PETase-PHL7 mutant and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and discloses a PETase-PHL7 mutant and an application thereof. The invention provides a PETase-PHL7 mutant, the amino acid sequence of the PETase-PHL7 mutant is shown as SEQ ID No.3, and the PETase-PHL7 mutant is marked as a mutant YPT. Compared with a mutant H185Y, the PET degradation activity of the mutant is improved by 55.6%, and the Tm is improved by 0.35 DEG C. On the basis of the mutant YPT, the amino acid of the mutant YPT is further subjected to mutation design, and six PETase-PHL7 mutants are provided. Compared with the mutant YPT, the PETase-PHL7 mutant provided by the invention has the advantages that the PET (Polyethylene Terephthalate) degradation activity is improved by 55.6 percent to 132.5 percent; meanwhile, the thermal stability of the mutant is improved to different extents. The method can be applied to the fields of PET enzyme preparation, PET degradation, PET degradation agent preparation or PET degradation product preparation and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and relates to a PET degrading enzyme, in particular to a PETase-PHL7 mutant and an application thereof. Background Art

[0002] Polyethylene terephthalate (PET) is a thermoplastic polyester made from the polycondensation of terephthalic acid and ethylene glycol. It is one of the most produced synthetic resins in the world. Due to its excellent transparency, impact and chemical resistance, ease of processing, and reasonable cost, PET is widely used in a variety of fields, including packaging, insulation, electronics, and medical devices.

[0003] However, the widespread and extensive use of PET has also led to the generation of significant amounts of PET waste. Due to the stable ester bonds in the PET molecular backbone, it is difficult to effectively degrade in the natural environment, taking hundreds of years to degrade in soil or oceans, and easily accumulating to form "white pollution." Furthermore, if discarded, it can break down into microplastics, which infiltrate ecosystems and threaten plant and animal life, as well as human health. Therefore, the development of efficient and sustainable solutions for PET waste is urgent.

[0004] Enzymatic degradation of PET, which utilizes specific microbial enzymes to catalyze the cleavage of PET molecular chains, offers advantages such as mild reaction conditions, low environmental impact, high-purity degradation products, and compatibility with a wide range of PET morphologies and physical states. It is expected to become a mainstream technology for PET recycling. Existing PET hydrolases are diverse, with significant differences in catalytic activity, thermal stability, and substrate adaptability. PETase-PHL7 (ENA No. LT571446) can degrade 90% of amorphous PET in 16 hours and completely degrade PET in 18 hours, making it a highly valuable and promising PET hydrolase. However, PETase-PHL7's core performance indicators, such as degradation activity and thermal stability, still fall short of the "high efficiency and stability" requirements for enzyme preparations in large-scale industrial applications, leaving significant room for improvement. Optimizing these key properties will better meet the actual needs of large-scale industrial production and lay the foundation for its industrialization. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the present invention is based on the modification of the PETase-PHL7 mutant H185Y to provide a PETase-PHL7 mutant with significantly improved degradation activity and thermal stability and its application.

[0006] To achieve the above-mentioned purpose, the embodiment of the present invention adopts the following technical solutions: In the first aspect, the present invention provides a PETase-PHL7 mutant, which is a mutant H185Y in which the alanine at position 188 is site-directedly mutated to proline, and the arginine at position 111 is site-directedly mutated to threonine, while the amino acid residues at other positions remain unchanged. The resulting amino acid sequence is shown in SEQ ID No. 3. The resulting PETase-PHL7 mutant is recorded as mutant YPT.

[0007] The present invention provides a PETase-PHL7 mutant, mutant YPT, derived from mutant H185Y by site-directed mutation of alanine at position 188 to proline and arginine at position 111 to threonine. Compared to mutant H185Y, mutant YPT exhibits a 55.6% increase in PET degradation activity and a 0.35°C increase in Tm.

[0008] Among them, the amino acid sequence of wild-type PETase-PHL7 is shown in SEQ ID No. 1, the amino acid sequence of mutant H185Y is shown in SEQ ID No. 2, and the amino acid sequence of mutant YPT is shown in SEQ ID No. 3.

[0009] In the second aspect, the present invention further performs single-point mutation on the basis of mutant YPT to provide three PETase-PHL7 mutants, whose amino acid sequences are any one of the following (1) to (3): (1) The serine at position 139 of the amino acid sequence shown in SEQ ID No. 3 was site-directedly mutated to alanine, while the amino acid residues at other positions remained unchanged. The resulting mutant was designated as mutant YPTA; (2) The glutamic acid at position 187 of the amino acid sequence shown in SEQ ID No. 3 was site-directedly mutated to isoleucine, while the amino acid residues at other positions remained unchanged. The resulting mutant was designated as mutant YPTI; (3) The asparagine at position 191 of the amino acid sequence shown in SEQ ID No. 3 was site-directedly mutated to histidine, while the amino acid residues at other positions remained unchanged. The resulting mutant was designated as mutant YPTH.

[0010] Testing revealed that compared to mutant YPT, the three mutants exhibited 19.5%-21.9% higher PET degradation activity. Compared to mutant H185Y, the three mutants exhibited 86%-90% higher PET degradation activity. Compared to mutant H185Y, the three mutants exhibited varying degrees of improved thermal stability. The Tm value of mutant YPTI was slightly increased, that of mutant YPTA increased by 1.57°C, and that of mutant YPTH exhibited the greatest increase, at 5.05°C.

[0011] In a third aspect, the present invention performs a single-point mutation on the basis of mutant YPTI, and site-directedly mutates the aspartic acid at position 198 in the amino acid sequence of the above mutant YPTI to proline, while the amino acid residues at other positions remain unchanged. The resulting mutant is recorded as mutant YPTIP.

[0012] The test found that compared with mutant YPTI, mutant YPTIP's PET degradation activity increased by 11.9%, and its Tm increased by 4.86°C. Compared with mutant H185Y, mutant YPTIP's PET degradation activity increased by 1.12 times, and its Tm value increased by 4.88°C.

[0013] In a fourth aspect, the present invention further performs a single-point mutation on the basis of the mutant YPTIP, i.e., replacing the amino acid residue at one position in the amino acid sequence of the mutant YPTIP, and obtaining an amino acid sequence as any one of (i) or (ii): (i) site-directed mutation of the histidine at position 109 in the amino acid sequence of the mutant YPTIP to leucine, while leaving the amino acid residues at other positions unchanged. The resulting amino acid sequence is shown in SEQ ID No. 8, and the resulting mutant is designated as mutant YPTIPL; (ii) The asparagine at position 191 in the amino acid sequence of the mutant YPTIP was site-directedly mutated to histidine, while the amino acid residues at other positions remained unchanged. The resulting amino acid sequence is shown in SEQ ID No. 9, and the resulting mutant is designated as mutant YPTIPH.

[0014] The tests found that compared with mutant YPTIP, mutant YPTIPL showed a 9.6% increase in PET degradation activity, with a 0.17°C increase in Tm. Mutant YPTIPH showed a 4.3% increase in PET degradation activity, with a 1.1°C increase in Tm. Compared with mutant H185Y, mutant YPTIPL showed a 1.32-fold increase in PET degradation activity, with a 5.05°C increase in Tm. YPTIPL also showed a 1.21-fold increase in PET degradation activity, with a 6.0°C increase in Tm.

[0015] In a fifth aspect, the present invention provides a DNA molecule encoding the PETase-PHL7 mutant according to any one of the first to fourth aspects.

[0016] In a sixth aspect, the present invention provides a recombinant plasmid capable of expressing the PETase-PHL7 mutant described in any one of the first to fourth aspects.

[0017] In a seventh aspect, the present invention provides a PETase-PHL7 mutant engineered strain comprising the recombinant plasmid described in the fifth aspect.

[0018] Preferably, the host cell of the engineered strain is Escherichia coli.

[0019] In an eighth aspect, the present invention provides the use of the PETase-PHL7 mutant described in any one of the first to fourth aspects above in degrading PET, preparing a PET degradation agent, or recovering PET degradation products.

[0020] In a ninth aspect, the present invention provides the use of the above-mentioned recombinant plasmid in degrading PET, preparing a PET degradation agent, or recovering PET degradation products.

[0021] In a tenth aspect, the present invention provides the use of the above-mentioned PETase-PHL7 mutant engineered strain in degrading PET, preparing PET degradation agents or recovering PET degradation products.

[0022] The present invention utilizes single-point or combined mutations based on the PETase-PHL7 mutant H185Y to obtain PETase-PHL7 mutants with significantly enhanced PET degradation activity and / or thermal stability. Compared to mutant H185Y, the seven PETase-PHL7 mutants provided herein exhibit 55.6% to 132.5% increased PET degradation activity. Among them, mutant YPTIPL exhibited a 1.32-fold increase in PET degradation activity and a 5.05°C increase in Tm compared to mutant H185Y. The PETase-PHL7 mutants provided herein can be used in fields such as PET degradation, preparation of PET degradation agents, and recovery of PET degradation products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Schematic diagram of the plasmid map of the recombinant plasmid pET-28a(+)-PETase-PHL7 in Example 1 of the present invention; Figure 2 1 is a comparison chart of the determination results of PET degradation activity and Tm value of PETase-PHL7 and its mutants in Example 1 of the present invention; Figure 3 Schematic diagram of the plasmid map of the recombinant plasmid pET-28a(+)-Y / A188P / R111T in Example 2 of the present invention; Figure 4This is a comparison chart of the PET degradation activity and Tm determination results of different PETase-PHL7 mutants in Example 2 of the present invention; Figure 5 This is a comparison chart of the PET degradation activity and Tm determination results of different PETase-PHL7 mutants in Example 3 of the present invention; Figure 6 This is a comparison chart of the PET degradation activity and Tm determination results of different PETase-PHL7 mutants in Example 4 of the present invention; Figure 7 This is a comparison chart of the PET degradation activity and Tm determination results of different PETase-PHL7 mutants in Example 5 of the present invention; Figure 8 This is a liquid phase diagram for determining the PET degradation activity of the mutant YPTIPL in Example 5 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] The mutants of the present invention are named according to conventional naming conventions used by those skilled in the art. For example, the mutant Y / T214F / T42R indicates that the threonine (T) at position 214 is site-directedly mutated to phenylalanine (F), and the threonine (T) at position 42 is site-directedly mutated to arginine (R) in the amino acid sequence of the mutant H185Y, while the amino acid residues at other positions remain unchanged. The mutant YPTIP-D196S represents a mutant YPTIP in which aspartic acid (D) at position 196 is site-directedly mutated to serine (S), while the amino acid residues at other positions remain unchanged.

[0027] Example 1 This example provides a method for preparing, expressing, purifying, and detecting the activity of a PETase-PHL7 mutant. The specific contents are as follows: 1. Construction of PETase-PHL7 mutant recombinant plasmid 1. Construction of recombinant plasmid PETase-PHL7 The amino acid sequence of wild-type PETase-PHL7 is shown in SEQ ID No. 1. The coding gene thereof was obtained by codon optimization, and the coding gene sequence thereof is shown in SEQ ID No. 4.

[0028] The recombinant plasmid PETase-PHL7 was synthesized by entrusting Jinweizhi Company. The specific process is as follows: after amplifying the gene with the nucleotide sequence shown in SEQ ID No.4, it was digested with NcoⅠ and XhoⅠ restriction endonucleases and ligated to the pET-28a(+) vector to obtain the recombinant plasmid pET-28a(+)-PETase-PHL7. The schematic diagram of the plasmid map is shown in Figure 1 shown.

[0029] Using site-directed mutagenesis technology, the recombinant plasmid PETase-PHL7 constructed above was used as a template and the primers shown in Table 1 were used to perform PCR to obtain a linearized plasmid fragment, which was then ligated using ABclonal 2X MultiF Seamless Assembly Mix and subsequently introduced into the cell line by heat shock. E. coliTrans -T1 competent cells, and Sanger sequencing was performed to ensure the correctness of the mutant construction to obtain pET-28a(+)-PETase-PHL7-H185Y.

[0030] The PCR reaction conditions in this step were: pre-denaturation at 98°C for 3 min; followed by 30 cycles, each cycle including: denaturation at 98°C for 15 s, annealing at 68°C for 15 s, extension at 72°C for 3 min; and final extension at 72°C for 5 min.

[0031] Table 1

[0032] The amino acid sequence of PETase-PHL7 is shown in SEQ ID No. 1, specifically: MANPYERGPDPTESSIEAVRGPFAVAQTTVSRLQADGFGGGTIYYPTDTSQGTFGAVAISPGFTAGQESIAWLGPRIASQGFVVITIDTITRLDQPDSRGRQLQAALDHLRTNSVVRNRIDPNRMAVMGH SMGGGGALSAAANNTSLEAAIPLQGWHTRKNWSSVRTPTLVVGAQLDTIAPVSSHSEAFYNSLPSDLDKAYMELRGASHLVSNTPDTTTAKYSIAWLKRFVDDDLRYEQFLCPAPDDFAISEYRSTCPF; The coding gene sequence of PETase-PHL7 is shown in SEQ ID No. 4, specifically: ATGGCGAACCCGTATGAACGCGGCCCGGATCCGACCGAAAGCAGCATTGAAGCGGTGCGCGGCCCGTTTGCGGTGGCGCAAACCACGGTTAGCCGCCTGCAAGCGGATGGCTTTGGCGGTGGCACCATTTATTATCCGACCGATACGAGCCAAGGCACCTTTGGCGCGGTGGCGATTAGCCCGGGCTTTACCGCGGGCCAAGAAAGCATTGCGTGGCTGGGCCCGCGCATTGCGAGCCAAGGCTTTGTGGTGATTACCATTGATACCATTACCCGCCTGGATCAGCCGGATAGCCGCGGCCGTCAGCTGCAAGCGGCGCTGGATCATCTGCGCACCAACAGCGTGGTGCGCAACCGCATTGATCCAAACCGTATGGCCGTGATGGGTCATAGTATGGGCGGTGGCGGCGCCCTGAGTGCGGCCGCGAACAACACGAGCCTGGAAGCGGCGATTCCGCTGCAAGGCTGGCATACCCGCAAAAACTGGAGCAGCGTGCGCACCCCGACCCTGGTGGTGGGCGCGCAGCTGGATACCATTGCGCCGGTGAGCAGCCATAGCGAAGCGTTTTATAACAGCCTGCCGAGCGATCTGGATAAAGCGTATATGGAACTGCGCGGCGCGAGCCATCTGGTGAGCAACACCCCGGATACCACGACCGCGAAATATAGTATTGCGTGGTTAAAGCGTTTTGTGGATGACGATCTGCGCTATGAACAGTTTCTGTGCCCGGCGCCGGATGATTTTGCGATTAGCGAATATCGCAGCACCTGCCCGTTT。

[0033] Among them, the amino acid sequence of mutant H185Y is shown in SEQ ID No. 2, specifically: MANPYERGPDPTESSIEAVRGPFAVAQTTVSRLQADGFGGGTIYYPTDTSQGTFGAVAISPGFTAGQESIAWLGPRIASQGFVVITIDTITRLDQPDSRGRQLQAALDHLRTNSVVRNRIDPNRMAVMGH SMGGGGALSAAANNTSLEAAIPLQGWHTRKNWSSVRTPTLVVGAQLDTIAPVSSYSEAFYNSLPSDLDKAYMELRGASHLVSNTPDTTTAKYSIAWLKRFVDDDLRYEQFLCPAPDDFAISEYRSTCPF.

[0034] The coding gene sequence of the mutant H185Y is shown in SEQ ID No. 5, specifically: ATGGCGAACCCGTATGAACGCGGCCCGGATCCGACCGAAAGCAGCATTGAAGCGGTGCGCGGCCCGTTTGCGGTGGCGCAAACCACGGTTAGCCGCCTGCAAGCGGATGGCTTTGGCGGTGGCACCATTTATTATCCGACCGATACGAGCCAAGGCACCTTTGGCGCGGTGGCGATTAGCCCGGGCTTTACCGCGGGCCAAGAAAGCATTGCGTGGCTGGGCCCGCGCATTGCGAGCCAAGGCTTTGTGGTGATTACCATTGATACCATTACCCGCCTGGATCAGCCGGATAGCCGCGGCCGTCAGCTGCAAGCGGCGCTGGATCATCTGCGCACCAACAGCGTGGTGCGCAACCGCATTGATCCAAACCGTATGGCCGTGATGGGTCATAGTATGGGCGGTGGCGGCGCCCTGAGTGCGGCCGCGAACAACACGAGCCTGGAAGCGGCGATTCCGCTGCAAGGCTGGCATACCCGCAAAAACTGGAGCAGCGTGCGCACCCCGACCCTGGTGGTGGGCGCGCAGCTGGATACCATTGCGCCGGTGAGCAGCTATAGCGAAGCGTTTTATAACAGCCTGCCGAGCGATCTGGATAAAGCGTATATGGAACTGCGCGGCGCGAGCCATCTGGTGAGCAACACCCCGGATACCACGACCGCGAAATATAGTATTGCGTGGTTAAAGCGTTTTGTGGATGACGATCTGCGCTATGAACAGTTTCTGTGCCCGGCGCCGGATGATTTTGCGATTAGCGAATATCGCAGCACCTGCCCGTTT。

[0035] 2. Screening of single mutants based on the mutant H185Y and construction of the recombinant plasmid of mutant YPT The present invention uses mutant H185Y as a basis for further modification. Through rational design methods such as evolutionary analysis and free energy calculation, the mutants YV19M, YA26R, YA26S, YV30I, YT42R, YI43V, YI90L, YH109L, YR111T, YV115T, YS139A, YE148Q, YT158M, YN161D, YI179V, YE187I, YA188P, YN191H, YD198P, YT214F, and YA249S with improved performance were screened. The amino acid sequence of mutant H185Y is shown in SEQ ID No. 2.

[0036] Among them, the mutant YV19M is a site-directed mutation of valine at position 19 in the amino acid sequence shown in SEQ ID No. 2 to methionine, while the amino acid residues at other positions remain unchanged; The mutant YA26R is a mutant in which the alanine at position 26 in the amino acid sequence shown in SEQ ID No. 2 is site-directedly mutated to arginine, while the amino acid residues at other positions remain unchanged; The mutant YA26S is a mutant in which the alanine at position 26 in the amino acid sequence shown in SEQ ID No. 2 is site-directedly mutated to serine, while the amino acid residues at other positions remain unchanged; The mutant YV30I is a site-directed mutation of valine at position 30 in the amino acid sequence shown in SEQ ID No. 2 to isoleucine, while the amino acid residues at other positions remain unchanged; The mutant YT42R is a site-directed mutation in which the threonine at position 42 in the amino acid sequence shown in SEQ ID No. 2 is converted to arginine, while the amino acid residues at other positions remain unchanged; The mutant YI43V is a site-directed mutation of isoleucine at position 43 in the amino acid sequence shown in SEQ ID No. 2 to valine, while the amino acid residues at other positions remain unchanged; The mutant YI90L is a site-directed mutation of isoleucine at position 90 in the amino acid sequence shown in SEQ ID No. 2 to leucine, while the amino acid residues at other positions remain unchanged; Mutant YH109L is a mutant in which the histidine at position 109 in the amino acid sequence shown in SEQ ID No. 2 is site-directedly mutated to leucine, while the amino acid residues at other positions remain unchanged; The mutant YR111T is a site-directed mutation of arginine at position 111 in the amino acid sequence shown in SEQ ID No. 2 to threonine, while the amino acid residues at other positions remain unchanged; The mutant YV115T is a site-directed mutation of valine at position 115 in the amino acid sequence shown in SEQ ID No. 2 to threonine, while the amino acid residues at other positions remain unchanged; Mutant YS139A is a site-directed mutation of serine at position 139 in the amino acid sequence shown in SEQ ID No. 2 to alanine, while the amino acid residues at other positions remain unchanged; Mutant YE148Q is a site-directed mutation of glutamic acid at position 148 in the amino acid sequence shown in SEQ ID No. 2 to glutamine, while the amino acid residues at other positions remain unchanged; The mutant YT158M is a site-directed mutation in which the threonine at position 158 in the amino acid sequence shown in SEQ ID No. 2 is converted to methionine, while the amino acid residues at other positions remain unchanged; Mutant YN161D is a mutant in which the asparagine at position 161 in the amino acid sequence shown in SEQ ID No. 2 is site-directedly mutated to aspartic acid, while the amino acid residues at other positions remain unchanged; Mutant YI179V is a site-directed mutation of isoleucine at position 179 in the amino acid sequence shown in SEQ ID No. 2 to valine, while the amino acid residues at other positions remain unchanged; Mutant YE187I is a site-directed mutation of glutamic acid at position 187 in the amino acid sequence shown in SEQ ID No. 2 to isoleucine, while the amino acid residues at other positions remain unchanged; The mutant YA188P is a site-directed mutation in which the alanine at position 188 in the amino acid sequence shown in SEQ ID No. 2 is converted to proline, while the amino acid residues at other positions remain unchanged; Mutant YN191H is a site-directed mutation of asparagine at position 191 in the amino acid sequence shown in SEQ ID No. 2 to histidine, while the amino acid residues at other positions remain unchanged; The mutant YD198P is a site-directed mutation in which the aspartic acid at position 198 in the amino acid sequence shown in SEQ ID No. 2 is converted to proline, while the amino acid residues at other positions remain unchanged; The mutant YT214F is a site-directed mutation of the threonine at position 214 in the amino acid sequence shown in SEQ ID No. 2 to phenylalanine, while the amino acid residues at other positions remain unchanged; The mutant YA249S is a mutant in which the alanine at position 249 in the amino acid sequence shown in SEQ ID No. 2 is site-directedly mutated to serine, while the amino acid residues at other positions remain unchanged.

[0037] The statistics of site-directed mutagenesis primers used in the construction of the above mutant recombinant plasmids are shown in Table 2 below.

[0038] Table 2

[0039] Using site-directed mutagenesis technology, the pET-28a(+)-PETase-PHL7-H185Y constructed above was used as a template and the primers shown in Table 2 were used to perform PCR to obtain linearized plasmid fragments, which were then ligated using ABclonal 2X MultiFSeamless Assembly Mix and introduced into E. coliTrans -T1 competent cells, and Sanger sequencing was used to ensure the correctness of the mutant construction, and the recombinant plasmids YV19M, YA26R, YA26S, YV30I, YT42R, YI43V, YI90L, YH109L, YR111T, YV115T, YS139A, YE148Q, YT158M, YN161D, YI179V, YE187I, YA188P, YN191H, YD198P, YT214F and YA249S were obtained.

[0040] The PCR reaction conditions in this step are the same as those for the construction of the recombinant plasmid PETase-PHL7.

[0041] 2. Construction of PETase-PHL7 mutant engineered strain The above recombinant plasmids PETase-PHL7, pET-28a(+)-PETase-PHL7-H185Y and recombinant plasmids YV19M~YA249S and other mutant recombinant plasmids were respectively introduced into Escherichia coli BL21 (DE3) competent cells were used to select and verify positive monoclonal strains, and corresponding PETase-PHL7 engineered strains and 22 PETase-PHL7 mutant engineered strains including mutants H185Y, YV19M, YA26R, YA26S, YV30I, YT42R, YI43V, YI90L, YH109L, YR111T, YV115T, YS139A, YE148Q, YT158M, YN161D, YI179V, YE187I, YA188P, YN191H, YD198P, YT214F and YA249S were obtained.

[0042] 3. Preparation and purification of PETase-PHL7 mutants The engineered strains constructed above were inoculated into 5 mL of LB medium and cultured at 37°C and 220 rpm for 12 h. Then, they were inoculated into 200 mL of ZYM medium shake flasks at a 1% inoculum for fermentation. Expression was induced at 21°C and 160 rpm for approximately 20 h to obtain a fermentation broth rich in the corresponding PET degrading enzyme.

[0043] The fermentation broths were centrifuged at 4000 rpm for 10 minutes to collect the cells. The cells were then resuspended in 20 mL of lysis buffer (50 mM Na₂HPO₄·12H₂O, 200 mM NaCl, and 10 mM imidazole per liter, pH 7.4). The cells were then disrupted using a high-pressure disruptor. After disruption, the cells were centrifuged at 10,000 rpm for 1 hour to remove cell debris. The supernatant contained the complete protein solution containing PETase-PHL7 and its mutants. This complete protein solution was filtered through a 0.45 μm filter to remove impurities, and then purified using a Ni-NTA column by gradient elution to obtain the target protein. The specific steps are as follows: first, the lysis buffer is balanced for 2 minutes, then the whole protein solution after passing through the membrane is repeatedly loaded on the column 3 times, and washed 3 times with washing buffer (each liter of washing buffer contains 50mM Na2HPO4·12H2O, 200mM NaCl and 50mM imidazole, pH=7.4) to remove impurities; then, the protein eluate is eluted with elution buffer (each 1L of elution buffer contains 50mM Na2HPO4·12H2O, 200mM NaCl and 250mM imidazole, pH=7.4); the protein is further concentrated, and the imidazole in the protein eluate is diluted to 1‰ of the original concentration by changing the washing buffer 3 times (each 1L of the washing buffer contains 50mM Na2HPO4·12H2O and 200mM NaCl, pH=7.4), thereby obtaining concentrated PETase-PHL7 and its mutant enzyme solution.

[0044] 4. Performance Characterization Methods 1. Determination of PET degradation activity The present invention uses amorphous PET film (purchased from Goodfellow) as the PET substrate; The concentrated PETase-PHL7 and its mutant enzyme solutions were added to 1.8 mL of a reaction solution (1 M potassium phosphate buffer, pH 8) at the corresponding concentration (0.6 mg enzyme / g PET). Approximately 45 mg of amorphous PET rectangular flakes (3 cm × 0.5 cm) were added to the reaction solution and reacted in a constant-temperature shaker at 700 rpm and 70°C for 8 hours. After the reaction, the TPA, MHET, and BHET produced were analyzed by high-performance liquid chromatography (HPLC). The total content of TPA, BHET, and MHET was used as an indicator of PET degradation activity.

[0045] 2. Tm determination method Protein melting temperatures were determined using differential scanning fluorimetry (DSF). Protein samples were loaded onto a 96-well plate, with each well containing 25 μL of the enzyme stock solution (15 μL of enzyme stock solution containing 50 mM Na₂HPO₄·12H₂O and 200 mM NaCl), 9 μL of protein solution (0.4 mg / mL), and 1 μL of SYPRO Orange dilution solution. DSF experiments were performed using a real-time fluorescence quantitative PCR system with a 465 nm excitation and 580 nm emission filter. Samples were heated from 25°C to 100°C at a rate of 0.3°C / s, and fluorescence was measured every 0.03 s. T m The Tm determination was repeated three times. Due to the small error, the error bars are no longer included in the histogram of the Tm determination results.

[0046] 5. Experimental Results Determination of PET degradation activity and T m The comparison chart of the measurement results is as follows: Figure 2 shown. Figure 2 Star-type representative T m value.

[0047] Depend on Figure 2 It can be seen that compared with mutant H185Y, mutants YV19M, YA26R, YA26S, YV30I, YT42R, YI43V, YI90L, YH109L, YR111T, YV115T, YS139A, YE148Q, YT158M, YN161D, YI179V, YE187I, YA188P, YN191H, YD198P, YT214F and YA249S have different degrees of improvement in the degradation activity of PET substrate at 70°C, with the degradation activity increased by 4.5% (mutant YN191H) to 57.9% (YT214F). Among them, mutants YA26R, YT42R, YR111T, YE148Q, YE187I, YA188P, YD198P, and YT214F showed outstanding PET degradation activity. In the future, we will further combine the above mutation sites in pairs to determine the best combination of mutants. In terms of thermal stability, mutants YE187I and YD198P have T m The values ​​were significantly improved compared with mutant H185Y, increasing by 3.78℃ and 2.82℃ respectively.

[0048] Example 2 On the basis of Example 1, in this example, the mutants YA26R, YT42R, YR111T, YE148Q, YE187I, YA188P, YD198P, and YT214F with significantly improved PET degradation activity were combined at two points to obtain 17 mutants, which were recorded as mutants Y / T214F / T42R, Y / T214F / E187I, Y / T214F / A188P, Y / T42R / E187I, Y / T42R / A 188P, Y / T214F / R111T, Y / A26R / T42R, Y / A26R / E187I, Y / A26R / A188P, Y / A26R / R111T, Y / A188P / R111T, Y / T214F / E148Q, Y / T214F / D198P, Y / R111T / E148Q, Y / R111T / D198P, Y / A188P / E148Q and Y / A188P / D198P.

[0049] In this example, the double-point combination mutants were constructed using known single-point mutation primers via sequential site-directed mutagenesis, eliminating the need for separate primer design. The specific method involves using a constructed single-point mutant plasmid as a template and performing site-directed PCR amplification with primers targeting a distant site to obtain the desired two-point combination mutant plasmid. For example, the mutant Y / T214F / T42R was obtained by PCR amplification using a recombinant plasmid of the single-point mutant YT214F as a template and primers targeting the T42R site. The remaining combination mutants were constructed using similar methods. Because the mutation sites involved are relatively distant from each other, the corresponding primers can efficiently and specifically introduce the mutations separately. All primer sequences are listed in Example 1 and are not repeated here.

[0050] By using site-directed mutagenesis technology, the pET-28a(+)-PETase-PHL7-H185Y constructed in Example 1 was used as a template and PCR was performed with the primers shown in Table 2 to prepare recombinant plasmids pET-28a(+)-Y / T214F / T42R, pET-28a(+)-Y / T214F / E187I, pET-28a(+)-Y / T214F / A188P, pET-28a(+)-Y / T42R / E187I, pET-28a(+)-Y / T42R / A188P, pET-28a(+)-Y / T214F / R111T, pET-28a(+)-Y / A26R / T42R, pET-28a(+)-Y / A26R / E187I, Seventeen PHL7-H185Y mutant recombinant plasmids, including pET-28a(+)-Y / A26R / A188P, pET-28a(+)-Y / A26R / R111T, pET-28a(+)-Y / A188P / R111T, pET-28a(+)-Y / T214F / E148Q, pET-28a(+)-Y / T214F / D198P, pET-28a(+)-Y / R111T / E148Q, pET-28a(+)-Y / R111T / D198P, pET-28a(+)-Y / A188P / E148Q and pET-28a(+)-Y / A188P / D198P, wherein the method for constructing the recombinant plasmid is the same as that in Example 1. The plasmid map of the recombinant plasmid pET-28a(+)-Y / A188P / R111T (also referred to as the recombinant plasmid pET-28a(+)-YPT) is shown in the figure below. Figure 3 shown.

[0051] Based on the 17 recombinant plasmids constructed above, the corresponding mutant engineering strains were further constructed according to the method described in Example 1, and the corresponding mutants Y / T214F / T42R, Y / T214F / E187I, Y / T214F / A188P, Y / T42R / E187I, Y / T42R / A188P, Y / T214F / R111T, Y / A26R / T42R, Y / A26R / E187I, Y / A26R / A188P, Y / A26R / R111T, Y / A188P / R111T, Y / T214F / E148Q, Y / T214F / D198P, Y / R111T / E148Q, Y / R111T / D198P, Y / A188P / E148Q and Y / A188P / D198P, and the PET degradation activity and Tm of different PETase-PHL7 mutants were determined. The comparison of the determination results is shown in the figure. Figure 4 shown.

[0052] Depend on Figure 4 It can be seen that compared with mutant H185Y, the PET degradation activity of mutants Y / T214F / R111T, Y / A26R / A188P, Y / A188P / R111T, and Y / A188P / D198P increased significantly, ranging from 41.9% to 51.9%. In terms of thermal stability, mutants Y / T214F / T42R, Y / T214F / E187I, Y / T214F / A188P, Y / T42R / A188P, Y / A26R / E187I, and Y / T214F / E148Q increased significantly. T m The values ​​were all 2.5°C to 4.4°C higher than those of mutant H185Y. However, these six mutants performed poorly in terms of PET degradation activity and were therefore not considered as a basis for subsequent modification. Among them, the amount of PET degradation product released by mutant Y / A188P / R111T (denoted as mutant YPT) increased from 47.4 mM in mutant H185Y to 73.8 mM, with a 55.6% increase in PET degradation activity and a 0.35°C increase in Tm.

[0053] The amino acid sequence of the mutant YPT is shown in SEQ ID No. 3, specifically: MANPYERGPDPTESSIEAVRGPFAVAQTTVSRLQADGFGGGTIYYPTDTSQGTFGAVAISPGFTAGQESIAWLGPRIASQGFVVITIDTITRLDQPDSRGRQLQAALDHLTTNSVVRNRIDPNRMAVMGH SMGGGGALSAAANNTSLEAAIPLQGWHTRKNWSSVRTPTLVVGAQLDTIAPVSSYSEPFYNSLPSDLDKAYMELRGASHLVSNTPDTTTAKYSIAWLKRFVDDDLRYEQFLCPAPDDFAISEYRSTCPF.

[0054] The coding gene sequence of the mutant YPT is shown in SEQ ID No. 6, specifically: ATGGCGAACCCGTATGAACGCGGCCCGGATCCGACCGAAAGCAGCATTGAAGCGGTGCGCGGCCCGTTTGCGGTGGCGCAAACCACGGTTAGCCGCCTGCAAGCGGATGGCTTTGGCGGTGGCACCATTTATTATCCGACCGATACGAGCCAAGGCACCTTTGGCGCGGTGGCGATTAGCCCGGGCTTTACCGCGGGCCAAGAAAGCATTGCGTGGCTGGGCCCGCGCATTGCGAGCCAAGGCTTTGTGGTGATTACCATTGATACCATTACCCGCCTGGATCAGCCGGATAGCCGCGGCCGTCAGCTGCAAGCGGCGCTGGATCATCTGACCACCAACAGCGTGGTGCGCAACCGCATTGATCCAAACCGTATGGCCGTGATGGGTCATAGTATGGGCGGTGGCGGCGCCCTGAGTGCGGCCGCGAACAACACGAGCCTGGAAGCGGCGATTCCGCTGCAAGGCTGGCATACCCGCAAAAACTGGAGCAGCGTGCGCACCCCGACCCTGGTGGTGGGCGCGCAGCTGGATACCATTGCGCCGGTGAGCAGCTATAGCGAACCGTTTTATAACAGCCTGCCGAGCGATCTGGATAAAGCGTATATGGAACTGCGCGGCGCGAGCCATCTGGTGAGCAACACCCCGGATACCACGACCGCGAAATATAGTATTGCGTGGTTAAAGCGTTTTGTGGATGACGATCTGCGCTATGAACAGTTTCTGTGCCCGGCGCCGGATGATTTTGCGATTAGCGAATATCGCAGCACCTGCCCGTTT。

[0055] Example 3 On the basis of Example 2, this example further superimposed the mutant YPT with the 19th, 26th, 30th, 43rd, 90th, 115th, 139th, 148th, 158th, 161st, 179th, 187th, 191st, 198th or 249th mutation sites to obtain mutants YPT-V19M, YPT-V21M, YPT-V31M, YPT-V40M, YPT-V51M, YPT-V60M, YPT-V71M, YPT-V81M, YPT-V91M, YPT-V19M, YPT-V19M, YPT-V19M, YPT-V19M, YPT-V21M, YPT-V19M, YPT-V19M, YPT-V21M, YPT-V31M, YPT-V40M, YPT-V51M, YPT-V61M, YPT-V71M, YPT-V81M, YPT-V91M, YPT-V19 ... -A26R, YPT-A26S, YPT-V30I, YPT-I43V, YPT-I90L, YPT-V115T, YPT-S139A, YPT-E148Q , YPT-T158M, YPT-N161D, YPT-I179V, YPT-E187I, YPT-N191H, YPT-D198P and YPT-A249S.

[0056] The site-directed mutagenesis primers used to construct the corresponding recombinant plasmids are shown in Table 2. Some primers need to be redesigned, as shown in Table 3.

[0057] Table 3

[0058] Using site-directed mutagenesis technology, the recombinant plasmid pET-28a(+)-YPT constructed in Example 2 was used as a template and the corresponding primers were used to perform PCR to prepare the corresponding recombinant plasmids. The method for preparing the recombinant plasmids was the same as that in Example 1. Furthermore, after constructing the corresponding engineered strains according to the method described in Example 1, the corresponding mutants were prepared, and the PET degradation activity and Tm of different PETase-PHL7 mutants were measured. The comparison of the measurement results is shown in the figure below. Figure 5 shown.

[0059] Depend on Figure 5 Compared to mutant YPT, mutants YPTS139A (denoted as mutant YPTA), YPTE187I (denoted as mutant YPTI), and YPTN191H (denoted as mutant YPTH) showed significantly enhanced PET degradation activity, while their Tm values ​​remained unchanged or increased to a certain extent. Compared to mutant YPT, mutant YPTA showed a 19.5% increase in PET degradation activity and a 1.2°C increase in Tm value. Mutant YPTI showed a 21.9% increase in PET degradation activity and a 0.3°C decrease in Tm value. Mutant YPTH showed a 20.5% increase in PET degradation activity and a 4.7°C increase in Tm value. Compared to mutant YPT, YPTD198P showed a 21.8% increase in PET degradation activity, but its Tm value decreased by 4.7°C. Mutant YPTI was selected as the basis for the next round of mutagenesis in the present invention.

[0060] Example 4 Based on Example 3, in this example, the A26R, V30I, I43V, I90L, S139A, D198P and D233N mutations were superimposed on the mutant YPTI to obtain mutants YPTI-A26R, YPTI-V30I, YPTI-I43V, YPTI-I90L, YPTI-S139A, YPTI-D198P, and YPTI-D233N, respectively, and the corresponding recombinant plasmids were constructed.

[0061] When constructing recombinant plasmids, the mutants require redesigned primers, as shown in Table 4.

[0062] Table 4

[0063] The site-directed mutagenesis technique was used, and the recombinant plasmid pET-28a(+)-YPTI constructed in Example 3 was used as a template. PCR was performed using the primers shown in Tables 2 and 4, respectively, to prepare the corresponding recombinant plasmids. The method for constructing the recombinant plasmid was the same as that in Example 1. After further constructing seven engineered strains, YPTI-A26R, YPTI-V30I, YPTI-I43V, YPTI-I90L, YPTI-S139A, YPTI-D198P, and YPTI-D233N, using the method described in Example 1, the corresponding mutants were fermented and purified to prepare the corresponding mutants. The PET degradation activity and Tm of different PETase-PHL7 mutants were measured, and the comparison of the measurement results is shown in the figure below. Figure 6 shown.

[0064] Depend on Figure 6 It can be seen that after further mutation of mutant YPTI, the PET degradation activity of mutants YPTI-A26R and YPTI-D198P increased by 7.0%~11.9%. In terms of thermal stability, mutants YPTI-V30I, YPTI-I90L and YPTI-D198P T m The values ​​were significantly higher than those of mutant YPTI, increasing by 6.9°C, 7.5°C, and 4.8°C, respectively. However, the PET degradation activity of mutants YPTI-V30I and YPTI-I90L decreased. Considering both thermal stability and degradation activity, mutant YPTI-D198P (also known as YPTIP) was selected as the basis for the next round of mutagenesis.

[0065] Example 5 Based on Example 4, this example superimposed the A26R, H109L, N191H, D196S, and A249S mutations on the mutant YPTIP to obtain mutants YPTIP-A26R, YPTIP-H109L, YPTIP-N191H, YPTIP-D196S, and YPTIP-A249S, respectively, and constructed the corresponding recombinant plasmids.

[0066] The site-directed mutagenesis primers used in constructing the recombinant plasmid are shown in Table 5 below.

[0067] Table 5

[0068] Using site-directed mutagenesis technology, the recombinant plasmid pET-28a(+)-YPTIP constructed in Example 4 was used as a template, and PCR was performed with corresponding primers to prepare the corresponding recombinant plasmids. The method for constructing the recombinant plasmid was the same as that in Example 1. The corresponding engineered strains of mutants YPTIP-A26R, YPTIP-H109L, YPTIP-N191H, YPTIP-D196S and YPTIP-A249S were further constructed using the method described in Example 1. The corresponding mutants were fermented and purified to prepare the corresponding mutants, and the PET degradation activity and Tm of different PETase-PHL7 mutants were measured. The comparison of the measurement results is shown in the figure below. Figure 7 shown.

[0069] Depend on Figure 7 It can be seen that after further mutation of mutant YPTIP, the PET degradation activity of mutant YPTIP-H109L (denoted as mutant YPTIPL) and YPTIP-N191H (denoted as mutant YPTIPH) increased by 4.3%-9.6%. T m Compared with mutant YPTIP, mutant YPTIPL showed a 9.6% increase in PET degradation activity and a 0.2°C increase in Tm value (the Tm value of mutant YPTIPL increased to 85.2°C), while mutant YPTIPH showed a 4.3% increase in PET degradation activity and a 1.1°C increase in Tm value (the Tm value of mutant YPTIPH increased to 86.2°C). Compared with mutant H185Y, mutant YPTIPL showed a 1.32-fold increase in PET degradation activity and a 5.0°C increase in Tm value, while mutant YPTIPH showed a 1.21-fold increase in PET degradation activity and a 6.0°C increase in Tm value.

[0070] Through comprehensive analysis of PET degradation activity and thermal stability, mutant YPTIPL is the optimal mutant in the present invention. The liquid phase diagram of the PET degradation activity of mutant YPTIPL is shown in FIG. Figure 8 shown. Figure 8 Peak 1 is TPA, peak 2 is MHET, and peak 3 is BHET. Figure 8 It can be seen that the response values ​​of TPA and MHET peaks are significantly higher than those of BHET peak, indicating that the mutant can efficiently catalyze the complete hydrolysis of PET substrate to generate the target product TPA, and the accumulation of intermediate products is low.

[0071] In summary, based on the mutant H185Y, the present invention site-directedly mutated the alanine at position 188 in its amino acid sequence to proline, and the arginine at position 111 to threonine, while the amino acid residues at other positions remained unchanged, to obtain the mutant YPT. Based on the mutant YPT, single-point mutations, two-point mutations, and combined mutations were further performed. In each round of iteration, the preferred mutant was used as the basis for the next round of mutations to obtain a series of PETase-PHL7 mutants. After multiple rounds of iterations, the degradation activity of the mutant was gradually improved. Compared with the mutant YPT, the PET degradation activity of the PETase-PHL7 mutant provided by the present invention was significantly improved, specifically by 55.6% to 132.5%; at the same time, the thermal stability of the mutant was also improved to a certain extent, with Tm increased by up to 6.0°C.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A PETase-PHL7 mutant, characterized in that: The alanine at position 188 of the mutant H185Y was site-directedly mutated to proline, and the arginine at position 111 was site-directedly mutated to threonine, while the amino acid residues at other positions remained unchanged. The resulting amino acid sequence is shown in SEQ ID No.

3.

2. A PETase-PHL7 mutant, characterized in that: The amino acid sequence is obtained by replacing the amino acid residue at one position in the amino acid sequence shown in SEQ ID No. 3, and the amino acid sequence is any one of the following (1) to (3): (1) The serine at position 139 of the amino acid sequence shown in SEQ ID No. 3 was site-directedly mutated to alanine, while the amino acid residues at other positions remained unchanged, and this was recorded as the mutant YPTA; (2) The glutamic acid at position 187 of the amino acid sequence shown in SEQ ID No. 3 was site-directedly mutated to isoleucine, while the amino acid residues at other positions remained unchanged, and this was recorded as mutant YPTI; (3) The asparagine at position 191 of the amino acid sequence shown in SEQ ID No. 3 was site-directedly mutated to histidine, while the amino acid residues at other positions remained unchanged, and the mutant was recorded as YPTH.

3. A PETase-PHL7 mutant, characterized in that: Its amino acid sequence is obtained by site-directed mutation of aspartic acid at position 198 in the amino acid sequence of mutant YPTI as claimed in claim 2 to proline, while the amino acid residues at other positions remain unchanged, and is recorded as mutant YPTIP.

4. A PETase-PHL7 mutant, characterized in that: The amino acid sequence is obtained by replacing the amino acid residue at one position in the amino acid sequence of the mutant YPTIP as claimed in claim 3, and the amino acid sequence is any one of (i) or (ii): (i) site-directed mutation of the histidine at position 109 in the amino acid sequence of the mutant YPTIP to leucine, while leaving the amino acid residues at other positions unchanged; the resulting mutant is designated as mutant YPTIPL; (ii) The asparagine at position 191 in the amino acid sequence of the mutant YPTIP was site-directedly mutated to histidine, while the amino acid residues at other positions remained unchanged. The resulting mutant was designated as mutant YPTIPL.

5. A DNA molecule encoding the PETase-PHL7 mutant according to any one of claims 1 to 4.

6. A recombinant plasmid, characterized in that: The recombinant plasmid can express the PETase-PHL7 mutant according to any one of claims 1 to 4.

7. A PETase-PHL7 mutant engineered strain, characterized by: It comprises the recombinant plasmid according to claim 6.

8. Use of the PETase-PHL7 mutant according to any one of claims 1 to 4 in degrading PET, preparing a PET degradation agent, or recovering PET degradation products.

9. Use of the recombinant plasmid according to claim 6 in degrading PET, preparing a PET degradation agent, or recovering PET degradation products.

10. Use of the PETase-PHL7 mutant engineered strain according to claim 7 in degrading PET, preparing PET degradation agents or recovering PET degradation products.

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