Efficient PET (Polyethylene Terephthalate) degrading enzyme mutant and application thereof in degrading PET material for milk

Abstract

The invention discloses an efficient PET (Polyethylene Terephthalate) degrading enzyme mutant and an application thereof in degrading a PET material for milk, and belongs to the technical field of biology. The method for high-throughput screening of the enzyme for degrading the PET material for milk is convenient to operate, high in throughput, low in cost and short in period. The PET degrading enzyme mutant screened by the method has higher degradation efficiency, the degradation rate of the PET degrading enzyme mutant to a PET material for milk within 6 hours can exceed 95%, and the PET degrading enzyme mutant has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to a highly efficient PET degrading enzyme mutant and its application in degrading dairy PET materials, belonging to the field of biotechnology. Background Art

[0002] Polyethylene terephthalate (PET) is a kind of plastic. Due to its advantages such as high mechanical strength, low air permeability, light weight, and low cost, it has important applications and significance in the dairy industry. As a packaging material, it is commonly used in liquid dairy products such as milk, yogurt, and milk tea. It is favored for its transparency, portability, and good sealing performance, effectively preventing oxidation and pollution. The excellent barrier properties of PET materials not only maintain the freshness and nutritional value of products but also pass the food-grade certification to ensure safety. In addition, PET can be recycled, which helps environmental protection. At the same time, the production cost is relatively low, suitable for large-scale applications, and reduces the packaging cost. Its light weight also fits the modern fast-paced life, playing a key role in product protection and meeting consumer needs, and promoting the sustainable development of the industry. Meanwhile, a large number of waste dairy PET materials are not effectively recycled and are difficult to degrade in the natural state, resulting in the accumulation in the global ecological system, causing the loss of soil nutrients and soil compaction, and a large amount of plastics floating in the ocean will have an adverse impact on the marine ecosystem. At present, enzymatic degradation is considered the most effective method to control plastic pollution. Various PET degrading enzymes have been successively identified and proven to be able to degrade PET to varying degrees, such as PETase of Ideonella sakaiensis (Science 351(6278):1196 - 1199), LC cutinase from the metagenome of plant compost (Appl Environ Microbiol 78(5):1556 - 1562), etc., but the degradation efficiency of dairy PET materials is low. Therefore, there is an urgent need for a rapid high-throughput screening method to effectively screen dairy PET material degrading enzymes to obtain their highly efficient mutants, so as to achieve the effect of efficiently degrading dairy PET materials. Summary of the Invention

[0003] The technical problem solved by the present invention is to provide a rapid and effective high-throughput screening method, which can screen highly efficient enzymes for degrading dairy PET materials based on the differences in the enzyme activities of degrading enzymes, and can be widely used in the screening of PET degrading enzyme mutant libraries.

[0004] The present invention provides the application of dairy PET powder in screening PET hydrolases. The application is to use dairy PET powder as a screening pressure agent for the culture medium, and characterize the degradation activity of dairy PET materials through the enzyme activity of microbial single colonies in the screening culture medium.

[0005] In one embodiment, the concentration of the milk-based PET powder in the culture medium is 30-50 g / L.

[0006] In one embodiment, the culture medium contains: 8-10 g / L of NaCl, 7-10 g / L of tryptone, and 3-5 g / L of yeast extract.

[0007] The present invention also provides a high-throughput screening method for an enzyme for degrading milk-based PET materials, and the method includes:

[0008] (1) Constructing a mutant library of PET-degrading enzymes;

[0009] (2) Culturing the PET-degrading enzyme mutants in the mutant library of PET-degrading enzymes in a liquid culture medium containing milk-based PET material powder;

[0010] (3) Selecting the PET-degrading enzyme mutants with enzyme activity higher than that of the wild-type PET-degrading enzyme.

[0011] In one embodiment, the constructing of the mutant library of PET-degrading enzymes includes, but is not limited to, mutating the wild-type PET-degrading enzyme gene, and then transforming the mutated fragment into a host cell to obtain a mutant library of PET-degrading enzymes.

[0012] In one embodiment, the mutation includes, but is not limited to, random mutation, directed mutation, etc.; the mutation method can be selected from chemical mutagenesis or physical mutagenesis.

[0013] In one embodiment, the mutant library is constructed by error-prone PCR method.

[0014] In one embodiment, the mutant uses pET24a as an expression vector and Escherichia coli BL21(DE3) as a host.

[0015] In one embodiment, the wild-type PET-degrading enzyme includes, but is not limited to, T. fusca cutinase, whose amino acid sequence is shown in SEQ ID NO.1 and nucleotide sequence is shown in SEQ ID NO.16.

[0016] In one embodiment, the wild-type PET-degrading enzyme includes, but is not limited to, BhrPETase of bacterium HR29, which has the amino acid sequence shown in SEQ ID NO.8 and nucleotide sequence shown in SEQ ID NO.17.

[0017] In one embodiment, the wild-type PET-degrading enzyme includes, but is not limited to, Phyllosticta compost cutinase, which has the amino acid sequence shown in SEQ ID NO.9 and nucleotide sequence shown in SEQ ID NO.18.

[0018] In one embodiment, the liquid screening medium contains 30 - 50 g / L of milk - used PET powder, 8 - 10 g / L of NaCl, 7 - 10 g / L of tryptone, and 3 - 5 g / L of yeast extract.

[0019] In one embodiment, after formulating the medium according to the recipe, the liquid screening medium is sterilized and added into a 96 - well deep - well plate (300 μL / well), and 10 μL of kanamycin with a final concentration of 30 μg / mL and an inducer IPTG with a final concentration of 0.1 mM are respectively added into each well containing the medium.

[0020] In one embodiment, the incubation is carried out at 25 °C for 24 h.

[0021] In one embodiment, a mutant with a ratio of mutant enzyme activity to wild - type PET - degrading enzyme activity greater than 1 is a positive mutant; the larger the ratio, the higher the activity of the mutant in degrading milk - used PET materials.

[0022] The present invention also provides a PET - degrading enzyme mutant obtained by screening using the above - mentioned method.

[0023] In one embodiment, the PET - degrading enzyme mutant has one or more mutations of P8H, N49S, S66N, W69S, D91G, P93H, N106S, R116S, D120G, L137P, R138S, S142N, W155S, N162S, S164N, A173V, A174V, D175G, D176G, V200A on the basis of the starting sequence.

[0024] In one embodiment, the starting sequence is derived from the cutinase of Thermobifida fusca and has the amino acid sequence shown in SEQ ID NO.1.

[0025] In one embodiment, on the basis of the starting sequence shown in SEQ ID NO.1, the prolines at positions 8 and 93 are mutated to histidines to obtain the mutant P8H / P93H.

[0026] In one embodiment, on the basis of the starting sequence shown in SEQ ID NO.1, the arginine at position 138 is mutated to serine to obtain the mutant R138S.

[0027] In one embodiment, on the basis of the starting sequence shown in SEQ ID NO.1, the aspartic acid at position 120 is mutated to glycine, the arginine at position 138 is mutated to serine, and the aspartic acid at position 120 is mutated to glycine to obtain the mutant D120G / R138S / D176G.

[0028] In one embodiment, the mutant is obtained by mutating asparagine at position 49 to serine, aspartic acid at position 120 to glycine, and aspartic acid at position 176 to glycine on the basis of the starting sequence shown in SEQ ID NO.1, to obtain the mutant N49S / D120G / D176G.

[0029] In one embodiment, the mutant is obtained by mutating aspartic acid at position 120 to glycine and alanine at position 173 to valine on the basis of the starting sequence shown in SEQ ID NO.1, to obtain the mutant D120G / A173V.

[0030] In one embodiment, the mutant is obtained by mutating serine at position 66 to asparagine, aspartic acid at position 91 to glycine, and serine at position 142 to asparagine on the basis of the starting sequence shown in SEQ ID NO.1, to obtain the mutant S66N / D91G / S142N.

[0031] In one embodiment, the starting sequence is BhrPETase of bacterium HR29, which has the amino acid sequence shown in SEQ ID NO.8, or has an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% homology with SEQ ID NO.8.

[0032] In one embodiment, the mutant is obtained by mutating proline at position 93 to histidine, arginine at position 116 to serine, and aspartic acid at position 175 to glycine on the basis of the starting sequence shown in SEQ ID NO.8, to obtain the mutant P93H / R116S / D175G.

[0033] In one embodiment, the mutant is obtained by mutating tryptophan at position 69 to serine and tryptophan at position 115 to serine on the basis of the starting sequence shown in SEQ ID NO.8, to obtain the mutant W69S / W155S.

[0034] In one embodiment, the mutant is obtained by mutating asparagine at position 106 to serine and serine at position 164 to asparagine on the basis of the starting sequence shown in SEQ ID NO.8, to obtain the mutant N106S / S164N.

[0035] In one embodiment, the starting sequence is a phyllosphere compost cutinase, having the amino acid sequence shown in SEQ ID NO. 12, or having an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% homology to SEQ ID NO. 12.

[0036] In one embodiment, the mutant is obtained by mutating the leucine at position 137 to proline on the basis of the starting sequence shown in SEQ ID NO. 12, to obtain the mutant L137P.

[0037] In one embodiment, the mutant is obtained by mutating the asparagine at position 162 to serine on the basis of the starting sequence shown in SEQ ID NO. 12, to obtain the mutant N162S.

[0038] In one embodiment, the mutant is obtained by mutating the alanine at position 174 to valine and the valine at position 200 to alanine on the basis of the starting sequence shown in SEQ ID NO. 12, to obtain the mutant A174V / V200A.

[0039] The present invention also provides a recombinant microorganism expressing the mutant.

[0040] In one embodiment, the recombinant microorganism uses pET24a as an expression vector and Escherichia coli BL21(DE3) as a host to express the mutant.

[0041] In one embodiment, the recombinant microorganism is a preparation method of the mutant provided by the present invention.

[0042] In one embodiment, the method is to culture the recombinant microorganism in a medium for a period of time and collect the PET-degrading enzyme mutant in the fermentation broth.

[0043] The present invention also provides an enzyme preparation containing the mutant.

[0044] In one embodiment, the enzyme preparation uses the PET-degrading enzyme mutant as the main enzyme component.

[0045] The present invention also provides the application of the mutant in degrading dairy PET materials or dairy packaging products containing PET.

[0046] Beneficial effects:

[0047] (1) The present invention provides a method for high-throughput screening of enzymes for degrading milk-use PET materials. By mutating PET-degrading enzymes, a mutant library is constructed, and further, the PET-degrading enzyme mutants are screened by a liquid screening medium containing milk-use PET powder through high-throughput screening to obtain mutants with high activity. The high-throughput screening method provided by the present invention is convenient to operate, has high throughput, low cost, and a short cycle (24 h), provides a new method for screening highly efficient enzymes for degrading milk-use PET materials, and has broad application prospects.

[0048] (2) The present invention provides an enzyme capable of efficiently degrading milk-use PET materials, and the PET degradation rate is significantly improved compared with that of the wild enzyme, and the degradation rate of milk-use PET materials can exceed 95% within 96 h.

[0049] (3) The present invention provides a method for degrading milk-use PET materials by using the screened PET-degrading enzyme, which can be used for degrading milk-use PET materials or milk-use packaging products containing PET. Compared with traditional pyrolysis or chemical depolymerization methods, it has a lower carbon footprint in terms of energy consumption, greenhouse gas emissions, and waste treatment, and is a more environmentally friendly treatment method. Brief Description of the Drawings

[0050] Figure 1 It is a schematic diagram of the high-throughput screening process. Detailed Embodiments

[0051] To facilitate the understanding of the present invention, the present invention will be described in more detail below in conjunction with the drawings and specific embodiments. The preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0052] (I) Technical Terms

[0053] As used herein, the term "mutant" refers to a polypeptide having PET-degrading enzyme activity and containing alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. Taking the mutant "R138S" as an example, the mutant "R138S" represents a polypeptide having PET-degrading enzyme activity in which arginine at position 138 is replaced by serine on the basis of the starting sequence.

[0054] (II) Reagents and Sources

[0055] The PCR enzyme, plasmid, and Escherichia coli BL21(DE3) involved in the following examples were purchased from Baori Biotechnology Co., Ltd. The gene sequencing involved in the following examples was undertaken by Genewiz Biotechnology Co., Ltd. The milk PET materials (such as Anmuxi bottles) involved in the following examples were donated by Yili Group. Ethylene terephthalate (BHET), 2-Hydroxyethyl terephthalate (MHET), and terephthalic acid (TPA) were purchased from Sigma Company.

[0056] (III) Detection Method

[0057] Determination of protein concentration:

[0058] The protein concentration was determined by the Coomassie Brilliant Blue method (Analytical Biochemistry 1976 72(1-2):248-54).

[0059] Determination of enzyme activity:

[0060] Tris-HCl buffer (10 mM pH 7.0): Weigh accurately 1.210 g of Tris and 0.584 g of NaCl, add approximately 800 mL of deionized water, stir well to dissolve, adjust the pH to 8.0 with HCl, and make up the volume to 1000 mL.

[0061] Substrate (50 mmol / L p-nitrophenyl butyrate solution): Weigh accurately 0.1046 g of p-nitrophenyl butyrate, make up the volume to 10 mL with acetonitrile, and store at -20 °C.

[0062] Preparation of the p-nitrophenol standard curve: Weigh 13.9 mg of p-nitrophenol, make up the volume to 1000 mL with 10 mM pH 7.0 Tris-HCl buffer to prepare a 100 μmol / L p-nitrophenol stock solution, and then dilute it with 10 mM pH 7.0 Tris-HCl buffer to 0, 20, 40, 60, 80, 100 μmol / L. Pipette 150 μL of the liquid into a 96-well plate (5 replicates / group) respectively, measure the absorbance at a wavelength of 405 nm using a microplate reader. Taking the p-nitrophenol concentration C as the abscissa and the absorbance A as the ordinate, plot the standard curve A = a×C + b.

[0063] Accurately pipette 150 μL of Tris-HCl buffer (pre-incubated at 37 °C for 10 min) into a 96-well plate and zero at an absorption wavelength of 405 nm. Take 144 μL of Tris-HCl buffer into the 96-well plate, add 3 μL of the diluted enzyme solution to be measured into the above 96-well plate, take 3 μL of the substrate solution, add it to the above 96-well plate (5 replicates / group), mix well and immediately place it in a microplate reader to measure the A value at an absorption wavelength of 405 nm. Record the A value every 5 seconds, and the reaction time is 1 minute.

[0064] Calculation:

[0065] Enzyme activity (U / mL) = ((K - b) × V1) × N / (a × V2 × 1000);

[0066] Specific enzyme activity (U / mg) = [((K - b) × V1) × N / (a × V2 × 1000)] / C;

[0067] Where: K: The slope of the curve formed by the A values measured at different times and the time (min) in the enzyme reaction;

[0068] V1: Reaction volume (mL);

[0069] V2: Enzyme addition volume (mL);

[0070] N: Dilution factor;

[0071] C: Enzyme concentration (mg / mL).

[0072] Specific enzyme activity: The number of enzyme activity units per milligram of protein, generally expressed as enzyme activity units / mg protein.

[0073] Determination of PET degradation rate:

[0074] Treatment of standards: Weigh the standards of TPA, MHET, and BHET separately and dissolve them in dimethyl sulfoxide (DMSO) to prepare a stock solution. Dilute the stock solution to a 0.1 mg / mL standard solution with sterile water, filter it with a 0.22 μM filter head, and inject it into a liquid phase bottle with a syringe for HPLC detection;

[0075] Treatment of samples: Let the culture solution stand for 10 min, take 5 mL of the supernatant, centrifuge it at 12000 rpm for 8 min, filter it with a 0.22 μM filter head, inject it into a liquid phase bottle with a syringe, and conduct HPLC detection.

[0076] Degradation rate (%) = ((M1 / X1 + M2 / X2 + M3 / X3) × M) / (PET mass before treatment) × 100;

[0077] M1, M2, and M3 represent the weights of TPA, MHET, and BHET in the reaction solution; M is the relative molecular mass of the PET unit; X1 is the relative molecular mass of TPA; X2 is the relative molecular mass of MHET; X3 is the relative molecular mass of BHET.

[0078] Example 1: Preparation of milk - used PET powder

[0079] Remove the lid and label from the Anmuxi milk bottle provided by Yili Group, clean it, and dry it in a drying oven at 40°C. Cut the clean PET bottle into PET flakes with a paper cutter. Put the cut PET flakes into a twin-screw extruder, set the hot melt temperature at 265 - 285°C, melt the plastic fragments and extrude them into a water tank, and quench and cool them through circulating water. The cooled plastic filaments are cut into particles about 0.5 cm long by a granulator, and then crushed into PET powder by a low-temperature grinder. This is the PET powder for milk use, which is added to the liquid medium for rapid screening of PET-degrading enzymes.

[0080] Example 2: Preparation of Liquid Screening Medium

[0081] Prepare the liquid screening medium according to the following formula: 30 g / L of PET material powder for milk use, 5 g / L of NaCl, 7 g / L of tryptone, and 5 g / L of yeast extract. After formulating the medium according to the formula, sterilize it and add it to a 96-well deep plate (500 μL / well), and add 10 μL each of kanamycin with a final concentration of 30 μg / mL and inducer IPTG with a final concentration of 0.1 mM to the medium.

[0082] Example 3: Construction of T. fusca Cutinase Mutant Library

[0083] Using the coding gene of T. fusca cutinase as the target PET-degrading enzyme (the nucleotide sequence is shown in SEQ ID NO. 16) as a template, randomly mutate its target gene fragment through error-prone PCR.

[0084] Forward primer: GAAGGAGATATACATATG;

[0085] Reverse primer: GTGCGGCCGCAAGCTTA;

[0086] The PCR reaction system is 50 μL, including 0.5 μL of template, 1.25 μL of 4 mM Mn 2+ 1.25 μL, 5 μL of 10*PCR buffer (Mg 2+ ), 1 μL each of 20 μM forward and reverse primers, 4 μL of dNTP, 0.5 μL of rTaq, and make up to 50 μL with ddH2O.

[0087] PCR amplification program: Pre-denaturation at 98°C for 5 min; (98°C for 15 s, 55°C for 30 s, 72°C for 1 min) × 30 cycles; Extension at 72°C for 5 min.

[0088] After recovering and purifying 50 μL of the PCR product by nucleic acid gel, a mutant gene fragment was obtained. Then, it was ligated to the pET24a vector through MegawhopPCR. The reaction system was 50 μL, containing 50 ng of template, 500 ng of the PCR recovery product, 25 μL of 10x SuperPfx MasterMix, and supplemented with ddH2O to 50 μL.

[0089] PCR amplification program: pre-denaturation at 98 °C for 5 min; (98 °C for 15 s, 55 °C for 30 s, 72 °C for 6.5 min) × 25 cycles; extension at 72 °C for 10 min.

[0090] Finally, 2.5 μL of DpnI was added to the PCR product and incubated overnight at room temperature to eliminate the template.

[0091] The digested PCR product was mixed with Escherichia coli BL21(DE3) competent cells, incubated on ice for 30 min, then heat-shocked at 42 °C for 90 s, and added with 1 mL of LB liquid medium to recover for 45 min, obtaining the mutant library.

[0092] Example 4: High-throughput screening of T. fusca cutinase mutants

[0093] The mutant library in Example 3 was spread on LB solid medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 15 g / L agar powder) containing 50 μg / mL kanamycin, and incubated inverted in a 37 °C constant temperature incubator for 8 - 12 h to obtain single colonies; using a high-throughput colony picking workstation, single colonies on the above solid plate were picked and inoculated into the liquid screening medium in a 96-deep well plate (1 colony / 1 well), a total of 359 single colonies, and cultured at 25 °C and 120 - 180 rpm for 8 - 12 h. 100 μL of the fermentation broth was aspirated, centrifuged at 8000 rpm for 5 min to take the supernatant, and the His-tagged purification gel beads (Ni-NTA, HY-K0210-10 mL) were used to purify the enzyme protein in the supernatant, and the enzyme activity was measured. Mutants with an enzyme activity ratio greater than 1 compared to the wild-type PET-degrading enzyme were positive mutants, a total of 121. Among them, there were 6 positive mutants with a ratio greater than 3.5, mutants M4, M57, M83, M92, M245, M283. There were 4 mutants with a ratio less than 1, mutants M7, M39, M67, M84, M92. The enzyme activity ratios of the remaining mutants were all within 1 - 3.5 (as shown in Table 1).

[0094] Table 1 Screening results of T. fusca cutinase and some mutants

[0095]

[0096]

[0097] Example 5: Degradation effect of T. fusca cutinase mutants on dairy PET materials

[0098] Preparation of enzyme solution: The mutants obtained by screening in Example 4 were selected and divided into: experimental groups (M4, M57, M83, M92, M245, and M283), wild-type WT, and control groups (M7, M39, M67, M84, and M92). The corresponding recombinant strains were inoculated into 5 mL of LB liquid medium containing 30 μg / mL kanamycin, and then inoculated into 100 mL of TB fermentation medium at an inoculation amount of 5%. After culturing at 37°C and 200 rpm for 2 - 3 h, an inducer IPTG with a final concentration of 0.1 mM was added, and the culture was continued at 25°C and 200 rpm for 24 h. The fermentation supernatant was obtained by centrifugation at 4°C and 4000 rpm, which was the crude enzyme solution. The above crude enzyme solution was first precipitated with ammonium sulfate to obtain a protein precipitate, which was then redissolved with 10 mM pH 7.0 Tris-HCl buffer. After heat treatment at 60°C for 1 h, the supernatant was centrifuged at 8000 rpm for 15 min to remove impurity proteins, and the supernatant was the purified enzyme solution.

[0099] Degradation of dairy PET materials: 5 mg of PET-degrading enzyme protein from each group and 100 mg of dairy PET powder were added to 35 mL of 100 mM phosphate buffer at pH 8.0, and the reaction was carried out at 60°C and 200 rpm for 96 h. 1 mL of the reaction solution was taken for HPLC analysis to calculate the degradation rate.

[0100] The results are shown in Table 2. The degradation rates of the mutants in the experimental groups for PET were significantly higher than those of the control group and the wild-type PET-degrading enzyme. After sequencing verification, the degradation rate of the M4 mutant (P8H / P93H, amino acid sequence as shown in SEQ ID NO.2) reached 87.6%; the degradation rate of the M57 mutant (R138S, amino acid sequence as shown in SEQ ID NO.3) reached 88.5%; the degradation rate of the M83 mutant (D120G / R138S / D176G, amino acid sequence as shown in SEQ ID NO.4) reached 93.6%; the degradation rate of the M92 mutant (N49S / D120G / D176G, amino acid sequence as shown in SEQ ID NO.5) reached 94.8%; the degradation rate of the M245 mutant (D120G / A173V, amino acid sequence as shown in SEQ ID NO.6) reached 92.8%; the degradation rate of the M283 mutant (S66N / D91G / S142N, amino acid sequence as shown in SEQ ID NO.7) reached 97.5%.

[0101] Table 2 Degradation effects of T. fusca cutinase and its mutants on dairy PET materials

[0102]

[0103]

[0104] Example 6: Determination of Enzymatic Properties of T. fusca Cutinase Mutants

[0105] Determination of the Optimal Temperature: Add 600 μL of 50 mM Tris-HCl buffer at pH 8.0 to a 1.5 mL EP tube, and then add 50 μL of the purified enzyme solution obtained in Example 5. Incubate them in a water bath at 20, 30, 40, 50, 60, 70, and 80 °C for preheating for 5 min, and then quickly add 50 μL of 100 mM substrate. Use a microplate reader to measure the absorbance value of the reaction system at a wavelength of 405 nm, record the A value every 5 seconds, and the reaction time is 1 minute. Take the value of the highest enzyme activity as 100%, and calculate the relative enzyme activity of the mutant at the remaining temperatures. The results are shown in Table 3. The optimal temperature of the forward mutant is 60 - 70 °C.

[0106] Determination of the Optimal pH: Add 600 μL of 50 mM buffer at different pH values (pH 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11) to a 1.5 mL EP tube, and then add 50 μL of the purified enzyme solution obtained in Example 5. Incubate it in a water bath at 60 °C for preheating for 5 min, and then quickly add 50 μL of substrate. Use a microplate reader to measure the absorbance value of the reaction system at a wavelength of 405 nm, record the A value every 5 seconds, and the reaction time is 1 minute. Take the value of the highest enzyme activity as 100%, and calculate the relative enzyme activity of the mutant at the remaining pH values. The results are shown in Table 4. The optimal pH of the forward mutant is 7.5 - 8.0.

[0107] Table 3 Optimal Temperature of T. fusca Cutinase Mutants

[0108]

[0109] Table 4 Optimal pH of T. fusca Cutinase Mutants

[0110]

[0111]

[0112] Example 7: Application of High-Throughput Screening Method in Other PET-Degrading Enzymes

[0113] BhrPETase (amino acid sequence shown in SEQ ID NO.8 and nucleotide sequence shown in SEQ ID NO.17) and LCC cutinase (amino acid sequence shown in SEQ ID NO.9 and nucleotide sequence shown in SEQ ID NO.18) derived from bacterium HR29 were selected as target enzymes for verification. Using methods similar to those in Examples 1 to 3, the genes encoding BhrPETase and LCC were randomly mutated respectively (the primers were the same as those of T. fusca cutinase), and the mutated gene fragments were inserted into plasmid pET24a respectively to obtain recombinant plasmids pET24a-BhrPETase and pET4a-LCC. The recombinant plasmids were transferred into Escherichia coli to form mutant libraries respectively. The enzyme proteins were prepared and the enzyme activities were measured according to the method described in Example 4. Three mutants with the largest enzyme activity ratios were selected respectively, and their degradation rates for dairy PET materials were further verified according to the method in Example 5.

[0114] The results are shown in Table 5. The degradation rates of the selected mutants for PET were significantly higher than those of the wild type. Among them, the highest degradation rate of the BhrPETase mutant Bhr_M34 (P93H / R116S / D175G, amino acid sequence shown in SEQ ID NO.9) could reach 98.5%. The highest degradation rate of the BhrPETase mutant Bhr_M45 (W69S / W155S, amino acid sequence shown in SEQ ID NO.10) could reach 95.4%. The highest degradation rate of the BhrPETase mutant Bhr_M49 (N106S / S164N, amino acid sequence shown in SEQ ID NO.11) could reach 95.8%. The degradation rate of the LCC mutant LCC_M54 (L137P, amino acid sequence shown in SEQ ID NO.13) could reach 94.5%. The degradation rate of the LCC mutant LCC_M76 (N162S, amino acid sequence shown in SEQ ID NO.14) could reach 95.4%. The degradation rate of the LCC mutant LCC_M85 (A174V / V200A, amino acid sequence shown in SEQ ID NO.15) could reach 98.4%.

[0115] Table 5 Degradation effects of PET-degrading enzyme mutants on PET

[0116]

[0117]

[0118] Example 8: Degradation efficiency of degrading enzymes on different PET products

[0119] According to the method described in Example 1, treat the bottles of Yili Jindian high-quality milk and the packaging bags of Yili Quancong student high-zinc and high-calcium milk powder to obtain the corresponding PET powder. At the same time, use Wahaha mineral water bottles and Watson's distilled water bottles as control groups. Add the mutants M283, Bhr_M34, and LCC_M85 screened in the aforementioned examples to the reaction system containing different dairy materials, and carry out the degradation of dairy PET materials according to the method described in Example 5. The results are shown in Table 6. The PET-degrading enzymes screened can achieve efficient degradation of plastic products.

[0120] Table 6 Degradation effects of PET-degrading enzyme mutants on PET

[0121]

[0122] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A PET degrading enzyme mutant, characterized in that, Based on the starting sequence, having one or more mutations among P8H, N49S, S66N, W69S, D91G, P93H, N106S, R116S, D120G, L137P, R138S, S142N, W155S, N162S, S164N, A173V, A174V, D175G, D176G, V200A; the starting sequence is as shown in SEQ ID NO.1, SEQ ID NO.8 or SEQ ID NO.

12.

2. The PET-degrading enzyme mutant according to claim 1, wherein Based on the starting sequence shown in SEQ ID NO.1, having any one of the mutations (a) - (f): (a) Mutating the prolines at positions 8 and 93 to histidines; (b) Mutating the arginine at position 138 to serine; (c) Mutating the aspartic acid at position 120 to glycine, mutating the arginine at position 138 to serine, and mutating the aspartic acid at position 120 to glycine; (d) Mutating the asparagine at position 49 to serine, mutating the aspartic acid at position 120 to glycine, and mutating the aspartic acid at position 176 to glycine; (e) Mutating the aspartic acid at position 120 to glycine and mutating the alanine at position 173 to valine; (f) Mutating the serine at position 66 to asparagine, mutating the aspartic acid at position 91 to glycine, and mutating the serine at position 142 to asparagine.

3. The PET-degrading enzyme mutant according to claim 1, wherein Based on the starting sequence shown in SEQ ID NO.8, having any one of the mutations (a) - (c): (a) Mutating the proline at position 93 to histidine, mutating the arginine at position 116 to serine, and mutating the aspartic acid at position 175 to glycine; (b) Mutating the tryptophan at position 69 to serine and mutating the tryptophan at position 115 to serine; (c) Mutating the asparagine at position 106 to serine and mutating the serine at position 164 to asparagine.

4. The PET degrading enzyme mutant according to claim 1, wherein Based on the starting sequence shown in SEQ ID NO.12, having any one of the mutations (a) - (c): (a) Mutating the leucine at position 137 to proline; (b) Mutating the asparagine at position 162 to serine to obtain the mutant N162S; (c) Mutating the alanine at position 174 to valine and mutating the valine at position 200 to alanine.

5. A gene encoding the PET - degrading enzyme mutant according to any one of claims 1 - 4.

6. A recombinant microorganism expressing the PET - degrading enzyme mutant according to any one of claims 1 - 4.

7. An enzyme preparation containing the mutant according to any one of claims 1 - 4.

8. Use of the PET - degrading enzyme mutant according to any one of claims 1 - 4, or the enzyme preparation according to claim 7, in degrading dairy PET materials or dairy packaging products containing PET.

9. The application according to claim 8, wherein React the PET - degrading enzyme mutant in a liquid environment containing PET material at 55°C - 70°C and 200 rpm for 48 - 96 hours.

10. A method for screening PET-degrading enzyme mutants, characterized in that, Comprising the following steps: (1) Construct a PET - degrading enzyme mutant library; (2) Culture the PET - degrading enzyme mutant in a liquid screening medium containing dairy PET powder; (3) Select a PET-degrading enzyme mutant with an enzyme activity higher than that of the wild-type PET-degrading enzyme.

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