Petase-phl7 mutants and uses thereof

Abstract

The application belongs to the technical field of enzyme engineering, and discloses a PETase-PHL7 mutant and application thereof. The application provides a PETase-PHL7 mutant, the amino acid sequence of which is shown as SEQ ID No. 3, and is recorded as mutant YPT. Compared with mutant H185Y, the PET degradation activity of the mutant YPT is increased by 55.6%, and Tm is increased by 0.35 DEG C. Based on the mutant YPT, the amino acid is further mutated and designed, and six kinds of PETase-PHL7 mutants are provided. Compared with the mutant YPT, the PET degradation activity of the PETase-PHL7 mutant provided by the application is increased by 55.6% to 132.5%, and at the same time, the thermal stability of the mutant is improved to different degrees. The application can be applied to the fields of preparation of PETase, degradation of PET, preparation of PET degrading agent or preparation of PET degradation product and the like.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a PET-degrading enzyme, specifically to a PETase-PHL7 mutant and its applications. Background Technology

[0002] Polyethylene terephthalate (PET) is a thermoplastic polyester made from terephthalic acid and ethylene glycol through a polycondensation reaction. It is also one of the world's most produced synthetic resins. Due to its advantages such as good transparency, impact resistance, chemical resistance, ease of processing, and moderate cost, PET is now widely used in many fields such as packaging materials, insulation materials, electronics, and medical devices.

[0003] However, with the widespread use of PET, a large amount of PET waste has also been generated. Due to the stable ester bond structure in the PET molecular backbone, it is difficult to degrade effectively in the natural environment, taking hundreds of years to degrade in soil or oceans, easily accumulating and forming "white pollution." Furthermore, if disposed of carelessly, it breaks down into microplastics, seeping into ecosystems and threatening the survival of plants and animals and human health. Therefore, developing efficient and sustainable PET waste solutions is urgently needed.

[0004] Enzymatic degradation of PET, utilizing specific microbial enzymes to catalyze the breaking of PET molecular chains, offers advantages such as mild reaction conditions, low environmental impact, high purity of degradation products, and broad compatibility with PET morphology and physical state, making it a promising mainstream technology for PET recycling. Existing PET hydrolases are diverse, exhibiting 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 still falls short of the "high efficiency and stability" requirements for enzyme preparations in large-scale industrial applications in terms of core performance indicators such as degradation activity and thermal stability, indicating significant room for improvement. Optimizing these key properties is crucial to better adapting to the actual needs of large-scale industrial production and laying the foundation for its industrialization. Summary of the Invention

[0005] In view of the above-mentioned problems in the prior art, the present invention modifies 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 objectives, the embodiments of the present invention employ the following technical solutions:

[0007] In a first aspect, the present invention provides a PETase-PHL7 mutant, which is obtained by site-directed mutation of alanine at position 188 of mutant H185Y to proline, and site-directed mutation of arginine at position 111 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 denoted as mutant YPT.

[0008] This invention provides a PETase-PHL7 mutant, namely mutant YPT, which is obtained by site-directed mutation of alanine at position 188 to proline and arginine at position 111 to threonine, based on mutant H185Y. Compared with mutant H185Y, mutant YPT has a 55.6% increase in PET degradation activity and a 0.35℃ increase in Tm.

[0009] 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.

[0010] Secondly, based on the mutant YPT, the present invention further provides three PETase-PHL7 mutants by single-point mutation, the amino acid sequences of which are any one of (1) to (3) below:

[0011] (1) The serine at position 139 of the amino acid sequence shown in SEQ ID No.3 is mutated to alanine, while the amino acid residues at other positions remain unchanged. The resulting mutant is denoted as mutant YPTA.

[0012] (2) The glutamic acid at position 187 of the amino acid sequence shown in SEQ ID No.3 is mutated to isoleucine, while the amino acid residues at other positions remain unchanged. The resulting mutant is denoted as mutant YPTI.

[0013] (3) The asparagine at position 191 of the amino acid sequence shown in SEQ ID No.3 is mutated to histidine, while the amino acid residues at other positions remain unchanged. The resulting mutant is denoted as mutant YPTH.

[0014] Tests revealed that, compared to the YPT mutant, the PET degradation activity of the three mutants was increased by 19.5%-21.9%. Compared to the H185Y mutant, the PET degradation activity of the three mutants was increased by 86%-90%. Compared to the H185Y mutant, the three mutants showed varying degrees of improvement in thermal stability. Among them, the Tm value of the YPTI mutant was slightly increased, the Tm of the YPTA mutant increased by 1.57℃, and the Tm value of the YPTH mutant increased the most, by 5.05℃.

[0015] Thirdly, the present invention performs a single-point mutation on the mutant YPTI, mutating the aspartic acid at position 198 in the amino acid sequence of the mutant YPTI to proline, while keeping the amino acid residues at other positions unchanged. The resulting mutant is denoted as mutant YPTIP.

[0016] Tests revealed that, compared to the mutant YPTI, the mutant YPTIP exhibited an 11.9% increase in PET degradation activity and a 4.86℃ increase in Tm. Compared to the mutant H185Y, the mutant YPTIP showed a 1.12-fold increase in PET degradation activity and a 4.88℃ increase in Tm.

[0017] Fourthly, the present invention further performs a single-point mutation on the mutant YPTIP, that is, replaces an amino acid residue at one position in the amino acid sequence of the mutant YPTIP, and its amino acid sequence is either (i) or (ii):

[0018] (i) The histidine at position 109 of the amino acid sequence of the mutant YPTIP is mutated to leucine, while the amino acid residues at other positions remain unchanged. The resulting amino acid sequence is shown in SEQ ID No. 8. The resulting mutant is denoted as mutant YPTIPL.

[0019] (ii) The asparagine at position 191 of the amino acid sequence of the mutant YPTIP is mutated to histidine, while the amino acid residues at other positions remain unchanged. The resulting amino acid sequence is shown in SEQ ID No. 9. The resulting mutant is denoted as mutant YPTIPH.

[0020] Tests revealed that, compared to the mutant YPTIP, the mutant YPTIPL exhibited a 9.6% increase in PET degradation activity and a 0.17℃ increase in Tm; the mutant YPTIPH showed a 4.3% increase in PET degradation activity and a 1.1℃ increase in Tm. Compared to the mutant H185Y, the mutant YPTIPL showed a 1.32-fold increase in PET degradation activity and a 5.05℃ increase in Tm; the mutant YPTIPH showed a 1.21-fold increase in PET degradation activity and a 6.0℃ increase in Tm.

[0021] Fifthly, the present invention provides a DNA molecule encoding the PETase-PHL7 mutant as described in any one of the first to fourth aspects.

[0022] In a sixth aspect, the present invention provides a recombinant plasmid that can express the PETase-PHL7 mutant described in any one of the first to fourth aspects.

[0023] In a seventh aspect, the present invention provides a PETase-PHL7 mutant engineered strain comprising the recombinant plasmid described in the fifth aspect.

[0024] Preferably, the host cell of the engineered strain is Escherichia coli.

[0025] Eighthly, the present invention provides the use of the PETase-PHL7 mutant described in any one of the first to fourth aspects in the degradation of PET, the preparation of PET degradation agents, or the recycling of PET degradation products.

[0026] In a ninth aspect, the present invention provides the application of the above-mentioned recombinant plasmid in the degradation of PET, the preparation of PET degradation agents, or the recycling of PET degradation products.

[0027] In a tenth aspect, the present invention provides the application of the above-mentioned PETase-PHL7 mutant engineered strain in the degradation of PET, the preparation of PET degradation agents, or the recycling of PET degradation products.

[0028] This invention involves single-point or combined mutations of the PETase-PHL7 mutant H185Y to obtain a PETase-PHL7 mutant with significantly improved PET degradation activity and / or thermal stability. Compared to mutant H185Y, the PET degradation activity of the seven PETase-PHL7 mutants provided by this invention is increased by 55.6% to 132.5%. Among them, the PET degradation activity of mutant YPTIPL is increased by 1.32 times and the thermal stability (Tm) is increased by 5.05℃ compared to mutant H185Y. The PETase-PHL7 mutants provided by this invention can be applied to the fields of PET degradation, preparation of PET degradation agents, or recycling of PET degradation products. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1This is a schematic diagram of the plasmid pET-28a(+)-PETase-PHL7 in Example 1 of the present invention;

[0031] Figure 2 This is a comparison chart of the PET degradation activity and Tm value of PETase-PHL7 and its mutants in Example 1 of the present invention;

[0032] Figure 3 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-28a(+)-Y / A188P / R111T in Example 2 of the present invention;

[0033] Figure 4 This is a comparison chart of the PET degradation activity and Tm measurement results of different PETase-PHL7 mutants in Example 2 of the present invention;

[0034] Figure 5 This is a comparison chart of the PET degradation activity and Tm measurement results of different PETase-PHL7 mutants in Example 3 of the present invention;

[0035] Figure 6 This is a comparison chart of the PET degradation activity and Tm measurement results of different PETase-PHL7 mutants in Example 4 of the present invention;

[0036] Figure 7 This is a comparison chart of the PET degradation activity and Tm measurement results of different PETase-PHL7 mutants in Example 5 of the present invention;

[0037] Figure 8 This is a liquid phase diagram showing the determination of the PET degradation activity of the mutant YPTIPL in Example 5 of the present invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] The mutants described in this invention are named in accordance with the conventional naming methods of those skilled in the art. For example, mutant Y / T214F / T42R means that the amino acid sequence of mutant H185Y is modified by site-directed mutation of threonine (T) at position 214 to phenylalanine (F) and threonine (T) at position 42 to arginine (R), while the amino acid residues at other positions remain unchanged.

[0040] The mutant YPTIP-D196S represents a site-directed mutation of the aspartic acid (D) at position 196 to serine (S) in the amino acid sequence of the mutant YPTIP, while the amino acid residues at other positions remain unchanged.

[0041] Example 1

[0042] This embodiment provides a method for preparing, expressing, purifying, and detecting the activity of the PETase-PHL7 mutant, as detailed below:

[0043] I. Construction of the PETase-PHL7 mutant recombinant plasmid

[0044] 1. Construction of recombinant plasmid PETase-PHL7

[0045] The amino acid sequence of wild-type PETase-PHL7 is shown in SEQ ID No. 1. Its coding gene was obtained through codon optimization, and its coding gene sequence is shown in SEQ ID No. 4.

[0046] The recombinant plasmid PETase-PHL7 was synthesized by Genewiz. 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 into the pET-28a(+) vector to obtain the recombinant plasmid pET-28a(+)-PETase-PHL7. A schematic diagram of its plasmid map is shown below. Figure 1 As shown.

[0047] Using site-directed mutagenesis, the recombinant plasmid PETase-PHL7 constructed above was used as a template, and PCR was performed with the primers shown in Table 1 to obtain a linearized plasmid fragment. This fragment was then ligated using ABclonal 2X MultiF Seamless AssemblyMix and subsequently introduced via heat shock. E. coliTrans -T1 competent cells were used, and the correctness of mutant construction was ensured by Sanger sequencing to obtain pET-28a(+)-PETase-PHL7-H185Y.

[0048] The PCR reaction conditions in this step are as follows: pre-denaturation at 98℃ for 3 min; followed by 30 cycles, each cycle consisting of: denaturation at 98℃ for 15 s, annealing at 68℃ for 15 s, extension at 72℃ for 3 min; and final extension at 72℃ for 5 min.

[0049] Table 1

[0050]

[0051] The amino acid sequence of PETase-PHL7 is shown in SEQ ID No. 1, specifically as follows:

[0052] MANPYERGPDPTESSIEAVRGPFAVAQTTVSRLQADGFGGGTIYYPTDTSQGTFGAVAISPGFTAGQESIAWLGPRIASQGFVVITIDTITRLDQPDSRGRQLQAALDHLRTNSVVRNRIDPNRMAVMGHSMGGGGALSAAANNTSLEAAIPLQGWHTRKNWSSVRTPTLVVGAQLDTIAPVSSHSEAFYNSLPSDLDKAYMELRGASHLVSNTPDTTTAKYSIAWLKRFVDDDLRYEQFLCPAPDDFAISEYRSTCPF;

[0053] The coding gene sequence of PETase-PHL7 is shown in SEQ ID No.4, specifically:

[0054] ATGGCGAACCCGTATGAACGCGGCCCGGATCCGACCGAAAGCAGCATTGAAGCGGTGCGCGGCCCGTTTGCGGTGGCGCAAACCACGGTTAGCCGCCTGCAAGCGGATGGCTTTGGCGGTGGCACCATTTATTATCCGACCGATACGAGCCAAGGCACCTTTGGCGCGGTGGCGATTAGCCCGGGCTTTACCGCGGGCCAAGAAAGCATTGCGTGGCTGGGCCCGCGCATTGCGAGCCAAGGCTTTGTGGTGATTACCATTGATACCATTACCCGCCTGGATCAGCCGGATAGCCGCGGCCGTCAGCTGCAAGCGGCGCTGGATCATCTGCGCACCAACAGCGTGGTGCGCAACCGCATTGATCCAAACCGTATGGCCGTGATGGGTCATAGTATGGGCGGTGGCGGCGCCCTGAGTGCGGCCGCGAACAACACGAGCCTGGAAGCGGCGATTCCGCTGCAAGGCTGGCATACCCGCAAAAACTGGAGCAGCGTGCGCACCCCGACCCTGGTGGTGGGCGCGCAGCTGGATACCATTGCGCCGGTGAGCAGCCATAGCGAAGCGTTTTATAACAGCCTGCCGAGCGATCTGGATAAAGCGTATATGGAACTGCGCGGCGCGAGCCATCTGGTGAGCAACACCCCGGATACCACGACCGCGAAATATAGTATTGCGTGGTTAAAGCGTTTTGTGGATGACGATCTGCGCTATGAACAGTTTCTGTGCCCGGCGCCGGATGATTTTGCGATTAGCGAATATCGCAGCACCTGCCCGTTT。

[0055] Among them, the amino acid sequence of mutant H185Y is shown in SEQ ID No.2, specifically:

[0056] MANPYERGPDPTESSIEAVRGPFAVAQTTVSRLQADGFGGGTIYYPTDTSQGTFGAVAISPGFTAGQESIAWLGPRIASQGFVVITIDTITRLDQPDSRGRQLQAALDHLRTNSVVRNRIDPNRMAVMGH SMGGGGALSAAANNTSLEAAIPLQGWHTRKNWSSVRTPTLVVGAQLDTIAPVSSYSEAFYNSLPSDLDKAYMELRGASHLVSNTPDTTTAKYSIAWLKRFVDDDLRYEQFLCPAPDDFAISEYRSTCPF.

[0057] The coding gene sequence of mutant H185Y is shown in SEQ ID No. 5, specifically:

[0058] ATGGCGAACCCGTATGAACGCGGCCCGGATCCGACCGAAAGCAGCATTGAAGCGGTGCGCGGCCCGTTTGCGGTGGCGCAAACCACGGTTAGCCGCCTGCAAGCGGATGGCTTTGGCGGTGGCACCATTTATTATCCGACCGATACGAGCCAAGGCACCTTTGGCGCGGTGGCGATTAGCCCGGGCTTTACCGCGGGCCAAGAAAGCATTGCGTGGCTGGGCCCGCGCATTGCGAGCCAAGGCTTTGTGGTGATTACCATTGATACCATTACCCGCCTGGATCAGCCGGATAGCCGCGGCCGTCAGCTGCAAGCGGCGCTGGATCATCTGCGCACCAACAGCGTGGTGCGCAACCGCATTGATCCAAACCGTATGGCCGTGATGGGTCATAGTATGGGCGGTGGCGGCGCCCTGAGTGCGGCCGCGAACAACACGAGCCTGGAAGCGGCGATTCCGCTGCAAGGCTGGCATACCCGCAAAAACTGGAGCAGCGTGCGCACCCCGACCCTGGTGGTGGGCGCGCAGCTGGATACCATTGCGCCGGTGAGCAGCTATAGCGAAGCGTTTTATAACAGCCTGCCGAGCGATCTGGATAAAGCGTATATGGAACTGCGCGGCGCGAGCCATCTGGTGAGCAACACCCCGGATACCACGACCGCGAAATATAGTATTGCGTGGTTAAAGCGTTTTGTGGATGACGATCTGCGCTATGAACAGTTTCTGTGCCCGGCGCCGGATGATTTTGCGATTAGCGAATATCGCAGCACCTGCCCGTTT。

[0059] 2. Screening of single point mutants based on mutant H185Y and construction of recombinant plasmid of mutant YPT

[0060] This invention uses the mutant H185Y as a basis for further modification. Through rational design methods such as evolutionary analysis and free energy calculation, mutants with improved performance, namely YV19M, YA26R, YA26S, YV30I, YT42R, YI43V, YI90L, YH109L, YR111T, YV115T, YS139A, YE148Q, YT158M, YN161D, YI179V, YE187I, YA188P, YN191H, YD198P, YT214F, and YA249S, were screened. The amino acid sequence of mutant H185Y is shown in SEQ ID No. 2.

[0061] Among them, the mutant YV19M is formed by site-directed mutation of valine at position 19 of the amino acid sequence shown in SEQ ID No.2 to methionine, while the amino acid residues at other positions remain unchanged;

[0062] The mutant YA26R is formed by site-directed mutation of alanine at position 26 in the amino acid sequence shown in SEQ ID No. 2 to arginine, while the amino acid residues at other positions remain unchanged;

[0063] The mutant YA26S is formed by site-directed mutation of alanine at position 26 in the amino acid sequence shown in SEQ ID No. 2 to serine, while the amino acid residues at other positions remain unchanged;

[0064] The mutant YV30I is formed by site-directed mutation of valine at position 30 of the amino acid sequence shown in SEQ ID No.2 to isoleucine, while the amino acid residues at other positions remain unchanged;

[0065] The mutant YT42R is formed by site-directed mutation of threonine at position 42 in the amino acid sequence shown in SEQ ID No.2 to arginine, while the amino acid residues at other positions remain unchanged;

[0066] The mutant YI43V is formed by 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;

[0067] The mutant YI90L is formed by 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;

[0068] The mutant YH109L is formed by site-directed mutation of histidine at position 109 in the amino acid sequence shown in SEQ ID No.2 to leucine, while the amino acid residues at other positions remain unchanged;

[0069] The mutant YR111T is formed by 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;

[0070] The mutant YV115T is formed by site-directed mutation of valine at position 115 of the amino acid sequence shown in SEQ ID No.2 to threonine, while the amino acid residues at other positions remain unchanged;

[0071] The mutant YS139A is formed by 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;

[0072] The mutant YE148Q is formed by 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;

[0073] The mutant YT158M is formed by site-directed mutation of threonine at position 158 in the amino acid sequence shown in SEQ ID No.2 to methionine, while the amino acid residues at other positions remain unchanged;

[0074] The mutant YN161D is formed by site-directed mutation of asparagine at position 161 of the amino acid sequence shown in SEQ ID No.2 to aspartic acid, while the amino acid residues at other positions remain unchanged;

[0075] The mutant YI179V is formed by 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;

[0076] The mutant YE187I is formed by 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;

[0077] The mutant YA188P is formed by site-directed mutation of alanine at position 188 in the amino acid sequence shown in SEQ ID No. 2 to proline, while the amino acid residues at other positions remain unchanged;

[0078] The mutant YN191H is formed by site-directed mutation of asparagine at position 191 of the amino acid sequence shown in SEQ ID No.2 to histidine, while the amino acid residues at other positions remain unchanged;

[0079] The mutant YD198P is formed by site-directed mutation of aspartic acid at position 198 in the amino acid sequence shown in SEQ ID No.2 to proline, while the amino acid residues at other positions remain unchanged.

[0080] The mutant YT214F is formed by site-directed mutation of 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;

[0081] The mutant YA249S is formed by site-directed mutation of alanine at position 249 of the amino acid sequence shown in SEQ ID No.2 to serine, while the amino acid residues at other positions remain unchanged.

[0082] The statistics of site-directed mutagenesis primers used in the construction of the above mutant recombinant plasmids are shown in Table 2 below.

[0083] Table 2

[0084]

[0085] Using site-directed mutagenesis, with the constructed pET-28a(+)-PETase-PHL7-H185Y as a template, PCR was performed using the primers shown in Table 2 to obtain linearized plasmid fragments. These fragments were then ligated using ABclonal 2X MultiFSeamless Assembly Mix and introduced into the target matrix. E. coliTrans -T1 competent cells were used, and the correctness of mutant construction was ensured by Sanger sequencing, resulting in 21 recombinant plasmids of PETase-PHL7 mutants: YV19M, YA26R, YA26S, YV30I, YT42R, YI43V, YI90L, YH109L, YR111T, YV115T, YS139A, YE148Q, YT158M, YN161D, YI179V, YE187I, YA188P, YN191H, YD198P, YT214F, and YA249S.

[0086] The PCR reaction conditions in this step are the same as those used when constructing the recombinant plasmid PETase-PHL7.

[0087] II. Construction of PETase-PHL7 mutant engineered strains

[0088] The above-mentioned recombinant plasmids PETase-PHL7, pET-28a(+)-PETase-PHL7-H185Y, and the mutant recombinant plasmids YV19M~YA249S were introduced into Escherichia coli BL21, respectively. (DE3) Competent cells were selected and verified to yield positive monoclonal strains, resulting in PETase-PHL7 engineered strains and twenty-two 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.

[0089] III. Preparation and Purification of PETase-PHL7 Mutant

[0090] 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% inoculation rate for fermentation. The expression was induced at 21 °C and 160 rpm for about 20 h to obtain fermentation broth rich in the corresponding PET degrading enzyme.

[0091] The different fermentation broths were centrifuged at 4000 rpm for 10 min using a high-speed refrigerated centrifuge to collect the cells. The cells were resuspended in 20 mL of lysis buffer (each 1 L of lysis buffer contains 50 mM Na₂HPO₄·12H₂O, 200 mM NaCl, and 10 mM imidazole, pH=7.4), and then the collected cells were lysed using a high-pressure homogenizer. After lysis, the cells were centrifuged at 10000 rpm for 1 h to remove cell debris. The supernatant was the total protein solution containing PETase-PHL7 and its mutants. The total protein solution was filtered through a 0.45 μm filter to remove impurities, and then purified using a Ni-NTA packed column via gradient elution to obtain the target protein. The specific steps are as follows: First, equilibrate with the lysis buffer for 2 minutes. Then, repeatedly attach the whole protein solution to the column 3 times after membrane transfer. Wash 3 times with washing buffer (each liter of washing buffer contains 50 mM Na2HPO4·12H2O, 200 mM NaCl, and 50 mM imidazole, pH=7.4) to remove impurities. Next, elute with elution buffer (each 1 L of elution buffer contains 50 mM Na2HPO4·12H2O, 200 mM NaCl, and 250 mM imidazole, pH=7.4) to obtain protein eluent. Further concentrate the protein by changing the wash buffer 3 times (each 1 L of wash buffer contains 50 mM Na2HPO4·12H2O and 200 mM NaCl, pH=7.4) to dilute the imidazole in the protein eluent to 1‰ of its original concentration, thereby obtaining concentrated PETase-PHL7 and its mutant enzyme solution.

[0092] IV. Performance Characterization Methods

[0093] 1. Determination of PET degradation activity

[0094] This invention uses amorphous PET film (purchased from Goodfellow) as the PET substrate;

[0095] The concentrated PETase-PHL7 and its mutant enzyme solutions were respectively placed in 1.8 mL of reaction solution (1M potassium phosphate buffer, pH=8) at the corresponding concentrations (0.6 mg enzyme / g PET). Approximately 45 mg of amorphous PET rectangular tablets (3 cm × 0.5 cm) were weighed and added to the above reaction solution. The mixture was reacted for 8 h in a constant temperature shaking mixer at 700 rpm and 70 °C. After the reaction, the contents of TPA, MHET, and BHET produced in the reaction were analyzed by high performance liquid chromatography (HPLC). The total contents of TPA, BHET, and MHET were used as the indicator to evaluate the PET degradation activity.

[0096] 2. Methods for determining Tm

[0097] Protein melting temperatures were determined using differential scanning fluorometry (DSF). Protein samples were loaded into 96-well plates at 25 μL per well, consisting of 15 μL of enzyme stock solution (containing 50 mM Na₂HPO₄·12H₂O and 200 mM NaCl per 1 L of enzyme stock solution), 9 μL of protein solution (protein concentration 0.4 mg / mL), and 1 μL of SYPRO Orange dilution solution. DSF experiments were performed using a real-time quantitative PCR system with 465 nm excitation and a 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 value of Tm was determined by the first derivative curve. The Tm measurement was repeated three times. Since the error was small, the error bar was no longer shown in the histogram of the Tm measurement results.

[0098] V. Experimental Results

[0099] The PET degradation activity of PETase-PHL7 and its mutants was determined in this example. T m Values. The comparison chart of the measurement results is shown below. Figure 2 As shown. Figure 2 Mid-star type T m value.

[0100] 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 showed varying degrees of increased degradation activity against PET substrates at 70℃, with the degradation activity increasing 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. Further research will involve pairwise combinations of these mutant sites to determine the optimal mutant combination. Regarding thermal stability, mutants YE187I and YD198P exhibited... T m The values ​​were significantly higher than those of the mutant H185Y, increasing by 3.78℃ and 2.82℃ respectively.

[0101] Example 2

[0102] Based on Example 1, this example combines the mutants YA26R, YT42R, YR111T, YE148Q, YE187I, YA188P, YD198P, and YT214F, which significantly enhance PET degradation activity, to obtain 17 mutants, denoted as mutants Y / T214F / T42R, Y / T214F / E187I, Y / T214F / A188P, Y / T42R / E187I, and 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.

[0103] In this embodiment, all two-site combined mutants are constructed using known single-site mutation primers via sequential site-directed mutagenesis, without the need for separately designed primers. Specifically, a pre-constructed single-site mutant plasmid is used as a template, and site-directed PCR amplification is performed using primers targeting another distant single-site mutation to obtain the desired two-site combined mutant plasmid. For example, the mutant Y / T214F / T42R is obtained by PCR amplification using the recombinant plasmid of the single-site mutant YT214F as a template and primers targeting the T42R site; other combined mutants are constructed using a similar method. Since the mutation sites involved are relatively far apart, the corresponding primers can efficiently and specifically introduce mutations separately, and all primer sequences have been listed in Example 1, therefore, they will not be repeated in this embodiment.

[0104] Using site-directed mutagenesis, and with pET-28a(+)-PETase-PHL7-H185Y constructed in Example 1 as a template, PCR was performed using 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, and pET-28a(+)-Y / A26R / E187I. Seventeen recombinant plasmids of the PHL7-H185Y mutant, 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, were constructed using the same method as in Example 1. The plasmid map of the recombinant plasmid pET-28a(+)-Y / A188P / R111T (also denoted as recombinant plasmid pET-28a(+)-YPT) is shown in the figure below. Figure 3 As shown.

[0105] Based on the 17 recombinant plasmids constructed above, corresponding mutant engineered strains were further constructed using the method described in Example 1. 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, and Y / T214F / R111T were prepared and purified. The mutants 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 were selected, and their PET degradation activity and Tm were measured. The comparison of the measurement results is shown in the figure below. Figure 4 As shown.

[0106] Depend on Figure 4 It can be seen that, compared with mutant H185Y, mutants Y / T214F / R111T, Y / A26R / A188P, Y / A188P / R111T, and Y / A188P / D198P showed a significant increase in PET degradation activity, ranging from 41.9% to 51.9%. Regarding thermal stability, mutants Y / T214F / T42R, Y / T214F / E187I, Y / T214F / A188P, Y / T42R / A188P, Y / A26R / E187I, and Y / T214F / E148Q exhibited… T m The values ​​were all 2.5℃~4.4℃ higher than those of mutant H185Y. However, these six mutants did not perform well in terms of PET degradation activity, so they were not considered as the basis for subsequent modification. Among them, the PET degradation product release of mutant Y / A188P / R111T (denoted as mutant YPT) increased from 47.4mM in mutant H185Y to 73.8mM, the PET degradation activity was improved by 55.6%, and the Tm was increased by 0.35℃.

[0107] The amino acid sequence of the mutant YPT is shown in SEQ ID No. 3, specifically:

[0108] MANPYERGPDPTESSIEAVRGPFAVAQTTVSRLQADGFGGGTIYYPTDTSQGTFGAVAISPGFTAGQESIAWLGPRIASQGFVVITIDTITRLDQPDSRGRQLQAALDHLTTNSVVRNRIDPNRMAVMGH SMGGGGALSAAANNTSLEAAIPLQGWHTRKNWSSVRTPTLVVGAQLDTIAPVSSYSEPFYNSLPSDLDKAYMELRGASHLVSNTPDTTTAKYSIAWLKRFVDDDLRYEQFLCPAPDDFAISEYRSTCPF.

[0109] The coding gene sequence of the mutant YPT is shown in SEQ ID No. 6, specifically:

[0110] ATGGCGAACCCGTATGAACGCGGCCCGGATCCGACCGAAAGCAGCATTGAAGCGGTGCGCGGCCCGTTTGCGGTGGCGCAAACCACGGTTAGCCGCCTGCAAGCGGATGGCTTTGGCGGTGGCACCATTTATTATCCGACCGATACGAGCCAAGGCACCTTTGGCGCGGTGGCGATTAGCCCGGGCTTTACCGCGGGCCAAGAAAGCATTGCGTGGCTGGGCCCGCGCATTGCGAGCCAAGGCTTTGTGGTGATTACCATTGATACCATTACCCGCCTGGATCAGCCGGATAGCCGCGGCCGTCAGCTGCAAGCGGCGCTGGATCATCTGACCACCAACAGCGTGGTGCGCAACCGCATTGATCCAAACCGTATGGCCGTGATGGGTCATAGTATGGGCGGTGGCGGCGCCCTGAGTGCGGCCGCGAACAACACGAGCCTGGAAGCGGCGATTCCGCTGCAAGGCTGGCATACCCGCAAAAACTGGAGCAGCGTGCGCACCCCGACCCTGGTGGTGGGCGCGCAGCTGGATACCATTGCGCCGGTGAGCAGCTATAGCGAACCGTTTTATAACAGCCTGCCGAGCGATCTGGATAAAGCGTATATGGAACTGCGCGGCGCGAGCCATCTGGTGAGCAACACCCCGGATACCACGACCGCGAAATATAGTATTGCGTGGTTAAAGCGTTTTGTGGATGACGATCTGCGCTATGAACAGTTTCTGTGCCCGGCGCCGGATGATTTTGCGATTAGCGAATATCGCAGCACCTGCCCGTTT。

[0111] Example 3

[0112] Based on Example 2, this example further combines the mutant YPT with mutation sites at positions 19, 26, 30, 43, 90, 115, 139, 148, 158, 161, 179, 187, 191, 198, or 249 to obtain mutants YPT-V19M and YPT, respectively. -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.

[0113] The site-directed mutagenesis primers used to construct the corresponding recombinant plasmids are shown in Table 2 above. Some primers need to be redesigned, as shown in Table 3 below.

[0114] Table 3

[0115]

[0116] Using site-directed mutagenesis, the recombinant plasmid pET-28a(+)-YPT constructed in Example 2 was used as a template, and PCR was performed with corresponding primers to prepare the corresponding recombinant plasmids. The method for preparing the recombinant plasmids was the same as in Example 1. Further, after constructing the corresponding engineered strains using the method described in Example 1, 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 As shown.

[0117] Depend on Figure 5 It can be seen that, compared with the mutant YPT, the mutants YPTS139A (denoted as mutant YPTA), YPTE187I (denoted as mutant YPTI), and YPTN191H (denoted as mutant YPTH) exhibit significantly enhanced PET degradation activity, while maintaining the same or slightly increasing Tm value. Compared with mutant YPT, mutant YPTA showed a 19.5% increase in PET degradation activity and a 1.2℃ increase in Tm value; mutant YPTI showed a 21.9% increase in PET degradation activity and a 0.3℃ decrease in Tm value; and mutant YPTH showed a 20.5% increase in PET degradation activity and a 4.7℃ increase in Tm value. Compared with mutant YPT, YPTD198P showed a 21.8% increase in PET degradation activity, but a 4.7℃ decrease in Tm value. This invention selects mutant YPTI as the basis for the next round of mutations.

[0118] Example 4

[0119] Based on Example 3, this example adds the mutations A26R, V30I, I43V, I90L, S139A, D198P, and D233N to the mutant YPTI to obtain the mutants YPTI-A26R, YPTI-V30I, YPTI-I43V, YPTI-I90L, YPTI-S139A, YPTI-D198P, and YPTI-D233N, respectively, and constructs the corresponding recombinant plasmids.

[0120] When constructing recombinant plasmids, the primers for mutants need to be redesigned, as shown in Table 4.

[0121] Table 4

[0122]

[0123] Using site-directed mutagenesis, 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 to prepare the corresponding recombinant plasmids. The method for constructing the recombinant plasmids was the same as in Example 1. Furthermore, seven engineered strains—YPTI-A26R, YPTI-V30I, YPTI-I43V, YPTI-I90L, YPTI-S139A, YPTI-D198P, and YPTI-D233N—were constructed using the method described in Example 1. These strains were then fermented and purified to prepare the corresponding mutants. The PET degradation activity and Tm of different PETase-PHL7 mutants were measured, and the comparison results are shown in the figure below. Figure 6 As shown.

[0124] Depend on Figure 6 It was found that further mutation of the YPTI mutant increased the PET degradation activity of mutants YPTI-A26R and YPTI-D198P by 7.0%–11.9%. Regarding thermal stability, mutants YPTI-V30I, YPTI-I90L, and YPTI-D198P showed… T m The values ​​were significantly higher than those of the 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 (i.e., YPTIP) was selected as the basis for the next round of mutations.

[0125] Example 5

[0126] Based on Example 4, this example adds the mutations A26R, H109L, N191H, D196S and A249S to the mutant YPTIP to obtain the mutants YPTIP-A26R, YPTIP-H109L, YPTIP-N191H, YPTIP-D196S and YPTIP-A249S, respectively, and constructs the corresponding recombinant plasmids.

[0127] The site-directed mutagenesis primers used in constructing recombinant plasmids are shown in Table 5 below.

[0128] Table 5

[0129]

[0130] Using site-directed mutagenesis, 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 plasmids was the same as in Example 1. Further, the corresponding engineered strains of mutants YPTIP-A26R, YPTIP-H109L, YPTIP-N191H, YPTIP-D196S, and YPTIP-A249S were constructed using the method described in Example 1. These mutants were then fermented and purified to prepare the corresponding mutants. The PET degradation activity and Tm of different PETase-PHL7 mutants were measured, and the comparison results are shown in the figure below. Figure 7 As shown.

[0131] Depend on Figure 7 It was found that further mutations of the YPTIP mutant resulted in a 4.3%-9.6% increase in PET degradation activity for mutants YPTIP-H109L (denoted as mutant YPTIPL) and YPTIP-N191H (denoted as mutant YPTIPH). Regarding thermal stability, different mutants... T m All values ​​were improved. Compared to the mutant YPTIP, the mutant YPTIPL showed a 9.6% increase in PET degradation activity and a 0.2℃ increase in Tm value (the Tm value of mutant YPTIPL increased to 85.2℃), while the mutant YPTIPH showed a 4.3% increase in PET degradation activity and a 1.1℃ increase in Tm value (the Tm value of mutant YPTIPH increased to 86.2℃). Compared to the mutant H185Y, the mutant YPTIPL showed a 1.32-fold increase in PET degradation activity and a 5.0℃ increase in Tm value, while the mutant YPTIPH showed a 1.21-fold increase in PET degradation activity and a 6.0℃ increase in Tm value.

[0132] Based on a comprehensive analysis considering PET degradation activity and thermal stability, the mutant YPTIPL is the optimal mutant in this invention. The liquid chromatography diagram for determining the PET degradation activity of the mutant YPTIPL is shown below. Figure 8 As shown. Figure 8 Peak 1 is TPA, peak 2 is MHET, and peak 3 is BHET. (By...) Figure 8 It can be seen that the peak response values ​​of TPA and MHET are significantly higher than those of BHET, 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.

[0133] In summary, this invention, based on the mutant H185Y, mutates alanine at position 188 to proline and arginine at position 111 to threonine, while keeping other amino acid residues unchanged, to obtain the mutant YPT. Further single-point, two-point, and combined mutations are performed on the YPT mutant, with the preferred mutant selected as the basis for the next round of mutations in each iteration, resulting in a series of PETase-PHL7 mutants. The degradation activity of the mutants gradually increases after multiple iterations. Compared to the YPT mutant, the PETase-PHL7 mutant provided by this invention shows a significant improvement in PET degradation activity, specifically an increase of 55.6% to 132.5%; simultaneously, the thermal stability of the mutant is also improved to some extent, with a maximum increase in Tm of 6.0℃.

[0134] 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 within the protection scope of the present invention.

Claims

1. A PETase-PHL7 mutant, characterized in that: The mutant H185Y has its alanine at position 188 mutated to proline, and its arginine at position 111 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.

2. A PETase-PHL7 mutant, characterized in that: Its amino acid sequence is obtained by replacing an amino acid residue at one position in the amino acid sequence shown in SEQ ID No. 3, and its 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 is mutated to alanine, while the amino acid residues at other positions remain unchanged. This mutant is denoted as YPTA. (2) The glutamic acid at position 187 of the amino acid sequence shown in SEQ ID No.3 is mutated to isoleucine, while the amino acid residues at other positions remain unchanged. This mutant is denoted as YPTI. (3) The asparagine at position 191 of the amino acid sequence shown in SEQ ID No.3 is mutated to histidine, while the amino acid residues at other positions remain unchanged. This mutant is called 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 to proline in the amino acid sequence of mutant YPTI as described in claim 2, while the amino acid residues at other positions remain unchanged, and is denoted as mutant YPTIP.

4. A PETase-PHL7 mutant, characterized in that: Its amino acid sequence is obtained by replacing an amino acid residue at one position in the amino acid sequence of the mutant YPTIP as described in claim 3, and its amino acid sequence is any one of (i) or (ii): (i) The histidine at position 109 in the amino acid sequence of the mutant YPTIP is mutated to leucine, while the amino acid residues at other positions remain unchanged. The resulting mutant is denoted as mutant YPTIPL. (ii) The asparagine at position 191 of the amino acid sequence of the mutant YPTIP is mutated to histidine, while the amino acid residues at other positions remain unchanged. The resulting mutant is denoted as mutant YPTIPH.

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 as described in any one of claims 1 to 4.

7. A PETase-PHL7 mutant engineered strain, characterized in that: It contains the recombinant plasmid as described in claim 6.

8. The use of the PETase-PHL7 mutant according to any one of claims 1 to 4 in the degradation of PET, the preparation of PET degradation agents, or the recycling of PET degradation products.

9. The application of the recombinant plasmid according to claim 6 in the degradation of PET, the preparation of PET degradation agents, or the recycling of PET degradation products.

10. The application of the PETase-PHL7 mutant engineered strain according to claim 7 in the degradation of PET, the preparation of PET degradation agents, or the recovery of PET degradation products.

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