Enzymatic degradation of polyethylene terephthalate
Variant Bhr-PETase enzymes with specific amino acid substitutions enhance PETase activity, enabling efficient enzymatic degradation of PET and facilitating PET recycling.
Patent Information
- Application Number
- JP2024508603
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- Priority Date
- 2021-08-11
- Filing Date
- 2022-08-11
- Publication Date
- 2026-01-13
- Estimated Expiration
- 2042-08-11
AI Technical Summary
Enzymatic degradation of polyethylene terephthalate (PET) has proven difficult, limiting its effective physical recycling and contributing to environmental pollution.
Development of variant Bhr-PETase enzymes with specific amino acid substitutions that maintain or enhance PETase activity, achieving at least 85% identity to SEQ ID NO: 1, allowing for efficient enzymatic degradation of PET.
The variant Bhr-PETase enzymes effectively degrade PET, addressing the challenge of enzymatic degradation difficulties and promoting the utilization of PET recycling.
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Abstract
Description
[Background technology]
[0001] The use of polyethylene terephthalate (PET) is widespread and ubiquitous. As such, PET is a major source of environmental pollution worldwide. Physical recycling is possible but remains underutilized in many situations. Enzymatic degradation has been investigated over the past few decades but has proven difficult.
[0002] Provided herein are variant enzymes for use in the enzymatic degradation of PET. Summary of the Invention
[0003] In one aspect, the disclosure provides a composition comprising a variant Bhr-PETase compared to SEQ ID NO: 1, wherein the variant is 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 1 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, 204, 206, 208, 209, 211, 212, 213, 216, 217, 218, 219, 221, 222, 223, 225, 227, 228, 229, 231, 236, 237, 241, 242, 243, 246, 249 , 250, 251, 252, 253, 254, 255, 258, 8, 31, 38, 95, 126, 137, 165, 169, 172, 191, 192 and 197, wherein said variant Bhr-PETase has at least 85% identity to SEQ ID NO: 1 and has PETase activity.In another aspect, the disclosure provides a composition comprising a variant Bhr-PETase compared to SEQ ID NO: 1, wherein the variant has an amino acid substitution at position 27 as well as at positions 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, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 8, 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, 204, 206, 208, 209, 211, 212, 213, 216, 217, 218, 219, 221, 222, 223, 225, 227, 228, 229, 231, 236, 237, 241, 242, 243, 246, 249 , 250, 251, 252, 253, 254, 255, 258, 8, 31, 38, 95, 126, 137, 165, 169, 172, 191, 192 and 197, wherein said variant Bhr-PETase has at least 85% identity to SEQ ID NO: 1 and has PETase activity.In another aspect, the disclosure provides a composition comprising a variant Bhr-PETase compared to SEQ ID NO:1, wherein the variant is 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, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 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, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 77, 179, 181, 182, 184, 185, 189, 190, 193, 194, 195, 198, 200, 203, 204, 206, 208, 209, 211, 212, 213, 216, 217, 218, 219, 221, 222, 223, 225, 227, 228, 229, 231, 236, 237, 241, 242, 243, 246, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 3 The present invention relates to a composition comprising at least one amino acid substitution at an amino acid position selected from the group consisting of 55, 258, 8, 31, 38, 95, 126, 137, 165, 169, 172, 191, 192 and 197, wherein said variant Bhr-PETase has at least 85% identity to SEQ ID NO: 1 and has PETase activity that is equivalent to or even greater than that of wild-type Bhr-PETase, SEQ ID NO: 1.In some embodiments, the amino acid substitutions above are 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, 151 3, 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, 204, 206, 208, 209, 211, 212, 213 and at an amino acid position selected from the group consisting of: 3, 216, 217, 218, 219, 221, 222, 223, 225, 227, 228, 229, 231, 236, 237, 241, 242, 243, 246, 249, 250, 251, 252, 253, 254, 255 and 258. In some embodiments, the amino acid substitution is at an amino acid position 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. In some embodiments, the amino acid substitution is at an amino acid position selected from the group consisting of 8, 126, 137, 165, 169, 172, 191, 192, and 197.
[0004] In some embodiments, the amino acid substitutions are 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, S22 P, S22R, S22V, V23L, V23T, A24D, A24G, A24H, A24N, A24S, A24T, A24V, T25A, T25F, T25H, T25Q, T25R, T25V, Y26C, Y26K, Y26L, Y26T, R30K, S32K, S32M, S32 Q, S32Y, V33G, V33Q, V33T, S34R, V40T, G46E, G46L, G46N, G46R, G46S, T48N, T48S, L49G, G53A, I54V, A55C, A55I, A55L, A55M, A55T, A55V, M56I, M56L, S57 C, S57E, S57F, S57I, S57L, S57M, S57T, S57V, Y60A, Y60H, Y60I, A62T, A68F, L70M, R72P, L74W, H77Q, I82F, I82L, I82M, V83I, V83L, V83T, N85D, N87F, N87 H, N87I, N87K, N87L, N87M, N87Q, N87R, N87V, N87W, N87Y, S88K, S88T, L90F, L90K, L90Y, F92G, F92I, F92K, F92L, F92N, F92Q, F92V, F92Y, A97C, A97E, A97 F, A97G, A97L, A97P, A97Q, A97S, A97T, A97V, S98A, S98D, S98E, S98L, S98M, S98N, S98Q, S98T, S98V, S101A, S101C, S101D, S101F, S101H, S101K, S101L, S 101M, S101N, S101Q, S101R, S101V, S101W, S101Y, A102V, N105D, R108C, R10 8E, R108H, R108K, R108N, R108P, R108Q, R108S, R108T, R108V, T109A, T109F,T109G、T109K、T109L、T109N、T109R、T109Y、S110D、S110G、S110H、S110K、S110N、S110R、S113A、S113K、S113N、S113P、S113Q、S113R、S113T、S113Y、A114K、A114L、A114S、A114V、A117F、A117G、A117L、A117N、A117Q、A117S、A117T、A117Y、L119I、L119M、A121S、N122A、N122E、N122H、N122P、N122R、N122S、A125S、A127M、A127S、A127V、A135G、T136A、T136M、T136S、T136V、R138E、R138L、I139A、I139T、S140A、Q142D、Q142E、Q142H、Q142L、Q142W、I143N、I143R、T145S、K147F、K147G、K147N、K147Q、G149A、G149C、G149D、G149N、G149S、G149T、G149V、V150I、V150L、T153L、H156N、T157A、T157G、D158E、D158I、D158K、D158L、T160K、T160Q、T160R、T160S、T160V、F161V、F161W、N162E、N162H、N162P、N162R、T163I、T163S、P164E、P164H、P164N、P164R、P164S、P164T、Q167I、Q167T、Q167V、V170L、E173R、A174K、A174R、V177A、P179Q、S181A、S181C、S181R、Q182T、A184C、A184G、A184S、I185A、I185E、I185G、I185L、I185Q、I185R、I185S、I185Y、Q189I、Q189L、Q189V、N190S、S193E、S193F、S193H、S193K、S193N、S193P、S193T、S193V、T194G、T194S、T195F、V198A、V200L、D203N、D203R、D203V、N204A、N204K、N204R、N204S、T206G、T206K、T206L、T206P、T206R、F208G、F208L、F208R、F208T、A209V、N211F、N211I、N211L、N211M、N211V、S212F, S212L, S212M, P213N, P213R, A216L, A216P, A216S, A216T, A216V, I217S, S218 A, V219F, V219I, V219K, V219L, V219R, T221S, I222L, S223A, S223C, M225L, L227R, W2 28F, V229C, V229I, V229L, N231L, N231Q, N231S, R236C, R236E, R236H, R236K, R236Q, Q237R, N241P, V242T, N243P, A246D, A246K, A246S, A246T, D249I, D249M, D249N, D249 S, D249T, F250I, F250L, F250V, F250Y, R251A, R251E, R251I, R251K, R251L, R251Q, R2 51T, R251V, S252T, N253S, N253Y, N254R, R255E, R255G, R255L, R255M, R255S, R255V, Selected from the group consisting of R255W, R255Y, Q258P, P8T, L31M, G38D, S95N, V126I, L137M, V165I, I169C, I169L, I169V, A172T, L191F, L191V, P192A, K197L, K197R, K197T, K197V and K197Y. In some embodiments, the amino acid substitutions are 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, A2 4V, T25A, T25F, T25H, T25Q, T25R, T25V, Y26K, Y26L, Y26T, R30K, S32K, S32M, S32Q, S32Y, V33G, V33Q, V33T, S34R, V40T, G46L, G46S, T 48N, T48S, L49G, G53A, I54V, A55L, A55V, M56I, M56L, S57I, S57M, S57V, Y60A, Y60H, Y60I, A62T, A68F, L70M, R72P, L74W, H77Q, I82F,I82L、I82M、V83T、N85D、N87F、N87H、N87I、N87K、N87L、N87M、N87R、N87V、N87W、N87Y、S88K、S88T、L90F、L90K、L90Y、F92G、F92I、F92K、F92L、F92N、F92Q、F92V、F92Y、A97F、A97G、S98A、S98D、S98E、S98L、S98M、S98N、S98Q、S98T、S98V、S101A、S101D、S101F、S101H、S101K、S101L、S101M、S101N、S101Q、S101R、S101V、S101W、A102V、N105D、R108C、R108E、R108H、R108K、R108N、R108P、R108Q、R108S、R108T、R108V、T109F、T109K、T109L、T109N、T109R、S110G、S110H、S110R、S113A、S113K、S113N、S113P、S113Q、S113R、S113T、S113Y、A114K、A114L、A114S、A114V、A117G、A117N、A117Q、A117S、L119I、L119M、A121S、N122A、N122E、N122H、N122P、N122R、N122S、A125S、A127M、A127S、A127V、A135G、T136A、T136M、T136S、T136V、R138E、R138L、I139A、I139T、S140A、Q142L、Q142W、I143N、I143R、T145S、K147F、K147G、K147N、K147Q、G149A、G149C、G149S、G149V、V150I、V150L、T153L、H156N、T157A、T157G、D158E、D158I、D158K、D158L、T160K、T160Q、T160R、T160S、T160V、F161W、N162E、N162H、N162P、N162R、T163S、P164S、Q167T、Q167V、V170L、E173R、A174K、A174R、V177A、P179Q、S181A、S181C、S181R、Q182T、A184S、I185L、I185Y、Q189I、Q189L、Q189V、N190S、S193E、S193F、S193H、S193K、S193N、S193P、S193T、S193V、T194G、T194S、T195F, V198A, V200L, D203N, D203R, D203V, N204A, N204K, N204R, N204S, T206 G, T206K, T206L, T206P, T206R, F208G, F208L, F208R, F208T, A209V, N211F, N2 11I, N211L, N211M, N211V, S212F, S212L, S212M, P213N, P213R, A216L, A216P, A216S, A216V, I217S, S218A, V219F, V219I, V219K, V219L, V219R, T221S, I222 L, S223A, S223C, M225L, L227R, W228F, V229C, V229I, N231L, N231Q, N231S, R236C, R236E, R236K, R236Q, Q237R, N241P, V242T, N243P, A246D, A246K, A246S, A246T, D249T, F250I, F250Y, R251I, R251K, R251V, S252T, N253Y, N254R, R255E, R255G, R255L, R255M, R255S, R255V, R255W, R255Y and Q258P. In some embodiments, the amino acid substitutions are 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, S101 R, S101V, S101W, A102V, T109F, T109K, T109L, T109N, T109R, S110G, S110 H, S110R, A114K, A114L, A114S, A114V, A117G, A117N, A117Q, A117S, A135G , T136A, T136M, T136S, T136V, I139A, I139T, S140A, Q142L, Q142W, I143N , I143R, G149A, G149C, G149S, G149V, F161W, T163S, P164S, Q167T, Q167V,In some embodiments, the amino acid substitutions are selected from the group consisting of A184S, I185L, I185Y, T195F, I222L, L227R, W228F, V229C, V229I, D249T, F250I, F250Y, R251I, R251K, and R251V. , 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, S 57T, V83I, V83L, N87Q, S95N, A97C, A97E, A97L, A97P, A97Q, A97S, A97T, A97V, S101C, S101Y, T 109A, T109G, T109Y, S110D, S110K, S110N, A117F, A117L, A117T, A117Y, V126I, L137M, Q142D, Q142E, Q142H, G149D, G149N, G149T, F161V, T163I, P164E, P164H, P164N, P164R, P164T, V165I , Q167I, I169C, I169L, I169V, A172T, A184C, A184G, I185A, I185E, I185G, I185Q, I185R, I185 S, L191F, L191V, P192A, K197L, K197R, K197T, K197V, K197Y, A216T, V229L, R236H, D249I, D249M, D249N, D249S, F250L, F250V, R251A, R251E, R251L, R251Q, R251T and N253S.
[0005] In some embodiments, the variant Bhr-PETase enzyme has one or more amino acid substitutions at position 1, at position 2, at position 3, at position 4, at position 5, at position 6, at position 7, at position 8, at position 9, at position 10, at position 11, at position 12, at position 13, at position 14, at position 15, at position 16, at position 17, at position 18, at position 19, or at position 20.
[0006] In some embodiments, the variant Bhr-PETase is 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 / R108 T / V150I / N253Y / N254R, D18R / I54V / M56I / R138E / I139A / T194S / D203N / V2 19K / F250I, D18R / M56I / I139A / L227R / W228F, D18R / M56I / R138E / I139A / N1 90S / D203N / M225L, D18R / M56I / R72P / H77Q / F92Q / A127M / V150I / A184S / D20 3N / N254R, D18R / N85D / R108T / L119I / N190S / T194S, D18R / V40T / I139A / D20 3N, D18R / V40T / I82F / S101L / N105D / L119I / H156N / F161W / N190S / T194S / D 203N, D18R / V40T / M56I / R108T / R138E / H156N, G53A / R108T / Q167T / A184S / T 194S / N243P, I143R / Q167T / V198A / W228F, I82F / R108T / L119I / V150I / F161 W / Q167T / T194S / D203N, L15I / D18R / R108T / R138E / I139A / F161W / A184S / N1 90S / D203N, M56I / I82F / R108T / I139A / F161W / T194S / D203N / N241P, M56I / S88T / R108T / N190S / L227R, N204A / Q237R, N9A / D18R / M56I / N85D / L119I / N2 54R, N9A / S22A / G46S / M56I / R72P / F92Q / L119I / T221S / M225L, N9A / S22A / Q1 67T, N9A / V40T / L49G / I54V / R108T / V150I / D203N / T221S / M225L, N9S / T25H,Q5E / N9A / M56I / F92Q / R108T / L119I / Q167T / N253Y、Q5E / S22A / R72P / H77Q / F92Q、R108T / L119I / K147Q / F161W / A184S / D203N、S101F / T136A、S22A / A24S / G46S / V150I / Q167T、S22A / A97G / V150I / D203N / M225L / R236E、S22A / G46S / S101L / F161W / D203N / R236E、S22A / I54V / R72P / F92Q / V150I / V200L、S22A / L49G / M56I / V83T / S101L / R108T / L119I / A127M / T194S、S22I / Y26K、S27T / I82L / P213N、S27T / T48S / I82L / F92Y / S252T、S27T / T48S / I82L / L90F / A135G / S140A / I143N / T145S / P213N、S27T / T48S / I82L / S140A / I143N / G149A、S32Y / A62T、S88T / R108T / V150I / A184S / D203N / T221S / M225L / N243P、S98Q / A209V、T16E / D18R / G53A / I54V / R72P / L119I / A127M / Q167T / V200L、T16E / D18R / M56I / A127M / R138E / T194S / V200L / M225L、T16E / D18R / M56I / K147Q / Q167T / A184S / D203N、T16E / D18R / M56I / N85D / S88T / R108T / F161W / Q167T、T16E / D18R / M56I / V150I / S218A / V219K、T16E / D18R / R108T / K147Q / Q167T / A184S / N190S / T194S、T16E / D18R / S22A / M56I / N85D / L119I / A184S、T16E / D18R / S22A / V40T / A97G / S101L / L119I / A127M / Q167T / D203N、T16E / D18R / S88T / R108T / W228F、T16E / D18R / V40T / M56I / I82F / R108T / L119I / F161W / L227R / Q258P、T16E / D18R / V40T / S88T / L119I / A127M / V150I / D203N、T16E / D18R / Y26T / S88T / S101L / H156N / V200L、T17A / I82L / F92Y / P213N、T17A / I82L / L90F / F92Y / A135G / S140A / I143N / G149A / Q167V、T17A / I82L / L90F / F92Y / Q167V、T17A / I82L / S140A / I143N / Q167V / P213N / S252T、T17A / Q167V、T17A / S27T、T17A / S27T / I82L、T17A / S27T / I82L / G149A / Q167V / P213N、T17A / S27T / I82L / L90F / F92Y / Q167V、T17A / S27T / I82L / P213N、T17A / S27T / I82L / P213N / S252T、T17A / S27T / I82L / S252T、T17A / S27T / I82L / T145S / G149A / P213N、T17A / S27T / L90F / A135G / S140A / Q167V / P213N / S252T、T17A / S27T / L90F / F92Y / A135G / Q167V / S252T、T17A / S27T / L90F / F92Y / P213N、T17A / S27T / T48S、T17A / S27T / T48S / A135G / Q167V / P213N、T17A / S27T / T48S / A135G / S140A、T17A / S27T / T48S / I82L、T17A / S27T / T48S / I82L / L90F / F92Y / Q167V / S252T、T17A / S27T / T48S / I82L / L90F / F92Y / S140A / T145S、T17A / S27T / T48S / I82L / L90F / F92Y / S140A / T145S / P213N、T17A / S27T / T48S / I82L / L90F / F92Y / S252T、T17A / S27T / T48S / I82L / L90F / P213N / S252T、T17A / S27T / T48S / I82L / L90F / Q167V、T17A / S27T / T48S / I82L / L90F / Q167V / P213N / S252T、T17A / S27T / T48S / I82L / P213N / S252T、T17A / S27T / T48S / L90F / F92Y、T17A / S27T / T48S / P213N、T17A / S27T / T48S / T145S / Q167V、T17A / T48S / I82L、T17A / T48S / I82L / F92Y / Q167V / S252T、T17A / T48S / I82L / L90F / A135G / S140A / Q167V、T17A / T48S / P213N、T17K / A125S、V177A / A216L、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 / T136A / D158E / A174R、L90F / F92G / T109K / A125S / A174K、S32K / L90F / F92L / T109K / A114K / Q142W、V23T / S32Q / N87I / L90F / F92Y / S101N / A114V / Q142W / A174R / D203R / I222L / V229I、S32Q / F92G / A114V / D203V、R12K / S32K / L90Y / F92L / T136A / A174K / D203V / I222L、R12K / A14K / V23T / L90Y / F92G / S101A / D203R、T17G / S32M / L90Y / F92Y / D203R / A216P、S32Q / F92L / A114V / A125S、P20T / F21W / S32M / L90Y / F92G / A114K / A125S / T136V / D158E / A174K / D203V / I222L、R12K / F21W / S32K / F92L / T109K / A125S / T136V / D158E / D203R、T17Q / L90Y / F92L / A174K / D203V / I222L、F21W / F92L / S101A / A117Q / D203R、T17A / S32M / Y60H / Q142L / D203V / I222L、R12K / Y60H / L90F / S101R / A114V / A117N / D158E、T17S / D18R / L90Y / F92G / A114V / A117Q / D203R、L90Y / F92L / S101A / S110R / A125S / Q142W / A174R / A216P、S32M / S110R / A125S / D158E、R12K / V23L / A24V / F92G / T136V / D203R、A24V / S32Q / F92L / T136V、S32K / L90Y / F92G / A114K / D203V、S32M / L90F / F92G / A114V / A117N / A125S / A174K / D203R、R12K / T17A / V23L / A24V / L90F / F92G / S101N / A125S / Q142W / D158I / D203R、P20T / F21W / Y60I / S101A / T109K / A174K、T17C / L90Y / F92L / S101N / T109L / S110R / A125S / A174K / D203R、T17Q / L90F / F92G / S101M / A117Q / T136A / D158I / A174R、V23L / L90F / F92G / A125S / D203V / I222L、T17C / L90Y / F92L / A125S / T136V / D158E / A174K、V23L / S32Q / Y60I / A117Q / T136A / A174K / V229C、V23T / L90F / F92Y / D203V、R12K / A24V / Y60H / A174R、T17G / P20T / Y60I / F92G / D203V、L90Y / F92L / S110R / A125S / D158E / D203V、T17Q / L90Y / F92L / T109L / T136A / D158E / A174R / D203V / V229C、R12K / V23T / A24V / S32Q / F92G / D158L / A174R / D203R、S32M / N87Y / L90F / F92G / A174K / D203V、T17S / V23L / S32Q / L90F / F92L / A114K / A117N / A125S / T136V / D158E / D203V / V229I、P20T / F21W / S32M / L90Y / F92G / T109L / S110R、A14K / S32M / L90Y / S101R / A114V / T136A / A216P、P20T / F21W / L90Y / F92G / A125S / A174K / V229I、V23L / S32K / L90F / F92L / S101R / T109R / T136V / I222L、A24V / F92L / Q142L / A216P、F92G / A125S / T136A / D158E / A216P、N87R / F92G / A117N / D203V / A216P、R12K / A14K / F92G / S110R / A117N / A125S / A174R / D203R、S32Q / N87F / L90F / F92G / S101M / A114V / A117Q / D203V、T17Q / A24V / N87M / F92G / A114K / A117N / D158E / D203R / I222L、N2R / S32Q / L90、 F / F92G / Q142L / D203V、A14K / S32Q / L90F / F92G / A114K / A117N / A174R / D203V / A216P / I222L、R12K / P20T / D158E / D203R / I222L、N2R / A14K / T17A / L90F / F92G / S101H / A174R / D203V、T17G / A24V / L90F / F92G / Q142W、S32K / L90Y / F92Y / S101A、V23T / N87I / F92G / A125S / T136A / D158L / D203R、T17S / N87L / V229I、A24T / L90F / F92L / A125S / T136A / D203V、R12K / A14K / F92G / T109R / T136A / Q142L / D158E / A174K / D203R / I222L、A24V / S32Q / Y60A / T136A、D18R / S32Q / N87H / L90Y / F92G / S101K / Q142L / D158I / A174R、T17A / F21W / S32K / N87F / L90F / F92Y / S101Q / T136A / D203R、A24V / L90F / F92G / A114V / A117N / T136V / Q142L / D203V / V229C、T17Q / L90Y / F92G / S101N / T109L / S110R / T136V / Q142L / D203V / A216P、S32Q / L90Y / F92L / T109K / A125S / T136V、Y60H / N87K / A114K / A117Q / D158E / A174K / A216P、P20T / L90Y / T109K / T136A / D158I / D203R、T17L / Y60A / D203V、N2R / S32Q / L90Y / F92Y / T109K / D203V、T17H / A24V / S32Q / L90Y / F92L / S101H / A114K / D158I / A174R / D203V、N2R / S32M / F92Y / T109K / S110R / I222L、P20T / F92Y / S101K / A125S / D158E / D203R、R12K / A14K / P20T / F21W / S32M / F92L、R12K / A14K / V23T / A24V / L90F / F92L / S101W / T136A / D203V、T17L / S32K / F92L / T109L / A114V / T136V / D158L / R236H、P20T / F21W / S32Q / N87F / F92L / D158E / D203V、F21W / L90F / F92G / S101A / A114V / A125S / T136V / Q142L / D203V / A216P, P20T / F21W / S32K / F92G / A125S / V229I, V23T / A24V / L90Y / F92G / A125S / T136V / D2 03V, P20T / S32K / L90Y / F92G / S101N / T109L / S110R / A125S / A174K / D203R / A 216P, V23L / L90F / F92L / T136A / D203R / I222L, R12K / A14K / A24V / N87L / L90Y / A125S / T136V / A216P, P20T / F21W / L90Y / A125S, N2R / L90Y / A114K / A117Q / D203V / V229I, F21W / S32K / L90Y / A125S / D158E / A216P / V229I, S27L / T136V, S27L / P8T / T17Q / F21W / S101A / T136V / Q142L, S27L / N2R / T17L, S27L / V23T / A24V / T136A / Q142W, S27L / F21W / T136V, S27L / T17Q / T109K / A114K / T136V / V 229I, S27L / T17Q, S27L / T136V / I222L / V229C, S27L / T17A / A114V / A117N / T 136A, S27L / Parent, S27L / N2R / T136A, S27L / N2R / T17A, S27L / T17C / S101A / T136V , S27L / P20T / T136A, S27L / T136V / Q142W / V229I, S27L / T109K / T136V / I222 L / V229I, S27L / T17L / S101H / A117N / Q142L / I222L / V229I, S27L / S101Q / T10 9L / A117N / T136A / Q142L, S27L / T109K / S110R / S193N / S252T / R255M, S27L / S22R / Y26K / R236Q, S27L / L90F / F92L / S98E / S113Y / A114K / T136A / D158E / S1 81R / T206G / S212M / V219I, S27L / N9S / S22P / T48N / L90Y / F92G / T109K / S110R / T136A / Q189V / N211F / R236Q, S27L / T17A / Y26L / T48N / I82M / S101D / R236Q,S27L / Y26T / S101D / D158E / V219I / S252T、S27L / S13R / S98E / T136V / S181R / T206G / V229I / N231S、S27L / F21W / F92L / S98N / S193P / I222L、S27L / S1G / Y26K / L90Y / S113Y / A114V / T136A / Q189V / N204R / N211L / R236Q、S27L / S22K / I82L / L90Y / F92G / R108S / A117N / D158E / S193N、S27L / S1G / N9S / T48S / L90Y / S98T / S101A / S113N / A114K / L119M / S193N / T206G / S252T、S27L / N9E / R12K / S22P / V23L / T160R / D203R / I222L、S27L / N9E / R12K / S22P / V23T / T48S / S98E / R108S / T160S / Q189V / T206G / S212L / V229I、S27L / S1A / N2R / N9S / T48N / L90F / F92L / D203V / S223A、S27L / N9E / R12K / V23T / I82M / L90Y / F92L / T136V / N204K / N231S / R255M、S27L / S1G / N9S / S22V / I82F / L90Y / F92L / A117N / L119M / Q142L / T206G / S212L / S223A、S27L / N9S / Y60H / R108C / S193P / V219L、S27L / N9E / S22K / S32M / L90F / F92G / R108T / L119M / Q189V / I222L / S223A / R236Q、S27L / T17Q / F92G / S98N / Q142L / Q189L / R236C、S27L / N9E / R12K / Q142W / T160Q / T206G / S212M、S27L / Y26T / S113N / A114V / T136A / D158E / D203R / N211F / R236Q / S252T / R255M、S27L / S1A / T17H / S22V / L90Y / F92G / S98E / A114V / T136A、S27L / N9E / T48S / I82M / L90Y / F92L / N122A / A127S / T160S / A174R / T206G / S212L / R255M、S27L / N9S / S22P / V23L / L90Y / F92G / A125S / T160S / N204R / I222L / S223A / R236Q、S27L / N9E / S22P / T48S / L90Y / F92L / T109R / E173R / A174K / D203V / S223A、S27L / R12K / Y26K / T48N / I82L / L90F / A125S / N211I / A216P、S27L / N9S / R12K / F92Y / T109K / S110R / T160K / Q189L / S223A / S252T / R255M、S27L / S1A / N2R / N9E / R12K / S32K / N87F / R108T / N211I / V219K / R255M、S27L / L90F / F92G / N122A / T136A / T160V / E173R / D203R / S252T、S27L / L90Y / F92G / S98T / T109L / D158I / S193K / V219K / R236Q / S252T、S27L / N9E / R12K / T48N / L90F / S98L / R108Q / A117Q / T136A / T206G / S212F / V219I / V229I / R255L、S27L / S1A / S22V / N87K / R108K / N122E / D158E / S193P / V219I / R255M、S27L / F21W / Y26T / S34R / R108H / L119M / N211M / R236C / S252T、S27L / S1A / N9E / R12K / V23T / T48S / N87H / S101Q / Q189L / N211M / V219I / R236Q / R255M、S27L / N9S / T25Q / L90Y / S101D / T136A / Q142L / Q189L / R236Q、S27L / A14K / I82F / F92G / S98E / R108C / A117N / L119M / Q189V / T206G / I222L / S223A、S27L / T17C / Y26T / N87V / R108C / N122E / T136V / S193P / S252T / R255M、S27L / L90Y / F92G / T109L / N122S / R255M、S27L / R12K / L90Y / T160V / A174R / R236Q、S27L / N9E / R12K / T48N / L90Y / F92G / S98T / S113R / N122R / A127S / T136A / A174K / N204K / S212L、S27L / V23T / L90Y / F92G / S98E / T109L / A125S / T160V / A174K / S181C / S193K / T206G / S212M / R255M、S27L / A24V / F92G / S101D / A114V / A117N / A125S / T136V / D203R、S27L / R12K / S32Q / S101Q、S27L / L90F / F92G / T136V / A174R / D203R、S27L / P20T / S32K / L90Y / D203V、S27L / N2R / A14K / T17S / L90Y / T109K / T136V / A216P、S27L / N2R / V23T / A24V / S32M / L90Y / F92G、S27L / A14K / L90Y / F92L / S101D / A117N / D158I / D203R、S27L / F21W / N87H / A114V / A117N / T136V、S27L / N2R / V23T / A24V / N87M / F92L / S101K / A125S / T136A、S27L / L90Y / F92L / S101N / T109R / S110R / Q142W、S27L / S32M / F92G / A216P、S27L / F21W / L90Y / F92L / A114K / A117N / T136A / D203V、S27L / N2R / V23T / S32M / N87M / F92L / T136A、S27L / R12K / L90F / F92G / S101A / D203V、S27L / A24V / L90F / F92G、S27L / R12K / F92L / S101A / A125S / T136A / D203R、S27L / R12K / V23L / L90F / F92L / A114K / T136A / D158E / D203V / I222L、S27L / L90F / F92G、S27L / A14K / P20T / S32Q / L90Y / A125S / D203V、S27L / T17L / L90F / F92G / A125S / A174R / D203V、S27L / N2R / N87K / A114K / A117Q / T136V / D203V、S27L / N2R / A114K / T136V / D203V、S27L / N2R / T17A / T136V / A216P、S27L / N2R / R12K / N87F / T136V / D158E / A174R、S27L / D203R、S27L / R12K / T17Q / T136V / D203V / A216P、S27L / N87F / T109L / Q142L、S27L / N2R / T17Q / A24V / A114K / A117Q / T136A / D158E / A174K / D203V / I222L、S27L / N2R / P20T / F21W / N87M / T109R / A117N / A125S / T136V / D203V / I222L、S27L / V23T / A24V / T136A / D158E / A174K / D203V / I222L、S27L / R12K / V23L / A114K / T136A / D158I / D203V / I222L、S27L / N87Y / T136V / D158E / D203V、S27L / T109K / S110R / D203R、S27L / N2R、 / T17L / A125S / T136A / D158E、S27L / A24V / D158E / A174K / A216P、S27L / T17G / V23L / N87L / A117Q / A125S / T136V / D158E / V229C、S27L / V23L / N87L / T109R / A114K / A117N / A125S / T136V / Q142L、S27L / N87Y / S101A / A114V / A117Q / T136V / A174K / A216P / I222L、S27L / R12K / T109R / Q142L、S27L / D203V、S27L / T17G / N87F / S101A / D203V、S27L / T17L / N87M / Q142L / D203V、S27L / N2R / R12K / S101D / A117N / T136A / D203V、S27L / R12K / P20T / F21W / A114K / Q142W / D203R、S27L / N2R / T17G / T109L / S110R / A114K / A117Q / T136V / Q142L / D203R、S27L / N2R、S27L / S32K / L90F / F92G / S101M / A114V / A117N / A125S / T136A / D158E / D203V、S27L / R12K / T17A / F21W / S32Q / Y60H / D203R、S27L / R12K / F92L / S110R / T136V / I222L、S27L / A14K / V23T / A24V / L90Y / F92G / A114V / A117Q / Q142L / A174R / I222L、S27L / F21W / S32Q / L90Y / S101D / T109K / A125S / T136A / A174K、S27L / V23T / L90Y / F92G / T136A / D203V / A216P、S27L / T17S / L90Y / F92G / A117N / T136A / D158L、S27L / A14K / V23L / A24V / F92L / Q142W / D203V、S27L / T17L / L90F / F92G / S101K / A174K、S27L / L90F / F92L / A125S / T136V / A174K / D203V / A216P / I222L、S27L / R12K / P20T / S32M / L90F / F92G / S101M / A114V / D203V、S27L / T17A / S32Q / L90F / F92L / T136V / Q142W / A174K / D203R、S27L / R12K / S32M / L90F / F92G / S101W / A114V / D158I / I222L、S27L / P20T / S32M / L90Y / F92L / A114V / A117N / I222L、S27L / T17C / L90Y / F92G / T136A / A174K / D203V、S27L / S32Q / N87Y / F92G / T109K / A114K / A117N / T136V / D203V / V229C、S27L / F21W / L90F / F92G / T136V / A174R、S27L / P20T / N87Y / T136A / D203V / I222L、S27L / A24V / L90Y / F92L / D158I / A174K / D203R / I222L、S27L / F21W / S32Q / L90Y / S110R / A114K / T136V / D203R / A216P / V229I、S27L / N87H / F92G / D203R、S27L / R12K / S32K / Y60A / A125S / T136V / D158L、S27L / V23T / A24V / S32Q / L90F / F92L / T136A、S27L / F92L / A114V / D158E / D203R、S27L / S32Q / L90Y / D203V / V229I、S27L / V23T / S32Q / Y60H / T109K / T136V / A216P / I222L、S27L / A14K / A24V / L90F / F92G / T109K / T136V / D158L / A174R / D203V / I222L / V229I、S27L / R12K / N87F / A174K、S27L / P20T / S32Q / Y60H / T109K / A114V / T136A / A174K、S27L / V23T / A24V / Y60A / A125S / A174R / A216P、S27L / F92Y / D158L / I222L、S27L / V23L / A24V / L90Y / F92G / D203V / I222L、S27L / V23T / A24V / S32Q / L90F / F92G / S101H / T136A / A174K / D203V / V229C、S27L / S32K / N87H / A125S、S27L / P20T / F21W / F92G / A114V、S27L / T17L / V23L / A24V / L90Y / F92L / A117N / T136V / L137M / Q142L、S27L / P20T / L90Y / D203R、S27L / S110R / Q142L / D158I / D203R / V229I、S27L / P20T / S32Q / L90F / F92L / T109K / S110R / A125S / D203V、S27L / V23T / A24V / L90F / F92G / D158E / D203R、S27L / F92G / T136A / A174R / D203V / V229C、S27L / P20T / L90Y / F92G / T109R / S110R / A125S / D203R、S27L / A14K / L90Y / F92L / D158L / A174R / D203V、S27L / F21W / S32Q / L90Y / F92G / T109L / S110R / A117N / D158I / A174K / D203V、S27L / R12K / A114K / A117N / D158E / D203V、S27L / T17S / N87H / T109R / A114K / A117N / T136V / Q142W / D203V、S27L / N2R / N87Y、S27L / T17S / V23T / A24V / S32Q / Y60H / N87K / T136A / D203R、S27L / P20T / F92Y / T109K / D158E / D203V、S27L / S32K / N87M / F92L / S101M / T109L / S110R / D203V、S27L / L90Y / F92G / T136A / D158E / D203V / A216P / V229I、S27L / A14K / S32M / F92L / A125S、S27L / L90Y / F92G / T136V / D158L / D203V、S27L / A24V / L90Y / F92G / S101A / T109L / S110R / A117Q / A174R / D203R、S27L / P20T / S101D / A114V / T136V / Q142W / D158E / D203R / A216P、S27L / N87Y / F92G / S101Q / A114K / A117N / T136A / D158L / D203R / A216P、S27L / V23T / A24V / N87Y / L90Y / F92L / S101D / T109R / A174K / D203R / A216P、S27L / N2R / P20T / F21W / L90F / F92Y、S27L / L90Y / F92G / D158I / D203V / V229C、S27L / S32Q / F92G / Q142W / I222L、S27L / N2R / R12K / S32M / N87Q / L90F / F92L / I222L、S27L / R12K / S32K / F92L / A114V / A117Q / T136A / D158E / A216P、S27L / L90Y / F92G、S27L / R12K / F21W / S32M / F92G / T136V / Q142L / D203R、S27L / R12K / A14K / L90Y / F92L / A125S / A174K、S27L / A24V / A114V / A117Q / T136V / D203V、S27L / R12K / T17Q / V23L / S32M / L90Y / F92G / S101D / T136V / I222L、S27L / V23T / A24V / S32Q / F92L / S101N / T109L / S110R / D203R、S27L / P20T / Y60H / N87K / T136A / A174R / D203V、S27L / P20T / F21W / L90Y / F92L / T136A / A174R / D203V / V229C、S27L / T17G / F21W / S32K / L90F / F92G / A125S / T136A / A174K、S27L / T17G / N87Y / T136A / D203V / A216P、S27L / F21W / L90F / F92G / S101D / A117N / T136A / A174K / D203V、S27L / R12K / A14K / A24V / L90Y / D203V、S27L / N2R / L90F / F92G / S101M / Q142L / D158L、S27L / N2R / T17C / A24V / N87K / A174K / A216P、S27L / N87M / Q142L / I222L、S27L / F92L / S98T / R108C / A117Q / A127S / Q142L / Q189V / A216P / R236Q、S27L / S22K / F92L / R108H / A127S / T136A / N211I、S27L / F21W / T48S / L90Y / F92L / T109L / A127S / T136V / N204K / S223A、S27L / T17L / L90Y / F92L / S98M / A174K / D203V / N211M / S212L / S223A、S27L / N87K / S98M / N211M、S27L / N9E / S22P / L90Y / N204K、S27L / S32M / T48S / F92G / A127S / Q142W、S27L / T17S / S22K / L90F / F92L / D158E / D203V / V219I / S252T、S27L / L90F / F92L / A127S / D203V、S27L / T25F / L90Y / F92G / N204R / S223A / N231S / R236C、S27L / F21W / S22V / T48S / L90F / F92L、S27L / S1A / S98T / S113K / A114V / L119M / A127S / Q142W / S193K / V219K / S252T / R255L、S27L / A24V / I82L / L90F / F92G / T109L / L119M / A127S / E173R / N204K、S27L / T17C / Y60H / L90F / A127S / E173R / A174R / N204K、S27L / L90F / F92L / Q142W / D203V / S223A、S27L / P20Q / L90Y / Q142L / S223A、S27L / N9S / Y60A / A216P / R236Q / R255M、S27L / F92G / R108C / S110R / A117Q / T136A / N211M、S27L / S22P / Y60H / S98A / S113K / A114K / T136V / Q189L / S193H、S27L / V23L / S98A / Q142L / N211M、S27L / V23L / Y26L / L31M / T48S / F92G / A174R / N204K / S252T、S27L / S13R / A14K / Y26T / T136A / S181R / N211M / S212M、S27L / F21W / I82F / L90Y / F92G / A127S / N211M、S27L / P20T / Y60H / S98L / T136A / R255L、S27L / P20T / S34R / N87M / D158E / S252T、S27L / T25A / L90F / F92G / Q189L / S193P / V229I、S27L / V219L / I222L、S27L / N9E / T48S / L90F / F92L / S98N / R108H / S110R / S113Y / N211V / S212L / S252T、S27L / L90F / F92Y / S113R / V219I / R255L、S27L / F21W / S22K / T48S / F92G / T109R / A127S、S27L / S1G / A14K / P20T / S32K / T48S / L90Y / F92L / T109L / T160R / V219L / I222L / S252T / R255L、S27L / L90F / F92G / D158E / T160R / S193N / N204K / S223A、S27L / A14K / Y26T / F92L / Q142L / S212L、S27L / S22R / L90Y / F92G / V126I / A127S / A174K / N204K、S27L / P20T / T25A / L90F / F92L / A117N / L119M / S252T / R255L, S27L / T17A / A24V / T48S / L90F / F92G / T109R / A127S / T160K / D203R / S223A, S27L / F21W / T48S / T109L / A127S / V219I / R255L, S27L / A14K / S22P / T48S / L90F / F92L / T109L / A127S / Q142L / A174R / N20 4K / S223A、S27L / T136A / S212M / N231S、S27L / F21W / L90F / F92G / A127S / S223A、S27L / T48S / L90Y / F92L / R108S / S110R / A127S / T136V / E173R / S223A、S27 L / T48S / F92G / S98E / Q142L / Q189V / S193P / N204R / S223A、S27L / T17S / L90F / F92Y / S101K / A127S / T136V / N204R / N211I / V229I / N231S、S27L / N9E / F21W / T 48S / L90Y / F92L / T109R / N204K / R236Q、S27L / N9E / S22R / T48S / F92L / S101N / A127S / E173R / N204R、S27L / S22K / N87F / A117Q / S181C / N204K / N211M / V229I / N231S / R236Q、S27L / N9S / T48S / L90F / F92G / S101N / A127S / N204K / A216P / S 252T / R255M、S27L / I82L / S193H / N211M / S212F / S223A、S27L / A14K / P20T / T4 8S / L90F / F92G / S98N / R108C / T136A / N204K / N211L / S212L / R236Q / S252T、S2 7L / S13R / T17L / T25Q / L90F / F92G / R108C / A127S / T160R / I222L / R255L、S27L / T48S / L90Y / F92G / T109L / S193N、S27L / S22V / T48S / L90Y / F92L / S98E / A127 S / T136A / N204K / A216P / S223A、S27L / V23L / N87H / F92L / R108C / S110R / N122 A / T160S / Q189L / S193P / N204K / S223A / R255L、S27L / F92G / A127S / A174R / S2 23A / V229I / N231S / R236C、S27L / S1G / L90Y / A127S / E173R / S223A、S27L / F92 Y / S98N / Q142W、S27L / P20T / S32M / S34R / T48S / L90Y / F92L / R108K / A127S / Q1 42W / A174R / N204K / V219I / S252T、S27L / F92G / A127S / Q189V / S193N / S223A、S27L / S34R / F92G / S110R / D158L / D203V / R255M、S27L / N9E / F21W / T48S / F92 G / T109L / A127S / R236C、S27L / S1A / S22P / T48S / L90Y / F92G / A127S / E173R / A 174R / N204K / S223A、S27L / S22K / T48S / T160S / N204K、S27L / F21W / T48S / L90 Y / F92G / S98V / R108C / A127S / N211L / S212M / N231S / R236C、S27L / V23L / A24V / A114K / T136A / I222L / V229I、S27L / P20T / S101H / T109R / A114V / T136A / Q1 42L / I222L / V229I、S27L / V23L / S101H / T109K / A114K / A117N / T136V / Q142L / I222L / V229I、S27L / T17L / F21W / S101H / T109R / A114V / T136A / Q142L / I222L / V229I、S27L / T17S / T136V / Q142L / I222L / V229C、S27L / T17C / S22P / T48S / L 90F / F92G / A127S / A174R / P192A / S193H / N204K / S223A / V229I / N231S / R236Q / R255L, S27L / N9E / T48N / N87H / F92L / A174K / Q189L / S193K / N204R / V219I S27L / A14K / V23L / Y26L / L90F / F92G / S98E / S113K / A114K / T136A / V229I / N231S, S27L / L90Y / F92L / S101D / A117N / Q142L / S193N, S27L / S22K / T25A / L90Y / Q142L / E173R / N204K / S223A, S27L / P20T / L90Y / T109L / A127S / A174K / V229C, S27L / S22V / L90Y / F92L / A117Q / R236C / R255L, S27L / V23L / A24G / Y26T / I82 F / L90Y / F92G / R108H / T136A / D158L / T160K / D203V、S27L / A14K / S22K / F92G / A127S / N204K / V229I、S27L / S22V / L90F / F92G / A127S / A174R / A216P / R236C、S27L / V23L / Y60A / S98T / T109L / A127S / T136V / T160Q / N204K / S223A / R255L, S27L / S13R / L90Y / S98L / T160V / Q189L / S193P / A216P / S252T / N253S / R2 55L, S27L / S22R / I82M / F92L / T109L / A127S / Q142W / Q189L / S223A, S27L / S1G / Y60A / L90F / F92L / N122E / T136A / N204K / S223A / N231S, S27L / S32K / F92 G / S98L / R108Q / S110R / R255L、S27L / L90Y / F92G / L119M / A127S / A174R / S2 52T、S27L / L90F / S110R / S181R / N211F、S27L / A14K / S22P / Y60H / S110R / N1 22S / D158I / N204K / S223A / R255M, S27L / F92L / S98V / T109L / N122A / E173R / R255L, S27L / S22R / N87L / F92G / R236C, S27L / S32M / S34R / I82L / F92G / N20 4K / S223A、S27L / A24V / T25Q / Y60I / A127S / A174K / N204K / V219I / S223A / S 252T / R255M、S27L / S98V / T136A / N211F、S27L / N9S / L90F / F92L / A127S / N2 04R / S212M、S27L / S22R / T48S / F92G / R108H / S110R / A127S / E173R / N204K / S223A / R236Q、S27L / A14K / L90Y / F92G / A127S、S27L / A14K / T48S / F92G / E17 3R / N204R / S223A / N231S, S27L / L90Y / T109R / S113N / V219L / I222L, S27L / N9S / T48S / L90Y / T136A / T206G / S223A, S27L / S22K / T48N / L90F / F92G / N20 4K / S223A / V229C / N231S、S27L / S22P / T48S / L90F / F92G / A127S / A174R / N2 04R / S223A、S27L / S1G / P20T / S32K / S34R / T48S / N87F / S98A / A127S / T136A、S27L / F21W / S22R / S98E / Q142L / T160V / S223A / S252T / R255L、S27L / P20T / T48S / F92Y / R108K / S110R / A127S / V219L / S223A / V229I / R236Q、S27L / Y26T / L90Y / F92L / A127S / V219L / S252T / R255L、S27L / N9S / F21W / T48S / F92L / A127S / D203V / S212F、S27L / F21W / Y60H / S98E / L119M / D158E / R236Q、S27L / F2 1W / S22R / L90F / F92L / N122S / Q142W / R236Q、S27L / F21W / T48S / L90F / F92G / T109K、S27L / L90Y / F92L / T109R / A127S / E173R / N204K / S223A、S27L / N9S / F 21W / T48S / L90Y / F92G / A127S / N204K / V219I / R255M, S27L / N9E / I82F / L90Y / F92L / S95N / T109L / A127S / A174R / S223A, S27L / L90F / F92L / S98L / N204K / A216P、S27L / A24V / T48S / A117Q / N211M / S212M / S223A、S27L / L90Y / F92G / T109L / A127S / N204R / S223A、S27L / N2R / S32Q / N87K / A125S / V219L、S27L / S 22P / T48S / L90F / F92L / S193N / V219L / S223A, S27L / T25Q / L119M / N211M / R236Q, S27L / L90Y / F92G / N122S / A127S / E173R / A216P / R236Q, S27L / P20T / S3 2K / L90F / F92G / N122E / D158E / T160S / Q189L / S223A / S252T、S27L / N9S / F21 W / T48S / N87H / L90F / F92L / S113N / V229C、S27L / T25Q / I82L / L90F / F92G / S9 8L / S113R / A114V / T136A / S181R、S27L / P20T / R108Q / S110R / D203V / N211I、 S27L / S1G / N2R / P20T / Y26T / S32M / T48S / L90Y / F92L / A127S / N204R / V219L、S27L / A14K / P20T / T48N / L90F / F92G / A117Q / A127S, S27L / N9S / T48N / L90Y / F92L / S98M / T109K / A127S / N204R / N211I / S212L / V219I, S27L / F21W / S22R / T48 S / L90F / F92L / R108Q / A174K / N204K / S212F / S223A、S27L / S22R / Y60H / S98T / T136V / S193K / R236Q、S27L / S22K / Y26K / N87Y / L90Y / F92G / T109R / A127S / S25 2T / R255L, S27L / L90F / F92L / T109L, S27L / F21W / F92L / N204K / S252T / R255L, S27L / V23L / L90Y / F92G / A127S / S193K / N204R / V219I / R236C / R255L, S27L / F 21W / L90Y / F92G / A114V / N122S / A127S / N204K / S223A / S252T、S27L / P20T / Y6 0A / T109L / Q189L / S212L、S27L / S22V / T48S / L90Y / F92G / N204K、S27L / S22V / S 32K / T48S / I82F / F92G / A127S / N204K / S223A, S27L / A55L, S27L / A97V, S27L / F250L, S27L / T109G, S27L / G38D, S27L / A97S, S27L / A55V / A216T, S27L / P20D S27L / A55V, S27L / T109Y, S27L / V165I, S27L / A184G, S27L / A97E, S27L / A184S, S27L / A97F, S27L / A97T, S27L / K197Y, S27L / A55I, S27L / A97P, S27L / A55M S27L / P20E, S27L / A117L, S27L / T109L, S27L / K197T, S27L / T136S, S27L / A97L, S27L / T109A, S27L / P20I, S27L / L191V, S27L / A184C, S27L / A97Q, S27L / F25 0V, S27L / K197V, S27L / A117S, S27L / K197R, S27L / T109K, S27L / A55C, S27L / N2S / V177A, S27L / Q142D, S27L / G149C, S27L / F21Y, S27L / G149A, S27L / P164ES27L / Y26C, S27L / T17N, S27L / S57M, S27L / D249I, S27L / T17S, S27L / P164T, S27L / Q142L, S27L / D249N, S27L / I185R, S27L / V83L, S27L / G149S, S27L / A24D, S27L / R251V, S27L / V83I, S27L / S110N, S27L / T17M, S27L / F161W, S27L / G46E, S27L / D249T, S2 7L / T17I, S27L / S57C, S27L / S57T, S27L / S57L, S27L / Q167T, S27L / S57E, S27L / T17K, S27L / G149N, S27L / Q142E, S27L / S57F, S27L / T17A, S27L / P164H, S27L / T17R, S27 L / G149T、S27L / R251T、S27L / S101Y、S27L / S57V、S27L / A24T、S27L / I185E、S27L / G 46N、S27L / P164N、S27L / P164S、S27L / Q167V、S27L / F161V、S27L / S57I、S27L / G149D S27L / Q167I、S27L / I185Q、S27L / N2L / T17I / T136S / P164H / D249M、S27L / P20E / F21Y / I139T / P164H / K197T / R251 E、S27L / A114V / A117N / T136A / I222L / V229I、S27L / P20T / A24V / S101Q / T109R / A117Q / I222L / V229I、S27L / S110 D / A184S / K197T, S27L / T136S / D249T, S27L / F21Y / A184S, S27L / N2F / P20E / S110D / A184S / L191V / R251V, S27L / T17G / V23T / A24V / T109L / A117Q / T136A / I222L / V229I, S27L / P20D / F21Y / A55T / F250L / R251V, S27L / T109R / A117 N / I222L / V229C、S27L / F21Y / A55T / T109A / A184S、S27L / V23L / A24V / S101K / A117N / I222L / V229C、S27L / S110K / A117Y / P164R、S27L / I139T / Q142E / I169V、S27L / A24H / R251V、S27L / F21W / A114V / T136A / I222L / V229I、S27L / N 2S / A55V / I185Q / R251E、S27L / N2F / S101Y / Q142L / G149T / Q167I / I169C / V229L、S27L / N2F / S101C / A117F / P164S 、S27L / Q142H / I185S / R251A、S27L / F21W / T109R / A117N / T136V / I222L / V229I、S27L / G46R / T109K / T136S / D249T、S27L / N2E / G46N / A55V / I185Y / D249I、S27L / V83L / F250V / R251E、S27L / F21W、S27L / S57C / R251E、S27L / V23L / A114V / A117N / T136A / I222L / V229I、S27L / A24N、S27L / T17A / P20T / S101Q / T109K / S110R / T136A / I222L / V229C、S27L / P164T / V165I / R251E、S27L / F21Y / G46E / A117T / T136S / G149C / R251Q、S27L / G46N / I139T / Q142E / P164T / I185A / V229L / F250V、S27L / V23L / T136A / I222L / V229I、S27L / V23T / A24V / T109K / A114K / A117Q / T136A / I222L / V229C、S27L / F250L / R251Q、S27L / P20E / F21Y / S57T / P164N / A184C / V229C、S27L / P20T / V23T / A24V / I222L / V229I、S27L / V83L / A97S / A184C / D249S、S27L / P20T / S101H / A114V / A117N / I222L / V229I、S27L / N2E / R251L、S27L / P20E / F21Y / S110D / A117S、S27L / T17A / V23T / A24V / T136A、S27L / P20D / S57E / S101C、S27L / N2S / V83L / A184S、S27L / T17S / S101N / T109K / A117N / T136V / Q142L / I222L / V229C、S27L / P20D / A55T / A117Y / K197R、S27L / P20T / I222L / V229C、S27L / T17I / A24D / S57I、S27L / N2F、S27L / T109K / A117N / T136V / I222L / V229C、S27L / N2L / A24T、S27L / N2F / P20I / F21Y / R251Q、S27L / N2F / G46E、S27L / T136V / Q142W / I222L / V229I、S27L / K197R / R251E、S27L / T17S / R251T、S27L / P20I / F21Y / A117F / I185G / D249N、S27L / T17L / S101M / T109K / S110R / A117Q / I222L / V229C、S27L / T17Q / T136A / I222L / V229C、S27L / V229I、S27L / T17A / F21W / S101Q / I222L / V229C、S27L / T17S / F21W / Q142L、S27L / T17L / T109L / S110R / A117Q / I222L / V229I、S27L / F21W / T136A、S27L / P20E / F21Y / G46E / V83L / A97Q、S27L / P20T / F21W / T136A / Q142L / I222L / V229I、S27L / T17G / F21W / A117N / I222L / V229I、S27L / F21W / T109K / S110R / T136A / V229I、S27L / T17C / T109K / A117N / T136A / I222L / V229I、S27L / P20T / T109L / S110R / A117N / I222L / V229I、S27L / A24N / P164S / K197V / R251T、S27L / V23L / A117Q、S27L / A55V / V83L / A117T、S27L / P20D / G46S / A55I / K197T、S27L / A117Y、S27L / F21W / S101M / I222L / V229C、S27L / N2L / T17N / S57C / S110D / A117L / P164N / I185G / V229L / R251T、S27L / N2E / G46S / V83L / T136S / A172T / V229C / D249N、S27L / P20T / T109R / A114K / A117Q / T136V / I222L / V229C、S27L / V23L / A24V / T136A / Q142W / I222L / V229I、S27L / N2L、S27L / T17A / F21W / T109R / S110R / I222L / V229I、S27L / T17C / A114K / A117N / T136V / I222L / V229I、S27L / V23L / I222L / V229C、S27L / S101N / A117Q / I222L、S27L / N2E / P20I / T109L / L191V / K197L / V229C、S27L / P20T / F21W / Q142W / I222L / V229C、S27L / N2L / G46E / T109K / F161V、S27L / T17S / V229I、S27L / T17Q / T136A / I222L / V229I、S27L / N2L / A172T、S27L / P20D / F21Y / F250V、S27L / N2L / A24H / A55C / V229L、S27L / N2L / P20E / F21Y / V229L、S27L / V23T / T109L / A114V / T136A / Q142L / I222L / V229C、S27L / P20T / F21W / I222L / V229C、S27L / T17H / F21W / T136V / Q142W / I222L / V229I、S27L / G46E / A55I / Q142E、S27L / N2F / R251Q、S27L / F21W / Q142L / I222L / V229C、S27L / I222L / V229I、S27L / P20T / S101M / A114V / A117N / Q142W / I222L / V229C、S27L / T17Q / A24V / S101W / T136A、S27L / N2S / P20D / A97C / A117F / F250L / R251E、S27L / S101M / A114V / A117Q / T136A、S27L / T163I、S27L / S101Q / Q142L / I222L / V229I、S27L / F21W / I222L / V229C、S27L / S101M / T136A / I222L / V229I、S27L / F21W / A24V、S27L / A24D / K197L、S27L / T17H / S110K、S27L / P20T / F21W / V229I、S27L / T17G / P20T / F21W / A117Q / I222L / V229C、S27L / F21W / A24V / A114V / A117Q / Q142W / I222L / V229C、S27L / P20T / A117Q / I222L / V229C、S27L / T17Q / V23T / A24V / I222L / V229I、S27L / N2F / D249N / F250L、S27L / V23T / Q142L / I222L / V229C、S27L / P20E / A24D / D249S / F250V、S27L / A24D / S110K、S27L / T109R / I222L / V229I、S27L / P20E / F21Y / K197T、S27L / T17A / V23L / S101M / T109K / S110R / A114V / A117Q / T136V / V229I、S27L / P20T / F21W / Q142W / I222L / V229I、S27L / F21W / I222L / V229I、S27L / P20T / V23T / A24V / T109L / S110R / I222L / V229I、S27L / A24D / L191F / R251Q、S27L / A24D / T136S / F250V, S27L / P20T / F21W / T109L / T136A / Q142W / I222L / V229C, S27L / N2E / I169L / F250L / R251Q, S27L / N2S / A24N / G46E / A55L / Q142D / V229C / R251L, S27L / N2E / P20E / F250V / R251L, S27L / N2F / P20E, S27L / A24V / V229C, S27L / P20D / F21Y / G46N / S110K / T163I / Q167V, S27L / P20T / T109K / A114K / Q142W / I222L / V229C, S27L / T17Q / S101D / T136A / I2 22L / V229I, S27L / F21W / T109R / A117N / T136V / Q142W / I222L / V229C, S27L / A24D / T109L / K197L, S27L / T17Q / Q142L / I222L / V229I, S27L / T17A / A24V / S101D / S110R / T136A, S27L / P20T / F21W / T109R / A117Q / T136V / Q142L / I222L / V229I, S27L / P2 0T / F21W / T109K / A117N / I222L / V229C, S27L / P20T / S101M / I222L / V229I, S27L / N2S / A24D / S101Y / P164E / V165I / I185E, S and S27L / T17N / A117S / V229L / R251A, S27L / S101W / A114V / T136V / V229C, S27L / P20D / G46N / A55C / S110N / T163I / A184S, S27L / A24H / G46E, S27L / T17H / S101Q / T136V / Q142L / V229C, and S27L / V23T / A24V / I222L / V229C. In some embodiments, the variant Bhr-PETase is 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 / I139A / L227R / W228F、D18R / M56I / R13、 8E / I139A / N190S / D203N / M225L、D18R / M56I / R72P / H77Q / F92Q / A127M / V150I / A184S / D203N / N254R、D18R / N85D / R108T / L119I / N190S / T194S、D18R / V40T / I139A / D203N、D18R / V40T / I82F / S101L / N105D / L119I / H156N / F161W / N190S / T194S / D203N、D18R / V40T / M56I / R108T / R138E / H156N、G53A / R108T / Q167T / A184S / T194S / N243P、I143R / Q167T / V198A / W228F、I82F / R108T / L119I / V150I / F161W / Q167T / T194S / D203N、L15I / D18R / R108T / R138E / I139A / F161W / A184S / N190S / D203N、M56I / I82F / R108T / I139A / F161W / T194S / D203N / N241P、M56I / S88T / R108T / N190S / L227R、N204A / Q237R、N9A / D18R / M56I / N85D / L119I / N254R、N9A / S22A / G46S / M56I / R72P / F92Q / L119I / T221S / M225L、N9A / S22A / Q167T、N9A / V40T / L49G / I54V / R108T / V150I / D203N / T221S / M225L、N9S / T25H、Q5E / N9A / M56I / F92Q / R108T / L119I / Q167T / N253Y、Q5E / S22A / R72P / H77Q / F92Q、R108T / L119I / K147Q / F161W / A184S / D203N、S101F / T136A、S22A / A24S / G46S / V150I / Q167T、S22A / A97G / V150I / D203N / M225L / R236E、S22A / G46S / S101L / F161W / D203N / R236E、S22A / I54V / R72P / F92Q / V150I / V200L、S22A / L49G / M56I / V83T / S101L / R108T / L119I / A127M / T194S、S22I / Y26K、S27T / I82L / P213N、S27T / T48S / I82L / F92Y / S252T、S27T / T48S / I82L / L90F / A135G / S140A / I143N / T145S / P213N、S27T / T48S / I82L / S140A / I143N / G149A、S32Y / A62T、S88T / R108T / V150I / A184S / D203N / T221S / M225L / N243P、S98Q / A209V、T16E / D18R / G53A / I54V / R72P / L119I / A127M / Q167T / V200L、T16E / D18R / M56I / A127M / R138E / T194S / V200L / M225L、T16E / D18R / M56I / K147Q / Q167T / A184S / D203N、T16E / D18R / M56I / N85D / S88T / R108T / F161W / Q167T、T16E / D18R / M56I / V150I / S218A / V219K、T16E / D18R / R108T / K147Q / Q167T / A184S / N190S / T194S、T16E / D18R / S22A / M56I / N85D / L119I / A184S、T16E / D18R / S22A / V40T / A97G / S101L / L119I / A127M / Q167T / D203N、T16E / D18R / S88T / R108T / W228F、T16E / D18R / V40T / M56I / I82F / R108T / L119I / F161W / L227R / Q258P、T16E / D18R / V40T / S88T / L119I / A127M / V150I / D203N、T16E / D18R / Y26T / S88T / S101L / H156N / V200L、T17A / I82L / F92Y / P213N、T17A / I82L / L90F / F92Y / A135G / S140A / I143N / G149A / Q167V、T17A / I82L / L90F / F92Y / Q167V、T17A / I82L / S140A / I143N / Q167V / P213N / S252T、T17A / Q167V、T17A / S27T、T17A / S27T / I82L、T17A / S27T / I82L / G149A / Q167V / P213N、T17A / S27T / I82L / L90F / F92Y / Q167V、T17A / S27T / I82L / P213N、T17A / S27T / I82L / P213N / S252T、T17A / S27T / I82L / S252T、T17A / S27T / I82L / T145S / G149A / P213N、T17A / S27T / L90F / A135G / S140A / Q167V / P213N / S252T, T17A / S27T / L90F / F92Y / A135G / Q167V / S252T, T17A / S27T / L90F / F92Y / P213N, T17A / S27T / T48S, T17A / S27T / T48S / A135G / Q167V / P213N, T17A / S27T / T48S / A135G / S140A, T17A / S27T / T48S / I82L, T17A / S27T / T48S / I82L / L90F / F92Y / Q167V / S252T, T17A / S27T / T48S / I82L / L90F / F92Y / S140A / T145S, T17A / S27T / T48S / I82L / L90F / F92Y / S140A / T145S / P213N, T17A / S27T / T48S / I82L / L90F / F92Y / S25 2T, T17A / S27T / T48S / I82L / L90F / P213N / S252T, T17A / S27T / T48S / I82L / L90F / Q167V, T17A / S27T / T48S / I82L / L 90F / Q167V / P213N / S252T, T17A / S27T / T48S / I82L / P213N / S252T, T17A / S27T / T48S / L90F / F92Y, T17A / S27T / T48S / P213N, T17A / S27T / T48S / T145S / Q167V, T17A / T48S / I82L, T17A / T48S / I82L / F92Y / Q167V / S252T, T17A / T48S / I82L / L90F / A135G / S140A / Q167V, T17A / T48S / P213N, T17K / A125S and V177A / A216L. In some embodiments, the variant Bhr-PETase is 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 / N190S / D203N / M225L、D18R / M56I / R72P / H77Q / F92Q / A127M / V150I / A184S / D203N / N254R、D18R / V40T / I139A / D203N、D18R / V40T / I82F / S101L / N105D / L119I / H156N / F161W / N190S / T194S / D203N、D18R / V40T / M56I / R108T / R138E / H156N、G53A / R108T / Q167T / A184S / T194S / N243P、I143R / Q167T / V198A / W228F、I82F / R108T / L119I / V150I / F161W / Q167T / T194S / D203N、L15I / D18R / R108T / R138E / I139A / F161W / A184S / N190S / D203N、M56I / I82F / R108T / I139A / F161W / T194S / D203N / N241P、M56I / S88T / R108T / N190S / L227R、N9A / S22A / G46S / M56I / R72P / F92Q / L119I / T221S / M225L、N9A / S22A / Q167T、N9A / V40T / L49G / I54V / R108T / V150I / D203N / T221S / M225L、Q5E / N9A / M56I / F92Q / R108T / L119I / Q167T / N253Y、Q5E / S22A / R72P / H77Q / F92Q、R108T / L119I / K147Q / F161W / A184S / D203N、S101F / T136A、S22A / A24S / G46S / V150I / Q167T、S22A / A97G / V150I / D203N / M225L / R236E、S22A / G46S / S101L / F161W / D203N / R236E、S22A / L49G / M56I / V83T / S101L / R108T / L119I / A127M / T194S、S27T / I82L / P213N、S27T / T48S / I82L / F92Y / S252T、S27T / T48S / I82L / L90F / A135G / S140A / I143N / T145S / P213N、S27T / T48S / I82L / S140A / I143N / G149A、S88T / R108T / V150I / A184S / D203N / T221S / M225L / N243P、T16E / D18R / G53A / I54V / R72P / L119I / A127M / Q167T / V200L、T16E / D18R / M56I / A127M / R138E / T194S / V200L / M225L、T16E / D18R / M56I / K147Q / Q167T / A184S / D203N、T16E / D18R / M56I / N85D / S88T / R108T / F161W / Q167T、T16E / D18R / R108T / K147Q / Q167T / A184S / N190S / T194S、T16E / D18R / S22A / M56I / N85D / L119I / A184S、T16E / D18R / S22A / V40T / A97G / S101L / L119I / A127M / Q167T / D203N、T16E / D18R / S88T / R108T / W228F、T16E / D18R / V40T / M56I / I82F / R108T / L119I / F161W / L227R / Q258P、T16E / D18R / V40T / S88T / L119I / A127M / V150I / D203N、T16E / D18R / Y26T / S88T / S101L / H156N / V200L、T17A / I82L / F92Y / P213N、T17A / I82L / L90F / F92Y / A135G / S140A / I143N / G149A / Q167V、T17A / I82L / L90F / F92Y / Q167V、T17A / I82L / S140A / I143N / Q167V / P213N / S252T、T17A / Q167V、T17A / S27T、T17A / S27T / I82L、T17A / S27T / I82L / G149A / Q167V / P213N、T17A / S27T / I82L / L90F / F92Y / Q167V、T17A / S27T / I82L / P213N、T17A / S27T / I82L / P213N / S252T、T17A / S27T / I82L / S252T、T17A / S27T / I82L / T145S / G149A / P213N、T17A / S27T / L90F / A135G / S140A / Q167V / P213N / S252T、T17A / S27T / L90F / F92Y / A135G / Q167V / S252T、T17A / S27T / L90F / F92Y / P213N、T17A / S27T / T48S、T17A / S27T / T48S / A135G / Q167V / P213N、T17A / S27T / T48S / A135G / S140A、T17A / S27T / T48S / I82L、T17A / S27T / T48S / I82L / L90F / F92Y / Q167V / S252T、T17A / S27T / T48S / I82L / L90F / F92Y / S140A / T145S、T17A / S27T / 、 T48S / I82L / L90F / F92Y / S140A / T145S / P213N, T17A / S27T / T48S / I82L / L90F / F92Y / S252T, T17A / S27T / T48S / I82L / L90F / P213N / S252T , T17A / S27T / T48S / I82L / L90F / Q167V, T17A / S27T / T48S / I82L / L90F / Q167V / P213N / S252T, T17A / S27T / T48S / I82L / P213N / S252T, T17 The set of amino acid substitutions is selected from the group consisting of A / S27T / T48S / L90F / F92Y, T17A / S27T / T48S / P213N, T17A / S27T / T48S / T145S / Q167V, T17A / T48S / I82L, T17A / T48S / I82L / F92Y / Q167V / S252T, T17A / T48S / I82L / L90F / A135G / S140A / Q167V, T17A / T48S / P213N, T17K / A125S and V177A / A216L. In some embodiments, the variant Bhr-PETase is 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 / S101 A / A114K / A117Q / D203R / I222L, S32Q / F92G / S101Q / A114K / Q142W / D158I / D203R / I22 2L, T17G / L90F / F92L / T109R / A114K / D158E / D203V / I222L, R12K / S32K / F92L / T136A / D158E / A174R, L90F / F92G / T109K / A125S / A174K, S32K / L90F / F92L / T109K / A114K / Q 142W, V23T / S32Q / N87I / L90F / F92Y / S101N / A114V / Q142W / A174R / D203R / I222L / V22 9I, S32Q / F92G / A114V / D203V, R12K / S32K / L90Y / F92L / T136A / A174K / D203V / I222L,R12K / A14K / V23T / L90Y / F92G / S101A / D203R、T17G / S32M / L90Y / F92Y / D203R / A216P、S32Q / F92L / A114V / A125S、P20T / F21W / S32M / L90Y / F92G / A114K / A125S / T136V / D158E / A174K / D203V / I222L、R12K / F21W / S32K / F92L / T109K / A125S / T136V / D158E / D203R、T17Q / L90Y / F92L / A174K / D203V / I222L、F21W / F92L / S101A / A117Q / D203R、T17A / S32M / Y60H / Q142L / D203V / I222L、R12K / Y60H / L90F / S101R / A114V / A117N / D158E、T17S / D18R / L90Y / F92G / A114V / A117Q / D203R、L90Y / F92L / S101A / S110R / A125S / Q142W / A174R / A216P、S32M / S110R / A125S / D158E、R12K / V23L / A24V / F92G / T136V / D203R、A24V / S32Q / F92L / T136V、S32K / L90Y / F92G / A114K / D203V、S32M / L90F / F92G / A114V / A117N / A125S / A174K / D203R、R12K / T17A / V23L / A24V / L90F / F92G / S101N / A125S / Q142W / D158I / D203R、P20T / F21W / Y60I / S101A / T109K / A174K、T17C / L90Y / F92L / S101N / T109L / S110R / A125S / A174K / D203R、T17Q / L90F / F92G / S101M / A117Q / T136A / D158I / A174R、V23L / L90F / F92G / A125S / D203V / I222L、T17C / L90Y / F92L / A125S / T136V / D158E / A174K、V23L / S32Q / Y60I / A117Q / T136A / A174K / V229C、V23T / L90F / F92Y / D203V、R12K / A24V / Y60H / A174R、T17G / P20T / Y60I / F92G / D203V、L90Y / F92L / S110R / A125S / D158E / D203V、T17Q / L90Y / F92L / T109L / T136A / D158E / A174R / D203V / V229C、R12K / V23T / A24V / S32Q / F92G / D158L / A174R / D203R、S32M / N87Y / L90F / F92G / A174K / D203V、T17S / V23L / S32Q / L90F / F92L / A114K / A117N / A125S / T136V / D158E / D203V / V229I、P20T / F21W / S32M / L90Y / F92G / T109L / S110R、A14K / S32M / L90Y / S101R / A114V / T136A / A216P、P20T / F21W / L90Y / F92G / A125S / A174K / V229I、V23L / S32K / L90F / F92L / S101R / T109R / T136V / I222L、A24V / F92L / Q142L / A216P、F92G / A125S / T136A / D158E / A216P、N87R / F92G / A117N / D203V / A216P、R12K / A14K / F92G / S110R / A117N / A125S / A174R / D203R、S32Q / N87F / L90F / F92G / S101M / A114V / A117Q / D203V、T17Q / A24V / N87M / F92G / A114K / A117N / D158E / D203R / I222L、N2R / S32Q / L90F / F92G / Q142L / D203V、A14K / S32Q / L90F / F92G / A114K / A117N / A174R / D203V / A216P / I222L、R12K / P20T / D158E / D203R / I222L、N2R / A14K / T17A / L90F / F92G / S101H / A174R / D203V、T17G / A24V / L90F / F92G / Q142W、S32K / L90Y / F92Y / S101A、V23T / N87I / F92G / A125S / T136A / D158L / D203R、T17S / N87L / V229I、A24T / L90F / F92L / A125S / T136A / D203V、R12K / A14K / F92G / T109R / T136A / Q142L / D158E / A174K / D203R / I222L、A24V / S32Q / Y60A / T136A、D18R / S32Q / N87H / L90Y / F92G / S101K / Q142L / D158I / A174R、T17A / F21W / S32K / N87F / L90F / F92Y / S101Q / T136A / D203R、A24V / L90F / F92G / A114V / A117N / T136V / Q142L / D203V / V229C、T17Q / L90Y / F92G / S101N / T109L / S110R / T136V / Q142L / D203V / A216P、S32Q / L90Y / F92L / T109K / A125S / T136V、Y60H / N87K / A114K / A117Q / D158E / A174K / A216P、P20T / L90Y / T109K / T136A / D158I / D203R、T17L / Y60A / D203V、N2R / S32Q / L90Y / F92Y / T109K / D203V、T17H / A24V / S32Q / L90Y / F92L / S101H / A114K / D158I / A174R / D203V、N2R / S32M / F92Y / T109K / S110R / I222L、P20T / F92Y / S101K / A125S / D158E / D203R、R12K / A14K / P20T / F21W / S32M / F92L、R12K / A14K / V23T / A24V / L90F / F92L / S101W / T136A / D203V、T17L / S32K / F92L / T109L / A114V / T136V / D158L / R236H、P20T / F21W / S32Q / N87F / F92L / D158E / D203V、F21W / L90F / F92G / S101A / A114V / A125S / T136V / Q142L / D203V / A216P、P20T / F21W / S32K / F92G / A125S / V229I、V23T / A24V / L90Y / F92G / A125S / T136V / D203V、P20T / S32K / L90Y / F92G / S101N / T109L / S110R / A125S / A174K / D203R / A216P、V23L / L90F / F92L / T136A / D203R / I222L、R12K / A14K / A24V / N87L / L90Y / A125S / T136V / A216P、P20T / F21W / L90Y / A125S、N2R / L90Y / A114K / A117Q / D203V / V229I、F21W / S32K / L90Y / A125S / D158E / A216P / V229I、S27L / T136V、S27L / P8T / T17Q / F21W / S101A / T136V / Q142L、S27L / N2R / T17L, S27L / V23T / A24V / T136A / Q142W, S27L / F21W / T136V, S27L / T17Q / T109K / A114K / T136V / V229I, S27L / T17Q, S27L / T136V / I222L / V229 C, S27L / T17A / A114V / A117N / T136A, S27L / Parent, S27L / N2R / T136A, S27L / N2R / T17A, S27L / T17C / S101A / T136V, S27L / P20T / T136A, S27L / T136V / Q142W / V2 29I, S27L / T109K / T136V / I222L / V229I, S27L / T17L / S101H / A117N / Q142L / I222L / V229I, S27L / S101Q / T109L / A117N / T136A / Q142L, S27L / T109K / S110 R / S193N / S252T / R255M, S27L / S22R / Y26K / R236Q, S27L / L90F / F92L / S98E / S113Y / A114K / T136A / D158E / S181R / T206G / S212M / V219I, S27L / N9S / S22P / T48N / L90Y / F92G / T109K / S110R / T136A / Q189V / N211F / R236Q, S27L / T17A / Y26L / T48N / I82M / S101D / R236Q, S27L / Y26T / S101D / D158E / V219I / S252T, S27L / S13R / S98E / T136V / S181R / T206G / V229I / N231S, S27L / F21W / F92L / S 98N / S193P / I222L, S27L / S1G / Y26K / L90Y / S113Y / A114V / T136A / Q189V / N20 4R / N211L / R236Q, S27L / S22K / I82L / L90Y / F92G / R108S / A117N / D158E / S19 3N, S27L / S1G / N9S / T48S / L90Y / S98T / S101A / S113N / A114K / L119M / S193N / T 206G / S252T, S27L / N9E / R12K / S22P / V23L / T160R / D203R / I222L, S27L / N9E / R12K / S22P / V23T / T48S / S98E / R108S / T160S / Q189V / T206G / S212L / V229I,S27L / S1A / N2R / N9S / T48N / L90F / F92L / D203V / S223A, S27L / N9E / R12K / V23T / I82M / L90Y / F92L / T136V / N204K / N231S / R255M, S27L / S1G / N9S / S22V / I82F / L90Y / F92L / A117N / L119M / Q142L / T206G / S212L / S223A, S27L / N9S / Y60H / R108 C / S193P / V219L、S27L / N9E / S22K / S32M / L90F / F92G / R108T / L119M / Q189V / I222L / S223A / R236Q、S27L / T17Q / F92G / S98N / Q142L / Q189L / R236C、S27L / N9E / R12K / Q142W / T160Q / T206G / S212M、S27L / Y26T / S113N / A114V / T136A / D158E / D203R / N211F / R236Q / S252T / R255M、S27L / S1A / T17H / S22V / L90Y / F92G / S98E / A114V / T136A、S27L / N9E / T48S / I82M / L90Y / F92L / N122A / A127S / T160S / A174R / T206G / S212L / R255M、S27L / N9S / S22P / V23L / L90Y / F92G / A125S / T160S / N204R / I222L / S223A / R236Q、S27L / N9E / S22P / T48S / L90Y / F92L / T109R / E173R / A174K / D203V / S223A、S27L / R12K / Y26K / T48N / I82L / L90F / A125S / N211I / A216P、S27L / N9S / R12K / F92Y / T109K / S110R / T160K / Q189L / S223A / S252T / R255M、S27L / S1A / N2R / N9E / R12K / S32K / N87F / R108T / N211I / V219K / R255M、S27L / L90F / F92G / N122A / T136A / T160V / E173R / D203R / S252T、S27L / L90Y / F92G / S98T / T109L / D158I / S193K / V219K / R236Q / S252T、S27L / N9E / R12K / T48N / L90F / S98L / R108Q / A117Q / T136A / T206G / S212F / V219I / V229I / R255L、S27L / S1A / S22V / N87K / R108K / N122E / D158E / S193P / V219I / R255M、S27L / F21W / Y26T / S34R / R108H / L119M / N211M / R236C / S252T、S27L / S1A / N9E / R12K / V23T / T48S / N87H / S101Q / Q189L / N211M / V219I / R236Q / R255M、S27L / N9S / T25Q / L90Y / S101D / T136A / Q142L / Q189L / R236Q、S27L / A14K / I82F / F92G / S98E / R108C / A117N / L119M / Q189V / T206G / I222L / S223A、S27L / T17C / Y26T / N87V / R108C / N122E / T136V / S193P / S252T / R255M、S27L / L90Y / F92G / T109L / N122S / R255M、S27L / R12K / L90Y / T160V / A174R / R236Q、S27L / N9E / R12K / T48N / L90Y / F92G / S98T / S113R / N122R / A127S / T136A / A174K / N204K / S212L、S27L / V23T / L90Y / F92G / S98E / T109L / A125S / T160V / A174K / S181C / S193K / T206G / S212M / R255M、S27L / A24V / F92G / S101D / A114V / A117N / A125S / T136V / D203R、S27L / R12K / S32Q / S101Q、S27L / L90F / F92G / T136V / A174R / D203R、S27L / P20T / S32K / L90Y / D203V、S27L / N2R / A14K / T17S / L90Y / T109K / T136V / A216P、S27L / N2R / V23T / A24V / S32M / L90Y / F92G、S27L / A14K / L90Y / F92L / S101D / A117N / D158I / D203R、S27L / F21W / N87H / A114V / A117N / T136V、S27L / N2R / V23T / A24V / N87M / F92L / S101K / A125S / T136A、S27L / L90Y / F92L / S101N / T109R / S110R / Q142W、S27L / S32M / F92G / A216P、S27L / F21W / L90Y / F92L / A114K / A117N / T136A / D203V、S27L / N2R / V23T / S32M / N87M / F92L / T136A、S27L / R12K / L90F / F92G / S101A / D203V、S27L / A24V / L90F / F92G、S27L / R12K / F92L / S101A / A125S / T136A / D203R、S27L / R12K / V23L / L90F / F92L / A114K / T136A / D158E / D203V / I222L、S27L / L90F / F92G、S27L / A14K / P20T / S32Q / L90Y / A125S / D203V、S27L / T17L / L90F / F92G / A125S / A174R / D203V、S27L / N2R / N87K / A114K / A117Q / T136V / D203V、S27L / N2R / A114K / T136V / D203V、S27L / N2R / T17A / T136V / A216P、S27L / N2R / R12K / N87F / T136V / D158E / A174R、S27L / D203R、S27L / R12K / T17Q / T136V / D203V / A216P、S27L / N87F / T109L / Q142L、S27L / N2R / T17Q / A24V / A114K / A117Q / T136A / D158E / A174K / D203V / I222L、S27L / N2R / P20T / F21W / N87M / T109R / A117N / A125S / T136V / D203V / I222L、S27L / V23T / A24V / T136A / D158E / A174K / D203V / I222L、S27L / R12K / V23L / A114K / T136A / D158I / D203V / I222L、S27L / N87Y / T136V / D158E / D203V、S27L / T109K / S110R / D203R、S27L / N2R / T17L / A125S / T136A / D158E、S27L / A24V / D158E / A174K / A216P、S27L / T17G / V23L / N87L / A117Q / A125S / T136V / D158E / V229C、S27L / V23L / N87L / T109R / A114K / A117N / A125S / T136V / Q142L、S27L / N87Y / S101A / A114V / A117Q / T136V / A174K / A216P / I222L、S27L / R12K / T109R / Q142L、S27L / D203V、S27L / T17G / N87F / S101A / D203V、S27L / T17L / N87M / Q142L / D203V、S27L / N2R / R12K / S101D / A117N / T136A / D203V、S27L / R12K / P20T / F21W / A114K / Q142W / D203R、S27L / N2R / T17G / T109L / S110R / A114K / A117Q / T136V / Q142L / D203R、S27L / N2R、S27L / S32K / L90F / F92G / S101M / A114V / A117N / A125S / T136A / D158E / D203V、S27L / R12K / T17A / F21W / S32Q / Y60H / D203R、S27L / R12K / F92L / S110R / T136V / I222L、S27L / A14K / V23T / A24V / L90Y / F92G / A114V / A117Q / Q142L / A174R / I222L、S27L / F21W / S32Q / L90Y / S101D / T109K / A125S / T136A / A174K、S27L / V23T / L90Y / F92G / T136A / D203V / A216P、S27L / T17S / L90Y / F92G / A117N / T136A / D158L、S27L / A14K / V23L / A24V / F92L / Q142W / D203V、S27L / T17L / L90F / F92G / S101K / A174K、S27L / L90F / F92L / A125S / T136V / A174K / D203V / A216P / I222L、S27L / R12K / P20T / S32M / L90F / F92G / S101M / A114V / D203V、S27L / T17A / S32Q / L90F / F92L / T136V / Q142W / A174K / D203R、S27L / R12K / S32M / L90F / F92G / S101W / A114V / D158I / I222L、S27L / P20T / S32M / L90Y / F92L / A114V / A117N / I222L、S27L / T17C / L90Y / F92G / T136A / A174K / D203V、S27L / S32Q / N87Y / F92G / T109K / A114K / A117N / T136V / D203V / V229C、S27L / F21W / L90F / F92G / T136V / A174R、S27L / P20T / N87Y / T136A / D203V / I222L、S27L / A24V / L90Y / F92L / D158I / A174K / D203R / I222L、S27L / F21W / S32Q / L90Y / S110R / A114K / T136V / D203R / A216P / V229I、S27L / N87H / F92G / D203R、S27L / R12K / S32K / Y60A / A125S / T136V / D158L、S27L / V23T / A24V / S32Q / L90F / F92L / T136A、S27L / F92L / A114V / D158E / D203R、S27L / S32Q / L90Y / D203V / V229I、S27L / V23T / S32Q / Y60H / T109K / T136V / A216P / I222L、S27L / A14K / A24V / L90F / F92G / T109K / T136V / D158L / A174R / D203V / I222L / V229I、S27L / R12K / N87F / A174K、S27L / P20T / S32Q / Y60H / T109K / A114V / T136A / A174K、S27L / V23T / A24V / Y60A / A125S / A174R / A216P、S27L / F92Y / D158L / I222L、S27L / V23L / A24V / L90Y / F92G / D203V / I222L、S27L / V23T / A24V / S32Q / L90F / F92G / S101H / T136A / A174K / D203V / V229C、S27L / S32K / N87H / A125S、S27L / P20T / F21W / F92G / A114V、S27L / T17L / V23L / A24V / L90Y / F92L / A117N / T136V / L137M / Q142L、S27L / P20T / L90Y / D203R、S27L / S110R / Q142L / D158I / D203R / V229I、S27L / P20T / S32Q / L90F / F92L / T109K / S110R / A125S / D203V、S27L / V23T / A24V / L90F / F92G / D158E / D203R、S27L / F92G / T136A / A174R / D203V / V229C、S27L / P20T / L90Y / F92G / T109R / S110R / A125S / D203R、S27L / A14K / L90Y / F92L / D158L / A174R / D203V、S27L / F21W / S32Q / L90Y / F92G / T109L / S110R / A117N / D158I / A174K / D203V、S27L / R12K / A114K / A117N / D158E / D203V、S27L / T17S / N87H / T109R / A114K / A117N / T136V / Q142W / D203V、S27L / N2R / N87Y、S27L / T17S / V23T / A24V / S32Q / Y60H / N87K / T136A / D203R、S27L / P20T / F92Y / T109K / D158E / D203V、S27L / S32K / N87M / F92L / S101M / T109L / S110R / D203V、S27L / L90Y / F92G / T136A / D158E / D203V / A216P / V229I、S27L / A14K / S32M / F92L / A125S、S27L / L90Y / F92G / T136V / D158L / D203V、S27L / A24V / L90Y / F92G / S101A / T、 109L / S110R / A117Q / A174R / D203R、S27L / P20T / S101D / A114V / T136V / Q142W / D158E / D203R / A216P、S27L / N87Y / F92G / S101Q / A114K / A117N / T136A / D158L / D203R / A216P、S27L / V23T / A24V / N87Y / L90Y / F92L / S101D / T109R / A174K / D203R / A216P、S27L / N2R / P20T / F21W / L90F / F92Y、S27L / L90Y / F92G / D158I / D203V / V229C、S27L / S32Q / F92G / Q142W / I222L、S27L / N2R / R12K / S32M / N87Q / L90F / F92L / I222L、S27L / R12K / S32K / F92L / A114V / A117Q / T136A / D158E / A216P、S27L / L90Y / F92G、S27L / R12K / F21W / S32M / F92G / T136V / Q142L / D203R、S27L / R12K / A14K / L90Y / F92L / A125S / A174K、S27L / A24V / A114V / A117Q / T136V / D203V、S27L / R12K / T17Q / V23L / S32M / L90Y / F92G / S101D / T136V / I222L、S27L / V23T / A24V / S32Q / F92L / S101N / T109L / S110R / D203R、S27L / P20T / Y60H / N87K / T136A / A174R / D203V、S27L / P20T / F21W / L90Y / F92L / T136A / A174R / D203V / V229C、S27L / T17G / F21W / S32K / L90F / F92G / A125S / T136A / A174K、S27L / T17G / N87Y / T136A / D203V / A216P、S27L / F21W / L90F / F92G / S101D / A117N / T136A / A174K / D203V、S27L / R12K / A14K / A24V / L90Y / D203V、S27L / N2R / L90F / F92G / S101M / Q142L / D158L、S27L / N2R / T17C / A24V / N87K / A174K / A216P、S27L / N87M / Q142L / I222L、S27L / F92L / S98T / R108C / A117Q / A127S / Q142L / Q189V / A216P / R236Q、S27 L / S22K / F92L / R108H / A127S / T136A / N211I、S27L / F21W / T48S / L90Y / F92L / T109L / A127S / T136V / N204K / S223A, S27L / T17L / L90Y / F92L / S98M / A174K / D203V / N211M / S212L / S223A, S27L / N87K / S98M / N211M, S27L / N9E / S22P / L 90Y / N204K、S27L / S32M / T48S / F92G / A127S / Q142W、S27L / T17S / S22K / L90 F / F92L / D158E / D203V / V219I / S252T、S27L / L90F / F92L / A127S / D203V、S27 L / T25F / L90Y / F92G / N204R / S223A / N231S / R236C, S27L / F21W / S22V / T48S / L90F / F92L, S27L / S1A / S98T / S113K / A114V / L119M / A127S / Q142W / S193K / V 219K / S252T / R255L, S27L / A24V / I82L / L90F / F92G / T109L / L119M / A127S / E173R / N204K, S27L / T17C / Y60H / L90F / A127S / E173R / A174R / N204K, S27L / L90F / F92L / Q142W / D203V / S223A, S27L / P20Q / L90Y / Q142L / S223A, S27L / N9S / Y60A / A216P / R236Q / R255M, S27L / F92G / R108C / S110R / A117Q / T136A / N 211M, S27L / S22P / Y60H / S98A / S113K / A114K / T136V / Q189L / S193H, S27L / V23L / S98A / Q142L / N211M, S27L / V23L / Y26L / L31M / T48S / F92G / A174R / N20 4K / S252T、S27L / S13R / A14K / Y26T / T136A / S181R / N211M / S212M、S27L / F21 W / I82F / L90Y / F92G / A127S / N211M、S27L / P20T / Y60H / S98L / T136A / R255L、S27L / P20T / S34R / N87M / D158E / S252T, S27L / T25A / L90F / F92G / Q189L / S193P / V229I, S27L / V219L / I222L, S27L / N9E / T48S / L90F / F92L / S98N / R1 08H / S110R / S113Y / N211V / S212L / S252T、S27L / L90F / F92Y / S113R / V219 I / R255L、S27L / F21W / S22K / T48S / F92G / T109R / A127S、S27L / S1G / A14K / P 20T / S32K / T48S / L90Y / F92L / T109L / T160R / V219L / I222L / S252T / R255L 、S27L / L90F / F92G / D158E / T160R / S193N / N204K / S223A、S27L / A14K / Y26T / F92L / Q142L / S212L、S27L / S22R / L90Y / F92G / V126I / A127S / A174K / N204K、S27L / P20T / T25A / L90F / F92L / A117N / L119M / S252T / R255L、S27L / T17 A / A24V / T48S / L90F / F92G / T109R / A127S / T160K / D203R / S223A、S27L / F2 1W / T48S / T109L / A127S / V219I / R255L、S27L / A14K / S22P / T48S / L90F / F9 2L / T109L / A127S / Q142L / A174R / N204K / S223A, S27L / T136A / S212M / N231S, S27L / F21W / L90F / F92G / A127S / S223A, S27L / T48S / L90Y / F92L / R108S / S110R / A127S / T136V / E173R / S223A、S27L / T48S / F92G / S98E / Q142L / Q189V / S193P / N204R / S223A、S27L / T17S / L90F / F92Y / S101K / A127S / T136V / N204R / N211I / V229I / N231S, S27L / N9E / F21W / T48S / L90Y / F92L / T109R / N204K / R236Q, S27L / N9E / S22R / T48S / F92L / S101N / A127S / E173R / N204RS27L / S22K / N87F / A117Q / S181C / N204K / N211M / V229I / N231S / R236Q、S27L / N9S / T48S / L90F / F92G / S101N / A127S / N204K / A216P / S252T / R255M、S27L / I 82L / S193H / N211M / S212F / S223A, S27L / A14K / P20T / T48S / L90F / F92G / S98N / R108C / T136A / N204K / N211L / S212L / R236Q / S252T, S27L / S13R / T17L / T25Q / L90F / F92G / R108C / A127S / T160R / I222L / R255L、S27L / T48S / L90Y / F92G / T109L / S193N、S27L / S22V / T48S / L90Y / F92L / S98E / A127S / T136A / N204K / A2 16P / S223A, S27L / V23L / N87H / F92L / R108C / S110R / N122A / T160S / Q189L / S193P / N204K / S223A / R255L, S27L / F92G / A127S / A174R / S223A / V229I / N231S / R236C, S27L / S1G / L90Y / A127S / E173R / S223A, S27L / F92Y / S98N / Q142W, S27L / P20T / S32M / S34R / T48S / L90Y / F92L / R108K / A127S / Q142W / A174R / N204K / V219I / S252T、S27L / F92G / A127S / Q189V / S193N / S223A、S27L / S34R / F92G / S110R / D158L / D203V / R255M、S27L / N9E / F21W / T48S / F92G / T109L / A127S / R2 36C、S27L / S1A / S22P / T48S / L90Y / F92G / A127S / E173R / A174R / N204K / S223 A、S27L / S22K / T48S / T160S / N204K、S27L / F21W / T48S / L90Y / F92G / S98V / R10 8C / A127S / N211L / S212M / N231S / R236C、S27L / V23L / A24V / A114K / T136A / I2 22L / V229I、S27L / P20T / S101H / T109R / A114V / T136A / Q142L / I222L / V229I、S27L / V23L / S101H / T109K / A114K / A117N / T136V / Q142L / I222L / V229I、S27L / T17L / F21W / S101H / T109R / A114V / T136A / Q142L / I222L / V229I、S27L / T17S / T136V / Q142L / I222L / V229C、S27L / T17C / S22P / T48S / L90F / F92G / A127S / A 174R / P192A / S193H / N204K / S223A / V229I / N231S / R236Q / R255L、S27L / N9E / T 48N / N87H / F92L / A174K / Q189L / S193K / N204R / V219I、S27L / A14K / V23L / Y26 L / L90F / F92G / S98E / S113K / A114K / T136A / V229I / N231S、S27L / L90Y / F92L / S 101D / A117N / Q142L / S193N, S27L / S22K / T25A / L90Y / Q142L / E173R / N204K / S223A, S27L / P20T / L90Y / T109L / A127S / A174K / V229C, S27L / S22V / L90Y / F92L / A117Q / R236C / R255L、S27L / V23L / A24G / Y26T / I82F / L90Y / F92G / R108H / T136A / D158L / T160K / D203V、S27L / A14K / S22K / F92G / A127S / N204K / V229I、S2 7L / S22V / L90F / F92G / A127S / A174R / A216P / R236C, S27L / V23L / Y60A / S98T / T109L / A127S / T136V / T160Q / N204K / S223A / R255L, S27L / S13R / L90Y / S98L / T 160V / Q189L / S193P / A216P / S252T / N253S / R255L、S27L / S22R / I82M / F92L / T 109L / A127S / Q142W / Q189L / S223A、S27L / S1G / Y60A / L90F / F92L / N122E / T136 A / N204K / S223A / N231S、S27L / S32K / F92G / S98L / R108Q / S110R / R255L、S27L / L90Y / F92G / L119M / A127S / A174R / S252T、S27L / L90F / S110R / S181R / N211F、S27L / A14K / S22P / Y60H / S110R / N122S / D158I / N204K / S223A / R255M、S27L / F92L / S98V / T109L / N122A / E173R / R255L、S27L / S22R / N87L / F92G / R236C、S27L / S32M / S34R / I82L / F92G / N204K / S223A、S27L / A24V / T25Q / Y60I / A127S / A174K / N204K / V219I / S223A / S252T / R255M、S27L / S98V / T136A / N211F、S27L / N9S / L90F / F92L / A、 127S / N204R / S212M、S27L / S22R / T48S / F92G / R108H / S110R / A127S / E173R / N204K / S223A / R236Q、S27L / A14K / L90Y / F92G / A127S、S27L / A14K / T48S / F 92G / E173R / N204R / S223A / N231S、S27L / L90Y / T109R / S113N / V219L / I222 L、S27L / N9S / T48S / L90Y / T136A / T206G / S223A、S27L / S22K / T48N / L90F / F9 2G / N204K / S223A / V229C / N231S, S27L / S22P / T48S / L90F / F92G / A127S / A174R / N204R / S223A, S27L / S1G / P20T / S32K / S34R / T48S / N87F / S98A / A127S / T136A、S27L / F21W / S22R / S98E / Q142L / T160V / S223A / S252T / R255L、S27L / P20T / T48S / F92Y / R108K / S110R / A127S / V219L / S223A / V229I / R236Q、S27L / Y26T / L90Y / F92L / A127S / V219L / S252T / R255L、S27L / N9S / F21W / T48S / F 92L / A127S / D203V / S212F、S27L / F21W / Y60H / S98E / L119M / D158E / R236Q、S 27L / F21W / S22R / L90F / F92L / N122S / Q142W / R236Q, S27L / F21W / T48S / L90F / F92G / T109K, S27L / L90Y / F92L / T109R / A127S / E173R / N204K / S223A, S27L / N9S / F21W / T48S / L90Y / F92G / A127S / N204K / V219I / R255M、S27L / N9E / I82F / L90Y / F92L / S95N / T109L / A127S / A174R / S223A、S27L / L90F / F92L / S98L / N204K / A216P、S27L / A24V / T48S / A117Q / N211M / S212M / S223A、S27L / L90Y / F92G / T109L / A127S / N204R / S223A、S27L / N2R / S32Q / N87K / A125S / V219L、S27L / S22P / T48S / L90F / F92L / S193N / V219L / S223A, S27L / T25Q / L119M / N211M / R236Q, S27L / L90Y / F92G / N122S / A127S / E173R / A216P / R236Q, S27L / P20 T / S32K / L90F / F92G / N122E / D158E / T160S / Q189L / S223A / S252T、S27L / N9S / F21W / T48S / N87H / L90F / F92L / S113N / V229C、S27L / T25Q / I82L / L90F / F92G / S 98L / S113R / A114V / T136A / S181R、S27L / P20T / R108Q / S110R / D203V / N211I、 S27L / S1G / N2R / P20T / Y26T / S32M / T48S / L90Y / F92L / A127S / N204R / V219L、S 27L / A14K / P20T / T48N / L90F / F92G / A117Q / A127S、S27L / N9S / T48N / L90Y / F9 2L / S98M / T109K / A127S / N204R / N211I / S212L / V219I、S27L / F21W / S22R / T48S / L90F / F92L / R108Q / A174K / N204K / S212F / S223A、S27L / S22R / Y60H / S98T / T136V / S193K / R236Q、S27L / S22K / Y26K / N87Y / L90Y / F92G / T109R / A127S / S25 2T / R255L, S27L / L90F / F92L / T109L, S27L / F21W / F92L / N204K / S252T / R255L, S27L / V23L / L90Y / F92G / A127S / S193K / N204R / V219I / R236C / R255L, S27L / F 21W / L90Y / F92G / A114V / N122S / A127S / N204K / S223A / S252T、S27L / P20T / Y6 0A / T109L / Q189L / S212L、S27L / S22V / T48S / L90Y / F92G / N204K、S27L / S22V / S 32K / T48S / I82F / F92G / A127S / N204K / S223A, S27L / A55L, S27L / A97V, S27L / F250L, S27L / T109G, S27L / G38D, S27L / A97S, S27L / A55V / A216T, S27L / P20DS27L / A55V, S27L / T109Y, S27L / V165I, S27L / A184G, S27L / A97E, S27L / A184S, S27L / A97F, S27L / A97T, S27L / K197Y, S27L / A55I, S 27L / A97P, S27L / A55M, S27L / P20E, S27L / A117L, S27L / T109L, S27L / K197T, S27L / T136S, S27L / A97L, S27L / T109A, S27L / P20I, S2 7L / L191V, S27L / A184C, S27L / A97Q, S27L / F250V, S27L / K197V, S27L / A117S, S27L / K197R, S27L / T109K, S27L / A55C, S27L / N2S / V1 77A, S27L / Q142D, S27L / G149C, S27L / F21Y, S27L / G149A, S27L / P164E, S27L / Y26C, S27L / T17N, S27L / S57M, S27L / D249I, S27L / T1 7S, S27L / P164T, S27L / Q142L, S27L / D249N, S27L / I185R, S27L / V83L, S27L / G149S, S27L / A24D, S27L / R251V, S27L / V83I, S27L / S1 10N, S27L / T17M, S27L / F161W, S27L / G46E, S27L / D249T, S27L / T17I, S27L / S57C, S27L / S57T, S27L / S57L, S27L / Q167T, S27L / S57E S27L / T17K, S27L / G149N, S27L / Q142E, S27L / S57F, S27L / T17A, S27L / P164H, S27L / T17R, S27L / G149T, S27L / R251T, S27L / S101Y S27L / S57V, S27L / A24T, S27L / I185E, S27L / G46N, S27L / P164N, S27L / P164S, S27L / Q167V, S27L / F161V, S27L / S57I, S27L / G149D S27L / Q167I, S27L / I185Q, S27L / N2L / T17I / T136S / P164H / D249M, S27L / P20E / F21Y / I139T / P164H / K197T / R251E, S27L / A114V / A117N / T136A / I222L / V229IS27L / P20T / A24V / S101Q / T109R / A117Q / I222L / V229I、S27L / S110D / A184S / K197T、S27L / T136S / D249T、S27L / F21Y / A184S、S27L / N2F / P20E / S110D / A184S / L191V / R251V、S27L / T17G / V23T / A24V / T109L / A117Q / T136A / I222L / V229I、S27L / P20D / F21Y / A55T / F250L / R251V、S27L / T109R / A117N / I222L / V229C、S27L / F21Y / A55T / T109A / A184S、S27L / V23L / A24V / S101K / A117N / I222L / V229C、S27L / S110K / A117Y / P164R、S27L / I139T / Q142E / I169V、S27L / A24H / R251V、S27L / F21W / A114V / T136A / I222L / V229I、S27L / N2S / A55V / I185Q / R251E、S27L / N2F / S101Y / Q142L / G149T / Q167I / I169C / V229L、S27L / N2F / S101C / A117F / P164S、S27L / Q142H / I185S / R251A、S27L / F21W / T109R / A117N / T136V / I222L / V229I、S27L / G46R / T109K / T136S / D249T、S27L / N2E / G46N / A55V / I185Y / D249I、S27L / V83L / F250V / R251E、S27L / F21W、S27L / S57C / R251E、S27L / V23L / A114V / A117N / T136A / I222L / V229I、S27L / A24N、S27L / T17A / P20T / S101Q / T109K / S110R / T136A / I222L / V229C、S27L / P164T / V165I / R251E、S27L / F21Y / G46E / A117T / T136S / G149C / R251Q、S27L / G46N / I139T / Q142E / P164T / I185A / V229L / F250V、S27L / V23L / T136A / I222L / V229I、S27L / V23T / A24V / T109K / A114K / A117Q / T136A / I222L / V229C、S27L / F250L / R251Q、S27L / P20E / F21Y / S57T / P164N / A184C / V229C、S27L / P20T / V23T / A24V / I222L / V229I、S27L / V83L / A97S / A184C / D249S、S27L / P20T / S101H / A114V / A117N / I222L / V229I、S27L / N2E / R251L、S27L / P20E / F21Y / S110D / A117S、S27L / T17A / V23T / A24V / T136A、S27L / P20D / S57E / S101C、S27L / N2S / V83L / A184S、S27L / T17S / S101N / T109K / A117N / T136V / Q142L / I222L / V229C、S27L / P20D / A55T / A117Y / K197R、S27L / P20T / I222L / V229C、S27L / T17I / A24D / S57I、S27L / N2F、S27L / T109K / A117N / T136V / I222L / V229C、S27L / N2L / A24T、S27L / N2F / P20I / F21Y / R251Q、S27L / N2F / G46E、S27L / T136V / Q142W / I222L / V229I、S27L / K197R / R251E、S27L / T17S / R251T、S27L / P20I / F21Y / A117F / I185G / D249N、S27L / T17L / S101M / T109K / S110R / A117Q / I222L / V229C、S27L / T17Q / T136A / I222L / V229C、S27L / V229I、S27L / T17A / F21W / S101Q / I222L / V229C、S27L / T17S / F21W / Q142L、S27L / T17L / T109L / S110R / A117Q / I222L / V229I、S27L / F21W / T136A、S27L / P20E / F21Y / G46E / V83L / A97Q、S27L / P20T / F21W / T136A / Q142L / I222L / V229I、S27L / T17G / F21W / A117N / I222L / V229I、S27L / F21W / T109K / S110R / T136A / V229I、S27L / T17C / T109K / A117N / T136A / I222L / V229I、S27L / P20T / T109L / S110R / A117N / I222L / V229I、S27L / A24N / P164S / K197V / R251T、S27L / V23L / A117Q、S27L / A55V / V83L / A117T、S27L / P20D / G46S / A55I / K197T、S27L / A117Y、S27L / F21W / S101M / I222L / V229C、S27L / N2L / T17N / S57C / S110D / A117L / P164N / I185G / V229L / R251T、S27L / N2E / G46S / V83L / T136S / A172T / V229C、 / D249N、S27L / P20T / T109R / A114K / A117Q / T136V / I222L / V229C、S27L / V23L / A24V / T136A / Q142W / I222L / V229I、S27L / N2L、S27L / T17A / F21W / T109R / S110R / I222L / V229I、S27L / T17C / A114K / A117N / T136V / I222L / V229I、S27L / V23L / I222L / V229C、S27L / S101N / A117Q / I222L、S27L / N2E / P20I / T109L / L191V / K197L / V229C、S27L / P20T / F21W / Q142W / I222L / V229C、S27L / N2L / G46E / T109K / F161V、S27L / T17S / V229I、S27L / T17Q / T136A / I222L / V229I、S27L / N2L / A172T、S27L / P20D / F21Y / F250V、S27L / N2L / A24H / A55C / V229L、S27L / N2L / P20E / F21Y / V229L、S27L / V23T / T109L / A114V / T136A / Q142L / I222L / V229C、S27L / P20T / F21W / I222L / V229C、S27L / T17H / F21W / T136V / Q142W / I222L / V229I、S27L / G46E / A55I / Q142E、S27L / N2F / R251Q、S27L / F21W / Q142L / I222L / V229C、S27L / I222L / V229I、S27L / P20T / S101M / A114V / A117N / Q142W / I222L / V229C、S27L / T17Q / A24V / S101W / T136A、S27L / N2S / P20D / A97C / A117F / F250L / R251E、S27L / S101M / A114V / A117Q / T136A、S27L / T163I、S27L / S101Q / Q142L / I222L / V229I、S27L / F21W / I222L / V229C、S27L / S101M / T136A / I222L / V229I、S27L / F21W / A24V、S27L / A24D / K197L、S27L / T17H / S110K、S27L / P20T / F21W / V229I、S27L / T17G / P20T / F21W / A117Q / I222L / V229C、S27L / F21W / A24V / A114V / A117Q / Q142W / I222L / V229C、S27L / P20T / A117Q / I222L / V229C、S27L / T17Q / V23T / A24V / I222L / V229I、S27L / N2F / D249N / F250L、S27L / V23T / Q142L / I222L / V229C、S27L / P20E / A24D / D249S / F250V、S27L / A24D / S110K、S27L / T109R / I222L / V229I、S27L / P20E / F21Y / K197T、S27L / T17A / V23L / S101M / T109K / S110R / A114V / A117Q / T136V / V229I、S27L / P20T / F21W / Q142W / I222L / V229I、S27L / F21W / I222L / V229I、S27L / P20T / V23T / A24V / T109L / S110R / I222L / V229I、S27L / A24D / L191F / R251Q、S27L / A24D / T136S / F250V、S27L / P20T / F21W / T109L / T136A / Q142W / I222L / V229C、S27L / N2E / I169L / F250L / R251Q、S27L / N2S / A24N / G46E / A55L / Q142D / V229C / R251L、S27L / N2E / P20E / F250V / R251L、S27L / N2F / P20E、S27L / A24V / V229C、S27L / P20D / F21Y / G46N / S110K / T163I / Q167V、S27L / P20T / T109K / A114K / Q142W / I222L / V229C、S27L / T17Q / S101D / T136A / I222L / V229I、S27L / F21W / T109R / A117N / T136V / Q142W / I222L / V229C、S27L / A24D / T109L / K197L、S27L / T17Q / Q142L / I222L / V229I、S27L / T17A / A24V / S101D / S110R / T136A、S27L / P20T / F21W / T109R / A117Q / T136V / Q142L / I222L / V229I、S27L / P20T / F21W / T109K / A117N / I222L / V229C、S27L / P20T / S101M / I222L / V229I、The set of amino acid substitutions is selected from the group consisting of S27L / N2S / A24D / S101Y / P164E / V165I / I185E, S27L / T17N / A117S / V229L / R251A, S27L / S101W / A114V / T136V / V229C, S27L / P20D / G46N / A55C / S110N / T163I / A184S, S27L / A24H / G46E, S27L / T17H / S101Q / T136V / Q142L / V229C, and S27L / V23T / A24V / I222L / V229C.
[0007] In another aspect, the disclosure relates to a nucleic acid encoding the variant Bhr-PETase enzyme of any one of the preceding claims. In another aspect, the disclosure relates to an expression vector comprising the nucleic acid. In another aspect, the disclosure relates to a host cell comprising the expression vector. In some embodiments, the cell is a bacterium, yeast, or fungus.
[0008] In another aspect, the disclosure relates to a method of making a variant Bhr-PETase enzyme, comprising culturing a host cell described herein under conditions such that said variant Bhr-PETase enzyme is produced, and recovering said variant Bhr-PETase enzyme.
[0009] In another aspect, the present disclosure relates to methods for pretreating PET prior to enzymatic degradation, including mechanical, thermomechanical, and / or chemical pretreatment of PET. In some embodiments, the mechanical pretreatment comprises grinding the PET into particles. In some embodiments, the thermomechanical pretreatment comprises extruding the PET at a temperature configured to amorphize the PET and reduce its crystallinity. In some embodiments, the chemical pretreatment comprises contacting the PET with an ionic solution, strong acid, base, or solvent configured to reduce the crystallinity or alter the surface structure of the PET.
[0010] In another aspect, the present disclosure relates to a method for degrading PET, comprising contacting the PET with a variant Bhr-PETase enzyme described herein. In some embodiments, the method further comprises pretreating the PET according to the methods described herein. In some embodiments, the method degrades PET in a mixed plastic composition. In some embodiments, the plastic composition comprises a biologically or chemically derived PET analog, PET-like material, or PET substitute. In some embodiments, the plastic composition comprises at least one selected from the group consisting of polybutylene terephthalate (PBT), polycarbonate (PC), polycaprolactone (PCL), polyethylene furanoate (PEF), and high-density polyethylene (HDPE). In some embodiments, the method omits sorting plastics to select PET from the mixed plastics. [Brief explanation of the drawings]
[0011] [Figure 1] FIG. 1 shows the sequence of an exemplary wild-type Bhr-PETase (also referred to herein as G1P Bhr-PETase; SEQ ID NO: 1). [Figures 2A-2I]
[0023] Figure 1 shows a sequence alignment of an exemplary wild-type Bhr-PETase (SEQ ID NO: 1), a wild-type Lcc-PETase (SEQ ID NO: 4), a wild-type Is-PETase (SEQ ID NO: 6), and 20 exemplary homologs of Bhr-PETase. The figure also discloses SEQ ID NOs: 8-34, respectively, in the order listed. [Figure 3] FIG. 1 shows the % sequence identity of an exemplary wild-type Bhr-PETase (SEQ ID NO: 1), a wild-type Lcc-PETase (SEQ ID NO: 4), a wild-type Is-PETase (SEQ ID NO: 6), and 20 exemplary homologs of Bhr-PETase relative to wild-type Bhr-PETase. [Figure 4] 1 is a graph showing a comparison of thermal stability among three wild-type enzymes, Lcc-PETase, Bhr-PETase, and Is-PETase. [Figure 5] 5 is a graph showing a comparison of Bhr-PETase and Lcc-PETase in amorphous PET at 65° C. (FIG. 5, graph A) and 72° C. (FIG. 5, graph B). [Figure 6] 6 is a graph showing a comparison of Bhr-PETase and Lcc-PETase at 65° C. (FIG. 6, graph A) and 72° C. (FIG. 6, graph B) in >40% crystalline PET. [Figure 7A-D]
[0033] Figure 1 shows Bhr-PETase G1 variants with improved total activity and thermal stability over Bhr-PETase G1P (wild-type Bhr-PETase). The Bhr-PETase G1 variant containing the amino acid substitution S27L exhibited 1.80-fold improved total activity and 3.36-fold improved thermal stability over Bhr-PETase G1P, and was therefore selected as Bhr-PETase G2P. [Figure 8A-B] FIG. 1 shows Bhr-PETase G1 variants with improved thermostability over Bhr-PETase G1P (wild-type Bhr-PETase). [Figure 9A-AA] Figures 9J-AA show Bhr-PETase G1 variants with improved total activity over Bhr-PETase G1P (wild-type Bhr-PETase). Figures 9J-AA show Bhr-PETase G2 variants with improved total activity over Bhr-PETase G2P (Bhr-PETase G1P with the amino acid substitution S27L). [Figure 10A-B] 7A-D, 8A-B, and 9A-AA depict position-specific variants of Bhr-PETase that demonstrate beneficial properties in total activity and / or thermostability. [Figure 11A-B] FIG. 1 shows the wild-type sequences of Bhr-PETase, Lcc-PETase, and Is-PETase, as well as the starting nucleic acid sequences and codon-optimized sequences for expression in bacteria, yeast, and fungi. DETAILED DESCRIPTION OF THE INVENTION
[0012] A. Introduction The present invention is directed to enzymes that hydrolyze polyethylene terephthalate (PET). PET is a polyester polymer made by combining two monomers: modified ethylene glycol and purified terephthalic acid. Plastics such as PET have found literally thousands of uses in modern society, but PET is essentially non-degradable. As a result, plastic pollution contaminates the entire planet and poses numerous serious problems for the planet and human health. PET is recyclable; however, this has not yet prevented large amounts of PET from being dumped into landfills and / or the ocean.
[0013] Plastics, including PET, are remarkably resistant to enzymatic degradation. There are two types of PET hydrolases: (i) PET-modifying enzymes, which confine degradation to the surface of PET without visible changes under electron microscopy, and (ii) PET-degrading enzymes, or PETases, which can significantly degrade the interior blocks of PET (e.g., at least 10%), with visible changes under electron microscopy. To date, many PET-modifying enzymes have been reported, but they cannot significantly degrade the bulk of PET and are therefore not applicable to PET biorecycling. As is well known in the art, there are a few enzyme types that exhibit limited ability to degrade the interior blocks of PET. The first enzyme reported to be capable of acting on the ester bonds of PET polymers was cutinase from Thermobifida fusca in 2005. Subsequent studies have identified additional enzymes, including the PET hydrolase Is-PETase from Ideonella sakaiensis in 2016 and the leaf compost cutinase (Lcc-PETase) in 2012. Although these PETases are active at ambient temperatures, these enzymes are not particularly thermostable and do not exhibit robust PET degradation.
[0014] PET exists as both an amorphous and a semi-crystalline material. Chain mobility may increase in the amorphous phase around PET's glass transition temperature, Tg (approximately 70°C), allowing for better access to ester bonds and thus faster degradation. The reaction temperature around Tg can be controlled to achieve efficient enzymatic PET degradation. In addition, the physical degradation process of PET at approximately 70°C may convert the mobile amorphous fraction into a recalcitrant microstructure that hinders further enzymatic hydrolysis of PET. Therefore, a thermostable and thermoactive PETase would preferably be able to cause the degradation reaction around the glass transition temperature, overcoming competing physical degradation processes.
[0015] As shown in Figures 4, 5, and 6, wild-type Bhr-PETase is a close homolog of Lcc-PETase, with 94% sequence identity between the two enzymes, but was found to be more thermostable and thermoactive than Lcc-PETase and Is-PETase. Thermostability and thermoactivity are important factors for efficient PET degradation. This disclosure relates to variants of PETase derived from bacterial HR29 (Bhr-PETase) that have been engineered to exhibit even higher activity and thermostability. The remarkable thermostability and high PET hydrolysis thermoactivity of Bhr-PETase variants make them of great potential for further analytical and industrial applications.
[0016] B. Definition As used herein, "modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence, or a change in a component chemically linked to a protein. For example, the modification may be a change in the carbohydrate or PEG structure attached to the protein. As used herein, "amino acid modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. For clarity, unless otherwise specified, the amino acid modification always refers to an amino acid encoded by DNA, for example, the 20 amino acids that have codons in DNA and RNA.
[0017] As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is for an amino acid that does not naturally occur in that organism or in all organisms at the particular position. For example, the substitution S27L refers to a variant polypeptide, in this case PETase, in which serine (S) at position 27 has been replaced with leucine L. For clarity, a protein that has been engineered to change the nucleic acid encoding the sequence but not change the starting amino acid (e.g., exchanging CGG (which encodes arginine) for CGA (which still encodes arginine) to increase host organism expression levels) is not an "amino acid substitution"; i.e., if a protein has the same amino acid as the starting one at a particular position despite the creation of a new gene encoding the same protein, it is not an amino acid substitution.
[0018] As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid sequence at a specific position in a parent polypeptide sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. Additionally, -233ADE or A233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.
[0019] As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid sequence at a specific position in a parent polypeptide sequence. For example, F250- or F250#, F250() or F250del indicates the deletion of glutamic acid at position 250. Additionally, FRS250- or FRS250# indicates the deletion of the sequence PheArgSer beginning at position 250.
[0020] As used herein, "parent polypeptide" refers to a starting polypeptide that is subsequently modified to generate a variant. A parent polypeptide can be a naturally occurring polypeptide or a variant or engineered version of a naturally occurring polypeptide. A parent polypeptide can refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence encoding it. In the present case, some embodiments utilize an exemplary wild-type Bhr-PETase (also referred to as G1P Bhr-PETase; SEQ ID NO: 1; the sequence shown in FIG. 1) as the parent polypeptide.
[0021] As used herein, "variant protein" or "protein variant" or "variant" refers to a protein that differs from that of a parent protein due to at least one amino acid modification. A protein variant can refer to the protein itself, a composition comprising the protein, or the amino acid sequence encoding it. Preferably, a protein variant has at least one amino acid modification compared to the parent protein, for example, about 1 to about 70 amino acid modifications, preferably about 1 to about 5 amino acid modifications, compared to the parent. As described below, in some embodiments, the parent polypeptide is a wild-type sequence; for example, an exemplary wild-type Bhr-PETase is referred to herein as "G1P." As discussed further below, a protein variant sequence herein preferably has 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 to the parent protein sequence, preferably at least about 90% identity and preferably at least about 95-98-99% identity. A variant protein can refer to the variant protein itself, a composition comprising the protein variant, or the DNA sequence encoding it. Thus, as used herein, "variant PETase" refers to a novel PETase that has at least one amino acid modification in its amino acid sequence compared to the parent PETase enzyme. Unless otherwise specified or apparent from the context, the variant PETases of the present invention are generally compared to the wild-type G1P sequence. Additionally, unless otherwise specified, the variant PETases of the present invention are enzymatically active, i.e., have detectable PETase activity using the PETase assay described in Example 9.
[0022] As used herein, the term "protein" refers to at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides. Generally, peptide groups contain naturally occurring amino acids and peptide bonds. In addition, polypeptides may contain 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 the addition of peptide tags or labels.
[0023] As used herein, "residue" refers to the position in a protein and the identity of its associated amino acid. For example, serine 27 (also called Ser27 or S27) is the 27th residue in the parent G1P enzyme.
[0024] As used herein, a "non-naturally occurring modification" means an amino acid modification that is not found in the parent (eg, G1P) enzyme in nature.
[0025] As used herein, "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids encoded by DNA and RNA.
[0026] As used herein, "position" means a location in the sequence of a protein. Generally, the position number (discussed more fully below) is relative to the first amino acid of the mature PETase sequence, e.g., with the signal peptide removed.
[0027] As used herein, "PETase" refers to a protein that has PETase activity. As used herein, "PETase activity" means that, in the absence of MHTase, the enzyme catalyzes the hydrolysis of PET to mono(hydroxyethyl) terephthalate (MHET) as the primary product. In the case of Example 9, in the presence of MHTase, MHTase further converts MHET to terephthalic acid (TPA) and ethylene glycol (EG) as the primary products of the enzymatic reaction. An enzyme that has detectable PETase activity in the assays outlined below and in Example 9 is considered a PETase herein. PETase activity can be measured as total PETase activity and / or PETase thermostability, as described herein.
[0028] "Identity" in reference to two sequences herein means that the same amino acids are in the same positions when considering alignment. The degree of identity between an amino acid sequence of the present invention ("invention sequence") and a parent amino acid sequence referred to in the claims (e.g., G1P, SEQ ID NO: 1) is calculated by dividing the number of exact matches in the alignment of the two sequences by the length of the "invention sequence" or the length of SEQ ID NO: 1, whichever is shorter. The result is expressed as a percent identity as calculated below.
[0029] For purposes of the present invention, 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) 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 a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
[0030] Identification of corresponding amino acid residues in other PETases can be performed using, 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., 2005, Nucleic Acids Research 33:51 1-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics), using their respective default parameters. 26:1899-1900), EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680), and EMBL-EBI using Clustal Omega (Sievers and Higgins, 2014, Methods Mol Biol. 2014;1079:105-16).
[0031] If other enzymes are distant from the polypeptide of SEQ ID NO: 1, such that traditional sequence-based comparisons fail to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used. Even greater sensitivity in sequence-based searches can be achieved using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and can detect distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even greater sensitivity can be achieved if a family or superfamily for a polypeptide has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from a variety of sources (PSI-BLAST, secondary structure predictions, structural alignment profiles, and solvation potentials) as input to neural networks that predict structural folds for query sequences. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919, can be used to align sequences of unknown structure with superfamily models present in the SCOP database. These alignments can then be used to generate homology models of the polypeptide, which can be assessed for accuracy using a variety of tools developed for this purpose.
[0032] For proteins of known structure, several tools and resources are available for searching and creating structural alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms, such as distance alignment matrix (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), and implementations of these algorithms can be further utilized to search structural databases with the structure of interest to find potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).
[0033] In describing the variants of the present invention, the nomenclature set out below is adapted for ease of reference: standard accepted IUPAC one-letter or three-letter amino acid abbreviations are used.
[0034] For amino acid substitutions, the following nomenclature is used herein: original amino acid, position, substituting amino acid. Thus, a substitution of serine with leucine at position 27 is designated "Ser27Leu" or "S27L." Multiple mutations are separated by a forward slash (" / "), e.g., "A102V / T136M" represents substitutions at positions 102 and 136, respectively.
[0035] [Table 1]
[0036] As used herein, "isolated" in the context of PETase means that the polypeptide is free from other proteins. In a specific embodiment, the PETase of the present invention is isolated. As used herein, the term "isolated" refers to a polypeptide that 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 95-98% pure, as determined by SDS-PAGE. In particular, it is preferred that the polypeptide be in "essentially pure form," i.e., that the polypeptide preparation is essentially free from other polypeptide material with which it is naturally associated. This can be achieved, for example, by preparing the polypeptide by means of known recombinant methods or by classical purification methods.
[0037] As used herein, "recombinant enzyme" means that the enzyme is produced by recombinant techniques, as outlined more fully below, and the nucleic acid encoding the enzyme of the invention is operably linked to at least one exogenous (e.g., not derived from the parent PETase) sequence, including, for example, a promoter, terminator, signal sequence, etc.
[0038] The term "nucleic acid construct" refers to a nucleic acid molecule, whether single-stranded or double-stranded, that is isolated from a naturally occurring gene or modified to contain a segment of nucleic acid in a manner that is not otherwise found in nature or is synthetic, and that includes one or more regulatory sequences.
[0039] The term "operably linked" refers to a configuration in which a regulatory sequence is appropriately positioned relative to a coding sequence of a polynucleotide so that the regulatory sequence directs the expression of the coding sequence.
[0040] As used herein, the term "about" is intended to modify values and ranges thereof, such as the length, degree of error, size, amount, concentration, volume, processing temperature, processing time, yield, flow rate, pressure, etc., of a nucleotide sequence, and refers to variations in quantity that may occur, for example, through typical measuring and handling procedures used to prepare a compound, composition, concentrate, or use formulation; through unintentional errors in these procedures; through differences in the purity of the manufacturing, raw materials, or starting materials or components used to carry out the method; and other such considerations. The term "about" also encompasses, for example, amounts that differ due to deterioration of a composition, formulation, or cell culture having a specific initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation having a specific initial concentration or mixture. Regardless of whether modified by the term "about," the claims appended hereto include equivalents of these quantities. Furthermore, the term "about" can 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 is within 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference value.
[0041] C. PETase of the Invention Thus, 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.
[0042] In general, the variant PETases of the present invention have altered and improved biochemical properties compared to wild-type Bhr-PETase, "G1P" (i.e., the "generation 1 parent"), herein SEQ ID NO: 1, as shown in Figure 1. The variant PETases of the present invention may also have altered and improved biochemical properties compared to "G2P" (i.e., the "generation 2 parent"), which has the amino acid substitution S27L. Biochemical properties of variant PETases that may be improved herein include, but are not limited to, thermostability, thermoactivity, specific activity, and production.
[0043] The variant Bhr-PETase of the present invention has one or more improved properties compared to G1P or G2P. As used herein, "improvement" refers to a desired change in at least one biochemical property. "Improvement in function" can be measured as a percentage increase or decrease in a specific activity, or as a "fold" change, with an improvement in a desired property (e.g., activity or thermostability). That is, a variant Bhr-PETase may have a 10% increase in thermostability or a 10% increase in PETase activity compared to G1P or G2P. Generally, a percentage change is used to describe a change in biochemical activity of less than 100%, and a fold change is used to describe a change in biochemical activity of more than 100% (often compared to the parent enzyme, in G1P or G2P). In the present invention, percentage changes (usually increases) in biochemical activity of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, and 99% can be achieved. In the present invention, a "fold increase" (or decrease) is measured relative to the starting or parent enzyme. For example, as shown in Figures 9A-AA, the variant T17A / S27T / T48S / I82L / L90F / Q167V / P213N / S252T exhibits a 1.6-fold increase in specific activity compared to G1P, calculated by [(activity of variant) / (activity of parent)]. In many embodiments, 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 more.
[0044] Generally, improvements are measured relative to a G1P or G2P enzyme using a Bhr-PETase activity assay under conditions that test the variant Bhr-PETase against the G1P or G2P enzyme.
[0045] 1. Increased total activity The present invention provides variant Bhr-PETases that have total activity equivalent to or greater than that of G1P (wild-type Bhr-PETase of SEQ ID NO: 1) or G2P (G1P with the amino acid substitution S27L). As used herein, "total activity" can be determined by monitoring the production of TPA (terephthalic acid) during the PET depolymerization reaction at elevated temperatures, such as 65°C, and quantified using the colorimetric assay or HPLC described in Example 9. Any improvement in total activity can be due to improved thermal activity, specific activity, and / or production of the variant PETase.
[0046] In many embodiments, the variant Bhr-PETase has at least a 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 more improved total activity.
[0047] Generally, improvements are measured relative to a G1P or G2P enzyme using a Bhr-PETase activity assay under conditions that test the variant Bhr-PETase against the G1P or G2P enzyme.
[0048] (i) Increased thermal activity In one aspect, a variant Bhr-PETase may have increased thermal activity. As used herein, "thermal activity" may be determined by monitoring the production of TPA (terephthalic acid), MHET (mono(hydroxyethyl) terephthalate), and BHET (bis(2-hydroxyl) terephthalate) during a PET depolymerization reaction at elevated temperatures, such as 65°C, and is expressed as mg of TPA equivalents (mg) produced per mg of enzyme per hour. TPA当量 h -1 mg 酵素 -1) as a measure of thermal activity. As used herein, "TPA equivalent" is calculated by the sum of TPA, MHET converted to TPA, BHET converted to TPA, and any other measurable oligomers converted to TPA. Thus, a PETase that exhibits increased activity per milligram of enzyme compared to G1P (wild-type Bhr-PETase of SEQ ID NO: 1) or G2P (G1P with the amino acid substitution S27L) may exhibit improved thermal activity.
[0049] In many embodiments, the variant Bhr-PETase has at least a 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 more improved thermal activity.
[0050] Generally, improvements are measured relative to a G1P or G2P enzyme using a Bhr-PETase activity assay under conditions that test the variant Bhr-PETase against the G1P or G2P enzyme.
[0051] (ii) Increased specific activity In another embodiment, a variant Bhr-PETase may have increased specific activity. As used herein, "specific activity" may be determined by monitoring the production of TPA (terephthalic acid), MHET (mono(hydroxyethyl) terephthalate), and BHET (bis(2-hydroxyl) terephthalate) during a PET depolymerization reaction at the optimal use temperature of the Bhr-PETase, and is expressed as mg of TPA equivalents (mg) produced per mg of enzyme per hour. TPA当量 h -1 mg 酵素 -1 ) is quantified. Thus, a PETase that shows increased activity per milligram of enzyme compared to G1P (wild-type Bhr-PETase of SEQ ID NO: 1) or G2P (G1P with the amino acid substitution S27L) may exhibit improved specific activity.
[0052] In many embodiments, the variant Bhr-PETase has at least a 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 more improved specific activity. In some embodiments, the variant Bhr-PETase exhibits at least a 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 more increase in TPA equivalents produced per mg of enzyme per hour.
[0053] Generally, the improvement is measured relative to a G1P or G2P enzyme under conditions in which the variant Bhr-PETase is tested against the G1P or G2P enzyme.
[0054] (iii) Increased production In one aspect, a variant Bhr-PETase may exhibit increased production. As used herein, "production" may be determined by monitoring the protein titer of Bhr-PETase in g / L. Thus, increased amounts of PETase per liter of enzyme supernatant compared to G1P (wild-type Bhr-PETase of SEQ ID NO: 1) or G2P (G1P with the amino acid substitution S27L) may indicate improved production.
[0055] In many embodiments, the variant Bhr-PETase has at least a 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 more improved thermal activity.
[0056] Generally, improvements are measured relative to a G1P or G2P enzyme using a Bhr-PETase activity assay under conditions that test the variant Bhr-PETase against the G1P or G2P enzyme.
[0057] 2.Thermal stability Additionally, as will be appreciated by those skilled in the art, it may be desirable to perform PET degradation near the glass transition temperature. Amorphous PET domains become more mobile near the glass transition temperature (approximately 67-72°C), making them more susceptible to enzymatic hydrolysis. At temperatures higher than the transition temperature, the PET matrix recrystallizes over time, which is unfavorable for PET degradation. Therefore, for example, approximately 65-72°C is believed to be the optimal temperature range for PET degradation.
[0058] Thus, in many embodiments, the variant Bhr-PETase has improved thermostability. In this context, "thermostable" means that the variant enzyme is more stable than G1P (wild-type Bhr-PETase of SEQ ID NO: 1) or G2P (G1P with the amino acid substitution S27L) under the same thermal challenge conditions, i.e., the activity of the variant is greater than that of the G1P or G2P enzyme under the same conditions (generally using the assays outlined herein and shown in Example 9).
[0059] In one embodiment, a variant Bhr-PETase is more stable than a G1P or G2P enzyme when exposed to temperatures of about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 79, 80, 81, 82, 83, 84, and / or 85°C for a period of time, e.g., from about 0.5, 1, 2, 3, 4, 5, or 6 hours to about 5, 6, 7, 8, 9, 10 hours or more, depending on the final conditions for use of the variant Bhr-PETase. In some embodiments, a variant Bhr-PETase is more stable than a G1P or G2P enzyme when exposed to temperatures, preferably from about 65°C to 85°C for at least about 0.5 hours, 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, and preferably at least 70°C for at least about 1.5 hours.
[0060] Thus, in many embodiments, the variant Bhr-PETase 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 more more thermostable than the G1P or G2P enzymes.
[0061] Generally, the improvement is measured relative to the G1P or G2P enzyme using a Bhr-PETase activity assay under conditions that measure the variant Bhr-PETase or G1P or G2P enzyme with or without heat treatment.
[0062] 3. PETase Assay 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 assays and in Example 5 for BHET-based assays. PETase activity can also be monitored by the pNPB (p-nitrophenyl butyrate) assay. In the pNPB assay, the surrogate substrate p-nitrophenyl butyrate can be hydrolyzed by PETase to p-nitrophenol and butanoic acid. Liberation of p-nitrophenol directly correlates to PETase activity and can be determined spectrophotometrically, for example, at 405 nm.
[0063] 4.Bhr-PETase The present invention provides a number of specific variant Bhr-PETases with improved activity and / or thermostability for use in the degradation of PET.
[0064] In some embodiments, the variant Bhr-PETase is 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, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155 7, 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, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 22 64, 167, 170, 173, 174, 177, 179, 181, 182, 184, 185, 189, 190, 193, 194, 195, 198, 200, 203, 204, 206, 208, 209, 211, 212, 213, 216, 217, 218, 219, 221, 222, 223, 225, 227, 228, 229, 231 , 236, 237, 241, 242, 243, 246, 249, 250, 251, 252, 253, 254, 255, 258, 8, 31, 38, 95, 126, 137, 165, 169, 172, 191, 192, and 197. In some embodiments, the variant Bhr-PETase has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions at positions (relative to G1P, SEQ ID NO: 1) selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions at positions (relative to G1P, SEQ ID NO: 1) selected from the same group.
[0065] In some embodiments, the variant Bhr-PETase 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, S 22K, S22P, S22R, S22V, V23L, V23T, A24D, A24G, A24H, A24N, A24S, A24T, A24 V, T25A, T25F, T25H, T25Q, T25R, T25V, Y26C, Y26K, Y26L, Y26T, R30K, S32K, S 32M, S32Q, S32Y, V33G, V33Q, V33T, S34R, V40T, G46E, G46L, G46N, G46R, G46 S, T48N, T48S, L49G, G53A, I54V, A55C, A55I, A55L, A55M, A55T, A55V, M56I, M 56L, S57C, S57E, S57F, S57I, S57L, S57M, S57T, S57V, Y60A, Y60H, Y60I, A62 T, A68F, L70M, R72P, L74W, H77Q, I82F, I82L, I82M, V83I, V83L, V83T, N85D, N 87F, N87H, N87I, N87K, N87L, N87M, N87Q, N87R, N87V, N87W, N87Y, S88K, S88 T, L90F, L90K, L90Y, F92G, F92I, F92K, F92L, F92N, F92Q, F92V, F92Y, A97C, A 97E, A97F, A97G, A97L, A97P, A97Q, A97S, A97T, A97V, S98A, S98D, S98E, S98 L, S98M, S98N, S98Q, S98T, S98V, S101A, S101C, S101D, S101F, S101H, S101K, S101L, S101M, S101N, S101Q, S101R, S101V, S101W, S101Y, A102V, N105D, R10 8C, R108E, R108H, R108K, R108N, R108P, R108Q, R108S, R108T, R108V, T109A,T109F、T109G、T109K、T109L、T109N、T109R、T109Y、S110D、S110G、S110H、S110K、S110N、S110R、S113A、S113K、S113N、S113P、S113Q、S113R、S113T、S113Y、A114K、A114L、A114S、A114V、A117F、A117G、A117L、A117N、A117Q、A117S、A117T、A117Y、L119I、L119M、A121S、N122A、N122E、N122H、N122P、N122R、N122S、A125S、A127M、A127S、A127V、A135G、T136A、T136M、T136S、T136V、R138E、R138L、I139A、I139T、S140A、Q142D、Q142E、Q142H、Q142L、Q142W、I143N、I143R、T145S、K147F、K147G、K147N、K147Q、G149A、G149C、G149D、G149N、G149S、G149T、G149V、V150I、V150L、T153L、H156N、T157A、T157G、D158E、D158I、D158K、D158L、T160K、T160Q、T160R、T160S、T160V、F161V、F161W、N162E、N162H、N162P、N162R、T163I、T163S、P164E、P164H、P164N、P164R、P164S、P164T、Q167I、Q167T、Q167V、V170L、E173R、A174K、A174R、V177A、P179Q、S181A、S181C、S181R、Q182T、A184C、A184G、A184S、I185A、I185E、I185G、I185L、I185Q、I185R、I185S、I185Y、Q189I、Q189L、Q189V、N190S、S193E、S193F、S193H、S193K、S193N、S193P、S193T、S193V、T194G、T194S、T195F、V198A、V200L、D203N、D203R、D203V、N204A、N204K、N204R、N204S、T206G、T206K、T206L、T206P、T206R、F208G、F208L、F208R、F208T、A209V、N211F、N211I、N211L、N211M、N211V, S212F, S212L, S212M, P213N, P213R, A216L, A216P, A216S, A216T, A216V, I217S, S218A, V219F, V219I, V219K, V219L, V219R, T221S, I222L, S223A, S223C, M225L, L227R, W 228F, V229C, V229I, V229L, N231L, N231Q, N231S, R236C, R236E, R236H, R236K, R236Q, Q2 37R, N241P, V242T, N243P, A246D, A246K, A246S, A246T, D249I, D249M, D249N, D249S, D24 9T, F250I, F250L, F250V, F250Y, R251A, R251E, R251I, R251K, R251L, R251Q, R251T, R25 1V, S252T, N253S, N253Y, N254R, R255E, R255G, R255L, R255M, R255S, R255V, R255W, R255 and one more amino acid substitution selected from the group consisting of Y, Q258P, P8T, L31M, G38D, S95N, V126I, L137M, V165I, I169C, I169L, I169V, A172T, L191F, L191V, P192A, K197L, K197R, K197T, K197V, and K197Y. In some embodiments, 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.
[0066] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 27 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S27L. In some embodiments, the amino acid substitution is S27F. In some embodiments, the amino acid substitution is S27H. In some embodiments, the amino acid substitution is S27T. In some embodiments, the amino acid substitution is S27W.
[0067] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 1 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S1A. In some embodiments, the amino acid substitution is S1G. In some embodiments, the amino acid substitution is S1M. In some embodiments, the amino acid substitution is S1R.
[0068] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 2 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0069] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glutamine at position 5 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is Q5E.
[0070] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 9 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is N9A. In some embodiments, the amino acid substitution is N9E. In some embodiments, the amino acid substitution is N9S.
[0071] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 12 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is R12K.
[0072] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 13 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S13L. In some embodiments, the amino acid substitution is S13R.
[0073] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 14 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A14K. In some embodiments, the amino acid substitution is A14S.
[0074] In some embodiments, the variant Bhr-PETase has an amino acid substitution of leucine at position 15 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L15I.
[0075] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 16 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T16E.
[0076] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 17 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric reasons). In some embodiments, 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.
[0077] In some embodiments, the variant Bhr-PETase has an amino acid substitution of aspartic acid at position 18 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is D18R.
[0078] In some embodiments, the variant Bhr-PETase has an amino acid substitution of proline at position 20 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, 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.
[0079] In some embodiments, the variant Bhr-PETase has an amino acid substitution of phenylalanine at position 21 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is F21W. In some embodiments, the amino acid substitution is F21Y.
[0080] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 22 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is S22A. In some embodiments, 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.
[0081] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 23 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V23L. In some embodiments, the amino acid substitution is V23T.
[0082] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 24 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0083] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 25 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T25A. In some embodiments, the amino acid substitution is T25F. In some embodiments, the amino acid substitution is T25H. In some embodiments, the amino acid substitution is T25Q. In some embodiments, the amino acid substitution is T25R. In some embodiments, the amino acid substitution is T25V.
[0084] In some embodiments, the variant Bhr-PETase has an amino acid substitution of tyrosine at position 26 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric effects). In some embodiments, the amino acid substitution is Y26C. In some embodiments, the amino acid substitution is Y26K. In some embodiments, the amino acid substitution is Y26L. In some embodiments, the amino acid substitution is Y26T.
[0085] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 30 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is R30K.
[0086] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 32 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0087] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 33 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V33G. In some embodiments, the amino acid substitution is V33Q. In some embodiments, the amino acid substitution is V33T.
[0088] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 34 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S34R.
[0089] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 40 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V40T.
[0090] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glycine at position 46 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0091] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 48 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T48N. In some embodiments, the amino acid substitution is T48S.
[0092] In some embodiments, the variant Bhr-PETase has an amino acid substitution of leucine at position 49 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L49G.
[0093] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glycine at position 53 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is G53A.
[0094] In some embodiments, the variant Bhr-PETase has an amino acid substitution of isoleucine at position 54 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is I54V.
[0095] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 55 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric effects). In some embodiments, 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.
[0096] In some embodiments, the variant Bhr-PETase has an amino acid substitution of methionine at position 56 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is M56I. In some embodiments, the amino acid substitution is M56L.
[0097] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 57 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric reasons). In some embodiments, the amino acid substitution is S57C. In some embodiments, 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.
[0098] In some embodiments, the variant Bhr-PETase has an amino acid substitution of tyrosine at position 60 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is Y60A. In some embodiments, the amino acid substitution is Y60H. In some embodiments, the amino acid substitution is Y60I.
[0099] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 62 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A62T.
[0100] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 68 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A68F.
[0101] In some embodiments, the variant Bhr-PETase has an amino acid substitution of leucine at position 70 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L70M.
[0102] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 72 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is R72P.
[0103] In some embodiments, the variant Bhr-PETase has an amino acid substitution of leucine at position 74 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L74W.
[0104] In some embodiments, the variant Bhr-PETase has an amino acid substitution of histidine at position 77 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is H77Q.
[0105] In some embodiments, the variant Bhr-PETase has an amino acid substitution of isoleucine at position 82 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is I82F. In some embodiments, the amino acid substitution is I82L. In some embodiments, the amino acid substitution is I82M.
[0106] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 83 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V83I. In some embodiments, the amino acid substitution is V83L. In some embodiments, the amino acid substitution is V83T.
[0107] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 85 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is N85D.
[0108] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 87 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0109] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 88 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S88K. In some embodiments, the amino acid substitution is S88T.
[0110] In some embodiments, the variant Bhr-PETase has an amino acid substitution of leucine at position 90 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L90F. In some embodiments, the amino acid substitution is L90K. In some embodiments, the amino acid substitution is L90Y.
[0111] In some embodiments, the variant Bhr-PETase has an amino acid substitution of phenylalanine at position 92 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0112] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 97 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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. In some embodiments, the amino acid substitution is A97C. In some embodiments, the amino acid substitution is A97E. In some embodiments, the amino acid substitution is A97F. In some embodiments, the amino acid substitution is A97G. In some embodiments, the amino acid substitution is A97L. In some embodiments, 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.
[0113] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 98 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S98A. In some embodiments, the amino acid substitution is S98D. In some embodiments, the amino acid substitution is S98E. In some embodiments, 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.
[0114] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 101 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric reasons). In some embodiments, the amino acid substitution is S101A. In some embodiments, the amino acid substitution is S101C. In some embodiments, the amino acid substitution is S101D. In some embodiments, the amino acid substitution is S101F. In some embodiments, 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.
[0115] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 102 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A102V.
[0116] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 105 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is N105D.
[0117] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 108 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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. In some embodiments, the amino acid substitution is R108C. In some embodiments, 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.
[0118] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 109 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T109A. In some embodiments, the amino acid substitution is T109F. In some embodiments, the amino acid substitution is T109G. In some embodiments, 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.
[0119] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 110 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S110D. In some embodiments, the amino acid substitution is S110G. In some embodiments, the amino acid substitution is S110H. In some embodiments, the amino acid substitution is S110K. In some embodiments, the amino acid substitution is S110N. In some embodiments, the amino acid substitution is S110R.
[0120] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 113 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is S113A. In some embodiments, the amino acid substitution is S113K. In some embodiments, the amino acid substitution is S113N. In some embodiments, the amino acid substitution is S113P. In some embodiments, 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.
[0121] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 114 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A114K. In some embodiments, the amino acid substitution is A114L. In some embodiments, the amino acid substitution is A114S. In some embodiments, the amino acid substitution is A114V.
[0122] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 117 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0123] In some embodiments, the variant Bhr-PETase has an amino acid substitution of leucine at position 119 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L119I. In some embodiments, the amino acid substitution is L119M.
[0124] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 121 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A121S.
[0125] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 122 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, 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.
[0126] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 125 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A125S.
[0127] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 127 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A127M. In some embodiments, the amino acid substitution is A127S. In some embodiments, the amino acid substitution is A127V.
[0128] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 135 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A135G.
[0129] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 136 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0130] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 138 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is R138E. In some embodiments, the amino acid substitution is R138L.
[0131] In some embodiments, the variant Bhr-PETase has an amino acid substitution of isoleucine at position 139 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is I139A. In some embodiments, the amino acid substitution is I139T.
[0132] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 140 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S140A.
[0133] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glutamine at position 142 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0134] In some embodiments, the variant Bhr-PETase has an amino acid substitution of isoleucine at position 143 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is I143N. In some embodiments, the amino acid substitution is I143R.
[0135] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 145 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T145S.
[0136] In some embodiments, the variant Bhr-PETase has an amino acid substitution of lysine at position 147 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0137] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glycine at position 149 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric reasons). In some embodiments, the amino acid substitution is G149A. In some embodiments, the amino acid substitution is G149C. In some embodiments, the amino acid substitution is G149D. In some embodiments, the amino acid substitution is G149N. In some embodiments, the amino acid substitution is G149S. In some embodiments, the amino acid substitution is G149T. In some embodiments, the amino acid substitution is G149V.
[0138] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 150 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V150I. In some embodiments, the amino acid substitution is V150L.
[0139] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 153 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T153L.
[0140] In some embodiments, the variant Bhr-PETase has an amino acid substitution of histidine at position 156 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is H156N.
[0141] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 157 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T157A. In some embodiments, the amino acid substitution is T157G.
[0142] In some embodiments, the variant Bhr-PETase has an amino acid substitution of aspartic acid at position 158 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is D158E. In some embodiments, the amino acid substitution is D158I. In some embodiments, the amino acid substitution is D158K. In some embodiments, the amino acid substitution is D158L.
[0143] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 160 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0144] In some embodiments, the variant Bhr-PETase has an amino acid substitution of phenylalanine at position 161 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is F161V. In some embodiments, the amino acid substitution is F161W.
[0145] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 162 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is N162E. In some embodiments, the amino acid substitution is N162H. In some embodiments, the amino acid substitution is N162P. In some embodiments, the amino acid substitution is N162R.
[0146] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 163 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T163I. In some embodiments, the amino acid substitution is T163S.
[0147] In some embodiments, the variant Bhr-PETase has an amino acid substitution of proline at position 164 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is P164E. In some embodiments, 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.
[0148] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glutamine at position 167 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is Q167I. In some embodiments, the amino acid substitution is Q167T. In some embodiments, the amino acid substitution is Q167V.
[0149] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 170 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V170L.
[0150] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glutamic acid at position 173 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is E173R.
[0151] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 174 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A174K. In some embodiments, the amino acid substitution is A174R.
[0152] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 177 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V177A.
[0153] In some embodiments, the variant Bhr-PETase has an amino acid substitution of proline at position 179 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine; some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is P179Q.
[0154] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 181 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric effects). In some embodiments, the amino acid substitution is S181A. In some embodiments, the amino acid substitution is S181C. In some embodiments, the amino acid substitution is S181R.
[0155] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glutamine at position 182 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is Q182T.
[0156] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 184 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric effects). In some embodiments, the amino acid substitution is A184C. In some embodiments, the amino acid substitution is A184G. In some embodiments, the amino acid substitution is A184S.
[0157] In some embodiments, the variant Bhr-PETase has an amino acid substitution of isoleucine at position 185 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is I185A. In some embodiments, the amino acid substitution is I185E. In some embodiments, the amino acid substitution is I185G. In some embodiments, 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.
[0158] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glutamine at position 189 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is Q189I. In some embodiments, the amino acid substitution is Q189L. In some embodiments, the amino acid substitution is Q189V.
[0159] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 190 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is N190S.
[0160] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 193 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, 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.
[0161] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 194 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T194G. In some embodiments, the amino acid substitution is T194S.
[0162] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 195 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T195F.
[0163] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 198 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V198A.
[0164] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 200 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V200L.
[0165] In some embodiments, the variant Bhr-PETase has an amino acid substitution of aspartic acid at position 203 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is D203N. In some embodiments, the amino acid substitution is D203R. In some embodiments, the amino acid substitution is D203V.
[0166] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 204 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is N204A. In some embodiments, the amino acid substitution is N204K. In some embodiments, the amino acid substitution is N204R. In some embodiments, the amino acid substitution is N204S.
[0167] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 206 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is T206G. In some embodiments, the amino acid substitution is T206K. 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.
[0168] In some embodiments, the variant Bhr-PETase has an amino acid substitution of phenylalanine at position 208 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is F208G. In some embodiments, the amino acid substitution is F208L. In some embodiments, the amino acid substitution is F208R. In some embodiments, the amino acid substitution is F208T.
[0169] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 209 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A209V.
[0170] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 211 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0171] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 212 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S212F. In some embodiments, the amino acid substitution is S212L. In some embodiments, the amino acid substitution is S212M.
[0172] In some embodiments, the variant Bhr-PETase has an amino acid substitution of proline at position 213 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is P213N. In some embodiments, the amino acid substitution is P213R.
[0173] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 216 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, 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.
[0174] In some embodiments, the variant Bhr-PETase has an amino acid substitution of isoleucine at position 217 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is I217S.
[0175] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 218 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S218A.
[0176] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 219 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0177] In some embodiments, the variant Bhr-PETase has an amino acid substitution of threonine at position 221 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T221S.
[0178] In some embodiments, the variant Bhr-PETase has an amino acid substitution of isoleucine at position 222 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is I222L.
[0179] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 223 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric effects). In some embodiments, the amino acid substitution is S223A. In some embodiments, the amino acid substitution is S223C.
[0180] In some embodiments, the variant Bhr-PETase has an amino acid substitution of methionine at position 225 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is M225L.
[0181] In some embodiments, the variant Bhr-PETase has an amino acid substitution of leucine at position 227 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L227R.
[0182] In some embodiments, the variant Bhr-PETase has an amino acid substitution of tryptophan at position 228 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is W228F.
[0183] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 229 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric effects). In some embodiments, the amino acid substitution is V229C. In some embodiments, the amino acid substitution is V229I. In some embodiments, the amino acid substitution is V229L.
[0184] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 231 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is N231L. In some embodiments, the amino acid substitution is N231Q. In some embodiments, the amino acid substitution is N231S.
[0185] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 236 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric effects). In some embodiments, the amino acid substitution is R236C. In some embodiments, 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.
[0186] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glutamine at position 237 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is Q237R.
[0187] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 241 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine; some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is N241P.
[0188] In some embodiments, the variant Bhr-PETase has an amino acid substitution of valine at position 242 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V242T.
[0189] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 243 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is N243P.
[0190] In some embodiments, the variant Bhr-PETase has an amino acid substitution of alanine at position 246 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0191] In some embodiments, the variant Bhr-PETase has an amino acid substitution of aspartic acid at position 249 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is D249I. In some embodiments, the amino acid substitution is D249M. In some embodiments, the amino acid substitution is D249N. In some embodiments, the amino acid substitution is D249S. In some embodiments, the amino acid substitution is D249T.
[0192] In some embodiments, the variant Bhr-PETase has an amino acid substitution of phenylalanine at position 250 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is F250I. In some embodiments, the amino acid substitution is F250L. In some embodiments, the amino acid substitution is F250V. In some embodiments, the amino acid substitution is F250Y.
[0193] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 251 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0194] In some embodiments, the variant Bhr-PETase has an amino acid substitution of serine at position 252 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: threonine, glutamine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S252T.
[0195] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 253 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is N253S. In some embodiments, the amino acid substitution is N253Y.
[0196] In some embodiments, the variant Bhr-PETase has an amino acid substitution of asparagine at position 254 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is N254R.
[0197] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 255 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, 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.
[0198] In some embodiments, the variant Bhr-PETase has an amino acid substitution of glutamine at position 258 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine; some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is Q258P.
[0199] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 8 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine; some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is P8T.
[0200] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 31 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L31M.
[0201] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 38 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is G38D.
[0202] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 95 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is S95N.
[0203] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 126 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V126I.
[0204] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 137 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L137M.
[0205] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 165 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is V165I.
[0206] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 169 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize proline (due to steric effects). In some embodiments, the amino acid substitution is I169C. In some embodiments, the amino acid substitution is I169L. In some embodiments, the amino acid substitution is I169V.
[0207] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 172 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, although some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is A172T.
[0208] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 191 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is L191F. In some embodiments, the amino acid substitution is L191V.
[0209] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 192 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids: serine, threonine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine, and tyrosine, and some embodiments do not utilize cysteine (due to potential disulfide formation). In some embodiments, the amino acid substitution is P192A.
[0210] In some embodiments, the variant Bhr-PETase has an amino acid substitution of arginine at position 197 of SEQ ID NO: 1. In some embodiments, the substitution is with any of the other 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, and some embodiments do not utilize cysteine (due to potential disulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is K197L. In some embodiments, the amino acid substitution is K197R. In some embodiments, the amino acid substitution is K197T. In some embodiments, the amino acid substitution is K197V. In some embodiments, the amino acid substitution is K197Y.
[0211] In some embodiments, the variant Bhr-PETase enzyme has one or more amino acid substitutions at positions 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 SEQ ID NO: 1 as described above.
[0212] D. Nucleic Acids of the Invention The present invention additionally provides nucleic acids encoding the variant Bhr-PETase of the present invention. As will be understood by those skilled in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids can be created that all encode the variant Bhr-PETase of the present invention. Thus, once a specific amino acid sequence is identified, one skilled in the art can create a large number of different nucleic acids by simply modifying the sequence of one or more codons without changing the amino acid sequence of the protein. Thus, providing an amino acid sequence allows for the generation of a large number of different nucleic acid sequences that encode the protein.
[0213] In some embodiments, the specific variant Bhr-PETase is encoded by the particular nucleic acid sequences listed in SEQ ID NOs: 2 and 3. In some embodiments, the specific variant Bhr-PETase is 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.
[0214] As is well known in the art, nucleic acids encoding the components of the present invention can be incorporated into expression vectors depending on the host cells used to produce the heterodimeric antibodies of the present invention, as is well known in the art. Generally, the nucleic acids are operably linked to a number of control elements (promoter, origin of replication, selectable marker, ribosome binding site, inducer, etc.). Expression vectors can be extrachromosomal or integrating vectors.
[0215] The nucleic acids and / or expression vectors of the invention are then transformed into many different types of host cells known in the art, including mammalian, bacterial, yeast, insect and / or fungal, with bacteria, yeast and fungi finding use in many embodiments.
[0216] 1. Variant Preparation Nucleic acids encoding the variant Bhr-PETases of the present 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.
[0217] Synthetic gene construction involves the in vitro synthesis of polynucleotide molecules designed to encode a polypeptide of interest. Gene synthesis can be performed using a number of techniques, including the multiplexed microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054) and similar techniques in which oligonucleotides are synthesized and assembled on a photo-programmable microfluidic chip. A preferred technique is GenScript®.
[0218] 2. Control arrays The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the invention operably linked to one or more regulatory sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the regulatory sequences.
[0219] Polynucleotides can be manipulated in a variety of ways to result in expression of variants. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides using recombinant DNA methods are known in the art.
[0220] The regulatory sequence may be a promoter, a polynucleotide recognized by a host cell for expression of a polynucleotide. The promoter contains a transcriptional regulatory sequence that mediates the expression of a variant. The promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous to or heterologous to the host cell.
[0221] Promoters for bacteria, yeast, and fungi are known in the art. Exemplary operons for expression in lactic acid bacteria include the S. thermophilus lactose operon or the L. lactic lac ABCDFEGX operon, which are successfully used to induce foreign gene expression in hosts (see, e.g., Simons et al., 1993, J. Bact. 175:5186-5175; Mollet et al., 1993, J. Bact. 175:4315-4324). Additional non-limiting examples of constitutive promoters for bacteria include the lac promoter, trp promoter, tac promoter, T7 promoter, erm promoter, tip promoter, nit promoter, and Sp6 promoter. Exemplary promoters for yeast include, but are not limited to, the AOX1 promoter, ADH promoter, PH05 promoter, gal10 promoter, PKG promoter, and GAP promoter. Other useful promoters for yeast host cells are described by Romanoset et al., 1992, Yeast 8:423-488. Further examples of useful promoters for fungal vectors include, but are not limited to, the Aspergillus niger GLA promoter, the Aspergillus nidulans GPD promoter, and those derived from Aspergillus nidulans glycolytic genes, such as the adh3 promoter (McKnight et al., EMBO J. 4:2093-2099, 1985).
[0222] 3. Codon optimization While the wild-type coding sequence for Bhr-PETase is set forth in SEQ ID NO: 1, those skilled in the art will recognize that codon optimization can be performed to increase expression in any specific host organism for heterologous expression. Codon optimization can be used with any of the variant Bhr-PETase polypeptides of the present invention to optimize expression in the host cell used. Such methods are known in the art and are described, for example, in WO 2007 / 142954. In heterologous expression systems, optimization steps can improve the host's ability to produce the desired variant Bhr-PETase polypeptide. Protein expression is governed by host factors, including those that affect transcription, mRNA processing, and translation stability and initiation. Polynucleotide optimization steps can include steps to improve the host's ability to produce foreign proteins and to assist researchers in efficiently designing expression constructs. Optimization strategies can include, for example, modifying the translation initiation region, altering mRNA structural elements, and using different codon biases. The following paragraphs discuss potential challenges that can result in reduced heterologous protein expression and techniques that can overcome these challenges.
[0223] In some embodiments, the reduction of heterologous protein expression results from rare codon-induced translational pauses. Rare codon-induced translational pauses involve the presence of codons that are rarely used in host organisms in the target polynucleotide, which can have a negative impact on protein translation due to their lack in the available tRNA pool. One method for improving optimal translation in host organisms includes performing codon optimization, which can result in rare host codons being modified in synthetic polynucleotide sequences.
[0224] In some embodiments, reduced heterologous protein expression results from alternate translation initiation. Alternate translation initiation may include synthetic polynucleotide sequences that inadvertently contain motifs that can function as ribosome binding sites (RBS). These sites may result in the initiation of translation of truncated proteins from sites within the gene. One method for reducing the likelihood of producing truncated proteins that may be difficult to remove during purification includes modifying the predicted internal RBS sequence from the optimized polynucleotide sequence.
[0225] In some embodiments, the reduction of heterologous protein expression occurs through repeat-induced polymerase slippage.Repeat-induced polymerase slippage involves nucleotide sequence repeats that have been shown to cause DNA polymerase slippage or stuttering, which can lead to frameshift mutations.Such repeat sequences can also cause RNA polymerase slippage.In organisms with high G+C content bias, there may be a higher degree of repeat sequences that are composed of G or C nucleotide repeat sequences.Therefore, one way to reduce the possibility of inducing RNA polymerase slippage includes changing the length of G or C nucleotide repeat sequences.
[0226] In some embodiments, the reduction of heterologous protein expression occurs through disrupting secondary structures. Secondary structures may isolate RBS sequences or start codons, which are associated with reduced protein expression. Stem-loop structures may also be involved in transcription pausing and attenuation. The optimized polynucleotide sequence may contain minimal secondary structures in the RBS and gene coding regions of the nucleotide sequence to enable improved transcription and translation.
[0227] In some embodiments, restriction sites may affect heterologous protein expression. The polynucleotide sequence may be optimized by modifying restriction sites that may prevent subsequent subcloning of the transcription unit into a host expression vector.
[0228] Optimizing DNA sequence can have a negative or positive effect on gene expression or protein production.For example, changing less common codons to more common codons can affect the half-life of mRNA or change its structure by introducing secondary structures that interfere with the translation of message.Therefore, in some cases, it may be necessary to modify the optimized message.
[0229] All or part of the gene may be optimized. In some embodiments, the desired modulation of expression is achieved by essentially optimizing the entire gene. In other embodiments, the desired modulation is achieved by optimizing part, but not all, of the gene.
[0230] The codon usage of any coding sequence can be adjusted to achieve desired properties, such as high-level expression in a particular cell type. The starting point for such optimization can be a coding sequence using 100% common codons, or a coding sequence containing a mixture of common and uncommon codons.
[0231] Two or more candidate sequences that differ in codon usage can be generated and tested to determine whether they have desired properties.Candidate sequences can be evaluated by using computer to check for the presence of control elements such as silencers or enhancers, and to check for the presence of the region of coding sequence that can be converted into such control elements by changing codon usage.Additional criteria include the abundance of specific nucleotides, such as A, C, G or U, the codon bias for specific amino acids, or the presence or absence of specific mRNA secondary or tertiary structure.Candidate sequence adjustments can be made based on a number of such criteria.
[0232] Promising candidate sequences are constructed and then experimentally evaluated. Multiple candidates may be evaluated independently of each other, or the process may be iterative, either using the most promising candidate as a new starting point or by combining regions of two or more candidates to produce novel hybrids. Further rounds of modification and evaluation may also be included.
[0233] Modifying the codon usage of candidate sequences can result in the creation or destruction of either positive or negative elements. Generally, positive elements refer to any element whose modification or removal from candidate sequences can result in a decrease in the expression of therapeutic proteins, or whose creation can result in an increase in the expression of therapeutic proteins. For example, positive elements include enhancers, promoters, downstream promoter elements, DNA binding sites for positive regulatory factors (e.g., transcriptional activators), or sequences that contribute to or modify mRNA secondary or tertiary structure. Negative elements refer to any element whose modification or removal from candidate sequences can result in an increase in the expression of therapeutic proteins, or whose creation can result in a decrease in the expression of therapeutic proteins. Negative elements include silencers, DNA binding sites for negative regulatory factors (e.g., transcriptional repressors), transcriptional pause sites, or sequences that contribute to or modify mRNA secondary or tertiary structure. Generally, negative elements occur more frequently than positive elements. Therefore, any change in codon usage that results in an increase in protein expression is more likely to occur by destroying a negative element rather than creating a positive element. In addition, altering a candidate sequence is more likely to destroy a positive element rather than creating a positive element. In some embodiments, candidate sequences are selected and modified to increase the production of a therapeutic protein. Candidate sequences can be modified, for example, by sequentially altering codons or by randomly altering codons in the candidate sequence. The modified candidate sequences are then evaluated by measuring the level of expression of the resulting therapeutic protein or by evaluating another parameter, for example, a parameter that correlates with the level of expression. Candidate sequences that result in an increase in the level of a therapeutic protein compared to the unaltered candidate sequence are selected.
[0234] In some embodiments, one or a group of codons can be modified and tested without referring to, for example, protein or message structure.Alternatively, one or more codons can be selected based on message-level characteristics, for example, location in a predetermined region, for example, location in a region with high or low GC content, structure such as enhancer or silencer, location in a region that can be modified to introduce structure such as enhancer or silencer, location in a region that has or is predicted to have secondary or tertiary structure, for example, intrachain pairing, interchain pairing, location in a region that lacks or is predicted to lack secondary or tertiary structure, for example, intrachain pairing or interchain pairing.A specific modified region is selected if it produces desired results.
[0235] A method for systematically generating candidate sequences is useful. For example, one or a group of codons, for example, a contiguous block of codons, at various positions of a synthetic nucleic acid sequence can be modified with common codons (or with uncommon codons, for example, if the starting sequence is optimized), and the resulting sequence can be evaluated. Candidates can be generated by optimizing (or deoptimizing) a given "window" of codons in a sequence to generate a first candidate, then moving the window to a new position in the sequence and optimizing (or deoptimizing) the codons at the new position under the window to generate a second candidate. Candidates can be evaluated by determining the level of expression they bring about, or by evaluating another parameter, for example, a parameter that correlates with the level of expression. Some parameters can be evaluated by research or computation, for example, high or low GC content; sequence elements such as enhancers or silencers; secondary or tertiary structure, for example, the presence or absence of intrastrand or interstrand pairing.
[0236] In some embodiments, an optimized nucleic acid sequence is capable of expressing a variant Bhr-PETase polypeptide of the present invention at a level that is at least about 110%, 150%, 200%, 500%, 1,000%, 5,000% or even 10,000% of the level expressed by a non-optimized nucleic acid sequence.
[0237] Starting from the amino acid sequence of the variant Bhr-PETase, candidate DNA sequences can be designed. When designing a synthetic DNA sequence, the frequency of codon usage can be compared with the codon usage of the host expression organism, and rare host codons can be modified in the synthetic sequence. Additionally, the synthetic candidate DNA sequence can be modified to remove undesirable enzyme restriction sites and add or modify any desired signal sequences, linkers, or untranslated regions. The synthetic DNA sequence can be analyzed for the presence of secondary structures that may interfere with the translation process, such as G / C repeats and stem-loop structures. Before the candidate DNA sequence is synthesized, the optimized sequence design can be examined to verify that the sequence accurately encodes the desired amino acid sequence. Finally, the candidate DNA sequence can be synthesized using DNA synthesis techniques, such as those well known in the art.
[0238] In some embodiments, the common codon usage in a host organism, such as any of those described herein, can be utilized to optimize the expression of heterologous polynucleotide sequences in the host organism.The percentage and distribution of codons that are rarely considered preferred for a particular amino acid in the host expression system can be evaluated.The values of 5% and 10% usage can be used as cutoff values for determining rare codons.
[0239] 4. Host Cells and Production Strains In one aspect, the present disclosure relates to an expression vector comprising a nucleic acid encoding a variant Bhr-PETase described herein. In another aspect, the present disclosure also relates to a host cell comprising the expression vector. In some embodiments, the host cell is a bacterium. In some embodiments, the host cell is a yeast. In some embodiments, the host cell is a fungus. In some embodiments, the host cell can be a bacterium, including but not limited to, E. coli or a Bacillus species. In some embodiments, the host cell can be a yeast, including but not limited to, Saccharomyces cerevisiae or a Pichia species. In some embodiments, the host cell can be a fungus, including but not limited to, A. niger, T. reseei, or Myceliophthora thermophila.
[0240] The expression vector can be any integrating vector that integrates into the genome or is an autonomously replicating plasmid in the selected host. In one embodiment, the vector can be stably maintained in the cell into which it is introduced, with the variant Bhr-PETase gene supported therein in a state suitable for expression. The expression vector can be selected to be suitable for the specific host cell into which it is introduced. Specific examples available for use include, but are not limited to, pBR322, pACYC184, pUC18, pKK223-2, pHSG398 (Takara Bio Inc.), pTrcHis (Invitrogen Corporation), and pET11a (Stratagene Corporation) when Escherichia coli is used as a host; pBBR122 (Mobiotech) and pBHR1 (Mobiotech) for other Gram-negative bacteria; pHW1520 (Mobiotech) and pHY300PLK (Takara Bio Inc.) for Bacillus spp.; and pSH19 (Herai et al., Proc. Natl. Acad. Sci., 101, 14031-14035, 2004), pIJ702 (John Innes Centre), pIJ943 (John Innes Centre), pIJ8600 (John Innes Centre) for actinomycetes. Centre), pIJ602 (John Innes Centre), 1, pTip-vectors (Nakashima et al., Appli. Environ. Microbiol., 70, 5557-5568, 2004), pTYM19 (Onaka et al., J. Antibiot., 56, 950-956, 2003); pPICZα and pPIC9 (Thermo Fisher Scientific) for Pichia genus, and pAO815 (Invitrogen Corporation), pAUR101 (Takara Bio Inc.), pAUR123 (Takara Bio Inc.), and pAUR316 (Takara Bio Inc.) for fungi.
[0241] In another aspect, the present disclosure also relates to a method of expressing a variant Bhr-PETase in a host cell, and a method of making said variant Bhr-PETase, comprising culturing a host cell under conditions such that said variant Bhr-PETase is produced, and recovering said variant Bhr-PETase.
[0242] The transformed organism can be cultured in a medium that can be the nutrient medium for the transformed host cell without affecting the transformation of the variant Bhr-PETase. Such a medium contains appropriate carbon sources, nitrogen sources, inorganic salts, natural organic nutrients, etc. As carbon sources, glucose, fructose, glycerin, sorbitol, and organic acids can be used individually or in combination. The concentration of the carbon source is not particularly limited and can be 1 to 10%. As nitrogen sources, ammonium, urea, ammonium sulfate, ammonium nitrate, ammonium acetate, etc. can be used individually or in combination of two or more of these. As inorganic salts, salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, manganese sulfate, and iron sulfate can be used. In addition, peptone, meat extract, yeast extract, corn steep liquor, and casamino acids can be used as organic nutrient sources that promote the growth of the used bacteria. Furthermore, small amounts of vitamins and nucleic acids may be contained in the medium.
[0243] 5. PETase Preparation and Use As will be appreciated by those skilled in the art, the formulation of the variant Bhr-PETase of the present invention will depend on its end use and associated conditions. Suitable formulations of the variant Bhr-PETase of the present invention include liquid, dried (including spray-dried), powder, granular, and pelleted formulations. Bhr-PETase may also be formulated "embedded in PET particles" for natural degradation.
[0244] In some embodiments, the enzyme compositions (i.e., polypeptide compositions) of the present invention may 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 enzyme.
[0245] In some embodiments, the enzyme composition may be a dry powder or granule, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme compositions may be stabilized by adding a stabilizer, such as, for example, a sugar, sugar alcohol, or another polyol, and / or lactic acid or another organic acid according to established processes.
[0246] In some embodiments, the dosage of the polypeptide compositions of the present invention and other conditions under which the compositions are used can be determined based on methods well known in the art.
[0247] The compositions are suitable for use in PET biodegradation, PET biocycling, PET upcycling and / or PET surface modification processes.
[0248] In some embodiments, the composition is used to degrade pretreated PET. PET pretreatment can be performed before the enzymatic degradation step. Commonly used PET pretreatments can be broadly classified into a) mechanical pretreatment, b) thermomechanical pretreatment, and c) chemical pretreatment. The mechanical process can involve sieving and then grinding the PET film into particles less than about 500 μm. This process can be combined with immersion of the PET film in liquid nitrogen to primarily facilitate the grinding process. The advantage of this type of mechanical pretreatment can be enzyme accessibility due to particle size reduction. In thermomechanical degradation, backbone scission reactions can affect the stability of cyclic oligomers. In thermomechanical pretreatment, highly crystalline PET flakes can be amorphized at very high temperatures (>260°C) using an extruder equipped with a melt pump, and then micronized to particle sizes less than 500 μm. An example of a commercial method for pretreating PET is to use an extrusion casting process, i.e., by molding molten polymer onto a chilled roll of PET to solidify it. Thermomechanical pretreatment can reduce the crystallinity and particle size of industrial-grade PET, allowing access for enzymes to depolymerize it. In the case of chemical pretreatment, ionic solutions, strong acids, bases, solvents, etc. can be used to reduce the crystallinity of PET or to alter the surface structure to facilitate access for enzymes that further depolymerize it to monomers.
[0249] Regardless of whether chemical or biological methods are used, two significant obstacles to recycling plastics are material variability and the costs associated with identifying and separating waste plastics into acceptable grade ranges. Different types and grades of plastics vary in density and molecular weight. Knowing the properties of a particular plastic during recycling can be advantageous, as it facilitates determining its value and durability when placed in valuable secondary-use applications. However, the sorting process poses a significant obstacle in terms of resource utilization, time management, and cost-effectiveness. Therefore, when focusing on biological mitigation to overcome the obstacles associated with differential plastic sorting, depolymerization of mixed plastics using robust enzymes with broad substrate specificity becomes a high priority. Mixed plastics is a term that encompasses all non-bottle plastic packaging sourced from the domestic waste stream, including rigid and flexible plastic products of various polymer types and colors typically found in household trash cans. Mixed plastics refers to a mixture of various plastics. The various polymer types may refer to PET and / or biologically or chemically derived PET analogs, PET-like materials, or PET substitutes. Examples of PET analogs, PET-like materials, or PET substitutes include, but are not limited to, polybutylene terephthalate (PBT), polycarbonate (PC), polycaprolactone (PCL), polyethylene furanoate (PEF), and high-density polyethylene (HDPE). Bhr-PETase, alone or in combination with other accessory enzymes, can revolutionize the biological depolymerization of mixed plastics. In contrast to chemical methodologies, the enzymatic depolymerization process of mixed plastics can be environmentally safe and may be able to preserve market value for secondary use applications. Such biological treatments may also be combined with mild chemical or thermomechanical pretreatments to achieve even higher depolymerization efficiencies.
[0250] After PET pretreatment and enzymatic degradation, the products can be recycled to produce other valuable chemicals. In one embodiment, TPA (terephthalic acid) is purified using an industrially suitable process that can also produce sodium sulfate, a chemical commonly used in the surfactant, paper, and glass industries. In another embodiment, recycled TPA is used as a starting material to synthesize virgin PET. PET synthesized from recycled TPA exhibits similar properties, such as average molecular weight and intrinsic viscosity, to PET synthesized using petrochemical TPA. In one embodiment, bottles made from recycled PET exhibit similar mechanical properties to regular PET bottles and even better brightness.
[0251] In some embodiments, the present invention provides methods for preparing enzyme cocktails comprising a variant Bhr-PETase described herein, along with other PET-degrading accessory enzymes and a downstream MHETase, to produce a PET-degrading enzyme cocktail for efficient turnover of pre-treated PET. [Example]
[0252] PET hydrolase gene selection, synthesis, and cloning Several novel PET hydrolases were selected based on bioinformatic analysis and synthesized using GeneWiz (https: / / www.genewiz.com / en / ). The synthetic genes were cloned into the pET28b(+) vector (Millipore Inc., catalog number 69865). Three hydrolases (Lcc-PETase, Bhr-PETase, Is-PETase) were selected for further purification and characterization. [Example]
[0253] Preparation of PET hydrolase produced by Escherichia coli in 250 ml shake flasks. A single colony of BL21(DE3) chemically competent E. coli (ThermoFisher Scientific, USA: Catalog No. C600003) containing the recombinant hydrolase-encoding gene was inoculated into an individual culture tube containing 5 mL of lysogeny broth (LB broth) containing 1% glucose and 50 μg / mL kanamycin. The culture was grown overnight at 30°C, 200 rpm, and 85% humidity. 1 mL of the overnight culture was transferred to a shake flask containing 50 mL of Terrific Broth (TB broth) containing 50 μg / mL kanamycin. The flask was then incubated for 2–2.5 hours at 37°C, 250 rpm, and 85% humidity. The pre-induction OD was measured at 2–2.5 hours, and when the OD600 reached approximately 0.6–0.8, induction with IPTG (isopropyl β-D-1-thiogalactopyranoside) was performed to obtain a final concentration of 0.5 mM IPTG in the flask. All flasks were then incubated for 18-24 hours at 30°C, 250 rpm, and 85% humidity, except for Is-PETase, which was grown at 16°C. The supernatant and lysate were transferred to 50 mL centrifuge tubes and stored at -20°C prior to activity assays. A total of three candidates (Lcc-PETase, Bhr-PETase, and Is-PETase) were grown according to the procedures described herein. [Example]
[0254] Ni column purification of His-tagged PET hydrolase produced in shake flasks Each sample was concentrated to 15-20 ml before use on a Thermo Scientific HisPur™ Ni-NTA Spin Column (Cat. No. 88226). Three PET hydrolases (Lcc-PETase, Bhr-PETase, and Is-PETase) were purified from cell cultures. The procedure followed the manufacturer's guidelines. Multiple elutions were performed in 250 mM imidazole, and based on the protein band intensity from the eluates in various fractions (measured using gel quantitation), the combined fraction pool was used for the desalting step. [Example]
[0255] Desalting of purified PET hydrolase produced in shake flasks Imidazole and NaCl were removed from the Ni-purified enzyme using disposable Thermo Scientific Zeba™ Spin Desalting Columns, 7K MWCO (Cat. No. 89892). The desalting protocol followed the manufacturer's guidelines. The desalted enzyme was then quantified to determine the protein concentration (g / L). [Example]
[0256] Thermostability study of Escherichia coli produced PET hydrolase using BHET (bis(2-hydroxyethyl) terephthalate) as a substrate Each candidate normalized PET hydrolase protein was subjected to a 1.5-hour treatment at either 30°C, 50°C, or 70°C. The enzyme was kept on ice for the same period and used as the untreated condition. After treatment, 250 μL of 10 mM BHET (bis(2-hydroxyethyl) terephthalate), 62.5 μL of untreated and treated enzymes, and 170 μL of 0.1 M sodium phosphate buffer, pH 8, were added to a Costar deep-well plate. The plate was incubated at 50°C for 2 hours. After 2 hours, the samples were centrifuged at 4,000 rpm for 2 minutes. To determine the amount of TPA produced in our reactions, the supernatant was analyzed using high-performance liquid chromatography (HPLC). A Zorbax Eclipse Plus C18 (Rapid Resolution HD 2.1 x 50 mm 1.8 micron) column, part number 959757-902, was used with a guard column, part number 82175-901. A flow rate of 0.6 mL / min and a column temperature of 35°C were set for the HPLC run. A gradient method was used to detect terephthalic acid (TPA), with mobile phase 1 consisting of water with 0.1% trifluoroacetic acid and mobile phase 2 consisting of acetonitrile with 0.1% trifluoroacetic acid. The % remaining of each PET hydrolase was calculated using the following formula: (MHET + TPA produced under treated conditions) / (MHET + TPA produced under untreated conditions) * 100.
[0257] The thermal stability results for the three candidates (Lcc-PETase, Bhr-PETase, and Is-PETase) are shown in Figure 4. Under the experimental conditions, Is-PETase was the most unstable candidate, losing 90% of its activity at 50°C. Bhr-PETase and Lcc-PETase retained ≥80% residual activity at all temperatures tested. [Example]
[0258] A large-scale PET film assay to evaluate PET hydrolases produced by Escherichia coli These experiments were carried out in 250 ml glass bottles. Two types of PET substrates were used in these experiments: A) Amorphous PET film was obtained from Goodfellow (catalog no. ES301445) at a thickness of 0.25 mm. The PET film was cut into 1 x 30 cm pieces using a paper cutter and then recut into approximately 1 x 0.25 cm pieces. The PET film pieces were ground to a fine powder using a mechanical grinder. B) Highly crystalline PET powder with a crystallinity of >40% was obtained from Goodfellow (catalog no. ES306031 / 1). 2-10 g / L PET powder was loaded into 0.1 M sodium phosphate buffer, pH 8.0, to a final volume of 50 ml. Various loadings of three candidate normalized proteins were used for evaluation at either 65 °C or 72 °C with 200 rpm shaking. Reactions were continued for periods ranging from 6 to 144 h. At various time points, bottles were sampled, and the reactions were analyzed using high-performance liquid chromatography (HPLC) to determine the amount of TPA produced in our reaction. A Zorbax Eclipse Plus C18 (Rapid Resolution HD 2.1 x 50 mm 1.8 micron) column, part number 959757-902, was used with a guard column, part number 82175-901. A flow rate of 0.6 mL / min and a column temperature of 35°C were set for the HPLC run. A gradient method was used to detect terephthalic acid (TPA), with mobile phase 1 consisting of water with 0.1% trifluoroacetic acid and mobile phase 2 consisting of acetonitrile with 0.1% trifluoroacetic acid.
[0259] The results for the PET assay are summarized in Figures 5 and 6. Bhr-PETase outperforms Lcc-PETase in both amorphous and >40% crystalline PET. The results further suggest that Lcc-PETase is less thermostable than Bhr-PETase, regardless of the substrate type. Compared to Bhr-PETase, which can depolymerize >90% amorphous PET at both 65°C and 72°C with an enzyme dosage of 2.4 mg / g PET (Figure 5), the depolymerization activity of Lcc-PETase at higher dosages is significantly lower (data not shown). Figure 6 further suggests that Bhr-PETase may not be able to completely degrade highly crystalline PET and may require additional pretreatment or accessory enzymes to do so.
[0260] Overall, Bhr-PETase was identified as the best PET hydrolase in all evaluated, and therefore was selected as a target for further improvement. [Example]
[0261] Design and construction of the Bhr-PETase collection To further improve the activity and thermostability of Bhr-PETase, a collection of G1 variants was designed based on sequence and structural analysis. The designs included one to multiple specific mutations per variant. The G1 variant collection was then constructed in Bhr-PETase G1P (wild-type Bhr-PETase, generation 1 parent) using standard site-directed mutagenesis techniques and then cloned into the pET28b(+) vector (Millipore Inc., catalog number 69865) for production in Escherichia coli.
[0262] To further improve the activity and thermostability of Bhr-PETase, a collection of G2 variants was designed based on sequence and structural analysis. The designs included one to multiple specific mutations per variant. The G2 variant collection was constructed in Bhr-PETase G2P (the generation 2 parent, i.e., G1P with the amino acid substitution S27L) using standard site-directed mutagenesis techniques and then cloned into an appropriate vector for production in the desired host. [Example]
[0263] HTP growth: Preparation of Bhr-PETase G1P, G1 variant, G2P, and G2 variant in microtiter plates A single colony of BL21(DE3) chemically competent E. coli (ThermoFisher Scientific, USA: Catalog No. C600003) containing the recombinant Bhr-PETase-encoding gene was inoculated into individual wells of a 96-well plate containing 180 μl of lysogeny broth (LB broth) containing 1% glucose and 50 μg / mL kanamycin. The culture was grown overnight at 30°C, 200 rpm, and 85% humidity. 20 μL of the overnight culture was transferred from each well to a 96-well plate containing 380 μL of Terrific Broth (TB broth) containing 50 μg / mL kanamycin. The plate was then incubated for 2–2.5 hours at 37°C, 250 rpm, and 85% humidity. The pre-induction OD was measured at 2–2.5 h, and when the OD reached approximately 0.6–0.8, induction with IPTG (isopropyl β-D-1-thiogalactopyranoside) was performed to achieve a final concentration of 0.5 mM IPTG in the wells. The plates were then incubated for 18–24 h at 30°C, 250 rpm, and 85% humidity. Supernatants were transferred to round-bottom plates and stored at -20°C prior to activity assays.
[0264] Recombinant Bhr-PETase-encoding gene-containing hosts from single colonies were inoculated into individual wells of 24- or 96-well plates containing the appropriate medium. Cultures were then grown overnight at 30°C, 200 rpm, and 85% humidity. The overnight cultures were subcultured into 24- or 96-well plates containing the appropriate medium. Plates were then incubated at 30°C, 200 rpm, and 85% humidity. Cultures were induced every 24 hours with the respective inducer for up to 120 hours. Supernatants were transferred to round-bottom plates at the desired harvest time and stored at -20°C prior to activity assays. [Example]
[0265] HTP PET film assay to assess Bhr-PETase G1P, G1 variant, G2P and G2 variant activity / thermal stability For total activity testing, amorphous PET film was obtained from Goodfellow (catalog number ES301445) at a thickness of 0.25 mm. The PET film was cut into 1 x 30 cm pieces using a paper cutter and then recut into approximately 1 x 0.25 cm pieces. The PET film pieces were ground to a fine powder using a mechanical grinder. The PET powder was loaded into a 96-well plate using a resin loader. Approximately 8-9 mg of PET powder was dispensed into each of 96 COSTER deep wells. 1.4 mL of 0.1 M sodium phosphate buffer, pH 8.0, was added to the COSTER deep wells containing the PET powder. 100 μL of enzyme was dispensed into the COSTER deep wells, the plate was sealed, and then incubated at 65°C for 72 hours. After 72 hours, the plate was centrifuged at 4,000 rpm for 2 minutes. 180 μL of the above reaction was transferred to a Costar round-bottom plate and 20 μL of in-house prepared Is-MHETase was added. The plate was incubated at 50°C for 30 minutes. After 30 minutes, the plate was centrifuged at 4,000 rpm for 2 minutes. The reaction mixture was diluted with sodium phosphate buffer, pH 7.2, for linear assay detection in Costar Deep by adding 50 μL of 10 mM EDTA and 50 μL of 10 mM FeSO4. The reaction was incubated in the dark for 10 minutes, and the plate was centrifuged at 4,000 rpm for 2 minutes before transferring 200 μL of the reaction to a black clear-bottom fluorometric quantitation plate. An endpoint read was taken after 10 minutes for TPA (terephthalic acid) activity at excitation 328 nm and emission 421 nm.
[0266] For the thermostability test, 150 μL of enzyme was transferred to a PCR plate. The plate was incubated at 86°C for 3 hours. After 3 hours, 100 μL of enzyme was transferred to a Costar deep-well plate containing 1.4 mL of 0.1 M sodium phosphate buffer, pH 8.0, and 8-9 mg / well of crushed PET powder. The plate was then placed at 65°C for 72 hours. After 72 hours, the plate was centrifuged at 4,000 rpm for 2 minutes. 180 μL of the above reaction mixture was transferred to a Costar round-bottom plate, and 20 μL of Is-MHETase was added. The plate was incubated at 50°C for 30 minutes. After 30 minutes, the plate was centrifuged at 4,000 rpm for 2 minutes. To determine the amount of TPA produced in our reactions, the reactions were analyzed using high-performance liquid chromatography (HPLC). A Zorbax Eclipse Plus C18 (Rapid Resolution HD 2.1 x 50 mm 1.8 micron) column, part number 959757-902, was used with a guard column, part number 82175-901. A flow rate of 0.6 mL / min and a column temperature of 35°C were set for the HPLC run. A gradient method was used to detect terephthalic acid (TPA), with mobile phase 1 consisting of water with 0.1% trifluoroacetic acid and mobile phase 2 consisting of acetonitrile with 0.1% trifluoroacetic acid.
[0267] The results are summarized in Figures 7, 8, and 9. All G1 variants showed improved total activity and / or thermostability compared to Bhr-PETase G1P (wild-type Bhr-PETase). The Bhr-PETase G1 variant with the amino acid substitution S27L showed a 1.80-fold improvement in total activity and a 3.36-fold improvement in thermostability compared to Bhr-PETase G1P, and was therefore selected as Bhr-PETase G2P. All G2 variants showed further improvement in total activity compared to Bhr-PETase G2P.
Claims
1. A composition comprising a variant Bhr-PETase comprising an amino acid sequence in which at least one amino acid substitution has been introduced into the amino acid sequence of wild-type Bhr-PETase (SEQ ID NO: 1), the at least one amino acid substitution is S27L; The amino acid sequence of the variant Bhr-PETase has at least 90% identity to the amino acid sequence of SEQ ID NO: 1, and The variant Bhr-PETase has PETase activity and exhibits improved total activity and / or thermostability compared to wild-type Bhr-PETase. composition.
2. The amino acid substitution is S27L / T136V, S27L / P8T / T17Q / F21W / S101A / T136V / Q142L, S27L / N2R / T17L, S27L / V23T / A24V / T136A / Q142W, S27L / F21W / T136V, S27L / T17Q / T109K / A114K / T136V / V229I, S27L / T17Q, S27L / T136V / I222L / V229C, S27L / T17A / A114V / A117N / T136A, S27L / N2R / T136A, S27L / N2R / T17A, S27L / T17C / S101A / T136V, S27L / P20T / T136A, S27L / T136V / Q142W / V229I, S27L / T109K / T136V / I222L / V229I, S27L / T17L / S101H / A117N / Q142L / I222L / V229I, S27L / S101Q / T109L / A117N / T136A / Q142L, S27L / T109K / S110R / S193N / S252T / R255M, S27L / S22R / Y26K / R236Q, S27L / L90F / F92L / S98E / S113Y / A114K / T136A / D158E / S181R / T206G / S212M / V219I, S27L / N9S / S22P / T48N / L90Y / F92G / T109K / S110R / T136A / Q189V / N211F / R236Q, S27L / T17A / Y26L / T48N / I82M / S101D / R236Q, S27L / Y26T / S101D / D158E / V219I / S252T, S27L / S13R / S98E / T136V / S181R / T206G / V229I / N231S, S27L / F21W / F92L / S98N / S193P / I222L, S27L / S1G / Y26K / L90Y / S113Y / A114V / T136A / Q189V / N204R / N211L / R236Q, S27L / S22K / I82L / L90Y / F92G / R108S / A117N / D158E / S193N, S27L / S1G / N9S / T48S / L90Y / S98T / S101A / S113N / A114K / L119M / S193N / T206G / S252T, S27L / N9E / R12K / S22P / V23L / T160R / D203R / I222L, S27L / N9E / R12K / S22P / V23T / T48S / S98E / R108S / T160S / Q189V / T206G / S212L / V229I, S27L / S1A / N2R / N9S / T48N / L90F / F92L / D203V / S223A, S27L / N9E / R12K / V23T / I82M / L90Y / F92L / T136V / N204K / N231S / R255M, S27L / S1G / N9S / S22V / I82F / L90Y / F92L / A117N / L119M / Q142L / T206G / S212L / S223A, S27L / N9S / Y60H / R108C / S193P / V219L, S27L / N9E / S22K / S32M / L90F / F92G / R108T / L119M / Q189V / I222L / S223A / R236Q, S27L / T17Q / F92G / S98N / Q142L / Q189L / R236C, S27L / N9E / R12K / Q142W / T160Q / T206G / S212M, S27L / Y26T / S113N / A114V / T136A / D158E / D203R / N211F / R236Q / S252T / R255M, S27L / S1A / T17H / S22V / L90Y / F92G / S98E / A114V / T136A, S27L / N9E / T48S / I82M / L90Y / F92L / N122A / A127S / T160S / A174R / T206G / S212L / R255M, S27L / N9S / S22P / V23L / L90Y / F92G / A125S / T160S / N204R / I222L / S223A / R236Q, S27L / N9E / S22P / T48S / L90Y / F92L / T109R / E173R / A174K / D203V / S223A, S27L / R12K / Y26K / T48N / I82L / L90F / A125S / N211I / A216P, S27L / N9S / R12K / F92Y / T109K / S110R / T160K / Q189L / S223A / S252T / R255M, S27L / S1A / N2R / N9E / R12K / S32K / N87F / R108T / N211I / V219K / R255M, S27L / L90F / F92G / N122A / T136A / T160V / E173R / D203R / S252T, S27L / L90Y / F92G / S98T / T109L / D158I / S193K / V219K / R236Q / S252T, S27L / N9E / R12K / T48N / L90F / S98L / R108Q / A117Q / T136A / T206G / S212F / V219I / V229I / R255L, S27L / S1A / S22V / N87K / R108K / N122E / D158E / S193P / V219I / R255M, S27L / F21W / Y26T / S34R / R108H / L119M / N211M / R236C / S252T, S27L / S1A / N9E / R12K / V23T / T48S / N87H / S101Q / Q189L / N211M / V219I / R236Q / R255M, S27L / N9S / T25Q / L90Y / S101D / T136A / Q142L / Q189L / R236Q, S27L / A14K / I82F / F92G / S98E / R108C / A117N / L119M / Q189V / T206G / I222L / S223A, S27L / T17C / Y26T / N87V / R108C / N122E / T136V / S193P / S252T / R255M, S27L / L90Y / F92G / T109L / N122S / R255M, S27L / R12K / L90Y / T160V / A174R / R236Q, S27L / N9E / R12K / T48N / L90Y / F92G / S98T / S113R / N122R / A127S / T136A / A174K / N204K / S212L, S27L / V23T / L90Y / F92G / S98E / T109L / A125S / T160V / A174K / S181C / S193K / T206G / S212M / R255M, S27L / A24V / F92G / S101D / A114V / A117N / A125S / T136V / D203R, S27L / R12K / S32Q / S101Q, S27L / L90F / F92G / T136V / A174R / D203R, S27L / P20T / S32K / L90Y / D203V, S27L / N2R / A14K / T17S / L90Y / T109K / T136V / A216P, S27L / N2R / V23T / A24V / S32M / L90Y / F92G, S27L / A14K / L90Y / F92L / S101D / A117N / D158I / D203R, S27L / F21W / N87H / A114V / A117N / T136V, S27L / N2R / V23T / A24V / N87M / F92L / S101K / A125S / T136A, S27L / L90Y / F92L / S101N / T109R / S110R / Q142W, S27L / S32M / F92G / A216P, S27L / F21W / L90Y / F92L / A114K / A117N / T136A / D203V, S27L / N2R / V23T / S32M / N87M / F92L / T136A, S27L / R12K / L90F / F92G / S101A / D203V, S27L / A24V / L90F / F92G, S27L / R12K / F92L / S101A / A125S / T136A / D203R, S27L / R12K / V23L / L90F / F92L / A114K / T136A / D158E / D203V / I222L, S27L / L90F / F92G, S27L / T17L / L90F / F92G / A125S / A174R / D203V, S27L / N2R / N87K / A114K / A117Q / T136V / D203V, S27L / N2R / A114K / T136V / D203V, S27L / N2R / T17A / T136V / A216P, S27L / N2R / R12K / N87F / T136V / D158E / A174R, S27L / D203R, S27L / R12K / T17Q / T136V / D203V / A216P, S27L / N87F / T109L / Q142L, S27L / N2R / T17Q / A24V / A114K / A117Q / T136A / D158E / A174K / D203V / I222L, S27L / N2R / P20T / F21W / N87M / T109R / A117N / A125S / T136V / D203V / I222L, S27L / V23T / A24V / T136A / D158E / A174K / D203V / I222L, S27L / R12K / V23L / A114K / T136A / D158I / D203V / I222L, S27L / N87Y / T136V / D158E / D203V, S27L / T109K / S110R / D203R, S27L / N2R / T17L / A125S / T136A / D158E, S27L / A24V / D158E / A174K / A216P, S27L / T17G / V23L / N87L / A117Q / A125S / T136V / D158E / V229C, S27L / V23L / N87L / T109R / A114K / A117N / A125S / T136V / Q142L, S27L / N87Y / S101A / A114V / A117Q / T136V / A174K / A216P / I222L, S27L / R12K / T109R / Q142L, S27L / D203V, S27L / T17G / N87F / S101A / D203V, S27L / T17L / N87M / Q142L / D203V, S27L / N2R / R12K / S101D / A117N / T136A / D203V, S27L / R12K / P20T / F21W / A114K / Q142W / D203R, S27L / N2R / T17G / T109L / S110R / A114K / A117Q / T136V / Q142L / D203R, S27L / N2R, S27L / S32K / L90F / F92G / S101M / A114V / A117N / A125S / T136A / D158E / D203V, S27L / R12K / T17A / F21W / S32Q / Y60H / D203R, S27L / R12K / F92L / S110R / T136V / I222L, S27L / A14K / V23T / A24V / L90Y / F92G / A114V / A117Q / Q142L / A174R / I222L, S27L / F21W / S32Q / L90Y / S101D / T109K / A125S / T136A / A174K, S27L / V23T / L90Y / F92G / T136A / D203V / A216P, S27L / T17S / L90Y / F92G / A117N / T136A / D158L, S27L / A14K / V23L / A24V / F92L / Q142W / D203V, S27L / T17L / L90F / F92G / S101K / A174K, S27L / L90F / F92L / A125S / T136V / A174K / D203V / A216P / I222L, S27L / R12K / P20T / S32M / L90F / F92G / S101M / A114V / D203V, S27L / T17A / S32Q / L90F / F92L / T136V / Q142W / A174K / D203R, S27L / R12K / S32M / L90F / F92G / S101W / A114V / D158I / I222L, S27L / P20T / S32M / L90Y / F92L / A114V / A117N / I222L, S27L / T17C / L90Y / F92G / T136A / A174K / D203V, S27L / S32Q / N87Y / F92G / T109K / A114K / A117N / T136V / D203V / V229C, S27L / F21W / L90F / F92G / T136V / A174R, S27L / P20T / N87Y / T136A / D203V / I222L, S27L / A24V / L90Y / F92L / D158I / A174K / D203R / I222L, S27L / F21W / S32Q / L90Y / S110R / A114K / T136V / D203R / A216P / V229I, S27L / N87H / F92G / D203R, S27L / R12K / S32K / Y60A / A125S / T136V / D158L, S27L / V23T / A24V / S32Q / L90F / F92L / T136A, S27L / F92L / A114V / D158E / D203R, S27L / S32Q / L90Y / D203V / V229I, S27L / V23T / S32Q / Y60H / T109K / T136V / A216P / I222L, S27L / A14K / A24V / L90F / F92G / T109K / T136V / D158L / A174R / D203V / I222L / V229I, S27L / R12K / N87F / A174K, S27L / P20T / S32Q / Y60H / T109K / A114V / T136A / A174K, S27L / V23T / A24V / Y60A / A125S / A174R / A216P, S27L / F92Y / D158L / I222L, S27L / V23L / A24V / L90Y / F92G / D203V / I222L, S27L / V23T / A24V / S32Q / L90F / F92G / S101H / T136A / A174K / D203V / V229C, S27L / S32K / N87H / A125S, S27L / P20T / F21W / F92G / A114V, S27L / T17L / V23L / A24V / L90Y / F92L / A117N / T136V / L137M / Q142L, S27L / P20T / L90Y / D203R, S27L / S110R / Q142L / D158I / D203R / V229I, S27L / P20T / S32Q / L90F / F92L / T109K / S110R / A125S / D203V, S27L / V23T / A24V / L90F / F92G / D158E / D203R, S27L / F92G / T136A / A174R / D203V / V229C, S27L / P20T / L90Y / F92G / T109R / S110R / A125S / D203R, S27L / A14K / L90Y / F92L / D158L / A174R / D203V, S27L / F21W / S32Q / L90Y / F92G / T109L / S110R / A117N / D158I / A174K / D203V, S27L / R12K / A114K / A117N / D158E / D203V, S27L / T17S / N87H / T109R / A114K / A117N / T136V / Q142W / D203V, S27L / N2R / N87Y, S27L / T17S / V23T / A24V / S32Q / Y60H / N87K / T136A / D203R, S27L / P20T / F92Y / T109K / D158E / D203V, S27L / S32K / N87M / F92L / S101M / T109L / S110R / D203V, S27L / L90Y / F92G / T136A / D158E / D203V / A216P / V229I, S27L / A14K / S32M / F92L / A125S, S27L / L90Y / F92G / T136V / D158L / D203V, S27L / A24V / L90Y / F92G / S101A / T109L / S110R / A117Q / A174R / D203R, S27L / P20T / S101D / A114V / T136V / Q142W / D158E / D203R / A216P, S27L / N87Y / F92G / S101Q / A114K / A117N / T136A / D158L / D203R / A216P, S27L / V23T / A24V / N87Y / L90Y / F92L / S101D / T109R / A174K / D203R / A216P, S27L / N2R / P20T / F21W / L90F / F92Y, S27L / L90Y / F92G / D158I / D203V / V229C, S27L / S32Q / F92G / Q142W / I222L, S27L / N2R / R12K / S32M / N87Q / L90F / F92L / I222L, S27L / R12K / S32K / F92L / A114V / A117Q / T136A / D158E / A216P, S27L / L90Y / F92G, S27L / R12K / F21W / S32M / F92G / T136V / Q142L / D203R, S27L / R12K / A14K / L90Y / F92L / A125S / A174K, S27L / A24V / A114V / A117Q / T136V / D203V, S27L / R12K / T17Q / V23L / S32M / L90Y / F92G / S101D / T136V / I222L, S27L / V23T / A24V / S32Q / F92L / S101N / T109L / S110R / D203R, S27L / P20T / Y60H / N87K / T136A / A174R / D203V, S27L / P20T / F21W / L90Y / F92L / T136A / A174R / D203V / V229C, S27L / T17G / F21W / S32K / L90F / F92G / A125S / T136A / A174K, S27L / T17G / N87Y / T136A / D203V / A216P, S27L / F21W / L90F / F92G / S101D / A117N / T136A / A174K / D203V, S27L / R12K / A14K / A24V / L90Y / D203V, S27L / N2R / L90F / F92G / S101M / Q142L / D158L, S27L / N2R / T17C / A24V / N87K / A174K / A216P, S27L / N87M / Q142L / I222L, S27L / F92L / S98T / R108C / A117Q / A127S / Q142L / Q189V / A216P / R236Q, S27L / S22K / F92L / R108H / A127S / T136A / N211I, S27L / F21W / T48S / L90Y / F92L / T109L / A127S / T136V / N204K / S223A, S27L / T17L / L90Y / F92L / S98M / A174K / D203V / N211M / S212L / S223A, S27L / N87K / S98M / N211M, S27L / N9E / S22P / L90Y / N204K, S27L / S32M / T48S / F92G / A127S / Q142W, S27L / T17S / S22K / L90F / F92L / D158E / D203V / V219I / S252T, S27L / L90F / F92L / A127S / D203V, S27L / T25F / L90Y / F92G / N204R / S223A / N231S / R236C, S27L / F21W / S22V / T48S / L90F / F92L, S27L / S1A / S98T / S113K / A114V / L119M / A127S / Q142W / S193K / V219K / S252T / R255L, S27L / A24V / I82L / L90F / F92G / T109L / L119M / A127S / E173R / N204K, S27L / T17C / Y60H / L90F / A127S / E173R / A174R / N204K, S27L / L90F / F92L / Q142W / D203V / S223A, S27L / P20Q / L90Y / Q142L / S223A, S27L / N9S / Y60A / A216P / R236Q / R255M, S27L / F92G / R108C / S110R / A117Q / T136A / N211M, S27L / S22P / Y60H / S98A / S113K / A114K / T136V / Q189L / S193H, S27L / V23L / S98A / Q142L / N211M, S27L / V23L / Y26L / L31M / T48S / F92G / A174R / N204K / S252T, S27L / S13R / A14K / Y26T / T136A / S181R / N211M / S212M, S27L / F21W / I82F / L90Y / F92G / A127S / N211M, S27L / P20T / Y60H / S98L / T136A / R255L, S27L / P20T / S34R / N87M / D158E / S252T, S27L / T25A / L90F / F92G / Q189L / S193P / V229I, S27L / V219L / I222L, S27L / N9E / T48S / L90F / F92L / S98N / R108H / S110R / S113Y / N211V / S212L / S252T, S27L / L90F / F92Y / S113R / V219I / R255L, S27L / F21W / S22K / T48S / F92G / T109R / A127S, S27L / S1G / A14K / P20T / S32K / T48S / L90Y / F92L / T109L / T160R / V219L / I222L / S252T / R255L, S27L / L90F / F92G / D158E / T160R / S193N / N204K / S223A, S27L / A14K / Y26T / F92L / Q142L / S212L, S27L / S22R / L90Y / F92G / V126I / A127S / A174K / N204K, S27L / P20T / T25A / L90F / F92L / A117N / L119M / S252T / R255L, S27L / T17A / A24V / T48S / L90F / F92G / T109R / A127S / T160K / D203R / S223A, S27L / F21W / T48S / T109L / A127S / V219I / R255L, S27L / A14K / S22P / T48S / L90F / F92L / T109L / A127S / Q142L / A174R / N204K / S223A, S27L / T136A / S212M / N231S, S27L / F21W / L90F / F92G / A127S / S223A, S27L / T48S / L90Y / F92L / R108S / S110R / A127S / T136V / E173R / S223A, S27L / T48S / F92G / S98E / Q142L / Q189V / S193P / N204R / S223A, S27L / T17S / L90F / F92Y / S101K / A127S / T136V / N204R / N211I / V229I / N231S, S27L / N9E / F21W / T48S / L90Y / F92L / T109R / N204K / R236Q, S27L / N9E / S22R / T48S / F92L / S101N / A127S / E173R / N204R, S27L / S22K / N87F / A117Q / S181C / N204K / N211M / V229I / N231S / R236Q, S27L / N9S / T48S / L90F / F92G / S101N / A127S / N204K / A216P / S252T / R255M, S27L / I82L / S193H / N211M / S212F / S223A, S27L / A14K / P20T / T48S / L90F / F92G / S98N / R108C / T136A / N204K / N211L / S212L / R236Q / S252T, S27L / S13R / T17L / T25Q / L90F / F92G / R108C / A127S / T160R / I222L / R255L, S27L / T48S / L90Y / F92G / T109L / S193N, S27L / S22V / T48S / L90Y / F92L / S98E / A127S / T136A / N204K / A216P / S223A, S27L / V23L / N87H / F92L / R108C / S110R / N122A / T160S / Q189L / S193P / N204K / S223A / R255L, S27L / F92G / A127S / A174R / S223A / V229I / N231S / R236C, S27L / S1G / L90Y / A127S / E173R / S223A, S27L / F92Y / S98N / Q142W, S27L / P20T / S32M / S34R / T48S / L90Y / F92L / R108K / A127S / Q142W / A174R / N204K / V219I / S252T, S27L / F92G / A127S / Q189V / S193N / S223A, S27L / S34R / F92G / S110R / D158L / D203V / R255M, S27L / N9E / F21W / T48S / F92G / T109L / A127S / R236C, S27L / S1A / S22P / T48S / L90Y / F92G / A127S / E173R / A174R / N204K / S223A, S27L / S22K / T48S / T160S / N204K, S27L / F21W / T48S / L90Y / F92G / S98V / R108C / A127S / N211L / S212M / N231S / R236C, S27L / V23L / A24V / A114K / T136A / I222L / V229I, S27L / P20T / S101H / T109R / A114V / T136A / Q142L / I222L / V229I, S27L / V23L / S101H / T109K / A114K / A117N / T136V / Q142L / I222L / V229I, S27L / T17L / F21W / S101H / T109R / A114V / T136A / Q142L / I222L / V229I, S27L / T17S / T136V / Q142L / I222L / V229C, S27L / T17C / S22P / T48S / L90F / F92G / A127S / A174R / P192A / S193H / N204K / S223A / V229I / N231S / R236Q / R255L, S27L / N9E / T48N / N87H / F92L / A174K / Q189L / S193K / N204R / V219I, S27L / A14K / V23L / Y26L / L90F / F92G / S98E / S113K / A114K / T136A / V229I / N231S, S27L / L90Y / F92L / S101D / A117N / Q142L / S193N, S27L / S22K / T25A / L90Y / Q142L / E173R / N204K / S223A, S27L / P20T / L90Y / T109L / A127S / A174K / V229C, S27L / S22V / L90Y / F92L / A117Q / R236C / R255L, S27L / V23L / A24G / Y26T / I82F / L90Y / F92G / R108H / T136A / D158L / T160K / D203V, S27L / A14K / S22K / F92G / A127S / N204K / V229I, S27L / S22V / L90F / F92G / A127S / A174R / A216P / R236C, S27L / V23L / Y60A / S98T / T109L / A127S / T136V / T160Q / N204K / S223A / R255L, S27L / S13R / L90Y / S98L / T160V / Q189L / S193P / A216P / S252T / N253S / R255L, S27L / S22R / I82M / F92L / T109L / A127S / Q142W / Q189L / S223A, S27L / S1G / Y60A / L90F / F92L / N122E / T136A / N204K / S223A / N231S, S27L / S32K / F92G / S98L / R108Q / S110R / R255L, S27L / L90Y / F92G / L119M / A127S / A174R / S252T, S27L / L90F / S110R / S181R / N211F, S27L / A14K / S22P / Y60H / S110R / N122S / D158I / N204K / S223A / R255M, S27L / F92L / S98V / T109L / N122A / E173R / R255L, S27L / S22R / N87L / F92G / R236C, S27L / S32M / S34R / I82L / F92G / N204K / S223A, S27L / A24V / T25Q / Y60I / A127S / A174K / N204K / V219I / S223A / S252T / R255M, S27L / S98V / T136A / N211F, S27L / N9S / L90F / F92L / A127S / N204R / S212M, S27L / S22R / T48S / F92G / R108H / S110R / A127S / E173R / N204K / S223A / R236Q, S27L / A14K / L90Y / F92G / A127S, S27L / A14K / T48S / F92G / E173R / N204R / S223A / N231S, S27L / L90Y / T109R / S113N / V219L / I222L, S27L / N9S / T48S / L90Y / T136A / T206G / S223A, S27L / S22K / T48N / L90F / F92G / N204K / S223A / V229C / N231S, S27L / S22P / T48S / L90F / F92G / A127S / A174R / N204R / S223A, S27L / S1G / P20T / S32K / S34R / T48S / N87F / S98A / A127S / T136A, S27L / F21W / S22R / S98E / Q142L / T160V / S223A / S252T / R255L, S27L / P20T / T48S / F92Y / R108K / S110R / A127S / V219L / S223A / V229I / R236Q, S27L / Y26T / L90Y / F92L / A127S / V219L / S252T / R255L, S27L / N9S / F21W / T48S / F92L / A127S / D203V / S212F, S27L / F21W / Y60H / S98E / L119M / D158E / R236Q, S27L / F21W / S22R / L90F / F92L / N122S / Q142W / R236Q, S27L / F21W / T48S / L90F / F92G / T109K, S27L / L90Y / F92L / T109R / A127S / E173R / N204K / S223A, S27L / N9S / F21W / T48S / L90Y / F92G / A127S / N204K / V219I / R255M, S27L / N9E / I82F / L90Y / F92L / S95N / T109L / A127S / A174R / S223A, S27L / L90F / F92L / S98L / N204K / A216P, S27L / A24V / T48S / A117Q / N211M / S212M / S223A, S27L / L90Y / F92G / T109L / A127S / N204R / S223A, S27L / N2R / S32Q / N87K / A125S / V219L, S27L / S22P / T48S / L90F / F92L / S193N / V219L / S223A, S27L / T25Q / L119M / N211M / R236Q, S27L / L90Y / F92G / N122S / A127S / E173R / A216P / R236Q, S27L / P20T / S32K / L90F / F92G / N122E / D158E / T160S / Q189L / S223A / S252T, S27L / N9S / F21W / T48S / N87H / L90F / F92L / S113N / V229C, S27L / T25Q / I82L / L90F / F92G / S98L / S113R / A114V / T136A / S181R, S27L / P20T / R108Q / S110R / D203V / N211I, S27L / S1G / N2R / P20T / Y26T / S32M / T48S / L90Y / F92L / A127S / N204R / V219L, S27L / A14K / P20T / T48N / L90F / F92G / A117Q / A127S, S27L / N9S / T48N / L90Y / F92L / S98M / T109K / A127S / N204R / N211I / S212L / V219I, S27L / F21W / S22R / T48S / L90F / F92L / R108Q / A174K / N204K / S212F / S223A, S27L / S22R / Y60H / S98T / T136V / S193K / R236Q, S27L / S22K / Y26K / N87Y / L90Y / F92G / T109R / A127S / S252T / R255L, S27L / L90F / F92L / T109L, S27L / F21W / F92L / N204K / S252T / R255L, S27L / V23L / L90Y / F92G / A127S / S193K / N204R / V219I / R236C / R255L, S27L / F21W / L90Y / F92G / A114V / N122S / A127S / N204K / S223A / S252T, S27L / P20T / Y60A / T109L / Q189L / S212L, S27L / S22V / T48S / L90Y / F92G / N204K, S27L / S22V / S32K / T48S / I82F / F92G / A127S / N204K / S223A, S27L / A55L, S27L / A97V, S27L / F250L, S27L / T109G, S27L / G38D, S27L / A97S, S27L / A55V / A216T, S27L / P20D, S27L / A55V, S27L / T109Y, S27L / V165I, S27L / A184G, S27L / A97E, S27L / A184S, S27L / A97F, S27L / A97T, S27L / K197Y, S27L / A55I, S27L / A97P, S27L / A55M, S27L / P20E, S27L / A117L, S27L / T109L, S27L / K197T, S27L / T136S, S27L / A97L, S27L / T109A, S27L / P20I, S27L / L191V, S27L / A184C, S27L / A97Q, S27L / F250V, S27L / K197V, S27L / A117S, S27L / K197R, S27L / T109K, S27L / A55C, S27L / N2S / V177A, S27L / Q142D, S27L / G149C, S27L / F21Y, S27L / G149A, S27L / P164E, S27L / Y26C, S27L / T17N, S27L / S57M, S27L / D249I, S27L / T17S, S27L / P164T, S27L / Q142L, S27L / D249N, S27L / I185R, S27L / V83L, S27L / G149S, S27L / A24D, S27L / R251V, S27L / V83I, S27L / S110N, S27L / T17M, S27L / F161W, S27L / G46E, S27L / D249T, S27L / T17I, S27L / S57C, S27L / S57T, S27L / S57L, S27L / Q167T, S27L / S57E, S27L / T17K, S27L / G149N, S27L / Q142E, S27L / S57F, S27L / T17A, S27L / P164H, S27L / T17R, S27L / G149T, S27L / R251T, S27L / S101Y, S27L / S57V, S27L / A24T, S27L / I185E, S27L / G46N, S27L / P164N, S27L / P164S, S27L / Q167V, S27L / F161V, S27L / S57I, S27L / G149D, S27L / Q167I, S27L / I185Q, S27L / P20D / F21Y / F250V, S27L / T17S / R251T, S27L / N2L / A24H / A55C / V229L, S27L / N2E / R251L, S27L / N2F / P20E, S27L / A117Y, S27L / P164T / V165I / R251E, S27L / A24H / G46E, S27L / N2S / V83L / A184S, S27L / A24H / R251V, S27L / N2F, S27L / A24D / L191F / R251Q, S27L / P20I / F21Y / A117F / I185G / D249N, S27L / N2S / A24N / G46E / A55L / Q142D / V229C / R251L, S27L / P20E / F21Y / S110D / A117S, S27L / I139T / Q142E / I169V, S27L / A24D / T136S / F250V, S27L / T17N / A117S / V229L / R251A, S27L / P20E / A24D / D249S / F250V, S27L / A24N, S27L / G46E / A55I / Q142E, S27L / A24D / K197L, S27L / N2L, S27L / V83L / A97S / A184C / D249S, S27L / F21Y / A184S, S27L / P20E / F21Y / S57T / P164N / A184C / V229C, S27L / N2L / T17I / T136S / P164H / D249M, S27L / A24D / S110K, S27L / N2S / A55V / I185Q / R251E, S27L / N2L / P20E / F21Y / V229L, S27L / F21Y / G46E / A117T / T136S / G149C / R251Q, S27L / T136S / D249T, S27L / F250L / R251Q, S27L / N2S / P20D / A97C / A117F / F250L / R251E, S27L / S57C / R251E, S27L / P20D / G46S / A55I / K197T, S27L / N2L / A24T, S27L / N2E / G46S / V83L / T136S / A172T / V229C / D249N, S27L / P20D / A55T / A117Y / K197R, S27L / N2F / P20I / F21Y / R251Q, S27L / N2E / P20I / T109L / L191V / K197L / V229C, S27L / S110D / A184S / K197T, S27L / N2E / I169L / F250L / R251Q, S27L / G46R / T109K / T136S / D249T, S27L / N2S / A24D / S101Y / P164E / V165I / I185E, S27L / Q142H / I185S / R251A, S27L / P20E / F21Y / K197T, S27L / G46N / I139T / Q142E / P164T / I185A / V229L / F250V, S27L / N2F / R251Q, S27L / T17H / S110K, S27L / F21Y / A55T / T109A / A184S, S27L / N2F / G46E, S27L / N2F / S101C / A117F / P164S, S27L / P20D / G46N / A55C / S110N / T163I / A184S, S27L / A24D / T109L / K197L, S27L / A55V / V83L / A117T, S27L / P20E / F21Y / I139T / P164H / K197T / R251E, S27L / V83L / F250V / R251E, S27L / T163I, S27L / N2L / G46E / T109K / F161V, S27L / P20D / F21Y / A55T / F250L / R251V, S27L / N2F / D249N / F250L, S27L / N2F / S101Y / Q142L / G149T / Q167I / I169C / V229L, S27L / A24N / P164S / K197V / R251T, S27L / N2L / T17N / S57C / S110D / A117L / P164N / I185G / V229L / R251T, S27L / N2E / P20E / F250V / R251L, S27L / P20D / F21Y / G46N / S110K / T163I / Q167V, S27L / N2E / G46N / A55V / I185Y / D249I, S27L / N2L / A172T, S27L / T17I / A24D / S57I, S27L / P20E / F21Y / G46E / V83L / A97Q, S27L / K197R / R251E, S27L / P20D / S57E / S101C, S27L / S110K / A117Y / P164R, S27L / N2F / P20E / S110D / A184S / L191V / R251V, S27L / P20T / V23T / A24V / I222L / V229I, S27L / F21W / T109K / S110R / T136A / V229I, S27L / A114V / A117N / T136A / I222L / V229I, S27L / T17H / F21W / T136V / Q142W / I222L / V229I, S27L / S101Q / Q142L / I222L / V229I, S27L / P20T / V23T / A24V / T109L / S110R / I222L / V229I, S27L / P20T / F21W / T109K / A117N / I222L / V229C, S27L / S101M / T136A / I222L / V229I, S27L / V23L / A24V / S101K / A117N / I222L / V229C, S27L / V23T / T109L / A114V / T136A / Q142L / I222L / V229C, S27L / F21W / A114V / T136A / I222L / V229I, S27L / T17Q / V23T / A24V / I222L / V229I, S27L / P20T / F21W / V229I, S27L / V23L / I222L / V229C, S27L / V229I, S27L / P20T / F21W / T109R / A117Q / T136V / Q142L / I222L / V229I, S27L / F21W / I222L / V229C, S27L / F21W / Q142L / I222L / V229C, S27L / T17A / F21W / S101Q / I222L / V229C, S27L / S101W / A114V / T136V / V229C, S27L / P20T / S101M / I222L / V229I, S27L / P20T / F21W / T109L / T136A / Q142W / I222L / V229C, S27L / T17A / V23T / A24V / T136A, S27L / T17A / F21W / T109R / S110R / I222L / V229I, S27L / T17S / S101N / T109K / A117N / T136V / Q142L / I222L / V229C, S27L / I222L / V229I, S27L / F21W / T136A, S27L / T17A / A24V / S101D / S110R / T136A, S27L / P20T / T109R / A114K / A117Q / T136V / I222L / V229C, S27L / V23T / A24V / I222L / V229C, S27L / T17L / T109L / S110R / A117Q / I222L / V229I, S27L / P20T / F21W / T136A / Q142L / I222L / V229I, S27L / T17G / V23T / A24V / T109L / A117Q / T136A / I222L / V229I, S27L / V23L / A24V / T136A / Q142W / I222L / V229I, S27L / P20T / T109L / S110R / A117N / I222L / V229I, S27L / T17H / S101Q / T136V / Q142L / V229C, S27L / T17A / V23L / S101M / T109K / S110R / A114V / A117Q / T136V / V229I, S27L / A24V / V229C, S27L / T17Q / A24V / S101W / T136A, S27L / S101M / A114V / A117Q / T136A, S27L / T17C / T109K / A117N / T136A / I222L / V229I, S27L / T109R / I222L / V229I, S27L / P20T / S101H / A114V / A117N / I222L / V229I, S27L / T136V / Q142W / I222L / V229I, S27L / F21W / I222L / V229I, S27L / V23L / T136A / I222L / V229I, S27L / T109R / A117N / I222L / V229C, S27L / T17G / F21W / A117N / I222L / V229I, S27L / T17C / A114K / A117N / T136V / I222L / V229I, S27L / P20T / I222L / V229C, S27L / F21W / A24V / A114V / A117Q / Q142W / I222L / V229C, S27L / P20T / S101M / A114V / A117N / Q142W / I222L / V229C, S27L / V23T / Q142L / I222L / V229C, S27L / F21W / A24V, S27L / T17Q / S101D / T136A / I222L / V229I, S27L / F21W, S27L / T109K / A117N / T136V / I222L / V229C, S27L / T17S / V229I, S27L / S101N / A117Q / I222L, S27L / T17Q / T136A / I222L / V229I, S27L / V23L / A117Q, S27L / F21W / S101M / I222L / V229C, S27L / F21W / T109R / A117N / T136V / I222L / V229I, S27L / V23T / A24V / T109K / A114K / A117Q / T136A / I222L / V229C, S27L / T17Q / Q142L / I222L / V229I, S27L / F21W / T109R / A117N / T136V / Q142W / I222L / V229C, S27L / V23L / A114V / A117N / T136A / I222L / V229I, S27L / P20T / F21W / I222L / V229C, S27L / T17Q / T136A / I222L / V229C, S27L / P20T / F21W / Q142W / I222L / V229I, S27L / T17L / S101M / T109K / S110R / A117Q / I222L / V229C, S27L / P20T / T109K / A114K / Q142W / I222L / V229C, S27L / P20T / F21W / Q142W / I222L / V229C, S27L / P20T / A24V / S101Q / T109R / A117Q / I222L / V229I, S27L / T17S / F21W / Q142L, S27L / P20T / A117Q / I222L / V229C, S27L / T17A / P20T / S101Q / T109K / S110R / T136A / I222L / V229C, and S27L / T17G / P20T / F21W / A117Q / I222L / V229C selected from the group consisting of The composition of claim 1.
3. A nucleic acid encoding the variant Bhr-PETase of claim 1 or 2.
4. An expression vector comprising the nucleic acid of claim 3.
5. A host cell comprising the expression vector of claim 4.
6. The host cell of claim 5 which is a bacterium, yeast or fungus.
7. A method for producing the variant Bhr-PETase of claim 1 or 2, comprising culturing the host cell of claim 5 under conditions in which the variant Bhr-PETase is produced, and recovering the variant Bhr-PETase.
8. A method for degrading PET, comprising contacting PET with the variant Bhr-PETase of claim 1 or 2.
9. 10. The method of claim 8, further comprising pretreating the PET by a method for pretreating PET, including mechanical, thermomechanical, and / or chemical pretreatment of the PET, prior to enzymatic degradation of the PET.
10. 10. The method of claim 9, wherein the mechanical pretreatment comprises grinding the PET into particles.
11. 10. The method of claim 9, wherein the thermomechanical pretreatment comprises extruding the PET at a temperature set to amorphize the PET and reduce its crystallinity.
12. 10. The method of claim 9, wherein the chemical pretreatment comprises contacting the PET with an ionic solution, a strong acid, a base, or a solvent configured to reduce the crystallinity or alter the surface structure of the PET.
13. 9. The method of claim 8, wherein PET in a mixed plastic composition is degraded.
14. 14. The method of claim 13, wherein the mixed plastic composition comprises a biologically or chemically derived PET analog, PET-like material, or PET substitute.
15. 14. The method of claim 13, wherein the mixed plastic composition comprises at least one selected from the group consisting of polybutylene terephthalate (PBT), polycarbonate (PC), polycaprolactone (PCL), polyethylene furanoate (PEF), and high density polyethylene (HDPE).
16. 10. The method of claim 8, which does not include separating plastics to select PET from a mixture of plastics.
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