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WO2019168811A1 - Enzymes pour dégradation de polymère - Google Patents

Enzymes pour dégradation de polymère Download PDF

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Publication number
WO2019168811A1
WO2019168811A1 PCT/US2019/019502 US2019019502W WO2019168811A1 WO 2019168811 A1 WO2019168811 A1 WO 2019168811A1 US 2019019502 W US2019019502 W US 2019019502W WO 2019168811 A1 WO2019168811 A1 WO 2019168811A1
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Prior art keywords
petase
pet
modified
polymer
panel
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Inventor
Gregg Tyler BECKHAM
Christopher W. Johnson
Bryon S. Donohoe
Nicholas Rorrer
John E. Mcgeehan
Harry P. AUSTIN
Mark D. Allen
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University of Portsmouth
Alliance for Sustainable Energy LLC
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University of Portsmouth
Alliance for Sustainable Energy LLC
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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
    • 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
    • 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

  • plastics have become essential to modern society, driven by their enormous versatility coupled to low production costs. It is, however, now widely recognized that plastics pose a dire global pollution threat, especially to marine wildlife and ecosystems, because of the ultra-long lifetimes of most synthetic plastics in the environment. In response to the accumulation of plastics in the biosphere, it is becoming increasingly recognized that microbes are adapting and evolving enzymes and catabolic pathways to partially degrade man-made plastics as carbon and energy sources. These evolutionary footholds offer promising starting points for industrial biotechnology and synthetic biology to help address the looming environmental threat posed by man-made synthetic plastics.
  • PET Polyethylene terephthalate
  • PET can be depolymerized to its constituents via chemistries able to cleave ester bonds.
  • few chemical recycling solutions have been deployed given the high processing costs relative to the purchase of inexpensive virgin PET. This in turn results in reclaimed PET primarily being mechanically recycled, ultimately resulting in a loss of material properties, and hence intrinsic value. Given the recalcitrance of PET, the fraction of this plastic stream that is landfilled or makes its way to the environment is projected to persist for hundreds of years.
  • Exemplary embodiments provide a modified poiytethylene terephthalate) (PET)- digesting enzyme (PETase) that exhibits improved polymer degradation capacity relative to wild-type PETase due to its narrowed binding cleft via mutation of two active-site residues is disclosed.
  • unmodified PETase is from a bacterium of the genus Ideonella.
  • the bacterium is a strain of Ideonella sakaiensis.
  • an amino residue at position 159 is mutated.
  • an amino residue at position 238 is mutated.
  • an amino residue at position 238 is mutated.
  • the modified PETases comprises the W159H/S238F double mutation.
  • a nucleic acid molecule encoding a modified PETase having mutations at two active-site residues is disclosed.
  • the amino acid residue at position 159 is mutated.
  • the amino acid residue at position 238 is mutated.
  • the modified PETase comprises the W159H/S238F double mutation.
  • an expression vector comprising the nucleic acid molecule encoding a modified PETase having mutations at two active-site residues is disclosed.
  • Exemplary embodiments provide a nucleic acid encoding the enzyme comprising the amino acid sequence depicted in FIG. 2(B).
  • PET poly(ethylene terephthalate)
  • PETase modified poly(ethylene terephthalate)-digesting enzyme
  • Exemplary embodiments provide method for degrading a polymer comprising contacting the modified PETase of claim 1 or the cell of claim 13 with the polymer.
  • the polymer is a polyester.
  • the polymer is an aromatic polymer or a semi-aromatic polymer.
  • the polymer is polyethylene terephtha!ate (PET).
  • PET polyethylene terephtha!ate
  • PET polyethylene terephtha!ate
  • the polymer is poly ethyl enefuranoate (PEF).
  • the polymer is from a recycled plastic material.
  • FIG. 1 shows the nucleotide (A) and amino acid (B) sequences of PETase from Ideonel la sakaiensi .
  • FIG. 2 shows the nucleotide (A) and amino acid (B) sequences of PETase from Ideonella sakaiensis containing the W159H and S238F mutations (bold and underlined).
  • FIG. 3 shows high resolution X-ray crystallography data collection and analysis of
  • FIG. 4 illustrates the structure of PETase.
  • FIG. 5 shows PETase sequence analysis
  • FIG. 6 illustrates the compari son of the active site cleft of PETase with eutinases
  • FIG. 7 show's multiple sequence alignments of PETase with lipase and cutinase family members.
  • FIG. 8 illustrates the structural and functional analysis of key residues in PETase.
  • FIG. 9 shows the chemical analysis of polymer substrates.
  • FIG. 10 illustrates a comparison of PETase and the engineered enzyme S238F/W159H with PET.
  • FIG. 1 1 shows the induced fit docking analysis of PETase and the engineered enzyme S238F/W159H with PET.
  • FIG. 12 show's degradation analysis of PBS and PLA by PETase.
  • FIG. 13 illustrates a comparison of PETase and the engineered enzyme S238F/W159H with PEF.
  • PET polyethylene terephthalate
  • Such enzymes include PET-degrading enzymes (PETases) wherein certain amino acid residues are mutated to different amino acids to improve enzymatic activity.
  • PETases PET-degrading enzymes
  • FIG. 2 provides nucleotide and amino acid sequences for a PETase from the bacterium Ideonella sakaiensis wherein the tryptophan residue at position 159 has been mutated to a histidine residue and the serine residue at position 238 has been mutated to a phenylalanine (W159H/S238F).
  • Ideonella sakaiensis 201-F6 is a bacterial strain with the ability to use PET as its major carbon and energy source for growth. Especially in the last decade, there have been multiple, foundational studies reporting such enzymes that can degrade PET, but previous work has not connected extracellular enzymatic PET degradation to catabolism in a single microbe. Previously, it had been demonstrated that an I. sakaiensis enzyme dubbed PETase converts PET to mono(2-hydroxyethyl) terephthalic acid (MHET), with trace amounts of terephthalic acid (TPA) and bis(2-hydroxyethyl)-TPA (BHET) as secondary products.
  • MHET mono(2-hydroxyethyl) terephthalic acid
  • TPA terephthalic acid
  • BHET bis(2-hydroxyethyl)-TPA
  • MHETase further converts MHET into the two monomers, TPA and ethylene glycol (EG). Both enzymes are secreted by I. sakaiensis and likely act synergistically to depolymerize PET. Sequence analysis of PETase highlights similar to ab-hydrolase enzymes, including the cutinase and lipase families, which catalyze hydrolysis of cutin and fatty acids, respectively.
  • PET is a semi-aromatic polyester.
  • Some aliphatic polyesters such as polylactic acid (PL A), poly butylene succinate (PBS), or polyhydroxyalkanoates can be produced from renewable sources and are marked as biodegradable plastics, given their relatively low crystallinity and glass transition temperatures, in turn providing relatively more direct enzymatic access to ester linkages.
  • Aromatic and semi-aromatic polyesters conversely, often exhibit enhanced thermal and material properties, and accordingly, have reached substantially higher market use, but are typically not as biodegradable as their aliphatic counterparts.
  • PEF polyethylene-2, 5-furandicarhoxylate (or polyethylenefuranoate, called FDCA).
  • FDCA sugar-derived 2, 5-fumandi carboxylic acid
  • PEF exhibits improved gas barrier properties over PET and is being pursued industrially.
  • PEF is a bio based semi-aromatic polyester, which is predicted to offset greenhouse gas emissions relative to PET, its lifetime in the environment, like that of PET, is likely to be quite long. Given that PETase has evolved to degrade crystalline PET, it potentially may have promiscuous activity across a range of polyesters.
  • Amino acids for modification may be selected from those found in or near a PETase active site - for example, as determined by reference to the PETase’ s crystal structure.
  • active site residues include T88, S238, H237, S160, D206, W159 or W185.
  • the modified enzymes may be from microorganisms such as bacteria, yeast, or fungi.
  • bacteria and fungi include species from the genera Ideonella (such as I. sakaiensi ), Thermohifida (such as T. fusca ) or Fusarium (such as F. solani).
  • Ideonella such as I. sakaiensi
  • Thermohifida such as T. fusca
  • Fusarium such as F. solani
  • other examples of modified enzymes or PETases from microbial sources are within the scope of this disclosure.
  • microorganisms engineered to express the modified enzymes disclosed herein and their use to degrade or depolymerize polymers are also presented. Polymer degradation/depolymerization may be carried out be culturing such microorganisms with a material containing a polymer and allowing the microorganisms to enzymatically degrade the conversion. Any microorganism capable of expressing the enzymes disclosed herein may be suitable. Exemplary microorganisms include bacteria, such as those from the genus Ideonella. Specific examples include strains of Ideonella sakaiensis, such as /. sakaiensis 201-F6.
  • Polymers or polymer-containing materials may be contacted with organisms at a concentration and a temperature for a time sufficient to achieve the desired amount of degradation or depolymerization. Suitable times range from a few hours to several days and may be selected to achieve a desired amount of conversion. Exemplary reaction times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours; and
  • reaction times may be one or more weeks.
  • Unpurified or semi-purified culture supernatants containing the modified enzymes may also be contacted with polymers or polymer-containing materials (e.g , in vitro) under similar reaction conditions suitable for allowing polymer degradation.
  • the degradation products may be further converted to additional products by further contact with enzymes.
  • PET may be contacted with a modified PETase to generate mono(2- hydroxyethyl) terephthalic acid (MHET), with trace amounts of terephthalic acid (TP A) and bis(2-hydroxyethyl)-TPA (BHET) as secondary' products.
  • MHET mono(2- hydroxyethyl) terephthalic acid
  • TP A terephthalic acid
  • BHET bis(2-hydroxyethyl)-TPA
  • MHET derived therefrom may be contacted with a second enzyme, MHETase, that further converts MHET into the two monomers, TPA and ethylene glycol (EG).
  • MHETase a second enzyme that further converts MHET into the two monomers, TPA and ethylene glycol (EG).
  • Methods of fractionating, isolating or purifying degradation products include a variety of biochemical engineering unit operations.
  • the reaction mixture or cell culture lysate may be filtered to separate solids from products present in a liquid portion. Products may be further extracted from a solvent and/or purified using conventional methods.
  • Exemplary methods for purification/i solation/ separation of products include at least one of affinity chromatography, ion exchange chromatography, solvent extraction, filtration, centrifugation, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing, differential solubilization, preparative disc-gel electrophoresis, isoelectric focusing, high performance liquid chromatography (HPLC), and/or or reversed-phase HPLC.
  • affinity chromatography ion exchange chromatography
  • solvent extraction filtration, centrifugation, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing, differential solubilization, preparative disc-gel electrophoresis, isoelectric focusing, high performance liquid chromatography (HPLC), and/or or reversed-phase HPLC.
  • HPLC high performance liquid chromatography
  • Exemplary' polymers include polyesters such as aromatic and semi-aromatic polyesters. Specific examples include polyethylene terephthalate (PET) or polyethylenefuranoate (PEF), which may be present in recycled materials such as beverage bottles, clothing, packaging, or earpetm
  • PET polyethylene terephthalate
  • PET polyethylenefuranoate
  • the sequences disclosed herein provide nucleic acid and amino acid sequences for exemplary enzymes for use in the disclosed methods.
  • Nucleic acid or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or poiydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides.
  • PNA protein nucleic acids
  • nucleic acids referred to herein as "isolated” are nucleic acids that have been removed from their natural milieu or separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library) and may have undergone further processing.
  • Isolated nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids that are isolated.
  • Nucleic acids referred to herein as "recombinant” are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures that rely upon a method of artificial replication, such as the polymerase chain reaction (PCR) and/or cloning or assembling into a vector using restriction enzymes.
  • Recombinant nucleic acids also include those that result from recombination events that occur through the natural mechanisms of cells but are selected for after the introduction to the cells of nucleic acids designed to allow or make probable a desired recombination event.
  • Portions of isolated nuclei c acids that code for polypeptides having a certain function can be identified and isolated by, for example, the method disclosed in U.S. Patent No. 4,952,501.
  • An isolated nucleic acid molecule can be isolated from its natural source or produced using recombinant DNA technology (e.g, polymerase chain reaction (PCR) amplification, cloning or assembling) or chemical synthesis.
  • Isolated nucleic acid molecules can include, for example, genes, natural allelic variants of genes, coding regions or portions thereof, and coding and/or regulatory regions modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a polypeptide or to form stable hybrids under stringent conditions with natural gene isolates.
  • An isolated nucleic acid molecule can include degeneracies.
  • nucleotide degeneracy refers to the phenomenon that one amino acid can be encoded by different nucleotide codons.
  • nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies.
  • nucleic acid molecule is not required to encode a protein having enzyme activity.
  • a nucleic acid molecule can encode a truncated, mutated or inactive protein, for example.
  • nucleic acid molecules may also be useful as probes and primers for the identification, isolation and/or purification of other nucleic acid molecules, independent of a protein-encoding function.
  • Suitable nucleic acids include fragments or variants that encode a functional enzyme or proteins disclosed herein.
  • a fragment can comprise the minimum nucleotides required to encode a functional PETase or component thereof.
  • Nucleic acid variants include nucleic acids with one or more nucleotide additions, deletions, substitutions, including transitions and transversions, insertion, or modifications (e.g., via RNA or DNA analogs). Alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a nucleic acid may be identical to a sequence represented herein.
  • the nucleic acids mav be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence represented herein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence represented herein. Sequence identity calculations can be performed using computer programs, hybridization methods, or calculations.
  • Exemplary computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, BLASTN, BLASTX, TBLASTX, and FASTA.
  • the BLAST programs are publicly available from NCBI and other sources.
  • nucleotide sequence identity can be determined by comparing query sequences to sequences in publicly available sequence databases (NCBI) using the BLASTN2 algorithm.
  • Embodiments of the nucleic acids include those that encode the polypeptides that possess the enzymatic activities described herein or functional equivalents thereof.
  • a functional equivalent includes fragments or variants of these that exhibit one or more of the enzymatic activities.
  • many nucleic acid sequences can encode a given polypeptide with a particular enzymatic activity. Such functionally equivalent variants are contemplated herein.
  • Nucleic acids may be derived from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA, or combinations thereof. Such sequences may comprise genomic DNA, which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions or poly (A) sequences. The sequences, genomic DNA, or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means.
  • recombinant vectors including expression vectors, containing nucleic acids encoding enzymes.
  • A“recombinant vector” is a nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice or for introducing such a nucleic acid sequence into a host ceil.
  • a recombinant vector may be suitable for use in cloning, assembling, sequencing, or otherwise manipulating the nucleic acid sequence of choice, such as by expressing or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell.
  • Such a vector typically contains heterologous nucleic acid sequences not naturally found adjacent to a nucleic acid sequence of choice, although the vector can also contain regulatory ' nucleic acid sequences (e.g., promoters, untranslated regions) that are naturally found adjacent to the nucleic acid sequences of choice or that are useful for expression of the nucleic acid molecules.
  • regulatory ' nucleic acid sequences e.g., promoters, untranslated regions
  • nucleic acids described herein may be used in methods for production of enzymes or proteins through incorporation into ceils, tissues, or organisms.
  • a nucleic acid may be incorporated into a vector for expression in suitable host cells.
  • the vector may then be introduced into one or more host cells by any method known in the art.
  • One method to produce an encoded protein includes transforming a host cell with one or more recombinant nucleic acids (such as expression vectors) to form a recombinant cell.
  • transformation is generally used herein to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell but can be used interchangeably with the term "transfection.”
  • Non-limiting examples of suitable host cells include cells from microorganisms such as bacteria, yeast, fungi, and filamentous fungi.
  • Exemplary microorganisms include, but are not limited to, bacteria such as E. coir, bacteria from the genera Ideonella (e.g., I. sakaiensis ), Thermobifidki (e.g., T. fused), Pseudomonas (e.g., P. pulida or P. fluorescens), Acinetobacter (e.g., strains of A. baylyi such as ADP1), Bacillus (e.g., B. subtilis, B. megaterium or B. brevis), Caulobacter (e.g., C.
  • bacteria such as E. coir
  • bacteria from the genera Ideonella e.g., I. sakaiensis
  • Thermobifidki e.g., T. fused
  • Pseudomonas
  • Lactoccocus e.g., L. lactis
  • Streptomyces e.g., S. coelicolor
  • Streptococcus e.g., S. lividans
  • Corynybacterium e.g., C. glutamicum
  • fungi from the 5 genera Fusarium (e.g., F. soiani), Trichoderma (e.g., T. reesei, T. viride, I koningu or T. harzianum)
  • PeniciUimn e.g., P. fimiculosum
  • Humicola e.g., H. insolens
  • Chrysosporium e.g., C.
  • Gliocladium Gliocladium
  • Aspergillus e.g, A. niger, A. nididans, A. aw amor i, or A. aculeatus
  • Neurospora Hypocrea (e.g., H. jecorind), and. Emericella
  • yeasts from the genera Saccharomyces e.g., S. cerevisiae
  • Pichia e.g., P. pastoris
  • Kluyveromyces e.g, K. lactis
  • Ceils from plants such as Arabidopsis, barley, citrus, cotton, maize, poplar, rice, soybean, sugarcane, wheat, switch grass, alfalfa, miscanthus, and trees such as hardwoods and softwoods are also contemplated herein as host cells.
  • Host cells can be transformed, transfected, or infected as appropriate by any suitable method including electroporation, calcium chloride-, lithium chloride-, lithium acetate/polyene glycol-, calcium phosphate-, DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting, injection, microinjection, microprojectile bombardment, phage infection, viral infection, or other established methods.
  • vectors containing the nucleic acids of interest can be transcribed in vitro , and the resulting RNA introduced into the host cell by well- known methods, for example, by injection.
  • Exemplary embodiments include a host cell or population of cells expressing one or more nucleic acid molecules or expression vectors described herein (for example, a genetically modified microorganism).
  • the cells into which nucleic acids have been introduced as described above also include the progeny of such cells.
  • Vectors may be introduced into host cells such as those from bacteria or fungi by direct transformation, in which DNA is mixed with the cells and taken up without any additional manipulation, by conjugation, electroporation, or other means known in the art.
  • Expression vectors may be expressed by bacteria or fungi or other host cells episomally or the gene of interest may be inserted into the chromosome of the host cell to produce cells that stably express the gene with or without the need for selective pressure.
  • expression cassettes may be targeted to neutral chromosomal sites by recombination.
  • Host cells carrying an expression vector may be selected using markers depending on the mode of the vector construction.
  • the marker may be on the same or a different DNA molecule.
  • the transformant may be selected, for example, by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
  • Host cells may be cultured in an appropriate fermentation medium.
  • An appropriate, or effective, fermentation medium refers to any medium in which a host cell, including a genetically modified microorganism, when cultured, is capable of growing or expressing the polypeptides described herein.
  • a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources, but can also include appropriate salts, minerals, metals and other nutrients.
  • Microorganisms and other cells can be cultured in conventional fermentation bioreactors and by any fermentation process, including batch, fed- batch, cell recycle, and continuous fermentation.
  • the pH of the fermentation medium is regulated to a pH suitable for growth of the particular organism.
  • Culture media and conditions for various host cells are known in the art. A wide range of media for culturing bacteria or fungi, for example, are available from ATCC. Media may be supplemented with aromatic substrates, or components of thermochemical waste streams as needed.
  • proteins are defined as fragments or portions of polypeptides, preferably fragments or portions having at least one functional activity ' ⁇ as the complete polypeptide sequence.
  • isolated proteins or polypeptides are proteins or polypeptides purified to a state beyond that in which they exist in cells. In certain embodiments, they may be at least 10% pure; in others, they may be substantially purified to 80% or 90% purity' or greater.
  • Isolated proteins or polypeptides include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides that are isolated. Proteins or polypeptides referred to herein as "recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids.
  • Proteins or polypeptides encoded by nucleic acids as well as functional portions or variants thereof are also described herein.
  • Polypeptide sequences may be identical to the amino acid sequences presented herein or may include up to a certain integer number of amino acid alterations.
  • Such protein or polypeptide variants retain enzymatic activity, and include mutants differing by the addition, deletion or substitution of one or more amino acid residues, or modified polypeptides and mutants comprising one or more modified residues.
  • the variant may have one or more conservative changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). Alterations may occur at the amino- or carboxy-terminai positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • polypeptides may be at least about 70%, 71%, 72%, 73%,
  • Percent sequence identity can be calculated using computer programs (such as the BLASTP and TBLASTN programs publicly available from NCBI and other sources) or direct sequence comparison.
  • Polypeptide variants can be produced using techniques known in the art including direct modifications to isolated polypeptides, direct synthesis, or modifications to the nucleic acid sequence encoding the polypeptide using, for example, recombinant DNA techniques.
  • Polypeptides may be retrieved, obtained, or used in "substantially pure” form, a purity that allows for the effective use of the protein in any method described herein or known in the art.
  • a protein to be most useful in any of the methods described herein or in any method utilizing enzymes of the types described herein, it is most often substantially free of contaminants, other proteins and/or chemicals that might interfere or that would interfere with its use in the method (e.g., that might interfere with enzyme activity), or that at least would be undesirable for inclusion with a protein.
  • Disclosed herein are multiple high-resolution X-ray crystal structures of PETase, which enable comparison to known cutinase structures.
  • PETase variants were produced and tested for PET degradation, including a double mutant distal to the catalytic center that altered important substrate-binding interactions.
  • this double mutant may have W159H/S238F mutations.
  • this double mutant inspired by cutinase architecture, exhibits improved PET degradation capacity relative to wild-type PETase.
  • silica docking and molecular dynamics (MD) simulations were deployed to characterize PET binding and dynamics, which provide insights into substrate binding and may suggest an explanation for the improved performance of the PETase double mutant.
  • PETase is capable of depoly merizing aliphatic polyesters, suggesting a broader role for PETase in degrading semi-aromatic polyesters.
  • SEM scanning electron microscopy
  • DSC differential scanning calorimetry
  • PETase a major carbon and energy source
  • PETase a major carbon and energy source
  • This present disclosure demonstrates that a collection of subtle variations on the surface of a lipase/cutinase-like fold has the ability to endow a modified and/or mutated PETase with a platform for aromatic polyester depolymerization.
  • the high-resolution X-ray crystal structure of the I. sakaiensis PETase was analyzed employing a synchrotron beamline capable of long-wavelength X-ray crystallography. Using single wavelength anomalous dispersion, phases were obtained from the native sulfur atoms present in the protein. The low background from the in vacuo setup and large curved detector resulted in exceptional diffraction data quality extending to a resolution of 0.92 A, with minimal radiation damage (Table l, FIG. 3).
  • Table 1 Crystallographic data and refinement statistics including crystallization conditions.
  • Ifh.ij is symmetry-related intensities and 1(h) is the mean intensity of the reflection with unique index h.
  • Cc 1 ⁇ 2 is the correlation coefficient of the mean intensities between two random half-datasets.
  • multiplicity refers to multiplicity for unique reflections.
  • the ratio Rwork/Rfree (expressed as a percentage) as shown in Table 1 w'as calculated by randomly selecting 5% of reflections for determination of the free R factor, prior to any refinement.
  • the B average is the temperature factors averaged for all atoms.
  • the line“geometry bond, angles” show RMS deviations from ideal geometry for bond lengths and restraint angles (Engh and Huber).
  • the Ramachandran is the percentage of residues in the‘most favored region’ of the Ramachandran plot and percentage of outliers (MOLPROBITY).
  • PDB ID refers to the protein data bank identifiers for coordinates.
  • the crystallography conditions analyzed were: long l, 0.1M MIB (Malonate, Imidazole, Borate), pH 5.0, 25% polyethylene glycol (PEG) 1500.
  • Native 1 0.2 M MgC , 0.1 M MES (2-(N-Morpholino)ethanesulfonic acid), pH 6.0, 20% PEG 6000.
  • Native 2 0.2 M NHrCl, 0.1 M MES, pH 6.0, 20% PEG 6000.
  • Native 3 0.2 M LbSOr, 0.1 M Bis-Tris (pH 5.5), 25% PEG 3350.
  • Native 4 1.6 M MgSOr, 0.1M MES, pH 6.5.
  • FIG. 3 shows high resolution X-ray crystallography data collection and analysis for PETase.
  • Panel A of FIG 3 illustrates a representative section of a diffraction image from the 0.92 A PETase structure determination.
  • the mean counts in the boxes are A (low resolution): 0.3 counts, B (solvent ring): 1.6 counts and C (high resolution): 0 1 counts.
  • the inset show's an enlarged portion around reflection -49 13 -11 at 1.0 A resolution (box D) where each box represents an individual pixel. Values range from zero (white) and 1 (light grey) background counts, through to the center of this refection at 16 counts.
  • FIG. 3 illustrates representative electron density from the high-resolution structure (6EQE) showing continuous density' across the disulfide bridge between Cys273 and Cys289.
  • the 2Fo-Fc map is contoured at 1s.
  • Panel C of FIG. 3 illustrates representative electron density quality is shown centered around Trp257.
  • the 2Fo-Fc map is contoured at 1 s.
  • PETase adopts a classical ab-hydrolase fold, with a core consisting of 8 b-strands and 6 a-helices (Panel A of FIG. 4).
  • PETase has sequence identity substantially close to bacterial cutinases, with Thermobifida fit sea cutinase being the closest known structural representative with 52% sequence identity (Panel B of FIG. 4, Panel A of FIG. 5), which is an enzyme that also degrades PET.
  • Thermobifida fit sea cutinase is the closest known structural representative with 52% sequence identity (Panel B of FIG. 4, Panel A of FIG. 5), which is an enzyme that also degrades PET.
  • the surface profile is quite different between the two enzymes.
  • PETase has a highly polarized surface charge (Panel C of FIG 4), creating a dipole across the molecule, and resulting in an overall isoelectric point (pi) of 9.6.
  • T. fiisca cutinase in common with other cutinases, has a number of small patches of both acidic and basic residues distributed over the surface conferring a more neutral pi of 6.3 (Panel D of FIG.
  • FIG. 4 illustrates the structure of PETase.
  • Panel A of FIG. 4 illustrates a cartoon representation of the PETase structure at 0.92 A resolution (PDB ID: 6EQE). The active site cleft is oriented at the top and highlighted with a dashed circle.
  • Panel B of FIG 4 illustrates a comparative structure of the T fiisca cutinase (PDB ID: 4CG1).
  • Panel C of FIG. 4 illustrates the electrostatic potential distribution mapped to the solvent accessible surface of PETase compared to the T. fiisca cutinase as a shaded gradient from black (acidic) -7 kTle to a lighter gray (basic) 7 kTle.
  • Panel D of FIG. 4 illustrates the T.
  • Panel E of FIG 4 shows the view along the active site cleft of PETase corresponding to the area highlighted with a dashed circle in A and C. The width of the cleft is shown between Thr88 and Ser238
  • Panel F of FIG. 4 shows the narrower cleft of T. fiisca cutinase active site is shown with the width between Thr61 and Phe209 in equivalent positions.
  • Panel G of FIG. 4 illustrates a close-up view of the PETase active site with the catalytic triad residues, His237, Seri 60, and Asp206, shaded. Residues Trpl59 and Trpl85 are lightly shaded.
  • 4H shows a comparative view' of the T. fusca cutinase active site with equivalent catalytic triad residues shaded. Residues Hisl29 and Trpl 55 are lightly shaded. The residues in PETase are also shaded that correspond to the site-directed mutagenesis targets S238F, W159H, and W185A.
  • FIG. 5 shows PETase sequence analysis.
  • Panel A of FIG. 5 show's the sequence alignment of PETase (labelled PET, accession number: A0A0K8P6T7), against a PET- degrading cutinase from T. fiuscia (TFcut, accession number: AET05798).
  • the signal sequences from both enzymes, as predicted by LipoP 1.0 Server, were excluded from the alignment for clarity.
  • the squares shaded dark gray indicate areas of identity with residues in text indicating moderate conservation.
  • the cartoons above the alignment denote secondary structure, with spirals representing alpha-helices, arrows representing beta-strands, and ⁇ ’ indicating turns.
  • Panel B of FIG. 5 shows the nucleotide sequence of the synthetic DNA fragment containing the gene encoding the PETase from I. sakaiensis optimized for expression in E. coli. Overlaps added for assembly into pET-21b(+) are underlined. The initiating ATG and stop codon of the His-tagged gene are in bold text. Ndel and Xhol restriction sites are capitalized.
  • PETase and the closest cutinase homologues Another difference between PETase and the closest cutinase homologues is the broader active-site cleft, which was hypothesized might be necessary to accommodate crystalline semi aromatic polyesters.
  • the cleft in PETase approaches three-times the width of the corresponding structure in the T. fusca cutinase.
  • the expansion is achieved with minimal rearrangement of the adjacent loops and secondary structure (Panels E and F of FIG. 4).
  • a single amino acid substitution from phenylalanine to serine in the lining of the active site cavity appears sufficient to cause this change, with the remaining deft formed between Trpl59 and Tip 185 (Panel G of FIG. 4)
  • This relative broadening of the active site cleft is also observed in comparisons with other known cutinase structures (Panels A-D of FIG. 6).
  • FIG. 6 illustrates the comparison of the active site cleft of PETase with cutinases.
  • Panel A of FIG. 6 show's a PETase cleft width calculated from the distance between the van der Waals surface of TBS and S238 PDB ID: 6EQE.
  • Panel B of FIG. 6 illustrates Tfusica cutinase with cleft width calculated from the distance between the van der Waals surface of T61 and F209 (PDB ID: 4CG1) (12).
  • Panel C of FIG. 6 show ' s Thermobifida cellulosilytica cutinase (PDB ID: code 5LUI) with cleft width calculated from the distance between the van der Waals surface of T63 and F212.
  • FIG. 6 shows leaf compost cutinase obtained from a metagenomics analysis of uncultured bacteria. Cleft width was calculated from the distance between the van der Waals surface of T61 and F210 (PDB ID: 4EBQ). Arrow's with labels denote the width of the active site as determined from equivalent residues in each enzyme.
  • Panel E of FIG. 6 shows hydrophobic adaptations in the active site of PETase with the catalytic residue, Seri 60, darkly shaded. Residues in a lighter shade indicate the hydrophobic residues surrounding the active site triad.
  • Panel F of FIG 6 shows a comparative view of the T. fusca hydrophobic distribution, with equivalent orientation and shading.
  • FIG. 7 show's multiple sequence alignments of PETase with lipase and cutinase family members.
  • Panel A of FIG. 7 illustrates multiple sequence alignment of PETase against members of the lipase family. Signal sequences, as predicted by LipoP 1.0 Server, were excluded from the alignment.
  • the box spanning residues 159 to 162 highlights the conserved lipase box, while the box spanning 237-238 indicates the serine in PETase, which is occupied by a phenylalanine or tyrosine in most lipases.
  • Aligned sequences with accession numbers are PET (PETase, I.
  • CTlip lipase, Ca!dimonas taiwanensis WP 06219554 -I ⁇
  • PSlip lipase, Pseudomonas saudimassiliensis CEA05385
  • OAlip lipase, Oleispira Antarctica, CCK74972.1
  • SSlip lipase, Saccharothrix sp, OKI36883.1
  • PYlip tricylglycerol lipase, Pseudomonas yangmingensis, SFM35944)
  • YPlip lipase, Vibro palustri , SJL84994
  • RGlip lipase, Rhizobacier gumtniphilus , ARN20166
  • VSlip Lipase, Vibro spartinae , 81095186
  • HClip lipase, Herbido
  • FIG. 7B show's the multiple sequence alignment of PETase against members of the cutinase family with the same scheme. Aligned sequences are PET (PETase, Ideonella sakaiensis, A0A0K8P6T7), PBeut (cutinase, Pseudomonas bauzanensis, SER72431), Picut (cutinase, Pseudomonase litoralis , SDS35700), PS cut (cutinase, Pseudomonas sale ge s, 81)1 28434), PXcut (cutinase, Pseudomonas xinjiangensis, SDS09569), MEcut (cutinase, Micromonospora echinospora, SCF30318), TAcut (cutinase, Thermobifida alba, ADV 92525), TFcut (cutinase, T.
  • the catalytic triad is conserved across the lipases and cutinase families.
  • the catalytic triad comprises Serl60, Asp206, and His237, suggesting a charge-relay system similar to that found in other a/b-fold hydrolases.
  • the specific location and geometry between the active site found in cutinases is also conserved in PETase (Panels G and H of FIG. 7).
  • the catalytic residues reside on loops, with the nucleophilic serine occupying a highly conserved position known as the nucleophilic elbow.
  • the nucleophilic serine sits in the consensus sequence (Gly-Xl-Ser-X2-Gly), and while this "lipase box” is common to most lipases (Panel A of FIG. 7) and cutinases (Panel B of FIG. 7), the XI position, usually occupied by a histidine or phenylalanine in cutinases and lipases contains a tryptophan residue, TrpJ 59, in PETase (Panel G of FIG. 4). This residue has the effect of extending the hydrophobic surface adjacent to the active site (Panels E and F of FIG. 6).
  • PETase In common with the Fusarium solani cutinase, PETase has two disulfide bonds, one adjacent to the active site and one near the C-terminus of the protein. MD simulations have predicted that the active site disulfide in F. solani cutinase is important for active site stability, and it may play a similar role in PETase.
  • FIG. 8 illustrates the structural and functional analysis of key residues in PETase.
  • Panel A of FIG. 8 shows the superposition of the PETase structures (PDB IDs: 6EQE, 6EQF, 6EQG, and 6EQH).
  • the RMSD of chains is 0.28A ⁇ 0.02. Overall, all the chains adopt the same fold with only slight variations in the loops.
  • the sideehains of the three residues in the catalytic triad, Serl60, Asp206, and His237 in addition to Trpl59 and Trpl85 are shown. All side-chain adopt the same rotameric state with the exception of Trpl85, where two residues adopted slightly different c-2 orientations, possibly reflecting a degree of mobility.
  • FIG. 8 illustrates a MD simulation demonstrating a wide range of movement for residue Trpl85 (shaded).
  • Panel G of FIG. 8 shows PET degradation after incubation with buffer only.
  • Panel H of FIG. 8 shows PET degradation after incubation with the W185A mutant PETase.
  • Panel I of FIG. 8 shows PEF degradation after incubation with buffer only.
  • Panel J of FIG. 8 shows PEF degradation after incubation with the Wl 85 A mutant PETase
  • three additional crystallography datasets ranging in resolution from 1.58 to 1.80 A provided a total of seven independent PETase chains (Table 1).
  • Example 2 Converting PETase to a cutinase-like active-site cleft enables improved crystalline PET degrada tion
  • FIG. 9 shows the chemical analysis of polymer substrates.
  • Panel A of FIG. 9 shows 3 ⁇ 4 nuclear magnetic resonance (NMR) spectra of the lab synthesized PET used in the study.
  • Panel B of FIG. 9 shows l H NMR spectra of the lab synthesized PEF used in the study.
  • Panel C of FIG. 9 shows 1 H NMR spectra of the lab synthesized PL A.
  • Panel D of FIG. 9 shows 1 H NMR spectra of poly(butylene succinate).
  • Panel E of FIG. 9 shows the reduction in crystallinity in samples before and after digestion as determined by DSC.
  • Panel F of FIG. 9 shows representative DSC trace for PET. After digestion, the melting transition is broadened indicating a reduction in both crystallinity and crystal domain size.
  • PETase digestions of amorphous PET films with a crystallinity of 1.9% which is lower than most PET samples that would be either encountered in the environment or in an industrial recycling context were examined.
  • this present disclosure examined PET digestion with coupons of higher crystallinity. Specifically, PET coupons with an initial crystallinity of 14.8 ⁇ 0.2% (for reference, a commercial soft drink botle examined via the same methods exhibits a crystallinity of 15.7% as measured by DSC) were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy to confirm their structure and DSC to determine their crystallinity (Panel A of FIG. 9).
  • NMR nuclear magnetic resonance
  • Panels A-D of Fig. 10 show' the results of PET degradation, including a buffer-only control, the wild-type PETase, and the double mutant. It is clear that PETase induces surface erosion and pitting of a PET film with a crystallinity of 13.3 ⁇ 0.2%, resulting in a 10.1% relative crystallinity reduction (absolute reduction of 1.5%; Table 2). Surprisingly, the PETase double mutant outperforms the wild- type PETase by both crystallinity reduction and product release.
  • Table 2 Crystallinity determined by differential scanning calorimetry for plastics with PETase and mutations for the aromatic polyesters.
  • the average cold crystallization for the PET samples was 24 J/g.
  • the error in PBS crystallinity was outside significant MG s
  • the results with the PETase double mutant exhibited no change in majored properties.
  • FIG. 10 illustrates a comparison of PETase and the engineered enzyme S238F/W159H with PET.
  • Panel A of FIG. 10 show's buffer-only control of PET coupon.
  • Panel B of FIG. 10 illustrates PET coupon after incubation with wild-type PETase.
  • Panel C of FIG. 10 shows PET coupon after incubation with the PETase double mutant, S238FVW 159H.
  • Ail SEM images were taken after 96 h of incubation at a PETase loading of 50 nM, pH 7.2 in phosphate buffer, or a buffer-only control.
  • Panel E of FIG. 10 shows predicted binding conformations of wild-type PETase from docking simulations demonstrate that PET is accommodated in an optimum position for the interaction of the carbon (black) with the nucleophilic hydroxyl group of Serl60, at a distance of 5.1 A (dark dashed line). His237 is positioned within 3.9 A of the Ser i 60 hydroxyl (lighter dashed line). Residues Trpl59 and Ser238 line the active site channel (both are shaded).
  • Panel F of FIG 10 show's the double mutant S238F/WI 59H adopts a more productive interaction with PET.
  • the S238 mutation provides new p-stacking and hydrophobic interactions to adjacent terephthalate moieties while the conversion to Hisl 59 from the bulkier Tip allows the PET polymer to sit deeper within the active site channel.
  • TWO aromatic interactions of interest between PET and Phe238 are at optimal distance (each at 5.4 A).
  • FIG. 11 show's the induced fit docking analysis. All distances shown are in angstroms. Panel A of FIG 11 shows the lowest energy, catalytically competent predicted pose of PEET tetramer in wild-type PETase, XP score of -8.23 kcal/mol; catalytic triad is intact, W185 and W159 stablize PET at optimal distances through parallel displaced and edge-to-face aromatic interactions, respectively. Panel B of FIG.
  • Panel C of FIG. 11 show's the lowest energy, catalytically competent predicted pose of PEF tetramer in wild-type PETase, XP score of -9.07 kcal/mol; catalytic triad is in act, WI 85 and W159 stablize PEF at optimal distances through parallel displaced and edge-to-face aromatic interactions, respectively.
  • Panel C of FIG. 11 show's the lowest energy, catalytically competent pose of PET tetramer in double mutant PETase, XP score of -11.25 kcal/mol; catalytic triad is intact.
  • PET is stabilized by four optimal aromatic contacts: edge-to-face to Trpl85, parallel displaced to Tyr87, point-to-face to Phe238, parallel displaced to Phe 238 (i.e., Phe238 participates in aromatic interactions from two terephthalate units).
  • Panel D of FIG. 11 shows the lowest energy, catalyticaHy competent pose of PEF tetramer in double mutant PETase, XI 3 score of -10.07 kcal/mole.
  • PEF is stabilized through three optimal aromatic interactions: parallel displaced to Trpl85, parallel displaced to Phe238, and a point-to-face interaction with His237.
  • FIG. 11 shows a surface view of PETdocked in PETase double mutant structure.
  • Panel G of FIG. 11 shows a surface view of PEF docked in PETase wild type.
  • Panel H of FIG. 11 shows a surface view of PEF docked in PETase double mutant structure.
  • Panels E-H of FIG 1 1 show how PET and PEF occupy the same binding channel in both wild type and mutant proteins; also the S238F mutation changes the nature of the binding cleft.
  • Panel I of FIG 1 1 shows an overlay of Trpl85 position in PETase crystal structure (shown in white), when PET is flexibly docked (shaded) and PEF is flexibly docked (lightly shaded).
  • Trpl 85 rotates to provide optimal aromatic interactions for stabilization.
  • the N-Ca-Cp-C dihedral is -177.5°, whereas when PET is docked the same dihedral has a value of 98.4°, and -146.4° with PEF docked.
  • This dihedral is also flexible in the double mutant structure, having values of 178.8 with PET and -155.5 with PEF flexibly docked.
  • Trpl85 is not the only flexible residue in the binding site, such rotations were also seen for H237, W159 (WT), H159 (mut), and F238 (mut). The rotation of these residues illustrates the importance of modeling induced fit effects for predicting ligand binding modes. W185’s flexibility was also captured with molecular dynamics simulations.
  • a PET carbonyl carbon is at a chemically relevant distance (5.1 A) for nucleophilic attack from the Seri 60 hydroxyl group
  • His237 is at an ideal distance (3.9 A) to activate Seri 60
  • Asp206 provides hydrogen bonding support to His237 (2 8 A).
  • This binding mode is predicted to have binding affinity (estimated by the docking score with descriptors in Table 3) of -8.23 kcal/moi (Table 3).
  • our IFD predicted binding modes are consistent with a productive Michaelis complex for PET chain cleavage.
  • XP descriptors are an energetic decomposition of the Glide XP score, which itself is an estimate of the binding affinity. Thus, XP descriptors provide insight into which energetic terms contribute most significantly to binding free energy. All values shown in Table 3 are in kcal/mol. It may be seen E vd w contributes most significantly to all poses and is even more favorable in mutant binding likely due to increased aromatic interactions with Phe238. Note that in Table 3“Mut” refers to the S238H/W159H double mutant PETase structure as described some embodiments herein.
  • PET aromatic rings are within ideal p-stacking distances to several binding site residues (W185, Y87), and in particular two aromatic interactions are formed to Phe238 (point-to-face interaction at 5 4 A, and parallel displaced interaction at 5.4 A).
  • the marked difference in predicted binding affinities between WT and double mutant enzymes for PET is consistent with the increased activity of the PETase double mutant on PET, as observed experimentally, and we can identify aromatic interactions supported by the S238F mutation as being integral to this enhancement All aromatic ring-ring distances for described binding modes are illustrated in Panels A and C of FIG. 11.
  • Trpl 85 is predicted to play an important role by contributing p-stacking interactions to PET aromatic groups. Additionally, in all productive binding modes (i.e., when the carbonyl is oriented to be in the oxyanion hole, and the carbonyl carbon at a catalytic distance from Seri 60), Trpl85 is predicted to reorient relative to the crystal structure, suggesting its movement opens the active site cleft, allowing PET binding (Panel I of FIG. 11).
  • Example 3 PETase depolymerizes PEE, but not aliphatic polyesters.
  • FIG. 12 shows degradation analysis of PBS and PLA by PETase.
  • Panel A of FIG 12 shows a PBS coupon before incubation.
  • Panel B of FIG. 12 shows a PBS coupon in buffer only.
  • FIG. 12 shows a PBS coupon in buffer with PETase.
  • Panel D of FIG. 12 show's PLA film before incubation.
  • Panel E of FIG. 12 shows a PLA film after incubation in buffer only.
  • Panel F of FIG 12 shows PLA after incubation in buffer with PETase. All images w ? ere taken after 96 hours of incubation at a PETase loading of 50 nM, pH 7.2 in phosphate buffer, or a buffer-only control. No surface pitting was observed.
  • PEF is another semi -aromatic polyester marketed as a bio-based PET replacement.
  • PETase may also depolymerize this substrate.
  • Panels A-D of FIG. 3 shows the results of PEF incubations with the wild-type PETase enzyme and the PETase double mutant, alongside a buffer-only control.
  • the surface morphology of PETase-treated PEF is even more modified than PET with SEM revealing the formation of large pits, suggesting that PETase is potentially much more active on this substrate than PET.
  • FIG. 13 illustrates a comparison of PETase and the engineered enzyme S238F/W159H with PEF
  • Panel A of FIG. 13 shows a buffer-only control of PEF coupon.
  • Panel B of FIG. 13 shows PEF coupon after incubation with wild-type PETase.
  • Panel C of FIG. 13 illustrates PEF coupon after incubation with the PETase double mutant, S238F/W 159H. All SEM images were taken after 96 h of incubation at a PETase loading of 50 nM, pH 7.2 in phosphate buffer, or a buffer-only control.
  • FIG. 13 shows the percent crystallinity change, coupon mass loss, and reaction product concentration after incubation with buffer, wild-type PETase, and the S238FAV159H double mutant.
  • Panel E of FIG. 13 illustrates the predicted binding conformations of wild-type PETase from docking simulations demonstrate that PEF is accommodated in an optimum position for the interaction of the carbon (black) with the nucleophilic hydroxyl group of Serl60, at a distance of 5.0 A (dashed line). His237 is positioned within 3.7 A of the Seri 60 hydroxyl (dashed line). Residues Trp 159 (lightly shaded) and Ser238 (more darkly shaded) line the active site channel. In contrast, the double mutant S238F/W159H significantly alters the architecture of the catalytic site for PEF binding.
  • Residue His237 rotates away from Seri 60 and instead forms an aromatic interaction with PEF chain, 5.1 A. Surprisingly, the mutated Hisl59 becomes an alternative productive H-bond partner, 3.2 A. Similar to interactions with PET, Phe238 also provides additional hydrophobic interactions to an adjacent furan ring of the extended PEF polymer, creating a more intimate binding mode with the cleft with a parallel displaced aromatic interaction at 5.2 A.
  • the high-resolution structure described here reveals the binding site architecture of the 1 sakaiensis 201-F6 PETase, while the IFD results provide a mechanistic basis for both the wild-type and PETase double mutant towards the crystalline semi-aromatic polyesters PET and PEF. Changes around the active site result in a widening of the cleft compared to structural representatives of three thermophilic cutinases (FIG 13), without other major changes in the underlying secondary or tertiary structure. Furthermore, w ?
  • PETase is active on PET of approximately 15% crystallinity, and while this observation is encouraging, it is envisaged that its performance would need to be enhanced substantially, perhaps via further active-site cleft engineering similar to ongoing work on thermophilic cutinases and lipases.
  • Enzyme scaffolds capable of PET breakdown above the glass transition temperature (>70°C for PET) will also be pursued in future studies. Coupling with other processes such as milling or grinding, which can increase the available surface area of the plastic, also merit investigation towards enzymatic solutions for PET and PEF recycling. Furthermore, in light of recent studies that demonstrate the impressive synergistic effect combining multiple PET-active lipases, we expect that incorporation of I.
  • the enzyme may interact with and cleave the substrate in an endo- fashion cleaving PET (or PEE) chains internal to a polymer or in an exo-fashion by only cleaving PET from the chain ends.
  • Methods employed in the cellulase and chitinase research community such as substrate labeling with easily detected reporter molecules or examination of product ratios, could potentially shed light on this question, and will be pursued in future efforts.
  • many polysaccharide-active enzymes rely on multi- modular architectures, with a carbohydrate-binding module attached to the catalytic domain.
  • polyesterase enzymes hydrophobias, carbohydrate-binding modules, and PHA-binding modules have been used to increase the catalytic efficiency of cutinases for PET degradation.
  • hydrophobias hydrophobias
  • carbohydrate-binding modules hydrophobias
  • PHA-binding modules PHA-binding modules
  • Example 1 A modified polyethylene terephthaiate (PET)-digesting enzyme (PETase) comprising at least one amino acid mutation of an active site residue, wherein the modified PETase has a narrowed binding cleft compared to the unmodified PETase.
  • PET polyethylene terephthaiate
  • PETase polyethylene terephthaiate-digesting enzyme
  • Example 2 The modified PETase of Example 1, wherein the unmodified PETase is from a bacterium of the genus Ideonella.
  • Example 3 The modified PETase Example 2, wherei n the bacterium is a strai n of Ideonella sakaiemis.
  • Example 4 The modified PETase Example 1, wherein an amino residue at position 159 is mutated.
  • Example 5 The modified PETase Example 4, wherein an amino residue at position 238 is mutated.
  • Example 6 The modified PETase Example 1, wherein an amino residue at position 238 is mutated.
  • Example 7 The modified PETase Example 1, wherein the modified PETases comprises the W159H/S238F double mutation.
  • Example 8 A nucleic acid molecule encoding a modified PETase having mutations at two active-site residues.
  • Example 9 The nucleic acid molecule of Example 8, wherein the amino acid residue at position 159 is mutated.
  • Example 10 The nucleic acid molecule of Example 8, wherein the amino acid residue at position 238 is mutated.
  • Example 1 The nucleic acid molecule ofExample 8, wherein the modified PETase comprises the W159H/S238F double mutation.
  • Example 12 An expression vector comprising the nucleic acid molecule of Example 8.
  • Example 13 A nucleic acid encoding the enzyme comprising the amino acid sequence depicted in FIG. 2(B).
  • Example 14 A cell that expresses the modified PETase of Example 1.
  • Example 15 A method for degrading a polymer comprising contacting the modified PETase of Example 1 with the polymer.
  • Example 16 The method ofExample 14, wherein the polymer is a polyester.
  • Example 17 The method of Example 14, wherein the polymer is an aromatic polymer or a semi-aromatic polymer.
  • Example 18 The method of Example 14, wherein the polymer is polyethylene terephthalate Example 19. The method ofExample 14, wherein the polymer is polyethylenefuranoate (PEF).
  • Example 20 The method of Example 14, wherein the polymer is from a recycled plastic material .
  • Example 21 The method of Example 14, wherein the PETase is expressed by a cell.
  • inventive aspects lie in less than all features of a single foregoing disclosed embodiment, configuration, or aspect. While certain aspects of conventional technology have been discussed to facilitate disclosure of some embodiments of the present invention, the Applicants in no disclaim these technical aspects, and it is contemplated that the claimed invention may encompass one or more of the conventional technical aspects discussed herein. Thus, the following claims are hereby incorporated into this Detailed Description, with each claim standing on its own as a separate aspect, embodiment, or configuration.

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Abstract

L'invention concerne des enzymes modifiées capables de dégrader des polymères tels que pe polyéthylène téréphtalate (PET). L'invention concerne également des acides nucléiques codant pour les enzymes et des cellules modifiées qui expriment les enzymes modifiées. L'invention concerne en outre des procédés de dégradation de polymères tels que des polyesters aromatiques et semi-aromatiques.
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WO2021148713A1 (fr) 2020-01-24 2021-07-29 Teknologian Tutkimuskeskus Vtt Oy Enzymes, micro-organismes et leurs utilisations, et procédé de dégradation d'un polyester
WO2022043545A1 (fr) * 2020-08-28 2022-03-03 Cyclezyme Ab Variant d'hydrolase de pet d'ideonella sakaiensis présentant une thermostabilité accrue.
WO2022090290A1 (fr) * 2020-10-27 2022-05-05 Carbios Nouvelles estérases et leurs utilisations
CN114480342A (zh) * 2021-06-22 2022-05-13 中国科学院苏州生物医学工程技术研究所 突变型pet水解酶、重组载体、重组工程菌及其应用
WO2022104434A1 (fr) * 2020-11-19 2022-05-27 Commonwealth Scientific And Industrial Research Organisation Variants d'hydrolase
WO2022135985A1 (fr) * 2020-12-24 2022-06-30 Société des Produits Nestlé S.A. Recyclage enzymatique de polyéthylène téréphtalate par des cutinases
WO2023019222A1 (fr) * 2021-08-11 2023-02-16 Biometis Technology, Inc. Dégradation enzymatique de polyéthylène téréphtalate
JP2023532838A (ja) * 2020-05-29 2023-08-01 ローディア ポリアミダ エ エスペシアリダデス エス．アー． エポキシ樹脂の化学酵素分解
WO2024040310A1 (fr) * 2022-08-26 2024-02-29 Samsara Eco Pty Limited Enzymes et leurs utilisations
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WO2022043545A1 (fr) * 2020-08-28 2022-03-03 Cyclezyme Ab Variant d'hydrolase de pet d'ideonella sakaiensis présentant une thermostabilité accrue.
WO2022090290A1 (fr) * 2020-10-27 2022-05-05 Carbios Nouvelles estérases et leurs utilisations
JP2023546500A (ja) * 2020-10-27 2023-11-02 キャルビオス 新規エステラーゼ及びその使用
WO2022104434A1 (fr) * 2020-11-19 2022-05-27 Commonwealth Scientific And Industrial Research Organisation Variants d'hydrolase
WO2022135985A1 (fr) * 2020-12-24 2022-06-30 Société des Produits Nestlé S.A. Recyclage enzymatique de polyéthylène téréphtalate par des cutinases
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CN114480342A (zh) * 2021-06-22 2022-05-13 中国科学院苏州生物医学工程技术研究所 突变型pet水解酶、重组载体、重组工程菌及其应用
WO2023019222A1 (fr) * 2021-08-11 2023-02-16 Biometis Technology, Inc. Dégradation enzymatique de polyéthylène téréphtalate
RU2843950C2 (ru) * 2021-08-11 2025-07-22 Байометис Текнолоджи, Инк. Ферментативная деградация полиэтилентерефталата
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WO2024040310A1 (fr) * 2022-08-26 2024-02-29 Samsara Eco Pty Limited Enzymes et leurs utilisations
EP4389864A1 (fr) * 2022-12-20 2024-06-26 Basf Se Cutinases
WO2024132625A1 (fr) * 2022-12-20 2024-06-27 Basf Se Cutinases
WO2025136384A1 (fr) * 2023-12-20 2025-06-26 Biometis Technology, Inc. Dégradation enzymatique de polyéthylène téréphtalate
WO2025206322A1 (fr) * 2024-03-29 2025-10-02 東洋紡株式会社 Dégradation enzymatique de pef

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