WO2023019222A1 - Enzymatic degradation of polyethylene terephthalate - Google Patents

Enzymatic degradation of polyethylene terephthalate Download PDF

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Publication number
WO2023019222A1
WO2023019222A1 PCT/US2022/074866 US2022074866W WO2023019222A1 WO 2023019222 A1 WO2023019222 A1 WO 2023019222A1 US 2022074866 W US2022074866 W US 2022074866W WO 2023019222 A1 WO2023019222 A1 WO 2023019222A1
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Prior art keywords
amino acid
acid substitution
petase
bhr
variant
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PCT/US2022/074866
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English (en)
French (fr)
Inventor
Jie Yang
Xiyun Zhang
Khin Oo
Goutami BANERJEE
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Biometis Technology Inc
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Biometis Technology Inc
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Priority to CN202280055812.9A priority Critical patent/CN117836407A/zh
Priority to US18/682,722 priority patent/US20250368971A1/en
Priority to CA3228057A priority patent/CA3228057A1/en
Priority to KR1020247004434A priority patent/KR20240032968A/ko
Priority to EP22856824.2A priority patent/EP4384607A4/en
Priority to JP2024508603A priority patent/JP7797623B2/ja
Application filed by Biometis Technology Inc filed Critical Biometis Technology Inc
Priority to AU2022325230A priority patent/AU2022325230B2/en
Priority to MX2024001814A priority patent/MX2024001814A/es
Publication of WO2023019222A1 publication Critical patent/WO2023019222A1/en
Anticipated expiration legal-status Critical
Priority to JP2025159783A priority patent/JP2026004384A/ja
Ceased legal-status Critical Current

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    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • 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

  • PET polyethylene terephthalate
  • the present disclosure relates to a composition
  • a composition comprising a variant Bhr-PETase as compared to SEQ ID NO:1, wherein said variant comprises at least one amino acid substitution compared to SEQ ID NO: 1 at an amino acid position(s) selected from the group consisting of 27, 1, 2, 5, 9, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 30, 32, 33, 34, 40, 46, 48, 49, 53, 54, 55, 56, 57, 60, 62, 68, 70, 72, 74, 77, 82, 83, 85, 87, 88, 90, 92, 97, 98, 101, 102, 105, 108, 109, 110, 113, 114, 117, 119, 121, 122, 125, 127, 135, 136, 138, 139, 140, 142, 143, 145, 147, 149, 150, 153, 156, 157,
  • the present disclosure relates to a composition
  • a composition comprising a variant Bhr-PETase as compared to SEQ ID NO:1, wherein said variant comprises a amino acid substitution at position 27 and at least one further amino acid substitution compared to SEQ ID NO: 1 at an amino acid position(s) selected from the group consisting of 1, 2, 5, 9, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 30, 32, 33, 34, 40, 46, 48, 49, 53, 54, 55, 56, 57, 60, 62, 68, 70, 72, 74, 77, 82, 83, 85, 87, 88, 90, 92, 97, 98, 101, 102, 105, 108, 109, 110, 113, 114, 117, 119, 121, 122, 125, 127, 135, 136, 138, 139, 140, 142, 143, 145, 147, 149, 150, 153, 156, 157, 158, 160, 16
  • the present disclosure relates to a composition
  • a composition comprising a variant Bhr-PETase as compared to SEQ ID NO:1, wherein said variant comprises at least one amino acid substitution compared to SEQ ID NO: 1 at an amino acid position(s) selected from the group consisting of 27, 1, 2, 5, 9, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 30, 32, 33, 34, 40, 46, 48, 49, 53, 54, 55, 56, 57, 60, 62, 68, 70, 72, 74, 77, 82, 83, 85, 87, 88, 90, 92, 97, 98, 101, 102, 105, 108, 109, 110, 113, 114, 117, 119, 121, 122, 125, 127, 135, 136, 138, 139, 140, 142, 143, 145, 147, 149, 150, 153, 156, 157, 158, 160, 161, 162, 163, 164,
  • said amino acid substitution above is at an amino acid position(s) selected from the group consisting of 27, 1, 2, 5, 9, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 30, 32, 33, 34, 40, 46, 48, 49, 53, 54, 55, 56, 57, 60, 62, 68, 70, 72, 74, 77, 82, 83, 85, 87, 88, 90, 92, 97, 98, 101, 102, 105, 108, 109, 110, 113, 114, 117, 119, 121, 122, 125, 127, 135, 136, 138, 139, 140, 142, 143, 145, 147, 149, 150, 153, 156, 157, 158, 160, 161, 162, 163, 164, 167, 170, 173, 174, 177, 179, 181, 182, 184, 185, 189, 190, 193, 194, 195, 198, 200, 203,
  • said amino acid substitution above is at an amino acid position(s) selected from the group consisting of 27, 2, 17, 20, 21, 23, 24, 40, 46, 49, 55, 57, 77, 83, 97, 101, 102, 109, 110, 114, 117, 135, 136, 139, 140, 142, 143, 149, 161, 163, 164, 167, 184, 185, 195, 222, 227, 228, 229, 249, 250 and 251.
  • said amino acid substitution above is at an amino acid position(s) selected from the group consisting of 8, 126, 137, 165, 169, 172, 191, 192 and 197.
  • said amino acid substitution is selected from the group consisting of S27L, S27F, S27H, S27T, S27W, S1A, S1G, S1M, S1R, N2E, N2F, N2L, N2R, N2S, Q5E, N9A, N9E, N9S, R12K, S13L, S13R, A14K, A14S, L15I, T16E, T17A, T17C, T17G, T17H, T17I, T17K, T17L, T17M, T17N, T17Q, T17R, T17S, D18R, P20D, P20E, P20I, P20Q, P20T, F21W, F21Y, S22A, S22I, S22K, S22P, S22R, S22V, V23L, V23T, A24D, A24G, A24H, A24N, A24S, A24
  • said amino acid substitution is selected from the group consisting of S27L, S27F, S27H, S27T, S27W, S1A, S1G, S1M, S1R, N2R, Q5E, N9A, N9E, N9S, R12K, S13L, S13R, A14K, A14S, L15I, T16E, T17A, T17C, T17G, T17H, T17K, T17L, T17Q, T17S, D18R, P20T, F21W, S22A, S22I, S22K, S22P, S22R, S22V, V23L, V23T, A24S, A24V, T25A, T25F, T25H, T25Q, T25R, T25V, Y26K, Y26L, Y26T, R30K, S32K, S32M, S32Q, S32Y, V33G, V33Q, V33T
  • said amino acid substitution is selected from the group consisting of S27L, S27F, S27H, S27T, S27W, N2R, T17A, T17C, T17G, T17H, T17K, T17L, T17Q, T17S, P20T, F21W, V23L, V23T, A24S, A24V, V40T, G46L, G46S, L49G, A55L, A55V, S57I, S57M, S57V, H77Q, V83T, A97F, A97G, S101A, S101D, S101F, S101H, S101K, S101L, S101M, S101N, S101Q, S101R, S101V, S101W, A102V, T109F, T109K, T109L, T109N, T109R, S110G, S110H, S110R, A114K, A114L, A114S, A114V, A117G, A117
  • said amino acid substitution is selected from the group consisting of N2E, N2F, N2L, N2S, P8T, T17I, T17M, T17N, T17R, P20D, P20E, P20I, P20Q, F21Y, A24D, A24G, A24H, A24N, A24T, Y26C, L31M, G38D, G46E, G46N, G46R, A55C, A55I, A55M, A55T, S57C, S57E, S57F, S57L, S57T, V83I, V83L, N87Q, S95N, A97C, A97E, A97L, A97P, A97Q, A97S, A97T, A97V, S101C, S101Y, T109A, T109G, T109Y, S110D, S110K, S110N, A117F, A117L, A117T, A117Y, V126I, L
  • said variant Bhr-PETase enzyme has one or more amino acid substitutions at one of said positions, two of said positions, three of said positions, four of said positions, five of said positions, six of said positions, seven of said positions, eight of said positions, nine of said positions, ten of said positions, eleven of said positions, twelve of said positions, thirteen of said positions, fourteen of said positions, fifteen of said positions, sixteen of said positions, seventeen of said positions, eighteen of said positions, nineteen of said positions or twenty of said positions.
  • said variant Bhr-PETase comprises a set of amino acid substitutions selected from the group consisting of A102V/T136M, A14S/L15I/S22A/F92Q/I143R/Q167T/V219K, D18R/G46S/M56I/R108T/A127M/R138E/I139A/N190S/L227R/W228F/R236E, D18R/I139A/F161W/W228F, D18R/I54V/I82F/N105D/A127M/A184S/S218A/V219K/M225L/N241P/N243P, D18R/I54V/I82F/R108T/V150I/N253Y/N254R, D18R/I54V/M56I/R138E/I139A/T194S/D203N/V219K/F250I, D18R/I54V/M56I
  • said variant Bhr-PETase comprises a set of amino acid substitutions selected from the group consisting of A102V/T136M, A14S/L15I/S22A/F92Q/I143R/Q167T/V219K, D18R/G46S/M56I/R108T/A127M/R138E/I139A/N190S/L227R/W228F/R236E, D18R/I139A/F161W/W228F, D18R/I54V/I82F/N105D/A127M/A184S/S218A/V219K/M225L/N241P/N243P, D18R/I54V/I82F/R108T/V150I/N253Y/N254R, D18R/I54V/M56I/R138E/I139A/T194S/D203N/V219K/F250I, D18R/M56I
  • said variant Bhr-PETase comprises a set of amino acid substitutions selected from the group consisting of A102V/T136M, A14S/L15I/S22A/F92Q/I143R/Q167T/V219K, D18R/G46S/M56I/R108T/A127M/R138E/I139A/N190S/L227R/W228F/R236E, D18R/I139A/F161W/W228F, D18R/I54V/I82F/N105D/A127M/A184S/S218A/V219K/M225L/N241P/N243P, D18R/I54V/M56I/R138E/I139A/T194S/D203N/V219K/F250I, D18R/M56I/I139A/L227R/W228F, D18R/M56I/R138E/I139A
  • said variant Bhr-PETase comprises a set of amino acid substitutions selected from the group consisting of L90F/F92G/D158L/A174R/D203V, V23L/A24V/S32K/N87F/F92Y/A125S/T136A, P20T/L90Y/F92L/S101M/D158E/A174K/I222L, D18R/S32K/Y60H/L90F, P20T/F21W/A24V/L90Y/F92L/T109K/A125S/T136A, Y60H/S101A/A114K/A117Q/D203R/I222L, S32Q/F92G/S101Q/A114K/Q142W/D158I/D203R/I222L, T17G/L90F/F92L/T109R/A114K/D158E/D203V/I222L, R12K/S32K/F92L
  • the present disclosure relates to a nucleic acid encoding the variant Bhr-PETase enzyme of any one of the preceding claims.
  • the present disclosure relates to an expression vector comprising the nucleic acid.
  • the present disclosure relates to a host cell comprising the expression vector.
  • the cell is bacteria, yeast or fungi.
  • the present disclosure relates to a method of making a variant Bhr- PETase enzyme comprising culturing the host cell described herein under conditions wherein said variant Bhr-PETase enzyme is produced, and recovering said variant Bhr-PETase enzyme.
  • the present disclosure relates to a method of pretreating PET, comprising a mechanical pretreatment, a thermo-mechanical pretreatment, and/or a chemical pretreatment of the PET prior to an enzymatic degradation of the PET.
  • the mechanical pretreatment comprises grinding of the PET into particles.
  • the thermo-mechanical pretreatment comprises extruding the PET at a temperature configured to amorphize and reduce crystallinity of the PET.
  • the chemical pretreatment comprises contacting the PET with an ionic liquid, strong acid, base, or solvent configured to reduce crystallinity or to change a surface structure of the PET.
  • the present disclosure relates to a method of degrading PET, comprising contacting the PET with the variant Bhr-PETase enzyme described herein.
  • the method further comprises pretreating the PET according to the method described herein.
  • the method degrades the PET in a mixed plastics composition.
  • the plastics composition comprises analog of PET, PET-like or PET substitute derived biologically or chemically.
  • the plastics composition comprises at least one selected from the group consisting of Polybutylene terephthalate (PBT), Polycabonate (PC), Polycaprolactone (PCL), Polyethylene Furanoate (PEF), and High Density Polyethylene (HDPE).
  • the method excludes sorting plastics to select PET from a mixture of plastics.
  • Figure 1 depicts the sequence of an exemplary wild type Bhr-PETase (also called G1P Bhr-PETase herein; SEQ ID NO:1).
  • Figures 2A-2I depicts the sequence alignment of the exemplary wild type Bhr-PETase (SEQ ID NO:1), wild type Lcc-PETase (SEQ ID NO:4), wild type Is-PETase (SEQ ID NO:6) and 20 exemplary homologs of Bhr-PETase.
  • Figure 3 depicts %Sequence identity of the exemplary wild type Bhr-PETase (SEQ ID NO:1), wild type Lcc-PETase (SEQ ID NO:4), wild type Is-PETase (SEQ ID NO:6) and 20 exemplary homologs of Bhr-PETase w.r.t. wild type Bhr-PETase.
  • Figure 4 depicts the comparison of thermostability between three wild type enzymes Lcc-PETase, Bhr-PETase and Is-PETase.
  • Figure 5 depicts the comparison of Bhr-PETase and Lcc-PETase on amorphous PET at 65°C (Fig 5 graph A) and at 72°C (Fig 5 graph B).
  • Figure 6 depicts the comparison of Bhr-PETase and Lcc-PETase on > 40% crystalline PET at 65°C (Fig 6 graph A) and at 72°C (Fig 6 graph B).
  • Figures 7A ⁇ D show the Bhr-PETase G1 variants with improved total activity and thermostability over Bhr-PETase G1P (wild type Bhr-PETase).
  • a Bhr-PETase G1 variant with amino acid substitution S27L exhibited 1.80 fold improved total activity and 3.36 fold improved thermostability over Bhr-PETase G1P and therefore selected as Bhr-PETase G2P.
  • Figures 8A ⁇ B show the Bhr-PETase G1 variants with improved thermostability over Bhr-PETase G1P (wild type Bhr-PETase).
  • Figures 9A ⁇ AA show the Bhr-PETase G1 variants with improved total activity over Bhr-PETase G1P (wild type Bhr-PETase).
  • Figures 9J ⁇ AA show the Bhr-PETase G2 variants with improved total activity over Bhr-PETase G2P (Bhr-PETase G1P with amino acid substitution S27L).
  • Figures 10A ⁇ B display particular variants of Bhr-PETase by position that demonstrate beneficial properties in total activity, and/or thermostability as displayed in Figures 7A ⁇ D, Figures 8A ⁇ B and Figures 9A ⁇ AA.
  • FIGS 11A ⁇ B depict the wild type sequences of Bhr-PETase, Lcc-PETase, and Is- PETase, as well as starting nucleic acids and codon optimized sequences for expression in Bacteria, Yeast and Fungi. DETAILED DESCRIPTION OF THE INVENTION A. Introduction.
  • the present invention is directed to enzymes that will hydrolyze polyethylene terephthalate (PET).
  • PET is a polyester polymer created by the combination of two monomers: modified ethylene glycol and purified terephthalic acid. While plastics such as PET find literally thousands of uses in modern society, PET, is essentially non-degradable.
  • PET plastic pollution has contaminated the entire planet, which poses a number of significant issues for the planet and human health. PET can be recycled; however this still does not prevent major amounts of PET from being dumped into landfill and/or the ocean.
  • Plastics including PET are remarkably resistant to enzymatic degradation.
  • PET hydrolases There are two categories of PET hydrolases: (i) PET-modifying enzymes that limit the degradation only at the surface of PET without visible change by electron microscope observations and (ii) PET-degrading enzymes or PETases that can significantly degrade the inner block of PET (e.g., by at least 10%) with visible change by electron microscope observations.
  • PET-modifying enzymes have been reported but they may not significantly degrade the body of PET and may not be applicable for biorecycling of PET. As is known in the art, there are a few enzyme types that show limited ability to degrade the inner block of PET.
  • the first reported enzyme able to act on ester bonds of PET polymers was a cutinase from Thermobifida fusca in 2005.
  • additional enzymes including a PET-hydrolyzing enzyme from Ideonella sakaiensis, Is-PETase, in 2016 and a leaf branch compost cutinase (Lcc-PETase) in 2012.
  • PET exists both as an amorphous and as a semi-crystalline material. Chain mobility may be increased in the amorphous phase around PET’s glass transition temperature Tg (around 70°C), which allows better access to ester linkages and hence faster degradation. The reaction temperature around Tg may be controlled to achieve efficient enzymatic PET degradation. In addition, the physical aging process of PET at around 70°C may convert the mobile amorphous fraction to recalcitrant microstructures which hinders further enzymatic hydrolysis of PET.
  • thermostable and thermoactive PETase is desirable to allow the degradation reaction to occur at around glass transition temperature and overcome the competing physical aging process.
  • wild type Bhr-PETase is a close homolog of Lcc-PETase, sharing 94% sequence identity between the two enzymes, it is found to be more thermostable and thermoactive than Lcc-PETase, as well as Is-PETase,. Thermostability and thermosactivity are key factors for efficient PET degradation.
  • the present disclosure relates to variants of a PETase from the bacterium HR29 (Bhr-PETase) that have been engineered to exhibit even greater activity and thermostability.
  • modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein.
  • a modification may be an altered carbohydrate or PEG structure attached to a protein.
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
  • the amino acid modification is always to an amino acid coded for by DNA, e.g. the 20 amino acids that have codons in DNA and RNA.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid.
  • the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism.
  • the substitution S27L refers to a variant polypeptide, in this case a PETase, in which the serine (S) at position 27 is replaced with leucine L.
  • amino acid insertion or "insertion” as used herein is meant the addition of an amino acid sequence at a particular position in a parent polypeptide sequence.
  • - 233E or 233E designates an insertion of glutamic acid after position 233 and before position 234.
  • -233ADE or A233ADE designates an insertion of AlaAspGlu after position 233 and before position 234.
  • amino acid deletion or “deletion” as used herein is meant the removal of an amino acid sequence at a particular position in a parent polypeptide sequence.
  • F250- or F250#, F250() or F250del designates a deletion of glutamic acid at position 250.
  • FRS250- or FRS250# designates a deletion of the sequence PheArgSer that begins at position 250.
  • parent polypeptide as used herein is meant a starting polypeptide that is subsequently modified to generate a variant.
  • the parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide.
  • Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it.
  • some embodiments utilize an exemplary wild-type Bhr-PETase (also called G1P Bhr- PETase; SEQ ID NO: 1; sequence shown in Figure 1) as the parent polypeptide.
  • variant protein or “protein variant”, or “variant” as used herein is meant a protein that differs from that of a parent protein by virtue of at least one amino acid modification.
  • Protein variant may refer to the protein itself, a composition comprising the protein, or the amino sequence that encodes it.
  • the protein variant has at least one amino acid modification compared to the parent protein, e.g. from about one to about seventy amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.
  • the parent polypeptide is a wild type sequence, for example the exemplary wild type Bhr-PETase designated “G1P” herein.
  • the protein variant sequence herein will preferably possess at least about 80, 81, 82, 83, 84, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with a parent protein sequence, preferably at least about 90% identity, and preferably at least about 95-98-99% identity.
  • Variant protein can refer to the variant protein itself, compositions comprising the protein variant, or the DNA sequence that encodes it.
  • variant PETase herein is meant a novel PETase that has at least one amino acid modification in the amino acid sequence as compared to a parent PETase enzyme.
  • the variant PETases of the invention generally are compared to the wild type G1P sequence. Additionally, unless otherwise noted, the variant PETases of the invention are enzymatically active, that is, there is detectable PETase activity using the PETase assay described in Example 9.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The peptidyl group generally comprise naturally occurring amino acids and peptide bonds.
  • polypeptides may include synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels.
  • residue as used herein is meant a position in a protein and its associated amino acid identity.
  • Serine 27 also referred to as Ser27 or S27
  • non-naturally occurring modification as used herein is meant an amino acid modification that is not found in the parent (e.g. G1P) enzyme in nature.
  • amino acid and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids that are coded for by DNA and RNA.
  • position as used herein is meant a location in the sequence of a protein. In general, the position number (which is more fully discussed below) is relative to the first amino acid of the mature PETase sequence, e.g. excluding the signal peptide.
  • PETase herein is meant a protein with PETase activity.
  • PETase activity herein is meant that in the absence of MHTase, the enzyme catalyzes the hydrolysis of PET to mono(hydroxyethyl)terephthalate (MHET) as the major product. In the presence of MHTase, which is the case of Example 9, MHTase will further convert MHET to terephthalic acid (TPA) and ethylene glycol (EG) as the major products of the enzymatic reaction. Enzymes having detectable PETase activity in the assay outlined below and in Example 9 are considered PETases herein. The PETase activity may be measured as PETase total activity and/or PETase thermostability as described herein.
  • invention sequence an amino acid sequence of the present invention
  • parent amino acid sequence referred to in the claims e.g. for G1P, SEQ ID NO:1
  • degree of identity is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "invention sequence,” or the length of the SEQ ID NO:1, whichever is the shortest.
  • the result is expressed in percent identity as calculated below.
  • the mature polypeptide disclosed in SEQ ID NO:1 is used to determine the corresponding amino acid residue in another PETase of the present invention.
  • the amino acid sequence of another PETase is aligned with the mature polypeptide disclosed in SEQ ID NO:1, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO:1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • Identification of the corresponding amino acid residue in another PETase can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log- expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059- 3066; Katoh et al.
  • MUSCLE multiple sequence comparison by log- expectation; version 3.5 or later
  • MAFFT version 6.857 or later
  • Katoh and Kuma 2002, Nucleic Acids Research 30: 3059- 3066; Katoh et al.
  • alignments can in turn be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.
  • tools and resources are available for retrieving and generating structural alignments.
  • SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable.
  • Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11 : 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567). [0043] In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The standardly accepted IUPAC single letter or three letter amino acid abbreviation is employed.
  • isolated in the context of a PETase herein is meant that the polypeptide is devoid of other proteins.
  • the PETase of the invention is isolated.
  • isolated refers to a polypeptide which is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, most preferably at least 90% pure, and even most preferably at least 95 to 98% pure, as determined by SDS-PAGE.
  • the polypeptides are in "essentially pure form", i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively associated.
  • recombinant enzyme herein is meant that the enzyme is produced by recombinant techniques and that nucleic acid encoding the enzyme of the invention is operably linked to at least one exogeneous (e.g. not native to the parent PETase) sequence, including, for examples, promoters, terminators, signal sequences, etc., as are more fully outlined below.
  • nucleic acid construct refers to a nucleic acid molecule, either single- stranded or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences.
  • operably linked refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
  • the term “about” means modifying, for example, lengths of nucleotide sequences, degrees of errors, dimensions, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, and like values, and ranges thereof, refers to variation in the numerical quantity that may occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations.
  • the term “about” also encompasses amounts that differ due to aging of, for example, a composition, formulation, or cell culture with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term “about” the claims appended hereto include equivalents to these quantities.
  • the term “about” further may refer to a range of values that are similar to the stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within 10, 9, 8,7, 6, 5,4, 3, 2, 1 percent or less of the stated reference value. C.
  • the present invention provides variant PETases with improved enzymatic activity that can be used in a variety of applications, most notably in the degradation of plastics made from PET.
  • the variant PETases of the invention have modified, improved biochemical properties as compared to the wild type Bhr-PETase, “G1P” (i.e. “Generation 1 Parent”), SEQ ID NO:1 herein, as shown in Figure 1.
  • the variant PETases of the invention can also have modified, improved biochemical properties as compared to the “G2P” (i.e. “Generation 2 Parent”), which has amino acid substitution S27L.
  • the biochemical properties of the variant PETases that can be improved herein include, but are not limited to, thermostability, thermoactivity, specific activity, and production.
  • the variant Bhr-PETases of the invention have one or more improved properties as compared to G1P or G2P.
  • improved herein is meant a desirable change of at least one biochemical property.
  • Improved function can be measured as a percentage increase or decrease of a particular activity, or as a “fold” change, with increases of desirable properties (e.g. activity or thermostability).
  • a variant Bhr-PETase may have a 10% increase in thermostability or a 10% increase in PETase activity, as compared to G1P or G2P.
  • percentage changes are used to describe changes in biochemical activity of less than 100%, and fold-changes are used to describe changes in biochemical activity of greater than 100% (as compared to the parental enzyme, in many cases G1P or G2P).
  • percentage changes usually increases of biochemical activity of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% and 99% can be accomplished.
  • a “fold increase” (or decrease) is measured as compared to the starting or parent enzyme.
  • the variant T17A/S27T/T48S/I82L/L90F/Q167V/P213N/S252T has 1.6 fold increase in specific activity as compared to G1P: this is calculated by [(activity of variant)/(activity of parent)].
  • the improvement is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold or higher.
  • the present invention provides variant Bhr-PETases that have total activity equal to or greater than the total activity of G1P (the wild type Bhr-PETase of SEQ ID NO:1) or G2P (G1P with amino acid substitution S27L).
  • total activity herein may be determined by monitoring the production of TPA (terephthalic acid) during the PET depolymerization reaction at an elevated temperature such as 65°C, quantified using a colorimetric assay or HPLC as described in Example 9. Any improvement in total activity may be due to the improvement of thermoactivity, specific activity, and/or production of the variant PETase.
  • the variant Bhr-PETases have improved total activity that is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold or higher.
  • the variant Bhr-PETases may have increased thermoactivity.
  • the “thermoactivity” herein may be determined by monitoring the production of TPA (terephthalic acid), MHET (mono(hydroxyethyl)terephthalate) and BHET (bis(2-hydroxyl) terephthalate during the PET depolymerization reaction at an elevated temperature such as 65°C, quantified in mg of equivalent TPA generated per h per mg of enzyme (mgTPAeq. h -1 mgenzyme -1 ).
  • Equivalent TPA is calculated by a sum of TPA, MHET converted to TPA, BHET converted to TPA, and any other measurable oligomers converted to TPA.
  • PETases that have increased activity per milligram of enzyme as compared to G1P (the wild type Bhr-PETase of SEQ ID NO:1) or G2P (G1P with amino acid substitution S27L) may show an improved thermoactivity.
  • the variant Bhr-PETases have improved thermoactivity that is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold or higher.
  • the “specific activity” herein may be determined by monitoring the production of TPA (terephthalic acid), MHET (mono(hydroxyethyl)terephthalate) and BHET (bis(2-hydroxyl) terephthalate during the PET depolymerization reaction at the optimal operating temperature of Bhr-PETases, quantified in mg of equivalent TPA generated per h per mg of enzyme (mg TPAeq. h -1 mg enzyme -1 ).
  • TPA terephthalic acid
  • MHET mono(hydroxyethyl)terephthalate
  • BHET bis(2-hydroxyl) terephthalate
  • the variant Bhr-PETases have improved specific activity that is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold or higher.
  • the variant Bhr-PETases have increased equivalent TPA generated per h per mg of enzyme that is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold or higher.
  • the variant Bhr-PETases may have increased production.
  • the “production” herein may be determined by monitoring the protein titer of Bhr-PETase in g/L.
  • PETases that have increased quantity per liter of enzyme supernatant as compared to G1P (the wild type Bhr-PETase of SEQ ID NO:1) or G2P (G1P with amino acid substitution S27L) may show an improved production.
  • the variant Bhr-PETases have improved thermoactivity that is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold or higher.
  • improvements are measured as compared to the G1P or G2P enzyme using a Bhr-PETase activity assay, under conditions that challenge the variant Bhr-PETase against the G1P or G2P enzyme. 2. Thermostability [0066] Additionally, as will be appreciated by those in the art, it can be desirable to run PET degradation at around the glass transition temperature.
  • Amorphous PET domains will increase the mobility at around the glass transition temperature (around 67-72°C), making it more accessible for enzyme hydrolysis. At higher temperatures above the transition temperature, the PET substrate will re-crystalize over time, which is not favorable for PET degradation. Therefore, for example, about 65-72°C may be considered an optimal temperature range for PET degradation. [0067] Accordingly, in many embodiments, the variant Bhr-PETases have improved thermostability.
  • “Thermostability” in this context means that the variant enzymes are more stable than G1P (the wild type Bhr-PETase of SEQ ID NO:1) or G2P (G1P with amino acid substitution S27L) under the same thermal challenge conditions, that is, the activity of the variant is higher than that of the G1P or G2P enzyme under identical conditions (generally using an assay as outlined herein and as shown in Example 9).
  • the variant Bhr-PETases are more stable than the G1P or G2P enzyme when exposed to temperatures of about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 and/or 85°C for a period of time, for example ranging from about 0.5, 1, 2, 3, 4, 5, or 6 hour to about 5, 6, 7, 8, 9, 10 hours or longer, depending on the ultimate conditions for the use of the variant Bhr-PETase.
  • the variant Bhr-PETases are more stable than the G1P or G2P enzyme when exposed to temperatures preferably from about 65°C to 85°C for at least about 0.5 hour, preferably from about 65°C to 72°C for at least about 1 hour, preferably at least 65°C for at least about 1 hour, preferably at least 70°C for at least about 1.5 hour.
  • the variant Bhr-PETases are more thermostable than the G1P or G2P enzyme by at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold or higher.
  • improvements are measured as compared to the G1P or G2P enzyme using a Bhr-PETase activity assay, under conditions that measure the variant Bhr-PETase or the G1P or G2P enzyme activity with and without thermal treatment. 3.
  • PETase Assays There are several PETase activity assays that can be used to determine activity, as generally outlined in Examples 6 and 9 for PET film-based assay, as well as in Example 5 for BHET-based assay. PETase activity can also be monitored by pNPB (p-Nitrophenyl Butyrate) assay. In pNPB assay, surrogate substrate p-nitrophenylbutyrate may be hydrolyzed by PETase to p-nitrophenol and butyric acid. The release of p-nitrophenol directly correlates to PETase activity and can be determined spectrophotometrically at 405 nm, for example. 4.
  • pNPB p-Nitrophenyl Butyrate
  • the present invention provides a number of specific variant Bhr-PETases with improved activity and/or thermostability for use in the degradation of PET.
  • the variant Bhr-PETase has one or more amino acid substitutions at a position (relative to G1P, SEQ ID NO:1) selected from the group consisting of 27, 1, 2, 5, 9, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 30, 32, 33, 34, 40, 46, 48, 49, 53, 54, 55, 56, 57, 60, 62, 68, 70, 72, 74, 77, 82, 83, 85, 87, 88, 90, 92, 97, 98, 101, 102, 105, 108, 109, 110, 113, 114, 117, 119, 121, 122, 125, 127, 135, 136, 138, 139, 140, 142, 143, 145, 147, 149,
  • the variant Bhr-PETase has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions at a position (relative to G1P, SEQ ID NO:1) selected from the same group. [0074] In some embodiments, the variant Bhr-PETase has one more amino acid substitutions selected from the group consisting of S27L, S27F, S27H, S27T, S27W, S1A, S1G, S1M, S1R, N2E, N2F, N2L, N2R, N2S, Q5E, N9A, N9E, N9S, R12K, S13L, S13R, A14K, A14S, L15I, T16E, T17A, T17C, T17G, T17H, T17I, T17K, T17L, T17M, T17N, T17Q, T17R, T17S, D18R, P20D, P20E, P20I, P
  • the variant Bhr-PETase has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions selected from the same group. [0075] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the serine at position 27 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S27L.
  • the amino acid substitution is S27F.
  • the amino acid substitution is S27H.
  • the amino acid substitution is S27T. In some embodiments, the amino acid substitution is S27W. [0076] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the serine at position 1 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S1A.
  • the amino acid substitution is S1G.
  • the amino acid substitution is S1M.
  • the amino acid substitution is S1R.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 2 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N2E. In some embodiments, the amino acid substitution is N2F. In some embodiments, the amino acid substitution is N2L. In some embodiments, the amino acid substitution is N2R. In some embodiments, the amino acid substitution is N2S. [0078] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the glutamine at position 5 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is Q5E.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 9 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N9A. In some embodiments, the amino acid substitution is N9E. In some embodiments, the amino acid substitution is N9S. [0080] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the arginine at position 12 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is R12K.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 13 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S13L. In some embodiments, the amino acid substitution is S13R. [0082] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the alanine at position 14 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A14K. In some embodiments, the amino acid substitution is A14S.
  • the variant Bhr-PETase has an amino acid substitution of the leucine at position 15 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L15I.
  • the variant Bhr-PETase has an amino acid substitution of the threonine at position 16 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine,asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T16E.
  • the variant Bhr-PETase has an amino acid substitution of the threonine at position 17 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine,asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is T17A. In some embodiments, the amino acid substitution is T17C. In some embodiments, the amino acid substitution is T17G. In some embodiments, the amino acid substitution is T17H. In some embodiments, the amino acid substitution is T17I. In some embodiments, the amino acid substitution is T17K. In some embodiments, the amino acid substitution is T17L. In some embodiments, the amino acid substitution is T17M. In some embodiments, the amino acid substitution is T17N. In some embodiments, the amino acid substitution is T17Q. In some embodiments, the amino acid substitution is T17R. In some embodiments, the amino acid substitution is T17S.
  • the variant Bhr-PETase has an amino acid substitution of the aspartic acid at position 18 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is D18R.
  • the variant Bhr-PETase has an amino acid substitution of the proline at position 20 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is P20D. In some embodiments, the amino acid substitution is P20E. In some embodiments, the amino acid substitution is P20I. In some embodiments, the amino acid substitution is P20Q. In some embodiments, the amino acid substitution is P20T. [0088] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the phenylalanine at position 21 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is F21W. In some embodiments, the amino acid substitution is F21Y.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 22 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is S22A.
  • the amino acid substitution is S22I. In some embodiments, the amino acid substitution is S22K. In some embodiments, the amino acid substitution is S22P. In some embodiments, the amino acid substitution is S22R. In some embodiments, the amino acid substitution is S22V. [0090] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the valine at position 23 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V23L. In some embodiments, the amino acid substitution is V23T.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 24 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A24D. In some embodiments, the amino acid substitution is A24G. In some embodiments, the amino acid substitution is A24H. In some embodiments, the amino acid substitution is A24N. In some embodiments, the amino acid substitution is A24S. In some embodiments, the amino acid substitution is A24T. In some embodiments, the amino acid substitution is A24V. [0092] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the threonine at position 25 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine,asparagine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T25A.
  • the amino acid substitution is T25F.
  • the amino acid substitution is T25H.
  • the amino acid substitution is T25Q. In some embodiments, the amino acid substitution is T25R. In some embodiments, the amino acid substitution is T25V. [0093] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the tyrosine at position 26 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and valine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is Y26C.
  • the amino acid substitution is Y26K.
  • the amino acid substitution is Y26L.
  • the amino acid substitution is Y26T.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 30 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is R30K.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 32 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S32K. In some embodiments, the amino acid substitution is S32M. In some embodiments, the amino acid substitution is S32Q. In some embodiments, the amino acid substitution is S32Y. [0096] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the valine at position 33 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V33G.
  • the amino acid substitution is V33Q.
  • the amino acid substitution is V33T.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 34 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S34R.
  • the variant Bhr-PETase has an amino acid substitution of the valine at position 40 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V40T.
  • the variant Bhr-PETase has an amino acid substitution of the glycine at position 46 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is G46E. In some embodiments, the amino acid substitution is G46L. In some embodiments, the amino acid substitution is G46N. In some embodiments, the amino acid substitution is G46R. In some embodiments, the amino acid substitution is G46S. [0100] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the threonine at position 48 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine,asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T48N. In some embodiments, the amino acid substitution is T48S.
  • the variant Bhr-PETase has an amino acid substitution of the leucine at position 49 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L49G.
  • the variant Bhr-PETase has an amino acid substitution of the glycine at position 53 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is G53A.
  • the variant Bhr-PETase has an amino acid substitution of the isoleucine at position 54 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is I54V.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 55 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is A55C. In some embodiments, the amino acid substitution is A55I. In some embodiments, the amino acid substitution is A55L. In some embodiments, the amino acid substitution is A55M. In some embodiments, the amino acid substitution is A55T. In some embodiments, the amino acid substitution is A55V. [0105] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the methionine at position 56 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is M56I. In some embodiments, the amino acid substitution is M56L.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 57 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is S57C.
  • the amino acid substitution is S57E. In some embodiments, the amino acid substitution is S57F. In some embodiments, the amino acid substitution is S57I. In some embodiments, the amino acid substitution is S57L. In some embodiments, the amino acid substitution is S57M. In some embodiments, the amino acid substitution is S57T. In some embodiments, the amino acid substitution is S57V. [0107] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the tyrosine at position 60 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and valine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is Y60A.
  • the amino acid substitution is Y60H.
  • the amino acid substitution is Y60I.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 62 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A62T.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 68 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A68F.
  • the variant Bhr-PETase has an amino acid substitution of the leucine at position 70 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L70M.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 72 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is R72P.
  • the variant Bhr-PETase has an amino acid substitution of the leucine at position 74 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L74W.
  • the variant Bhr-PETase has an amino acid substitution of the histidine at position 77 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is H77Q.
  • the variant Bhr-PETase has an amino acid substitution of the isoleucine at position 82 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is I82F. In some embodiments, the amino acid substitution is I82L. In some embodiments, the amino acid substitution is I82M. [0115] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the valine at position 83 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V83I.
  • the amino acid substitution is V83L.
  • the amino acid substitution is V83T.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 85 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N85D.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 87 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N87F. In some embodiments, the amino acid substitution is N87H. In some embodiments, the amino acid substitution is N87I. In some embodiments, the amino acid substitution is N87K. In some embodiments, the amino acid substitution is N87L. In some embodiments, the amino acid substitution is N87M. In some embodiments, the amino acid substitution is N87Q. In some embodiments, the amino acid substitution is N87R. In some embodiments, the amino acid substitution is N87V. In some embodiments, the amino acid substitution is N87W. In some embodiments, the amino acid substitution is N87Y.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 88 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S88K. In some embodiments, the amino acid substitution is S88T. [0119] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the leucine at position 90 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L90F.
  • the amino acid substitution is L90K.
  • the amino acid substitution is L90Y.
  • the variant Bhr-PETase has an amino acid substitution of the phenylalanine at position 92 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is F92G. In some embodiments, the amino acid substitution is F92I. In some embodiments, the amino acid substitution is F92K. In some embodiments, the amino acid substitution is F92L. In some embodiments, the amino acid substitution is F92N. In some embodiments, the amino acid substitution is F92Q. In some embodiments, the amino acid substitution is F92V. In some embodiments, the amino acid substitution is F92Y. [0121] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the alanine at position 97 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine.
  • the amino acid substitution is A97C.
  • the amino acid substitution is A97E.
  • the amino acid substitution is A97F.
  • the amino acid substitution is A97G.
  • the amino acid substitution is A97L.
  • the amino acid substitution is A97P. In some embodiments, the amino acid substitution is A97Q. In some embodiments, the amino acid substitution is A97S. In some embodiments, the amino acid substitution is A97T. In some embodiments, the amino acid substitution is A97V. [0122] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the serine at position 98 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S98A.
  • the amino acid substitution is S98D.
  • the amino acid substitution is S98E.
  • the amino acid substitution is S98L. In some embodiments, the amino acid substitution is S98M. In some embodiments, the amino acid substitution is S98N. In some embodiments, the amino acid substitution is S98Q. In some embodiments, the amino acid substitution is S98T. In some embodiments, the amino acid substitution is S98V. [0123] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the serine at position 101 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is S101A.
  • the amino acid substitution is S101C.
  • the amino acid substitution is S101D.
  • the amino acid substitution is S101F.
  • the amino acid substitution is S101H. In some embodiments, the amino acid substitution is S101K. In some embodiments, the amino acid substitution is S101L. In some embodiments, the amino acid substitution is S101M. In some embodiments, the amino acid substitution is S101N. In some embodiments, the amino acid substitution is S101Q. In some embodiments, the amino acid substitution is S101R. In some embodiments, the amino acid substitution is S101V. In some embodiments, the amino acid substitution is S101W. In some embodiments, the amino acid substitution is S101Y. [0124] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the alanine at position 102 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A102V.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 105 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N105D.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 108 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine.
  • the amino acid substitution is R108C.
  • the amino acid substitution is R108E. In some embodiments, the amino acid substitution is R108H. In some embodiments, the amino acid substitution is R108K. In some embodiments, the amino acid substitution is R108N. In some embodiments, the amino acid substitution is R108P. In some embodiments, the amino acid substitution is R108Q. In some embodiments, the amino acid substitution is R108S. In some embodiments, the amino acid substitution is R108T. In some embodiments, the amino acid substitution is R108V. [0127] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the threonine at position 109 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T109A.
  • the amino acid substitution is T109F.
  • the amino acid substitution is T109G.
  • the amino acid substitution is T109K. In some embodiments, the amino acid substitution is T109L. In some embodiments, the amino acid substitution is T109N. In some embodiments, the amino acid substitution is T109R. In some embodiments, the amino acid substitution is T109Y. [0128] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the serine at position 110 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S110D.
  • the amino acid substitution is S110G.
  • the amino acid substitution is S110H.
  • the amino acid substitution is S110K. In some embodiments, the amino acid substitution is S110N. In some embodiments, the amino acid substitution is S110R. [0129] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the serine at position 113 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is S113A.
  • the amino acid substitution isS113K.
  • the amino acid substitution is S113N.
  • the amino acid substitution is S113P.
  • the amino acid substitution is S113Q. In some embodiments, the amino acid substitution is S113R, S113T. In some embodiments, the amino acid substitution is S113Y. [0130] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the alanine at position 114 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A114K.
  • the amino acid substitution is A114L.
  • the amino acid substitution is A114S.
  • the amino acid substitution is A114V.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 117 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A117F. In some embodiments, the amino acid substitution is A117G. In some embodiments, the amino acid substitution is A117L. In some embodiments, the amino acid substitution is A117N. In some embodiments, the amino acid substitution is A117Q. In some embodiments, the amino acid substitution is A117S. In some embodiments, the amino acid substitution is A117T. In some embodiments, the amino acid substitution is A117Y. [0132] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the leucine at position 119 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L119I. In some embodiments, the amino acid substitution is L119M.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 121 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A121S.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 122 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is N122A. In some embodiments, the amino acid substitution is N122E. In some embodiments, the amino acid substitution is N122H. In some embodiments, the amino acid substitution is N122P. In some embodiments, the amino acid substitution is N122R. In some embodiments, the amino acid substitution is N122S. [0135] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the alanine at position 125 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A125S.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 127 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A127M. In some embodiments, the amino acid substitution is A127S. In some embodiments, the amino acid substitution is A127V. [0137] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the alanine at position 135 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A135G.
  • the variant Bhr-PETase has an amino acid substitution of the threonine at position 136 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T136A. In some embodiments, the amino acid substitution is T136M. In some embodiments, the amino acid substitution is T136S. In some embodiments, the amino acid substitution is T136V. [0139] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the arginine at position 138 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is R138E. In some embodiments, the amino acid substitution is R138L.
  • the variant Bhr-PETase has an amino acid substitution of the isoleucine at position 139 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is I139A. In some embodiments, the amino acid substitution is I139T. [0141] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the serine at position 140 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S140A.
  • the variant Bhr-PETase has an amino acid substitution of the glutamine at position 142 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is Q142D. In some embodiments, the amino acid substitution is Q142E. In some embodiments, the amino acid substitution is Q142H. In some embodiments, the amino acid substitution is Q142L. In some embodiments, the amino acid substitution is Q142W. [0143] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the isoleucine at position 143 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is I143N. In some embodiments, the amino acid substitution is I143R.
  • the variant Bhr-PETase has an amino acid substitution of the threonine at position 145 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T145S.
  • the variant Bhr-PETase has an amino acid substitution of the lysine at position 147 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is K147F. In some embodiments, the amino acid substitution is K147G. In some embodiments, the amino acid substitution is K147N. In some embodiments, the amino acid substitution is K147Q. [0146] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the glycine at position 149 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is G149A.
  • the amino acid substitution is G149C.
  • the amino acid substitution is G149D.
  • the amino acid substitution is G149N.
  • the amino acid substitution is G149S. In some embodiments, the amino acid substitution is G149T. In some embodiments, the amino acid substitution is G149V. [0147] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the valine at position 150 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V150I. In some embodiments, the amino acid substitution is V150L.
  • the variant Bhr-PETase has an amino acid substitution of the threonine at position 153 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T153L.
  • the variant Bhr-PETase has an amino acid substitution of the histidine at position 156 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is H156N.
  • the variant Bhr-PETase has an amino acid substitution of the threonine at position 157 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T157A. In some embodiments, the amino acid substitution is T157G. [0151] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the aspartic acid at position 158 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is D158E.
  • the amino acid substitution is D158I.
  • the amino acid substitution is D158K.
  • the amino acid substitution is D158L.
  • the variant Bhr-PETase has an amino acid substitution of the threonine at position 160 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T160K. In some embodiments, the amino acid substitution is T160Q. In some embodiments, the amino acid substitution is T160R. In some embodiments, the amino acid substitution is T160S. In some embodiments, the amino acid substitution is T160V. [0153] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the phenylalanine at position 161 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is F161V. In some embodiments, the amino acid substitution is F161W.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 162 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is N162E.
  • the amino acid substitution is N162H. In some embodiments, the amino acid substitution is N162P. In some embodiments, the amino acid substitution is N162R. [0155] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the threonine at position 163 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T163I. In some embodiments, the amino acid substitution is T163S.
  • the variant Bhr-PETase has an amino acid substitution of the proline at position 164 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is P164E.
  • the amino acid substitution is P164H. In some embodiments, the amino acid substitution is P164N. In some embodiments, the amino acid substitution is P164R. In some embodiments, the amino acid substitution is P164S. In some embodiments, the amino acid substitution is P164T. [0157] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the glutamine at position 167 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is Q167I.
  • the amino acid substitution is Q167T.
  • the amino acid substitution is Q167V.
  • the variant Bhr-PETase has an amino acid substitution of the valine at position 170 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V170L.
  • the variant Bhr-PETase has an amino acid substitution of the glutamic acid at position 173 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is E173R.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 174 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A174K. In some embodiments, the amino acid substitution is A174R. [0161] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the valine at position 177 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V177A.
  • the variant Bhr-PETase has an amino acid substitution of the proline at position 179 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is P179Q.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 181 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is S181A.
  • the amino acid substitution is S181C. In some embodiments, the amino acid substitution is S181R. [0164] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the glutamine at position 182 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is Q182T.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 184 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is A184C.
  • the amino acid substitution is A184G. In some embodiments, the amino acid substitution is A184S. [0166] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the isoleucine at position 185 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is I185A.
  • the amino acid substitution is I185E.
  • the amino acid substitution is I185G.
  • the amino acid substitution is I185L. In some embodiments, the amino acid substitution is I185Q. In some embodiments, the amino acid substitution is I185R. In some embodiments, the amino acid substitution is I185S. In some embodiments, the amino acid substitution is I185Y. [0167] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the glutamine at position 189 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is Q189I.
  • the amino acid substitution is Q189L.
  • the amino acid substitution is Q189V.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 190 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N190S.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 193 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is S193E. In some embodiments, the amino acid substitution is S193F. In some embodiments, the amino acid substitution is S193H. In some embodiments, the amino acid substitution is S193K. In some embodiments, the amino acid substitution is S193N. In some embodiments, the amino acid substitution is S193P. In some embodiments, the amino acid substitution is S193T. In some embodiments, the amino acid substitution is S193V. [0170] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the threonine at position 194 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T194G. In some embodiments, the amino acid substitution is T194S.
  • the variant Bhr-PETase has an amino acid substitution of the threonine at position 195 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T195F.
  • the variant Bhr-PETase has an amino acid substitution of the valine at position 198 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V198A.
  • the variant Bhr-PETase has an amino acid substitution of the valine at position 200 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V200L.
  • the variant Bhr-PETase has an amino acid substitution of the aspartic acid at position 203 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is D203N. In some embodiments, the amino acid substitution is D203R. In some embodiments, the amino acid substitution is D203V. [0175] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the asparagine at position 204 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N204A.
  • the amino acid substitution is N204K.
  • the amino acid substitution is N204R.
  • the amino acid substitution is N204S.
  • the variant Bhr-PETase has an amino acid substitution of the threonine at position 206 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is T206G. In some embodiments, the amino acid substitution isT206K. In some embodiments, the amino acid substitution is T206L. In some embodiments, the amino acid substitution is T206P. In some embodiments, the amino acid substitution is T206R. [0177] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the phenylalanine at position 208 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is F208G.
  • the amino acid substitution is F208L.
  • the amino acid substitution is F208R.
  • the amino acid substitution is F208T.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 209 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A209V.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 211 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N211F. In some embodiments, the amino acid substitution is N211I. In some embodiments, the amino acid substitution is N211L. In some embodiments, the amino acid substitution is N211M. In some embodiments, the amino acid substitution is N211V. [0180] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the serine at position 212 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S212F.
  • the amino acid substitution is S212L.
  • the amino acid substitution is S212M.
  • the variant Bhr-PETase has an amino acid substitution of the proline at position 213 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is P213N.
  • the amino acid substitution is P213R.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 216 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is A216L. In some embodiments, the amino acid substitution is A216P. In some embodiments, the amino acid substitution is A216S. In some embodiments, the amino acid substitution is A216T. In some embodiments, the amino acid substitution is A216V. [0183] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the isoleucine at position 217 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is I217S.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 218 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S218A.
  • the variant Bhr-PETase has an amino acid substitution of the valine at position 219 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V219F. In some embodiments, the amino acid substitution is V219I. In some embodiments, the amino acid substitution is V219K. In some embodiments, the amino acid substitution is V219L. In some embodiments, the amino acid substitution is V219R. [0186] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the threonine at position 221 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is T221S.
  • the variant Bhr-PETase has an amino acid substitution of the isoleucine at position 222 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is I222L.
  • the variant Bhr-PETase has an amino acid substitution of the serine at position 223 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is S223A. In some embodiments, the amino acid substitution is S223C. [0189] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the methionine at position 225 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is M225L.
  • the variant Bhr-PETase has an amino acid substitution of the leucine at position 227 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L227R.
  • the variant Bhr-PETase has an amino acid substitution of the tryptophan at position 228 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is W228F.
  • the variant Bhr-PETase has an amino acid substitution of the valine at position 229 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is V229C. In some embodiments, the amino acid substitution is V229I. In some embodiments, the amino acid substitution is V229L. [0193] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the asparagine at position 231 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N231L.
  • the amino acid substitution is N231Q.
  • the amino acid substitution is N231S.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 236 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is R236C.
  • the amino acid substitution is R236E. In some embodiments, the amino acid substitution is R236H. In some embodiments, the amino acid substitution is R236K. In some embodiments, the amino acid substitution is R236Q. [0195] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the glutamine at position 237 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is Q237R.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 241 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is N241P.
  • the variant Bhr-PETase has an amino acid substitution of the valine at position 242 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V242T.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 243 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is N243P.
  • the variant Bhr-PETase has an amino acid substitution of the alanine at position 246 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A246D. In some embodiments, the amino acid substitution is A246K. In some embodiments, the amino acid substitution is A246S. In some embodiments, the amino acid substitution is A246T. [0200] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the aspartic acid at position 249 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is D249I.
  • the amino acid substitution is D249M.
  • the amino acid substitution is D249N.
  • the amino acid substitution is D249S. In some embodiments, the amino acid substitution is D249T. [0201] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the phenylalanine at position 250 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is F250I.
  • the amino acid substitution is F250L.
  • the amino acid substitution is F250V.
  • the amino acid substitution is F250Y.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 251 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is R251A. In some embodiments, the amino acid substitution is R251E. In some embodiments, the amino acid substitution is R251I. In some embodiments, the amino acid substitution is R251K. In some embodiments, the amino acid substitution is R251L. In some embodiments, the amino acid substitution is R251Q. In some embodiments, the amino acid substitution is R251T. In some embodiments, the amino acid substitution is R251V. [0203] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the serine at position 252 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S252T.
  • the variant Bhr-PETase has an amino acid substitution of the asparagine at position 253 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N253S. In some embodiments, the amino acid substitution is N253Y. [0205] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the asparagine at position 254 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is N254R.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 255 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is R255E. In some embodiments, the amino acid substitution is R255G. In some embodiments, the amino acid substitution is R255L. In some embodiments, the amino acid substitution is R255M. In some embodiments, the amino acid substitution is R255S. In some embodiments, the amino acid substitution is R255V. In some embodiments, the amino acid substitution is R255W. In some embodiments, the amino acid substitution is R255Y. [0207] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the glutamine at position 258 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is Q258P.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 8 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is P8T.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 31 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L31M.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 38 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is G38D.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 95 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is S95N.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 126 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V126I.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 137 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L137M.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 165 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is V165I.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 169 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing proline (due to steric effects).
  • the amino acid substitution is I169C. In some embodiments, the amino acid substitution is I169L. In some embodiments, the amino acid substitution is I169V. [0216] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the arginine at position 172 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is A172T.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 191 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is L191F. In some embodiments, the amino acid substitution is L191V. [0218] In some embodiments, the variant Bhr-PETase has an amino acid substitution of the arginine at position 192 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation).
  • the amino acid substitution is P192A.
  • the variant Bhr-PETase has an amino acid substitution of the arginine at position 197 of SEQ ID NO:1.
  • the substitution is with any other of the 19 naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine, with some embodiments not utilizing cysteine (due to possible disulfide formation) or proline (due to steric effects).
  • the amino acid substitution is K197L.
  • the amino acid substitution is K197R.
  • the amino acid substitution is K197T.
  • the amino acid substitution is K197V. In some embodiments, the amino acid substitution is K197Y.
  • the variant Bhr-PETase enzyme has one or more amino acid substitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 of the positions of SEQ ID NO: 1 as described above.
  • D. Nucleic Acids of the Invention [0221] The present invention additional provides nucleic acids encoding the variant Bhr- PETases of the invention.
  • nucleic acids may be made, all of which encode the variant Bhr-PETases of the present invention.
  • those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein.
  • providing the amino acid sequence allows the generation of a very large number of different nucleic acid sequences encoding the proteins.
  • specific variant Bhr-PETases are encoded by specific nucleic acid sequences, as are listed in SEQ ID NOs 2 and 3.
  • specific variant Bhr-PETases are encoded by a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NOs 2 and 3.
  • the nucleic acids encoding the components of the invention can be incorporated into expression vectors as is known in the art, and depending on the host cells used to produce the heterodimeric antibodies of the invention.
  • nucleic acids are operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.).
  • the expression vectors can be extra-chromosomal or integrating vectors.
  • the nucleic acids and/or expression vectors of the invention are then transformed into any number of different types of host cells as is well known in the art, including mammalian, bacterial, yeast, insect and/or fungal cells, with bacteria, yeast and fungi finding use in many embodiments. 1.
  • nucleic acids encoding the variant Bhr-PETases of the invention can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis and synthetic gene construction as are well known in the art.
  • Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips. A preferred technique is GenScript®. 2.
  • the present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • the polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
  • the control sequence may be a promoter, a polynucleotide which is recognized by a host cell for expression of the polynucleotide.
  • the promoter contains transcriptional control sequences that mediate the expression of the variant.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • Promoters for bacteria, yeast and fungi are well known in the art. Exemplary operons for expression in lactic acid bacteria include S. thermophilus lactose operons or L.
  • lactic lac ABCDFEGX operons which have been successfully used to induce foreign gene expression in hosts (eg, Simons et al., 1993, J. Bact.175: 5186-5175; see Mollet et al., 1993, J. Bact. 175: 4315-4324).
  • constitutive promoters for bacteria include lac promoter, trp promoter, tac promoter, T7 promoter, erm promoter, tip promoter, nit promoter, and Sp6 promoter.
  • Exemplary promoters for yeasts include, but not limited to, AOX1 promoter, ADH promoter, PH05 promoter, gal10 promoter, PKG promoter and GAP promoter.
  • useful promoters for yeast host cells are described by Romanoset al., 1992, Yeast 8: 423-488.
  • examples of useful promoters for fungi vectors include, but not limit to, Aspergillus niger GLA promoter, Aspergillus nidulans GPD promoter, those derived from Aspergillus nidulans glycolytic genes, such as the adh3 promoter (McKnight et al., EMBO J.4:2093-2099,1985). 3.
  • Codon Optimization can be done to increase expression in any particular host organism. Codon optimization can be employed with any of the variant Bhr-PETase polypeptides of the present invention, in order to optimize expression in the host cell employed. Such methods are well known in the art and described in, for example, WO 2007/142954. In heterologous expression systems, optimization steps can improve the ability of the host to produce the desired variant Bhr- PETase polypeptides. Protein expression is governed by a host of factors including those that affect transcription, mRNA processing, and stability and initiation of translation.
  • the polynucleotide optimization steps can include steps to improve the ability of the host to produce the foreign protein as well as steps to assist the researcher in efficiently designing expression constructs.
  • Optimization strategies can include, for example, the modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases.
  • the following paragraphs discuss potential problems that may result in reduced heterologous protein expression, and techniques that may overcome these problems.
  • reduced heterologous protein expression results from a rare codon-induced translational pause.
  • a rare codon-induced translational pause includes the presence of codons in the polynucleotide of interest that are rarely used in the host organism can have a negative effect on protein translation due to their scarcity in the available tRNA pool.
  • One method of improving optimal translation in the host organism includes performing includes performing codon optimization which can result in rare host codons being modified in the synthetic polynucleotide sequence.
  • reduced heterologous protein expression results from by alternate translational initiation.
  • Alternate translational initiation can include a synthetic polynucleotide sequence inadvertently containing motifs capable of functioning as a ribosome binding site (RBS). These sites can result in initiating translation of a truncated protein from a gene-internal site.
  • RBS ribosome binding site
  • One method of reducing the possibility of producing a truncated protein, which can be difficult to remove during purification includes modifying putative internal RBS sequences from an optimized polynucleotide sequence.
  • reduced heterologous protein expression occurs through repeat-induced polymerase slippage.
  • Repeat-induced polymerase slippage involves nucleotide sequence repeats that have been shown to cause slippage or stuttering of DNA polymerase which can result in frameshift mutations. Such repeats can also cause slippage of RNA polymerase.
  • RNA polymerase slippage In an organism with a high G+C content bias, there can be a higher degree of repeats composed of G or C nucleotide repeats. Therefore, one method of reducing the possibility of inducing RNA polymerase slippage includes altering extended repeats of G or C nucleotides.
  • reduced heterologous protein expression occurs through interfering secondary structures.
  • Secondary structures can sequester the RBS sequence or initiation codon and have been correlated to a reduction in protein expression. Stemloop structures can also be involved in transcriptional pausing and attenuation.
  • An optimized polynucleotide sequence can contain minimal secondary structures in the RBS and gene coding regions of the nucleotide sequence to allow for improved transcription and translation.
  • restriction sites can affect heterologous protein expression. By modifying restriction sites that could interfere with subsequent sub-cloning of transcription units into host expression vectors a polynucleotide sequence can be optimized.
  • Optimizing a DNA sequence can negatively or positively affect gene expression or protein production.
  • modifying a less-common codon with a more common codon may affect the half-life of the mRNA or alter its structure by introducing a secondary structure that interferes with translation of the message. It may therefore be necessary, in certain instances, to alter the optimized message.
  • All or a portion of a gene can be optimized.
  • the desired modulation of expression is achieved by optimizing essentially the entire gene. In other embodiments, the desired modulation will be achieved by optimizing part but not all of the gene.
  • the codon usage of any coding sequence can be adjusted to achieve a desired property, for example high levels of expression in a specific cell type.
  • the starting point for such an optimization may be a coding sequence with 100% common codons, or a coding sequence which contains a mixture of common and non-common codons.
  • Two or more candidate sequences that differ in their codon usage can be generated and tested to determine if they possess the desired property.
  • Candidate sequences can be evaluated by using a computer to search for the presence of regulatory elements, such as silencers or enhancers, and to search for the presence of regions of coding sequence which could be converted into such regulatory elements by an alteration in codon usage.
  • Additional criteria can include enrichment for particular nucleotides, e.g., A, C, G or U, codon bias for a particular amino acid, or the presence or absence of particular mRNA secondary or tertiary structure. Adjustment to the candidate sequence can be made based on a number of such criteria.
  • Promising candidate sequences are constructed and then evaluated experimentally. Multiple candidates may be evaluated independently of each other, or the process can be iterative, either by using the most promising candidate as a new starting point, or by combining regions of two or more candidates to produce a novel hybrid. Further rounds of modification and evaluation can be included.
  • Modifying the codon usage of a candidate sequence can result in the creation or destruction of either a positive or negative element.
  • a positive element refers to any element whose alteration or removal from the candidate sequence could result in a decrease in expression of the therapeutic protein, or whose creation could result in an increase in expression of a therapeutic protein.
  • a positive element can include an enhancer, a promoter, a downstream promoter element, a DNA binding site for a positive regulator (e.g., a transcriptional activator), or a sequence responsible for imparting or modifying an mRNA secondary or tertiary structure.
  • a negative element refers to any element whose alteration or removal from the candidate sequence could result in an increase in expression of the therapeutic protein, or whose creation would result in a decrease in expression of the therapeutic protein.
  • a negative element includes a silencer, a DNA binding site for a negative regulator (e.g., a transcriptional repressor), a transcriptional pause site, or a sequence that is responsible for imparting or modifying an mRNA secondary or tertiary structure.
  • a negative element arises more frequently than a positive element.
  • any change in codon usage that results in an increase in protein expression is more likely to have arisen from the destruction of a negative element rather than the creation of a positive element.
  • alteration of the candidate sequence is more likely to destroy a positive element than create a positive element.
  • a candidate sequence is chosen and modified so as to increase the production of a therapeutic protein.
  • the candidate sequence can be modified, e.g., by sequentially altering the codons or by randomly altering the codons in the candidate sequence.
  • a modified candidate sequence is then evaluated by determining the level of expression of the resulting therapeutic protein or by evaluating another parameter, e.g., a parameter correlated to the level of expression.
  • a candidate sequence which produces an increased level of a therapeutic protein as compared to an unaltered candidate sequence is chosen.
  • one or a group of codons can be modified, e.g., without reference to protein or message structure and tested.
  • one or more codons can be chosen on a message-level property, e.g., location in a region of predetermined, e.g., high or low GC content, location in a region having a structure such as an enhancer or silencer, location in a region that can be modified to introduce a structure such as an enhancer or silencer, location in a region having, or predicted to have, secondary or tertiary structure, e.g., intra-chain pairing, inter-chain pairing, location in a region lacking, or predicted to lack, secondary or tertiary structure, e.g., intra-chain or inter-chain pairing.
  • a particular modified region is chosen if it produces the desired result.
  • one or a group, e.g., a contiguous block of codons, at various positions of a synthetic nucleic acid sequence can be modified with common codons (or with non common codons, if for example, the starting sequence has been optimized) and the resulting sequence evaluated.
  • Candidates can be generated by optimizing (or de-optimizing) a given “window” of codons in the sequence to generate a first candidate, and then moving the window to a new position in the sequence, and optimizing (or de-optimizing) the codons in the new position under the window to provide a second candidate.
  • Candidates can be evaluated by determining the level of expression they provide, or by evaluating another parameter, e.g., a parameter correlated to the level of expression.
  • the optimized nucleic acid sequence can express the variant Bhr-PETase polypeptide of the invention, at a level which is at least about 110%, 150%, 200%, 500%, 1,000%, 5,000% or even 10,000% of that expressed by nucleic acid sequence that has not been optimized.
  • a candidate DNA sequence can be designed.
  • the frequency of codon usage can be compared to the codon usage of the host expression organism and rare host codons can be modified in the synthetic sequence.
  • the synthetic candidate DNA sequence can be modified in order to remove undesirable enzyme restriction sites and add or alter any desired signal sequences, linkers or untranslated regions.
  • the synthetic DNA sequence can be analyzed for the presence of secondary structure that may interfere with the translation process, such as G/C repeats and stem-loop structures.
  • the optimized sequence design can be checked to verify that the sequence correctly encodes the desired amino acid sequence.
  • the candidate DNA sequence can be synthesized using DNA synthesis techniques, such as those known in the art.
  • the general codon usage in a host organism can be utilized to optimize the expression of the heterologous polynucleotide sequence in the host organism.
  • the percentage and distribution of codons that rarely would be considered as preferred for a particular amino acid in the host expression system can be evaluated. Values of 5% and 10% usage can be used as cutoff values for the determination of rare codons. 4.
  • Host Cells and Production Strains [0248]
  • the present disclosure relates to an expression vector comprising the nucleic acid encoding the variant Bhr-PETase described herein.
  • the present disclosure also relates to a host cell comprising the expression vector.
  • the host cell is bacteria.
  • the host cell is yeast. In some embodiments, the host cell is fungi. In some embodiments, the host cell may be bacteria, including but not limited to E. coli, Bacillus. In some embodiments, the host cell may be yeast, including but not limited to Saccharomyces cerevisiae, Pichia. In some embodiments, the host cell may be fungi, including but not limited to A. niger, T. reseei, or Myceliophthora thermophila. [0249] The expression vector may be any of integration vectors which are to be integrated into genome or autonomously replicating plasmids in the selected host.
  • the vector can be stably maintained in the introduced cell, with the variant Bhr-PETase gene supported thereon in a fit state for expression.
  • the expression vector may be selected to be suitable for the specific host cells to which the vector is introduced. Specific examples available for use include but not limit to pBR322, pACYC184, pUC18, pKK223-2, pHSG398 (Takara Bio Inc.), pTrcHis (Invitrogen Corporation) and pET11a (Stratagene Corporation) in the case where Escherichia coli is used as a host; pBBR122 (Mobiotech) and pBHR1 (Mobiotech) for the other gram-negative bacteria; pHW1520 (Mobiotech) and pHY300PLK (Takara Bio Inc.) for Bacillus; pSH19 (Herai et al., Proc.
  • the present disclosure also relates to a method of expressing the variant Bhr-PETase in the host cell.
  • the present disclosure also relates to a method of making the variant Bhr-PETase comprising culturing the host cell under conditions wherein said variant Bhr-PETase is produced, and recovering said variant Bhr-PETase.
  • the culture of a transformed organism may be performed in the medium which can be a nutritive medium of the transformed host cell without affecting the transformation of the variant Bhr-PETase.
  • a medium comprises an appropriate carbon source, nitrogen source, inorganic salt, natural organic nutrient and the like.
  • a carbon source glucose, fructose, glycerol, sorbitol, organic acids can be used individually or in combination.
  • the concentration of the carbon source is not particularly limited and may be 1 to 10 %.
  • a nitrogen source ammonium, urea, ammonium sulfate, ammonium nitrate, ammonium acetate and the like can be used individually or in combination of two or more members thereof.
  • an inorganic salt salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, manganese sulfate and ferrous sulfate can be used.
  • peptone, meat extract, yeast extract, corn steep liquor and casamino acids can be used and furthermore, a small amount vitamins and nucleic acids may be contained in the medium. 5.
  • the formulation of the variant Bhr-PETases of the invention depends on its end use and the associated conditions. Suitable formulations for the variant Bhr-PETases of the invention include liquid formulations, dried formulations (including spray dried formulations), powdered formulations, granular formulations, and pelleted formulations. Bhr-PETases can also be formulated as “embedded in PET particles” for natural degradation.
  • the enzyme composition i.e., polypeptide compositions
  • the enzyme composition can be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme composition, or a host cell, as a source of the enzymes.
  • the enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme.
  • Liquid enzyme compositions may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
  • stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
  • the dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.
  • the above compositions are suitable for use in PET biodegradation, PET biocycling, PET upcycling, and/or PET surface modification processes.
  • the above compositions are used to degrade pretreated PET. PET pretreatment may be performed before the enzymatic degradation step.
  • PET pretreatment can be broadly classified into a) mechanical pretreatment, b) thermo- mechanical pretreatment, and c) chemical pretreatment.
  • the mechanical process may involve grinding of the PET film into particles less than about 500 ⁇ m post sieving. This process could be combined with immersing the PET film into liquid nitrogen mainly for ease of the process of grinding.
  • the advantage of this type of mechanical pretreatment may be enzyme accessibility due to reduced particle size.
  • the main- chain scission reactions may affect the stability of the cyclic oligomers.
  • highly crystalline PET flakes may be amorphized at very high temperatures (> 260°C) using extruder equipped with melt pump and later micronized to particle size of less than 500 ⁇ m.
  • thermo-mechanical pretreatment may lower the crystallinity as well as particle size of the industrial grade PET allowing accessibility of the enzymes to depolymerize it.
  • chemical pretreatment ionic liquid, strong acid, base, solvents etc. can be used to reduce crystallinity or to change the surface structure of the PET to facilitate access of the enzymes to further depolymerize it to its monomers.
  • the two significant stumbling blocks in recycling plastics irrespective of using chemical or biological method may be material variability and the costs associated with identifying and separating waste plastics into recognizable grade ranges.
  • the mixed plastic refers to a mixture of different plastics.
  • Various polymer types may refer to PET and/or analog of PET, PET-like or PET substitute derived biologically or chemically.
  • analog of PET, PET-like or PET substitute include but are not limited to Polybutylene terephthalate (PBT), Polycabonate (PC), Polycaprolactone (PCL), Polyethylene Furanoate (PEF) and High Density Polyethylene (HDPE). Bhr-PETase alone or in conjunction with other accessory enzyme (s) can revolutionize the biological method of depolymerization of mixed plastics.
  • the process of enzymatic depolymerization of mixed plastics as opposed to chemical methodologies may be environmentally safer and capable of retaining the market value of the second use applications.
  • TPA terephthalic acid
  • PET synthesized from recycled TPA may demonstrate similar properties, such as average molecular weight and intrinsic viscosity, as PET synthesized using petrochemical TPA.
  • bottles blown from recycled PET exhibits similar mechanical property and better lightness value than regular PET bottles.
  • the present invention provides a method of preparing enzyme cocktail comprising the variant Bhr-PETase as described herein with other PET degrading accessory enzymes and downstream MHETase to produce a PET degrading enzyme cocktail for the efficient turn-over of pretreated PET.
  • Example 2 Preparation of PET Hydrolases Produced by Escherichia coli in 250ml Shake flask [0262] The BL21(DE3) Chemically Competent E. coli, (ThermoFisher Scientific, USA: Catalogue #C600003) containing recombinant hydrolase-encoding genes from single colonies were inoculated into individual culture tubes containing 5mL lysogeny broth (LB broth) with 1% glucose and 50 ⁇ g/mL of Kanamycin. The cultures were grown overnight at 30°C, 200 rpm and 85% humidity.
  • Example 3 Ni-Column Purification of the His-tag PET Hydrolases Produced from Shake flasks [0263] Each sample was concentrated to 15-20 ml before using Thermo Scientific HisPurTM Ni-NTA Spin Columns (catalog number 88226). The three PET hydrolases (Lcc-PETase, Bhr-PETase, Is-PETase) were purified from the cell culture.
  • Example 4 Desalting of purified PET Hydrolases Produced from Shake flasks [0264] Using one time use Thermo Scientific ZebaTM Spin Desalting Columns, 7K MWCO (catalog number 89892), imidazole and NaCl were removed from Ni Purified enzyme. Desalting protocol was followed according to manufacturer’s guidelines. Desalted enzyme was then quantified to determine protein concentration (g/L).
  • Example 5 Thermostability testing of Escherichia coli Produced PET Hydrolases using BHET (Bis (2-Hydroxyehtyl) terephthalate) as a substrate
  • BHET Bis (2-Hydroxyehtyl) terephthalate
  • Normalized PET hydrolase proteins of each candidate were subjected to a 1.5 hr challenge at either 30°C, 50°C, or 70°C. Enzymes kept on ice for the same time served as unchallenged condition.
  • thermostability results of the three candidates are shown in Figure 4. Under the current experimental condition, Is-PETase is the most unstable candidate that looses 90% activity at 50°C. Bhr-PETase and Lcc-PETase retain ⁇ 80% residual activity at all temperature tested.
  • Example 6 Large scale PET Film Assay to Evaluate Escherichia coli Produced PET Hydrolases [0267] These experiments were carried out in 250 ml glass bottles. Two types of PET substrate were used in these experiments. A) Amorphous PET film was obtained from Goodfellow (Catalog # ES301445) with 0.25 mm thickness.
  • PET film was cut using paper trimmer into 1 x 30 cm strips, and then cut again to approximately 1 x 0.25 cm strips. Strips of PET film were grinded into fine powder with mechanical grinder.
  • Bhr-PETase cannot fully degrade highly crystalline PET and might require additional pretreatment or accessory enzymes to do so.
  • Bhr-PETase was identified as the best performing PET hydrolases among all that have been evaluated. Therefore, Bhr-PETase was selected as a target for further improvement.
  • Example 7 Design and Construction of Bhr-PETase collections [0270] To further improve the activity and thermostability of Bhr-PETase, multiple G1 variant collections were designed based on sequence and structure analysis. The design includes one to multiple specific mutations per variant.
  • the G1 variant collections were subsequently constructed on Bhr-PETase G1P (wild type Bhr-PETase, Generation 1 Parent) using standard site-directed mutagenesis methods and subsequently cloned into the pET28b (+) vector (Millipore Inc., Catalog # 69865) for production in Escherichia coli. [0271]
  • Bhr-PETase G1P wild type Bhr-PETase, Generation 1 Parent
  • pET28b (+) vector Millipore Inc., Catalog # 69865
  • multiple G2 variant collections were designed based on sequence and structure analysis. The design includes one to multiple specific mutations per variant.
  • the G2 variant collections were constructed on Bhr-PETase G2P (Generation 2 Parent, i.e.
  • Example 8 HTP growth: Preparation of Bhr-PETase G1P, G1 variants, G2P and G2 Variants in Microtiter Plates [0272] The BL21(DE3) Chemically Competent E.
  • coli (ThermoFisher Scientific, USA: Catalogue #C600003) containing recombinant Bhr-PETase encoding genes from single colonies were inoculated into individual wells of 96 well plates containing 180 ⁇ l lysogeny broth (LB broth) with 1% glucose and 50 ⁇ g/mL of Kanamycin. The cultures were grown overnight at 30°C, 200 rpm and 85% humidity. 20 ⁇ L of overnight culture was transferred from each well into 96 wells plate containing 380 ⁇ L of terrific broth (TB broth) with 50 ⁇ g/mL Kanamycin. The plates were then incubated for 2-2.5 hrs at 37°C, 250 rpm and 85% humidity.
  • Pre-induction OD was measured at 2-2.5 hrs and if OD600 reached around 0.6- 0.8, induction with IPTG (Isopropyl ⁇ -D-1-thiogalactopyranoside) was carried out to obtain final concentration of 0.5 mM IPTG in wells.
  • IPTG Isopropyl ⁇ -D-1-thiogalactopyranoside
  • the plates were then incubated for 18-24 hrs at 30°C, 250 rpm and 85% humidity. The supernatants were transferred to round bottom plates and stored at -20 oC prior to activity assay.
  • the host containing recombinant Bhr-PETase encoding genes from single colonies were inoculated into individual wells of 24 or 96 well plates containing relevant medium.
  • Example 9 HTP PET Film Assay to Evaluate Bhr-PETase G1P, G1 variants, G2P and G2 Variants Activity/Thermostability [0274] For total activity testing, amorphous PET film was obtained from Goodfellow (Catalog # ES301445) with 0.25 mm thickness.
  • PET film was cut using paper trimmer into 1 x 30 cm strips, and then cut again to approximately 1 x 0.25 cm strips. Strips of PET film were grinded into fine powder with mechanical grinder. PET powder was loaded into 96- well plates using resin loader. Approximately 8-9 mg of PET powder were dispensed in each of the 96 costar deep well. Into costar deep well containing PET powder, 1.4 mL of 0.1M sodium phosphate buffer was added, pH 8.0. 100 ⁇ L of enzyme was dispensed into costar deep wells, the plates were sealed, and then incubated at 65°C for 72 hours. After 72 hours, plates were centrifuged at 4,000 rpm for 2 minutes.
  • reaction mixture was diluted to linear assay detection using sodium phosphate buffer, pH 7.2 in Costar Deep with addition of 50 ⁇ L of 10 mM EDTA, and 50 ⁇ L of 10 mM FeSO4. Incubate the reaction in dark for 10 minutes and centrifuge plates at 4,000 rpm for 2 minutes before transferring 200 ⁇ L of reaction into black clear-bottom fluorometric plate.
  • Endpoint reading was measured after 10 minutes with 328 nm for excitation and 421 emission for TPA (Terephthalic Acid) activity.
  • TPA Tephthalic Acid
  • 150 ⁇ L of enzyme was transferred into PCR plates. The plates were incubated at 86 °C for 3 hours. After 3 hours, 100 ⁇ L of enzyme was transferred into costar deep well plates containing 1.4 mL of 0.1M sodium phosphate buffer, pH 8.0, and 8-9 mg/well of grinded PET powder. Plates were at 65°C for 72 hours. After 72 hours, plates were centrifuged at 4,000 rpm for 2 minutes. Into costar round bottom plates, 180 ⁇ L of above reaction was transferred and 20 ⁇ L of Is-MHETase was added.
  • the plates were incubated at 50 °C for 30 minutes. After 30 minutes, the plates were centrifuged at 4,000 rpm for 2 minutes. The reactions were analyzed using High Performance Liquid Chromatography (HPLC) to determine the amount of TPA produced in our reactions.
  • HPLC High Performance Liquid Chromatography
  • Zorbax Eclipase Plus C18 (Rapid Resolution HD 2.1x50mm 1.8-Micron) column with part number 959757-902 was used along with guard column part number 82175-901. Flow rate of 0.6 mL/minute with column temperature of 35°C is set up for running in HPLC.
  • the gradient method was used to detect Terephthalic Acid (TPA) with mobile phase 1 consist of water with 0.1% trifluoroacetic acid and mobile phase 2 consist of Acetonitrile with 0.1% trifluoroacetic acid.
  • TPA Terephthalic Acid
  • mobile phase 1 consist of water with 0.1% trifluoroacetic acid
  • mobile phase 2 consist of Acetonitrile with 0.1% trifluoroacetic acid.
  • All G1 variants showed improved total activity and/or thermostability than Bhr-PETase G1P (wild type Bhr-PETase).
  • a Bhr- PETase G1 variant with amino acid substitution S27L exhibited 1.80 fold improved total activity and 3.36 fold improved thermostability over Bhr-PETase G1P and therefore selected as Bhr-PETase G2P.
  • All G2 variants showed further improved total activity than Bhr- PETase G2P.

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WO2025159530A1 (ko) * 2024-01-24 2025-07-31 씨제이제일제당 (주) 폴리에스테르 효소 분해 공정에서 TPA(terephthalic acid)를 효율적으로 회수하는 공정
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WO2024236009A3 (en) * 2023-05-15 2025-01-09 Carbios Novel esterases and uses thereof
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IT202300014223A1 (it) * 2023-07-07 2025-01-07 Milano Politecnico Enzima ingegnerizzato termostabile
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CN119842650A (zh) * 2023-10-16 2025-04-18 中国科学院天津工业生物技术研究所 一种合成聚酯中间体的酰基转移酶及其应用
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WO2025159530A1 (ko) * 2024-01-24 2025-07-31 씨제이제일제당 (주) 폴리에스테르 효소 분해 공정에서 TPA(terephthalic acid)를 효율적으로 회수하는 공정
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WO2026034323A1 (ja) * 2024-08-06 2026-02-12 東洋紡株式会社 エチレンテレフタレート化合物分解酵素
JP7818252B1 (ja) * 2024-08-06 2026-02-20 東洋紡株式会社 エチレンテレフタレート化合物分解酵素

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