PET hydrolase mutants and their applications

Abstract

The present invention belongs to the field of genetic engineering, and in particular relates to a PET hydrolase mutant with high catalytic activity and its application. The present invention utilizes structural analysis and site-directed mutagenesis technology to modify the amino acid sequence of the PET hydrolase PES-H1 shown in SEQ ID NO.2. L92F / Q94Y Mutations at amino acids 62, 65, 66, 71, 154, 156, 175, 177, 183, 184, 209, 213, 214, and 215 of the amino acid sequence yielded multiple mutants, including mutants with enhanced catalytic activity. These PET hydrolase mutants enhance the degradation of PET plastics by promoting the hydrolysis of ester bonds in PET. They could potentially be used in the enzymatic depolymerization of PET plastics in our daily lives.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to a PET hydrolase mutant and an application thereof. Background Art

[0002] Polyethylene terephthalate (PET) is widely used in food packaging, fibers, films, and other fields due to its lightweight, corrosion-resistant, and transparent properties. However, its chemical inertness makes it difficult to degrade naturally, resulting in millions of tons of waste annually. Landfill or incineration pollutes soil, air, and water, and releases greenhouse gases and toxic substances (such as dioxins).

[0003] Currently, the main methods for treating PET waste include direct landfill, incineration, mechanical recycling, and chemical recycling. Landfill, as the most primitive disposal method, not only occupies land, but also pollutes water and soil resources, and consumes significant manpower and material resources. Incineration produces toxic and hazardous substances such as nitrogen oxides, benzene, and formaldehyde, which pose a potential threat to human health. Mechanical recycling primarily involves grinding, pulverizing, or cutting PET waste into small pieces for reuse in the manufacture of new PET products. However, this process requires high temperatures and pressures, which presents significant limitations. Chemical recycling requires complex processes and equipment, resulting in high recycling costs. In contrast, biological methods (microorganisms or enzymes) to degrade PET plastics not only avoid the harmful gases or byproducts produced by traditional physical or chemical methods, but also meet environmental sustainability requirements and reduce negative environmental impacts.

[0004] Using bio-enzymatic methods to degrade waste PET into low molecular weight monomers and recycle them is a potential green solution. Among the many PET hydrolases, PET hydrolase PES-H1 is a thermophilic hydrolase derived from the metagenome. It has a high starting point for the balance between thermal stability and activity. Although its thermal stability is slightly inferior to LCC, its substrate affinity is higher, and its unique "multiple substrate binding mode" also provides a multi-dimensional target for design. Based on the understanding of the structure and mechanism of action of PES-H1, researchers have modified it through protein engineering technology to obtain mutants with better performance. PES-H1 L92F / Q94Y The variant not only has improved hydrolysis activity on amorphous PET film and pretreated real PET waste, but also has the highest efficiency in hydrolyzing low-crystallinity PET reported in the literature. ICCG However, PES-H1 L92F / Q94Y The enzyme activity is still low, which limits its industrial application in plastic recycling and requires further improvement of its catalytic efficiency. L92F / Q94YThe enzyme was used as the research object, and a series of mutants were constructed through site-directed mutagenesis technology in order to improve the catalytic activity of the enzyme on PET plastics and accelerate the process of applying the enzyme to the enzymatic depolymerization of industrial PET plastics. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a PET hydrolase mutant and its application in view of the shortcomings of the existing technology.

[0006] In order to solve the above technical problems, the present invention discloses a PET hydrolase mutant and its application. The specific technical solution is as follows:

[0007] A PET hydrolase mutant, wherein the PET hydrolase mutant is obtained by subjecting the PET hydrolase having an amino acid sequence as shown in SEQ ID NO.2 to any one of the following amino acid mutations (1) to (7):

[0008] (1) H184Y,

[0009] (2) H184F,

[0010] (3) S183E,

[0011] (4) S183Q,

[0012] (5) L209G,

[0013] (6) P214N,

[0014] (7) P214Q.

[0015] The PET hydrolase mutants shown are proteins having PET hydrolase activity, wherein the homology of the remaining amino acid residues is greater than 99%, greater than 95%, greater than 90%, greater than 85% or greater than 80% except for the mutation site in any of the proteins (1) to (7).

[0016] Preferably, the PET hydrolase mutant protein is a protein having PET hydrolase activity obtained by adding a 6xHis tag sequence to the C-terminus of the amino acid sequence of any one of the proteins (1) to (7).

[0017] Further preferably, the PET hydrolase mutant is obtained by subjecting the PET hydrolase having the amino acid sequence shown in SEQ ID NO. 2 to an amino acid mutation such as H184Y.

[0018] In a second aspect, the present invention provides a gene encoding the PET hydrolase mutant described in the first aspect.

[0019] In a third aspect, the present invention provides an expression cassette or a recombinant vector containing the gene described in the second aspect.

[0020] In a fourth aspect, the present invention provides a recombinant bacterium containing the expression cassette or recombinant vector described in the third aspect.

[0021] In a fifth aspect, the present invention provides use of the PET hydrolase mutant described in the first aspect in the depolymerization of polyethylene terephthalate (PET).

[0022] The method for depolymerizing polyethylene terephthalate using the PET hydrolase mutant comprises the following steps: mixing the PET hydrolase mutant with the substrate polyethylene terephthalate in a buffer solution and then performing a depolymerization reaction.

[0023] The amount of the PET hydrolase mutant used is 0.5-10 mg per gram of polyethylene terephthalate, preferably 0.5-2 mg per gram of polyethylene terephthalate.

[0024] The concentration of the substrate polyethylene terephthalate in the reaction system is 2-200 g / L, preferably 10-30 g / L. Furthermore, preferably, the PET is in powder form. More preferably, the PET is amorphous PET powder (Gf-PET).

[0025] The buffer is a phosphate buffer of 0.5-2 M and pH 7.5-9, preferably a phosphate buffer of 1 M and pH 8.0.

[0026] The depolymerization reaction has a depolymerization time of 2-36 h and a depolymerization temperature of 60-75°C, preferably 65°C.

[0027] Beneficial effects:

[0028] The present invention utilizes site-directed mutagenesis technology to modify PET hydrolase (PES-H1 L92F / Q94Y ) were mutated to obtain 26 mutants, of which 7 variants effectively improved PES-H1 L92F / Q94Y PET degradation activity. The mutants of the PET hydrolase designed in the present invention, obtained by subjecting the amino acid sequence of SEQ ID NO. 2 to an amino acid mutation such as H184Y, exhibited a 2.4-fold increase in enzymatic activity and a nearly 4-fold increase in degradation efficiency, demonstrating significant degradation effects. PET is an insoluble polymer that is difficult to degrade. The various mutants of the present invention enhance the degradation efficiency of PET plastics and have promising prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1 Analysis of the hydrolytic activity of 26 mutant proteins designed for the present invention.

[0031] Figure 2 Comparison of the concentrations of TPA and MHET released by the hydrolysis of PET by the 26 mutants designed for the present invention.

[0032] Figure 3 HPLC liquid phase diagram of TPA and MHET, the products released during the proteolysis of PET by seven mutants with enhanced activity.

[0033] Figure 4 T of the 26 mutants designed for this invention m Value comparison. DETAILED DESCRIPTION

[0034] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0035] The PTE substrates described in the following examples are all amorphous PET powders (good-fellow PET).

[0036] Example 1 Construction of PET hydrolase mutant plasmid

[0037] Using the existing PET hydrolase PES-H1 L92F / Q94Y The original PET hydrolase was studied to increase its industrial application value. In this example, PES-H1 L92F / Q94Y Cloning, expression and purification were carried out. By studying the structure of this PET hydrolase, the amino acids in its active region involved in substrate interaction were mutated to increase the enzyme's catalytic activity on the substrate PET.

[0038] In this example, PET hydrolase PES-H1 was first constructed. L92F / Q94Y The recombinant plasmid and its mutant recombinant plasmid. Original PET hydrolase PES-H1 L92F / Q94Y The amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence encoded by it is shown in SEQ ID NO.1.

[0039] With molecular chaperones E. coli BL21(DE3) competent cells were prepared as follows: pKJE7 plasmid (dnaK-dnaJ-grpE molecular chaperone vector plasmid) was transformed into E. coliBL21 (DE3) competent cells were then cultured on LB plates containing chloramphenicol (20 μg / mL) to screen for chaperone protein plasmid transformants. Single colonies were selected and transferred to a test tube containing 5 mL of fresh LB liquid medium and cultured at 37°C, 200 rpm, and shaken for 12 hours. The above bacterial solution was transferred to a shake flask containing 100 mL of fresh LB medium at a 1% v / v inoculum and cultured at 37°C, 200 rpm, and the OD value was calculated. 600 A value of 0.6-0.8 is ideal. Place the shake flask culture on ice for 10 minutes, then aliquot into sterile centrifuge tubes and centrifuge at 4°C, 4100 rpm, for 10 minutes. Discard the supernatant and repeat the centrifugation. Add 4 mL of pre-chilled 0.1M CaCl₂ solution to each centrifuge tube. Shake the tubes on ice until the cells are suspended and no bacterial sludge adheres to the tube walls. Place on ice for 30 minutes, centrifuge at 4°C, 4100 rpm, for 10 minutes, and remove the supernatant. Add 2 mL of the same pre-chilled 0.05M CaCl₂ solution (containing 15% v / v glycerol) to the centrifuge tubes. Resuspend the cells as above. Add 200 μL of the stock solution to each sterile 1.5mL EP tube. These are the competent cells and store in a -80°C freezer until ready to use.

[0040] Amplification of PES-H1 L92F / Q94Y The gene was cloned into the linearized plasmid pET-28a(+) with NcoI and XhoI restriction sites at its 5' and 3' ends, and then transferred into E. coli DH5α, and the recombinant plasmid pET-28a(+)-PES-H1 was constructed L92F / Q94Y , take 1 μL of recombinant plasmid pET-28a(+)-PES-H1 L92F / Q94Y Added to the chaperone (dnaK-dnaJ-grpE) E. coli Transformation was performed in BL21(DE3) competent cells. Single clones were picked and transferred to a test tube containing LB liquid containing kanamycin sulfate. Plasmids were extracted and used as PCR templates for PCR amplification using the primers described in Table 1 to obtain plasmids expressing different PET hydrolase mutants. The purified reaction products were transformed into E. coli competent cells and initially screened with kanamycin sulfate. DNA sequencing was performed to confirm the successful mutation of the gene, thereby obtaining plasmids expressing different PET hydrolase mutants.

[0041] PES-H1 L92F / Q94Y Rationally designed site-directed mutagenesis primers were used to perform site-directed mutagenesis on 14 sites, resulting in 26 mutants, namely:

[0042] l) mutating the phenylalanine at position 62 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to tryptophan, while leaving the amino acid residues at other positions unchanged, to obtain a protein having PET hydrolase activity, namely F62W;

[0043] 2) Mutating the glycine at position 65 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to aspartic acid, while leaving the amino acid residues at other positions unchanged, to obtain a protein having PET hydrolase activity, namely G65D;

[0044] 3) Mutating the glycine at position 65 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to glutamic acid, while leaving the amino acid residues at other positions unchanged, to obtain a protein having PET hydrolase activity, namely G65E;

[0045] 4) Mutating the glutamine at position 66 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to alanine, while leaving the amino acid residues at other positions unchanged, to obtain a protein having PET hydrolase activity, namely Q66A;

[0046] 5) Mutating the tryptophan at position 71 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to phenylalanine, while leaving the amino acid residues at other positions unchanged, to obtain a protein having PET hydrolase activity, namely W71F;

[0047] 6) Mutating the tryptophan at position 71 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to tyrosine, while leaving the amino acid residues at other positions unchanged, to obtain a protein having PET hydrolase activity, namely W71Y;

[0048] 7) Mutating the tryptophan at position 71 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to aspartic acid, while leaving the amino acid residues at other positions unchanged, to obtain a protein having PET hydrolase activity, namely W71D;

[0049] 8) Mutating the tryptophan at position 71 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to histidine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely W71H;

[0050] 9) Mutating the tryptophan at position 71 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to alanine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely W71A;

[0051] 10) Mutating the glycine at position 154 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to proline, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely G154P;

[0052] 11) Mutating the histidine at position 156 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to serine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely H156S;

[0053] 12) Mutating the leucine at position 175 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to alanine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely L175A;

[0054] 13) The leucine at position 175 of the PET hydrolase amino acid sequence shown in SEQ ID NO.2 was mutated to arginine, and the amino acid residues at other positions remained unchanged to obtain a protein having PET hydrolase activity, namely L175R;

[0055] 14) Mutating the threonine at position 177 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to serine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely T177S;

[0056] 15) Mutating the serine at position 183 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to glycine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely S183G;

[0057] 16) Mutating the serine at position 183 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to glutamic acid, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely S183E;

[0058] 17) Mutating the serine at position 183 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to glutamine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely S183Q;

[0059] 18) Mutating the histidine at position 184 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to tyrosine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely H184Y;

[0060] 19) Mutating the histidine at position 184 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to phenylalanine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely H184F;

[0061] 20) Mutating the leucine at position 209 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to glycine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely L209G;

[0062] 21) Mutating the threonine at position 213 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to glutamic acid, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely T213E;

[0063] 22) Mutating the threonine at position 213 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to arginine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely T213R;

[0064] 23) Mutating the proline at position 214 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to aspartic acid, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely P214D;

[0065] 24) Mutating the proline at position 214 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to asparagine, while leaving the other amino acid residues unchanged, to obtain a protein having PET hydrolase activity, namely P214N;

[0066] 25) Mutating the proline at position 214 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 to glutamine, while leaving the amino acid residues at other positions unchanged, to obtain a protein having PET hydrolase activity, namely P214Q;

[0067] 26) The aspartic acid at position 215 in the amino acid sequence of the PET hydrolase shown in SEQ ID NO. 2 was mutated to asparagine, while the amino acid residues at other positions remained unchanged, to obtain a protein having PET hydrolase activity, namely D215N.

[0068] Table 1 Site-directed mutagenesis primers

[0069]

[0070] Table 2 Site-directed mutagenesis primers continued

[0071]

[0072] The preparation process of mutant plasmid and pure enzyme is as follows: upstream and downstream primers are designed and synthesized according to the nucleotide sequence corresponding to the pre-mutation site, as shown in Table 1 and Table 2, and then the plasmid pET-28a(+)-PES-H1 is used to generate the mutant plasmid. L92F / Q94Y Site-directed mutagenesis was performed using the template as the template to obtain a PCR product. The PCR procedure was as follows: (1) initial denaturation: 95°C, 3 min; (2) denaturation: 95°C, 30 s; annealing: 55°C, 30 s; extension: 68°C, 3 min; this step was cycled 20 times; (3) final extension: 68°C, 7 min. After the PCR procedure was completed, the PCR product was digested with the digestion enzyme Dpn I at a ratio of 2% w / w. The digested PCR products were transformed into competent E. coli DH5α cells, plated onto LB plates containing kanamycin sulfate, screened for positive clones, and cultured. Positive clones were then transferred to 5 mL of liquid LB medium containing 50 μg / mL kanamycin sulfate for in vitro culture. Plasmids were extracted and verified. Plasmids containing correct mutations were transformed into competent E. coli BL21 (DE3) cells containing the aforementioned molecular chaperone (dnaK-dnaJ-grpE). Positive clones were then plated onto LB plates containing kanamycin sulfate and chloramphenicol for in vitro culture. Positive clones were then transferred to 5 mL of liquid LB medium containing 50 μg / mL kanamycin sulfate and 20 μg / mL chloramphenicol. The seed culture was transferred to 100 mL of LB medium containing 0.5 mg / mL L-arabinose and cultured at 37°C. When the OD600 reached 0.6-0.8, 0.1 mM IPTG was added for induction for 4 h. Crude enzyme was obtained by sonication.

[0073] To obtain a highly pure enzyme protein, the bacterial suspension was sonicated and centrifuged at 8000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter and purified using a Ni-NTA column. The target protein was eluted using buffers containing varying concentrations of imidazole (50 mM Na₂HPO₄, 50 mM KH₂PO₄, 300 mM NaCl, and 50 mM, 100 mM, 200 mM, 300 mM, or 500 mM imidazole). Contaminants were eluted at 50 mM imidazole, while the target protein was eluted at 100–300 mM imidazole. The eluted mutant protein (molecular weight 29.1 kDa) was concentrated using an ultrafiltration centrifuge tube and dialyzed to remove imidazole. The resulting protein concentration was 1 mg / mL.

[0074] Example 2 PET hydrolase mutants and original PES-H1 L92F / Q94Y Relative enzyme activity analysis

[0075] To verify the difference between the original PET hydrolase and the PET hydrolase mutant, this example further measured the degradation activity of the two on PET. The PET hydrolase activity test method is mainly:

[0076] The PET substrate p-nitrophenylbutyrate ( p NPB) was dissolved in methanol to prepare a 10 mM test solution. 10 μL of the test solution and 10 μL of the pure enzyme prepared in Example 1 were added to 980 μL of 50 mM PB buffer (pH 8.0). The solution was incubated at 37°C for 5 min. The absorbance change at 410 nm was measured on a microplate reader to calculate the amount of product generated. Enzyme activity was defined as the amount of product generated at 37°C, which is 1 μmol of p-nitrophenol (NPB) per minute. p The amount of enzyme required for NP) is regarded as one unit of enzyme activity.

[0077] The results are as follows Figure 1 As shown, G65D, G154P, S183E, S183Q, H184Y, L209G, P214N, P214Q relative to wild-type PES-H1 L92F / Q94Y The relative enzyme activities of the ester bond enzymes were improved, among which the activity of H184Y increased by 2.4 times, the activity of P214N increased by 2.8 times, and the activity of P214Q increased by 1.4 times.

[0078] Example 3 PET hydrolase mutants and original PES-H1 L92F / Q94Y Analysis of the degradation performance of PET by depolymerase

[0079] The reaction system was 2 mL: the reaction temperature for enzymatic depolymerization was 65°C, and the buffer was a phosphate buffer with a concentration of 1 M and a pH of 8.0. The amount of PET substrate added was 20 mg / mL, and the amount of enzyme added was 0.07 wt% of the substrate. Therefore, the final enzymatic depolymerization system was 2 mL of phosphate buffer, 28 μg of pure enzyme, and 40 mg of PET powder as substrate. The enzymatic degradation reaction was carried out in a metal bath at 65°C and the speed was set to 800 r / min for 3 h. After the reaction, the mixture was centrifuged at 12,000 r / min for 10 min. The PET degradation product was filtered using a 0.22 μm filter, diluted appropriately according to the product concentration, and then analyzed using an Agilent 1260 Infinity II high-performance liquid chromatography system through a C18 column (Agilent 5 HC-C18 (2) 150×4.6 mm). A solvent consisting of 20% acetonitrile, 1% formic acid and 79% water was used as the mobile phase, the temperature was 30°C, the injection volume was 10 μL, the detection time was 12 min, the flow rate was 0.8 mL / min, and the detection wavelength was 240 nm.

[0080] PES-H1 L92F / Q94Y The depolymerization of PET plastic by the enzyme and its variants does not produce dimers, and its hydrolysis products are only terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and ethylene glycol (EG). Therefore, the depolymerization rate of PET plastic can be calculated by detecting the content of TPA and MHET in the depolymerization system. The activity of the original and mutant enzymes can be determined by comparing the peak area of ​​the hydrolysis product TPA of the original PET hydrolase or its mutants. During the HPLC detection process, the amount of compound in the solution is linearly related to the peak area, so the concentration of the hydrolysis product in the solution can be calculated based on the peak area. The depolymerization rate calculation formula is:

[0081]

[0082] The results are as follows Figure 2 As shown, it can be seen that 7 of the 26 mutants listed have higher PET hydrolysis activity than the wild-type protein, among which H184Y has the highest activity, and the concentration of the product produced after 3 h of catalysis is nearly 4 times higher than that of the wild-type WT. Figure 3 The HPLC detection results of the seven activity-enhanced mutant proteins showed that the peak time of TPA in liquid chromatography was 4.7 min, and the peak time of MHET in liquid chromatography was 6.2 min. Figure 3 It can be seen that both the wild-type and mutant proteins peaked at 4.7 min and 6.2 min, indicating that both the wild-type and mutant proteins had degradation activity on PET and terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected after degradation.

[0083] Example 4 PET hydrolase mutants and original PES-H1 L92F / Q94Y Thermal stability analysis of depolymerase

[0084] Proteins undergo thermal denaturation and unfolding during heating. The melting temperature (Tm) of a protein refers to the temperature at which 50% of the protein is unfolded. The nanoDSF method is used to determine the melting temperature (Tm) of a protein. m ), PET hydrolase PES-H1 L92F / Q94Y and its mutant T mValues ​​were determined by nano-differential scanning fluorimetry (NanoDSF) using a Prometheus NT.48 and differential scanning calorimetry (DSC). Measurements were performed using protein concentrated to the desired concentration (approximately 0.5-2 mg / mL), with temperature profiles measured from 20°C to 95°C and from 20°C to 100°C, respectively, at a rate of 1°C per minute. The nanoDSF instrument had a fixed excitation wavelength of 285 nm, and emission was recorded at 330 nm and 350 nm.

[0085] The results are as follows Figure 4 As shown, wild-type protein T m The value is 75.8℃, and the T m The value is 78.1℃, and the T of G154P m The value is 79.9℃, and the T m The value is 77.7℃, and the T of S183E m The value is 79℃, and the T m The value is 79.3℃, and the T of H184Y m The value is 79.2℃, and the T m The value is 78.9℃, T213R m The value is 77.4℃, and the T of P214Q m The value was 76.6℃, and the T value of mutant H184Y was 2.377℃ compared with that of wild-type protein. m The value increased by 3.4℃.

[0086] The present invention provides a PET hydrolase mutant and its application concept and method. There are many methods and approaches to implement this technical solution. The above 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 PET hydrolase mutant, characterized in that The PET hydrolase mutant is obtained by subjecting the PET hydrolase having an amino acid sequence as shown in SEQ ID NO.2 to any one of the following amino acid mutations (1) to (3): (1) H184Y, (2) P214N, (3) P214Q.

2. A gene encoding the PET hydrolase mutant according to claim 1.

3. An expression cassette or recombinant vector containing the gene according to claim 2.

4. A recombinant bacterium containing the expression cassette or recombinant vector according to claim 3.

5. Use of the PET hydrolase mutant according to claim 1 in the depolymerization of polyethylene terephthalate.

6. The use according to claim 5, characterized in that The method for depolymerizing polyethylene terephthalate using a PET hydrolase mutant comprises the following steps: mixing the PET hydrolase mutant with a substrate polyethylene terephthalate in a buffer solution and then performing a depolymerization reaction.

7. The use according to claim 6, characterized in that The dosage of the PET hydrolase mutant is calculated as 0.5-10 mg of the PET hydrolase mutant per gram of polyethylene terephthalate.

8. The use according to claim 6, characterized in that The concentration of the substrate polyethylene terephthalate in the reaction system is 2-200 g / L.

9. The use according to claim 6, characterized in that The buffer solution is a phosphate buffer solution with a concentration of 0.5-2 M and a pH of 7.5-9.

10. The use according to claim 6, characterized in that The depolymerization reaction has a depolymerization time of 2-36 h and a depolymerization temperature of 60-75°C.

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