Ispetase mutants and their use in degrading pet plastics

Abstract

This invention discloses a Is PETase mutants and their application in the degradation of PET plastics. This invention addresses existing... Is The PETase mutant cannot tolerate high substrate (PET plastic) concentrations, thus providing a method to improve the degradation activity of PET plastic. Is PETase mutant. For the aforementioned Is The present invention also optimizes the reaction system of the PETase mutant, thereby providing a method for depolymerizing PET plastics. Using the mutants described in this invention, such as M16-G, the degradation of high-concentration (20%) PET plastics can be completed at a Tg temperature, and it can be used for the degradation and recycling of PET plastics under industrial conditions. Furthermore, the present invention... Is PETase mutants, such as M16-G, can tolerate seawater. Seawater can be used to prepare the reaction buffer required for its degradation of PET plastics, which helps to save freshwater resources and protect the environment.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and PET plastic degradation. More specifically, it relates to a... Is PETase mutants and their application in the degradation of PET plastics. Background Technology

[0002] Polyethylene terephthalate (PET) is a common polyester plastic widely used in food packaging and other fields due to its advantages such as good heat resistance and high transparency. However, PET plastic is difficult to degrade, and large amounts of waste PET plastic continue to accumulate in the environment, causing serious environmental pollution.

[0003] From Ideonella sakaiensis PET plastic degrading enzyme of strain 201-F6 ( Is PETase exhibits a 5-120 times greater ability to degrade PET plastics at room temperature than other PET-degrading enzymes, demonstrating significant application potential. In Chinese patent publication CN120082534A, technicians mutated it, resulting in enzymes with significantly improved thermal stability and activity. Is Mutants of PETase were developed; among them, mutants M16 and M17 were able to hydrolyze PET plastic at the glass transition temperature (Tg), thus improving the degradation effect of PET plastic. However, according to reports from researchers such as Luisana Avilan and Gregory Arnal, Is PETase exhibits a high concentration-dependent limitation; even with sufficient substrate, Is The ability of PETase to hydrolyze PET plastic does not increase with increasing enzyme concentration, thus... IsPETase and its mutants are not suitable for large-scale industrial applications (Luisana A, RBL, Gerhard K, et al. Concentration dependent inhibition of mesophilic PETases on poly(ethyleneterephthalate) can be eliminated by enzyme engineering. [J]. ChemSusChem, 2023, 16(8):e202202277 and Grégory A, Julien A, Sabine G, et al. Assessment of Four Engineered PET Degrading Enzymes Considering Large-Scale Industrial Applications. [J]. ACS catalysis, 2023, 13(20):13156-1316).

[0004] Therefore, it is necessary to develop more active [products / technologies]. Is PETase mutants are developed to meet the need for biodegrading PET plastics at high substrate concentrations. Summary of the Invention

[0005] This invention addresses the problems existing in the prior art by providing a solution. Is PETase mutants, whose PET plastic degradation activity is compared to currently reported... Is The PETase mutant significantly increased.

[0006] The first objective of this invention is to provide Is PETase mutant.

[0007] A second object of the present invention is to provide an encoding of the above. Is The gene of the PETase mutant.

[0008] A third objective of this invention is to provide a recombinant vector containing the said gene.

[0009] The fourth object of the present invention is to provide a method capable of expressing the above. Is Recombinant bacteria with PETase mutant.

[0010] The fifth object of the present invention is to provide a product containing the aforementioned Is Preparations of PETase mutants and / or the recombinant bacteria.

[0011] The sixth object of the present invention is to provide the aforementioned Is The use of PETase mutants, the recombinant bacteria, or the formulation in the degradation of polyethylene terephthalate or polyethylene terephthalate-containing plastic articles.

[0012] The seventh object of the present invention is to provide the aforementioned Is The use of the PETase mutant, the gene, the recombinant vector, the recombinant bacteria, and the formulation in the preparation of products for the degradation of polyethylene terephthalate or polyethylene terephthalate-containing plastic articles.

[0013] The eighth object of the present invention is to provide a method for depolymerizing plastics.

[0014] The above-mentioned objective of this invention is achieved through the following technical solution: This invention is for further improvement Is PETase exhibits PET plastic degradation activity as a protein with the amino acid sequence shown in SEQ ID NO.1 (i.e., Is The PETase mutant M12 was used as the parent, and enhanced enzyme activity was obtained through mutation. Is PETase mutant.

[0015] Specifically, the present invention described Is The PETase mutant is obtained by first mutating the protein with the amino acid sequence shown in SEQ ID NO.1 to leucine at position 90, alanine at position 106 to cysteine, threonine at position 122 to cysteine, and aspartic acid at position 191 to glutamic acid, and then mutating the asparagine at position 66 to glycine. Alternatively, using the protein with the amino acid sequence shown in SEQ ID NO.1 as the parent, the following mutations can be performed: glutamine at position 90 is mutated to leucine, alanine at position 106 to cysteine, threonine at position 122 to cysteine, serine at position 185 to valine, and aspartic acid at position 191 to glutamic acid. The 66th asparagine position is mutated to leucine, alanine, glycine, or glutamine; Or, the 90th leucine residue could be mutated to glycine; Alternatively, the valine at position 185 could be mutated to a serine; Alternatively, the isoleucine at position 189 could be mutated to leucine; Alternatively, the asparagine at position 66 could be mutated to alanine, and the leucine at position 90 to glycine; Alternatively, the asparagine at position 66 could be mutated to histidine, and the isoleucine at position 189 to leucine. Alternatively, the asparagine at position 66 could be mutated to histidine, the isoleucine at position 189 to leucine, and the leucine at position 90 to glycine.

[0016] Specifically, encoding Is The nucleotide sequence of the gene for the PETase mutant M12 is shown in SEQ ID NO.2.

[0017] The present invention also provides an improvement Is A method for assessing the PET plastic degradation activity of PETase, wherein the method first involves wild-type... Is PETase is mutated to mutant M16 or M17, and then, based on M16, the asparagine at position 66 of M16 is mutated to glycine; or, based on M17, the asparagine at position 66 of M17 is mutated to leucine, alanine, glycine, or glutamine. Or, the 90th leucine residue could be mutated to glycine; Alternatively, the valine at position 185 could be mutated to a serine; Alternatively, the isoleucine at position 189 could be mutated to leucine; Alternatively, the asparagine at position 66 could be mutated to alanine, and the leucine at position 90 to glycine; Alternatively, the asparagine at position 66 could be mutated to histidine, and the isoleucine at position 189 to leucine. Alternatively, the asparagine at position 66 could be mutated to histidine, the isoleucine at position 189 to leucine, and the leucine at position 90 to glycine.

[0018] Specifically, the amino acid sequence of the gene encoding mutant M16 is shown in SEQ ID NO.3.

[0019] Specifically, the amino acid sequence of the gene encoding mutant M17 is shown in SEQ ID NO.4.

[0020] Although the present invention does not specifically describe each of the above, Is The amino acid or gene sequence of the PETase mutant is known, but given that the amino acid and gene sequences of the parent protein are known, the amino acid sequence of the mutant can be obtained by knowing the amino acid sequence resulting from the mutation at the mutation site. Besides mutating the parent protein, the corresponding mutant can also be obtained through artificial synthesis or other methods based on its sequence. Therefore, this invention seeks protection for the encoding described above. Is The gene of the PETase mutant.

[0021] Using the aforementioned gene, the corresponding [gene] can be obtained through recombination expression. Is PETase mutant, i.e., the gene described in the preparation of the present invention IsApplications involving PETase mutants are also within the scope of this invention.

[0022] This invention also claims protection for the code described herein. Is Recombinant vectors containing the gene sequence of PETase mutants.

[0023] The recombinant vector is used in the preparation of the present invention. Is Applications of PETase mutants are also within the scope of protection of this invention.

[0024] This invention also claims protection for a method capable of expressing the description of this invention. Is Recombinant bacteria with PETase mutant.

[0025] Optionally, the recombinant bacteria contain the recombinant vector described in this invention.

[0026] Optionally, when constructing the recombinant bacteria, Escherichia coli, Bacillus, Pseudomonas, Halophilic yeast, or other similar strains may be selected as the starting strain.

[0027] The recombinant bacteria are used in the preparation of the present invention. Is Applications involving PETase mutants are also within the scope of protection of this invention.

[0028] This invention also claims protection for a formulation containing the ingredients described in this invention. Is PETase mutants and / or the recombinant bacteria.

[0029] Specifically, the formulation also contains Tris-HCl buffer.

[0030] Specifically, the solvent used to prepare the Tris-HCl buffer solution is fresh water or seawater.

[0031] Specifically, when using the formulation described in this invention to degrade PET plastic, the concentration of the Tris-HCl buffer solution is 180–220 mM.

[0032] This invention also claims protection for the aforementioned Is The use of PETase mutants, the recombinant bacteria, or the formulation in the degradation of polyethylene terephthalate plastics or polyethylene terephthalate-containing plastics.

[0033] This invention also claims protection for the aforementioned Is The use of the PETase mutant, the gene, the recombinant vector, the recombinant bacteria, and the formulation in the preparation of products for the degradation of polyethylene terephthalate plastics or polyethylene terephthalate-containing plastics.

[0034] The present invention also provides a method for depolymerizing plastics, the method comprising:Is The PETase mutant, the recombinant bacteria, or the preparation were placed with plastic powder in a Tris-HCl buffer solution with a concentration of 180–220 mM and a pH of 8.8–9.2, and reacted at 64–66 °C.

[0035] Specifically, the plastic is polyethylene terephthalate plastic or plastic containing polyethylene terephthalate.

[0036] Specifically, the concentration of the Tris-HCl buffer is 190–210 mM, and the pH is 8.9–9.1.

[0037] Preferably, the concentration of the Tris-HCl buffer is 200 mM and the pH is 9.

[0038] The present invention has the following beneficial effects: This invention is aimed at Is PETase has a high concentration-dependent limitation, and existing... Is The PETase mutant cannot tolerate high substrate (PET plastic) concentrations, thus providing a method to improve the degradation activity of PET plastic. Is PETase mutant. Targeting the obtained... Is The present invention also optimizes the reaction system of the PETase mutant, thereby providing a method for depolymerizing PET plastics. Using the mutants described in this invention, such as M16-G, the degradation of high-concentration (20%) PET plastics can be completed at a Tg temperature, and it can be used for the degradation and recycling of PET plastics under industrial conditions. Furthermore, the present invention... Is PETase mutants, such as M16-G, can tolerate seawater. Seawater can be used to prepare the reaction buffer required for its degradation of PET plastics, which helps to save freshwater resources and protect the environment. Attached Figure Description

[0039] Figure 1 The figure shows the activity comparison and Tm value determination results of mutants M17-AG and M16-G with their respective parents; different letters in the figure represent significant differences. p <0.05.

[0040] Figure 2 Figure A shows the results of the optimal reaction temperature determination for mutants M17-AG and M16-G, as well as the results of their PET plastic degradation activity tests with HotPETase and LCC-ICCG. Figure B shows the results of the optimal reaction temperature determination for mutants M17-AG and M16-G, and the results of the PET plastic degradation activity tests for mutants M17-AG, M16-G, HotPETase, and LCC-ICCG.

[0041] Figure 3 The results show the optimized hydrolysis conditions for the mutants. In the figure, A represents the relative activity of mutant M16-G in five other buffers with different pH (8-9) or different salt concentrations, relative to Gly-NaOH (pH 9.0, 100 mM) buffer. In the figure, B and C represent the concentration-dependent inhibition of mutants M17-AG and M16-G in four different buffers: Gly-NaOH (pH 9.0, 100 mM), NaH2PO4-Na2HPO4 (pH 8.5, 50 mM), KH2PO4-K2HPO4 (pH 9.0, 50 mM), and Tris-HCl (pH 9.0, 200 mM).

[0042] Figure 4 The figures show the degradation curves of mutant M16-G at different substrate (PET plastic) concentrations; Figure A represents the degradation curve of M16-G in PET plastic in Tris-HCl (pH 9.0, 200 mM) buffer prepared from fresh water and seawater at a substrate concentration of 5%; Figure B represents the degradation curve of M16-G in PET plastic in Tris-HCl (pH 9.0, 200 mM) buffer prepared from seawater at a substrate concentration of 20%; the red arrows in the figures indicate the enzyme addition time points (for 5% substrate concentration, each addition resulted in a final concentration of 2 μM of purified enzyme of mutant M16-G, and for 20% substrate concentration, each addition resulted in a final concentration of 6 μM of purified enzyme of mutant M16-G). Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0044] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0045] The formulations of the non-commercially available reagents used in the embodiments of this invention are as follows: LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl; LB solid medium is obtained by adding 20 g / L agar to the LB liquid medium.

[0046] 8× Binding buffer: NaCl 23.376 g, Tris base 1.9676 g, imidazole 0.2723 g, add ultrapure water to a final volume of 100 mL, pH 7.9.

[0047] 8× Wash buffer: NaCl 23.376 g, Tris base 1.9676 g, imidazole 3.2678 g, add ultrapure water to a final volume of 100 mL, pH 7.9.

[0048] 4× Elute buffer: NaCl 11.668 g, Tris base 0.96912 g, imidazole 27.232 g, add ultrapure water to a final volume of 100 mL, pH 7.9.

[0049] 1× Binding buffer, 1× Wash buffer and 1× Elute buffer were all obtained by diluting their mother liquor.

[0050] The detection methods involved in the embodiments of the present invention are as follows: Construction of the standard curve for terephthalic acid (TPA): A 5 mM stock solution of TPA was prepared and diluted with buffer (50 mM Glycine-NaOH, pH 9.0) to prepare TPA standard solutions of different concentrations. The TPA standard solutions of different concentrations were analyzed by HPLC and the peak areas were read. The standard curve was plotted with the concentration of TPA standard solution as the x-axis and the peak area as the y-axis. The resulting formula is: y = 0.0499x + 0.3569, R² = 0.9998.

[0051] Construction of the standard curve for monohydroxyethyl terephthalate (MHET): MHET was prepared into a 5 mM stock solution, and then diluted with buffer (50 mM Glycine-NaOH, pH 9.0) to prepare MHET standard solutions of different concentrations. The MHET standard solutions of different concentrations were analyzed by HPLC and the peak areas were read. The standard curve was plotted with the concentration of the MHET standard solution as the x-axis and the peak area as the y-axis. The resulting formula is: y = 0.0566x + 0.0099, R² = 0.9999.

[0052] Half-deactivation temperature (T) 50 10 The half-inactivation temperature is defined as the temperature at which the PET plastic degrading enzyme loses 50% of its initial activity within 10 minutes.

[0053] Half-deactivation temperature (T) 50 10 Determination of enzyme activity: The purified enzyme was incubated at different temperatures for 10 min, and the remaining enzyme activity was measured.

[0054] Half-deactivation temperature change (ΔT) 50 10: This represents the temperature difference between the mutant and the original enzyme (parent) that results in a 50% loss of initial activity within 10 minutes.

[0055] PET plastic degradation activity assay: Purified enzyme was added to the buffer solution to make a final volume of 300 μL, with a final enzyme concentration of 200 nM or 400 nM. 8 mg of PET plastic microparticles were added and mixed thoroughly. The mixture of purified enzyme and PET substrate was incubated at a specific temperature for 30 min. After the reaction, the mixture was centrifuged at 14000 rpm for 1 min, and the supernatant was collected. An equal volume of methanol was added, and the mixture was mixed and then inactivated at 98 ℃ for 15 min. The supernatant was centrifuged at 14000×g for 1 min, and filtered through a 0.22 μm filter. The filtered sample was used for HPLC analysis.

[0056] Depolymerization of PET plastic: In a 2 L bioreactor, 500 mL of buffer solution was added, and after the temperature reached 65 °C, 5% or 20% (w / v) of PET substrate was added. Simultaneously, purified enzyme was added in batches to a final concentration of 2 μM (5% substrate concentration) or 6 μM (20% substrate concentration). The rotation speed was set to 400 rpm, and the pH of the reaction system was maintained at 9.0 using 2 M (5% substrate concentration) or 3 M (20% substrate concentration) NaOH solution. During the reaction, a 200 μL sample was taken periodically, and an equal volume of methanol was added. After mixing, the sample was inactivated at 98 °C for 15 min. After centrifugation at 14000 ×g for 1 min, the supernatant was filtered through a 0.22 μm filter. The filtered sample was used for HPLC analysis. The consumption of NaOH was recorded, and the degradation rate of PET was calculated based on the NaOH consumption. 2 mol of NaOH consumed corresponded to 1 mol of TPA generated.

[0057] HPLC analysis method: The mobile phase used for product separation was 0.1% (v / v) formic acid solution (phase A) and acetonitrile (phase B); the total flow rate was 0.8 mL / min, the detection wavelength was 260 nm, and the column temperature was 30 ℃; the separation method was as follows: the initial proportion of phase B was 1%, and from 1 to 6 min, the proportion of phase B increased from 1% to 5% in a linear gradient; from 6 to 16 min, the proportion of phase B increased from 5% to 70% in a linear gradient; from 16 to 22 min, the proportion of phase B increased from 70% to 100% in a linear gradient; from 22 to 26 min, the proportion of phase B remained at 100%; from 26 to 31 min, the proportion of phase B decreased from 100% to 1% in a gradient; and from 31 to 36 min, the proportion of phase B remained at 1%.

[0058] Melting temperature (T) mAssay: Real-time quantitative PCR was used for the assay. First, 5 μg of purified enzyme was mixed with 1× protein thermal shift dye, with a total volume of 20 μL. The program was set to Melt Curve, and the temperature was increased from 25 °C to 99 °C at a rate of 1.6 °C / s. A curve was plotted by differentiating fluorescence intensity from temperature to determine the Tg. m value.

[0059] Example 1 Is Obtaining PETase mutants This invention is based on previously obtained Is Degradation performance of PETase mutants M16 and M17 at high substrate concentrations (20%) was tested. While both mutants could degrade PET at the specified Tg, they could not complete degradation at high substrate concentrations (20%), limiting their potential for industrial application. To address this, this invention, based on mutants M16 and M17, further mutated them to obtain a mutant with enhanced enzyme activity. Is PETase mutant.

[0060] The mutant M16 is defined in the amino acid sequence shown in SEQ ID NO. 1. Is Based on the PETase mutant M12 (whose gene sequence is shown in SEQ ID NO.2), the mutant M17 was obtained by mutating glutamine at position 90 to leucine, alanine at position 106 to cysteine, threonine at position 122 to cysteine, and aspartic acid at position 191 to glutamic acid; the mutant M17 is derived from... Is Based on the PETase mutant M12, the following mutations were made: glutamine at position 90 was replaced with leucine, alanine at position 106 was replaced with cysteine, threonine at position 122 was replaced with cysteine, serine at position 185 was replaced with valine, and aspartic acid at position 191 was replaced with glutamic acid.

[0061] Based on mutant M16, this invention introduces mutated amino acids at the corresponding sites through PCR amplification, resulting in a mutant with further enhanced enzyme activity than M16, namely M16-N66G.

[0062] Based on mutant M17, this invention introduces mutated amino acids at corresponding sites via PCR amplification, obtaining mutants with further enhanced enzyme activity than M17, including M17-N66L, M17-N66A, M17-N66G, M17-N66Q, M17-L90G, M17-V185T, and M17-I189L. Furthermore, this invention also performs combined mutations on M17 at the above sites, obtaining mutants with further enhanced enzyme activity, including M17-N66A-L90G, M17-N66H-I189L, and M17-N66H-L90G-I189L.

[0063] The process of obtaining the above mutants is as follows: The mutation was introduced by PCR amplification using either the recombinant plasmid pET-28a(+)-TAC-M16 containing the gene sequence encoding mutant M16 (SEQ ID NO.3) or the recombinant plasmid pET-28a(+)-TAC-M17 containing the gene sequence encoding mutant M17 (SEQ ID NO.4). The PCR primers used for PCR amplification are shown in Table 1. Table 1 PCR primer sequences

[0064] The PCR amplification reaction system is shown in Table 2.

[0065] Table 2 PCR amplification reaction system

[0066] The PCR amplification reaction conditions were as follows: Stage 1: 98 ℃ pre-denaturation for 3 min; Stage 2: 98 ℃ denaturation for 10 s, 55 ℃ annealing for 5 s, 72 ℃ extension for 1 min 30 s, for a total of 30 cycles; Stage 3: 72 ℃ extension for 3 min.

[0067] After PCR amplification, the PCR products were analyzed by gel electrophoresis. Bands of similar size were cut and recovered from the gel. The recovered products were purified using the FastPure Gel DNA Extraction mini kit and then purified using the Accure Seamless Cloning Ligation Kit. OK Clon The recovered DNA was ligated into the pET-28a(+)-TAC vector and reacted at 50 °C for 15 min.

[0068] After the ligation reaction was complete, the ligation product was cooled on ice. 10 μL of the ligation product was then mixed with 100 μL of... E .coliBL21(DE3) competent cells were mixed and heat-treated in a 42°C water bath for 90 s. The competent cells mixed with the ligation product were placed on ice and incubated for 2 min. 900 μL of LB liquid medium was added, and the mixture was incubated at 37°C and 200 rpm for 1 h. The culture was then evenly spread on LB agar plates containing 50 μg / mL Kana and incubated overnight at 37°C. Three morphologically normal single colonies were picked from the incubated plates and transferred to 20 mL of LB liquid medium, and incubated overnight at 37°C and 220 rpm. The overnight culture was sent to Guangzhou Ruibo Biotechnology Co., Ltd. for sequencing to obtain the DNA sequence of the ligation product. The culture containing the DNA sequence of the correct mutation was preserved for subsequent experiments.

[0069] Take sequencing samples containing the correct single-point mutation E.coli BL21(DE3) strain was cultured overnight at 37 ℃ and 220 rpm to obtain sufficient bacterial cells. The overnight culture was inoculated into fresh LB liquid medium (containing 50 μg / mL Kana) at a volume ratio of 1% and cultured at 37 ℃ and 220 rpm with shaking until the OD600 of the bacterial culture reached 0.8–1.0. IPTG (final concentration 0.6 mM) was added to induce protein expression, and the culture was continued at 16 ℃ and 180 rpm for 24 h. The cells were collected and lysed by centrifugation at 8000 g for 10 min to obtain crude enzyme solution. The crude enzyme solution was purified to obtain mutant protein (purified enzyme).

[0070] The purification steps for the crude enzyme solution are as follows: (1) The collected bacterial cells were resuspended in 10 mL of 1× Binding Buffer and the cells were disrupted by an ultrasonic disruptor. After disruption, the cells were centrifuged at 8000 g for 10 min, the supernatant was collected, and the supernatant was filtered through a 0.45 μm filter membrane to obtain crude enzyme solution. (2) Take 4 mL of His Bin resin and add it to the filter column to form a purification column; (3) Wash the purification column sequentially with 10 mL of ultrapure water and 10 mL of 1× Binding Buffer; (4) Slowly add the crude enzyme solution to the purification column; (5) Add 20 mL of 1× Binding Buffer and 12 mL of 1× Wash Buffer, elute with 5 mL of 1× Elute Buffer, and collect the protein eluent; (6) Replace the eluted protein with potassium phosphate buffer (50 mM, pH 7.0) using a 10 kDa ultrafiltration tube and store at 4°C.

[0071] The half-inactivation temperature of the purified enzyme was determined and the change in half-inactivation temperature (ΔT) was calculated. 50 10 Meanwhile, the degradation activity of the purified enzyme in PET plastic after reacting at 65 °C for 30 min was measured, and its relative activity with respect to the parent (M16 / M17) was calculated. The results are shown in Table 3.

[0072] Table 3. Relative activity and half-inactivation temperature change of purified enzymes

[0073] Note: The mutants shown in the table exhibit significantly different PET plastic degradation activity compared to their parents. p <0.05).

[0074] Table 3 shows that the PET plastic degradation activity of the mutants obtained based on M16 in this invention is 15% higher than that of their parents (50% higher than that of M17), and the PET plastic degradation activity of the mutants obtained based on M17 is also more than 10% higher than that of their parents. The ΔT values ​​of mutants M17-N66A, M17-N66G, M17-L90G, and M17-N66A-L90G are also shown. 50 10 >0 ℃, meaning that while the degradation activity of PET plastic is improved, its thermal stability is also improved.

[0075] In this invention, the mutant M16-N66G is named M16-G and the mutant M17-N66A-L90G is named M17-AG. The enzymatic properties of these two mutants will be further analyzed in the future.

[0076] Example 2 Enzymatic Properties Analysis of Mutants M16-G and M17-AG 1. Degradation activity and melting temperature of PET plastic (T) m ) Measurement In addition to testing the PET plastic degradation activity of the mutants M16, M17, M16-G, and M17-AG purification enzymes, this invention also tested the melting temperatures of the M16, M17, M16-G, and M17-AG purification enzymes, respectively. The results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the degradation activity of M17-AG and M16-G in PET plastics is higher than that of their corresponding parents, and the differences are significant. p <0.05), the Tm values ​​of M17-AG and M16-G were 90.85 ℃ and 85.15 ℃, respectively. The former was 1.62 ℃ higher than the parent, while the latter remained basically unchanged.

[0077] 2. Determination of optimal reaction temperature The mutants M16-G and M17-AG were purified and placed in Glycine-NaOH buffer (100 mM, pH 9.0), mixed with the substrate PET plastic, and then subjected to catalytic reactions at 62 ℃, 65 ℃, 68 ℃, 70 ℃, 72 ℃, and 75 ℃, respectively. All conditions except the reaction temperature remained constant. The optimal reaction temperatures for M17-AG and M16-G were determined as follows: Figure 2 As shown in Figure A, the optimal reaction temperatures for mutants M17-AG and M16-G are 72 ℃ and 65 ℃, respectively.

[0078] 3. Degradation activity test of PET plastic This invention commissioned Genewiz Biotechnology Co., Ltd. to synthesize Is The gene sequences of the PETase mutant HotPETase and the LCC-ICCG mutant of the cutinase LCC derived from the leaf and branch compost metagenomics were ligated into the vector pET-28a(+)-TAC according to the method described in Example 1, and then transformed into Escherichia coli to obtain strains that can express HotPETase and LCC-ICCG, thereby obtaining purified enzymes of HotPETase and LCC-ICCG.

[0079] The mutant HotPETase and LCC-ICCG are reported PET depolymerases with good overall performance, and their sequences were obtained from the references. Specifically, the mutant HotPETase was obtained from reference 1 (Bell EL, Smithson R, Kilbride S. Directed evolution of an efficient and thermostable PET depolymerase[J]. Nature Catalysis,2022(8):5.); the mutant LCC-ICCG was obtained from reference 2 (Tournier V, Topham CM, Gilles A, et al. An engineered PET depolymerase to break down and recycle plastic bottles[J]. Nature, 2020,580(7802):216-219.).

[0080] The present invention uses purified enzymes of the mutants Hot-PETase, M16-G, M17-AG, and LCC-ICCG as experimental materials to detect the PET plastic degradation activity of the four mutants.

[0081] The purified enzymes of the mutants Hot-PETase, M16-G, M17-AG, and LCC-ICCG were added to different buffer solutions. Hot-PETase was prepared with Gly-NaOH (pH 9.2, 50 mM), M16-G and M17-AG with Gly-NaOH (pH 9.2, 100 mM), and LCC-ICCG with NaH2PO4-Na2HPO4 (pH 9.2, 100 mM). The final volume of the purified enzyme and buffer solution was 300 μL, the substrate (PET plastic) concentration was 26.7 g / L, and the final concentration of the purified enzyme was 400 Nm. After thorough mixing, the enzymes were reacted at their respective optimum temperatures (65 ℃ for Hot-PETase and M16-G, and 72 ℃ for M17-AG and LCC-ICCG). After the reaction, the content of the reaction products was detected by HPLC, and the PET plastic degradation activity of the mutant purified enzymes was determined. The results are as follows: Figure 2 As shown in Figure B, within a 2-hour reaction period, M16-G produced the highest amount of product from PET degradation, reaching 9.11 mM, which is 1.65 times that of HotPEtase and 1.18 times that of LCC-ICCG. M17-AG produced the second highest amount of product, at 8.18 mM, which is 1.49 times that of HotPEtase and 1.06 times that of LCC-ICCG. These results indicate that the mutants M16-G and M17-AG can efficiently degrade PET at temperatures above 65 °C, demonstrating good application potential in the industrial biodegradation of PET.

[0082] Example 3: Optimization of PET hydrolysis conditions Referring to Luisana Avilan's research, it was found that Is PETase exhibits a high concentration-dependent inhibition, and hydrolysis conditions significantly affect this phenomenon. Therefore, this invention focuses on the purified enzyme of the mutant M16-G, optimizing the conditions for its PET hydrolysis reaction, including buffer type, buffer pH, and buffer salt ion concentration, to determine the optimal conditions for the PET hydrolysis reaction and reduce the high concentration-dependent limitation.

[0083] Using the hydrolytic activity of mutant M16-G in Gly-NaOH buffer (pH 9.0, 100 mM) as a control, five different buffers were selected, including NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, KH2PO4-NaOH, Na2HPO4-HCl and Tris-HCl, to determine the relative activity of M16-G in different buffers, different pH (8-9), and different salt concentrations (10-200 mM). Except for the hydrolysis conditions, other conditions remained unchanged.

[0084] Subsequently, the conditions with the highest relative activity (>80%) in each buffer solution were selected, and the high concentration-dependent limitation of the enzyme was studied by measuring the amount of PET hydrolysis products generated at different enzyme concentrations (0–3 μM). All conditions remained constant except for the enzyme concentration.

[0085] The relative activities of mutant M16-G in five other buffers with different pH (8–9) or different salt concentrations, relative to Gly-NaOH (pH 9.0, 100 mM) buffer, are as follows: Figure 3 As shown in Figure A, at lower enzyme concentrations (200 nM), the activity of PET hydrolase in Gly-NaOH buffer is greater than that in the other five buffers. However, as the enzyme concentration increases (>1 μM), due to... Is The high concentration dependence of PETase means that the hydrolysis products in Gly-NaOH buffer do not increase with increasing enzyme concentration.

[0086] Furthermore, this invention also investigated the concentration-dependent inhibition of the mutants M17-AG and M16-G in four different buffer solutions: Gly-NaOH (pH 9.0, 100 mM), NaH2PO4-Na2HPO4 (pH 8.5, 50 mM), KH2PO4-K2HPO4 (pH 9.0, 50 mM), and Tris-HCl (pH 9.0, 200 mM). The results are as follows: Figure 3 Figures B and C in the figure show that, as can be seen from the figure, in Tris-HCl and phosphate buffer, M16-G ( Figure 3 B) and M17-AG ( Figure 3 C) in the study showed greater hydrolytic activity, with Tris-HCl being superior.

[0087] The results indicate that, compared to Gly-NaOH and phosphate buffer, Tris-HCl buffer can alleviate the high concentration-dependent inhibition of mutants to a greater extent, with the optimal conditions being pH 9.0 and salt ion concentration of 200 mM.

[0088] Example 4: PET depolymerization reaction This embodiment investigates the effect of replacing fresh water with seawater on the depolymerization performance of PET plastic, using the highly active mutant M16-G as an example.

[0089] The specific method is as follows: First, 500 mL of Tris-HCl (pH 9.0, 200 mM) buffer prepared with fresh water / seawater was added to a 2 L bioreactor. 25 g of PET plastic powder was then added, followed by the addition of purified enzyme of mutant M16-G at a final concentration of 2 μM in batches. The reaction was carried out at 65 ℃ and 200 rpm, with the pH of the reaction system maintained at 9.0 using 2 M NaOH. Samples were taken at different time points for HPLC analysis to determine the content of reaction products and the degradation ability of the purified enzyme of mutant M16-G on PET. The consumption of NaOH was also recorded to calculate the degradation rate of PET (2 mol of NaOH consumed corresponds to 1 mol of TPA generated).

[0090] Subsequently, in order to determine the hydrolytic ability of mutant M16-G to high-concentration substrates under seawater conditions, the above method was followed, except that the substrate concentration was 20% (100 g PET added to 500 mL buffer), the final concentration of purified enzyme was 6 μM each time, and the NaOH concentration was 3 M.

[0091] The degradation curves of mutant M16-G at different substrate (PET plastic) concentrations are as follows: Figure 4 As shown. By Figure 4 It can be seen that at a low substrate concentration (5%), M16-G in Tris-HCl prepared with fresh water can hydrolyze 50% of PET plastic within 6 hours, 90% of PET plastic within 18 hours, and after 24 hours, it can almost completely hydrolyze 25g of PET plastic. Figure 4 (A) The above data demonstrates that the mutant M16-G can completely hydrolyze post-consumer PET plastic within 24 hours, showing good application potential in the industrial biodegradation of PET plastic. When the Tris-HCl buffer is prepared with seawater, M16-G can also hydrolyze 90% of PET within 24 hours. Figure 4 (A) demonstrates the feasibility of M16-G degradation in seawater, which is beneficial for conserving freshwater resources and is environmentally friendly. To verify the degradation of M16-G under high substrate concentrations, the substrate concentration was further increased (5%→20%), such as... Figure 4 As shown in B, M16-G can hydrolyze 50% of PET within 10 h and 90% of PET within 48 h in a seawater-prepared buffer solution.

[0092] The above results indicate that M16-G can be used for the degradation of PET at high substrate concentrations in industrial applications, while also saving freshwater resources and being environmentally friendly.

[0093] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A kind Is PETase mutant, characterized by, The mutant is obtained by using the protein with the amino acid sequence shown in SEQ ID NO.1 as the parent protein, by mutating glutamine at position 90 to leucine, alanine at position 106 to cysteine, threonine at position 122 to cysteine, and aspartic acid at position 191 to glutamic acid, and then mutating asparagine at position 66 to glycine. Alternatively, using the protein with the amino acid sequence shown in SEQ ID NO.1 as the parent, the following mutations can be performed: glutamine at position 90 is mutated to leucine, alanine at position 106 is mutated to cysteine, threonine at position 122 is mutated to cysteine, serine at position 185 is mutated to valine, and aspartic acid at position 191 is mutated to glutamic acid. The 66th asparagine position is mutated to leucine, alanine, glycine, or glutamine; Or, the 90th leucine residue could be mutated to glycine; Alternatively, the valine at position 185 could be mutated to a serine; Alternatively, the isoleucine at position 189 could be mutated to leucine; Alternatively, the asparagine at position 66 could be mutated to alanine, and the leucine at position 90 to glycine; Alternatively, the asparagine at position 66 could be mutated to histidine, and the isoleucine at position 189 to leucine. Alternatively, the asparagine at position 66 could be mutated to histidine, the isoleucine at position 189 to leucine, and the leucine at position 90 to glycine.

2. A coding scheme as described in claim 1 Is The gene of the PETase mutant.

3. A recombinant vector containing the gene of claim 2.

4. A device capable of expressing the description of claim 1 Is Recombinant bacteria with PETase mutant.

5. A formulation, characterized in that, The formulation contains the product of claim 1. Is PETase mutants and / or the recombinant bacteria of claim 4.

6. The formulation according to claim 5, characterized in that, It also contains Tris-HCl buffer.

7. The formulation according to claim 6, characterized in that, The solvent used to prepare the Tris-HCl buffer solution is fresh water or seawater.

8. The claim 1 Is The use of PETase mutants, the recombinant bacteria of claim 4, or the formulations of any one of claims 5 to 7 in the degradation of polyethylene terephthalate plastics or polyethylene terephthalate-containing plastics.

9. The claim 1 Is The use of the PETase mutant, the gene of claim 2, the recombinant vector of claim 3, the recombinant bacteria of claim 4, and the formulation of any one of claims 5 to 7 in the preparation of products for degrading polyethylene terephthalate plastics or polyethylene terephthalate-containing plastics.

10. A method for depolymerizing plastics, characterized in that, The claim 1 Is The PETase mutant, the recombinant bacteria of claim 4, or any of the preparations of claims 5 to 7 are placed with plastic powder in a Tris-HCl buffer solution with a concentration of 180 to 220 mM and a pH of 8.8 to 9.2, and reacted at 64 to 66 °C; the plastic is polyethylene terephthalate or a plastic containing polyethylene terephthalate.

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