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WO2025060302A1 - Pet-mh mutant of pet degrading enzyme, and encoding gene, recombinant plasmid, engineered strain and use thereof - Google Patents

Pet-mh mutant of pet degrading enzyme, and encoding gene, recombinant plasmid, engineered strain and use thereof Download PDF

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Publication number
WO2025060302A1
WO2025060302A1 PCT/CN2023/143044 CN2023143044W WO2025060302A1 WO 2025060302 A1 WO2025060302 A1 WO 2025060302A1 CN 2023143044 W CN2023143044 W CN 2023143044W WO 2025060302 A1 WO2025060302 A1 WO 2025060302A1
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pet
amino acid
seq
acid sequence
mutant
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Chinese (zh)
Inventor
齐崴
尤生萍
王梦凡
郑昀欣
林伟
苏荣欣
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Yuantian Biotechnology Tianjin co ltd
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Yuantian Biotechnology Tianjin co ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
    • B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
    • B09B3/60—Biochemical treatment, e.g. by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J11/00—Recovery or working-up of waste materials
    • C08J11/04—Recovery or working-up of waste materials of polymers
    • C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
    • B09B2101/00—Type of solid waste
    • B09B2101/75—Plastic waste
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/62—Plastics recycling; Rubber recycling

Definitions

  • the present application relates to the field of enzyme engineering technology, and in particular to a PET-degrading enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineering bacteria and application.
  • PET Polyethylene terephthalate
  • PET plastic is one of the most widely used plastics at present. It has the advantages of strong chemical resistance, high thermal stability, light weight, and convenient storage. It can be used to produce packaging materials, mineral water bottles, mobile phone cases and many other daily necessities. It is a common engineering polymer with an annual output of about 50 million tons. Because of the chemical inertness of the ester bond and aromatic nucleus in polyethylene terephthalate, PET plastic is difficult to degrade. With the increase in the use of PET plastics, PET waste has accumulated in the environment in large quantities, which is one of the culprits of the "white pollution" problem. It is reported that micron-level PET has been found in food and has become part of the human food chain, which may threaten human health.
  • the PET degrading enzyme PET-mh mutant has an amino acid sequence: at least one of the amino acids at positions 63, 68, 152, 183, 208, and 219 in the amino acid sequence shown in SEQ ID No. 1 is mutated.
  • the amino acid sequence of the PET degrading enzyme PET-mh mutant is as follows: any one of the amino acids at positions 63, 68, 152, 176, and 208 and the amino acids at positions 124 and 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together. More preferably, the amino acid sequence of the PET degrading enzyme PET-mh mutant is as follows: the amino acid at position 208 and the amino acids at positions 124 and 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together.
  • High crystallinity PET is more difficult to degrade than low crystallinity PET, and has a worse degradation effect under the same conditions.
  • the amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 is mutated, or the amino acid at position 124 or 152 and the amino acid at position 183 are mutated together, or the amino acids at positions 124, 183 and 208 are mutated together.
  • the resulting mutant can significantly improve the degradation efficiency of low crystallinity PET and the degradation efficiency of high crystallinity PET relative to the protein, and has better practicality.
  • the present application also provides the use of the above-mentioned PET degrading enzyme PET-mh mutant in hydrolyzing PET: adding the PET degrading enzyme PET-mh mutant to a potassium phosphate buffer with a concentration of 80mM ⁇ 120mM and a pH value of 7.5 ⁇ 8.5 until the final concentration of the PET degrading enzyme PET-mh mutant is 5 ⁇ g/mL ⁇ 15 ⁇ g/mL, and performing PET degradation at a temperature of 65°C ⁇ 75°C and a rotation speed of 100rpm ⁇ 220rpm.
  • amino acid sequence of the PET degrading enzyme PET-mh mutant is shown as SEQ ID No.2 to SEQ ID No.23, and its nucleotide sequence is shown as SEQ ID No.24 to SEQ ID No.45 respectively.
  • the present application also provides a recombinant plasmid comprising the nucleotide sequence of the above-mentioned encoding gene.
  • the present application also provides a method for constructing the above-mentioned recombinant plasmid: using the nucleotide sequence encoding the amino acid shown in SEQ ID No.1 as a template, a single point mutation is constructed using the corresponding primers, the target gene is first exponentially amplified by PCR reaction, and then the template chain is removed using the restriction endonuclease Dpn I, and then the PCR product is quickly circularized using DNA ligase to obtain a recombinant plasmid.
  • the present application also provides an engineered bacterium, including a host cell having the above-mentioned recombinant plasmid.
  • the engineered bacteria is Escherichia coli.
  • the present application also provides a method for constructing the above-mentioned engineered bacteria: transforming the above-mentioned recombinant plasmid into BL21 (DE3) competent cells, spreading them on ampicillin plate culture medium, and obtaining positive recombinants, namely the engineered bacteria.
  • the method for degrading PET using the above-mentioned engineered bacteria can refer to the following operation: the above-mentioned engineered bacteria are cultured in LB liquid culture medium until the OD600 value reaches the range of 0.8 to 1.0, the inducer IPTG (isopropyl ⁇ -D-1-thiogalactopyranoside) is added to the concentration of the inducer IPTG to be 0.1 mM to 0.15 mM, and the expression is induced at a temperature of 15° C. to 17° C. for 20 h to 24 h to obtain a fermentation culture;
  • the inducer IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • the fermentation culture is centrifuged, resuspended by pipetting with a bacteria-breaking buffer, and centrifuged again to obtain a crude enzyme solution containing a PET-mh mutant of a PET-degrading enzyme; the crude enzyme solution is protein purified and then concentrated to obtain a concentrated solution as a biocatalyst for degrading PET;
  • the PET film substrate to be degraded is added into the PET reaction solution, and a biocatalytic reaction is carried out at 65° C.-75° C.
  • the PET-mh mutant of the PET degrading enzyme provided by the present application has higher activity and thermal stability than the protein with an amino acid sequence as shown in SEQ ID No. 1, and the catalytic efficiency is greatly improved, and it can more effectively degrade PET plastic waste, which is beneficial to the resource regeneration and secondary development of PET waste, and can not only alleviate the pressure of petroleum resources required for the production of PET, but also solve the serious environmental pollution problem caused by PET waste, which is in line with the concept of sustainable development, and can also increase the recycling value of PET and promote the realization of the plastic economy.
  • FIG1 shows the degradation effect of the PET-mh mutant of the PET degrading enzyme after the amino acids at positions 63, 68, 124, and 183 are mutated separately in Test Example 1 of the present application on low-crystallinity PET;
  • FIG2 shows the degradation effect of the PET-mh mutant of the PET degrading enzyme on low-crystallinity PET after mutation of any one of the amino acids at positions 63, 68, 124, 152, 176, 177 and the amino acid at position 183 in Test Example 1 of the present application;
  • FIG3 shows the degradation effect of the PET-mh mutant of the PET degrading enzyme on low-crystallinity PET after mutation of any one of the amino acids at positions 63, 68, 152, 176, 208 and the amino acids at positions 124 and 183 in Test Example 1 of the present application;
  • Figure 4 shows the degradation effect of the PET-mh mutant of the PET degrading enzyme in which the amino acid at position 183 is mutated alone, the PET-mh mutant of the PET degrading enzyme in which the amino acid at position 124 or 152 and the amino acid at position 183 are mutated together, and the PET-mh mutant of the PET degrading enzyme in which the amino acids at positions 124, 183 and 208 are mutated together on high crystallinity PET in Test Example 2 of the present application.
  • ICCG represents the parent PET degrading enzyme, i.e., the enzyme including the amino acid sequence shown in SEQ ID No. 1.
  • the numbers in the abscissa represent the numbers of the mutation points, for example, 63 and 68 represent that the amino acids at positions 63 and 68 are mutated, respectively; 176-183 represents that the amino acids at positions 176 and 183 are mutated together; and 124-183-208 represents that the amino acids at positions 124, 183, and 208 are mutated together.
  • the numbering of the mutation point position of the PET degrading enzyme PET-mh mutant corresponds to the amino acid sequence numbering of the original PET degrading enzyme SEQ ID No. 1.
  • Amino acids are represented by single-letter abbreviations, such as S29A, which means that the S at position 29 in the amino acid sequence of the original PET degrading enzyme SEQ ID No. 1 is replaced (mutated) with A.
  • PET products are being produced and used in large quantities due to their low price, light weight, convenience, and strong plasticity.
  • the resulting PET waste will cause unimaginable pollution.
  • the biological method of using enzymes or microorganisms to degrade PET into monomer molecules terephthalic acid (TPA) and ethylene glycol is considered to be the most promising method to reduce PET plastic pollution.
  • TPA terephthalic acid
  • ethylene glycol ethylene glycol
  • Modifying the amino acid sequence of an enzyme is a common way to obtain new functions, but not all modifications can produce mutants with ideal functions.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, which is obtained by mutating at least one of the amino acids at positions 29, 62, 63, 68, 124, 152, 176, 177, 183, 208, and 219 in the amino acid sequence shown in SEQ ID No. 1.
  • mutation of the amino acid at position 177 can increase the melting point (melting temperature, Tm) of the crystalline polymer to 99.6°C; mutation of the amino acids at other positions can improve the thermal stability to varying degrees while also improving the efficiency of PET degradation.
  • the embodiments of the present application also provide a method for constructing the above-mentioned PET degrading enzyme PET-mh mutant and its application in hydrolyzing PET.
  • the examples of the present application also provide a gene encoding the above mutant, and its nucleotide sequence is shown in SEQ ID No.24 to SEQ ID No.45.
  • the embodiments of the present application also provide a recombinant plasmid containing the above nucleotide sequence and a construction method thereof, as well as an engineered bacterium constructed using the recombinant plasmid.
  • the examples of the present application also provide a method for constructing the engineered bacteria and its application in degrading PET.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-serine at position 29 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-alanine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.2, and the nucleotide sequence is shown in SEQ ID No.24.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 62 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.25.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-aspartic acid at position 63 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.4, and the nucleotide sequence is shown in SEQ ID No.26.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 68 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-asparagine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.27.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.6, and the nucleotide sequence is shown in SEQ ID No.28.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 152 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-methionine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.7, and the nucleotide sequence is shown in SEQ ID No.29.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-threonine at position 176 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-arginine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.8, and the nucleotide sequence is shown in SEQ ID No.30.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-valine at position 177 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-isoleucine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.9, and the nucleotide sequence is shown in SEQ ID No.31.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-histidine at position 183 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-tyrosine, and the amino acid sequence of the obtained mutant is shown in SEQ ID No.10, and the nucleotide sequence is shown in SEQ ID No.32.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-isoleucine at position 208 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.11, and the nucleotide sequence is shown in SEQ ID No.33.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-valine at position 219 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-methionine, and the amino acid sequence of the obtained mutant is shown in SEQ ID No.12, and the nucleotide sequence is shown in SEQ ID No.34.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-aspartic acid at position 63 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.13, and the nucleotide sequence is shown in SEQ ID No.35.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 68 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-asparagine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.14, and the nucleotide sequence is shown in SEQ ID No.36.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.15, and the nucleotide sequence is shown in SEQ ID No.37.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 152 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-methionine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.16, and the nucleotide sequence is shown in SEQ ID No.38.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-threonine at position 176 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-arginine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.17, and the nucleotide sequence is shown in SEQ ID No.39.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-valine at position 177 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-isoleucine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.18, and the nucleotide sequence is shown in SEQ ID No.40.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-aspartic acid at position 63 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the L-leucine at position 124 is mutated to S-glycine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.19, and the nucleotide sequence is shown in SEQ ID No.41.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 68 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-asparagine, the L-leucine at position 124 is mutated to S-glycine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.20, and the nucleotide sequence is shown in SEQ ID No.42.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the L-alanine at position 152 is mutated to L-asparagine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.21, and the nucleotide sequence is shown in SEQ ID No.43.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the L-threonine at position 176 is mutated to L-arginine, and the L-histidine at position 183 is mutated to L-tyrosine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.22, and the nucleotide sequence is shown in SEQ ID No.44.
  • the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the L-histidine at position 183 is mutated to L-tyrosine, and the L-isoleucine at position 208 is mutated to L-threonine.
  • the amino acid sequence of the obtained mutant is shown in SEQ ID No.23, and the nucleotide sequence is shown in SEQ ID No.45.
  • the present application provides the use of the PET-mh mutant of the PET degrading enzyme in Examples 1 to 22 in hydrolyzing PET:
  • the PET-mh mutant of the PET degrading enzyme of each example was added to 300 ⁇ L of potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as the PET reaction solution, and PET degradation was performed in a water bath at 60° C. to 75° C.
  • the present application provides a recombinant plasmid containing a gene encoding the PET-mh mutant of the PET degrading enzyme in Examples 1 to 22 and a construction method thereof:
  • Example 1 Take the recombinant plasmid containing the gene encoding the PET-mh mutant of the PET degrading enzyme in Example 1 as an example:
  • PET-mh-1 Using the nucleotide sequence encoding the amino acids shown in SEQ ID No.1 as a template and the corresponding primers, the target gene was exponentially amplified by PCR reaction to construct a PET-mh mutant (PET-mh-1).
  • the primers were designed as follows:
  • the amplification reaction system is:
  • the amplification program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s, annealing at Tm-5°C for 20 s, extension at 72°C for 2 min, 30 cycles; and extension at 72°C for 5 min.
  • the PCR amplification products were subjected to 1% agarose gel electrophoresis, and the PCR products were recovered using a DNA recovery kit to obtain the gene of the PET-mh mutant containing a single point mutation.
  • the template strand was then removed using restriction endonuclease Dpn I, followed by rapid circularization of the PCR product using DNA ligase to obtain a recombinant plasmid containing the gene encoding the PET-mh mutant of the PET degrading enzyme in Example 1.
  • the same method can be used to construct a recombinant plasmid containing a gene encoding a PET-mh mutant of the PET degrading enzyme in Examples 2 to 22.
  • the PCR amplification system and procedure are the same, and the difference lies in the designed upstream and downstream primers and templates.
  • the nucleotide sequence encoding the amino acid shown in SEQ ID No. 1 SEQ ID No. 1 (SEQ ID No.
  • PET-mh-1 to PET-mh-11 the corresponding primers were used to exponentially amplify the target gene by PCR reaction to construct the corresponding PET-mh mutants (PET-mh-1 to PET-mh-11); in the process of constructing a recombinant plasmid containing a gene encoding a PET-mh mutant of the PET degrading enzyme in Examples 12 to 17, the nucleotide sequence encoding the amino acid shown in SEQ ID No. 10 (SEQ ID No.
  • the present application provides an engineering bacterium containing the recombinant plasmid of Example 24 and a method for constructing the same:
  • the recombinant plasmid of Example 24 was transformed into BL21 (DE3) (full gold, CD601) competent cells and spread on ampicillin plate culture medium to obtain positive recombinants, which are Escherichia coli engineered bacteria containing the recombinant plasmids in Example 24.
  • the present application example provides the use of the engineered bacteria in Example 25 in degrading PET:
  • the E. coli engineered bacteria constructed in Example 25 were first cultured in 5 mL LB liquid medium (containing 100 mg/L ampicillin resistance) for 12 h-16 h, and then transferred to a shake flask for culture at a 1% inoculum. When the OD600 value reached the range of 0.8-1.0, the inducer IPTG (isopropyl ⁇ -D-1-thiogalactopyranoside) was added to a concentration of 0.1 mM, and the expression was induced at 16 ° C for 22 h to obtain a fermentation culture.
  • inducer IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • the broken bacterial solution was centrifuged at 4 °C and 10000 rpm for 1 h, and the supernatant was taken to obtain a crude enzyme solution containing the PET-mh mutant.
  • elution buffer each liter of elution buffer contained 50mM Tris-HCl, 300mM NaCl and 300mM imidazole, pH
  • the eluted liquid was collected, i.e., the protein purification liquid.
  • the protein purification liquid was concentrated to 5 mg/mL through a protein concentrator tube, and the concentrated liquid was collected and stored in a refrigerator at 4 °C.
  • the concentrated liquid was then added to the catalytic system (final concentration: 10 ⁇ g/mL) as a biocatalyst for degrading PET.
  • This comparative example provides a PET degrading enzyme mutant, in which the L-alanine at position 211 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-arginine.
  • This comparative example provides a PET degradation enzyme mutant, in which the L-serine at position 212 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-arginine.
  • This comparative example provides a PET degrading enzyme mutant, in which the L-proline at position 179 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-glutamic acid.
  • This comparative example provides a PET degradation enzyme mutant, in which the L-serine at position 66 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-alanine.
  • This comparative example provides a PET degrading enzyme mutant, in which the L-alanine at position 66 in the amino acid sequence shown in SEQ ID No. 1 is mutated to S-glycine.
  • Example 23 According to the method of Example 23, the effects of the protein with an amino acid sequence as shown in SEQ ID No. 1, the PET degrading enzyme mutants of Examples 1 to 22 and Comparative Examples 1 to 5 on the degradation of low-crystallinity PET for 5 h were investigated.
  • a PET film (6 mm in diameter) with a crystallinity of 7.3% was used as a substrate.
  • a protein with an amino acid sequence as shown in SEQ ID No. 1, a PET degrading enzyme PET-mh mutant of each embodiment, or a PET degrading enzyme mutant of each comparative example was added independently to 300 ⁇ L of a potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as a PET reaction solution, and PET degradation was performed under a water bath condition of 60° C.-75° C.
  • BHET terephthalic acid bis(2-hydroxyethyl) ester
  • MHET terephthalic acid 2-hydroxyethyl monoester
  • This test example investigated the effect of the protein with the amino acid sequence shown in SEQ ID No. 1 and the PET-mh mutant of the PET degrading enzyme of Examples 9, 14, 15 and 22 on the degradation of high-crystallinity PET for 5 h according to the method of Example 23.
  • PET particles (100 mg) obtained by grinding a PET plastic bottle with a crystallinity of 26% were used as a substrate.
  • the protein with an amino acid sequence as shown in SEQ ID No. 1 or the PET degrading enzyme PET-mh mutant of each embodiment was independently added to 300 ⁇ L of potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as a PET reaction solution.
  • PET degradation was carried out in a water bath at 70° C. After the reaction for 5 hours, methanol was added to terminate the reaction, and the generated amounts of TPA, BHET and MHET were tested by reverse phase HPLC analysis.
  • the mutants obtained by mutating the amino acid at position 183 alone, or mutating the amino acid at positions 124 or 152 and the amino acid at position 183, or mutating the amino acids at positions 124, 183 and 208 together can achieve a higher degradation effect on high-crystalline PET than the protein with the amino acid sequence shown in SEQ ID No. 1 when degrading for 5 hours.
  • the yields of MHET and TPA are shown in Figure 4.
  • This test example investigated the effect of the protein with an amino acid sequence as shown in SEQ ID No. 1 and the PET-mh mutant of the PET degrading enzyme of Examples 1, 2, 6, 7, 10, and 11 on the degradation of PET with different crystallinity for 24 hours according to the method of Example 23.
  • the protein with amino acid sequence as shown in SEQ ID No. 1 or the PET-mh mutant of each embodiment was added to 300 ⁇ L potassium phosphate buffer with pH 8 to a final concentration of 500 nM as PET reaction solution, and PET degradation was carried out in a water bath at 60° C.-75° C. After 24 hours of reaction, methanol was added to terminate the reaction, and the amount of TPA and MHET generated was tested by reverse phase HPLC analysis.

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Abstract

The present application belongs to the technical field of enzyme engineering, and specifically relates to a PET-mh mutant of a PET degrading enzyme, and an encoding gene, a recombinant plasmid, an engineered strain and the use thereof. The PET-mh mutant of a PET degrading enzyme provided in the present application is prepared by means of mutating amino acids at positions 29, 62, 63, 68, 124, 152, 176, 177, 183, 208 and 219 in a protein having an amino acid sequence as shown in SEQ ID NO: 1. Compared with the protein having the amino acid sequence as shown in SEQ ID NO: 1, the mutant has significantly improved activity or thermal stability, and has relatively high application value for implementing efficient degradation of PET. Further provided in the present application are an encoding gene encoding the mutant, a recombinant plasmid containing the encoding gene, an engineered strain containing the recombinant plasmid, and the use thereof.

Description

PET降解酶PET-mh突变体及其编码基因、重组质粒、工程菌和应用PET-degrading enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineering bacteria and application

本专利申请要求于2023年09月19日提交的中国专利申请No.CN202311211045.9的优先权。在先申请的公开内容通过整体引用并入本申请。This patent application claims the priority of Chinese patent application No. CN202311211045.9 filed on September 19, 2023. The disclosure of the prior application is incorporated into this application by reference in its entirety.

技术领域Technical Field

本申请涉及酶工程技术领域，具体涉及一种PET降解酶PET-mh突变体及其编码基因、重组质粒、工程菌和应用。The present application relates to the field of enzyme engineering technology, and in particular to a PET-degrading enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineering bacteria and application.

背景技术Background Art

聚对苯二甲酸乙二醇酯（polyethylene terephthalate, PET）是目前应用最广泛的塑料之一。其具有耐化学性强、热稳定性高、重量轻、储存方便等优点，可用于生产包装材料、矿泉水瓶、手机壳等众多生活用品，是一种常见的工程聚合物，年产量约为5000万吨。因为聚对苯二甲酸乙二醇酯中酯键和芳香族核的化学惰性，所以PET塑料的降解困难。随着PET塑料使用量的增加，PET废弃物在环境中大量累积，是造成“白色污染”问题的罪魁祸首之一。据报道，微米级PET已存在于食物中，成为人类食物链的一部分，可能威胁人类健康。Polyethylene terephthalate (PET) is one of the most widely used plastics at present. It has the advantages of strong chemical resistance, high thermal stability, light weight, and convenient storage. It can be used to produce packaging materials, mineral water bottles, mobile phone cases and many other daily necessities. It is a common engineering polymer with an annual output of about 50 million tons. Because of the chemical inertness of the ester bond and aromatic nucleus in polyethylene terephthalate, PET plastic is difficult to degrade. With the increase in the use of PET plastics, PET waste has accumulated in the environment in large quantities, which is one of the culprits of the "white pollution" problem. It is reported that micron-level PET has been found in food and has become part of the human food chain, which may threaten human health.

目前对PET废弃物的回收策略主要包括物理回收和化学回收，但这两种回收方式都容易对环境造成二次污染。利用酶或微生物将PET降解成单体分子对苯二甲酸（terephthalic acid, TPA）和乙二醇的生物法，具有降解条件温和（常温常压）、降解产物单一且易于回收、绿色环保等优势，符合可持续发展的理念，被认为是减少PET塑料污染最具潜力的方法。但生物法降解PET塑料过程中存在着酶稳定性差、催化效率低等问题。通过生物合成技术，研究人员已得到一些降解活性提高的PET降解酶，但用这些PET降解酶降解PET时需要先通过高温熔融重结晶和破碎机的微粉化，来降低PET的高结晶度和疏水表面对PET酶解的影响，使该降解过程复杂且成本较高。At present, the recycling strategies for PET waste mainly include physical recycling and chemical recycling, but both recycling methods are prone to secondary pollution to the environment. The biological method of using enzymes or microorganisms to degrade PET into monomer molecules of terephthalic acid (TPA) and ethylene glycol has the advantages of mild degradation conditions (normal temperature and pressure), single degradation products and easy recycling, and green environmental protection. It conforms to the concept of sustainable development and is considered to be the most promising method to reduce PET plastic pollution. However, there are problems such as poor enzyme stability and low catalytic efficiency in the biological degradation of PET plastics. Through biosynthesis technology, researchers have obtained some PET degradation enzymes with improved degradation activity, but when using these PET degradation enzymes to degrade PET, it is necessary to first reduce the high crystallinity and hydrophobic surface of PET on PET enzymatic hydrolysis through high-temperature melt recrystallization and crusher micronization, making the degradation process complicated and costly.

技术问题Technical issues

本申请提供了一种PET降解酶PET-mh突变体及其编码基因、重组质粒、工程菌和应用，该PET降解酶PET-mh突变体具有较高的活性和热稳定性，能够提高PET的降解效率，并且对高结晶度PET也能高效降解，从而能够简化PET降解工艺，降低成本。The present application provides a PET degrading enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineered bacteria and application. The PET degrading enzyme PET-mh mutant has high activity and thermal stability, can improve the degradation efficiency of PET, and can also efficiently degrade high-crystallinity PET, thereby simplifying the PET degradation process and reducing costs.

技术解决方案Technical Solutions

为达到上述申请目的，本申请采用了如下的技术方案：In order to achieve the above application purpose, this application adopts the following technical solutions:

第一方面，本申请提供一种PET降解酶PET-mh突变体，该PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第29、62、63、68、124、152、176、177、183、208、219位的氨基酸中的至少一个进行突变。In a first aspect, the present application provides a PET degrading enzyme PET-mh mutant, wherein the amino acid sequence of the PET degrading enzyme PET-mh mutant is as follows: at least one of the amino acids at positions 29, 62, 63, 68, 124, 152, 176, 177, 183, 208, and 219 in the amino acid sequence shown in SEQ ID No. 1 is mutated.

氨基酸序列如SEQ ID No.1所示的蛋白质对PET具有良好的降解活性，本申请通过对该氨基酸序列中特定位置的氨基酸进行突变，可获得活性和热稳定性优于该蛋白质的突变体，所得突变体能够提高降解PET的效率，为PET生物法规模转化和单体回收提供新的选择。The protein with an amino acid sequence as shown in SEQ ID No. 1 has good degradation activity for PET. The present application obtains a mutant with better activity and thermal stability than the protein by mutating the amino acid at a specific position in the amino acid sequence. The obtained mutant can improve the efficiency of PET degradation, providing a new option for PET bio-scale conversion and monomer recovery.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第29位的氨基酸突变为L-丙氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 29 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-alanine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第62位的氨基酸突变为L-苏氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 62 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-threonine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第63位的氨基酸突变为L-苏氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 63 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-threonine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第68位的氨基酸突变为L-天冬酰胺。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 68 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-asparagine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第124位的氨基酸突变为S-甘氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 124 in the amino acid sequence shown in SEQ ID No. 1 is mutated to S-glycine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第152位的氨基酸突变为L-甲硫氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 152 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-methionine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第176位的氨基酸突变为L-精氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 176 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-arginine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第177位的氨基酸突变为L-异亮氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 177 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-isoleucine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第183位的氨基酸突变为L-酪氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-tyrosine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第208位的氨基酸突变为L-苏氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 208 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-threonine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第219位的氨基酸突变为L-甲硫氨酸。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 219 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-methionine.

结合第一方面，在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第63、68、152、183、208、219位的氨基酸中的至少一个进行突变。In combination with the first aspect, in some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence: at least one of the amino acids at positions 63, 68, 152, 183, 208, and 219 in the amino acid sequence shown in SEQ ID No. 1 is mutated.

在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：至少将如SEQ ID No.1所示的氨基酸序列中第183位氨基酸进行突变。实验证明，即使仅对如SEQ ID No.1所示的氨基酸序列中第183位氨基酸进行突变，所得突变体的性能相对于该蛋白质也会获得大幅度提升。并且，将如SEQ ID No.1所示的氨基酸序列中第183位氨基酸与其他位点的氨基酸共同进行突变，还有可能获得协同效果。In some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence of at least mutating the amino acid at position 183 in the amino acid sequence as shown in SEQ ID No.1. Experiments have shown that even if only the amino acid at position 183 in the amino acid sequence as shown in SEQ ID No.1 is mutated, the performance of the resulting mutant is greatly improved relative to the protein. In addition, mutating the amino acid at position 183 in the amino acid sequence as shown in SEQ ID No.1 together with amino acids at other positions may also obtain a synergistic effect.

具体地，作为更优选的技术方案，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第63、68、124、152、176、177位的任何一个氨基酸以及第183位的氨基酸共同进行突变。上述组合突变的形式能够进一步提高降解PET的效率。Specifically, as a more preferred technical solution, the amino acid sequence of the PET degrading enzyme PET-mh mutant is as follows: any one of the amino acids at positions 63, 68, 124, 152, 176, 177 and the amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together. The above-mentioned combined mutation form can further improve the efficiency of PET degradation.

在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第124或152位的氨基酸以及第183位的氨基酸共同进行突变。In some embodiments, the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 124 or 152 and the amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together.

在一些实施例中，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第63、68、152、176、208位的任何一个氨基酸以及第124和第183位的氨基酸共同进行突变。其中更进一步优选地，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第208位氨基酸以及第124和第183位的氨基酸共同进行突变。In some embodiments, the amino acid sequence of the PET degrading enzyme PET-mh mutant is as follows: any one of the amino acids at positions 63, 68, 152, 176, and 208 and the amino acids at positions 124 and 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together. More preferably, the amino acid sequence of the PET degrading enzyme PET-mh mutant is as follows: the amino acid at position 208 and the amino acids at positions 124 and 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together.

高结晶度PET相对于低结晶度PET更难降解，在相同条件下降解效果更差。将如SEQ ID No.1所示的氨基酸序列中第183位的氨基酸进行突变，或第124或152位的氨基酸以及第183位的氨基酸共同进行突变，或第124、183和208位的氨基酸共同进行突变，所得突变体相对于该蛋白质来说，不仅能够显著提高对低结晶度PET的降解效率，还能大大提高对高结晶度PET的降解效率，具有更好的实用性。High crystallinity PET is more difficult to degrade than low crystallinity PET, and has a worse degradation effect under the same conditions. The amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 is mutated, or the amino acid at position 124 or 152 and the amino acid at position 183 are mutated together, or the amino acids at positions 124, 183 and 208 are mutated together. The resulting mutant can significantly improve the degradation efficiency of low crystallinity PET and the degradation efficiency of high crystallinity PET relative to the protein, and has better practicality.

第二方面，本申请还提供上述PET降解酶PET-mh突变体在水解PET中的应用：向浓度为80mM~120mM、pH值为7.5～8.5的磷酸钾缓冲液中加入所述PET降解酶PET-mh突变体至所述PET降解酶PET-mh突变体的终浓度为5μg/mL~15μg/mL，在温度为65℃～75℃，转速为100rpm～220rpm条件下进行PET降解。In a second aspect, the present application also provides the use of the above-mentioned PET degrading enzyme PET-mh mutant in hydrolyzing PET: adding the PET degrading enzyme PET-mh mutant to a potassium phosphate buffer with a concentration of 80mM~120mM and a pH value of 7.5~8.5 until the final concentration of the PET degrading enzyme PET-mh mutant is 5μg/mL~15μg/mL, and performing PET degradation at a temperature of 65°C~75°C and a rotation speed of 100rpm~220rpm.

第三方面，本申请还提供一种编码基因，包含编码上述PET降解酶PET-mh突变体的核苷酸序列。In a third aspect, the present application also provides a coding gene comprising a nucleotide sequence encoding the above-mentioned PET degrading enzyme PET-mh mutant.

结合第三方面，所述PET降解酶PET-mh突变体的氨基酸序列如SEQ ID No.2~SEQ ID No.23所示，其核苷酸序列分别如SEQ ID No.24~SEQ ID No.45所示。In combination with the third aspect, the amino acid sequence of the PET degrading enzyme PET-mh mutant is shown as SEQ ID No.2 to SEQ ID No.23, and its nucleotide sequence is shown as SEQ ID No.24 to SEQ ID No.45 respectively.

第四方面，本申请还提供一种重组质粒，包含上述编码基因的核苷酸序列。In a fourth aspect, the present application also provides a recombinant plasmid comprising the nucleotide sequence of the above-mentioned encoding gene.

第五方面，本申请还提供上述重组质粒的构建方法：以编码SEQ ID No.1所示氨基酸的核苷酸序列为模板，利用对应引物构建单点突变，先通过PCR反应对目的基因进行指数扩增，然后利用限制性内切酶Dpn I去除模板链，接着利用DNA连接酶快速环化PCR产物，获得重组质粒。In the fifth aspect, the present application also provides a method for constructing the above-mentioned recombinant plasmid: using the nucleotide sequence encoding the amino acid shown in SEQ ID No.1 as a template, a single point mutation is constructed using the corresponding primers, the target gene is first exponentially amplified by PCR reaction, and then the template chain is removed using the restriction endonuclease Dpn I, and then the PCR product is quickly circularized using DNA ligase to obtain a recombinant plasmid.

第六方面，本申请还提供一种工程菌，包括具有上述重组质粒的宿主细胞。In a sixth aspect, the present application also provides an engineered bacterium, including a host cell having the above-mentioned recombinant plasmid.

结合第六方面，所述工程菌为大肠杆菌。In combination with the sixth aspect, the engineered bacteria is Escherichia coli.

第七方面，本申请还提供上述工程菌的构建方法：将上述重组质粒转化入BL21(DE3)感受态细胞，涂布于氨苄青霉素平板培养基，得到阳性重组子，即为所述工程菌。In a seventh aspect, the present application also provides a method for constructing the above-mentioned engineered bacteria: transforming the above-mentioned recombinant plasmid into BL21 (DE3) competent cells, spreading them on ampicillin plate culture medium, and obtaining positive recombinants, namely the engineered bacteria.

第八方面，本申请还提供上述工程菌在降解PET中的应用。In an eighth aspect, the present application also provides the use of the above-mentioned engineered bacteria in the degradation of PET.

示例性地，用上述工程菌降解PET的方法可参考如下操作：将上述工程菌在LB液体培养基中培养至OD600值达到0.8~1.0范围内，添加诱导剂IPTG（异丙基β-D-1-硫代半乳糖吡喃糖苷）至所述诱导剂IPTG的浓度为0.1mM ~0.15mM，在温度为15℃~17℃条件下诱导表达20h~24h，得到发酵培养物；Exemplarily, the method for degrading PET using the above-mentioned engineered bacteria can refer to the following operation: the above-mentioned engineered bacteria are cultured in LB liquid culture medium until the OD600 value reaches the range of 0.8 to 1.0, the inducer IPTG (isopropyl β-D-1-thiogalactopyranoside) is added to the concentration of the inducer IPTG to be 0.1 mM to 0.15 mM, and the expression is induced at a temperature of 15° C. to 17° C. for 20 h to 24 h to obtain a fermentation culture;

将发酵培养物离心，用破菌缓冲液吹吸重悬，再次离心，获得含PET降解酶PET-mh突变体的粗酶液；将粗酶液进行蛋白纯化后浓缩，得到浓缩液，作为降解PET的生物催化剂；The fermentation culture is centrifuged, resuspended by pipetting with a bacteria-breaking buffer, and centrifuged again to obtain a crude enzyme solution containing a PET-mh mutant of a PET-degrading enzyme; the crude enzyme solution is protein purified and then concentrated to obtain a concentrated solution as a biocatalyst for degrading PET;

将待降解的PET薄膜底物，加入PET反应液中，在65℃-75℃下进行生物催化反应，PET反应液中含有pH=8.0磷酸钾缓冲液以及降解PET的生物催化剂。The PET film substrate to be degraded is added into the PET reaction solution, and a biocatalytic reaction is carried out at 65° C.-75° C. The PET reaction solution contains a pH=8.0 potassium phosphate buffer and a biocatalyst for degrading PET.

有益效果Beneficial Effects

本申请的有益效果在于：本申请提供的PET降解酶PET-mh突变体相对于氨基酸序列如SEQ ID No.1所示的蛋白质而言，具有更高的活性和热稳定性，催化效率大幅提高，能够更有效地降解PET塑料废弃物，有利于对PET废弃物进行资源再生和二次开发，不仅能够缓解生产PET所需的石油资源的压力，同时也能解决PET废弃物造成的严重的环境污染问题，符合可持续的发展理念，并且还能提高PET的回收价值，推进塑料经济的实现。The beneficial effects of the present application are as follows: the PET-mh mutant of the PET degrading enzyme provided by the present application has higher activity and thermal stability than the protein with an amino acid sequence as shown in SEQ ID No. 1, and the catalytic efficiency is greatly improved, and it can more effectively degrade PET plastic waste, which is beneficial to the resource regeneration and secondary development of PET waste, and can not only alleviate the pressure of petroleum resources required for the production of PET, but also solve the serious environmental pollution problem caused by PET waste, which is in line with the concept of sustainable development, and can also increase the recycling value of PET and promote the realization of the plastic economy.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为本申请检验例1中单独对第63、68、124、183位氨基酸进行突变后的PET降解酶PET-mh突变体对低结晶度PET的降解效果；FIG1 shows the degradation effect of the PET-mh mutant of the PET degrading enzyme after the amino acids at positions 63, 68, 124, and 183 are mutated separately in Test Example 1 of the present application on low-crystallinity PET;

图2为本申请检验例1中对第63、68、124、152、176、177位的任何一个氨基酸以及第183位氨基酸共同突变后的PET降解酶PET-mh突变体对低结晶度PET的降解效果；FIG2 shows the degradation effect of the PET-mh mutant of the PET degrading enzyme on low-crystallinity PET after mutation of any one of the amino acids at positions 63, 68, 124, 152, 176, 177 and the amino acid at position 183 in Test Example 1 of the present application;

图3为本申请检验例1中对第63、68、152、176、208位的任何一个氨基酸以及第124和第183位氨基酸共同突变后的PET降解酶PET-mh突变体对低结晶度PET的降解效果；FIG3 shows the degradation effect of the PET-mh mutant of the PET degrading enzyme on low-crystallinity PET after mutation of any one of the amino acids at positions 63, 68, 152, 176, 208 and the amino acids at positions 124 and 183 in Test Example 1 of the present application;

图4为本申请检验例2中单独对第183位氨基酸进行突变的PET降解酶PET-mh突变体，对第124或152位氨基酸以及第183位氨基酸共同突变的PET降解酶PET-mh突变体，对第124、183和208位氨基酸共同突变后的PET降解酶PET-mh突变体对高结晶度PET的降解效果。Figure 4 shows the degradation effect of the PET-mh mutant of the PET degrading enzyme in which the amino acid at position 183 is mutated alone, the PET-mh mutant of the PET degrading enzyme in which the amino acid at position 124 or 152 and the amino acid at position 183 are mutated together, and the PET-mh mutant of the PET degrading enzyme in which the amino acids at positions 124, 183 and 208 are mutated together on high crystallinity PET in Test Example 2 of the present application.

图中，ICCG表示亲本PET降解酶，即包括如SEQ ID No .1所示氨基酸序列的酶。横坐标中的数字表示突变点位置的编号，例如，63、68分别表示第63、68位氨基酸进行了突变；176-183表示第176和183位氨基酸共同进行了突变；124-183-208表示第124、183和208位氨基酸共同进行了突变。In the figure, ICCG represents the parent PET degrading enzyme, i.e., the enzyme including the amino acid sequence shown in SEQ ID No. 1. The numbers in the abscissa represent the numbers of the mutation points, for example, 63 and 68 represent that the amino acids at positions 63 and 68 are mutated, respectively; 176-183 represents that the amino acids at positions 176 and 183 are mutated together; and 124-183-208 represents that the amino acids at positions 124, 183, and 208 are mutated together.

本发明的实施方式Embodiments of the present invention

为了使本申请的目的、技术方案及优点更加清楚明白，以下结合具体实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本申请，并不用于限定本申请。In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

本申请中，PET降解酶PET-mh突变体的突变点位置的编号对应于原始PET降解酶SEQ ID No .1所示的氨基酸序列编号。氨基酸采用单字母简称进行表示，如S29A表示原始PET降解酶SEQ ID No .1所示的氨基酸序列中第29位的S替换（突变）为A。In the present application, the numbering of the mutation point position of the PET degrading enzyme PET-mh mutant corresponds to the amino acid sequence numbering of the original PET degrading enzyme SEQ ID No. 1. Amino acids are represented by single-letter abbreviations, such as S29A, which means that the S at position 29 in the amino acid sequence of the original PET degrading enzyme SEQ ID No. 1 is replaced (mutated) with A.

随着我国经济的快速发展，PET制品以价格低廉、轻质、方便、可塑性强等特点被大量地生产和使用，而由此产生的PET废弃物将造成难以想象的污染。利用酶或微生物将PET降解成单体分子对苯二甲酸（TPA）和乙二醇的生物法，被认为是减少PET塑料污染最具潜力的方法。但生物法降解PET塑料过程中存在着酶稳定性差、催化效率低等问题。With the rapid development of my country's economy, PET products are being produced and used in large quantities due to their low price, light weight, convenience, and strong plasticity. The resulting PET waste will cause unimaginable pollution. The biological method of using enzymes or microorganisms to degrade PET into monomer molecules terephthalic acid (TPA) and ethylene glycol is considered to be the most promising method to reduce PET plastic pollution. However, there are problems such as poor enzyme stability and low catalytic efficiency in the biological degradation of PET plastics.

对酶的氨基酸序列进行改造是获得新功能的常用方式，但并非任何改造都能获得具有理想功能的突变体。Modifying the amino acid sequence of an enzyme is a common way to obtain new functions, but not all modifications can produce mutants with ideal functions.

为了解决上述问题，本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第29、62、63、68、124、152、176、177、183、208、219位的氨基酸中的至少一个进行突变而得。其中，对第177位的氨基酸进行突变，可将结晶聚合物的熔点（melting temperature, Tm）提升至99.6℃；对其他位置的氨基酸进行突变，能够在不同程度地提高热稳定性的同时，还提升其降解PET的效率。In order to solve the above problems, the embodiment of the present application provides a PET degrading enzyme PET-mh mutant, which is obtained by mutating at least one of the amino acids at positions 29, 62, 63, 68, 124, 152, 176, 177, 183, 208, and 219 in the amino acid sequence shown in SEQ ID No. 1. Among them, mutation of the amino acid at position 177 can increase the melting point (melting temperature, Tm) of the crystalline polymer to 99.6°C; mutation of the amino acids at other positions can improve the thermal stability to varying degrees while also improving the efficiency of PET degradation.

本申请实施例还提供了上述PET降解酶PET-mh突变体的构建方法及其在水解PET中的应用。The embodiments of the present application also provide a method for constructing the above-mentioned PET degrading enzyme PET-mh mutant and its application in hydrolyzing PET.

本申请实施例还提供了编码上述突变体的基因，其核苷酸序列如SEQ ID No.24~SEQ ID No.45所示。The examples of the present application also provide a gene encoding the above mutant, and its nucleotide sequence is shown in SEQ ID No.24 to SEQ ID No.45.

本申请实施例还提供了含有上述核苷酸序列的重组质粒及其构建方法以及用该重组质粒构建的工程菌。The embodiments of the present application also provide a recombinant plasmid containing the above nucleotide sequence and a construction method thereof, as well as an engineered bacterium constructed using the recombinant plasmid.

本申请实施例还提供了该工程菌的构建方法及其在降解PET中的应用。The examples of the present application also provide a method for constructing the engineered bacteria and its application in degrading PET.

以下通过具体实施例来对本申请的方案进行说明。The solution of the present application is described below through specific embodiments.

以下实施例中的试剂如无特殊说明，均来源于市售或按照本领域公知方法取得。Unless otherwise specified, the reagents in the following examples are commercially available or obtained according to methods known in the art.

实施例1Example 1

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第29位的L-丝氨酸突变为L-丙氨酸，所得突变体的氨基酸序列如SEQ ID No.2所示，核苷酸序列如SEQ ID No.24所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-serine at position 29 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-alanine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.2, and the nucleotide sequence is shown in SEQ ID No.24.

实施例2Example 2

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第62位的L-丙氨酸突变为L-苏氨酸，所得突变体的氨基酸序列如SEQ ID No.3所示，核苷酸序列如SEQ ID No.25所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 62 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.25.

实施例3Example 3

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第63位的L-天冬氨酸突变为L-苏氨酸，所得突变体的氨基酸序列如SEQ ID No.4所示，核苷酸序列如SEQ ID No.26所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-aspartic acid at position 63 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.4, and the nucleotide sequence is shown in SEQ ID No.26.

实施例4Example 4

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第68位的L-丙氨酸突变为L-天冬酰胺，所得突变体的氨基酸序列如SEQ ID No.5所示，核苷酸序列如SEQ ID No.27所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 68 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-asparagine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.27.

实施例5Example 5

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第124位的L-亮氨酸突变为S-甘氨酸，所得突变体的氨基酸序列如SEQ ID No.6所示，核苷酸序列如SEQ ID No.28所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.6, and the nucleotide sequence is shown in SEQ ID No.28.

实施例6Example 6

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第152位的L-亮氨酸突变为L-甲硫氨酸，所得突变体的氨基酸序列如SEQ ID No.7所示，核苷酸序列如SEQ ID No.29所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 152 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-methionine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.7, and the nucleotide sequence is shown in SEQ ID No.29.

实施例7Example 7

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第176位的L-苏氨酸突变为L-精氨酸，所得突变体的氨基酸序列如SEQ ID No.8所示，核苷酸序列如SEQ ID No.30所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-threonine at position 176 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-arginine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.8, and the nucleotide sequence is shown in SEQ ID No.30.

实施例8Example 8

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第177位的L-缬氨酸突变为L-异亮氨酸，所得突变体的氨基酸序列如SEQ ID No.9所示，核苷酸序列如SEQ ID No.31所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-valine at position 177 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-isoleucine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.9, and the nucleotide sequence is shown in SEQ ID No.31.

实施例9Embodiment 9

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.10所示，核苷酸序列如SEQ ID No.32所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-histidine at position 183 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-tyrosine, and the amino acid sequence of the obtained mutant is shown in SEQ ID No.10, and the nucleotide sequence is shown in SEQ ID No.32.

实施例10Example 10

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第208位的L-异亮氨酸突变为L-苏氨酸，所得突变体的氨基酸序列如SEQ ID No.11所示，核苷酸序列如SEQ ID No.33所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-isoleucine at position 208 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the amino acid sequence of the obtained mutant is shown in SEQ ID No.11, and the nucleotide sequence is shown in SEQ ID No.33.

实施例11Embodiment 11

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第219位的L-缬氨酸突变为L-甲硫氨酸，所得突变体的氨基酸序列如SEQ ID No.12所示，核苷酸序列如SEQ ID No.34所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-valine at position 219 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-methionine, and the amino acid sequence of the obtained mutant is shown in SEQ ID No.12, and the nucleotide sequence is shown in SEQ ID No.34.

实施例12Example 12

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第63位的L-天冬氨酸突变为L-苏氨酸，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.13所示，核苷酸序列如SEQ ID No.35所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-aspartic acid at position 63 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.13, and the nucleotide sequence is shown in SEQ ID No.35.

实施例13Embodiment 13

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第68位的L-丙氨酸突变为L-天冬酰胺，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.14所示，核苷酸序列如SEQ ID No.36所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 68 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-asparagine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.14, and the nucleotide sequence is shown in SEQ ID No.36.

实施例14Embodiment 14

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第124位的L-亮氨酸突变为S-甘氨酸，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.15所示，核苷酸序列如SEQ ID No.37所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.15, and the nucleotide sequence is shown in SEQ ID No.37.

实施例15Embodiment 15

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第152位的L-亮氨酸突变为L-甲硫氨酸，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.16所示，核苷酸序列如SEQ ID No.38所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 152 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-methionine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.16, and the nucleotide sequence is shown in SEQ ID No.38.

实施例16Example 16

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第176位的L-苏氨酸突变为L-精氨酸，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.17所示，核苷酸序列如SEQ ID No.39所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-threonine at position 176 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-arginine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.17, and the nucleotide sequence is shown in SEQ ID No.39.

实施例17Embodiment 17

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第177位的L-缬氨酸突变为L-异亮氨酸，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.18所示，核苷酸序列如SEQ ID No.40所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-valine at position 177 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-isoleucine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.18, and the nucleotide sequence is shown in SEQ ID No.40.

实施例18Embodiment 18

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第63位的L-天冬氨酸突变为L-苏氨酸，第124位的L-亮氨酸突变为S-甘氨酸，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.19所示，核苷酸序列如SEQ ID No.41所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-aspartic acid at position 63 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-threonine, the L-leucine at position 124 is mutated to S-glycine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.19, and the nucleotide sequence is shown in SEQ ID No.41.

实施例19Embodiment 19

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第68位的L-丙氨酸突变为L-天冬酰胺，第124位的L-亮氨酸突变为S-甘氨酸，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.20所示，核苷酸序列如SEQ ID No.42所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-alanine at position 68 in the amino acid sequence shown in SEQ ID No.1 is mutated to L-asparagine, the L-leucine at position 124 is mutated to S-glycine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.20, and the nucleotide sequence is shown in SEQ ID No.42.

实施例20Embodiment 20

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第124位的L-亮氨酸突变为S-甘氨酸，第152位的L-丙氨酸突变为L-天冬酰胺，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.21所示，核苷酸序列如SEQ ID No.43所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the L-alanine at position 152 is mutated to L-asparagine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.21, and the nucleotide sequence is shown in SEQ ID No.43.

实施例21Embodiment 21

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第124位的L-亮氨酸突变为S-甘氨酸，第176位的L-苏氨酸突变为L-精氨酸，第183位的L-组氨酸突变为L-酪氨酸，所得突变体的氨基酸序列如SEQ ID No.22所示，核苷酸序列如SEQ ID No.44所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the L-threonine at position 176 is mutated to L-arginine, and the L-histidine at position 183 is mutated to L-tyrosine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.22, and the nucleotide sequence is shown in SEQ ID No.44.

实施例22Example 22

本申请实施例提供了一种PET降解酶PET-mh突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第124位的L-亮氨酸突变为S-甘氨酸，第183位的L-组氨酸突变为L-酪氨酸，第208位的L-异亮氨酸突变为L-苏氨酸，所得突变体的氨基酸序列如SEQ ID No.23所示，核苷酸序列如SEQ ID No.45所示。The embodiment of the present application provides a PET degrading enzyme PET-mh mutant, in which the L-leucine at position 124 in the amino acid sequence shown in SEQ ID No.1 is mutated to S-glycine, the L-histidine at position 183 is mutated to L-tyrosine, and the L-isoleucine at position 208 is mutated to L-threonine. The amino acid sequence of the obtained mutant is shown in SEQ ID No.23, and the nucleotide sequence is shown in SEQ ID No.45.

实施例23Embodiment 23

本申请实施例提供了实施例1~22中的PET降解酶PET-mh突变体在水解PET中的应用：The present application provides the use of the PET-mh mutant of the PET degrading enzyme in Examples 1 to 22 in hydrolyzing PET:

以待降解的PET作为底物，向300μL、pH值为8的磷酸钾缓冲液中加入各实施例的PET降解酶PET-mh突变体至终浓度为500nM，作为PET反应液，在60℃～75℃的水浴条件下进行PET降解。Using the PET to be degraded as a substrate, the PET-mh mutant of the PET degrading enzyme of each example was added to 300 μL of potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as the PET reaction solution, and PET degradation was performed in a water bath at 60° C. to 75° C.

实施例24Embodiment 24

本申请实施例提供了含有编码实施例1~22中PET降解酶PET-mh突变体的基因的重组质粒以及构建方法：The present application provides a recombinant plasmid containing a gene encoding the PET-mh mutant of the PET degrading enzyme in Examples 1 to 22 and a construction method thereof:

以含有编码实施例1中PET降解酶PET-mh突变体的基因的重组质粒为例：Take the recombinant plasmid containing the gene encoding the PET-mh mutant of the PET degrading enzyme in Example 1 as an example:

1. 以编码SEQ ID No.1所示氨基酸的核苷酸序列为模板，利用对应引物，先通过PCR反应对目的基因进行指数扩增，构建PET-mh突变体（PET-mh-1），设计引物如下：1. Using the nucleotide sequence encoding the amino acids shown in SEQ ID No.1 as a template and the corresponding primers, the target gene was exponentially amplified by PCR reaction to construct a PET-mh mutant (PET-mh-1). The primers were designed as follows:

上游引物P1Upstream primer P1

5’- CACCGTTGCGCGGCT -3’5’- CACCGTTGCGCGGCT -3’

下游引物P2Downstream primer P2

5’- CAAACGGTGTAGGTTGCCAC -3’5’- CAAACGGTGTAGGTTGCCAC-3’

在PCR反应体系中，以P1和P2作为上、下游引物，以ICCG酶基因（SEQ ID No.1）为模板，进行PCR反应，对目的基因进行指数扩增。In the PCR reaction system, P1 and P2 were used as upstream and downstream primers, and the ICCG enzyme gene (SEQ ID No. 1) was used as a template to perform a PCR reaction and exponentially amplify the target gene.

其扩增的反应体系为：The amplification reaction system is:

扩增程序为：95℃预变性2min；95℃变性20s，Tm-5℃退火20s，72℃延伸2min，反应30个循环；72℃延伸5min。The amplification program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s, annealing at Tm-5°C for 20 s, extension at 72°C for 2 min, 30 cycles; and extension at 72°C for 5 min.

PCR扩增产物经1%琼脂糖凝胶电泳，用DNA回收试剂盒回收PCR产物，得到含有单点突变的PET-mh突变体的基因。The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the PCR products were recovered using a DNA recovery kit to obtain the gene of the PET-mh mutant containing a single point mutation.

2. 然后利用限制性内切酶Dpn I去除模板链，接着利用DNA连接酶快速环化PCR产物，获得含有编码实施例1中PET降解酶PET-mh突变体的基因的重组质粒。2. The template strand was then removed using restriction endonuclease Dpn I, followed by rapid circularization of the PCR product using DNA ligase to obtain a recombinant plasmid containing the gene encoding the PET-mh mutant of the PET degrading enzyme in Example 1.

按同样方法可构建含有编码实施例2~22中PET降解酶PET-mh突变体的基因的重组质粒。构建含有编码不同实施例中PET降解酶PET-mh突变体的基因的重组质粒过程中，PCR扩增体系和程序相同，区别在于设计的上、下游引物以及模板。在构建含有编码实施例1~11中PET降解酶PET-mh突变体的基因的重组质粒过程时，以编码SEQ ID No.1所示氨基酸的核苷酸序列（SEQ ID No.46）为模板，利用对应引物，先通过PCR反应对目的基因进行指数扩增，构建相应的PET-mh突变体（PET-mh-1~PET-mh-11）；在构建含有编码实施例12~17中PET降解酶PET-mh突变体的基因的重组质粒过程时，以编码SEQ ID No.10所示氨基酸的核苷酸序列（SEQ ID No.32）为模板，利用对应引物，先通过PCR反应对目的基因进行指数扩增，构建相应的PET-mh突变体（PET-mh-12~PET-mh-17）；在构建含有编码实施例18~22中PET降解酶PET-mh突变体的基因的重组质粒过程时，以编码SEQ ID No.15所示氨基酸的核苷酸序列（SEQ ID No.37）为模板，利用对应引物，先通过PCR反应对目的基因进行指数扩增，构建相应的PET-mh突变体（PET-mh-18~ PET-mh-22）。具体如表1所示：The same method can be used to construct a recombinant plasmid containing a gene encoding a PET-mh mutant of the PET degrading enzyme in Examples 2 to 22. In the process of constructing a recombinant plasmid containing a gene encoding a PET-mh mutant of the PET degrading enzyme in different embodiments, the PCR amplification system and procedure are the same, and the difference lies in the designed upstream and downstream primers and templates. In the process of constructing a recombinant plasmid containing a gene encoding a PET-mh mutant of the PET degrading enzyme in Examples 1 to 11, the nucleotide sequence encoding the amino acid shown in SEQ ID No. 1 (SEQ ID No. 46) was used as a template, and the corresponding primers were used to exponentially amplify the target gene by PCR reaction to construct the corresponding PET-mh mutants (PET-mh-1 to PET-mh-11); in the process of constructing a recombinant plasmid containing a gene encoding a PET-mh mutant of the PET degrading enzyme in Examples 12 to 17, the nucleotide sequence encoding the amino acid shown in SEQ ID No. 10 (SEQ ID No. 32) was used as a template, and the corresponding primers were used to exponentially amplify the target gene by PCR reaction to construct the corresponding PET-mh mutants (PET-mh-12 to PET-mh-17); in the process of constructing a recombinant plasmid containing a gene encoding a PET-mh mutant of the PET degrading enzyme in Examples 18 to 22, the nucleotide sequence encoding the amino acid shown in SEQ ID No. 15 (SEQ ID No.37) as a template, using the corresponding primers, firstly exponentially amplify the target gene through PCR reaction, and construct the corresponding PET-mh mutants (PET-mh-18~PET-mh-22). The details are shown in Table 1:

表1 构建含有编码实施例2~22中PET降解酶突变体的基因的重组质粒设计的引物Table 1 Primers designed for constructing recombinant plasmids containing genes encoding PET degrading enzyme mutants in Examples 2 to 22

实施例25Embodiment 25

本申请实施例提供了含有实施例24重组质粒的工程菌及其构建方法：The present application provides an engineering bacterium containing the recombinant plasmid of Example 24 and a method for constructing the same:

将实施例24的重组质粒转化入BL21(DE3)（全式金，CD601）感受态细胞，涂布于氨苄青霉素平板培养基，得到阳性重组子，即为含有实施例24中各重组质粒的大肠杆菌工程菌。The recombinant plasmid of Example 24 was transformed into BL21 (DE3) (full gold, CD601) competent cells and spread on ampicillin plate culture medium to obtain positive recombinants, which are Escherichia coli engineered bacteria containing the recombinant plasmids in Example 24.

实施例26Embodiment 26

本申请实施例提供了实施例25中的工程菌在降解PET中的应用：The present application example provides the use of the engineered bacteria in Example 25 in degrading PET:

将实施例25构建的大肠杆菌工程菌首先在5 mL LB液体培养基（含100 mg/L氨苄抗性）中培养12h-16h，然后按1 %接种量转接至摇瓶中培养。当OD600值达到0.8-1.0范围内时，添加诱导剂IPTG（异丙基β-D-1-硫代半乳糖吡喃糖苷）至浓度0.1 mM，16 ℃诱导表达22 h，得到发酵培养物。The E. coli engineered bacteria constructed in Example 25 were first cultured in 5 mL LB liquid medium (containing 100 mg/L ampicillin resistance) for 12 h-16 h, and then transferred to a shake flask for culture at a 1% inoculum. When the OD600 value reached the range of 0.8-1.0, the inducer IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to a concentration of 0.1 mM, and the expression was induced at 16 ° C for 22 h to obtain a fermentation culture.

将发酵培养物在4000 rpm下离心20 min，获得含PET-mh突变体的菌体浆状物，用破菌缓冲液（每升破菌缓冲液含有50mM Tris-HCl、150mM NaCl和10mM 咪唑，pH=7.5）吹吸重悬，通过超高压连续流细胞破碎机破碎细胞得到破碎菌液。该破碎菌液在4 ℃、10000 rpm下离心1 h，取上清液，获得含PET-mh突变体的粗酶液。将粗酶液用0.45 μm滤膜过滤，与Ni介质混匀后放入4 ℃、180 rpm全温震荡培养箱中混匀至少1 h。然后通过Ni-NTA填充柱柱层析进行蛋白纯化，先用洗杂缓冲液（每升洗杂缓冲液含有50mM Tris-HCl、150mM NaCl和20mM 咪唑，pH=7.5）洗去杂蛋白，然后用洗脱缓冲液（每升洗脱缓冲液含有50mM Tris-HCl、300mM NaCl和300mM咪唑，pH=7.5）洗脱，收集洗脱液体，即蛋白纯化液。通过蛋白浓缩管将蛋白纯化液的浓度浓缩至5 mg/mL，收集浓缩液，保存在4 ℃冰箱中。之后将浓缩液添加至催化体系中（终浓度为：10μg/mL）作为降解PET的生物催化剂。The fermentation culture was centrifuged at 4000 rpm for 20 min to obtain a bacterial slurry containing the PET-mh mutant, which was resuspended by aspiration in a bacterial lysis buffer (each liter of bacterial lysis buffer contained 50mM Tris-HCl, 150mM NaCl and 10mM imidazole, pH=7.5), and the cells were broken by an ultra-high pressure continuous flow cell disruptor to obtain a broken bacterial solution. The broken bacterial solution was centrifuged at 4 ℃ and 10000 rpm for 1 h, and the supernatant was taken to obtain a crude enzyme solution containing the PET-mh mutant. The crude enzyme solution was filtered with a 0.45 μm filter membrane, mixed with the Ni medium, and placed in a full-temperature shaking incubator at 4 ℃ and 180 rpm for at least 1 h. Then, the protein was purified by Ni-NTA column chromatography. The impurities were first washed with washing buffer (each liter of washing buffer contained 50mM Tris-HCl, 150mM NaCl and 20mM imidazole, pH = 7.5), and then eluted with elution buffer (each liter of elution buffer contained 50mM Tris-HCl, 300mM NaCl and 300mM imidazole, pH = 7.5). The eluted liquid was collected, i.e., the protein purification liquid. The protein purification liquid was concentrated to 5 mg/mL through a protein concentrator tube, and the concentrated liquid was collected and stored in a refrigerator at 4 °C. The concentrated liquid was then added to the catalytic system (final concentration: 10μg/mL) as a biocatalyst for degrading PET.

取待降解的PET底物，加入300μL的PET反应液中（每升PET反应液中含有100 mM磷酸钾缓冲液，pH=8.0，添加PET-mh突变体蛋白至终浓度为10μg/mL），将其置于65℃-75℃下进行生物催化反应。Take the PET substrate to be degraded, add it to 300 μL of PET reaction solution (each liter of PET reaction solution contains 100 mM potassium phosphate buffer, pH=8.0, and PET-mh mutant protein is added to a final concentration of 10 μg/mL), and place it at 65°C-75°C for biocatalytic reaction.

对比例1Comparative Example 1

本对比例提供了一种PET降解酶突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第211位的L-丙氨酸突变为L-精氨酸。This comparative example provides a PET degrading enzyme mutant, in which the L-alanine at position 211 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-arginine.

对比例2Comparative Example 2

本对比例提供了一种PET降解酶突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第212位的L-丝氨酸突变为L-精氨酸。This comparative example provides a PET degradation enzyme mutant, in which the L-serine at position 212 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-arginine.

对比例3Comparative Example 3

本对比例提供了一种PET降解酶突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第179位的L-脯氨酸突变为L-谷氨酸。This comparative example provides a PET degrading enzyme mutant, in which the L-proline at position 179 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-glutamic acid.

对比例4Comparative Example 4

本对比例提供了一种PET降解酶突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第66位的L-丝氨酸突变为L-丙氨酸。This comparative example provides a PET degradation enzyme mutant, in which the L-serine at position 66 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-alanine.

对比例5Comparative Example 5

本对比例提供了一种PET降解酶突变体，该突变体是将如SEQ ID No.1所示的氨基酸序列中第66位的L-丙氨酸突变为S-甘氨酸。This comparative example provides a PET degrading enzyme mutant, in which the L-alanine at position 66 in the amino acid sequence shown in SEQ ID No. 1 is mutated to S-glycine.

检验例1Test Example 1

按实施例23的方法考察氨基酸序列如SEQ ID No.1所示的蛋白质、实施例1~22以及对比例1~5的PET降解酶突变体对低结晶度PET降解5h的效果。According to the method of Example 23, the effects of the protein with an amino acid sequence as shown in SEQ ID No. 1, the PET degrading enzyme mutants of Examples 1 to 22 and Comparative Examples 1 to 5 on the degradation of low-crystallinity PET for 5 h were investigated.

以结晶度为7.3%的PET膜（直径6mm）作为底物，向300μL、pH值为8的磷酸钾缓冲液中分别独立地加入氨基酸序列如SEQ ID No.1所示的蛋白质、各实施例的PET降解酶PET-mh突变体或各对比例的PET降解酶突变体至终浓度为500nM，作为PET反应液，在60℃-75℃的水浴条件下进行PET降解。反应5h后，加入甲醇终止反应，利用反相HPLC分析测试TPA、双（2-羟乙基）对苯二甲酸酯（terephthalic acid bis(2-hydroxyethyl) ester, BHET）和对苯二甲酸2-羟乙基单酯（4-((2-Hydroxyethoxy)carbonyl)benzoic acid, MHET）生成量。BHET和MHET是酶在降解PET过程中的中间产物。A PET film (6 mm in diameter) with a crystallinity of 7.3% was used as a substrate. A protein with an amino acid sequence as shown in SEQ ID No. 1, a PET degrading enzyme PET-mh mutant of each embodiment, or a PET degrading enzyme mutant of each comparative example was added independently to 300 μL of a potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as a PET reaction solution, and PET degradation was performed under a water bath condition of 60° C.-75° C. After reacting for 5 hours, methanol was added to terminate the reaction, and the production of TPA, terephthalic acid bis(2-hydroxyethyl) ester (BHET) and terephthalic acid 2-hydroxyethyl monoester (4-((2-Hydroxyethoxy)carbonyl)benzoic acid, MHET) was tested by reverse phase HPLC analysis. BHET and MHET are intermediate products of the enzyme in the process of degrading PET.

结果如表2所示。The results are shown in Table 2.

表2 TPA、BHET和MHET生成量（低结晶度，5h）Table 2 TPA, BHET and MHET production (low crystallinity, 5h)

由以上结果可见，单独对第63、68、124、183位氨基酸进行突变，或对第63、68、124、152、176、177位的任何一个氨基酸以及第183位氨基酸共同突变，或对第63、68、152、176、208位的任何一个氨基酸以及第124和183位氨基酸共同突变，所得突变体在降解5h时即可获得高于氨基酸序列如SEQ ID No.1所示的蛋白质的降解效果。MHET和TPA的产量见图1~图3。From the above results, it can be seen that by mutating the amino acids at positions 63, 68, 124, and 183 alone, or mutating any one of the amino acids at positions 63, 68, 124, 152, 176, and 177 and the amino acid at position 183 together, or mutating any one of the amino acids at positions 63, 68, 152, 176, and 208 and the amino acids at positions 124 and 183 together, the resulting mutants can achieve a higher degradation effect than the protein with the amino acid sequence shown in SEQ ID No. 1 when degraded for 5 hours. The yields of MHET and TPA are shown in Figures 1 to 3.

检验例2Test Example 2

本检验例按实施例23的方法考察了氨基酸序列如SEQ ID No.1所示的蛋白质和实施例9、14、15、22的PET降解酶PET-mh突变体对高结晶度PET降解5h的效果。This test example investigated the effect of the protein with the amino acid sequence shown in SEQ ID No. 1 and the PET-mh mutant of the PET degrading enzyme of Examples 9, 14, 15 and 22 on the degradation of high-crystallinity PET for 5 h according to the method of Example 23.

以结晶度为26%的PET塑料瓶研磨得到PET颗粒（100mg）作为底物，向300μL、pH值为8的磷酸钾缓冲液中分别独立地加入氨基酸序列如SEQ ID No.1所示的蛋白质或各实施例的PET降解酶PET-mh突变体至终浓度为500nM，作为PET反应液，在70℃的水浴条件下进行PET降解，反应5h后，加入甲醇终止反应，利用反相HPLC分析测试TPA、BHET和MHET生成量。PET particles (100 mg) obtained by grinding a PET plastic bottle with a crystallinity of 26% were used as a substrate. The protein with an amino acid sequence as shown in SEQ ID No. 1 or the PET degrading enzyme PET-mh mutant of each embodiment was independently added to 300 μL of potassium phosphate buffer with a pH value of 8 to a final concentration of 500 nM as a PET reaction solution. PET degradation was carried out in a water bath at 70° C. After the reaction for 5 hours, methanol was added to terminate the reaction, and the generated amounts of TPA, BHET and MHET were tested by reverse phase HPLC analysis.

结果如表3所示。The results are shown in Table 3.

表3 TPA、BHET和MHET生成量（高结晶度，5h）Table 3 TPA, BHET and MHET production (high crystallinity, 5h)

由以上结果可见，单独对第183位氨基酸进行突变，或对第124或152位氨基酸以及第183位氨基酸共同突变，或对第124、183和208位氨基酸共同突变，所得突变体对高结晶度PET在降解5h时均能获得高于氨基酸序列如SEQ ID No.1所示的蛋白质的降解效果。MHET和TPA的产量见图4。From the above results, it can be seen that the mutants obtained by mutating the amino acid at position 183 alone, or mutating the amino acid at positions 124 or 152 and the amino acid at position 183, or mutating the amino acids at positions 124, 183 and 208 together, can achieve a higher degradation effect on high-crystalline PET than the protein with the amino acid sequence shown in SEQ ID No. 1 when degrading for 5 hours. The yields of MHET and TPA are shown in Figure 4.

检验例3Test Example 3

本检验例按实施例23的方法考察了氨基酸序列如SEQ ID No.1所示的蛋白质和实施例1、2、6、7、10、11的PET降解酶PET-mh突变体对不同结晶度PET降解24h的效果。This test example investigated the effect of the protein with an amino acid sequence as shown in SEQ ID No. 1 and the PET-mh mutant of the PET degrading enzyme of Examples 1, 2, 6, 7, 10, and 11 on the degradation of PET with different crystallinity for 24 hours according to the method of Example 23.

以不同结晶度的PET作为底物，向300μL、pH值为8的磷酸钾缓冲液中分别独立地加入氨基酸序列如SEQ ID No.1所示的蛋白质或各实施例的PET降解酶PET-mh突变体至终浓度为500nM，作为PET反应液，在60℃-75℃的水浴条件下进行PET降解。反应24h后，加入甲醇终止反应，利用反相HPLC分析测试TPA和MHET生成量。Using PET with different crystallinity as substrate, the protein with amino acid sequence as shown in SEQ ID No. 1 or the PET-mh mutant of each embodiment was added to 300 μL potassium phosphate buffer with pH 8 to a final concentration of 500 nM as PET reaction solution, and PET degradation was carried out in a water bath at 60° C.-75° C. After 24 hours of reaction, methanol was added to terminate the reaction, and the amount of TPA and MHET generated was tested by reverse phase HPLC analysis.

结果如表4所示。The results are shown in Table 4.

表4 TPA和MHET生成量（24h）Table 4 TPA and MHET production (24h)

由以上结果可见，单独对第29、62、152、176、208或219位氨基酸进行突变，所得突变体对不同形态和结晶度的PET在降解24h时均能获得高于氨基酸序列如SEQ ID No.1所示的蛋白质的降解效果。From the above results, it can be seen that by mutating the amino acid at position 29, 62, 152, 176, 208 or 219 alone, the mutants obtained can achieve a higher degradation effect on PET of different morphologies and crystallinities than the protein with the amino acid sequence shown in SEQ ID No. 1 when degrading for 24 hours.

以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本申请的保护范围之内。The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims (17)

一种PET降解酶PET-mh突变体，其特征在于，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第29、62、63、68、124、152、176、177、183、208、219位的氨基酸中的至少一个进行突变。A PET degrading enzyme PET-mh mutant, characterized in that the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: at least one of the amino acids at positions 29, 62, 63, 68, 124, 152, 176, 177, 183, 208, and 219 in the amino acid sequence shown in SEQ ID No.1 is mutated. 根据权利要求1所述的PET降解酶PET-mh突变体，其特征在于，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第29位的氨基酸突变为L-丙氨酸；和/或The PET degrading enzyme PET-mh mutant according to claim 1, characterized in that the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 29 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-alanine; and/or 将如SEQ ID No.1所示的氨基酸序列中第62位的氨基酸突变为L-苏氨酸；和/或The amino acid at position 62 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-threonine; and/or 将如SEQ ID No.1所示的氨基酸序列中第63位的氨基酸突变为L-苏氨酸；和/或The amino acid at position 63 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-threonine; and/or 将如SEQ ID No.1所示的氨基酸序列中第68位的氨基酸突变为L-天冬酰胺；和/或The amino acid at position 68 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-asparagine; and/or 将如SEQ ID No.1所示的氨基酸序列中第124位的氨基酸突变为S-甘氨酸；和/或The amino acid at position 124 in the amino acid sequence shown in SEQ ID No. 1 is mutated to S-glycine; and/or 将如SEQ ID No.1所示的氨基酸序列中第152位的氨基酸突变为L-甲硫氨酸；和/或The amino acid at position 152 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-methionine; and/or 将如SEQ ID No.1所示的氨基酸序列中第176位的氨基酸突变为L-精氨酸；和/或The amino acid at position 176 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-arginine; and/or 将如SEQ ID No.1所示的氨基酸序列中第177位的氨基酸突变为L-异亮氨酸；和/或The amino acid at position 177 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-isoleucine; and/or 将如SEQ ID No.1所示的氨基酸序列中第183位的氨基酸突变为L-酪氨酸；和/或The amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-tyrosine; and/or 将如SEQ ID No.1所示的氨基酸序列中第208位的氨基酸突变为L-苏氨酸；和/或The amino acid at position 208 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-threonine; and/or 将如SEQ ID No.1所示的氨基酸序列中第219位的氨基酸突变为L-甲硫氨酸。The amino acid at position 219 in the amino acid sequence shown in SEQ ID No. 1 is mutated to L-methionine. 根据权利要求1或2所述的PET降解酶PET-mh突变体，其特征在于，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第63、68、152、183、208、219位的氨基酸中的至少一个进行突变。The PET degrading enzyme PET-mh mutant according to claim 1 or 2, characterized in that the PET degrading enzyme PET-mh mutant has an amino acid sequence in which at least one of the amino acids at positions 63, 68, 152, 183, 208, and 219 in the amino acid sequence shown in SEQ ID No. 1 is mutated. 根据权利要求3所述的PET降解酶PET-mh突变体，其特征在于，所述PET降解酶PET-mh突变体具有的氨基酸序列为：至少将如SEQ ID No.1所示的氨基酸序列中第183位氨基酸进行突变。The PET degrading enzyme PET-mh mutant according to claim 3 is characterized in that the amino acid sequence of the PET degrading enzyme PET-mh mutant is: at least the 183rd amino acid in the amino acid sequence shown in SEQ ID No.1 is mutated. 根据权利要求4所述的PET降解酶PET-mh突变体，其特征在于，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第63、68、124、152、176、177位的任何一个氨基酸以及第183位的氨基酸共同进行突变。The PET degrading enzyme PET-mh mutant according to claim 4 is characterized in that the amino acid sequence of the PET degrading enzyme PET-mh mutant is as follows: any one of the amino acids at positions 63, 68, 124, 152, 176, 177 and the amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together. 根据权利要求5所述的PET降解酶PET-mh突变体，其特征在于，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第124或152位的氨基酸以及第183位的氨基酸共同进行突变。The PET degrading enzyme PET-mh mutant according to claim 5, characterized in that the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the amino acid at position 124 or 152 and the amino acid at position 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together. 根据权利要求4所述的PET降解酶PET-mh突变体，其特征在于，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第63、68、152、176、208位的任何一个氨基酸以及第124和第183位的氨基酸共同进行突变。The PET degrading enzyme PET-mh mutant according to claim 4 is characterized in that the amino acid sequence of the PET degrading enzyme PET-mh mutant is as follows: any one of the amino acids at positions 63, 68, 152, 176, and 208 and the amino acids at positions 124 and 183 in the amino acid sequence shown in SEQ ID No. 1 are mutated together. 根据权利要求7所述的PET降解酶PET-mh突变体，其特征在于，所述PET降解酶PET-mh突变体具有的氨基酸序列为：将如SEQ ID No.1所示的氨基酸序列中第208位氨基酸以及第124和第183位的氨基酸共同进行突变。The PET degrading enzyme PET-mh mutant according to claim 7 is characterized in that the PET degrading enzyme PET-mh mutant has an amino acid sequence as follows: the 208th amino acid and the 124th and 183rd amino acids in the amino acid sequence shown in SEQ ID No. 1 are mutated together. 权利要求1~8任一项所述的PET降解酶PET-mh突变体在水解PET中的应用，其特征在于，向浓度为80mM~120mM、pH值为7.5～8.5的磷酸钾缓冲液中加入所述PET降解酶PET-mh突变体至所述PET降解酶PET-mh突变体的终浓度为5μg/mL ~15μg/mL，在温度为65℃～75℃，转速为100rpm～220rpm条件下进行PET降解。The use of the PET degrading enzyme PET-mh mutant according to any one of claims 1 to 8 in the hydrolysis of PET is characterized in that the PET degrading enzyme PET-mh mutant is added to a potassium phosphate buffer having a concentration of 80mM to 120mM and a pH value of 7.5 to 8.5 until the final concentration of the PET degrading enzyme PET-mh mutant is 5μg/mL to 15μg/mL, and PET degradation is carried out at a temperature of 65°C to 75°C and a rotation speed of 100rpm to 220rpm. 一种编码基因，其特征在于，包含编码权利要求1~8任一项所述的PET降解酶PET-mh突变体的核苷酸序列。A coding gene, characterized in that it comprises a nucleotide sequence encoding the PET-mh mutant of the PET degrading enzyme according to any one of claims 1 to 8. 根据权利要求10所述的编码基因，其特征在于，所述PET降解酶PET-mh突变体的氨基酸序列如SEQ ID No.2~SEQ ID No.23所示，其对应的核苷酸序列分别如SEQ ID No.24~SEQ ID No.45所示。The encoding gene according to claim 10, characterized in that the amino acid sequence of the PET degrading enzyme PET-mh mutant is shown in SEQ ID No.2 to SEQ ID No.23, and the corresponding nucleotide sequences are shown in SEQ ID No.24 to SEQ ID No.45, respectively. 一种重组质粒，其特征在于，包含权利要求10或11所述编码基因的核苷酸序列。A recombinant plasmid, characterized in that it contains the nucleotide sequence encoding the gene according to claim 10 or 11. 权利要求12所述的重组质粒的构建方法，其特征在于，以编码SEQ ID No.1所示氨基酸的核苷酸序列为模板，利用对应引物构建单点突变，先通过PCR反应对目的基因进行指数扩增，然后利用限制性内切酶Dpn I去除模板链，接着利用DNA连接酶快速环化PCR产物，获得重组质粒。The method for constructing a recombinant plasmid according to claim 12 is characterized in that a nucleotide sequence encoding the amino acid shown in SEQ ID No. 1 is used as a template, a single point mutation is constructed using corresponding primers, the target gene is first exponentially amplified by a PCR reaction, and then the template chain is removed using a restriction endonuclease Dpn I, and then the PCR product is quickly circularized using a DNA ligase to obtain a recombinant plasmid. 一种工程菌，其特征在于，包括具有权利要求12所述的重组质粒的宿主细胞。An engineered bacterium, characterized in that it comprises a host cell having the recombinant plasmid according to claim 12. 根据权利要求14所述的工程菌，其特征在于，所述工程菌为大肠杆菌。The engineered bacteria according to claim 14, characterized in that the engineered bacteria is Escherichia coli. 权利要求15所述的工程菌的构建方法，其特征在于，将权利要求12所述重组质粒转化入BL21(DE3)感受态细胞，涂布于氨苄青霉素平板培养基，得到阳性重组子，即为所述工程菌。The method for constructing the engineered bacteria according to claim 15 is characterized in that the recombinant plasmid according to claim 12 is transformed into BL21 (DE3) competent cells, spread on ampicillin plate culture medium, and obtain positive recombinants, namely the engineered bacteria. 权利要求14或15所述的工程菌在降解PET中的应用。Use of the engineered bacteria described in claim 14 or 15 in degrading PET.
PCT/CN2023/143044 2023-09-19 2023-12-29 Pet-mh mutant of pet degrading enzyme, and encoding gene, recombinant plasmid, engineered strain and use thereof Pending WO2025060302A1 (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018011284A1 (en) * 2016-07-12 2018-01-18 Carbios Novel esterases and uses thereof
CN112159478A (en) * 2020-09-28 2021-01-01 江南大学 Fusion protein and application thereof in polymer degradation
CN112673104A (en) * 2018-07-27 2021-04-16 卡比奥斯公司 Novel esterases and their use
CN113454213A (en) * 2019-02-21 2021-09-28 汉高股份有限及两合公司 Improvement of care properties for polyester textiles by using cutinases
CN116286727A (en) * 2023-03-06 2023-06-23 山东大学 A kind of cutinase variant and its preparation method and application in plastic degradation
CN117187212A (en) * 2023-09-19 2023-12-08 源天生物科技(天津)有限公司 PET-degrading enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineering bacteria and applications

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018011284A1 (en) * 2016-07-12 2018-01-18 Carbios Novel esterases and uses thereof
CN112673104A (en) * 2018-07-27 2021-04-16 卡比奥斯公司 Novel esterases and their use
CN113454213A (en) * 2019-02-21 2021-09-28 汉高股份有限及两合公司 Improvement of care properties for polyester textiles by using cutinases
CN112159478A (en) * 2020-09-28 2021-01-01 江南大学 Fusion protein and application thereof in polymer degradation
CN116286727A (en) * 2023-03-06 2023-06-23 山东大学 A kind of cutinase variant and its preparation method and application in plastic degradation
CN117187212A (en) * 2023-09-19 2023-12-08 源天生物科技(天津)有限公司 PET-degrading enzyme PET-mh mutant and its encoding gene, recombinant plasmid, engineering bacteria and applications

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