A method for polyester material degradation using a heat-stable branch pile lignin esterase based on coordination modification of rare earth ions

Abstract

The application relates to the technical field of biodegradation, in particular to a method for degrading polyester materials by using a heat-stable leaf branch compost lignin esterase based on coordination modification of rare earth ions. The application significantly improves the thermal stability and catalytic efficiency of the enzyme by using the coordination strategy of rare earth ions and developing enzyme mutants with enhanced coordination, and the polyester materials are completely degraded within 8 hours. Meanwhile, the application simplifies the synthesis and purification steps of the enzyme, directly uses the crude enzyme to efficiently degrade the polyester materials at high temperatures, reduces the reaction cost, and realizes the biological recovery of polyester monomer raw materials. Compared with the traditional biodegradation method, the application has higher efficiency, lower cost and better environmental compatibility. The enzyme modification strategy not only promotes the progress of enzyme engineering technology, but also provides a new perspective for the fields of environmental protection and new material development, and has broad application potential and social value.

Description

Technical Field

[0001] This application relates to the field of biodegradation technology, and in particular to a polyester degrading enzyme modified by rare earth ion coordination and its application for industrial waste plastic degradation. Background Technology

[0002] Polyester polymers such as polyethylene terephthalate (PET) and polyurethane (PU) are high-performance synthetic plastics widely used in textiles, packaging materials, and other fields. However, due to the high crystallinity and glass transition temperature of polyester materials, their degradation process is extremely difficult. Traditional chemical degradation methods suffer from high energy consumption and severe pollution, while existing enzymatic degradation technologies also face bottlenecks such as low catalytic efficiency and poor thermal stability when degrading highly crystalline materials. Although some lipolytic enzymes (such as Ideonella sakaiensis PETase and leaf-branch compost lignin esterase (LCC)) have been developed, their thermal stability and catalytic efficiency are still insufficient to meet the needs of large-scale industrial applications. Therefore, developing enzymes with higher thermal stability and catalytic efficiency, especially those that efficiently degrade highly crystalline polyester materials, has become an urgent technical problem to be solved. Summary of the Invention

[0003] In view of this, this application provides a design method for modifying the metal ion binding sites of enzymes, which significantly improves the thermal stability and catalytic efficiency of enzymes. Specifically, this invention uses rare earth ions to replace calcium ions in enzyme molecules, utilizing the high coordination number and strong bond energy characteristics of rare earth ions to improve the stability and catalytic activity of enzymes, especially at high temperatures (above 65°C) and in neutral environments (pH 7.5), enabling efficient degradation of highly crystalline polyester materials.

[0004] To achieve the above-mentioned objectives, this application provides the following technical solution:

[0005] This application provides a method for improving the thermal stability and / or catalytic efficiency of lignin esterase in leaf and branch compost, comprising coordinating the lignin esterase in leaf and branch compost with rare earth ions.

[0006] In some specific embodiments of this application, the above method further includes site-directed mutagenesis of the lignin esterase in the leaf and branch compost to enhance its ability to coordinate with rare earth ions.

[0007] In some specific embodiments of this application, the site-directed mutation of the above method is at least one of N204D, S206D, N211D, N214D, S248D, and N253D.

[0008] This application also provides the application of site-directed mutagenesis in enhancing the following properties of lignin esterases in foliage compost:

[0009] (i) Thermal stability;

[0010] (ii) Ability to coordinate with rare earth ions;

[0011] The site-directed mutation is at least one of N204D, S206D, N211D, N214D, S248D, and N253D.

[0012] In some specific embodiments of this application, the leaf and branch compost lignin esterase described in the above method and application has the following characteristics:

[0013] (1) The amino acid sequence as shown in SEQ ID NO: 1; or

[0014] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0015] (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2);

[0016] The plurality refers to any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any combination thereof.

[0017] In some specific embodiments of this application, the rare earth ions in the above methods and applications are at least one of the following:

[0018] (I) Trivalent lanthanum ion;

[0019] (II) Trivalent praseodymium ion;

[0020] (III) Neodymium trivalent ion.

[0021] This application also provides mutants of lignin esterase in leaf and branch compost, including at least one of the following:

[0022] The 204th residue is D;

[0023] The 206th residue is D;

[0024] The 211th residue is D;

[0025] Residue at position 214 is D;

[0026] Residue at position 248 is D;

[0027] The 253rd residue is D.

[0028] In some specific embodiments of this application, the above-mentioned mutant is coordinated with rare earth ions;

[0029] The rare earth ion is at least one of the following:

[0030] (I) Trivalent lanthanum ion;

[0031] (II) Trivalent praseodymium ion;

[0032] (III) Neodymium trivalent ion.

[0033] In some specific embodiments of this application, the above-mentioned mutant has:

[0034] (4) The amino acid sequence as shown in SEQ ID NO: 2; or

[0035] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or

[0036] (6) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (4) or (5);

[0037] The plurality refers to any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any combination thereof.

[0038] This application also provides an expression vector having gene elements encoding the above-mentioned mutants.

[0039] In some specific embodiments of this application, the expression vector may be pET-25b.

[0040] This application also provides a recombinant strain containing the above-described expression vector.

[0041] In some specific embodiments of this application, the chassis of the recombinant strain can be a eukaryote or a prokaryote;

[0042] The prokaryote can be Escherichia coli.

[0043] This application also provides methods for degrading polyester materials, including:

[0044] (a) Mix the polyester material, acceptable auxiliaries, and the above-mentioned mutant, react, and degrade the polyester material; or

[0045] (b) Introduce the above expression vector into the expression strain, culture the expression strain, break it to obtain a crude enzyme solution, mix the polyester material and acceptable auxiliaries with the crude enzyme solution, react, and degrade the polyester material; or

[0046] (c) Cultivate the above recombinant strain, break it to obtain crude enzyme solution, mix the polyester material and acceptable auxiliaries with the crude enzyme solution, react and degrade the polyester material.

[0047] This application also provides a method for obtaining degradable monomers for polyester materials, including:

[0048] (A) Mix the polyester material, acceptable auxiliaries, and the above mutant; react to degrade the polyester material; and recover the degraded monomers; or

[0049] (B) Introduce the above expression vector into the expression strain, culture the expression strain, break it to obtain a crude enzyme solution, mix the polyester material and acceptable auxiliaries with the crude enzyme solution, react to degrade the polyester material, and recover the degraded monomers; or

[0050] (C) Cultivate the above recombinant strain, break it to obtain crude enzyme solution, mix the polyester material and acceptable auxiliaries with the crude enzyme solution, react to degrade the polyester material, and recover the degraded monomers.

[0051] In some specific embodiments of this application, the auxiliaries in the above methods may contain lanthanides;

[0052] The lanthanide elements include at least one of lanthanum, cerium, praseodymium, neodymium, europium, gadolinium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0053] In some specific embodiments of this application, the adjuvant in the above method may contain at least one of the following:

[0054] (I) Trivalent lanthanum ion;

[0055] (II) Trivalent praseodymium ion;

[0056] (III) Neodymium trivalent ion.

[0057] In some specific embodiments of this application, the cultivation in the above method includes an induction step. The induction agent used may be IPTG, and the concentration of the induction agent may be one or any combination of 0.09 mM, 0.1 mM, 0.11 mM, etc. The induction temperature may be one or any combination of 16°C, 20°C, 24°C, etc. The induction rotation speed may be one or any combination of 200 rpm, 210 rpm, 220 rpm, etc. The induction time may be one or any combination of 16 h, 20 h, 24 h, etc.

[0058] In some specific embodiments of this application, the crushing in the above method can be high-pressure homogenization crushing. The specific steps can be to first wash or not wash the expressed strain or the recombinant strain, then homogenize the bacterial cells under high pressure to obtain the crude enzyme solution. The crushing pressure can be one of 1000 bar, 1200 bar, 1400 bar or any combination of both.

[0059] In some specific embodiments of this application, the reaction temperature in the above method can be a range of one or any two of 60℃, 65℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 85℃, and 90℃, and the reaction rotation speed can be a range of one or any two of 200 rpm, 210 rpm, and 220 rpm.

[0060] In some specific embodiments of this application, the recycling in the above method may include: centrifuging the reaction solution after the reaction, collecting the supernatant, filtering, collecting the filtrate, adjusting the filtrate to alkaline, stirring, centrifuging, collecting the supernatant, filtering, collecting the filtrate, adjusting the filtrate to acidic, stirring, vacuum filtering the supernatant, collecting the insoluble matter, washing the insoluble matter, drying, and obtaining the degraded monomer of the polyester material;

[0061] The alkaline pH value is a range consisting of one or any two of 8, 9, 10, 11, 12, 13, and 14;

[0062] The acidic pH value is a range consisting of one or any two of the following: 0, 1, 2, 3, 4, 5, and 6.

[0063] In some specific embodiments of this application, the polyester material described in the above method includes at least one of polyethylene terephthalate and polyester-type polyurethane.

[0064] The rare-earth ion-coordinated thermostable enzyme and the enhanced rare-earth ion-coordinated thermostable enzyme mutant provided by this invention both contribute to improving the enzyme's thermal stability. This invention also provides a simplified method for degrading polyester materials under high-temperature conditions. By simplifying the enzyme synthesis and purification steps, the crude enzyme can be directly used for highly efficient polyester material degradation, thereby giving the LCC enzyme higher thermal stability, stronger catalytic efficiency, and lower reaction costs. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0066] Figure 1 This is a schematic diagram of a thermally stable leaf and branch compost lignin esterase based on rare earth ion coordination modification for the degradation of polyester materials.

[0067] Figure 2 A comparison of the three-dimensional structures of wild-type leaf and branch compost lignin esterase (LCC) and rare earth coordination mutant (DD) in Example 1 of this invention;

[0068] Figure 3 This is a comparison of the protein thermal denaturation temperatures of wild-type leaf and branch compost lignin esterase (LCC) and rare earth coordination mutant (DD) in Example 1 of the present invention.

[0069] Figure 4 This is a comparison of the efficiency of wild-type leaf and branch compost lignin esterase (LCC) and rare earth coordination mutant (DD) in degrading polyethylene terephthalate (PET) after coordination with different metals in Example 2 of the present invention. In this example, A represents the reaction formula and B represents the efficiency comparison bar chart.

[0070] Figure 5 This is a comparison of the degradation efficiency of polyethylene terephthalate (PET) by wild-type (WT) lignin esterase in leaf and branch compost and different mutants (N204D, S206D, N211D, N214D, S248D, N253D) after coordination with rare earth elements in Example 3 of the present invention.

[0071] Figure 6 This is a comparison of the efficiency of wild-type leaf and branch compost lignin esterase (LCC) and rare earth coordination mutant (DD) in degrading polyester polyurethane (PU) after coordination with different metals in Example 4 of the present invention. In this example, A represents the reaction formula and B represents the efficiency comparison bar chart.

[0072] Figure 7 Example 5 of this invention compares the degradation efficiency of waste plastic boxes by wild-type leaf and branch compost lignin esterase (LCC), rare earth coordination mutant (DD), and reported high-temperature resistant polyester hydrolases (ICCG and HotPETase). In this paper, A is a line graph of catalytic efficiency, B is a photograph of polyester material degradation, and C is a micrograph of polyester material.

[0073] Figure 8 This is a comparison of the trends of total MHET (ethylene glycol terephthalate) and TPA (terephthalic acid) content in the degradation system of mixed waste plastics by the lysate of the rare earth coordination mutant (DD) expression strain in Example 6 of the present invention as a function of degradation reaction time.

[0074] Figure 9 This is the process of extracting polyester material monomer TPA (terephthalic acid) from the degradation reaction solution of the strain lysate in Example 6 of the present invention. In this example, A is a photo of the strain lysate degradation reaction system, B is a photo of the purification process of the monomer product after degradation, C is a photo of the purified and recovered monomer powder, and D is a diagram of the chemical reaction mechanism in the degradation and recovery process of polyester material.

[0075] Figure 10 The nuclear magnetic properties of the recovered polyester monomer TPA (terephthalic acid) in Example 6 of this invention. Detailed Implementation

[0076] This application discloses a method for modifying heat-stable leaf and branch compost lignin esterase (LCC), a mutant sequence, and a polyester degradation reaction method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this application to realize and apply the technology of this invention.

[0077] This application provides a method for improving the thermal stability of lignin esterase (LCC) in leaf and branch compost, used to degrade polyester materials under heating conditions, achieving the goal of biologically degrading polyester materials and realizing the green treatment and recycling of plastic waste (such as...). Figure 1 (As shown).

[0078] Specifically, the method for improving the thermal stability of lignin esterase (LCC) in leaf and branch compost includes: replacing calcium ions on the enzyme's metal binding sites with rare earth ions to enhance the enzyme's structural stability; and using genetic engineering technology to construct enzyme mutants to enhance the binding capacity of rare earth ions, thereby further improving the enzyme's structural stability and high-temperature tolerance.

[0079] Furthermore, the polyester degradation method of the heat-stable leaf and branch compost lignin esterase (LCC) includes, but is not limited to, using purified enzyme to coordinate rare earth ions for polyester material degradation, or using enzyme-expressed bacterial cell lysate to coordinate rare earth ions for polyester material degradation, and recovering polyester material monomers through the degradation solution treatment method, thereby achieving the biodegradation and recycling of polyester.

[0080] This invention provides a rare-earth ion-coordinated thermostable enzyme:

[0081] Natural leaf and branch compost lignin esterase (LCC) was expressed and purified, then mixed with a rare earth ion solution to coordinate at least one of the following rare earth ions at the calcium ion binding site (D203 / E173 / S248):

[0082] 1) Trivalent lanthanum ion;

[0083] 2) Trivalent praseodymium ion;

[0084] 3) Trivalent neodymium ions.

[0085] This invention provides a thermostable enzyme mutant with enhanced rare earth ion coordination:

[0086] By modifying the calcium ion binding site of the natural LCC enzyme, the modified site includes at least one of the following:

[0087] 1) Replace amino acid N at position 204 with amino acid D;

[0088] 2) Replace amino acid S at position 206 with amino acid D;

[0089] 3) Replace amino acid N at position 211 with amino acid D;

[0090] 4) Replace amino acid N at position 214 with amino acid D;

[0091] 5) Replace amino acid S at position 248 with amino acid D;

[0092] 6) Replace amino acid N at position 253 with amino acid D.

[0093] This invention provides a simple method for degrading polyester materials under high-temperature conditions:

[0094] The crude enzyme catalysis based on the aforementioned thermostable enzyme mutant with enhanced rare earth ion coordination includes the following steps:

[0095] Step A: Express the thermostable enzyme mutant with enhanced rare earth ion coordination;

[0096] 1) The gene encoding the above mutant was constructed in the pET-25b expression vector using genetic engineering technology;

[0097] 2) Use the BL21(DE3) expression strain;

[0098] 3) Induction was performed using 0.1 mM IPTG as an inducer;

[0099] 4) Expression was induced at 20℃ and 220 rpm;

[0100] 5) The induction time is 20 h;

[0101] Step B: Polyester degradation was performed using the expressed thermostable enzyme mutant with enhanced rare earth ion coordination;

[0102] 1) Collect Escherichia coli cells expressing thermostable enzyme mutants by centrifugation at 4℃ and 5000 rpm;

[0103] 2) Resuspend E. coli cells in 1 M MES-hydrochloric acid buffer (pH 7.5) containing 20 mM rare earth ions;

[0104] 3) Rare earth ions include, but are not limited to, trivalent neodymium ions;

[0105] 4) The bacterial cells were broken down using a pressure of 1200 bar to obtain a crude enzyme solution;

[0106] 5) Degrade polyester materials using crude enzyme solution at 75°C and 220 rpm;

[0107] 6) Polyester materials include, but are not limited to, polyethylene terephthalate (PET) and polyester polyurethane (PU);

[0108] Step C: Use the degradation reaction solution to recover the polyester degradation monomers in the solution;

[0109] 1) Centrifuge the reaction mixture at room temperature using 8000 rpm and collect the supernatant;

[0110] 2) Filter the supernatant using a 0.45 μm filter membrane;

[0111] 3) Adjust the pH of the filtrate to 12.0 using sodium hydroxide (NaOH) solid powder;

[0112] 4) Stir the solution at 750 rpm for 4 hours at room temperature;

[0113] 5) Centrifuge the stirred solution at 8000 rpm at room temperature and collect the supernatant;

[0114] 6) Filter the supernatant using a 0.45 μm filter membrane;

[0115] 7) Adjust the pH of the filtrate to 2.0 using a 37% hydrochloric acid (HCl) solution;

[0116] 8) Stir the solution at 750 rpm for 10 min at room temperature;

[0117] 9) Use a 0.22 μm filter membrane to vacuum filter the supernatant and collect the insoluble matter;

[0118] 10) After resuspending the insoluble matter in ultrapure water, filter and wash the insoluble matter again, and repeat three times;

[0119] 11) Use 45℃ vacuum drying to dry insoluble matter and collect the degradation monomers of polyester material.

[0120] In summary, this invention provides a technology for the degradation of polyester materials using thermally stable leaf and branch compost lignin esterase based on rare earth ion coordination modification, comprising:

[0121] 1) Enhances the thermal stability of lignin esterase in leaf and branch compost;

[0122] 2) Mutate the metal binding sites of lignin esterase in leaf and branch compost to enhance rare earth ion coordination ability.

[0123] 3) Use mutant leaf and branch compost lignin esterase to complete the degradation and recycling of polyester materials.

[0124] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0125] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0126] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0127] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0128] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0129] The reagents and materials used in the modification method, mutant sequence, and polyester degradation reaction method of the heat-stable leaf and branch compost lignin esterase (LCC) provided by this invention are all commercially available.

[0130] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for the degradation of lignin esterase and polyester materials in thermally stable leaf and branch compost based on rare earth ion coordination modification.

[0131] Example 1

[0132] See Figure 2The lignin esterase in leaf and branch compost was mutated from the wild type to a rare earth ion coordination mutant using site-directed mutagenesis. The wild type of lignin esterase in leaf and branch compost (LCC) described in this embodiment has the following amino acid sequence: SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDYPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELDNASHFAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALSDFRTNNRHCQ, SEQ ID NO: 1) The natural calcium ion binding site is D203 / E173 / S248. The point-mutated rare earth ion coordination mutant (DD) has the following amino acid sequence: SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDYPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELD D ASHFAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPAL D The rare earth ion binding sites of DFRTNNRHCQ (SEQ ID NO: 2) are E173 / D203 / N204D and E201 / S248D / D249.

[0133] Analysis of the docking results with polyester molecules (taking methyl terephthalate trimer as an example) shows that the catalytic triplet of the enzyme reaction site of wild-type leaf and branch compost lignin esterase (LCC) is S130 / D175 / H207, while the catalytic triplet of the mutant remains S130 / D175 / H207. Therefore, the mutant and the wild type have the same reaction mechanism when carrying out enzyme reactions.

[0134] See Figure 3The thermal stability of wild-type enzymes and mutants was compared. The thermal denaturation temperature of wild-type lignin esterase (LCC) was 73.4℃, while that of rare earth ion coordination mutant (DD) was 82.8℃. The thermal stability of the modified mutant was improved by 9.4℃.

[0135] Example 2

[0136] See Figure 4 In section A, this invention employs a purified enzyme product coordinated with rare earth elements, used in a high-temperature reaction to degrade polyethylene terephthalate (PET). The hydrolysis products of PET mainly include MHET (ethylene terephthalate diol), TPA (terephthalic acid), and EG (ethylene glycol). In this embodiment, enzyme activity is determined by detecting the amount of EG produced after 6 hours of degradation of a 5 mg / mL PET substrate at 65°C with 0.01 mg / mL enzyme and 1 mM metal ions.

[0137] The effects of different ions on the activity of rare earth coordination mutants were further analyzed, including divalent alkaline earth metal ions (such as Ca²⁺, Mg²⁺) and light rare earth ions (such as La³⁺, Ce³⁺, Pr³⁺, Nd³⁺). Results are shown below. Figure 4 In the B group (corresponding data are shown in Table 1), metal ions have a certain degree of enhancement on the activity of wild-type enzymes at 65℃. In particular, Nd³⁺ significantly improved the high-temperature catalytic efficiency of both wild-type and mutant enzymes, increasing it by 2.67 times and 10.15 times, respectively, compared with calcium coordinate enzymes.

[0138] Table 1

[0139]

[0140] Example 3

[0141] See Figure 5 (See Table 2 for corresponding data). To verify the effect of rare earth coordination mutations on enzyme activity, this example demonstrates a comparison of the degradation activities of LCC (labeled WT in the figure) and its various mutants (labeled N204D, S206D, N211D, N214D, S248D, and N253D) on polyethylene terephthalate (PET). In this example, the enzyme activity was determined by detecting the amount of EG produced after 6 hours of degradation of 5 mg / mL PET substrate at 65°C with 0.01 mg / mL enzyme and the participation of 1 mM mixed rare earth ions (lanthanum, cerium, praseodymium, and neodymium).

[0142] Table 2

[0143]

[0144] The effects of different mutation sites on enzyme activity were further analyzed. All mutants showed some improvement in enzyme activity compared to wild-type LCC, with the N204D and S248D mutants showing the highest catalytic efficiency, increasing by 4.90 and 4.15 times, respectively, compared to the wild-type enzyme.

[0145] Example 4

[0146] See Figure 6 In section A, to further investigate the degradation ability of LCC and its mutant polyurethane-based polyester materials, this example demonstrates the degradation of polyester polyurethane (PU) into MDI (diisocyanate) monomers and ethylene glycol (EG). In this example, enzyme activity was determined by detecting the amount of EG produced from the degradation of a 5 mg / mL PU substrate by the enzyme at 65°C with 0.01 mg / mL enzyme under 1 mM metal ion involvement for 6 h. See [link to results]. Figure 6 B in the table (see Table 3 for corresponding data).

[0147] Table 3

[0148]

[0149] The effects of different ions on the activity of rare-earth coordination mutants were further analyzed, including divalent alkaline earth metal ions (such as Ca²⁺ and Mg²⁺) and light rare-earth ions (such as La³⁺, Ce³⁺, Pr³⁺, and Nd³⁺). Metal ions enhanced the activity of the wild-type enzyme at 65 °C, with Nd³⁺ significantly improving the high-temperature catalytic efficiency of both the wild-type and mutant enzymes. Compared to the wild-type enzyme, the rare-earth-coordinated mutants, especially those coordinated with Nd³⁺, exhibited significantly higher catalytic activity. Compared to the calcium-coordinated enzyme, LCC and DD showed 1.23-fold and 2.00-fold increases in ethylene glycol production, respectively. The maximum degradation efficiency reached 87.36%. These findings highlight the potential of the wild-type LCC enzyme and its rare-earth-coordinated mutants in broad-spectrum polyester degradation, particularly providing strong support for the efficient degradation of various polyester substrates.

[0150] Example 5

[0151] Analysis of the hydrolysis time of polyethylene terephthalate (PET) further confirms the rare earth coordination mutant lignin esterase (DD) provided in this invention, which in this example coordinates Nd. 3+ At 75°C, it significantly outperformed previously reported thermostable esterases (ICCG and HotPETase) and wild-type enzymes (LCC) in catalytic efficiency (see [link to relevant documentation]). Figure 7(See Table 4 for data corresponding to A in the table). All tested enzymes maintained activity during the 8-hour reaction period. DD exhibited the fastest degradation rate and remained active throughout the experiment. Notably, even after 5 hours, DD continued to degrade the remaining PET molecules, highlighting its sustained catalytic efficiency. In contrast, the thermostable wild-type enzyme LCC showed reduced activity at high temperatures, resulting in minimal increase in product formation over time. For both ICCG and HotPETase, their activities were lower than DD in the initial 5 hours, but their activities were similar. Of these two thermostable enzymes, HotPETase showed slightly better thermostability, maintaining degradation activity after 5 hours, similar to DD. This indicates that although ICCG and HotPETase have higher thermostability than LCC, their catalytic efficiency is still lower than DD. In particular, after 8 hours of reaction, the molded polyester material was completely degraded by the DD enzyme (see Table 4 for the data). Figure 7 In the diagram, the dotted circle represents the size of the plastic sheet before degradation, and the scale represents 1 cm.

[0152] Table 4

[0153]

[0154] To further evaluate the degradation capabilities of these four enzymes, PET substrate was removed from the reaction system after 2 hours of reaction, washed, and its surface morphology was analyzed (see [link to reaction details]). Figure 7 (C in the figure represents 50 μm). Among all enzymes, LCC exhibited the weakest degradation ability, only producing tiny cracks on the PET surface with a crack length of less than 5 μm. In contrast, ICCG and HotPETase showed moderate degradation abilities, forming corrosion pits with diameters between 5 and 20 μm on the PET surface. Notably, HotPETase produced more corrosion pits than ICCG, indicating its slightly superior performance. DD showed the strongest degradation ability, completely eroding the PET surface and leaving a deep and continuous pitted structure. These microstructural observations further confirm the excellent degradation ability of DD-Nd under high-temperature conditions, further highlighting its potential in industrial applications for PET recycling.

[0155] Example 6

[0156] Rare earth-coordinated mutant lignin esterase (DD) from leaf and branch compost exhibited excellent thermal stability and significantly improved catalytic efficiency at high temperatures. To fully utilize the stability of the mutant enzyme and reduce usage costs by simplifying the enzyme purification process, the degradation ability of the enzyme expression strain's lysate on polyester materials was investigated, demonstrating the high stability and strong catalytic activity of this method in degrading PET materials.

[0157] In this example, 150 g of bacteria (fermentation method: 10 L of LB medium was inoculated with enzyme expression culture at a volume ratio of 100:1, cultured at 37°C and 220 rpm for 3 h, followed by induction with 0.1 mM isopropyl-β-D-thiogalactopyranoside at 20°C for 20 h, and harvested by low-temperature high-speed centrifugation) were used for lysis via high-pressure homogenization. Each reaction system contained 150 mL of lysis buffer and 150 mg of PET waste, and was incubated at 65°C with shaking at 220 rpm. Figure 8 As shown in Table 5 (corresponding data), the lysis buffer containing DD enzyme exhibited sustained activity, degrading PET within 24 hours with an actual degradation efficiency of 94.64%. During the reaction, the concentrations of TPA and MHET changed over time. The MHET level initially increased and then decreased, indicating incomplete PET degradation in the early stages of the reaction. As the long-chain polymers depolymerized, the remaining MHET further degraded, ultimately producing only TPA as a degradation product. These results indicate that higher temperatures and longer reaction times are beneficial for PET degradation, resulting in fewer byproducts. Finally, following the aforementioned polyester monomer degradation and recovery method, TPA monomer was successfully recovered from the PET degradation products in the lysis buffer (see Table 5). Figure 9 The chemical reaction mechanism of A~C in the equation is shown in D). Nuclear magnetic resonance (NMR) detection... 13 C and 1 H spectrum (see) Figure 10 As shown in the image: 13 C166.60 ppm (s, 2C), 134.36 ppm (s, 2C), 129.38 ppm (s, 4C); 1 H spectrum: 8.04 ppm (s, 4H), confirming successful monomer recovery. These examples demonstrate that PET degradation using bacterial lysate is a cost-effective and practical enzymatic method for PET recovery. Furthermore, this method enables efficient PET degradation and monomer recovery under scalable conditions.

[0158] Table 5

[0159]

[0160] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. Application of site-directed mutagenesis in enhancing the following properties of lignin esterases in leaf and branch compost: (i) Thermal stability; (ii) Ability to coordinate with rare earth ions; The site-directed mutation is at least one of N204D, S206D, N211D, N214D, S248D, and N253D; The amino acid sequence of the lignin esterase in the leaf and branch compost is shown in SEQ ID NO: 1; The rare earth ion is at least one of the following: (I) Trivalent lanthanum ion; (II) Trivalent praseodymium ion; (III) Neodymium trivalent ion.

2. A mutant of lignin esterase from leaf and branch compost, wherein the amino acid sequence of the lignin esterase from leaf and branch compost is shown in SEQ ID NO: 1, characterized in that, Includes at least one of the following: The 204th residue is D; The 206th residue is D; The 211th residue is D; Residue at position 214 is D; Residue at position 248 is D; The 253rd residue is D.

3. The mutant as described in claim 2, characterized in that, The amino acid sequence is shown in SEQ ID NO:

2.

4. The mutant as described in claim 2 or 3, characterized in that, Coordination with rare earth ions; The rare earth ion is at least one of the following: (I) Trivalent lanthanum ion; (II) Trivalent praseodymium ion; (III) Neodymium trivalent ion.

5. An expression vector, characterized in that, It has a gene element that encodes the mutant of claim 2 or 3.

6. A recombinant strain, characterized in that, It includes the expression vector as described in claim 5.

7. A method for improving the thermal stability and / or catalytic efficiency of the mutant according to claim 2 or 3, characterized in that, The mutant is coordinated with rare earth ions; The rare earth ion is at least one of the following: (I) Trivalent lanthanum ion; (II) Trivalent praseodymium ion; (III) Neodymium trivalent ion.

8. A method for degrading polyester materials, characterized in that, include: (a) Mixing the polyester material with the mutant according to any one of claims 2 to 4, reacting, and degrading the polyester material; or (b) Introduce the expression vector according to claim 5 into the expression strain, culture the expression strain, break it to obtain a crude enzyme solution, mix the polyester material with the crude enzyme solution, react, and degrade the polyester material; or (c) Cultivate the recombinant strain according to claim 6, break it to obtain a crude enzyme solution, mix the polyester material with the crude enzyme solution, react and degrade the polyester material.

9. A method for obtaining degradable monomers for polyester materials, characterized in that, include: (A) Mixing the polyester material with the mutant according to any one of claims 2 to 4, reacting, degrading the polyester material, and recovering the degradation monomers; or (B) Introduce the expression vector according to claim 5 into the expression strain, culture the expression strain, break it to obtain a crude enzyme solution, mix the polyester material with the crude enzyme solution, react to degrade the polyester material, and recover the degradation monomers; or (C) Cultivate the recombinant strain according to claim 6, break it to obtain crude enzyme solution, mix the polyester material with the crude enzyme solution, react to degrade the polyester material, and recover the degraded monomers; The polyester material includes at least one of polyethylene terephthalate and polyester-type polyurethane.

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