[go: up one dir, main page]

WO2025129034A2 - Estérases et procédés associés - Google Patents

Estérases et procédés associés Download PDF

Info

Publication number
WO2025129034A2
WO2025129034A2 PCT/US2024/060086 US2024060086W WO2025129034A2 WO 2025129034 A2 WO2025129034 A2 WO 2025129034A2 US 2024060086 W US2024060086 W US 2024060086W WO 2025129034 A2 WO2025129034 A2 WO 2025129034A2
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
esterase
petase
substitution
terephthalate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/060086
Other languages
English (en)
Other versions
WO2025129034A3 (fr
Inventor
Pengfei TIAN
Trine Holst SORENSEN
Stephen Merritt JONES
Molly Krisann GIBSON
Alexandra Rachael SNEIDER
Brett Adam BOGHIGIAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Flagship Pioneering Innovations VII Inc
Original Assignee
Flagship Pioneering Innovations VII Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Flagship Pioneering Innovations VII Inc filed Critical Flagship Pioneering Innovations VII Inc
Publication of WO2025129034A2 publication Critical patent/WO2025129034A2/fr
Publication of WO2025129034A3 publication Critical patent/WO2025129034A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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)
    • 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
    • C08J11/105—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 by treatment with enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01001—Carboxylesterase (3.1.1.1)
    • 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
    • C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
    • C08J2367/02—Polyesters derived from dicarboxylic acids and dihydroxy compounds
    • 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

  • esterases e.g., PETases
  • polynucleotides encoding the same fusions and conjugates comprising an esterase (e.g., PETase); methods of manufacturing; compositions; and methods of use including, e.g., methods of hydrolyzing a terephthalic acid ester, hydrolyzing a polyester, and degrading a polyester containing product (e.g., a plastic product).
  • the degradation results in the production of any one or more of terephthalic acid (TPA), mono-(2-hydroxyethyl) terephthalate (MHET), bis-(2-hydroxyethyl) terephthalate (BHET), and/or and ethylene glycol (EG) (or any combination thereof).
  • TPA terephthalic acid
  • MHET mono-(2-hydroxyethyl) terephthalate
  • BHET bis-(2-hydroxyethyl) terephthalate
  • EG ethylene glycol
  • the plastic product comprises a textile, container ⁇ e.g., bag, bottle), film, or powder.
  • compositions comprising the recovered MHET, BHET, TPA, and/or EG, or any combination thereof.
  • kits for producing PET comprising utilizing the MHET, BHET, TPA, and/or EG, or any combination thereof, produced by any one or more of the methods described herein or recovered, isolated, and/or purified by according to any one or more of the methods described herein.
  • the method comprises a polycondensation process.
  • compositions comprising the PET made by any one or more of the methods described herein.
  • the composition comprises a polyester.
  • the composition is a plastic product.
  • the plastic product is a textile, container ⁇ e.g., bag, bottle), film, or powder.
  • kits for producing a plastic product comprising utilizing the MHET, BHET, TPA, and/or EG, or any combination thereof, produced by any one or more of the methods described herein or recovered, isolated, and/or purified by any one or more of the methods described herein.
  • the method comprises a polycondensation process.
  • the plastic product comprises PET.
  • the plastic product comprises a textile, container ⁇ e.g., bag, bottle), film, or powder.
  • PET Polyethylene terephthalate
  • PET is one of the most commonly used and a major contributor to global plastics waste pollution.
  • PET is commonly used for example in e.g., textiles, packing materials, plastic bottles, food jars, and films.
  • PET does not readily decompose in nature, contributing, at least in pail, to its major role in environmental plastic pollution.
  • Approaches to deal with the problem of plastic waste products have typically included incineration, disposal in landfills, and mechanical disintegration. However, these approaches also have a significant environmental impact. Enzymatic and chemical methods of degradation have been considered but are relatively inefficient with the enzymes tested or require the use of relatively hazardous chemicals, respectively.
  • PET degrading enzymes i.e., PETases
  • the inventors have, inter alia, discovered novel esterases (e.g., PETases).
  • the esterases e.g., PETases
  • the current disclosure provides, inter alia, esterases (e.g., PETases) capable of hydrolyzing PET; as well as nucleic acids encoding the same, cell and compositions comprising the same.
  • the activity (e.g., hydrolase activity, esterase activity, PETase activity) of the esterase is increased by at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, 10000- fold relative to the activity (e.g., esterase activity, hydrolase activity, PETase activity, cutinase activity, lipase activity, acyltransferase activity) of a reference esterase (e.g., PETase) (e.g., a reference esterase comprising the amino acid sequence set forth in SEQ ID NO: 1).
  • a reference esterase e.g., PETase
  • a reference esterase comprising the amino acid sequence set forth in SEQ ID NO: 1
  • the esterase e.g., PETase
  • the esterase has enzymatic activity (e.g., esterase activity, hydrolase activity, PETase activity, cutinase activity, lipase activity, acyltransferase activity) at a range of temperatures.
  • the esterase e.g., PETasc
  • has enzymatic activity e.g., esterase activity, hydrolase activity, PETase activity, cutinase activity, lipase activity, acyltransferase activity
  • enzymatic activity e.g., esterase activity, hydrolase activity, PETase activity, cutinase activity, lipase activity, acyltransferase activity
  • Suitable methods of determining or measuring the activity e.g., enzymatic activity (e.g., esterase activity, hydrolase activity, PETase activity, cutinase activity, lipase activity, acyltransferase activity) of a protein will be familial’ to persons skilled in the art.
  • enzymatic activity e.g., esterase activity, hydrolase activity, PETase activity, cutinase activity, lipase activity, acyltransferase activity
  • Example 2 set forth herein describes an exemplary method of determining PETase activity.
  • the PETase activity of a protein described herein is determined as set forth in Example 2.
  • fusion proteins comprising an esterase (e.g., PETase) (e.g., described herein) (or a functional fragment, functional variant, or domain thereof) and one or more heterologous protein (or a functional fragment, functional variant, or domain thereof).
  • conjugates comprising an esterase (e.g., PETase) (e.g., described herein) (or a functional fragment, functional variant, or domain thereof) (or a nucleic acid molecule encoding an esterase (e.g., PETasc) (or a functional fragment, functional variant, or domain thereof) described herein) and one or more heterologous moiety.
  • Heterologous moieties include, but are not limited to, proteins, peptides, small molecules, nucleic acid molecules e.g., DNA, RNA, DNA/RNA hybrid molecules), carbohydrates, lipids, and polymers (e.g., synthetic polymers).
  • the esterase e.g., PETase
  • the esterase is operably connected to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more heterologous moieties.
  • the esterase e.g., PETase
  • the esterase is operably connected to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, but no more than 10 heterologous moieties.
  • the esterase e.g., PETase
  • the esterase e.g., PETase
  • a functional fragment, functional variant, or domain thereof is operably connected to no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, heterologous moieties.
  • the heterologous moiety is a protein.
  • fusion proteins comprising an esterase (e.g., PETase) (e.g., described herein) (or a functional fragment, functional variant, or domain thereof) and one or more heterologous protein.
  • esterase e.g., PETase
  • heterologous protein e.g., any heterologous protein described herein
  • the fusion protein comprises more than one heterologous protein. In some embodiments, the fusion protein comprises a plurality of heterologous proteins.
  • the esterase e.g., PETase
  • the esterase is operably connected to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more heterologous proteins. In some embodiments, the esterase (e.g., PETase) (or a functional fragment, functional variant, or domain thereof) is operably connected to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, but no more than 10 heterologous proteins. In some embodiments, the esterase (e.g., PETase) (or a functional fragment, functional variant, or domain thereof) is operably connected to no more than
  • the esterase e.g., PETase
  • the esterase is operably connected to from about 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 heterologous proteins (or a functional fragment, functional variant, or domain thereof).
  • the esterase e.g., PETase
  • the esterase is operably connected to about 1 ,
  • the heterologous protein is a cellular export signal peptide. In some embodiments, the heterologous protein is an N-terminal cellular export signal peptide.
  • the heterologous protein is an enzyme. In some embodiments, the heterologous protein exhibits enzymatic activity (e.g., MHETase activity).
  • a heterologous moiety e.g., heterologous protein
  • an esterase e.g., PETase
  • the heterologous protein is directly operably connected to an esterase (e.g., PETase) (e.g., described herein).
  • a heterologous polypeptide is directly operably connected to an esterase (e.g., PETase) (e.g., described herein) via a peptide bond.
  • a heterologous protein is indirectly operably connected to an esterase (e.g., PETase) (e.g., described herein). In some embodiments, a heterologous protein is indirectly operably connected to an esterase (e.g., PETase) (e.g., described herein) via a linker.
  • an esterase e.g., PETase
  • a heterologous protein is indirectly operably connected to an esterase (e.g., PETase) (e.g., described herein) via a peptide linker.
  • an esterase e.g., PETase
  • a peptide linker is one or any combination of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and/or a non-hclical linker.
  • an amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers set forth in Table 3.
  • the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers set forth in Table 3, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions).
  • the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers set forth in Table 3, comprising 1, 2, or 3 amino acid variations e.g., substitutions, deletions, additions).
  • the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers set forth in Table 3, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers set forth in Table 3, comprising 1, 2, or 3 amino acid substitutions.
  • the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 26-85, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 26-85, comprising 1, 2, or 3 amino acid substitutions.
  • a heterologous moiety e.g., heterologous protein
  • the N-terminus of the esterase e.g., PETase
  • a heterologous moiety e.g., heterologous protein
  • the C-terminus of the esterase e.g., PETase
  • Proteins described herein may be produced using standard methods known in the art. For example, each may be produced by recombinant technology in host cells (e.g., insect cells, mammalian cells, bacteria) that have been transfected or transduced with a nucleic acid expression vector (e.g., plasmid, viral vector e.g., a baculoviral expression vector)) encoding the protein (e.g., the esterase, fusion protein, etc.).
  • host cells e.g., insect cells, mammalian cells, bacteria
  • a nucleic acid expression vector e.g., plasmid, viral vector e.g., a baculoviral expression vector
  • a nucleic acid expression vector e.g., plasmid, viral vector e.g., a baculoviral expression vector
  • the expression vector typically contains an expression cassette that includes nucleic acid sequences capable of bringing about expression of the nucleic acid molecule encoding the protein of interest (e.g., the esterase (e.g., PETase), fusion protein, etc.), such as promoter(s), enhancer(s), polyadenylation signals, and the like.
  • the protein of interest e.g., the esterase (e.g., PETase), fusion protein, etc.
  • promoter(s), enhancer(s), polyadenylation signals e.g., the esterase (e.g., PETase), fusion protein, etc.)
  • promoter(s), enhancer(s), polyadenylation signals e.g., polyadenylation signals, and the like.
  • promoters can be constitutive or regulated, and can be obtained from various sources, e.g., viruses, prokaryotic or eukaryotic sources, or artificially designed.
  • the produced protein may be isolated from the cell cultures, by, for example, column chromatography in either flow-flow through or bind-and-elute modes. Examples include, but are not limited to, ion exchange resins and affinity resins, such as lentil lectin Sepharose, and mixed mode cation exchange-hydrophobic interaction columns (CEX-HIC).
  • the protein may be concentrated, buffer exchanged by ultrafiltration, and the rctcntatc from the ultrafiltration may be filtered through an appropriate filter, e.g., a 0.22pm filter. See, e.g., hacker, David (Ed.), Recombinant Protein Expression in Mammalian Cells: Methods and Protocols (Methods in Molecular Biology), Humana Press (2016).
  • polynucleotides e.g., DNA, RNA
  • any protein described herein including, e.g., an esterase (e.g., a PETase) described herein (see, e.g., ⁇
  • a fusion protein described herein see, e.g., ⁇ 4.3
  • a conjugate described herein see, e.g., ⁇
  • the polynucleotide is a DNA polynucleotide or an RNA polynucleotide. In some embodiments, the polynucleotide is a translatable RNA polynucleotide. In some embodiments, the polynucleotide is an mRNA polynucleotide. In some embodiments, the polynucleotide is a circular- RNA polynucleotide. In some embodiments, the polynucleotide is a translatable circular RNA polynucleotide.
  • the vector is a non-viral vector. In some embodiments, the vector is a minicirclc. In some embodiments, the vector is a plasmid.
  • plasmid DNA may be generated to allow efficient production of the encoded esterases in cell lines, e.g., in insect cell lines, for example using vectors as described in W02009150222A2 and as defined in PCT claims 1 to 33, the disclosure relating to claim 1 to 33 of W02009150222A2 the entire contents of which is incorporated by reference herein for all purposes. 4.6.2 Viral Vectors
  • Exemplary viral vectors include, but are not limited to, adenovirus vectors, adeno- associated virus vectors, lentivirus vectors, retrovirus vectors, poxvirus vectors, parapoxivirus vectors, vaccinia virus vectors, fowlpox virus vectors, herpes virus vectors, adeno-associated vims vectors, alphavirus vectors, lentivirus vectors, rhabdovirus vectors, measles vims, Newcastle disease vims vectors, picornavimses vectors, or lymphocytic choriomeningitis virus vectors.
  • the vector is an adenoviral vector (e.g., human adenoviral vector, e.g., HAdV or AdHu).
  • the adenovirus vector has the El region deleted, rendering it replication-deficient in human cells. Other regions of the adenovirus such as E3 and E4 may also be deleted.
  • Exemplary adenovirus vectors include, but are not limited to, those described in e.g., W02005071093 or WQ2006048215, the entire contents of each of which is incorporated by reference herein for all purposes.
  • the adenovirus-based vector used is a simian adenovims, thereby avoiding dampening of the immune response after vaccination by pre-existing antibodies to common human entities such as AdHu5.
  • simian adenovirus vectors include AdCh63 (see, e.g., W02005071093, the entire contents of which is incorporated by reference herein for all purposes) or AdCh68.
  • Viral vectors can be generated through the use of a packaging/produccr cell line (e.g., a mammalian cell line) using standard methods known to the person of ordinary skill in the art.
  • a nucleic acid construct e.g., a plasmid
  • the transgene e.g., an immunogenic peptide or protein described herein
  • additional elements e.g., a promoter, inverted terminal repeats (ITRs) flanking the transgene
  • a plasmid encoding e.g., viral replication and structural proteins along with one or more helper plasmids
  • a host cell e.g., a host cell line
  • a host cell line i.e., the packing/producer cell line
  • helper plasmid may also be needed that include helper genes from another virus (e.g., in the instance of adeno-associated viral vectors).
  • Eukaryotic expression plasmids are commercially available from a variety of suppliers, for example the plasmid series: pcDNATM, pCR3.1 TM, pCMVTM, pFRTTM, pVAXl TM, pCITM, NanoplasmidTM, and Pcaggs.
  • the person of ordinary skill in the art is aware of numerous transfection methods and any suitable method of transfection may be employed (e.g., using a biochemical substance as carrier (e.g., lipofectamine), by mechanical means, or by electroporation,).
  • the cells are cultured under conditions suitable and for a sufficient time for plasmid expression.
  • the viral particles may be purified from the cell culture medium using standard methods known to the person of ordinary skill in the art. For example, by centrifugation followed by e.g., chromatography or ultrafiltration.
  • cells e.g., host cells
  • a population of cells e.g., a population of host cells
  • an esterase e.g., PETase
  • a fusion protein described herein
  • a conjugate described herein see, e.g., ⁇ 4.3
  • a polynucleotide described herein see, e.g., ⁇ 4.5
  • a vector described herein see, e.g., ⁇ 4.6.
  • the cell e.g., host cell
  • an esterase e.g., PETase
  • the cell can be useful, e.g., in the production (expression) of an esterase (e.g., PETase) described herein.
  • the cell is a prokaryotic cell.
  • the cell is a bacteria cell.
  • the bacterium is a Gram-positive bacterium.
  • the bacterium is a Gram-negative bacterium.
  • the cell is a eukaryotic cell.
  • Exemplary eukaryotic cells include, e.g., yeast, fungal, mammalian, insect, and plant cells.
  • the cell is mammalian cell.
  • the cell is an animal cell.
  • the cell is a yeast cell.
  • the cell is an insect cell.
  • the cell is a fungal cell.
  • Suitable host cells will be familiar to persons of ordinary skill in the ait, illustrative examples of which include, but are not limited to a recombinant Bacillus, recombinant E. coli, recombinant Pseudomonas, recombinant Aspergillus, recombinant Trichoderma, recombinant Streptomyces, recombinant Saccharomyces, recombinant Pichia, recombinant Thermus or recombinant Yarrowia.
  • Standard methods known in the art can be utilized to deliver any one of the foregoing (e.g., esterase e.g., PETase) protein, fusion protein, vector, polynucleotide, carrier, etc.) into a cell (e.g., a host cell).
  • Standard methods known in the art can be utilized to culture cells (e.g., host cells) in vitro or ex vivo.
  • the composition may comprise the esterase (e.g., PETase) protein, polynucleotide, vector, cell, etc. and optionally one or more excipient and /or reagent etc.
  • excipients may include buffers commonly used in biochemistry, agents for adjusting pH, preservatives such as sodium benzoate, sodium sorbate or sodium ascorbate, conservatives, protective or stabilizing agents such as starch, dextrin, arabic gum, salts, sugars e.g., sorbitol, trehalose or lactose, glycerol, polyethyleneglycol, polyethene glycol, polypropylene glycol, propylene glycol, divalent ions such as calcium, sequestering agent such as EDTA, reducing agents (c-g- , bcta-mcrcaptocthanol, dithiothrcitol, ascorbic acid, tris(2- carboxyethyl)phosphin
  • the composition comprises an esterase (e.g., PETase) protein described herein (the protein may be present in the composition in an isolated or at least partially purified form).
  • the esterase (e.g., PETase) described herein is solubilized in an aqueous medium together with one or more excipients, such as excipients that may suitably stabilize or protect the esterase (e.g., PETase) from degradation.
  • esterase e.g., PETase
  • the esterase may be solubilized in water and then admixed with excipients such as glycerol, sorbitol, dextrin, starch, glycol such as propanediol, salt, etc.
  • excipients such as glycerol, sorbitol, dextrin, starch, glycol such as propanediol, salt, etc.
  • the resulting admixture may then be dried so as to obtain a powder.
  • Methods for drying such mixture are well known to the one skilled in the art and include, without limitation, lyophilization, freeze-drying, spraydrying, supercritical drying, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyer drying, fluidized bed drying, drum drying or any combination thereof.
  • kits comprising any one or more of an esterase (e.g., PETase) described herein (see, e.g., ⁇ 4.2), a fusion protein described herein (see, e.g., ⁇ 4.3), a conjugate described herein (see, e.g., ⁇ 4.3), a polynucleotide described herein (see, e.g., ⁇ 4.5), a vector described herein (see, e.g., ⁇ 4.6), a cell (or population of cells) described herein (see, e.g., ⁇ 4.7)), and/or a composition described herein (see, e.g., ⁇ 4.8).
  • the kit may comprise a technical instruction.
  • the technical instructions of the kit may contain information about preparation and/or use of the any of the foregoing.
  • a terephthalic acid ester comprising contacting a terephthalic acid ester with an esterase (e.g., PETase) described herein (see, e.g., ⁇ 4.2), a fusion protein described herein (see, e.g., ⁇ 4.3), a conjugate described herein (see, e.g., ⁇ 4.3), a polynucleotide described herein (see, e.g., ⁇ 4.5), a vector described herein (see, e.g., ⁇ 4.6), a cell (or population of cells) described herein (see, e.g., ⁇ 4.7)), and/or a composition described herein (see, e.g., ⁇ 4.8), to thereby hydrolyze the terephthalic acid ester.
  • an esterase e.g., PETase
  • a fusion protein described herein see, e.g., ⁇ 4.3
  • the method is carried out under conditions sufficient to hydrolyze the terephthalic acid ester. In some embodiments, the method is carried out under conditions sufficient to convert the terephthalic acid ester to any one or more of terephthalic acid, ethylene glycol, mono-(2- hydroxyethyl) terephthalate, and/or bis-(2-hydroxyethyl) terephthalate.
  • the terephthalic acid ester is a mono-terephthalic acid ester, a di-terephthalic acid ester, or a poly-terephthalic acid ester.
  • the terephthalic acid ester is a polyester.
  • the polyester comprises polyethylene terephthalate (PET), polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycapro lactone (PCL), polyethylene adipate (PEA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), or any combination of any of the foregoing.
  • the polyester comprises PET.
  • the hydrolysis results in the conversion of the terephthalic acid ester to terephthalic acid, mono-(2-hydroxyethyl) terephthalate (MHET), bis-(2-hydroxyethyl) terephthalate (BHET), and/or and ethylene glycol (EG).
  • the hydrolysis results in the production of any one or more of terephthalic acid (TPA), mono-(2-hydroxyethyl) terephthalate (MHET), bis-(2-hydroxyethyl) terephthalate (BHET), and/or and ethylene glycol (EG).
  • the hydrolysis results in the production of mono-(2-hydroxyethyl) terephthalate (MHET).
  • the method further comprises recovering, isolating, and/or purifying the MHET, BHET, TP A, and/or EG, or any combination thereof.
  • a polyethylene terephthalate comprising contacting a PET with an esterase (e.g., PETase) described herein (see, e.g., ⁇ 4.2), a fusion protein described herein (see, e.g., ⁇ 4.3), a conjugate described herein (see, e.g., ⁇ 4.3), a polynucleotide described herein (see, e.g., ⁇ 4.5), a vector described herein (see, e.g., ⁇ 4.6), a cell (or population of cells) described herein (see, e.g., ⁇ 4.7)), and/or a composition described herein (see, e.g., ⁇ 4.8), to thereby hydrolyze the PET.
  • an esterase e.g., PETase
  • a fusion protein described herein see, e.g., ⁇ 4.3
  • a conjugate described herein see, e.g., ⁇ 4.3
  • the method is carried out under conditions sufficient to hydrolyze the PET. In some embodiments, the method is carried out under conditions sufficient to convert the PET to any one or more of terephthalic acid (TPA), ethylene glycol, mono-(2- hydroxyethyl) terephthalate, and/or bis-(2-hydroxyethyl) terephthalate.
  • TPA terephthalic acid
  • ethylene glycol mono-(2- hydroxyethyl) terephthalate
  • bis-(2-hydroxyethyl) terephthalate bis-(2-hydroxyethyl) terephthalate.
  • the PET is part of a polyester.
  • the polyester additionally comprises polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycapro lactone (PCL), polyethylene adipate (PEA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), or any combination of any of the foregoing.
  • PLA polylactic acid
  • PTT polytrimethylene terephthalate
  • PBT polybutylene terephthalate
  • PEIT polyethylene isosorbide terephthalate
  • PET polyethylene terephthalate
  • PBS poly
  • the PET is part of a mixture or blend of more than one material. Additional materials include, for example, but are not limited to, metal, ceramic, glass, wood and polymers (e.g., a polyamide (e.g., nylon), polyolefin (e.g., polyethylene and polypropylene), or polyvinylchloride (PVC)).
  • the PET is selectively hydrolyzed.
  • the PET is an admixture with one or more co-material (e.g., as a melt blend or laminated structure).
  • the hydrolysis results in the conversion of the PET to any one or more of terephthalic acid (TPA), mono-(2-hydroxyethyl) terephthalate (MHET), bis-(2- hydroxyethyl) terephthalate (BHET), and/or and ethylene glycol (EG).
  • TPA terephthalic acid
  • MHET mono-(2-hydroxyethyl) terephthalate
  • BHET bis-(2- hydroxyethyl) terephthalate
  • EG ethylene glycol
  • the hydrolysis results in the production of mono-(2-hydroxyethyl) terephthalate (MHET).
  • MHET mono-(2-hydroxyethyl) terephthalate
  • a single type of product or several different types of products may be recovered, depending on the starting substrate.
  • the method further comprises recovering, isolating, and/or purifying the MHET, BHET, TPA, and/or EG, or any combination thereof.
  • the method further comprises simultaneously or sequentially (e.g., before and/or after) contacting the PET with one or more of a mono-(2-hydroxyethyl) terephthalate hydrolase (MHETase), a bis-2-hydroxyethyl terephthalate hydrolase (BHETase), an esterase, a PETase, a carboxylesterase, and/or a cutinase.
  • MHETase mono-(2-hydroxyethyl) terephthalate hydrolase
  • BHETase bis-2-hydroxyethyl terephthalate hydrolase
  • an esterase e.g., a PETase, a carboxylesterase, and/or a cutinase.
  • the method further comprises simultaneously or sequentially (e.g., before and/or after) contacting the PET with a mono-(2-hydroxy ethyl) terephthalate hydrolase (MHETase).
  • MHET is degraded into terephthalic acid (TPA) and/or EG.
  • the method further comprises simultaneously or sequentially contacting the PET with a bis-2-hydroxyethyl terephthalate hydrolase (BHETase).
  • BHET is degraded into MHET.
  • a polyester comprising contacting a polyester with an esterase (e.g., PETase) described herein (see, e.g., ⁇
  • the method is carried out under conditions sufficient to convert the polyester to any one or more of terephthalic acid, ethylene glycol, mono-(2- hydroxyethyl) terephthalate, and/or bis-(2-hydroxyethyl) terephthalate.
  • the hydrolysis results in the production of mono-(2-hydroxyethyl) terephthalate (MHET).
  • MHET mono-(2-hydroxyethyl) terephthalate
  • a single type of product or several different types of products may be recovered, depending on the starting substrate.
  • the method further comprises recovering, isolating, and/or purifying the MHET, BHET, TPA, and/or EG, or any combination thereof.
  • a plastic product comprising a polyester
  • the method comprising the plastic product with an esterase (e.g., PETase) described herein (see, e.g., ⁇ 4.2), a fusion protein described herein (see, e.g., ⁇ 4.3), a conjugate described herein (see, e.g., ⁇ 4.3), a polynucleotide described herein (see, e.g., ⁇ 4.5), a vector described herein (see, e.g., ⁇ 4.6), a cell (or population of cells) described herein (see, e.g., ⁇ 4.7)), and/or a composition described herein (see, e.g., ⁇ 4.8), to thereby recycle the plastic product.
  • an esterase e.g., PETase
  • a fusion protein described herein see, e.g., ⁇ 4.3
  • a conjugate described herein see, e.g., ⁇
  • the polyester comprises polyethylene terephthalate (PET), poly lactic acid (PL A), poly trimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylcnc adipate terephthalate (PBAT), polyethylene furanoatc (PEF), polycapro lactone (PCL), polyethylene adipate (PEA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), or any combination of any of the foregoing.
  • the polyester comprises PET.
  • the method further comprises simultaneously or sequentially (e.g., before and/or after) contacting the plastic product with one or more of a mono-(2- hydroxyethyl) terephthalate hydrolase (MHETase), a bis-2-hydroxyethyl terephthalate hydrolase (BHETase), an esterase, a PETase, a carboxylesterase, and/or a cutinase.
  • MHETase mono-(2- hydroxyethyl) terephthalate hydrolase
  • BHETase bis-2-hydroxyethyl terephthalate hydrolase
  • an esterase e.g., a PETase, a carboxylesterase, and/or a cutinase.
  • the method further comprises simultaneously or sequentially (e.g., before and/or after) contacting the plastic product with a mono-(2-hydroxyethyl) terephthalate hydrolase (MHETase).
  • MHET is degraded into terephthalic acid (TPA) and/or EG.
  • the method further comprises simultaneously or sequentially contacting the plastic product with a bis-2-hydroxyethyl terephthalate hydrolase (BHETase).
  • BHET is degraded into MHET.
  • the plastic product is subject to thermal processing, washing and/or mechanical grinding prior to the contacting with the esterase (e.g., PETase), fusion protein, conjugate, polynucleotide, vector, cell (or population of cells), composition.
  • the thermal processing renders a crystalline or semi-crystalline structure of the polyester (e.g., PET) amorphous.
  • MHET terephthalic acid
  • MHET mono-(2-hydroxyethyl) terephthalate
  • BHET bis-(2-hydroxyethyl) terephthalate
  • EG ethylene glycol
  • the recovered products may be further purified, using any suitable purifying method and conditioned in a repolymerizable form.
  • suitable purifying methods include stripping process, separation by aqueous solution, steam selective condensation, filtration and concentration of the medium after the bioprocess, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalyst treatment, semi continuous mode distillation or continuous mode distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid/liquid extraction, hydrogenation, azeotropic distillation process, adsorption, column chromatography, simple vacuum distillation and microfiltration, combined or not.
  • the repolymerizable products may be used to synthesize new polyesters.
  • polyesters of same nature are repolymerized. It is further possible to mix the recovered products with other products, for example, in order to synthesize new copolymers. Alternatively, the recovered products may be used as chemical intermediates in order to produce new chemical compounds of interest.
  • compositions comprising the recovered MHET, BHET, TPA, and/or EG, or any combination thereof described in ⁇ 4.13.6.
  • the composition comprises PET.
  • the composition is a plastic product.
  • the plastic product comprises PET.
  • the plastic product comprises a textile, container (e.g., bag, bottle), film, or powder.
  • masterbatch compositions comprising esterase (e.g., PETase), composition, or cell expressing the esterase (e.g., PETase) or an extract thereof containing the esterase (e.g., PETase).
  • esterase e.g., PETase
  • Such plastic compound or masterbatch composition described herein can be used for the production of a polyester containing material.
  • the resulting plastic compound or masterbatch composition is a biodegradable plastic compound or masterbatch composition complying with at least one of the relevant standards and/or labels known by the person skilled in the art, such as standard EN 13432, standard ASTM D6400, OK Biodegradation Soil (Label Vincotte), OK Biodegradation Water (Label Vincotte), OK Compost (Label Vincotte), OK Home Compost (Label Vincotte).
  • MHET MHET
  • BHET BHET
  • TPA TPA
  • EG EG
  • any one of the methods described herein e.g., any one of the methods described in ⁇ 4.13.1, 4.13.3, or 4.13.4
  • a method described herein as described in ⁇ 4.13.6
  • the method comprises a polycondensation process.
  • MHET MHET
  • BHET BHET
  • TPA TPA
  • EG EG
  • any one of the methods described herein e.g., any one of the methods described in ⁇ 4.13.1, 4.13.3, or 4.13.4
  • a method described herein as described in ⁇ 4.13.6
  • the method comprises a polycondensation process.
  • the plastic product comprises a polyester.
  • the polyester comprises polyethylene terephthalate (PET), polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycapro lactone (PCL), polyethylene adipate (PEA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), or any combination of any of the foregoing.
  • the polyester comprises PET.
  • the plastic product comprises PET.
  • the plastic product comprises PET.
  • the plastic product comprises a textile, container (e
  • HIS tagged versions of each of esterases e.g., PETases 1 -24 (SEQ ID NOS: 2-25) and Reference esterases 1-24 (SEQ ID NOS: 2-25) were expressed and purified according to standard methods known in the art.
  • the HIS tagged version comprised a MGSSHHHHHH (SEQ ID NO: 84) peptide or a MGSSHHHHHH (SEQ ID NO: 85) peptide fused to the terminus of the esterase.
  • plasmids were designed comprising a codon optimized coding sequence for each of the esterases (e.g., PETases), a T7 promoter to drivee protein expression in bacteria, a transcription termination signal, and a carbenicillin-resistance gene.
  • esterases e.g., PETases
  • T7 promoter to drivee protein expression in bacteria
  • transcription termination signal e.g., a transcription termination signal
  • carbenicillin-resistance gene e.g., a codon optimized coding sequence for each of the esterases (e.g., PETases), a T7 promoter to drivee protein expression in bacteria, a transcription termination signal, and a carbenicillin-resistance gene.
  • Each of the generated plasmids was transformed into a BL21(DE3) competent variant of E.coli (C2527H, New England Biolabs) using a standard transformation protocol. Following transformation, the E.coli cells were spread on carbenicillin plates and maintained at 37 °C
  • the culture tube was subsequently placed in a shaking incubator set at 800 rpm and held at a consistent 37°C for 3-4 hours. Upon achieving an optical density between 0.4 and 0.8, the samples were incubated at 20°C with shaking (constant rpm) for 20 hours. Following incubation, the culture tubes were centrifuged for 20 minutes at 4000 x g to pellet bacterial cells. The separated supernatant was discarded, and the cell pellet resuspended in sonication buffer. The cells were lysed with sonication using a Branson Sonifier. The proteins were purified using standard His tag purification as detailed by Spriestersbach A, Kubicek J, Schafer F, Block H, Maertens B.
  • the esterase (e.g., PETase) activity of each of the HIS tagged esterases (e.g., PETases) 1-24 (SEQ ID NOS: 2-25) generated in Example 1 was assessed using standard methods known in the art. Briefly, coupons (approx. 8 mg) of amorphous PET film (Goodfellow, ES30-FM- 000145) was transferred to specific wells of a 96 well polystyrene plate. Typical reaction mixtures were assembled by combining the following components in the following order: MilliQ water (140 pl), and 460 mM HEPES buffer pH 8 with 2% (w/v) NaCl (60 pl), and 0.5 pM purified enzyme stock solution (50 pl).
  • MilliQ water 140 pl
  • 460 mM HEPES buffer pH 8 with 2% (w/v) NaCl 60 pl
  • 0.5 pM purified enzyme stock solution 50 pl.
  • Enzyme Diluent Purified enzyme (esterase (e.g., PETase)) stocks were prepared in 5 mM HEPES buffer pH 8 with 0.5% NaCl (referred to as Enzyme Diluent). In the no-enzyme negative controls, Enzyme Diluent was added in place of the enzyme stock. In the no-substrate controls, the reaction mixture was added to a well lacking the PET substrate coupon (the amorphous PET film). In some experiments the final enzyme concentration was increased 20-fold by reducing the MilliQ water volume (5 pl) and replacing the 0.5 pM enzyme stock with 2.7 pM enzyme stock (185 pl) in Enzyme Diluent.
  • Enzyme Diluent Purified enzyme
  • esterases e.g., PETases
  • esterase activity demonstrated by the presence of soluble aromatic compounds (e.g., MHET) liberated by the breakdown of polyethylene terephthalate (PET) fragments.
  • MHET soluble aromatic compounds liberated by the breakdown of polyethylene terephthalate

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Sustainable Development (AREA)
  • Polymers & Plastics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Separation, Recovery Or Treatment Of Waste Materials Containing Plastics (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des estérases (par exemple, PETases) (et leurs fragments fonctionnels, variants fonctionnels et domaines), des molécules d'acide nucléique codant pour celles-ci, et des compositions les comprenant. La divulgation concerne en outre des procédés d'utilisation des estérases (par exemple, PETases) (ou des molécules d'acide nucléique codant pour celles-ci), y compris, par exemple, dans des procédés de dégradation de produits contenant du polyester (par exemple, produits plastiques).
PCT/US2024/060086 2023-12-15 2024-12-13 Estérases et procédés associés Pending WO2025129034A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363610675P 2023-12-15 2023-12-15
US63/610,675 2023-12-15

Publications (2)

Publication Number Publication Date
WO2025129034A2 true WO2025129034A2 (fr) 2025-06-19
WO2025129034A3 WO2025129034A3 (fr) 2025-07-31

Family

ID=94383659

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/060086 Pending WO2025129034A2 (fr) 2023-12-15 2024-12-13 Estérases et procédés associés

Country Status (1)

Country Link
WO (1) WO2025129034A2 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5762939A (en) 1993-09-13 1998-06-09 Mg-Pmc, Llc Method for producing influenza hemagglutinin multivalent vaccines using baculovirus
WO2005071093A2 (fr) 2004-01-23 2005-08-04 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Porteurs de vaccin adenoviral de chimpanze
WO2006048215A1 (fr) 2004-11-02 2006-05-11 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Amplicon adenoviral et cellules productrices de vecteurs adenoviraux a defaut de replication, et leurs methodes de preparation et d'utilisation
WO2009150222A2 (fr) 2008-06-12 2009-12-17 Affitech A/S Système amélioré d'expression de protéine
US11446344B1 (en) 2018-12-12 2022-09-20 Flagship Pioneering Innovations V, Inc. Anellovirus compositions and methods of use

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140187468A1 (en) * 2011-08-31 2014-07-03 Danisco Us Inc. Compositions and Methods Comprising a Lipolytic Enzyme Variant
US20150017700A1 (en) * 2011-12-22 2015-01-15 Danisco Us Inc. Compositions and methods comprising a lipolytic enzyme variant
US20250368971A1 (en) * 2021-08-11 2025-12-04 Biometis Technology, Inc. Enzymatic Degradation of Polyethylene Terephthalate

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5762939A (en) 1993-09-13 1998-06-09 Mg-Pmc, Llc Method for producing influenza hemagglutinin multivalent vaccines using baculovirus
WO2005071093A2 (fr) 2004-01-23 2005-08-04 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Porteurs de vaccin adenoviral de chimpanze
WO2006048215A1 (fr) 2004-11-02 2006-05-11 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Amplicon adenoviral et cellules productrices de vecteurs adenoviraux a defaut de replication, et leurs methodes de preparation et d'utilisation
WO2009150222A2 (fr) 2008-06-12 2009-12-17 Affitech A/S Système amélioré d'expression de protéine
US11446344B1 (en) 2018-12-12 2022-09-20 Flagship Pioneering Innovations V, Inc. Anellovirus compositions and methods of use

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"Recombinant Protein Expression in Mammalian Cells: Methods and Protocols (Methods in Molecular Biology", 2018, HUMANA PRESS
ALTSCHUL SF ET AL., J MOL BIOL, vol. 215, 1990, pages 403
ALTSCHUL SF ET AL., NUC ACIDS RES, vol. 25, 1997, pages 3389 - 3402
KARLIN SALTSCHUL SF, PNAS, vol. 87, 1990, pages 2264 - 2268
KARLIN SALTSCHUL SF, PNAS, vol. 90, 1993, pages 5873 - 5877
MYERSMILLER, CABIOS, vol. 4, 1988, pages 11 - 17
PALM ET AL., NAT. COMM., vol. 10, 2019, pages 1717
SAGONG ET AL., ACS CATAL., vol. 10, 2020, pages 4805
SPRIESTERSBACH AKUBICEK JSCHÄFER FBLOCK HMAERTENS B: "Methods Enzymol.", vol. 559, 2015, article "Purification of His-Tagged Proteins", pages: 1 - 15
YOSHIDA ET AL., SCIENCE, vol. 352, no. 6278, 2016, pages 1196
ZHONG-JOHNSON, E. Z. L.VOIGT, C. A.SINSKEY, A. J.: "An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films", SCIENTIFIC REPORTS, vol. 11, no. 1, 2021, pages 928

Also Published As

Publication number Publication date
WO2025129034A3 (fr) 2025-07-31

Similar Documents

Publication Publication Date Title
JP7392029B2 (ja) 新規エステラーゼ及びその使用
CN112654701B (zh) 新酯酶及其用途
CN112673104B (zh) 新酯酶及其用途
CN114096665B (zh) 新型酯酶及其用途
US10508269B2 (en) Polypeptide having a polyester degrading activity and uses thereof
CN114096664B (zh) 新型酯酶及其用途
US10829754B2 (en) Plastic degrading proteases
CN120098957A (zh) 新酯酶及其用途
CN116368225A (zh) 新型酯酶及其用途
WO2025129034A2 (fr) Estérases et procédés associés
WO2025129001A1 (fr) Estérases et procédés associés
WO2025128936A1 (fr) Estérases et procédés associés
WO2025128953A1 (fr) Estérases et procédés associés
WO2025128954A1 (fr) Estérases et procédés associés
WO2025128943A1 (fr) Estérases et procédés associés
WO2025128934A1 (fr) Estérases et procédés associés
JP2025531043A (ja) 酵素バリアント及びその使用
RU2829537C2 (ru) Эстеразы и их применение
JP2025528449A (ja) 酵素及びその使用
JP2024525117A (ja) 酵素バリアント及びその使用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24846769

Country of ref document: EP

Kind code of ref document: A2