[0155] CCCCGTGGCACACCGATAAAACGTTTAATACCAGTGTCCCGGTGCTGATTG

[0156] TTGGCGCAGAAGCTGACACCGTGGCGCCGGTTTCGCAGTATGCCATCCCGT

[0157] TTTATCAAAACCTGCCGAGCACCACGCCGAAAGTTTACGTCGAACTGTGCA

[0158] ACGCATCGCACATTGCTCCGAATAGCGATAATGCGGCCATTTCCGTTTATAC

[0159] GATCTCATGGATGAAACTGTGGGTCGATAATGACACCCGTTACCGCCAGTT

[0160] CCTGTGTAATGTGAACGACCCGGCTCTGTGCGACTTCCGCACCAATAATCG

[0161] CCACTGCCAActcgagcaccaccaccaccaccac(SEQ ID NO.6)

[0162] MSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGG

[0163] IAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNY

[0164] LRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTD

[0165] KTFNTSVPVLIVGAEADTVAPVSQYAIPFYQNLPSTTPKVYVELCNASHIAPNA

[0166] DNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQLEHHHHHH(SEQ ID NO.7)

[0167] atgAGCAACCCGTACCAGCGTGGCCCGAATCCGACCCGCAGCGCACTGACC

[0168] GCAGATGGCCCGTTTAGCGTGGCAACCTACACCGTCTCACGCCTGTCAGTC

[0169] TCGGGTTTTGGCGGTGGCGTGATTTATTACCCGACCGGCACGTCTCTGACG

[0170] TTCGGTGGCATCGCGATGAGTCCGGGTTATACCGCAGATGCTAGCTCTCTGG

[0171] CATGGCTGGGTCGTCGCCTGGCTTCCCATGGCTTTGTGGTTCTGGTGATTAA

[0172] CACGAATTCACGTTTCGATGGTCCGGACAGCCGCGCCTCTCAGCTGAGTGC

[0173] CGCCCTGAACTACCTGCGTACCAGTTCCCCGAGCGCCGTTCGCGCACGTCT

[0174] GGATGCAAATCGTCTGGCGGTTGCCGGTCATTCTATGGGTGGCGGTGGCAC

[0175] CCTGCGTATTGCAGAACAAAACCCGAGCCTGAAAGCGGCTGTCCCGCTGA

[0176] CCCCGTGGCACACCGATAAAACGTTTAATACCAGTGTCCCGGTGCTGATTG

[0177] TTGGCGCAGAAGCTGACACCGTGGCGCCGGTTTCGCAGTATGCCATCCCGT

[0178] TTTATCAAAACCTGCCGAGCACCACGCCGAAAGTTTACGTCGAACTGTGCA

[0179] ACGCATCGCACATTGCTCCGAATGCAGATAATGCGGCCATTTCCGTTTATAC

[0180] GATCTCATGGATGAAACTGTGGGTCGATAATGACACCCGTTACCGCCAGTT

[0181] CCTGTGTAATGTGAACGACCCGGCTCTGTGCGACTTCCGCACCAATAATCGCCACTGCCAActcgagcaccaccaccaccaccac(SEQ ID NO.8)

[0182] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A cutinase variant, characterized in that The cutinase variant is mutated based on the cutinase ICCG shown in SEQ ID NO. 1, and the cutinase variant is selected from the following group of mutants: Single mutants: H183Y or N213D or S212A; LCC-A2:H183Y / N213D; LCC-A3:H183Y / N213D / S212A.

2. The Cutinase variant according to claim 1, characterized in that The cutinase variant is modified with a His tag at the carboxyl terminus, and the His tag amino acid sequence is shown in SEQ ID NO.

2.

3. A polynucleotide, characterized in that The polynucleotide encodes the Cutinase variant according to any one of claims 1-2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the polynucleotide according to claim 3; The recombinant expression vector is obtained by effectively connecting the polynucleotide to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid or an artificial chromosome.

5. The recombinant expression vector according to claim 4, wherein The expression vector is any one or more of a phage or a cosmid.

6. The recombinant expression vector according to claim 4, wherein The expression vector is a plasmid, and the plasmid is PET-26b.

7. A host cell, characterized in that The host cell contains the recombinant expression vector according to any one of claims 4 to 6 or has the polynucleotide according to claim 3 integrated into its chromosome or expresses the cutinase variant according to any one of claims 1 to 2.

8. A method for preparing the Cutinase variant according to any one of claims 1 to 2, characterized in that: include: Cultivating the host cell according to claim 7 to express the cutinase variant; and isolating and purifying to obtain the cutinase variant.

9. Use of the cutinase variant according to any one of claims 1-2, the polynucleotide according to claim 3, the recombinant expression vector according to any one of claims 4-6, and the host cell according to claim 7 in the field of plastic product degradation and catalysis; in, The plastic product is a product comprising polyester plastic, and the polyester is polyethylene terephthalate; The reaction temperature was controlled to be no less than 70°C.

10. The use according to claim 9, characterized in that The reaction temperature is controlled to be no less than 72°C.

11. The use according to claim 10, characterized in that The reaction temperature is 72-84°C.

12. A method for degrading polyester, characterized in that: Applying the cutinase variant according to any one of claims 1 to 2 or the host cell according to claim 7 to polyester for reaction; The polyester is polyethylene terephthalate; The reaction temperature was controlled to be no less than 70°C.

13. The method according to claim 12, wherein: The reaction temperature is controlled to be no less than 72°C.

14. The method according to claim 13, wherein The reaction temperature is 72-84°C.

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