Lignin peroxidase and application of lignin peroxidase mutant in degradation of thermoplastic polyurethane plastic

Abstract

The invention relates to the field of environmental science, in particular to lignin peroxidase and application of a mutant of lignin peroxidase in degradation of thermoplastic polyurethane plastics. The invention provides a lignin peroxidase mutant, the amino acid sequence of the lignin peroxidase mutant is as shown in SEQ ID NO. 1, and the coding gene sequence is as shown in SEQ ID NO. 2. The lignin peroxidase mutant can be used independently or combined with other enzymes for degrading thermoplastic polyurethane plastics, and the molecular weight degradation efficiency of the lignin peroxidase mutant and esterase combined for degrading the thermoplastic polyurethane plastics can reach 30% or above. The research provides a new insight for green and efficient degradation of TPU plastics, provides a more sustainable solution for plastic waste management, and has a huge application prospect.

Description

Technical Field

[0001] The invention relates to the field of environmental science, and in particular to application of a lignin peroxidase and a mutant thereof in degrading thermoplastic polyurethane plastics. Background Art

[0002] Thermoplastic polyurethane (TPU) has been widely used in many fields such as films, coatings, automotive parts, medical devices and 3D printing due to its excellent flexibility, wear resistance and chemical stability. In 2021, the annual production and demand of global TPU reached 18.6 million tons, and as the demand increases, the accumulation of TPU waste in the environment has become increasingly concerned ( T., et al., Critical evaluation of biodegradation studies on synthetic plastics through a systematic literature review. Sci Total Environ, 2021. 752: p. 141959). Due to the complex chemical structure of TPU, such as extensive hydrogen bonds forming physical crosslinks, it is difficult to completely degrade under natural conditions, resulting in its continued existence in the environment (Molina, GA, et al., Synthesis and Characterization of Inulin-Based Responsive Polyurethanes for Breast Cancer Applications. Polymers (Basel), 2020. 12 (4)). Traditional waste management methods, such as landfill and incineration, not only release toxic gases (such as isocyanates), but also produce microplastics, causing continuous pollution to soil, water and food chains. Therefore, the development of efficient and environmentally friendly TPU degradation technology is imminent.

[0003] At present, the degradation technology of TPU is mainly divided into two categories: chemical degradation and biodegradation. Chemical degradation methods include hydrolysis, alcoholysis, photodegradation and strong oxidation, but these methods usually require high temperature and high pressure operating conditions, and may produce complex by-products, causing secondary pollution. In contrast, biodegradation has attracted much attention due to its mild conditions and strong environmental compatibility. However, due to the stable polyurethane bonds and aromatic structures in the TPU polymer chain, it is difficult for microorganisms to directly utilize them, resulting in low biodegradation efficiency. Although studies have shown that certain fungi (such as Penicillium and Aspergillus) and bacteria (such as Pseudomonas) can partially degrade TPU by secreting extracellular enzymes such as proteases, these methods still have the defects of low efficiency and unclear mechanisms. Although esterases and ureases have been shown to be able to degrade the lipid bonds and polyurethane bonds of TPU, their activity is significantly reduced when faced with complex cross-linked structures ( J., et al., Biodegradative Activities of Selected Environmental Fungi on a Polyester Polyurethane Varnish and Polyether Polyurethane Foams. Appl Environ Microbiol, 2016. 82(17): p. 5225-35). Existing studies have mostly focused on the role of a single enzyme, ignoring the mechanism of multi-enzyme synergistic degradation in nature. This further highlights the importance of developing new degradation enzymes and optimizing enzyme functions.

[0004] Lignin peroxidase (LiP) is a common oxidase that exhibits excellent performance in the degradation of refractory aromatic compounds such as lignin and dyes. Given that TPU and lignin have similar oxygen-containing functional groups, LiP may have great potential in the degradation of TPU plastics. Although the degradation efficiency of wild-type LiP for polymers is limited, its catalytic performance can be significantly improved through protein engineering. For example, Joo et al. improved the thermal stability of LiP by changing the heme coordination, and the conversion rate of their mutant PcLiP at 40°C was three times that of the wild type (Park, JY, et al., Enhancing the thermostability of lignin peroxidase: Heme as a keystone cofactor driving stability changes in heme enzymes. Heliyon, 2024.10(17): p.e37235.). Therefore, this study proposed an innovative method for the degradation of TPU based on LiP, and enhanced its catalytic performance through protein engineering and the construction of a multi-enzyme system, providing a new idea for solving the environmental problems of TPU waste. Summary of the Invention

[0005] Purpose of the Invention: This invention addresses the technical problem of existing methods failing to efficiently and environmentally degrade TPU, a ubiquitous component of the environment. The present invention provides an innovative method for degrading TPU using LiP, enhancing its catalytic performance through protein engineering and the construction of a multi-enzyme system. This method primarily involves constructing a recombinant yeast that efficiently expresses LiP, screening for potential stable mutants based on molecular docking, and combining this method with other enzymes to construct a multi-enzyme system for efficient TPU degradation, demonstrating both environmental and high efficiency.

[0006] To solve the above technical problems, the present invention discloses the use of a lignin peroxidase and its mutants in the degradation of thermoplastic polyurethane plastics. The specific technical solution is as follows:

[0007] The present invention provides a lignin peroxidase mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention provides a gene encoding the lignin peroxidase mutant described in the first aspect, preferably, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] In a third aspect, the present invention provides a method for degrading thermoplastic polyurethane plastic, comprising the following steps: mixing thermoplastic polyurethane plastic, an oxidant, and a degrading enzyme in a first solvent to perform a degradation reaction;

[0010] Wherein, the degradation enzyme includes the lignin peroxidase mutant described in the first aspect.

[0011] The mutant lignin peroxidase is prepared by amplifying the gene encoding the mutant lignin peroxidase, cloning it into a plasmid vector, introducing it into Pichia pastoris to produce a recombinant bacterium, fermenting it to obtain a fermentation broth, and then centrifuging and ultrafiltration to obtain the mutant lignin peroxidase. Preferably, the plasmid vector is pPIC9K, and the Pichia pastoris is Pichia pastoris GS115. The nucleotide sequence of the gene encoding the mutant lignin peroxidase is shown in SEQ ID NO. 2.

[0012] The amount of the lignin peroxidase mutant is calculated as 1 to 8 U of the lignin peroxidase mutant per mg of thermoplastic polyurethane plastic. The enzyme activity (U) of the lignin peroxidase mutant is defined as the amount of enzyme required to oxidize 1 μmol of resveratrol per minute at 30°C and pH 2.5, which is defined as 1 U. Preferably, the amount of the degradative enzyme is calculated as 1 to 8 U of the degradative enzyme per mg of thermoplastic polyurethane plastic. More preferably, when the degradative enzyme only comprises the lignin peroxidase mutant, the amount of the lignin peroxidase mutant is calculated as 5 U of the lignin peroxidase mutant per mg of thermoplastic polyurethane plastic.

[0013] The oxidant is H2O2 at a concentration of 0.5 to 2 mM; the first solvent is a 200 to 300 mM tartaric acid buffer solution at a pH of 2 to 3; the degradation reaction is carried out for a reaction time of 24 to 72 hours at a temperature of 25 to 35° C. Preferably, the oxidant is added at a concentration of 1.0 mM; the first solvent is a 250 mM tartaric acid buffer solution at a pH of 2.5; the degradation reaction is carried out for a reaction time of 36 to 72 hours at a temperature of 30° C.

[0014] Wherein, the degradation enzyme further comprises a combination of manganese peroxidase and laccase; wherein the PDB ID of the manganese peroxidase is 3m5q; the PDB ID of the laccase is 1kya;

[0015] The dosage of the manganese peroxidase is calculated as 1 to 8 U of manganese peroxidase per mg of thermoplastic polyurethane plastic; the enzyme activity U of the manganese peroxidase is defined as the amount of enzyme required to oxidize 1 μmol MnSO4 per minute at 30°C and pH 2.5, which is defined as 1 U.

[0016] The laccase is used in an amount of 1 to 8 U per mg of thermoplastic polyurethane. The enzyme activity (U) of the laccase is defined as the amount of enzyme required to oxidize 1 μmol of ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] per minute at 30°C and pH 2.5.

[0017] Preferably, when the degradative enzyme comprises a lignin peroxidase mutant, a manganese peroxidase and a laccase, the total amount of the degradative enzyme is calculated as 5 U of degradative enzyme per mg of thermoplastic polyurethane plastic, wherein the amount of the lignin peroxidase mutant is calculated as 1 to 2 U of lignin peroxidase mutant per mg of thermoplastic polyurethane plastic, and the amount of the manganese peroxidase is calculated as 1 to 2 U of manganese peroxidase per mg of thermoplastic polyurethane plastic; the amount of the laccase is calculated as 1 to 2 U of laccase per mg of thermoplastic polyurethane plastic, and the enzyme activity ratio of the lignin peroxidase mutant, manganese peroxidase and laccase is 1:1:1.

[0018] Wherein, the reaction system of the degradation reaction also includes 0.5-1.5mM Mn 2+ ; preferably 1 mM MnSO4. After the degradation reaction is completed, the resulting solid is mixed with a second solvent and an esterase and degraded further at 25-35°C for 24-48 hours, preferably at 30°C for 36 hours. In this case, the degradation time of the degradation enzyme is the same as the degradation time of the esterase, thereby achieving degradation of the thermoplastic polyurethane. Further preferably, the solid obtained by the reaction of the lignin peroxidase mutant and the esterase can be further degraded multiple times (1-5 times) using the lignin peroxidase mutant degradation system and the esterase degradation system.

[0019] The esterase is carboxylesterase EC 3.1.1.1; the amount of the esterase used is 1-3 mg per mg of thermoplastic polyurethane; and the second solvent is a 5-15 mM phosphate buffer solution at a pH of 6-7. Preferably, the amount of the esterase used is 1 mg per mg of thermoplastic polyurethane; and the second solvent is a 10 mM phosphate buffer solution at a pH of 6.5.

[0020] Beneficial effects:

[0021] The lignin peroxidase mutant described in this study can be used alone or in combination with other enzymes to degrade thermoplastic polyurethane (TPU). The combination of the lignin peroxidase mutant and esterase achieved a molecular weight degradation efficiency exceeding 30% at room temperature, exceeding that of previously reported biodegradation methods. This research provides new insights into the green and efficient degradation of TPU plastics, offering a more sustainable solution for plastic waste management and promising applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0023] Figure 1 The degradation effect of wild-type LiP on TPU plastic after 72 hours of treatment. TPU represents the control group, and WT represents the wild-type LiP degradation group.

[0024] Figure 2 is the degradation effect of mutant LiP on TPU plastic, Figure 2 a in the figure is the result of degradation of TPU plastic by several different mutants for 72 hours. Figure 2 Figure b shows the degradation of TPU plastic by the mutant LiP F46W for 28 days.

[0025] Figure 3 This is the degradation result of TPU plastic by multi-enzyme combined system. Figure 3 a in the equation is the molecular weight change of TPU after degradation by LiP-Mnp-Lac for 72 hours. Figure 3 b in the figure is the molecular weight change of TPU degradation after continuous treatment with LiP-EC for 15 days. DETAILED DESCRIPTION

[0026] The present invention discloses an innovative method for degrading TPU based on LiP, and enhances its catalytic performance through protein engineering and the construction of a multi-enzyme system. The present invention can be better understood according to the following examples.

[0027] The sources of the following examples are as follows:

[0028] The lignin peroxidase LiP is derived from Trametes cervine, and its PDB ID is 3q3u. The nucleotide sequence of the encoding gene after base optimization is shown in SEQ ID NO. 3, and is named TrcLiP.

[0029] The manganese peroxidase (Mnp) is derived from Phanerochaete chrysosprium and has a PDB ID of 3m5q. The nucleotide sequence of the gene encoding it is shown in SEQ ID NO. 4 and is named PhcMnp. The enzyme activity (U) of the manganese peroxidase is defined as the amount of enzyme required to oxidize 1 μmol of MnSO₄ per minute at 30°C and pH 2.5.

[0030] The laccase Lac is derived from Trametes versicolor, with a PDB ID of 1kya. The nucleotide sequence of the gene encoding it is shown in SEQ ID NO. 5 and is named TrvLac. The enzyme activity (U) of the laccase is defined as the amount of enzyme required to oxidize 1 μmol of ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) per minute at 30°C and pH 2.5.

[0031] The commercial esterase is esterase EC 3.1.1.1, purchased from Shanghai Meirui Biochemical Technology Co., Ltd., with the product number S23705-5KU. The enzyme activity U of the esterase is defined as: at 25° C. and pH 8.0, 1 U is defined as the amount of ethyl butyrate hydrolyzed to butyric acid and ethanol per minute at 1.0 μmol.

[0032] The TPU plastic comes from Nanjing Jiangdao Environmental Science and Technology Research Institute, has a weight-average molecular weight of 69,680 Da, and the structural formula of its monomer is shown in Formula I.

[0033]

[0034] The formula of BMMY medium is: yeast powder 10 g / L, peptone 20 g / L, (NH4)2SO4 10 g / L, YNB 3.4 g / L, methanol 10 g / L, potassium phosphate buffer 100 mmol / L.

[0035] The formula of YPD liquid culture medium is: 20 g / L peptone, 10 g / L yeast powder, 20 g / L glucose, and dilute to 1 L with distilled water (YPD solid culture medium is YPD liquid culture medium with 20 g / L agar added).

[0036] The main components of BMGY medium include: yeast powder 10g / L, peptone 20g / L, (NH4)2SO4 10g / L, YNB3.4g / L, glycerol 10g / L, potassium phosphate buffer 100mmol / L.

[0037] The molecular degradation rate of the present invention is calculated as follows:

[0038]

[0039] Where M0 is the initial weight average molecular weight (average molecular weight), and M1 is the weight average molecular weight (average molecular weight) after degradation.

[0040] The detection equipment used in the following examples is as follows: gel permeation chromatograph (Waters).

[0041] The present invention adopts gel permeation chromatography (HLC-8321GPC / HT) to analyze molecular weight, and the specific method is:

[0042] The molecular weight of TPU polymers was analyzed using gel permeation chromatography (HLC-8321GPC / HT). A 2 mg TPU sample was weighed and placed in a sample vial. 2 mL of tetrahydrofuran was added and the solution was shaken on a high-temperature shaker until the sample was completely or mostly dissolved. The solution was then filtered through a 0.22 μm PTFE filter to obtain a 1 mg / mL polymer solution. 1 mL of the solution was injected into the GPC analyzer at 145°C and a flow rate of 1.8 mL / min.

[0043] Example 1

[0044] The TPU plastic film used in this example is 1×1 cm in size. This example provides an innovative method for degrading TPU based on LiP, and enhances its catalytic performance through protein engineering and the construction of a multi-enzyme system. The specific steps are as follows:

[0045] (1) Recombinant Pichia pastoris GS115 (pPIC9K-Trclip) was streaked onto a YPD plate for recovery culture. A single colony was selected from the plate and inoculated into BMGY medium. The culture was conducted at 30°C and 200 rpm for 24 hours. The recombinant Pichia pastoris GS115 (pPIC9K-Trclip) was prepared as follows:

[0046] Construction of LiP recombinant plasmid pPIC9K-Trclip: EcoR I and Not I restriction enzyme sites were introduced at the 5'-end and 3'-end of the LiP gene (also known as Trclip), respectively, and cloned into the plasmid vector pPIC9K. The recombinant plasmid pPIC9K-Trclip was synthesized by Nanjing KingSher Co., Ltd. and cloned into the Escherichia coli strain TOP10. The TOP10 glycerol bacteria containing the recombinant plasmid pPIC9K-Trclip were streaked on LB plates for activation, and then cultured at 37°C on LB plates containing ampicillin to screen transformants. The screened transformants were transferred to liquid LB culture medium for expansion. The plasmid was extracted using a plasmid extraction kit, and the integrity of the plasmid was detected by agarose gel electrophoresis. The successfully constructed recombinant plasmid was named pPIC9K-Trclip.

[0047] The recombinant plasmid pPIC9K-Trclip was linearized using SacI rapid endonuclease. Subsequently, the linearized plasmid was transformed into P. pastoris GS115 competent cells by electroporation. A single recombinant yeast colony was selected from the plate, inoculated into 15.0 mL of YPD liquid medium, and cultured overnight at 30°C and 200 r / min. The recombinant yeast genomic DNA was then extracted, and PCR was used to verify whether the recombinant plasmid was successfully integrated into the GS115 competent cells. The samples verified correct by PCR were sequenced. The recombinant yeast colony that was successfully sequenced was named GS115 (pPIC9K-Trclip).

[0048] (2) The cells obtained after BMGY expansion culture in step (1) were collected by centrifugation at 7000 r / min for 5 minutes, resuspended in potassium phosphate buffer (pH = 6), and transferred to BMMY medium containing 1 mmol / L lignin (purchased from Bidex Pharmaceuticals, BD136340). The cells were induced and cultured at 30°C and 200 r / min for 120 hours, and 0.7% v / v methanol was added every 24 hours.

[0049] (3) After the fermentation is completed, the fermentation supernatant is collected by centrifugation at 8000 r / min, and the lignin peroxidase LiP in the supernatant is concentrated using a 10 kDa ultrafiltration centrifuge tube, and the protein is quantitatively analyzed using a BCA protein quantification kit.

[0050] (4) Molecular docking of LiP and TPU monomer 4,4'-diphenylmethane diisocyanate (MDI) was performed using Autodock to predict the optimal binding mode between the ligand molecule and the receptor protein. Alanine scanning was performed after molecular docking to identify the key amino acids that interact with the TPU receptor molecule. By replacing key amino acid residues in the target protein with alanine (the smallest natural amino acid), their importance to protein function was evaluated. The affinity decreased after amino acid replacement, indicating the important role of the residue in maintaining function. Among them, eight amino acid residues caused functional instability when mutated, indicating their importance, as shown in Table 1.

[0051] Table 1 Amino acid scanning mutagenesis

[0052]

[0053] (5) The eight important amino acids predicted in Table 1 were mutated into the other 19 standard amino acids, and mutants with stable affinity were screened out (as shown in Table 2). After the mutated amino acids were determined, the mutated sequence was base-optimized and then submitted to Nanjing GenScript for synthesis. The amino acid sequence of the mutant LiP F46W is shown in SEQ ID NO.1, and the nucleotide sequence after base optimization is shown in SEQ ID NO.2.

[0054] Table 2 Amino acid saturation mutation

[0055]

[0056] (6) Repeat steps (1) to (3) to obtain a concentrated supernatant containing the LiP mutant. Specifically, the concentrated supernatant of the mutant LiP F46W was prepared as follows: EcoRI and Not I restriction enzyme sites were introduced into the 5'-end and 3'-end of the LiP F46W coding gene (the amino acid sequence of the LiP F46W mutant is shown in SEQ ID NO.1, and the nucleotide sequence of its coding gene is shown in SEQ ID NO.2), respectively, and cloned into the plasmid vector pPIC9K, synthesized by Nanjing GenScript Co., Ltd., and cloned into the Escherichia coli strain TOP10. The TOP10 glycerol bacteria plate containing the recombinant plasmid was activated and then cultured at 37°C on an LB plate containing ampicillin to screen transformants. The selected transformants were transferred to liquid LB culture medium for expansion culture. The plasmid was extracted using a plasmid extraction kit, and the integrity of the plasmid was detected by agarose gel electrophoresis. The successfully constructed recombinant plasmid was named pPIC9K-TrclipF46W. The recombinant plasmid pPIC9K-TrclipF46W was linearized using SacI rapid endonuclease. Subsequently, the linearized plasmid was transformed into P. pastoris GS115 competent cells using the electroporation method. Recombinant yeast colonies were selected from the plate, inoculated into 15.0 mL YPD liquid culture medium, and cultured overnight at 30°C and 200 r / min. The recombinant yeast genomic DNA was then extracted, and PCR was used to verify whether the plasmid was successfully integrated into the GS115 competent cells. The verified samples were sequenced. After successful sequencing, the recombinant yeast was named GS115 (pPIC9K-TrclipF46W), and the constructed recombinant bacteria were cultured and ultrafiltrated according to steps (2) to (3) to obtain a concentrated supernatant containing the mutant LiP F46W. The construction methods of other mutant LiP (including LiP H47W, LiPH47F, LiP P147F, LiP H175R, LiP H175K, LiP H175W, and LiP L233Y) are the same as above.

[0057] (7) The wild-type LiP obtained in step (3) and the mutant LiP obtained in step (6) were used to degrade TPU plastic. The specific reaction system is as follows: 250 mM tartaric acid buffer solution (pH 2.5), 1.0 mM H2O2, 5 U / (mg TPU) LiP or its mutant, 1 cm×1 cm TPU film (2 mg), and a total reaction volume of 2 mL. In the blank control group, 250 mM tartaric acid buffer solution (pH 2.5) was used instead of the enzyme-containing reaction solution. The reaction was carried out at 30°C and 200 rpm with shaking for 72 h to 28 days, and the reaction was terminated by adding 3.0 mL of methanol. Among them, one enzyme activity unit (U) of LiP or its mutant is defined as: 30°C, pH 2.5, the amount of enzyme required to oxidize 1 μmol of resveratrol per minute is defined as 1 U. Among them, 5 U / (mg TPU) LiP means that 5 U of LiP enzyme is used per mg of TPU, and the other interpretations of the U / (mg TPU) unit in this article are the same as here.

[0058] The degraded TPU plastic particles were dissolved in tetrahydrofuran and the degradation efficiency of the TPU molecular weight was analyzed by gel permeation chromatography (HLC-8321GPC / HT). All experiments were repeated three times to improve the reliability of the experimental results.

[0059] The degradation efficiency of wild-type LiP and mutant LiP is shown in Figure 2. Figure 1 and Figure 2 shown.

[0060] Figure 1 The WT group is the degradation result of wild-type LiP on TPU plastic. Compared with the control group (TPU) treated only with tartaric acid, the weight-average molecular weight (average molecular weight) of TPU was significantly reduced after 72 hours of degradation by wild-type LiP, and the molecular weight degradation rate was 5.44%.

[0061] Figure 2 is the degradation result of mutant LiP on TPU plastic, such as Figure 2 As shown in Figure a, compared with the control group (TPU) treated with tartaric acid only and several other mutant LiPs, the LiP mutant LiP F46W screened by molecular docking technology has the highest degradation efficiency. After 72 hours of degradation, the molecular weight degradation efficiency of TPU plastic is 11.97%, which is 2.2 times that of wild-type LiP. Figure 2 As shown in b, in this example, after the degradation time of the LiP mutant LiP F46W was extended to 28 days, the molecular weight degradation efficiency of TPU was increased to 26.58%.

[0062] Example 2

[0063] The nucleotide sequence of the gene encoding manganese peroxidase Mnp described in this example is shown in SEQ ID NO.4, and is named PhcMnp; the nucleotide sequence of the gene encoding laccase Lac is shown in SEQ ID NO.5, and is named TrvLac.

[0064] In this example, a multi-enzyme combined system LiP-Mnp-Lac was designed to optimize the degradation reaction, as follows:

[0065] (1) The recombinant Pichia pastoris GS115 (pPIC9K-PhcMnp) and GS115 (pPIC9K-TrvLac) were revived and cultured in the same manner as step (1) in Example 1. The recombinant Pichia pastoris GS115 (pPIC9K-PhcMnp) and GS115 (pPIC9K-TrvLac) were revived and cultured in the same manner as step (1) in Example 1. The recombinant plasmids pPIC9K-PhcMnp and pPIC9K-TrvLac were constructed in the same manner as pPIC9K-Trclip, and the restriction enzyme cleavage sites were still EcoR I and Not I.

[0066] (2) Using BMMY medium containing 1.5 mmol / L MnSO4 and BMMY medium containing 1.5 mmol / L CuSO4, respectively, the expression of Mnp and Lac in recombinant Pichia pastoris GS115 (pPIC9K-PhcMnp) and GS115 (pPIC9K-TrvLac) was induced. The induction method was the same as step (2) in Example 1, and ultrafiltration and concentration were performed according to the method described in step (3) in Example 1 to obtain manganese peroxidase Mnp and laccase Lac, respectively.

[0067] (3) Multi-enzyme system LiP-Mnp-Lac: The LiP F46W mutant with the best degradation effect in Example 1 was combined with the Mnp and Lac obtained in this example to degrade TPU plastic. The specific reaction system is as follows: 250mM tartaric acid buffer solution (pH 2.5), 1.0mM H2O2, 1.67U / (mg TPU)LiP, 1.67U / (mg TPU)Mnp, and 1.67U / (mg TPU)Lac, that is, the total enzyme amount used per mg TPU is 5U, 1cm×1cm TPU film (2mg), and the total reaction volume is 2mL. In the blank control, 250mM tartaric acid buffer solution (pH 2.5) was used instead of the enzyme-containing reaction solution. The reaction was carried out at 30°C and shaken at 200rpm for 72 hours, and the reaction was terminated by adding 3.0mL of methanol.

[0068] In addition, the present invention also designed a multi-enzyme system, LiP-EC, which combines the LiP F46W mutant, which showed the best degradation effect in Example 1, with a commercial esterase (EC) to degrade TPU plastic. The reaction proceeds according to the following steps 1 and 2, which constitute one reaction cycle. Specifically, the following steps are as follows:

[0069] Step 1: 250 mmol / L tartaric acid buffer (pH 2.5), 1.0 mmol / L H₂O₂, 1.0 mmol / L MnSO₄, 5 U / (mg TPU) LiP F46W, and 1 cm × 1 cm TPU membrane (2 mg) in a total reaction volume of 2.0 mL. The system was reacted at 30°C and 200 rpm for 36 hours. The remaining solids were then transferred to an esterase degradation system for step 2.

[0070] Step 2: 2 mL of 10 mmol / L, pH 6.5 phosphate buffer and 2 mg of esterase were added. The reaction was continued at 30°C and 200 rpm for 36 hours. The combined degradation reaction of Steps 1 and 2 lasted for 72 hours (3 days). The reaction was terminated by adding 3.0 mL of methanol. In a control group, 250 mM tartaric acid buffer (pH 2.5) was used instead of the enzyme-containing reaction solution.

[0071] For the degradation experiment of the multi-enzyme combined system LiP-EC, the test time was extended to 15 days for testing. During the test, each cycle was 3 days. Specifically, the reaction solution was replaced every 3 days. After the total degradation reaction of steps 1 and 2 was completed (i.e., a cycle of 3 days), the TPU degradation product obtained continued to be degraded according to the system of steps 1 and 2, and the cycle was repeated. That is, the reaction product after every 3 days of treatment was repeated once for the above reaction cycle to reduce the substrate inhibition effect and perform testing.

[0072] The degraded TPU plastic particles were dissolved in tetrahydrofuran and the degradation efficiency of the TPU molecular weight was analyzed by gel permeation chromatography (HLC-8321GPC / HT). All experiments were repeated three times to improve the reliability of the experimental results.

[0073] The degradation effects of the two multi-enzyme combined systems of LiP-Mnp-Lac and LiP-EC on TPU plastics are as follows: Figure 3 As shown. Figure 3 It can be seen that the multi-enzyme combined system LiP-Mnp-Lac and LiP-EC significantly improved the degradation efficiency of TPU. After 72 hours of degradation, the molecular weight degradation efficiency of TPU plastic in the LiP-Mnp-Lac system reached 15.05%, and the molecular weight degradation efficiency of TPU plastic in the LiP-EC system reached 29.20% ( Figure 3b shows the data of LiP-EC-1). After the degradation time is extended to 15 days, the degradation efficiency of the LiP-EC system on TPU plastic is further improved to 31.16% ( Figure 3 b shows the data of LiP-EC-5), which shows a significant degradation effect on TPU plastics. Figure 3 In panel b, LiP-EC-1, LiP-EC-2, LiP-EC-3, LiP-EC-4, and LiP-EC-5 represent continuous degradation for 3, 6, 9, 12, and 15 days, respectively.

[0074] The present invention provides a method and concept for the application of lignin peroxidase and its mutants in the degradation of thermoplastic polyurethane. There are numerous methods and approaches for implementing this technical solution. The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A lignin peroxidase mutant, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the lignin peroxidase mutant according to claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A method for degrading thermoplastic polyurethane plastics, characterized in that: The method comprises the following steps: mixing thermoplastic polyurethane plastic, an oxidant and a degradation enzyme in a first solvent to carry out a degradation reaction; Wherein, the degradation enzyme includes the lignin peroxidase mutant according to claim 1.

4. The method according to claim 3, characterized in that The lignin peroxidase mutant is prepared according to the following method: amplifying the gene encoding the lignin peroxidase mutant, cloning it into a plasmid vector, introducing it into Pichia pastoris to obtain a recombinant bacterium, fermenting it to obtain a fermentation broth, and then centrifuging and ultrafiltration to obtain the lignin peroxidase mutant.

5. The method according to claim 3, characterized in that The dosage of the lignin peroxidase mutant is calculated as 1 to 8 U of the lignin peroxidase mutant per mg of thermoplastic polyurethane plastic.

6. The method according to claim 3, characterized in that The oxidant is H2O2, and the added concentration is 0.5-2mM; The first solvent is a 200-300 mM, pH 2-3 tartaric acid buffer solution; The degradation reaction has a reaction time of 24 to 72 hours and a reaction temperature of 25 to 35°C.

7. The method according to any one of claims 3 to 6, characterized in that: The degradation enzyme comprises a combination of manganese peroxidase and laccase; wherein the PDB ID of the manganese peroxidase is 3m5q; and the PDB ID of the laccase is 1kya.

8. The method according to claim 7, characterized in that The dosage of the manganese peroxidase is calculated as 1 to 8 U of manganese peroxidase per mg of thermoplastic polyurethane plastic; the dosage of the laccase is calculated as 1 to 8 U of laccase per mg of thermoplastic polyurethane plastic.

9. The method according to any one of claims 3 to 6, characterized in that: The degradation reaction system also includes 0.5-1.5 mM Mn 2+ ; After the degradation reaction is completed, the solid obtained by the reaction is mixed with the second solvent and esterase, and the degradation is continued at 25-35° C. for 24-48 hours to achieve further degradation of the thermoplastic polyurethane plastic.

10. The method according to claim 9, characterized in that The esterase is carboxylesterase EC 3.1.1.1； The esterase is used in an amount of 1 to 3 mg per mg of thermoplastic polyurethane plastic. The second solvent is a phosphate buffer solution with a concentration of 5 to 15 mM and a pH of 6 to 7.

Images