Enzymatic degradation of polyethylene terephthalate

Abstract

To provide a variant enzyme for use in the enzymatic degradation of polyethylene terephthalate (PET).SOLUTION: To provide a variant PETase with improved enzyme activity that can be used in the degradation of plastics made of PET. The biochemical properties of a variant PETase that may be improved herein include, but are not limited to, thermostability, thermoactivity, specific activity and production.SELECTED DRAWING: None

Description

[Background technology]

[0001] The use of polyethylene terephthalate (PET) is widespread and ubiquitous. PET is a major source of pollution worldwide. While it can be physically recycled, in many circumstances 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 includes a variant Bhr-PETase compared to SEQ ID NO:1 10. A composition comprising: a) a variant of SEQ ID NO: 1; b) a variant of SEQ ID NO: 1; c) a variant of SEQ ID NO: 1; d) a variant of SEQ ID NO: 1; 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 3 0, 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, 11 7, 119, 121, 122, 125, 127, 135, 136, 138, 139, 14 0, 142, 143, 145, 147, 149, 150, 153, 156, 157, 15 8, 160, 161, 162, 163, 164, 167, 170, 173, 174, 17 7, 179, 181, 182, 184, 185, 189, 190, 193, 194, 19 5, 198, 200, 203, 204, 206, 208, 209, 211, 212, 21 3, 216, 217, 218, 219, 221, 222, 223, 225, 227, 22 8, 229, 231, 236, 237, 241, 242, 243, 246, 249, 25 0, 251, 252, 253, 254, 255, 258, 8, 31, 38, 95, 126 , 137, 165, 169, 172, 191, 192 and 197 and wherein the variant Bhr-PE comprises at least one amino acid substitution at an amino acid position where Tase has at least 85% identity to SEQ ID NO: 1 and has PETase activity In another aspect, the present disclosure relates to a composition comprising a variant Bhr- compared to SEQ ID NO: 1. A composition comprising PETase, wherein the variant is at position 27 compared to SEQ ID NO: 1. Amino acid substitutions and 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, 8 2, 83, 85, 87, 88, 90, 92, 97, 98, 101, 102, 105, 10 8, 109, 110, 113, 114, 117, 119, 121, 122, 125, 12 7, 135, 136, 138, 139, 140, 142, 143, 145, 147, 14 9, 150, 153, 156, 157, 158, 160, 161, 162, 163, 16 4, 167, 170, 173, 174, 177, 179, 181, 182, 184, 18 5, 189, 190, 193, 194, 195, 198, 200, 203, 204, 20 6, 208, 209, 211, 212, 213, 216, 217, 218, 219, 22 1, 222, 223, 225, 227, 228, 229, 231, 236, 237, 24 1, 242, 243, 246, 249, 250, 251, 252, 253, 254, 25 5, 258, 8, 31, 38, 95, 126, 137, 165, 169, 172, 191 at least one additional amino acid at an amino acid position selected from the group consisting of 192 and 197; and wherein the variant Bhr-PETase has at least the amino acid substitution In another embodiment, the composition has 85% identity and has PETase activity. The present 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, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 5, 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, 1 01, 102, 105, 108, 109, 110, 113, 114, 117, 119, 1 21, 122, 125, 127, 135, 136, 138, 139, 140, 142, 1 43, 145, 147, 149, 150, 153, 156, 157, 158, 160, 1 61, 162, 163, 164, 167, 170, 173, 174, 177, 179, 1 81, 182, 184, 185, 189, 190, 193, 194, 195, 198, 2 00, 203, 204, 206, 208, 209, 211, 212, 213, 216, 2 17, 218, 219, 221, 222, 223, 225, 227, 228, 229, 2 31, 236, 237, 241, 242, 243, 246, 249, 250, 251, 2 52, 253, 254, 255, 258, 8, 31, 38, 95, 126, 137, 16 amino acid positions selected from the group consisting of 5, 169, 172, 191, 192 and 197 and wherein said variant Bhr-PETase comprises at least one amino acid substitution at the sequence SEQ ID NO: 1, and having at least 85% identity to wild-type Bhr-PETase, SEQ ID NO: 1 In some embodiments, the composition has a PETase activity equal to or even higher than The amino acid substitutions 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, 7 2, 74, 77, 82, 83, 85, 87, 88, 90, 92, 97, 98, 101, 1 02, 105, 108, 109, 110, 113, 114, 117, 119, 121, 1 22, 125, 127, 135, 136, 138, 139, 140, 142, 143, 1 45, 147, 149, 150, 153, 156, 157, 158, 160, 161, 1 62, 163, 164, 167, 170, 173, 174, 177, 179, 181, 1 82, 184, 185, 189, 190, 193, 194, 195, 198, 200, 2 03, 204, 206, 208, 209, 211, 212, 213, 216, 217, 2 18, 219, 221, 222, 223, 225, 227, 228, 229, 231, 2 36, 237, 241, 242, 243, 246, 249, 250, 251, 252, 2 at an amino acid position selected from the group consisting of 53, 254, 255 and 258 In some embodiments, the amino acid substitutions are 27, 2, 17, 20, 21, 2 3, 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 substitutions are 8, 126, 137, 165 , 169, 172, 191, 192 and 197 In.

[0004] In some embodiments, the amino acid substitutions are S27L, S27F, S27H, S27T , S27W, S1A, S1G, S1M, S1R, N2E, N2F, N2L, N2R, N2 S, Q5E, N9A, N9E, N9S, R12K, S13L, S13R, A14K, A1 4S, L15I, T16E, T17A, T17C, T17G, T17H, T17I, T1 7K, T17L, T17M, T17N, T17Q, T17R, T17S, D18R, P2 0D, P20E, P20I, P20Q, P20T, F21W, F21Y, S22A, S2 2I, S22K, S22P, S22R, S22V, V23L, V23T, A24D, A2 4G, A24H, A24N, A24S, A24T, A24V, T25A, T25F, T2 5H, T25Q, T25R, T25V, Y26C, Y26K, Y26L, Y26T, R3 0K, S32K, S32M, S32Q, S32Y, V33G, V33Q, V33T, S3 4R, V40T, G46E, G46L, G46N, G46R, G46S, T48N, T4 8S, L49G, G53A, I54V, A55C, A55I, A55L, A55M, A5 5T, A55V, M56I, M56L, S57C, S57E, S57F, S57I, S5 7L, S57M, S57T, S57V, Y60A, Y60H, Y60I, A62T, A6 8F、L70M、R72P、L74W、H77Q、I82F、I82L、I82M、V8 3I, V83L, V83T, N85D, N87F, N87H, N87I, N87K, N8 7L, N87M, N87Q, N87R, N87V, N87W, N87Y, S88K, S8 8T、L90F、L90K、L90Y、F92G、F92I、F92K、F92L、F9 2N, F92Q, F92V, F92Y, A97C, A97E, A97F, A97G, A9 7L, A97P, A97Q, A97S, A97T, A97V, S98A, S98D, S9 8E、S98L、S98M、S98N、S98Q、S98T、S98V、S101A、S 101C、S101D、S101F、S101H、S101K、S101L、S101M 、S101N、S101Q、S101R、S101V、S101W、S101Y、A10 2V、N105D、R108C、R108E、R108H、R108K、R108N、R 108P、R108Q、R108S、R108T、R108V、T109A、T109F 、T109G、T109K、T109L、T109N、T109R、T109Y、S11 0D、S110G、S110H、S110K、S110N、S110R、S113A、S 113K、S113N、S113P、S113Q、S113R、S113T、S113Y 、A114K、A114L、A114S、A114V、A117F、A117G、A11 7L, A117N, A117Q, A117S, A117T, A117Y, L119I, L 119M、A121S、N122A、N122E、N122H、N122P、N122R 、N122S、A125S、A127M、A127S、A127V、A135G、T13 6A、T136M、T136S、T136V、R138E、R138L、I139A、I 139T、S140A、Q142D、Q142E、Q142H、Q142L、Q142W 、I143N、I143R、T145S、K147F、K147G、K147N、K14 7Q、G149A、G149C、G149D、G149N、G149S、G149T、G 149V、V150I、V150L、T153L、H156N、T157A、T157G 、D158E、D158I、D158K、D158L、T160K、T160Q、T16 0R、T160S、T160V、F161V、F161W、N162E、N162H、N 162P、N162R、T163I、T163S、P164E、P164H、P164N 、P164R、P164S、P164T、Q167I、Q167T、Q167V、V17 0L、E173R、A174K、A174R、V177A、P179Q、S181A、S 181C、S181R、Q182T、A184C、A184G、A184S、I185A 、I185E、I185G、I185L、I185Q、I185R、I185S、I18 5Y、Q189I、Q189L、Q189V、N190S、S193E、S193F、S 193H、S193K、S193N、S193P、S193T、S193V、T194G 、T194S、T195F、V198A、V200L、D203N、D203R、D20 3V、N204A、N204K、N204R、N204S、T206G、T206K、T 206L、T206P、T206R、F208G、F208L、F208R、F208T 、A209V、N211F、N211I、N211L、N211M、N211V、S21 2F, S212L, S212M, P213N, P213R, A216L, A216P, A 216S, A216T, A216V, I217S, S218A, V219F, V219I , V219K, V219L, V219R, T221S, I222L, S223A, S22 3C, M225L, L227R, W228F, V229C, V229I, V229L, N 231L, N231Q, N231S, R236C, R236E, R236H, R236K , R236Q, Q237R, N241P, V242T, N243P, A246D, A24 6K, A246S, A246T, D249I, D249M, D249N, D249S, D 249T, F250I, F250L, F250V, F250Y, R251A, R251E , R251I, R251K, R251L, R251Q, R251T, R251V, S25 2T, N253S, N253Y, N254R, R255E, R255G, R255L, R 255M, R255S, R255V, R255W, R255Y, Q258P, P8T, L 31M, G38D, S95N, V126I, L137M, V165I, I169C, I1 69L, I169V, A172T, L191F, L191V, P192A, K197L, Selected from the group consisting of K197R, K197T, K197V and K197Y. In one embodiment, the amino acid substitutions are S27L, S27F, S27H, S27T, ...H, S27H, S27H, S27H, S2 7W, S1A, S1G, S1M, S1R, N2R, Q5E, N9A, N9E, N9S, R 12K, S13L, S13R, A14K, A14S, L15I, T16E, T17A, T 17C, T17G, T17H, T17K, T17L, T17Q, T17S, D18R, P 20T、F21W、S22A、S22I、S22K、S22P、S22R、S22V、V 23L、V23T、A24S、A24V、T25A、T25F、T25H、T25Q、T 25R、T25V、Y26K、Y26L、Y26T、R30K、S32K、S32M、S 32Q、S32Y、V33G、V33Q、V33T、S34R、V40T、G46L、G 46S、T48N、T48S、L49G、G53A、I54V、A55L、A55V、M 56I、M56L、S57I、S57M、S57V、Y60A、Y60H、Y60I、A 62T、A68F、L70M、R72P、L74W、H77Q、I82F、I82L、I 82M、V83T、N85D、N87F、N87H、N87I、N87K、N87L、N 87M、N87R、N87V、N87W、N87Y、S88K、S88T、L90F、L 90K、L90Y、F92G、F92I、F92K、F92L、F92N、F92Q、F 92V、F92Y、A97F、A97G、S98A、S98D、S98E、S98L、S 98M、S98N、S98Q、S98T、S98V、S101A、S101D、S101 F、S101H、S101K、S101L、S101M、S101N、S101Q、S1 01R、S101V、S101W、A102V、N105D、R108C、R108E、 R108H、R108K、R108N、R108P、R108Q、R108S、R108 T、R108V、T109F、T109K、T109L、T109N、T109R、S1 10G、S110H、S110R、S113A、S113K、S113N、S113P、 S113Q、S113R、S113T、S113Y、A114K、A114L、A114 S、A114V、A117G、A117N、A117Q、A117S、L119I、L1 19M、A121S、N122A、N122E、N122H、N122P、N122R、 N122S、A125S、A127M、A127S、A127V、A135G、T136 A、T136M、T136S、T136V、R138E、R138L、I139A、I1 39T、S140A、Q142L、Q142W、I143N、I143R、T145S、 K147F、K147G、K147N、K147Q、G149A、G149C、G149 S、G149V、V150I、V150L、T153L、H156N、T157A、T1 57G、D158E、D158I、D158K、D158L、T160K、T160Q、 T160R、T160S、T160V、F161W、N162E、N162H、N162 P、N162R、T163S、P164S、Q167T、Q167V、V170L、E1 73R、A174K、A174R、V177A、P179Q、S181A、S181C、 S181R、Q182T、A184S、I185L、I185Y、Q189I、Q189 L、Q189V、N190S、S193E、S193F、S193H、S193K、S1 93N、S193P、S193T、S193V、T194G、T194S、T195F、 V198A、V200L、D203N、D203R、D203V、N204A、N204 K、N204R、N204S、T206G、T206K、T206L、T206P、T2 06R、F208G、F208L、F208R、F208T、A209V、N211F、 N211I、N211L、N211M、N211V、S212F、S212L、S212 M、P213N、P213R、A216L、A216P、A216S、A216V、I2 17S, S218A, V219F, V219I, V219K, V219L, V219R, T221S, I222L, S223A, S223C, M225L, L227R, W228 F, V229C, V229I, N231L, N231Q, N231S, R236C, R2 36E, R236K, R236Q, Q237R, N241P, V242T, N243P, A246D, A246K, A246S, A246T, D249T, F250I, F250 Y, R251I, R251K, R251V, S252T, N253Y, N254R, R2 55E, R255G, R255L, R255M, R255S, R255V, R255W, In some embodiments, the amino acid sequence is selected from the group consisting of R255Y and Q258P. The 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, S101R, S101V, S101 W, A102V, T109F, T109K, T109L, T109N, T109R, S1 10G, S110H, S110R, A114K, A114L, A114S, A114V, A117G, A117N, A117Q, A117S, A135G, T136A, T136 M, T136S, T136V, I139A, I139T, S140A, Q142L, Q1 42W, I143N, I143R, G149A, G149C, G149S, G149V, F161W, T163S, P164S, Q167T, Q167V, A184S, I185 L, I185Y, T195F, I222L, L227R, W228F, V229C, V2 29I, D249T, F250I, F250Y, R251I, R251K and R251 In some embodiments, the amino acid substitution is selected from the group consisting of N2E, N2V, ... F , N2L, N2S, P8T, T17I, T17M, T17N, T17R, P20D, P2 0E, P20I, P20Q, F21Y, A24D, A24G, A24H, A24N, A2 4T, Y26C, L31M, G38D, G46E, G46N, G46R, A55C, A5 5I, A55M, A55T, S57C, S57E, S57F, S57L, S57T, V8 3I, V83L, N87Q, S95N, A97C, A97E, A97L, A97P, A9 7Q, A97S, A97T, A97V, S101C, S101Y, T109A, T109 G, T109Y, S110D, S110K, S110N, A117F, A117L, A1 17T, A117Y, V126I, L137M, Q142D, Q142E, Q142H, G149D, G149N, G149T, F161V, T163I, P164E, P164 H, P164N, P164R, P164T, V165I, Q167I, I169C, I1 69L, I169V, A172T, A184C, A184G, I185A, I185E, I185G, I185Q, I185R, I185S, L191F, L191V, P192 A, K197L, K197R, K197T, K197V, K197Y, A216T, V2 29L, R236H, D249I, D249M, D249N, D249S, F250L, F250V, R251A, R251E, R251L, R251Q, R251T and N2 53S.

[0005] In some embodiments, the variant Bhr-PETase enzyme has at one of said positions , at two of the positions, at three of the positions, at four of the positions, at five of the positions, At position 6, at position 7, at position 8, at position 9, at position 10 11 of the positions 12 of the positions 13 of the positions 14 of the positions 15 of said positions, 16 of said positions, 17 of said positions, 18 of said positions one or more amino acid substitutions at 19 of said positions or at 20 of said positions Has.

[0006] In some embodiments, the variant Bhr-PETase is A102V / T136 M, A14S / L15I / S22A / F92Q / I143R / Q167T / V219K, D18R / G46S / M56I / R108T / A127M / R138E / I139A / N 190S / L227R / W228F / R236E, D18R / I139A / F161W / W228F, D18R / I54V / I82F / N105D / A127M / A184S / S 218A / V219K / M225L / N241P / N243P, D18R / I54V / I 82F / R108T / V150I / N253Y / N254R, D18R / I54V / M5 6I / R138E / I139A / T194S / D203N / V219K / F250I, D 18R / M56I / I139A / L227R / W228F, D18R / M56I / R13 8E / I139A / N190S / D203N / M225L、D18R / M56I / R72 P / H77Q / F92Q / A127M / V150I / A184S / D203N / N254 R、D18R / N85D / R108T / L119I / N190S / T194S、D18R / V40T / I139A / D203N、D18R / V40T / I82F / S101L / N 105D / L119I / H156N / F161W / N190S / T194S / D203N 、D18R / V40T / M56I / R108T / R138E / H156N、G53A / R 108T / Q167T / A184S / T194S / N243P、I143R / Q167T / V198A / W228F、I82F / R108T / L119I / V150I / F161 W / Q167T / T194S / D203N、L15I / D18R / R108T / R138 E / I139A / F161W / A184S / N190S / D203N、M56I / I82 F / R108T / I139A / F161W / T194S / D203N / N241P、M5 6I / S88T / R108T / N190S / L227R、N204A / Q237R、N9 A / D18R / M56I / N85D / L119I / N254R、N9A / S22A / G4 6S / M56I / R72P / F92Q / L119I / T221S / M225L、N9A / S22A / Q167T、N9A / V40T / L49G / I54V / R108T / V150 I / D203N / T221S / M225L、N9S / T25H、Q5E / N9A / M56 I / F92Q / R108T / L119I / Q167T / N253Y、Q5E / S22A / R72P / H77Q / F92Q、R108T / L119I / K147Q / F161W / A 184S / D203N、S101F / T136A、S22A / A24S / G46S / V1 50I / Q167T、S22A / A97G / V150I / D203N / M225L / R2 36E, S22A / G46S / S101L / F161W / D203N / R236E, S2 2A / 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 / S1 40A / I143N / T145S / P213N、S27T / T48S / I82L / S14 0A / I143N / G149A、S32Y / A62T、S88T / R108T / V150 I / A184S / D203N / T221S / M225L / N243P、S98Q / A20 9V、T16E / D18R / G53A / I54V / R72P / L119I / A127M / Q167T / V200L、T16E / D18R / M56I / A127M / R138E / T 194S / V200L / M225L、T16E / D18R / M56I / K147Q / Q1 67T / 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 / N8 5D / L119I / A184S、T16E / D18R / S22A / V40T / A97G / S101L / L119I / A127M / Q167T / D203N、T16E / D18R / S88T / R108T / W228F、T16E / D18R / V40T / M56I / I82 F / R108T / L119I / F161W / L227R / Q258P、T16E / D18 R / V40T / S88T / L119I / A127M / V150I / D203N、T16E / D18R / Y26T / S88T / S101L / H156N / V200L、T17A / I 82L / F92Y / P213N、T17A / I82L / L90F / F92Y / A135G / S140A / I143N / G149A / Q167V、T17A / I82L / L90F / F92Y / Q167V、T17A / I82L / S140A / I143N / Q167V / P 213N / S252T、T17A / Q167V、T17A / S27T、T17A / S27 T / I82L、T17A / S27T / I82L / G149A / Q167V / P213N、 T17A / S27T / I82L / L90F / F92Y / Q167V、T17A / S27T / I82L / 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N122 R / A127S / T136A / A174K / N204K / S212L、S27L / V23 T / L90Y / F92G / S98E / T109L / A125S / T160V / A174K / S181C / S193K / T206G / S212M / R255M、S27L / A24V / F92G / S101D / A114V / A117N / A125S / T136V / D203 R、S27L / R12K / S32Q / S101Q、S27L / L90F / F92G / T1 36V / A174R / D203R, S27L / P20T / S32K / L90Y / D203 V, S27L / N2R / A14K / T17S / L90Y / T109K / T136V / A2 16P, S27L / N2R / V23T / A24V / S32M / L90Y / F92G, S2 7L / A14K / L90Y / F92L / S101D / A117N / D158I / D203 R、S27L / F21W / N87H / A114V / A117N / T136V、S27L / N2R / V23T / A24V / N87M / F92L / S101K / A125S / T136 A、S27L / L90Y / F92L / S101N / T109R / S110R / Q142W S27L / S32M / F92G / A216P, S27L / F21W / L90Y / F92 L / A114K / A117N / T136A / D203V、S27L / N2R / V23T / S32M / N87M / F92L / T136A、S27L / R12K / L90F / F92G / S101A / D203V、S27L / A24V / L90F / F92G、S27L / R1 2K / F92L / S101A / A125S / T136A / D203R、S27L / R12 K / V23L / L90F / F92L / A114K / T136A / D158E / D203V / I222L、S27L / L90F / F92G、S27L / A14K / P20T / S32 Q / L90Y / A125S / D203V、S27L / T17L / L90F / F92G / A 125S / A174R / D203V、S27L / N2R / N87K / A114K / A11 7Q / T136V / D203V, S27L / N2R / A114K / T136V / D203 V、S27L / N2R / T17A / T136V / A216P、S27L / N2R / R12 K / N87F / T136V / D158E / A174R、S27L / D203R、S27L / R12K / T17Q / T136V / D203V / A216P、S27L / N87F / T 109L / Q142L、S27L / N2R / T17Q / A24V / A114K / A117 Q / T136A / D158E / A174K / D203V / I222L、S27L / N2R / P20T / F21W / N87M / T109R / A117N / A125S / T136V / D203V / I222L、S27L / V23T / A24V / T136A / D158E / A 174K / D203V / I222L、S27L / R12K / V23L / A114K / T1 36A / D158I / D203V / I222L、S27L / N87Y / T136V / D1 58E / D203V、S27L / T109K / S110R / D203R、S27L / N2 R / T17L / A125S / T136A / D158E、S27L / A24V / D158E / A174K / A216P, S27L / T17G / V23L / N87L / A117Q / A1 25S / T136V / D158E / V229C, S27L / V23L / N87L / T10 9R / A114K / A117N / A125S / T136V / Q142L、S27L / N8 7Y / S101A / A114V / A117Q / T136V / A174K / A216P / I 222L, 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Q142W / A174K / D203R、S27L / R12K / S32M / L90F / F92G / S101W / A1 14V / D158I / I222L、S27L / P20T / S32M / L90Y / F92L / A114V / A117N / I222L、S27L / T17C / L90Y / F92G / T 136A / A174K / D203V、S27L / S32Q / N87Y / F92G / T10 9K / A114K / A117N / T136V / D203V / V229C, S27L / F2 1W / L90F / F92G / T136V / A174R、S27L / P20T / N87Y / T136A / D203V / I222L、S27L / A24V / L90Y / F92L / D1 58I / A174K / D203R / I222L、S27L / F21W / S32Q / L90 Y / S110R / A114K / T136V / D203R / A216P / V229I、S2 7L / N87H / F92G / D203R, S27L / R12K / S32K / Y60A / A 125S / T136V / D158L、S27L / V23T / A24V / S32Q / L90 F / F92L / T136A、S27L / F92L / A114V / D158E / D203R 、S27L / S32Q / L90Y / D203V / V229I、S27L / V23T / S3 2Q / Y60H / T109K / T136V / A216P / I222L、S27L / A14 K / A24V / L90F / F92G / T109K / T136V / D158L / A174R / D203V / I222L / V229I、S27L / R12K / N87F / A174K、 S27L / P20T / S32Q / Y60H / T109K / A114V / T136A / A1 74K, S27L / V23T / A24V / Y60A / A125S / A174R / A216 P、S27L / F92Y / D158L / I222L、S27L / V23L / A24V / L 90Y / F92G / D203V / I222L、S27L / V23T / A24V / S32Q / L90F / F92G / S101H / T136A / A174K / D203V / V229C S27L / S32K / N87H / A125S, S27L / P20T / F21W / F92 G / A114V、S27L / T17L / V23L / A24V / L90Y / F92L / A1 17N / T136V / L137M / Q142L、S27L / P20T / L90Y / D20 3R, S27L / S110R / Q142L / D158I / D203R / V229I, S2 7L / P20T / S32Q / L90F / F92L / T109K / S110R / A125S / D203V、S27L / V23T / A24V / L90F / F92G / D158E / D2 03R, S27L / F92G / T136A / A174R / D203V / V229C, S2 7L / P20T / L90Y / F92G / T109R / S110R / A125S / D203 R、S27L / A14K / L90Y / F92L / D158L / A174R / D203V、 S27L / F21W / S32Q / L90Y / F92G / T109L / S110R / A11 7N / D158I / A174K / D203V、S27L / R12K / A114K / A11 7N / D158E / D203V、S27L / T17S / N87H / T109R / A114 K / 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 / D2 03V、S27L / L90Y / F92G / T136A / D158E / D203V / A21 6P / V229I、S27L / A14K / S32M / F92L / A125S、S27L / L90Y / F92G / T136V / D158L / D203V、S27L / A24V / L9 0Y / F92G / S101A / T109L / S110R / A117Q / A174R / D2 03R、S27L / P20T / S101D / A114V / T136V / Q142W / D1 58E / D203R / A216P、S27L / N87Y / F92G / S101Q / A11 4K / A117N / T136A / D158L / D203R / A216P、S27L / V2 3T / A24V / N87Y / L90Y / F92L / S101D / T109R / A174K / D203R / A216P、S27L / N2R / P20T / F21W / L90F / F92 Y、S27L / L90Y / F92G / D158I / D203V / V229C、S27L / S32Q / F92G / Q142W / I222L、S27L / N2R / R12K / S32M / N87Q / L90F / F92L / I222L、S27L / R12K / S32K / F92 L / A114V / A117Q / T136A / D158E / A216P、S27L / L90 Y / F92G, S27L / R12K / F21W / S32M / F92G / T136V / Q1 42L / D203R、S27L / R12K / A14K / L90Y / F92L / A125S / A174K、S27L / A24V / A114V / A117Q / T136V / D203V 、S27L / R12K / T17Q / V23L / S32M / L90Y / F92G / S101 D / T136V / I222L、S27L / V23T / A24V / S32Q / F92L / S 101N / T109L / S110R / D203R、S27L / P20T / Y60H / N8 7K / T136A / A174R / D203V, S27L / P20T / F21W / L90Y / F92L / T136A / A174R / D203V / V229C、S27L / T17G / F21W / S32K / L90F / F92G / A125S / T136A / A174K, S2 7L / 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 / T1 7C / A24V / N87K / A174K / A216P、S27L / N87M / Q142L / I222L、S27L / F92L / S98T / R108C / A117Q / A127S / Q142L / Q189V / A216P / R236Q、S27L / S22K / F92L / R 108H / A127S / T136A / N211I、S27L / F21W / T48S / L9 0Y / F92L / T109L / A127S / T136V / N204K / S223A、S2 7L / T17L / L90Y / F92L / S98M / A174K / D203V / N211M / S212L / S223A、S27L / N87K / S98M / N211M、S27L / N 9E / S22P / L90Y / N204K、S27L / S32M / T48S / F92G / A 127S / Q142W、S27L / T17S / S22K / L90F / F92L / D158 E / D203V / V219I / S252T、S27L / L90F / F92L / A127S / D203V、S27L / T25F / L90Y / F92G / N204R / S223A / N 231S / R236C、S27L / F21W / S22V / T48S / L90F / F92L 、S27L / S1A / S98T / S113K / A114V / L119M / A127S / Q 142W / S193K / V219K / S252T / R255L、S27L / A24V / I 82L / L90F / F92G / T109L / L119M / A127S / E173R / N2 04K、S27L / T17C / Y60H / L90F / A127S / E173R / A174 R / N204K, S27L / L90F / F92L / Q142W / D203V / S223A S27L / P20Q / L90Y / Q142L / S223A, S27L / N9S / Y60 A / A216P / R236Q / R255M、S27L / F92G / R108C / S110 R / A117Q / T136A / N211M、S27L / S22P / Y60H / S98A / S113K / A114K / T136V / Q189L / S193H, S27L / V23L / S98A / Q142L / N211M, S27L / V23L / Y26L / L31M / T48 S / F92G / A174R / N204K / S252T、S27L / S13R / A14K / Y26T / T136A / S181R / N211M / S212M、S27L / F21W / I 82F / L90Y / F92G / A127S / N211M、S27L / P20T / Y60H / S98L / T136A / R255L、S27L / P20T / S34R / N87M / D1 58E / S252T, S27L / T25A / L90F / F92G / Q189L / S193 P / V229I、S27L / V219L / I222L、S27L / N9E / T48S / L 90F / F92L / S98N / R108H / S110R / S113Y / N211V / S2 12L / S252T, S27L / L90F / F92Y / S113R / V219I / R25 5L, S27L / F21W / S22K / T48S / F92G / T109R / A127S, S27L / S1G / A14K / P20T / S32K / T48S / L90Y / F92L / T 109L / T160R / V219L / I222L / S252T / R255L、S27L / L90F / F92G / D158E / T160R / S193N / N204K / S223A、 S27L / A14K / Y26T / F92L / Q142L / S212L、S27L / S22 R / L90Y / F92G / V126I / A127S / A174K / N204K、S27L / P20T / T25A / L90F / F92L / A117N / L119M / S252T / R 255L、S27L / T17A / A24V / T48S / L90F / F92G / T109R / A127S / T160K / D203R / S223A、S27L / F21W / T48S / T109L / A127S / V219I / R255L、S27L / A14K / S22P / T 48S / L90F / F92L / T109L / A127S / Q142L / A174R / N2 0 4K / S223A、S27L / T136A / S212M / N231S、S27L / F21 W / L90F / F92G / A127S / S223A、S27L / T48S / L90Y / F 92L / R108S / S110R / A127S / T136V / E173R / S223A、 S27L / T48S / F92G / S98E / Q142L / Q189V / S193P / N2 04R / S223A、S27L / T17S / L90F / F92Y / S101K / A127 S / T136V / N204R / N211I / V229I / N231S、S27L / N9E / F21W / T48S / L90Y / F92L / T109R / N204K / R236Q、S 27L / N9E / S22R / T48S / F92L / S101N / A127S / E173R / N204R、S27L / S22K / N87F / A117Q / S181C / N204K / N211M / V229I / N231S / R236Q、S27L / N9S / T48S / L9 0F / F92G / S101N / A127S / N204K / A216P / S252T / R2 55M, S27L / I82L / S193H / N211M / S212F / S223A, S2 7L / A14K / P20T / T48S / L90F / F92G / S98N / R108C / T 136A / N204K / N211L / S212L / R236Q / S252T, S27L / S13R / T17L / T25Q / L90F / F92G / R108C / A127S / T16 0R / I222L / R255L、S27L / T48S / L90Y / F92G / T109L / S193N、S27L / S22V / T48S / L90Y / F92L / S98E / A12 7S / T136A / N204K / A216P / S223A、S27L / V23L / N87 H / F92L / R108C / S110R / N122A / T160S / Q189L / S19 3P / N204K / S223A / R255L, S27L / F92G / A127S / A17 4R / 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、S27 L / F92G / A127S / Q189V / S193N / S223A、S27L / S34R / F92G / S110R / D158L / D203V / R255M、S27L / N9E / F 21W / T48S / F92G / T109L / A127S / R236C、S27L / S1A / S22P / T48S / L90Y / F92G / A127S / E173R / A174R / N 204K / S223A, S27L / S22K / T48S / T160S / N204K, S2 7L / F21W / T48S / L90Y / F92G / S98V / R108C / A127S / N211L / S212M / N231S / R236C、S27L / V23L / A24V / A 114K / T136A / I222L / V229I、S27L / P20T / S101H / T 109R / 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 / T 48S / L90F / F92G / A127S / A174R / P192A / S193H / N2 04K / S223A / V229I / N231S / R236Q / R255L、S27L / N 9E / T48N / N87H / F92L / A174K / Q189L / S193K / N204 R / V219I、S27L / A14K / V23L / Y26L / L90F / F92G / S9 8E / S113K / A114K / T136A / V229I / N231S、S27L / L9 0Y / F92L / S101D / A117N / Q142L / S193N、S27L / S22 K / T25A / L90Y / Q142L / E173R / N204K / S223A、S27L / P20T / L90Y / T109L / A127S / A174K / V229C、S27L / S22V / L90Y / F92L / A117Q / R236C / R255L, S27L / V2 3L / A24G / Y26T / I82F / L90Y / F92G / R108H / T136A / D158L / T160K / D203V、S27L / A14K / S22K / F92G / A1 27S / N204K / V229I, S27L / S22V / L90F / F92G / A127 S / A174R / A216P / R236C、S27L / V23L / Y60A / S98T / T109L / A127S / T136V / T160Q / N204K / S223A / R255 L、S27L / S13R / L90Y / S98L / T160V / Q189L / S193P / A216P / S252T / N253S / R255L, S27L / S22R / I82M / F 92L / T109L / A127S / Q142W / Q189L / S223A、S27L / S 1G / Y60A / L90F / F92L / N122E / T136A / N204K / S223 A / N231S、S27L / S32K / F92G / S98L / R108Q / S110R / R255L、S27L / L90Y / F92G / L119M / A127S / A174R / S 252T, S27L / L90F / S110R / S181R / N211F, S27L / A1 4K / S22P / Y60H / S110R / N122S / D158I / N204K / S22 3A / R255M, S27L / F92L / S98V / T109L / N122A / E173 R / R255L、S27L / S22R / N87L / F92G / R236C、S27L / S 32M / 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 / S22 R / T48S / F92G / R108H / S110R / A127S / E173R / N204 K / S223A / R236Q、S27L / A14K / L90Y / F92G / A127S、 S27L / A14K / T48S / F92G / E173R / N204R / S223A / N2 31S, S27L / L90Y / T109R / S113N / V219L / I222L, S2 7L / N9S / T48S / L90Y / T136A / T206G / S223A, S27L / S22K / T48N / L90F / F92G / N204K / S223A / V229C / N2 31S、S27L / S22P / T48S / L90F / F92G / A127S / A174R / N204R / S223A、S27L / S1G / P20T / S32K / S34R / T48 S / N87F / S98A / A127S / T136A、S27L / F21W / S22R / S 98E / Q142L / T160V / S223A / S252T / R255L、S27L / P 20T / T48S / F92Y / R108K / S110R / A127S / V219L / S2 23A / V229I / R236Q, S27L / Y26T / L90Y / F92L / A127 S / V219L / S252T / R255L、S27L / N9S / F21W / T48S / F 92L / A127S / D203V / S212F、S27L / F21W / Y60H / S98 E / L119M / D158E / R236Q、S27L / F21W / S22R / L90F / F92L / N122S / Q142W / R236Q、S27L / F21W / T48S / L9 0F / F92G / T109K、S27L / L90Y / F92L / T109R / A127S / E173R / N204K / S223A、S27L / N9S / F21W / T48S / L9 0Y / F92G / A127S / N204K / V219I / R255M、S27L / N9E / I82F / L90Y / F92L / S95N / T109L / A127S / A174R / S 223A, S27L / L90F / F92L / S98L / N204K / A216P, S27 L / 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 / L 119M / N211M / R236Q、S27L / L90Y / F92G / N122S / A1 27S / E173R / A216P / R236Q, S27L / P20T / S32K / L90 F / F92G / N122E / D158E / T160S / Q189L / S223A / S25 2T、S27L / N9S / F21W / T48S / N87H / L90F / F92L / S11 3N / V229C, S27L / T25Q / I82L / L90F / F92G / S98L / S 113R / A114V / T136A / S181R、S27L / P20T / R108Q / S 110R / D203V / N211I、S27L / S1G / N2R / P20T / Y26T / S32M / T48S / L90Y / F92L / A127S / N204R / V219L、S2 7L / A14K / P20T / T48N / L90F / F92G / A117Q / A127Sぁ S27L / N9S / T48N / L90Y / F92L / S98M / T109K / A127S / N204R / N211I / S212L / V219I、S27L / F21W / S22R / T48S / L90F / F92L / R108Q / A174K / N204K / S212F / S 223A, S27L / S22R / Y60H / S98T / T136V / S193K / R23 6Q、S27L / S22K / Y26K / N87Y / L90Y / F92G / T109R / A 127S / S252T / R255L、S27L / L90F / F92L / T109L、S2 7L / F21W / F92L / N204K / S252T / R255L, S27L / V23L / L90Y / F92G / A127S / S193K / N204R / V219I / R236C / R255L、S27L / F21W / L90Y / F92G / A114V / N122S / A 127S / N204K / S223A / S252T、S27L / P20T / Y60A / T1 09L / Q189L / S212L、S27L / S22V / T48S / L90Y / F92G / N204K、S27L / S22V / S32K / T48S / I82F / F92G / A12 7S / N204K / S223A、S27L / A55L、S27L / A97V、S27L / F250L, S27L / T109G, S27L / G38D, S27L / A97S, S27 L / A55V / A216T、S27L / P20D、S27L / A55V、S27L / T1 09Y、S27L / V165I、S27L / A184G、S27L / A97E、S27L / A184S、S27L / A97F、S27L / A97T、S27L / K197Y、S2 7L / A55I, S27L / A97P, S27L / A55M, S27L / P20E, S2 7L / A117L, S27L / T109L, S27L / K197T, S27L / T136 S, S27L / A97L, S27L / T109A, S27L / P20I, S27L / L1 91V, 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、S 27L / Y26C, S27L / T17N, S27L / S57M, S27L / D249I S27L / T17S, S27L / P164T, S27L / Q142L, S27L / D24 9N、S27L / I185R、S27L / V83L、S27L / G149S、S27L / A24D, S27L / R251V, S27L / V83I, S27L / S110N, S27 L / T17M、S27L / F161W、S27L / G46E、S27L / D249T、S 2 7L / T17I, S27L / S57C, S27L / S57T, S27L / S57L, S2 7L / Q167T, S27L / S57E, S27L / T17K, S27L / G149N S27L / Q142E, S27L / S57F, S27L / T17A, S27L / P164 H, S27L / T17R, S27L / G149T, S27L / R251T, S27L / S 101Y、S27L / S57V、S27L / A24T、S27L / I185E、S27L / G46N、S27L / P164N、S27L / P164S、S27L / Q167V、S 27L / F161V, S27L / S57I, S27L / G149D, S27L / Q167 I, S27L / I185Q, S27L / N2L / T17I / T136S / P164H / D 249M、S27L / P20E / F21Y / I139T / P164H / K197T / R2 51E、S27L / A114V / A117N / T136A / I222L / V229I、S 27L / P20T / A24V / S101Q / T109R / A117Q / I222L / V2 29I、S27L / S110D / A184S / K197T、S27L / T136S / D2 49T、S27L / F21Y / A184S、S27L / N2F / P20E / S110D / A184S / L191V / R251V、S27L / T17G / V23T / A24V / T1 09L / A117Q / T136A / I222L / V229I、S27L / P20D / F2 1Y / A55T / F250L / R251V、S27L / T109R / A117N / I22 2L / 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 / I 222L / V229I、S27L / N2S / A55V / I185Q / R251E、S27 L / N2F / S101Y / Q142L / G149T / Q167I / I169C / V229 L、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、S 27L / V23L / A114V / A117N / T136A / I222L / V229I、S 27L / A24N、S27L / T17A / P20T / S101Q / T109K / S110 R / T136A / I222L / V229C、S27L / P164T / V165I / R25 1E、S27L / F21Y / G46E / A117T / T136S / G149C / R251 Q、S27L / G46N / I139T / Q142E / P164T / I185A / V229 L / F250V、S27L / V23L / T136A / I222L / V229I、S27L / V23T / A24V / T109K / A114K / A117Q / T136A / I222L / V229C、S27L / F250L / R251Q、S27L / P20E / F21Y / S 57T / P164N / A184C / V229C、S27L / P20T / V23T / A24 V / I222L / V229I、S27L / V83L / A97S / A184C / D249S 、S27L / P20T / S101H / A114V / A117N / I222L / V229I 、S27L / N2E / R251L、S27L / P20E / F21Y / S110D / A11 7S、S27L / T17A / V23T / A24V / T136A、S27L / P20D / S 57E / S101C、S27L / N2S / V83L / A184S、S27L / T17S / S101N / T109K / A117N / T136V / Q142L / I222L / V229 C、S27L / P20D / A55T / A117Y / K197R、S27L / P20T / I 222L / V229C、S27L / T17I / A24D / S57I、S27L / N2F、 S27L / T109K / A117N / T136V / I222L / V229C、S27L / N2L / A24T、S27L / N2F / P20I / F21Y / R251Q、S27L / N 2F / G46E、S27L / T136V / Q142W / I222L / V229I、S27 L / K197R / R251E、S27L / T17S / R251T、S27L / P20I / F21Y / A117F / I185G / D249N、S27L / T17L / S101M / T 109K / S110R / A117Q / I222L / V229C、S27L / T17Q / T 136A / I222L / V229C, S27L / V229I, S27L / T17A / F2 1W / S101Q / I222L / V229C、S27L / T17S / F21W / Q142 L、S27L / T17L / T109L / S110R / A117Q / I222L / V229 I、S27L / F21W / T136A、S27L / P20E / F21Y / G46E / V8 3L / A97Q, S27L / P20T / F21W / T136A / Q142L / I222L / V229I、S27L / T17G / F21W / A117N / I222L / V229I、 S27L / F21W / T109K / S110R / T136A / V229I、S27L / T 17C / T109K / A117N / T136A / I222L / V229I、S27L / P 20T / T109L / S110R / A117N / I222L / V229I、S27L / A 24N / P164S / K197V / R251T、S27L / V23L / A117Q、S2 7L / A55V / V83L / A117T, S27L / P20D / G46S / A55I / K 197T, S27L / A117Y, S27L / F21W / S101M / I222L / V2 29C、S27L / N2L / T17N / S57C / S110D / A117L / P164N / I185G / V229L / R251T、S27L / N2E / G46S / V83L / T1 36S / A172T / V229C / D249N, S27L / P20T / T109R / A1 14K / A117Q / T136V / I222L / V229C, S27L / V23L / A2 4V / 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、S2 7L / P20T / F21W / Q142W / I222L / V229C、S27L / N2L / G46E / T109K / F161V、S27L / T17S / V229I、S27L / T1 7Q / T136A / I222L / V229I、S27L / N2L / A172T、S27L / P20D / F21Y / F250V、S27L / N2L / A24H / A55C / V229 L、S27L / N2L / P20E / F21Y / V229L、S27L / V23T / T10 9L / A114V / T136A / Q142L / I222L / V229C、S27L / P2 0T / F21W / I222L / V229C、S27L / T17H / F21W / T136V / Q142W / I222L / V229I、S27L / G46E / A55I / Q142E、 S27L / N2F / R251Q, S27L / F21W / Q142L / I222L / V22 9C、S27L / I222L / V229I、S27L / P20T / S101M / A114 V / A117N / Q142W / I222L / V229C、S27L / T17Q / A24V / S101W / T136A、S27L / N2S / P20D / A97C / A117F / F2 50L / R251E、S27L / S101M / A114V / A117Q / T136A、S 27L / T163I、S27L / S101Q / Q142L / I222L / V229I、S 27L / F21W / I222L / V229C、S27L / S101M / T136A / I2 22L / V229I, S27L / F21W / A24V, S27L / A24D / K197L S27L / T17H / S110K, S27L / P20T / F21W / V229I, S2 7L / T17G / P20T / F21W / A117Q / I222L / V229C、S27L / F21W / A24V / A114V / A117Q / Q142W / I222L / V229C 、S27L / P20T / A117Q / I222L / V229C、S27L / T17Q / V 23T / A24V / I222L / V229I、S27L / N2F / D249N / F250 L、S27L / V23T / Q142L / I222L / V229C、S27L / P20E / A24D / D249S / F250V、S27L / A24D / S110K、S27L / T1 09R / I222L / V229I、S27L / P20E / F21Y / K197T、S27 L / T17A / V23L / S101M / T109K / S110R / A114V / A117 Q / T136V / V229I、S27L / P20T / F21W / Q142W / I222L / V229I、S27L / F21W / I222L / V229I、S27L / P20T / V 23T / A24V / T109L / S110R / I222L / V229I、S27L / A2 4D / L191F / R251Q、S27L / A24D / T136S / F250V、S27 L / P20T / F21W / T109L / T136A / Q142W / I222L / V229 C, S27L / N2E / I169L / F250L / R251Q, S27L / N2S / A2 4N / G46E / A55L / Q142D / V229C / R251L、S27L / N2E / P20E / F250V / R251L、S27L / N2F / P20E、S27L / A24V / V229C, S27L / P20D / F21Y / G46N / S110K / T163I / Q 167V, S27L / P20T / T109K / A114K / Q142W / I222L / V 229C, S27L / T17Q / S101D / T136A / I222L / V229I, S 27L / F21W / T109R / A117N / T136V / Q142W / I222L / V 229C, S27L / A24D / T109L / K197L, S27L / T17Q / Q14 2L / I222L / V229I, S27L / T17A / A24V / S101D / S110 R / T136A, S27L / P20T / F21W / T109R / A117Q / T136V / Q142L / I222L / V229I, S27L / P20T / F21W / T109K / A117N / I222L / V229C, S27L / P20T / S101M / I222L / V229I, S27L / N2S / A24D / S101Y / P164E / V165I / I1 85E, S27L / T17N / A117S / V229L / R251A, S27L / S10 1W / A114V / T136V / V229C, S27L / P20D / G46N / A55C / S110N / T163I / A184S, S27L / A24H / G46E, S27L / T 17H / S101Q / T136V / Q142L / V229C and S27L / V23T / A24V / I222L / V229C. In some embodiments, the variant Bhr-PETase is A102V / T13 6M, 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 / M 56I / R138E / I139A / T194S / D203N / V219K / F250I、 D18R / M56I / I139A / L227R / W228F、D18R / M56I / R1 3 8E / I139A / N190S / D203N / M225L、D18R / M56I / R72 P / H77Q / F92Q / A127M / V150I / A184S / D203N / N254 R、D18R / N85D / R108T / L119I / N190S / T194S、D18R / V40T / I139A / D203N、D18R / V40T / I82F / S101L / N 105D / L119I / H156N / F161W / N190S / T194S / D203N 、D18R / V40T / M56I / R108T / R138E / H156N、G53A / R 108T / Q167T / A184S / T194S / N243P、I143R / Q167T / V198A / W228F、I82F / R108T / L119I / V150I / F161 W / Q167T / T194S / D203N、L15I / D18R / R108T / R138 E / I139A / F161W / A184S / N190S / D203N、M56I / I82 F / R108T / I139A / F161W / T194S / D203N / N241P、M5 6I / S88T / R108T / N190S / L227R、N204A / Q237R、N9 A / D18R / M56I / N85D / L119I / N254R、N9A / S22A / G4 6S / M56I / R72P / F92Q / L119I / T221S / M225L、N9A / S22A / Q167T、N9A / V40T / L49G / I54V / R108T / V150 I / D203N / T221S / M225L、N9S / T25H、Q5E / N9A / M56 I / F92Q / R108T / L119I / Q167T / N253Y、Q5E / S22A / R72P / H77Q / F92Q、R108T / L119I / K147Q / F161W / A 184S / D203N、S101F / T136A、S22A / A24S / G46S / V1 50I / Q167T、S22A / A97G / V150I / D203N / M225L / R2 36E、S22A / G46S / S101L / F161W / D203N / R236E、S2 2A / 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 / S1 40A / I143N / T145S / P213N、S27T / T48S / I82L / S14 0A / I143N / G149A、S32Y / A62T、S88T / R108T / V150 I / A184S / D203N / T221S / M225L / N243P、S98Q / A20 9V、T16E / D18R / G53A / I54V / R72P / L119I / A127M / Q167T / V200L、T16E / D18R / M56I / A127M / R138E / T 194S / V200L / M225L、T16E / D18R / M56I / K147Q / Q1 67T / 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 / N8 5D / L119I / A184S、T16E / D18R / S22A / V40T / A97G / S101L / L119I / A127M / Q167T / D203N、T16E / D18R / S88T / R108T / W228F、T16E / D18R / V40T / M56I / I82 F / R108T / L119I / F161W / L227R / Q258P、T16E / D18 R / V40T / S88T / L119I / A127M / V150I / D203N、T16E / D18R / Y26T / S88T / S101L / H156N / V200L、T17A / I 82L / F92Y / P213N、T17A / I82L / L90F / F92Y / A135G / S140A / I143N / G149A / Q167V、T17A / I82L / L90F / F92Y / Q167V、T17A / I82L / S140A / I143N / Q167V / P 213N / S252T、T17A / Q167V、T17A / S27T、T17A / S27 T / I82L、T17A / S27T / I82L / G149A / Q167V / P213N、 T17A / S27T / I82L / L90F / F92Y / Q167V、T17A / S27T / I82L / P213N、T17A / S27T / I82L / P213N / S252T、T 17A / S27T / I82L / S252T、T17A / S27T / I82L / T145S / G149A / P213NぁT17A / S27T / L90F / A135G / S140A / Q167V / P213N / S252T, T17A / S27T / L90F / F92Y / A1 35G / Q167V / S252T, T17A / S27T / L90F / F92Y / P213 N、T17A / S27T / T48S、T17A / S27T / T48S / A135G / Q1 67V / P213N、T17A / S27T / T48S / A135G / S140A、T17 A / S27T / T48S / I82L、T17A / S27T / T48S / I82L / L90 F / F92Y / Q167V / S252T、T17A / S27T / T48S / I82L / L 90F / F92Y / S140A / T145S、T17A / S27T / T48S / I82L / L90F / F92Y / S140A / T145S / P213N、T17A / S27T / T 48S / I82L / L90F / F92Y / S252T、T17A / S27T / T48S / I82L / L90F / P213N / S252T、T17A / S27T / T48S / I82 L / L90F / Q167V、T17A / S27T / T48S / I82L / L90F / Q1 67V / P213N / S252T, T17A / S27T / T48S / I82L / P213 N / S252T, T17A / S27T / T48S / L90F / F92Y, T17A / S2 7T / T48S / P213N、T17A / S27T / T48S / T145S / Q167V 、T17A / T48S / I82L、T17A / T48S / I82L / F92Y / Q167 V / S252T、T17A / T48S / I82L / L90F / A135G / S140A / Q167V、T17A / T48S / P213N、T17K / A125SおよびV177A / A216L. The variant Bhr-PETase has the sequence A102V / T136M, A14S / L1 5I / S22A / F92Q / I143R / Q167T / V219K, D18R / G46S / M56I / R108T / A127M / R138E / I139A / N190S / L227 R / W228F / R236E, D18R / I139A / F161W / W228F, D18 R / I54V / I82F / N105D / A127M / A184S / S218A / V219 K / 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 / F 92Q / A127M / V150I / A184S / D203N / N254R, D18R / V 40T / I139A / D203N, D18R / V40T / I82F / S101L / N10 5D / L119I / H156N / F161W / N190S / T194S / D203N, D 18R / V40T / M56I / R108T / R138E / H156N, G53A / R10 8T / Q167T / A184S / T194S / N243P, I143R / Q167T / V 198A / 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 / M5 6I / R72P / F92Q / L119I / T221S / M225L、N9A / S22A / Q167T、N9A / V40T / L49G / I54V / R108T / V150I / D20 3N / T221S / M225L, Q5E / N9A / M56I / F92Q / R108T / L 119I / Q167T / N253Y、Q5E / S22A / R72P / H77Q / F92Q 、R108T / L119I / K147Q / F161W / A184S / D203N、S10 1F / T136A、S22A / A24S / G46S / V150I / Q167T、S22A / A97G / V150I / D203N / M225L / R236E、S22A / G46S / S101L / F161W / D203N / R236E、S22A / L49G / M56I / V 83T / S101L / R108T / L119I / A127M / T194S、S27T / I 82L / P213N、S27T / T48S / I82L / F92Y / S252T、S27T / T48S / I82L / L90F / A135G / S140A / I143N / T145S / P213N、S27T / T48S / I82L / S140A / I143N / G149A、S 88T / R108T / V150I / A184S / D203N / T221S / M225L / N243P、T16E / D18R / G53A / I54V / R72P / L119I / A12 7M / Q167T / V200L、T16E / D18R / M56I / A127M / R138 E / T194S / V200L / M225L、T16E / D18R / M56I / K147Q / Q167T / A184S / D203N、T16E / D18R / M56I / N85D / S 88T / R108T / F161W / Q167T、T16E / D18R / R108T / K1 47Q / Q167T / A184S / N190S / T194S、T16E / D18R / S2 2A / M56I / N85D / L119I / A184S、T16E / D18R / S22A / V40T / A97G / S101L / L119I / A127M / Q167T / D203N、 T16E / D18R / S88T / R108T / W228F、T16E / D18R / V40 T / M56I / I82F / R108T / L119I / F161W / L227R / Q258 P、T16E / D18R / V40T / S88T / L119I / A127M / V150I / D203N, T16E / D18R / Y26T / S88T / S101L / H156N / V2 00L、T17A / I82L / F92Y / P213N、T17A / I82L / L90F / F92Y / A135G / S140A / I143N / G149A / Q167V、T17A / I82L / L90F / F92Y / Q167V、T17A / I82L / S140A / I14 3N / Q167V / P213N / S252T、T17A / Q167V、T17A / S27 T、T17A / S27T / I82L、T17A / S27T / I82L / G149A / Q1 67V / P213N, T17A / S27T / I82L / L90F / F92Y / Q167V T17A / S27T / I82L / P213N, T17A / S27T / I82L / P21 3N / S252T、T17A / S27T / I82L / S252T、T17A / S27T / I82L / T145S / G149A / P213N, T17A / S27T / L90F / A1 35G / S140A / Q167V / P213N / S252T、T17A / S27T / L9 0F / F92Y / A135G / Q167V / S252T、T17A / S27T / L90F / F92Y / P213N, T17A / S27T / T48S, T17A / S27T / T48 S / A135G / Q167V / P213N, T17A / S27T / T48S / A135G / S140A, T17A / S27T / T48S / I82L, T17A / S27T / T48 S / I82L / L90F / F92Y / Q167V / S252T, T17A / S27T / T 48S / I82L / L90F / F92Y / S140A / T145S, T17A / S27T / T48S / I82L / L90F / F92Y / S140A / T145S / P213N, T1 7A / S27T / T48S / I82L / L90F / F92Y / S252T, T17A / S 27T / T48S / I82L / L90F / P213N / S252T, T17A / S27T / T48S / I82L / L90F / Q167V, T17A / S27T / T48S / I82 L / L90F / Q167V / P213N / S252T, T17A / S27T / T48S / I82L / P213N / S252T, T17A / S27T / T48S / L90F / F92 Y, T17A / S27T / T48S / P213N, T17A / S27T / T48S / T1 45S / Q167V, T17A / T48S / I82L, T17A / T48S / I82L / F92Y / Q167V / S252T, T17A / T48S / I82L / L90F / A13 5G / S140A / Q167V, T17A / T48S / P213N, T17K / A125 S and V177A / A216L In some embodiments, the variant Bhr-PETase is L90F / F92G / D158L / A174R / D203V、V23L / A24V / S32K / N87F / F9 2Y / A125S / T136A, P20T / L90Y / F92L / S101M / D158 E / A174K / I222L、D18R / S32K / Y60H / L90F、P20T / F 21W / A24V / L90Y / F92L / T109K / A125S / T136A、Y60 H / S101A / A114K / A117Q / D203R / I222L、S32Q / F92 G / S101Q / A114K / Q142W / D158I / D203R / I222L、T1 7G / L90F / F92L / T109R / A114K / D158E / D203V / I22 2L, R12K / S32K / F92L / T136A / D158E / A174R, L90F / F92G / T109K / A125S / A174K、S32K / L90F / F92L / T 109K / A114K / Q142W、V23T / S32Q / N87I / L90F / F92 Y / S101N / A114V / Q142W / A174R / D203R / I222L / V2 29I、S32Q / F92G / A114V / D203V、R12K / S32K / L90Y / F92L / T136A / A174K / D203V / I222L、R12K / A14K / V23T / L90Y / F92G / S101A / D203R, T17G / S32M / L90 Y / F92Y / D203R / A216P、S32Q / F92L / A114V / A125S 、P20T / F21W / S32M / L90Y / F92G / A114K / A125S / T1 36V / D158E / A174K / D203V / I222L、R12K / F21W / S3 2K / F92L / T109K / A125S / T136V / D158E / D203R、T1 7Q / L90Y / F92L / A174K / D203V / I222L、F21W / F92L / S101A / A117Q / D203RぁT17A / S32M / Y60H / Q142L / D203V / I222L、R12K / Y60H / L90F / S101R / A114V / A 117N / D158E、T17S / D18R / L90Y / F92G / A114V / A11 7Q / D203R、L90Y / F92L / S101A / S110R / A125S / Q14 2W / A174R / A216P、S32M / S110R / A125S / D158E、R1 2K / V23L / A24V / F92G / T136V / D203R、A24V / S32Q / F92L / T136V, S32K / L90Y / F92G / A114K / D203V, S3 2M / L90F / F92G / A114V / A117N / A125S / A174K / D20 3R、R12K / T17A / V23L / A24V / L90F / F92G / S101N / A 125S / Q142W / D158I / D203R、P20T / F21W / Y60I / S1 01A / T109K / A174K、T17C / L90Y / F92L / S101N / T10 9L / S110R / A125S / A174K / D203R、T17Q / L90F / F92 G / S101M / A117Q / T136A / D158I / A174R、V23L / L90 F / F92G / A125S / D203V / I222L、T17C / L90Y / F92L / A125S / T136V / D158E / A174K、V23L / S32Q / Y60I / A 117Q / T136A / A174K / V229C、V23T / L90F / F92Y / D2 03V、R12K / A24V / Y60H / A174R、T17G / P20T / Y60I / F92G / D203V、L90Y / F92L / S110R / A125S / D158E / D 203V、T17Q / L90Y / F92L / T109L / T136A / D158E / A1 74R / D203V / V229C、R12K / V23T / A24V / S32Q / F92G / D158L / A174R / D203R、S32M / N87Y / L90F / F92G / A 174K / D203V、T17S / V23L / S32Q / L90F / F92L / A114 K / A117N / A125S / T136V / D158E / D203V / V229I、P2 0T / F21W / S32M / L90Y / F92G / T109L / S110R、A14K / S32M / L90Y / S101R / A114V / T136A / A216P、P20T / F 21W / L90Y / F92G / A125S / A174K / V229I、V23L / S32 K / 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 / D2 03V、T17Q / A24V / N87M / F92G / A114K / A117N / D158 E / D203R / I222LぁN2R / S32Q / L90F / F92G / Q142L / D 203V、A14K / S32Q / L90F / F92G / A114K / A117N / A17 4R / D203V / A216P / I222L、R12K / P20T / D158E / D20 3R / I222L, N2R / A14K / T17A / L90F / F92G / S101H / A 174R / D203V、T17G / A24V / L90F / F92G / Q142W、S32 K / L90Y / F92Y / S101A、V23T / N87I / F92G / A125S / T 136A / D158L / D203R、T17S / N87L / V229I、A24T / L9 0F / F92L / A125S / T136A / D203V、R12K / A14K / F92G / T109R / T136A / Q142L / D158E / A174K / D203R / I22 2L, A24V / S32Q / Y60A / T136A, D18R / S32Q / N87H / L 90Y / F92G / S101K / Q142L / D158I / A174R、T17A / F2 1W / S32K / N87F / L90F / F92Y / S101Q / T136A / D203R 、A24V / L90F / F92G / A114V / A117N / T136V / Q142L / D203V / V229C、T17Q / L90Y / F92G / S101N / T109L / S 110R / T136V / Q142L / D203V / A216P、S32Q / L90Y / F 92L / T109K / A125S / T136V、Y60H / N87K / A114K / A1 17Q / D158E / A174K / A216P、P20T / L90Y / T109K / T1 36A / D158I / D203R、T17L / Y60A / D203V、N2R / S32Q / L90Y / F92Y / T109K / D203V、T17H / A24V / S32Q / L9 0Y / F92L / S101H / A114K / D158I / A174R / D203V、N2 R / S32M / F92Y / T109K / S110R / I222L、P20T / F92Y / S101K / A125S / D158E / D203R、R12K / A14K / P20T / F 21W / S32M / F92L、R12K / A14K / V23T / A24V / L90F / F 92L / S101W / T136A / D203V, T17L / S32K / F92L / T10 9L / A114V / T136V / D158L / R236H, P20T / F21W / S32 Q / N87F / F92L / D158E / D203V, F21W / L90F / F92G / S 101A / A114V / A125S / T136V / Q142L / D203V / A216P , P20T / F21W / S32K / F92G / A125S / V229I, V23T / A2 4V / 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 / T13 6V / A216P, P20T / F21W / L90Y / A125S, N2R / L90Y / A 114K / A117Q / D203V / V229I, F21W / S32K / L90Y / A1 25S / D158E / A216P / V229I, S27L / T136V, S27L / P8 T / T17Q / F21W / S101A / T136V / Q142L, S27L / N2R / T 17L, S27L / V23T / A24V / T136A / Q142W, S27L / F21W / T136V, S27L / T17Q / T109K / A114K / T136V / V​​​​​​​​ 27L / T109K / T136V / I222L / V229I、S27L / T17L / S1 01H / A117N / Q142L / I222L / V229I、S27L / S101Q / T 109L / A117N / T136A / Q142L、S27L / T109K / S110R / S193N / S252T / R255M、S27L / S22R / Y26K / R236Q、S 27L / L90F / F92L / S98E / S113Y / A114K / T136A / D15 8E / S181R / T206G / S212M / V219I、S27L / N9S / S22P / T48N / L90Y / F92G / T109K / S110R / T136A / Q189V / N211F / R236Q、S27L / T17A / Y26L / T48N / I82M / S10 1D / R236Q、S27L / Y26T / S101D / D158E / V219I / S25 2T、S27L / S13R / S98E / T136V / S181R / T206G / V229 I / N231S、S27L / F21W / F92L / S98N / S193P / I222L、 S27L / S1G / Y26K / L90Y / S113Y / A114V / T136A / Q18 9V / N204R / N211L / R236Q、S27L / S22K / I82L / L90Y / F92G / R108S / A117N / D158E / S193N、S27L / S1G / N 9S / T48S / L90Y / S98T / S101A / S113N / A114K / L119 M / S193N / T206G / S252T、S27L / N9E / R12K / S22P / V 23L / T160R / D203R / I222L、S27L / N9E / R12K / S22P / V23T / T48S / S98E / R108S / T160S / Q189V / T206G / S212L / V229I、S27L / S1A / N2R / N9S / T48N / L90F / F 92L / D203V / S223A、S27L / N9E / R12K / V23T / I82M / L90Y / F92L / T136V / N204K / N231S / R255M、S27L / S 1G / N9S / S22V / I82F / L90Y / F92L / A117N / L119M / Q 142L / T206G / S212L / S223A、S27L / N9S / Y60H / R10 8 C / S193P / V219L、S27L / N9E / S22K / S32M / L90F / F9 2G / R108T / L119M / Q189V / I222L / S223A / R236Q、S 27L / T17Q / F92G / S98N / Q142L / Q189L / 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N2R / P20T / F21W / N87M / T109R / A117N / A125S / T136V / D203V / I222L、S27 L / V23T / A24V / T136A / D158E / A174K / D203V / I222 L、S27L / R12K / V23L / A114K / T136A / D158I / D203V / I222L、S27L / N87Y / T136V / D158E / D203V、S27L / T109K / S110R / D203R、S27L / N2R / T17L / A125S / T1 36A / D158E, S27L / A24V / D158E / A174K / A216P, S2 7L / T17G / V23L / N87L / A117Q / A125S / T136V / D158 E / 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 / S 101A / D203V, S27L / T17L / N87M / Q142L / D203V, S2 7L / N2R / R12K / S101D / A117N / T136A / D203V、S27L / R12K / P20T / F21W / A114K / Q142W / D203R、S27L / N 2R / T17G / T109L / S110R / A114K / A117Q / T136V / Q1 42L / D203R、S27L / N2R、S27L / S32K / L90F / F92G / S 101M / A114V / A117N / A125S / T136A / D158E / D203V S27L / R12K / T17A / F21W / S32Q / Y60H / D203R S27 L / R12K / F92L / 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F21W / L90F / F92Y、S27L / L90Y / F92 G / D158I / D203V / V229C、S27L / S32Q / F92G / Q142W / I222L、S27L / N2R / R12K / S32M / N87Q / L90F / F92L / I222L、S27L / R12K / S32K / F92L / A114V / A117Q / T 136A / D158E / A216P、S27L / L90Y / F92G、S27L / R12 K / F21W / S32M / F92G / T136V / Q142L / D203R、S27L / R12K / A14K / L90Y / F92L / A125S / A174K, S27L / A24 V / A114V / A117Q / T136V / D203V、S27L / R12K / T17Q / V23L / S32M / L90Y / F92G / S101D / T136V / I222L、S 27L / V23T / A24V / S32Q / F92L / S101N / T109L / S110 R / D203R、S27L / P20T / Y60H / N87K / T136A / A174R / D203V、S27L / P20T / F21W / L90Y / F92L / T136A / A17 4R / D203V / V229C、S27L / T17G / F21W / S32K / L90F / F92G / A125S / T136A / A174K、S27L / T17G / N87Y / T1 36A / D203V / A216P、S27L / F21W / L90F / F92G / S101 D / A117N / T136A / A174K / D203V、S27L / R12K / A14K / A24V / L90Y / D203V、S27L / N2R / L90F / F92G / S101 M / Q142L / D158L、S27L / N2R / T17C / A24V / N87K / A1 74K / A216P、S27L / N87M / Q142L / I222L、S27L / F92 L / S98T / R108C / A117Q / A127S / Q142L / Q189V / A21 6P / R236Q、S27L / S22K / F92L / R108H / A127S / T136 A / N211I、S27L / F21W / T48S / L90Y / F92L / T109L / A 127S / T136V / N204K / S223A、S27L / T17L / L90Y / F9 2L / S98M / A174K / D203V / N211M / S212L / S223A、S2 7L / N87K / S98M / N211M, S27L / N9E / S22P / L90Y / N2 04K、S27L / S32M / T48S / F92G / A127S / Q142W、S27L / T17S / S22K / L90F / F92L / D158E / D203V / V219I / S 252T, S27L / L90F / F92L / A127S / D203V, S27L / T25 F / L90Y / F92G / N204R / S223A / N231S / R236C、S27L / F21W / S22V / T48S / L90F / F92L、S27L / S1A / S98T / S113K / A114V / L119M / A127S / Q142W / S193K / V219 K / S252T / R255L、S27L / A24V / I82L / L90F / F92G / T 109L / L119M / A127S / E173R / N204K、S27L / T17C / Y 60H / L90F / A127S / E173R / A174R / N204K、S27L / L9 0F / F92L / Q142W / D203V / S223A、S27L / P20Q / L90Y / Q142L / S223A、S27L / N9S / Y60A / A216P / R236Q / R 255M、S27L / F92G / R108C / S110R / A117Q / T136A / N 211M、S27L / S22P / Y60H / S98A / S113K / A114K / T13 6V / Q189L / S193H、S27L / V23L / S98A / Q142L / N211 M、S27L / V23L / Y26L / L31M / T48S / F92G / A174R / N2 04K / S252T、S27L / S13R / A14K / Y26T / T136A / S181 R / N211M / S212M、S27L / F21W / I82F / L90Y / F92G / A 127S / N211M、S27L / P20T / Y60H / S98L / T136A / R25 5L, S27L / P20T / S34R / N87M / D158E / S252T, S27L / T25A / L90F / F92G / Q189L / S193P / V229I、S27L / V2 19L / I222L, S27L / N9E / T48S / L90F / F92L / S98N / R 108H / S110R / S113Y / N211V / S212L / S252T、S27L / L90F / F92Y / S113R / V219I / R255L, S27L / F21W / S2 2K / T48S / F92G / T109R / A127S、S27L / S1G / A14K / P 20T / S32K / T48S / L90Y / F92L / T109L / T160R / V219 L / I222L / S252T / R255L, S27L / L90F / F92G / D158E / T160R / S193N / N204K / S223A、S27L / A14K / Y26T / F92L / Q142L / S212L, S27L / S22R / L90Y / F92G / V12 6I / A127S / A174K / N204K、S27L / P20T / T25A / L90F / F92L / A117N / L119M / S252T / R255LぁS27L / T17A / A24V / T48S / L90F / F92G / T109R / A127S / T160K / D2 03R / S223A、S27L / F21W / T48S / T109L / A127S / V21 9I / R255L、S27L / A14K / S22P / T48S / L90F / F92L / T 109L / A127S / Q142L / A174R / N204K / S223A、S27L / T136A / S212M / N231S、S27L / F21W / L90F / F92G / A1 27S / S223A、S27L / T48S / L90Y / F92L / R108S / S110 R / 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 / L90 Y / F92L / T109R / N204K / R236Q、S27L / N9E / S22R / T 48S / F92L / S101N / A127S / E173R / N204R、S27L / S2 2K / N87F / A117Q / S181C / N204K / N211M / V229I / N2 31S / R236Q、S27L / N9S / T48S / L90F / F92G / S101N / A127S / N204K / A216P / S252T / R255M, S27L / I82L / S193H / N211M / S212F / S223A、S27L / A14K / P20T / T 48S / L90F / F92G / S98N / R108C / T136A / N204K / N21 1L / S212L / R236Q / S252T, S27L / S13R / T17L / T25Q / L90F / F92G / R108C / A127S / T160R / I222L / R255L S27L / T48S / L90Y / F92G / T109L / S193N, S27L / S2 2V / T48S / L90Y / F92L / S98E / A127S / T136A / N204K / A216P / S223A、S27L / V23L / N87H / F92L / R108C / S 110R / N122A / T160S / Q189L / S193P / N204K / S223A / R255L、S27L / F92G / A127S / A174R / S223A / V229I / N231S / R236C、S27L / S1G / L90Y / A127S / E173R / S 223A, S27L / F92Y / S98N / Q142W, S27L / P20T / S32M / S34R / T48S / L90Y / F92L / R108K / A127S / Q142W / A 174R / N204K / V219I / S252T、S27L / F92G / A127S / Q 189V / S193N / S223A, S27L / S34R / F92G / S110R / D1 58L / D203V / R255M、S27L / N9E / F21W / T48S / F92G / T109L / A127S / R236C、S27L / S1A / S22P / T48S / L90 Y / F92G / A127S / E173R / A174R / N204K / S223A、S27 L / S22K / T48S / T160S / N204K、S27L / F21W / T48S / L 90Y / F92G / S98V / R108C / A127S / N211L / S212M / N2 31S / R236C、S27L / V23L / A24V / A114K / T136A / I22 2L / V229I, S27L / P20T / S101H / T109R / A114V / T13 6A / Q142L / I222L / V229I、S27L / V23L / S101H / T10 9K / A114K / A117N / T136V / Q142L / I222L / V229I、S 27L / T17L / F21W / S101H / T109R / A114V / T136A / Q1 42L / I222L / V229I、S27L / T17S / T136V / Q142L / I2 22L / V229C, S27L / T17C / S22P / T48S / L90F / F92G / A127S / A174R / P192A / S193H / N204K / S223A / V229 I / N231S / R236Q / R255L、S27L / N9E / T48N / N87H / F 92L / A174K / Q189L / S193K / N204R / V219I、S27L / A 14K / V23L / Y26L / L90F / F92G / S98E / S113K / A114K / T136A / V229I / N231S、S27L / L90Y / F92L / S101D / A117N / Q142L / S193N, S27L / S22K / T25A / L90Y / Q1 42L / E173R / N204K / S223A, S27L / P20T / L90Y / T10 9L / A127S / A174K / V229C, S27L / S22V / L90Y / F92L / A117Q / R236C / R255L、S27L / V23L / A24G / Y26T / I 82F / L90Y / F92G / R108H / T136A / D158L / T160K / D2 03V、S27L / A14K / S22K / F92G / A127S / N204K / V229 I、S27L / S22V / L90F / F92G / A127S / A174R / A216P / R236C, S27L / V23L / Y60A / S98T / T109L / A127S / T1 36V / T160Q / N204K / S223A / R255L, S27L / S13R / L9 0Y / S98L / T160V / Q189L / S193P / A216P / S252T / N2 53S / R255L, S27L / S22R / I82M / F92L / T109L / A127 S / Q142W / Q189L / S223A、S27L / S1G / Y60A / L90F / F 92L / N122E / T136A / N204K / S223A / N231S、S27L / S 32K / F92G / S98L / R108Q / S110R / R255L、S27L / L90 Y / F92G / L119M / A127S / A174R / S252TぁS27L / L90F / S110R / S181R / N211F、S27L / A14K / S22P / Y60H / S 110R / N122S / D158I / N204K / S223A / R255M、S27L / F92L / S98V / T109L / N122A / E173R / R255L、S27L / S 22R / N87L / F92G / R236C, S27L / S32M / S34R / I82L / F92G / N204K / S223A, S27L / A24V / T25Q / Y60I / A12 7S / A174K / N204K / V219I / S223A / S252T / R255M、S 27L / S98V / T136A / N211F, S27L / N9S / L90F / F92L / A 127S / N204R / S212M、S27L / S22R / T48S / F92G / R10 8H / S110R / A127S / E173R / N204K / S223A / R236Q、S 27L / A14K / L90Y / F92G / A127S、S27L / A14K / T48S / F92G / E173R / N204R / S223A / N231S、S27L / L90Y / T 109R / S113N / V219L / I222L、S27L / N9S / T48S / L90 Y / T136A / T206G / S223A, S27L / S22K / T48N / L90F / F92G / N204K / S223A / V229C / N231S、S27L / S22P / T 48S / L90F / F92G / A127S / A174R / N204R / S223A、S2 7L / S1G / P20T / S32K / S34R / T48S / N87F / S98A / A12 7S / T136A、S27L / F21W / S22R / S98E / Q142L / T160V / S223A / S252T / R255L、S27L / P20T / T48S / F92Y / R 108K / S110R / A127S / V219L / S223A / V229I / R236Q 、S27L / Y26T / L90Y / F92L / A127S / V219L / S252T / R 255L、S27L / N9S / F21W / T48S / F92L / A127S / D203V / S212F、S27L / F21W / Y60H / S98E / L119M / D158E / R 236Q, S27L / F21W / S22R / L90F / F92L / N122S / Q142 W / R236Q、S27L / F21W / T48S / L90F / F92G / T109K、S 27L / L90Y / F92L / T109R / A127S / E173R / N204K / S2 23A, S27L / N9S / F21W / T48S / L90Y / F92G / A127S / N 204K / V219I / R255M, S27L / N9E / I82F / L90Y / F92L / S95N / T109L / A127S / A174R / S223A、S27L / L90F / F92L / S98L / N204K / A216P、S27L / A24V / T48S / A11 7Q / N211M / S212M / S223A、S27L / L90Y / F92G / T109 L / A127S / N204R / S223A, S27L / N2R / S32Q / N87K / A 125S / V219L、S27L / S22P / T48S / L90F / F92L / S193 N / V219L / S223A, S27L / T25Q / L119M / N211M / R236 Q, S27L / L90Y / F92G / N122S / A127S / E173R / A216P / R236Q、S27L / P20T / S32K / L90F / F92G / N122E / D1 58E / T160S / Q189L / S223A / S252T, S27L / N9S / F21 W / T48S / N87H / L90F / F92L / S113N / V229C、S27L / T 25Q / I82L / L90F / F92G / S98L / S113R / A114V / T136 A / S181R、S27L / P20T / R108Q / S110R / D203V / N211 I、S27L / S1G / N2R / P20T / Y26T / S32M / T48S / L90Y / F92L / A127S / N204R / V219L、S27L / A14K / P20T / T4 8N / L90F / F92G / A117Q / A127S、S27L / N9S / T48N / L 90Y / F92L / S98M / T109K / A127S / N204R / N211I / S2 12L / V219I、S27L / F21W / S22R / T48S / L90F / F92L / R108Q / A174K / N204K / S212F / S223A, S27L / S22R / Y60H / S98T / T136V / S193K / R236Q, S27L / S22K / Y2 6K / N87Y / L90Y / F92G / T109R / A127S / S252T / R255 L、S27L / L90F / F92L / T109L、S27L / F21W / F92L / N2 04K / S252T / R255L, S27L / V23L / L90Y / F92G / A127 S / S193K / N204R / V219I / R236C / R255L、S27L / F21 W / L90Y / F92G / A114V / N122S / A127S / N204K / S223 A / S252T、S27L / P20T / Y60A / T109L / Q189L / S212L 、S27L / S22V / T48S / L90Y / F92G / N204K、S27L / S22 V / S32K / T48S / I82F / F92G / A127S / N204K / S223A、 S27L / A55L, S27L / A97V, S27L / F250L, S27L / T109 G, S27L / G38D, S27L / A97S, S27L / A55V / A216T, S2 7L / P20D, S27L / A55V, S27L / T109Y, S27L / V165I S27L / A184G, S27L / A97E, S27L / A184S, S27L / A97 F、S27L / A97T、S27L / K197Y、S27L / A55I、S27L / A9 7P、S27L / A55M、S27L / P20E、S27L / A117L、S27L / T 109L, S27L / K197T, S27L / T136S, S27L / A97L, S27 L / T109A、S27L / P20I、S27L / L191V、S27L / A184C、 S27L / A97Q, S27L / F250V, S27L / K197V, S27L / A11 7S、S27L / K197R、S27L / T109K、S27L / A55C、S27L / N2S / V177A, S27L / Q142D, S27L / G149C, S27L / F21 Y, S27L / G149A, S27L / P164E, S27L / Y26C, S27L / T 17N、S27L / S57M、S27L / D249I、S27L / T17S、S27L / P164T、S27L / Q142L、S27L / D249N、S27L / I185R、S 27L / V83L, S27L / G149S, S27L / A24D, S27L / R251V S27L / V83I, S27L / S110N, S27L / T17M, S27L / F16 1W、S27L / G46E、S27L / D249T、S27L / T17I、S27L / S 57C、S27L / S57T、S27L / S57L、S27L / Q167T、S27L / S57E, S27L / T17K, S27L / G149N, S27L / Q142E, S27 L / S57F, S27L / T17A, S27L / P164H, S27L / T17R, S2 7L / G149T, S27L / R251T, S27L / S101Y, S27L / S57V S27L / A24T, S27L / I185E, S27L / G46N, S27L / P16 4N、S27L / P164S、S27L / Q167V、S27L / F161V、S27L / S57I、S27L / G149D S27L / Q167I、S27L / I185Q、S 27L / N2L / T17I / T136S / P164H / D249M、S27L / P20E / F21Y / I139T / P164H / K197T / R251E、S27L / A114V / A117N / T136A / I222L / V229I、S27L / P20T / A24V / S101Q / T109R / A117Q / I222L / V229I、S27L / S110D / A184S / K197T、S27L / T136S / D249T、S27L / F21Y / A184S、S27L / N2F / P20E / S110D / A184S / L191V / R2 51V、S27L / T17G / V23T / A24V / T109L / A117Q / T136 A / I222L / V229I、S27L / P20D / F21Y / A55T / F250L / R251V, S27L / T109R / A117N / I222L / V229C, S27L / F21Y / A55T / T109A / A184S, S27L / V23L / A24V / S10 1K / A117N / I222L / V229C、S27L / S110K / A117Y / P1 64R, S27L / I139T / Q142E / I169V, S27L / A24H / R25 1V、S27L / F21W / A114V / T136A / I222L / V229I、S27 L / N2S / A55V / I185Q / R251E、S27L / N2F / S101Y / Q1 42L / G149T / Q167I / I169C / V229L、S27L / N2F / S10 1C / A117F / P164S、S27L / Q142H / I185S / R251A、S2 7L / F21W / T109R / A117N / T136V / I222L / V229I、S2 7L / G46R / T109K / T136S / D249T、S27L / N2E / G46N / A55V / I185Y / D249I、S27L / V83L / F250V / R251E、S 27L / 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 / G 46E / A117T / T136S / G149C / R251Q、S27L / G46N / I1 39T / Q142E / P164T / I185A / V229L / F250V、S27L / V 23L / T136A / I222L / V229I、S27L / V23T / A24V / T10 9K / A114K / A117Q / T136A / I222L / V229C、S27L / F2 50L / R251Q、S27L / P20E / F21Y / S57T / P164N / A184 C / V229C、S27L / P20T / V23T / A24V / I222L / V229I、 S27L / V83L / A97S / A184C / D249S, S27L / P20T / S10 1H / A114V / A117N / I222L / V229I、S27L / N2E / R251 L, S27L / P20E / F21Y / S110D / A117S, S27L / T17A / V 23T / A24V / T136A, S27L / P20D / S57E / S101C, S27L / N2S / V83L / A184S、S27L / T17S / S101N / T109K / A1 17N / T136V / Q142L / I222L / V229C、S27L / P20D / A5 5T / A117Y / K197R、S27L / P20T / I222L / V229C、S27 L / T17I / A24D / S57I、S27L / N2F、S27L / T109K / A11 7N / T136V / I222L / V229C、S27L / N2L / A24T、S27L / N2F / P20I / F21Y / R251Q、S27L / N2F / G46E、S27L / T 136V / Q142W / I222L / V229I、S27L / K197R / R251E、 S27L / T17S / R251T、S27L / P20I / F21Y / A117F / I18 5G / D249N、S27L / T17L / S101M / T109K / S110R / A11 7Q / I222L / V229C、S27L / T17Q / T136A / I222L / V22 9C、S27L / V229I、S27L / T17A / F21W / S101Q / I222L / V229C、S27L / T17S / F21W / Q142L、S27L / T17L / T1 09L / S110R / A117Q / I222L / V229I、S27L / F21W / T1 36A、S27L / P20E / F21Y / G46E / V83L / A97Q、S27L / P 20T / F21W / T136A / Q142L / I222L / V229I、S27L / T1 7G / F21W / A117N / I222L / V229I、S27L / F21W / T109 K / S110R / T136A / V229I、S27L / T17C / T109K / A117 N / T136A / I222L / V229I、S27L / P20T / T109L / S110 R / A117N / I222L / V229I、S27L / A24N / P164S / K197 V / R251T、S27L / V23L / A117Q、S27L / A55V / V83L / A 117T、S27L / P20D / G46S / A55I / K197T、S27L / A117 Y、S27L / F21W / S101M / I222L / V229C、S27L / N2L / T 17N / S57C / S110D / A117L / P164N / I185G / V229L / R 251T, S27L / N2E / G46S / V83L / T136S / A172T / V229 C / D249N、S27L / P20T / T109R / A114K / A117Q / T136V / I222L / V229C、S27L / V23L / A24V / T136A / Q142W / I222L / V229I, S27L / N2L, S27L / T17A / F21W / T109 R / S110R / I222L / V229I、S27L / T17C / A114K / A117 N / T136V / I222L / V229I、S27L / V23L / I222L / V229 C、S27L / S101N / A117Q / I222L、S27L / N2E / P20I / T 109L / L191V / K197L / V229C、S27L / P20T / F21W / Q1 42W / I222L / V229C、S27L / N2L / G46E / T109K / F161 V、S27L / T17S / V229I、S27L / T17Q / T136A / I222L / V229I, S27L / N2L / A172T, S27L / P20D / F21Y / F250 V、S27L / N2L / A24H / A55C / V229L、S27L / N2L / P20E / F21Y / V229L、S27L / V23T / T109L / A114V / T136A / Q142L / I222L / V229C、S27L / P20T / F21W / I222L / V 229C, S27L / T17H / F21W / T136V / Q142W / I222L / V2 29I、S27L / G46E / A55I / Q142E、S27L / N2F / R251Q、 S27L / F21W / Q142L / I222L / V229C、S27L / I222L / V 229I、S27L / P20T / S101M / A114V / A117N / Q142W / I 222L / V229C, S27L / T17Q / A24V / S101W / T136A, S2 7L / 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 / S110 K、S27L / P20T / F21W / V229I、S27L / T17G / P20T / F2 1W / A117Q / I222L / V229C、S27L / F21W / A24V / A114 V / A117Q / Q142W / I222L / V229C、S27L / P20T / A117 Q / I222L / V229C、S27L / T17Q / V23T / A24V / I222L / V229I, S27L / N2F / D249N / F250L, S27L / V23T / Q14 2L / I222L / V229C, S27L / P20E / A24D / D249S / F250 V、S27L / A24D / S110K、S27L / T109R / I222L / V229I S27L / P20E / F21Y / K197T, S27L / T17A / V23L / S10 1M / T109K / S110R / A114V / A117Q / T136V / V229I、S 27L / P20T / F21W / Q142W / I222L / V229I、S27L / F21 W / I222L / V229I、S27L / P20T / V23T / A24V / T109L / S110R / I222L / V229I、S27L / A24D / L191F / R251Q、 S27L / A24D / T136S / F250V、S27L / P20T / F21W / T10 9L / T136A / Q142W / I222L / V229C、S27L / N2E / I169 L / F250L / R251Q, S27L / N2S / A24N / G46E / A55L / Q1 42D / V229C / R251L、S27L / N2E / P20E / F250V / R251 L、S27L / N2F / P20E、S27L / A24V / V229C、S27L / P20 D / 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 / P2 0T / F21W / T109R / A117Q / T136V / Q142L / I222L / V2 29I、S27L / P20T / F21W / T109K / A117N / I222L / V22 9C, S27L / P20T / S101M / I222L / V229I, S27L / N2S / A24D / S101Y / P164E / V165I / I185E、S27L / T17N / A 117S / V229L / R251A、S27L / S101W / A114V / T136V / V229C, S27L / P20D / G46N / A55C / S110N / T163I / A1 84S, S27L / A24H / G46E, S27L / T17H / S101Q / T136V / Q142L / V229C and S27L / V23T / A24V / I222L / V229 C.

[0007] In another aspect, the present disclosure provides a variant Bhr- In another aspect, the present disclosure relates to an expression vector comprising a nucleic acid encoding the PETase enzyme. In another aspect, the present 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 present disclosure provides a method for the preparation of a host cell as described herein, comprising the step of: and culturing the variant Bhr-PETa under conditions in which the enzyme Bhrase is produced. and recovering the Bhr-PETase enzyme. do.

[0009] In another aspect, the present disclosure provides a method for the mechanical pretreatment of PET, such as thermomechanical pretreatment, prior to enzymatic degradation of PET. Some embodiments relate to methods of pretreating PET, including pretreatment and / or chemical pretreatment. In some embodiments, the mechanical pretreatment comprises grinding the PET into particles. Mechanical pretreatment is performed at a temperature set to amorphize PET and reduce its crystallinity. In some embodiments, the chemical pretreatment comprises extruding the PET. Ion dissolving agents configured to reduce the crystallinity of or change the surface structure of This involves contacting the PET with a liquid, strong acid, base, or solvent.

[0010] In another aspect, the present disclosure provides a method for converting PET to a variant Bhr-PETase as described herein. In some embodiments, the method comprises contacting PET with an enzyme. further comprising pretreating the PET by a method described herein. In some embodiments, the method degrades PET in a mixed plastic composition. The plastic composition is a biologically or chemically derived analogue of PET, a PET-like material. Contains PET-like or PET substitute. In some embodiments, the plastic composition comprises polybutylene terephthalate (PBT), Polycarbonate (PC), Polycaprolactone (PCL) ), polyethylene furanoate (PEF) and high density polyethylene (HDPE) In some embodiments, the method comprises at least one selected from the group consisting of: This eliminates the need to sort plastics to select PET from a mixture of 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 made from a combination of two monomers: modified ethylene glycol and purified terephthalic acid. PET and other plastics are widely used in modern society. PET has found literally thousands of uses in the world, but it is essentially non-degradable. Therefore, plastic pollution is polluting the entire planet and posing many serious problems to the planet and human health. PET is recyclable; however, this means that a large amount of PET ends up in landfills. still does not prevent the release of waste into the atmosphere and / or the sea.

[0013] Plastics containing PET are remarkably resistant to enzymatic degradation. Enzymes include: (i) PET-modifying enzymes; Decomposition is limited to the surface of PET, with no visible changes observed under an electron microscope. and (ii) PET-degrading enzyme, or PETase, visible by electron microscopy. The inner blocks of PET can be significantly degraded (e.g., at least 10%) with To date, many PET-modifying enzymes have been reported, but they do not significantly reduce the mass of PET. It is not biodegradable and is not applicable to biorecycling of PET. There are a few enzyme types that show limited ability to degrade the inner blocks of PET. The first enzyme reported to be able to act on the ester bond of T polymers was The cutinase was derived from R. tschonoskii fusca. PET hydrolase from Ideonella sakaiensis, Is-PETa se and 2012 leaf compost containing cutinase (Lcc-PETase) These PETases are active at ambient temperature, but is not particularly heat stable and does not exhibit robust PET degradation.

[0014] PET exists as both an amorphous and a semi-crystalline material. The mobility increases in the amorphous phase around the glass transition temperature Tg of PET (approximately 70°C). may be added, allowing better access to the ester bond and therefore 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 converts the mobile amorphous fraction into P ET may be transformed into a refractory microstructure that prevents further enzymatic hydrolysis. Therefore, thermostable and thermoactive PETases have a glass transition temperature It is preferable to be able to cause decomposition reactions to occur around the temperature, overcoming competing material degradation processes. do.

[0015] As shown in Figures 4, 5, and 6, wild-type Bhr-PETase inhibited Lcc-PETase Although Lcc-PET is a close homologue of PET, with 94% sequence identity between the two enzymes, It was found to be more thermostable and thermoactive than Is-PETase and Is-PETase. The thermal stability and thermoactivity are important factors for efficient PET decomposition. These are important factors for solving the problem. The present disclosure provides a method for engineering the enzyme to exhibit even higher activity and thermal stability. The PETase variant derived from the bacterium HR29 (Bhr-PETase) was Remarkable thermostability and high PET hydrolysis activity of Bhr-PETase variants makes 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 modification in a polypeptide sequence. or deletion, or alteration of components chemically linked to the protein. The modification can be a change in the carbohydrate or PEG structure attached to the protein. In the present context, "amino acid modification" refers to an amino acid substitution, insertion and / or For clarity, unless otherwise stated, amino acid modifications are always expressed as DN Amino acids coded by A, e.g., have codons in DNA and RNA This applies to 20 types of amino acids.

[0017] As used herein, an "amino acid substitution" or "substitution" refers to a specific amino acid in a parent polypeptide sequence. It refers to the replacement of an amino acid at a certain position with a different amino acid. In this case, the substitution is at a particular position and is not naturally occurring in that organism or in all organisms. For example, the substitution S27L is for an amino acid that does not occur naturally in the A variant polypeptide in which serine (S) at position 7 is replaced with leucine (L), For clarity, the nucleic acid encoding the starting sequence is modified. engineered to not alter amino acids (e.g., to increase expression levels in the host organism) Exchange CGG (which encodes arginine) for CGA (which still encodes arginine) The proteins encoded by the nucleotide sequences are not "amino acid substitutions"; i.e., the same proteins are encoded by the nucleotide sequences. Despite the creation of a new gene, the protein has the same amino acid sequence as the starting one at a specific position. If it has an amino acid, it is not an amino acid substitution.

[0018] As used herein, an "amino acid insertion" or "insertion" refers to an amino acid sequence that is inserted into a parent polypeptide. This refers to the addition of an amino acid sequence at a specific position in the sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. 233ADE or A233ADE is AlaAsp after position 233 and before position 234 The insertion of Glu is indicated.

[0019] As used herein, an "amino acid deletion" or "deletion" refers to a deletion of an amino acid from a parent polypeptide. This refers to the deletion of an amino acid sequence at a specific position in the sequence. For example, F250- or F250#, F250() or F250del indicates deletion of glutamic acid at position 250 Additionally, FRS250- or FRS250# indicates the sequence Phe starting at position 250. The deletion of ArgSer is shown.

[0020] As used herein, a "parent polypeptide" refers to a polypeptide from which a variant is subsequently generated. The parent polypeptide refers to a starting polypeptide that is modified to be a naturally occurring polypeptide. polypeptide or a variant or engineered version of a naturally occurring polypeptide The parent polypeptide may be the polypeptide itself, a composition comprising the parent polypeptide, or In this case, some embodiments may refer to the amino acid sequence encoding it. Type Bhr-PETase (also called G1P Bhr-PETase; SEQ ID NO: 1; Figure 1 1) is used as the parent polypeptide.

[0021] As used herein, "variant protein" or "protein variant" refers to a protein that is A "mutant" or "variant" is a variant of a parent protein by virtue of at least one amino acid modification. A protein variant refers to a protein that is different from the original protein. It can refer to a composition containing a protein or an amino acid sequence encoding it. Protein variants have at least one amino acid modification compared to the parent protein, e.g., about 1 to about 70 amino acid modifications, preferably about 1 to about 5 amino acid modifications, compared to As described below, in some embodiments, the parent polypeptide has the wild-type sequence For example, an exemplary wild-type Bhr-PETase is referred to herein as "G1P." As discussed further below, protein variant sequences as used herein include: Preferably, the parent protein sequence 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, preferably at least about 90% identity and preferably at least The variant protein has at least about 95 to 98 to 99% identity. the protein itself, compositions containing protein variants, or DNA sequences encoding same. Therefore, as used herein, "variant PETase" may refer to a variant of the parent PETase. A novel PETa having at least one amino acid modification in the amino acid sequence compared to the se enzyme Unless otherwise stated or clear from the context, the Ant PETase is generally compared to the wild-type G1P sequence. In addition, unless otherwise noted As long as the variant PETase of the present invention is enzymatically active, i.e., as described in Example 9, There is detectable PETase activity using the PETase assay described above.

[0022] As used herein, a "protein" refers to a compound having at least two refers to amino acids linked by two covalent bonds, and constitutes proteins, polypeptides, and oligopeptides. and peptides. Generally, peptide groups are made up of naturally occurring amino acids and peptide bonds. In addition, the polypeptide may be modified with one or more side chain or terminal synthetic derivatizations, Glycosylation, PEGylation, circular permutation, ring as linkers to other molecules, fusions to proteins or protein domains, and peptides. This may include the addition of a tag or label.

[0023] As used herein, a "residue" refers to a position in a protein and its associated For example, serine 27 (also known as Ser27 or S27) (also called G1P) is the 27th residue in the parent G1P enzyme.

[0024] As used herein, a "non-naturally occurring modification" refers to a modification that occurs in nature but is different from the parent (e.g., G1P) enzyme.

[0025] As used herein, "amino acid" and "amino acid identity" refer to DNA and one of the 20 naturally occurring amino acids encoded by RNA. do.

[0026] As used herein, "position" means a location in the sequence of a protein. Generally, the position numbers (discussed more fully below) are used to identify, for example, the position of a signal peptide. This is relative to the first amino acid of the mature PETase sequence with the following removed:

[0027] As used herein, "PETase" refers to a protein that has PETase activity. As used herein, "PETase activity" refers to the amount of PETase activity that an enzyme can produce in the absence of MHTase. The conversion of PET to mono(hydroxyethyl) terephthalate (MHET) as a potential product In Example 9, MHTas in the presence of MHTase catalyzes water decomposition. e produces terephthalic acid (TPA) and ethylene glycol ( MHET is further converted to EG) using the assays outlined below and in Example 9. An enzyme that has detectable PETase activity in PETase activity may be determined by measuring the total PETase activity and / or It can be measured as PETase thermostability.

[0028] "Identity" as used herein in reference to two sequences refers to the degree to which the same amino acids align. The amino acid sequences of the present invention ("invention sequences") and patents Between the parent amino acid sequence referred to in the claims (e.g., G1P, SEQ ID NO: 1) The degree of identity is determined by the number of exact matches in the alignment of two sequences. or the length of SEQ ID NO: 1, whichever is shorter. The results are expressed as percent identity as calculated below.

[0029] For purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: 1 is It is used to determine the corresponding amino acid residues in other PETases. The amino acid sequence of Tase aligns with the mature polypeptide disclosed in SEQ ID NO:1. and based on the alignment, the sequence of the sequence in the mature polypeptide disclosed in SEQ ID NO: 1 The amino acid position number corresponding to any amino acid residue in the sequence is calculated by the Needleman-Wunsch algorithm. (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) and the needle program of the EMBOSS package (EM BOSS:The European Molecular Biology Open Software Suite,Rice et al., 2000,Trends Genet. 16:276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty 10, gap extension penalty 10, and NARTI 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) ) permutation matrix.

[0030] Identification of the corresponding amino acid residues in other PETases includes, but is not limited to, Using these respective initialization parameters, MUSCLE (multiple logarithmic expectation) Sequence comparison (multiple sequence comparison by log -expectation); version 3.5 or later; Edgar, 2004, Nucl eic Acids Research 32:1792~1797), MAFFT Version 6.857 and 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,Bioinforma tics 26:1899~1900), EMBOSS E using ClustalW MMA (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680) and Clustal Omega. EMBL-EBI (Sievers and Higgins, 2014, Met hods Mol Biol.2014;1079:105~16) The computer program is used to align multiple polypeptide sequences. It can be determined that:

[0031] Other enzymes may be involved in the synthesis of SEQ ID NO: 1, such that comparison based on traditional sequences fails to detect their relationship. When separated from the polypeptide (Lindahl and Elofsson, 2000 , J. Mol. Biol. 295:613~615), other pairwise sequence comparison algorithms Even greater sensitivity in sequence-based searches can be achieved by searching databases. A search program that uses probabilistic representations of polypeptide families (profiles) to For example, the PSI-BLAST program can be used to generate repetitive data. Through a database search process, profiles can be created to detect distant homologs. (Atschul et al., 1997, Nucleic Acids Res. 25:3 389-3402). Even greater sensitivity can be achieved by using family or series of polypeptides. Parfamilies have one or more representatives in protein structure databases This can be achieved in the case of GenTHREADER (Jones, 1999, J. Mol. B iol.287:797~815;McGuffin and Jones,2003, Bioinformatics 19:874-881) As input to a neural network that predicts structural folds for a given sequence, Various sources (PSI-BLAST, secondary structure prediction, structural alignment profile and Uses information from solvation potentials Similarly, Gough et al., 2000, J. Mol. Biol. 313:903-91 The 9 method uses superfamily models present in the SCOP database to identify unknown These alignments can be used to align the sequences of the polypeptides. Such a model can then be used to generate a homology model of the The various tools developed can be used to evaluate accuracy.

[0032] For proteins of known structure, several tools and sources provide structural alignments. For example, SCOP supergroups of proteins can be used to search for and create supergroups. Milli is structurally aligned and these alignments are accessible and available for download. Two or more protein structures can be aligned using a distance alignment matrix. ce alignment matrix)(Holm and Sander,199 8, Proteins 33:88~96) or combinatorial extension ( combinatorial extension)(Shindyalov and Bourne, 1998, Protein Engineering 11:739~7 47) and various other algorithms can be used to align the Search a structural database using the structure of interest to find potential structural homologs. can be further utilized to search for ,Bioinformatics 16:566~567).

[0033] In describing variants of the present invention, the nomenclature set forth below is for ease of reference. The standard accepted IUPAC one-letter or three-letter amino acid abbreviations are used. will be done.

[0034] For amino acid substitutions, the following nomenclature is used herein: original amino acid , position, substituted amino acid. Thus, the substitution of serine with leucine at position 27 is Multiple mutations are indicated by a forward slash ( " / "). For example, "A102V / T136M" means that positions 102 and 1 Each represents a substitution at position 36.

[0035] [Table 1]

[0036] As used herein, "isolated" in the context of PETase means that the polypeptide is isolated from other proteins. In a specific embodiment, the PETase of the present invention is free of As used herein, the term "isolated" refers to a protein isolated from a sample containing SDS-PA. At least 20% pure, preferably at least 40% pure, as determined by GE. More preferably, it is at least 60% pure, even more preferably it is at least 80% pure, and most preferably it is at least 60% pure. Preferably at least 90% pure and even most preferably at least 95 to 9% pure. 8%. In particular, the polypeptide is in "essentially pure form." That is, the polypeptide preparation is essentially free of other polypeptide material with which it is naturally associated. This can be achieved, for example, by means of known recombinant methods or by classical methods. This can be achieved by preparing the polypeptide by conventional purification methods.

[0037] As used herein, a "recombinant enzyme" refers to an enzyme that is The nucleic acid produced by recombinant technology and encoding the enzyme of the present invention is, for example, At least one exogenous (e.g., parent) element, such as a sequence encoding a nucleotide sequence, a terminator, a signal sequence, etc. By "PETase" we mean that the enzyme is operably linked to a sequence (not derived from PETase).

[0038] The term "nucleic acid construct" refers to a nucleic acid that has been isolated from a naturally occurring gene or that is not otherwise found in nature. modified to contain segments of nucleic acid in a manner that is absent or synthetic refers to a nucleic acid molecule, whether single-stranded or double-stranded, that contains one or more regulatory sequences. .

[0039] The term "operably linked" refers to a regulatory sequence that is operatively linked so as to direct the expression of the coding sequence. The term "conformation" refers to the conformation in which the nucleotide sequence is properly positioned relative to the coding sequence of the polynucleotide. vinegar.

[0040] As used herein, the term "about" refers to, for example, the length of a nucleotide sequence, Degree of error, size, amount of ingredients in the composition, concentration, volume, processing temperature, processing time, yield The values ​​and ranges of the compounds, compositions, etc., are modified. , used to make concentrates or use formulations through typical measurement and handling procedures; through unintentional errors in these procedures; in the purity of the manufacturing, raw materials or starting materials or components used to carry out the process; and quantities that may arise through such considerations. The term "about" refers to a variation in, for example, a composition having a particular initial concentration or mixture. amounts that vary due to deterioration of the substance, formulation, or cell culture, and the specific initial concentration or mixture This also includes amounts that vary due to mixing or processing of the composition or formulation having the formula: The claims appended hereto, whether or not modified by the term "about," Ranges include equivalents of these amounts. Additionally, the term "about" refers to the same as the stated reference value. In certain embodiments, the term "about" refers to a range of values ​​within one of the stated reference values. Refers to a range of values ​​that fall within 0, 9, 8, 7, 6, 5, 4, 3, 2, and 1 percent.

[0041] C. PETase of the Invention Thus, the present invention provides a method for manufacturing plastics in a variety of applications, most notably plastics made from PET. Variant PETase with improved enzymatic activity that can be used in the degradation of PET sticks provide.

[0042] Generally, the variant PETase of the present invention has a structure similar to that of wild-type Bhr-PE, as shown in FIG. Tase, "G1P" (i.e., "generation 1 parent"), compared herein with SEQ ID NO: 1 The variant PETase of the present invention has been modified to have improved biochemical properties. Modified compared to "G2P" (i.e., "generation 2 parent"), which has the amino acid substitution S27L The variant PEs that can be improved herein may also have improved biochemical properties. The biochemical properties of Tase include, but are not limited to, thermostability, thermoactivity, specific activity, and and production.

[0043] The variant Bhr-PETase of the present invention has one or more G1P or G2P-dependent As used herein, "improved" means that the product has at least one biochemical "Improved function" refers to a desired change in a biological property. or as a percentage increase or decrease in a specific activity, accompanied by an improvement in the activity (e.g., thermal stability). The fold change can be measured as the number of times the variant Bhr-PETase increases G1P. or the thermostability may be increased by 10% compared to G2P, or the PETase activity may be increased by 10%. Generally, a percentage change describes a less than 100% change in biochemical activity. fold change is used to describe a change in biochemical activity of more than 100%. used to compare the parent enzymes (often in G1P or G2P). In the light, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% %, 90%, 95%, 98% and 99% percent change (usually an increase) in biochemical activity In the present invention, a "fold increase" (or decrease) is a decrease in the starting or parent enzyme. For example, as shown in Figures 9A-AA, the variant T17A / S27T / T48S / I82L / L90F / Q167V / P213N / S252T are G There was a 1.6-fold increase in specific activity compared to 1P, which is consistent with the activity of the variant In many embodiments, the improvement is at least 1. .1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1 9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x or 10x or more High.

[0044] In general, improvements were observed when variant Bhr-PETase was used against G1P or G2P enzymes. G1P or G2P enzymes using Bhr-PETase activity assay under conditions to be validated It is measured relative to

[0045] 1. Increased total activity The present invention relates to the use of G1P (wild-type Bhr-PETase of SEQ ID NO: 1) or G2P (amino acid Bhr-PETase of SEQ ID NO: 1) The total activity of G1P (with the acid substitution S27L) was equal to or higher than that of G1P. As used herein, "total activity" refers to activity at temperatures, such as 65°C. Monitoring TPA (terephthalic acid) production during PET depolymerization at elevated temperatures can be determined by the colorimetric assay described in Example 9 or by HPLC. Any improvement in total activity is quantified using thermal activity, specific activity and / or This may be due to improved production of variant PETase.

[0046] In many embodiments, the variant Bhr-PETase is at least 1.1-fold, 1. 2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x , 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or higher improvement It has a total activity.

[0047] In general, improvements were observed when variant Bhr-PETase was used against G1P or G2P enzymes. G1P or G2P enzymes using Bhr-PETase activity assay under conditions to be validated It is measured relative to

[0048] (i) Increased thermal activity In one aspect, the variant Bhr-PETase may have increased thermal activity. As used herein, "thermal activity" refers to the TP activity during the PET depolymerization reaction at high temperatures, such as 65°C. A (terephthalic acid), MHET (mono(hydroxyethyl) terephthalate) and BH By monitoring the production of ET (bis(2-hydroxyl) terephthalate) The amount of TPA produced per mg of enzyme per hour can be determined by the following formula: TPA当 量 h -1 mg 酵素 -1As used herein, "TPA equivalent" refers to the amount of TPA, T MHET converted to PA, BHET converted to TPA and any other It is calculated by the sum of measurable oligomers. compared with wild-type Bhr-PETase) or G2P (G1P with the amino acid substitution S27L) PETase exhibits improved thermal activity, demonstrating increased activity per milligram of enzyme compared to PETase. obtain.

[0049] In many embodiments, the variant Bhr-PETase is at least 1.1-fold, 1. 2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x , 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or higher improvement It has thermal activity.

[0050] In general, improvements were observed when variant Bhr-PETase was used against G1P or G2P enzymes. G1P or G2P enzymes using Bhr-PETase activity assay under conditions to be validated It is measured relative to

[0051] (ii) Increased specific activity In another embodiment, the variant Bhr-PETase may have increased specific activity. As used herein, the term "specific activity" refers to the PET decomposition activity of Bhr-PETase at the optimum operating temperature. TPA (terephthalic acid), MHET (mono(hydroxyethyl) terephthalic acid) during the polymerization reaction The production of bis(2-hydroxyl) terephthalate (BHET) and BHET (bis(2-hydroxyl) terephthalate) was monitored. The amount of TPA equivalents produced per mg of enzyme per hour can be determined by mg(mg TPA当量 h -1 mg 酵素-1 ) is quantified. wild-type Bhr-PETase (No. 1) or G2P (G1 with the amino acid substitution S27L) PETase exhibits increased activity per milligram of enzyme compared to specific activity (P). can show improvements.

[0052] In many embodiments, the variant Bhr-PETase is at least 1.1-fold, 1. 2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x , 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or higher improvement In some embodiments, the variant Bhr-PETase has at least one specific activity. At most 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1. 8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x or 10x indicates a higher increase in TPA equivalents produced per mg of enzyme per hour.

[0053] In general, improvements were observed when variant Bhr-PETase was used against G1P or G2P enzymes. It is measured relative to the G1P or G2P enzyme under the conditions to be tested.

[0054] (iii) Increased production In one aspect, the variant Bhr-PETase may have increased production. "Production" was measured by monitoring the protein titer of Bhr-PETase in g / L. Therefore, G1P (wild-type Bhr-PETas of SEQ ID NO: 1) e) or G2P (G1P with the amino acid substitution S27L) compared with 1 liter of enzyme supernatant. Increased amounts of PETase per unit may indicate improved production.

[0055] In many embodiments, the variant Bhr-PETase is at least 1.1-fold, 1. 2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x , 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or higher improvement It has excellent thermal activity.

[0056] In general, improvements were observed when variant Bhr-PETase was used against G1P or G2P enzymes. G1P or G2P enzymes using Bhr-PETase activity assay under conditions to be validated It is measured relative to

[0057] 2.Thermal stability Additionally, as will be appreciated by those skilled in the art, PET degradation can be performed around the glass transition temperature. Amorphous PET domains tend to form around the glass transition temperature ( At temperatures between approximately 67 and 72°C, the mobility increases and the enzyme hydrolysis becomes more readily accessible. At higher temperatures, the PET matrix recrystallizes over time, resulting in increased PET degradation. Therefore, for example, about 65 to 72°C is the optimum temperature range for PET decomposition. It is thought to be within the range.

[0058] Thus, in many embodiments, the variant Bhr-PETase has improved thermostability. In this context, "thermal stability" means the ability to withstand the same thermal load conditions. The variant enzyme was found to be G1P (wild type of SEQ ID NO: 1) under the challenge condition. Bhr-PETase) or G2P (G1P with the amino acid substitution S27L) The conditions are constant, i.e., the same conditions (generally as outlined herein and shown in Example 9) The activity of the variant is higher than that of the G1P or G2P enzymes under the This means that

[0059] In one embodiment, the variant Bhr-PETase has a sequence similar to that of about 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, 9, 70, 71, 72, 73, 74, 75, 76, 77, 79, 80, 81, 82, 83 , for example, variant Bhr-PETas, at a temperature of 84 and / or 85 °C for a certain period of time. Depending on the final conditions for use, from about 0.5, 1, 2, 3, 4, 5 or 6 hours to about G1P or G2P when exposed for 5, 6, 7, 8, 9, 10 hours or longer In some embodiments, the variant Bhr-PETase is more stable than the temperature , preferably at about 65°C to 85°C for at least about 0.5 hours, preferably at about 65°C to 7 2°C for at least about 1 hour, preferably at least 65°C for at least about 1 hour, or G1P or G2P when exposed to at least 70°C for at least about 1.5 hours. It is more stable than enzymes.

[0060] Thus, in many embodiments, the variant Bhr-PETase is G1P or at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.0 times higher than G2P enzymes. 6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9 10 times or more more thermostable.

[0061] In general, improvements were observed with the use of variant Bhr-PETase or used the Bhr-PETase activity assay under conditions that measure G1P or G2P enzymes. and measured relative to the G1P or G2P enzymes.

[0062] 3. PETase Assay PET film-based assays are described in Examples 6 and 9, and BHET-based assays are described in Examples 7 and 8. Assays for serotonin were used to determine activity as generally outlined in Example 5. There are several PETase activity assays available. PETase activity is measured using pNPB (p- It can also be monitored by the pNPB assay. The alternative substrate p-nitrophenyl butyrate is converted to p-nitrophenyl by PETase. The release of p-nitrophenol is catalyzed by PETase. It directly correlates to activity and can be determined spectrophotometrically, for example, at 405 nm.

[0063] 4.Bhr-PETase The present invention provides a method for the preparation of PET-degrading polymers having improved activity and / or thermal stability for use in the degradation of PET. A number of specific variant Bhr-PETases are provided.

[0064] In some embodiments, the variant Bhr-PETase is 27, 1, 2, 5, 9, 1 2, 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, 9 2, 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, 1 Selected from the group consisting of 26, 137, 165, 169, 172, 191, 192 and 197 1) with one or more amino acid substitutions at selected positions (relative to G1P, SEQ ID NO: 1) In some embodiments, the variant Bhr-PETase is (G1P, SEQ ID NO: 1) 1, 2, 3, 4, 5, 6, 7, 8, It has 9, 10 or more amino acid substitutions.

[0065] In some embodiments, the variant Bhr-PETase is S27L, S27F, S2 7H, S27T, S27W, S1A, S1G, S1M, S1R, N2E, N2F, N2L , N2R, N2S, Q5E, N9A, N9E, N9S, R12K, S13L, S13R, A14K, A14S, L15I, T16E, T17A, T17C, T17G, T17H, T17I, T17K, T17L, T17M, T17N, T17Q, T17R, T17S, D18R, P20D, P20E, P20I, P20Q, P20T, F21W, F21Y, S22A, S22I, S22K, S22P, S22R, S22V, V23L, V23T, A24D, A24G, A24H, A24N, A24S, A24T, A24V, T25A, T25F、T25H、T25Q、T25R、T25V、Y26C、Y26K、Y26L、 Y26T、R30K、S32K、S32M、S32Q、S32Y、V33G、V33Q、 V33T、S34R、V40T、G46E、G46L、G46N、G46R、G46S、 T48N, T48S, L49G, G53A, I54V, A55C, A55I, A55L A55M, A55T, A55V, M56I, M56L, S57C, S57E, S57F S57I、S57L、S57M、S57T、S57V、Y60A、Y60H、Y60I、 A62T、A68F、L70M、R72P、L74W、H77Q、I82F、I82L、 I82M, V83I, V83L, V83T, N85D, N87F, N87H, N87I N87K, N87L, N87M, N87Q, N87R, N87V, N87W, N87Y S88K, S88T, L90F, L90K, L90Y, F92G, F92I, F92K F92L, F92N, F92Q, F92V, F92Y, A97C, A97E, A97F A97G, A97L, A97P, A97Q, A97S, A97T, A97V, S98A S98D、S98E、S98L、S98M、S98N、S98Q、S98T、S98V、 S101A、S101C、S101D、S101F、S101H、S101K、S101 L、S101M、S101N、S101Q、S101R、S101V、S101W、S1 01Y、A102V、N105D、R108C、R108E、R108H、R108K、 R108N、R108P、R108Q、R108S、R108T、R108V、T109 A、T109F、T109G、T109K、T109L、T109N、T109R、T1 09Y、S110D、S110G、S110H、S110K、S110N、S110R、 S113A、S113K、S113N、S113P、S113Q、S113R、S113 T、S113Y、A114K、A114L、A114S、A114V、A117F、A1 17G、A117L、A117N、A117Q、A117S、A117T、A117Y、 L119I、L119M、A121S、N122A、N122E、N122H、N122 P、N122R、N122S、A125S、A127M、A127S、A127V、A1 35G、T136A、T136M、T136S、T136V、R138E、R138L、 I139A、I139T、S140A、Q142D、Q142E、Q142H、Q142 L、Q142W、I143N、I143R、T145S、K147F、K147G、K1 47N、K147Q、G149A、G149C、G149D、G149N、G149S、 G149T、G149V、V150I、V150L、T153L、H156N、T157 A、T157G、D158E、D158I、D158K、D158L、T160K、T1 60Q、T160R、T160S、T160V、F161V、F161W、N162E、 N162H、N162P、N162R、T163I、T163S、P164E、P164 H、P164N、P164R、P164S、P164T、Q167I、Q167T、Q1 67V、V170L、E173R、A174K、A174R、V177A、P179Q、 S181A、S181C、S181R、Q182T、A184C、A184G、A184 S、I185A、I185E、I185G、I185L、I185Q、I185R、I1 85S、I185Y、Q189I、Q189L、Q189V、N190S、S193E、 S193F, S193H, S193K, S193N, S193P, S193T, S193 V, T194G, T194S, T195F, V198A, V200L, D203N, D2 03R, D203V, N204A, N204K, N204R, N204S, T206G, T206K, T206L, T206P, T206R, F208G, F208L, F208 R, F208T, A209V, N211F, N211I, N211L, N211M, N2 11V, S212F, S212L, S212M, P213N, P213R, A216L, A216P, A216S, A216T, A216V, I217S, S218A, V219 F, V219I, V219K, V219L, V219R, T221S, I222L, S2 23A, S223C, M225L, L227R, W228F, V229C, V229I, V229L, N231L, N231Q, N231S, R236C, R236E, R236 H, R236K, R236Q, Q237R, N241P, V242T, N243P, A2 46D, A246K, A246S, A246T, D249I, D249M, D249N, D249S, D249T, F250I, F250L, F250V, F250Y, R251 A, R251E, R251I, R251K, R251L, R251Q, R251T, R? 51V, S252T, N253S, N253Y, N254R, R255E, R255G, R255L, R255M, R255S, R255V, R255W, R255Y, Q258 P, P8T, L31M, G38D, S95N, V126I, L137M, V165I, I 169C, I169L, I169V, A172T, L191F, L191V, P192A selected from the group consisting of K197L, K197R, K197T, K197V and K197Y In some embodiments, the variant Bhr-P has one or more selected amino acid substitutions. ETase is 1, 2, 3, 4, 5, 6, 7, 8 selected from the same group , 9, 10 or more amino acid substitutions.

[0066] In some embodiments, the variant Bhr-PETase is a variant of the serine amino acid sequence at position 27 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the diet are threonine, glutamine, asparagine, lysine, and arginine. histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is S27L. 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. do.

[0067] In some embodiments, the variant Bhr-PETase has a nucleotide sequence similar to serine at position 1 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. amino acids, namely threonine, glutamine, asparagine, lysine, and arginine , histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, ara Nine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan, and tyrosine, and some embodiments use cysteine ​​(for disulfide formation). Some experiments do not utilize α- and β-glucan (due to steric effects) 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. The acid substitution is S1R.

[0068] In some embodiments, the variant Bhr-PETase has an aspartate residue at position 2 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. Amino acids present in the , histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, ara Nine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan, and tyrosine, and some embodiments use cysteine ​​(for disulfide formation). Some experiments do not utilize α- and β-glucan (due to steric effects) 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 N2S.

[0069] In some embodiments, the variant Bhr-PETase is a variant of glutamate at position 5 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan In some embodiments, the amino acid sequence is substituted with phenylalanine, valine, and tyrosine, and in some embodiments, the amino acid sequence is substituted with cysteine ​​(disulfide). (due to the possibility of amide 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 aspartate residue at position 9 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. Amino acids present in the , histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, ara Nine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan, and tyrosine, and some embodiments use cysteine ​​(for disulfide formation). Some experiments do not utilize α- and β-glucan (due to steric effects) and proline (due to steric effects). In 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 aryl group at position 12 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids. The amino acids present in histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In an embodiment, the amino acid substitution is R12K.

[0072] In some embodiments, the variant Bhr-PETase is a variant of the serine amino acid sequence at position 13 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the diet are threonine, glutamine, asparagine, lysine, and arginine. histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is S13L. It is the 13th race.

[0073] In some embodiments, the variant Bhr-PETase has a sequence similar to that of aryl nucleotides at position 14 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is A14K. It's 14S.

[0074] In some embodiments, the variant Bhr-PETase has a leucovorin at position 15 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In an embodiment, the amino acid substitution is L15I.

[0075] In some embodiments, the variant Bhr-PETase is a variant of the Bhr-PETase at position 16 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is T16E.

[0076] In some embodiments, the variant Bhr-PETase is a variant of the Bhr-PETase at position 17 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, Some embodiments use proline (due to steric effects) and tyrosine. 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 aspartate residue at position 18 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, asparagine, Lysine, arginine, histidine, glutamic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is D18R.

[0078] In some embodiments, the variant Bhr-PETase comprises a protease having a sequence similar to that at position 20 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming In some embodiments, amino acid substitutions are not utilized in 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 an embodiment, the amino acid substitution is P20T.

[0079] In some embodiments, the variant Bhr-PETase has a phenotype at position 21 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. of the naturally occurring amino acids, namely, serine, threonine, glutamine, and asparagine , lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycolipid Leucine, proline, alanine, isoleucine, leucine, methionine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is F21W. In some embodiments, the amino acid substitution is F It's 21Y.

[0080] In some embodiments, the variant Bhr-PETase is a variant of the serine amino acid sequence at position 22 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the diet are threonine, glutamine, asparagine, lysine, and arginine. histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming 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. It's the S22V.

[0081] In some embodiments, the variant Bhr-PETase comprises a variant Bhr-PETase at position 23 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the Arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline Phosphorus, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, and some embodiments use cysteine ​​(disulfide-forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is V23L. It is 23T.

[0082] In some embodiments, the variant Bhr-PETase has a sequence similar to that of aryl nucleotides at position 24 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is A24D. 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. The acid substitution is A24V.

[0083] In some embodiments, the variant Bhr-PETase is a variant of the Bhr-PETase at position 25 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in Cysteine, glutamic acid, aspartic acid, cysteine, glycine, proline, alanine, Isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is T25A. 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 a tyrosine residue at position 26 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan Some embodiments use phenylalanine and valine, and some embodiments use 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 sequence similar to that of position 30 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids. The amino acids present in histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is R30K.

[0086] In some embodiments, the variant Bhr-PETase is a variant of the serine amino acid sequence at position 32 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the diet are threonine, glutamine, asparagine, lysine, and arginine. histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is S32K. In some embodiments, the amino acid substitution is S32Q. The amino acid substitution is S32Y.

[0087] In some embodiments, the variant Bhr-PETase comprises a variant Bhr-PETase at position 33 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the Arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline Phosphorus, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, and some embodiments use cysteine ​​(disulfide-forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is V33G. 33Q. In some embodiments, the amino acid substitution is V33T.

[0088] In some embodiments, the variant Bhr-PETase is a variant of the serinoceptor at position 34 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the diet are threonine, glutamine, asparagine, lysine, and arginine. histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is S34R.

[0089] In some embodiments, the variant Bhr-PETase comprises a variant Bhr-PETase at position 40 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the Arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline Phosphorus, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, and some embodiments use cysteine ​​(disulfide-forming (due to possible side effects) and proline (due to steric effects). In an embodiment, the amino acid substitution is V40T.

[0090] In some embodiments, the variant Bhr-PETase has a glycan at position 46 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is G46E. 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. do.

[0091] In some embodiments, the variant Bhr-PETase is a variant of the Bhr-PETase at position 48 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, alanine, isoleucine, leucine, methionine, phenylalanine, triphosphatase Some embodiments use cysteine ​​(dysprosine), valine, and tyrosine. due to the possibility of sulfide formation) and proline (due to steric effects). In some embodiments, the amino acid substitution is T48N. The amino acid substitution is T48S.

[0092] In some embodiments, the variant Bhr-PETase has a leucovorin at position 49 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is L49G.

[0093] In some embodiments, the variant Bhr-PETase has a glycan at position 53 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is G53A.

[0094] In some embodiments, the variant Bhr-PETase comprises the isoform at position 54 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine , proline, alanine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In an embodiment, the amino acid substitution is I54V.

[0095] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase having a sequence similar to that of aryl nucleotides at position 55 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, Some embodiments use proline (due to steric effects) and tyrosine. 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. The amino acid substitution is A55T. In some embodiments, the amino acid substitution is A55V.

[0096] In some embodiments, the variant Bhr-PETase has a methyl group at position 56 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, alanine, isoleucine, leucine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is M56I. It is 56L.

[0097] In some embodiments, the variant Bhr-PETase is a variant of the serine amino acid sequence at position 57 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the diet are threonine, glutamine, asparagine, lysine, and arginine. histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, Some embodiments use proline (due to steric effects) and tyrosine. 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. is S57V.

[0098] In some embodiments, the variant Bhr-PETase is a tyrosine kinase inhibitor (TKI) at position 60 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and valine, and some embodiments use cysteine ​​(disulfide-forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is Y60A. In some embodiments, the amino acid substitution is Y 60H. In some embodiments, the amino acid substitution is Y60I.

[0099] In some embodiments, the variant Bhr-PETase has a sequence similar to that of aryl nucleotides at position 62 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is A62T.

[0100] In some embodiments, the variant Bhr-PETase has a sequence similar to that of aryl nucleotides at position 68 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is A68F.

[0101] In some embodiments, the variant Bhr-PETase has a nucleotide sequence similar to that of position 70 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is L70M.

[0102] In some embodiments, the variant Bhr-PETase has an amino acid sequence similar to that of position 72 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids. The amino acids present in histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming In some embodiments, the amino acid substitution is R72P. be.

[0103] In some embodiments, the variant Bhr-PETase has a leucovorin residue at position 74 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is L74W.

[0104] In some embodiments, the variant Bhr-PETase has a His residue at position 77 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids. The amino acids present in arginine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In an embodiment, the amino acid substitution is H77Q.

[0105] In some embodiments, the variant Bhr-PETase comprises the isoform at position 82 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine , proline, alanine, isoleucine, leucine, methionine, phenylalanine, tri with tyrosine, valine, and cysteine ​​(diamine). due to the possibility of sulfide formation) and proline (due to steric effects) In some embodiments, the amino acid substitution is I82F. The amino acid substitution is I82L. In some embodiments, the amino acid substitution is I82M.

[0106] In some embodiments, the variant Bhr-PETase comprises a variant Bhr-PETase at position 83 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the Arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline Phosphorus, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine, and some embodiments use cysteine ​​(disulfide-forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is V83I. In some embodiments, the amino acid substitution is V 83L. In some embodiments, the amino acid substitution is V83T.

[0107] In some embodiments, the variant Bhr-PETase has an aspartate residue at position 85 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, lysine, and arginine histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In an embodiment, the amino acid substitution is N85D.

[0108] In some embodiments, the variant Bhr-PETase has an aspartate residue at position 87 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, lysine, and arginine histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is N87F. 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 embodiments, the amino acid substitution is N87V. 87W. In some embodiments, the amino acid substitution is N87Y.

[0109] In some embodiments, the variant Bhr-PETase is a variant of the serine amino acid sequence at position 88 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the diet are threonine, glutamine, asparagine, lysine, and arginine. histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is S88K. It is 88T.

[0110] In some embodiments, the variant Bhr-PETase has a leucovorin at position 90 of SEQ ID NO:1. In some embodiments, the substitutions are any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is L90F. 90K. In some embodiments, the amino acid substitution is L90Y.

[0111] In some embodiments, the variant Bhr-PETase has a phenotype at position 92 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. of the naturally occurring amino acids, namely, serine, threonine, glutamine, and asparagine , lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycolipid Leucine, proline, alanine, isoleucine, leucine, methionine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is F92G. 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. The amino acid substitution is F92V. In some embodiments, the amino acid substitution is F92Y.

[0112] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase having a sequence similar to that of aryl nucleotides at position 97 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, 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 embodiments, the amino acid substitution is A97L. 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 is a variant of the serinoceptor at position 98 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present in the diet are threonine, glutamine, asparagine, lysine, and arginine. histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is S98A. 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 an embodiment, the amino acid substitution is S98V.

[0114] In some embodiments, the variant Bhr-PETase has a sequence similar to that of position 101 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, Valine and tyrosine are used, and some embodiments use proline (due to steric effects). 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 S101F. In some embodiments, the amino acid substitution is S101H. 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. The amino acid substitution is S101R. In some embodiments, the amino acid substitution is S101V. In some embodiments, the amino acid substitution is S101W. The acid substitution is S101Y.

[0115] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 102 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is A102V.

[0116] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 105 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is N105D.

[0117] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 108 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, In some embodiments, the amino acid substitution is with valine and tyrosine. In some embodiments, the amino acid substitution is R108E. 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 R108Q. In some embodiments, the amino acid substitution is R108S. The substitution is R108T. In some embodiments, the amino acid substitution is R108V.

[0118] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 109 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 T 109L. In some embodiments, the amino acid substitution is T109N. In some embodiments, the amino acid substitution is T109R. It's 9Y.

[0119] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 110 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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. 110N. In some embodiments, the amino acid substitution is S110R.

[0120] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 113 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is at S113. In some embodiments, the amino acid substitution is S113K. 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. It's 13Y.

[0121] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 114 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 an embodiment, the amino acid substitution is A114V.

[0122] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 117 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 A117S. In some embodiments, the amino acid substitution is A117T. It's 7Y.

[0123] In some embodiments, the variant Bhr-PETase has a locus at position 119 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is L119I. is L119M.

[0124] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 121 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is A121S.

[0125] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 122 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is N122. In some embodiments, the amino acid substitution is N122E. 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. The amino acid substitution is N122S.

[0126] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 125 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is A125S.

[0127] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 127 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is A127M. is A127S. In some embodiments, the amino acid substitution is A127V.

[0128] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 135 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is A135G.

[0129] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 136 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 an embodiment, the amino acid substitution is T136V.

[0130] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 138 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is R138E. is R138L.

[0131] In some embodiments, the variant Bhr-PETase has a nucleotide sequence similar to that of position 139 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, asparagine, Lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine leucine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is I139A. is I139T.

[0132] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 140 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is S140A.

[0133] In some embodiments, the variant Bhr-PETase comprises the nucleotide sequence at position 142 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids of glutamine. Naturally occurring amino acids, namely, serine, threonine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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. It is 142W.

[0134] In some embodiments, the variant Bhr-PETase has a nucleotide sequence similar to that of position 143 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, asparagine, Lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine leucine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is I143N. is I143R.

[0135] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 145 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is T145S.

[0136] In some embodiments, the variant Bhr-PETase comprises a nucleotide sequence similar to that at position 147 of SEQ ID NO:1. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present, namely, serine, threonine, glutamine, asparagine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 an embodiment, the amino acid substitution is K147Q.

[0137] In some embodiments, the variant Bhr-PETase comprises the nucleotide sequence at position 149 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, Valine and tyrosine are used, and some embodiments use proline (due to steric effects). In some embodiments, the amino acid substitution is G149A. In some embodiments, the amino acid substitution is G149C. 149D. In some embodiments, the amino acid substitution is G149N. In some embodiments, the amino acid substitution is G149S. 9T. In some embodiments, the amino acid substitution is G149V.

[0138] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase at position 150 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan In some embodiments, cysteine ​​(in disulfide form) is used. (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is V150I. is the V150L.

[0139] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 153 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is T153L.

[0140] In some embodiments, the variant Bhr-PETase comprises a human Bhr-PETase having a nucleotide sequence similar to that of position 156 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, arginine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is H156N.

[0141] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 157 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is T157A. is the T157G.

[0142] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 158 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. of the naturally occurring amino acids, namely, serine, threonine, glutamine, and asparagine , lysine, arginine, histidine, glutamic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 an embodiment, the amino acid substitution is D158L.

[0143] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 160 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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. It is 160V.

[0144] In some embodiments, the variant Bhr-PETase has a nucleotide sequence similar to that of position 161 of SEQ ID NO:1. In some embodiments, the substitution is any other 19 amino acid sequence. Naturally occurring amino acids of the species, namely, serine, threonine, glutamine, asparagine lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, Lysine, proline, alanine, isoleucine, leucine, methionine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is F161V. is F161W.

[0145] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 162 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is N162. In some embodiments, the amino acid substitution is N162H. 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. be.

[0146] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 163 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is T163I. is the T163S.

[0147] In some embodiments, the variant Bhr-PETase has a sequence similar to that of position 164 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is P164 In some embodiments, the amino acid substitution is P164H. 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. The amino acid substitution is P164T.

[0148] In some embodiments, the variant Bhr-PETase comprises the nucleotide sequence at position 167 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids of glutamine. Naturally occurring amino acids, namely, serine, threonine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is Q167I. is Q167T. In some embodiments, the amino acid substitution is Q167V.

[0149] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase at position 170 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan In some embodiments, cysteine ​​(in disulfide form) is used. (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is V170L.

[0150] In some embodiments, the variant Bhr-PETase comprises the nucleotide sequence at position 173 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, asparagine, Lysine, arginine, histidine, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is E173R.

[0151] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 174 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is A174K. is A174R.

[0152] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase at position 177 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan In some embodiments, cysteine ​​(in disulfide form) is used. (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is V177A.

[0153] In some embodiments, the variant Bhr-PETase comprises the polypeptide at position 179 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is P179. It's Q.

[0154] In some embodiments, the variant Bhr-PETase has a sequence similar to that of position 181 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, Valine and tyrosine are used, and some embodiments use proline (due to steric effects). In some embodiments, the amino acid substitution is S181A. In some embodiments, the amino acid substitution is S181C. It is 181R.

[0155] In some embodiments, the variant Bhr-PETase comprises the nucleotide sequence at position 182 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids of glutamine. Naturally occurring amino acids, namely, serine, threonine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is Q182T.

[0156] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 184 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, Valine and tyrosine are used, and some embodiments use proline (due to steric effects). In some embodiments, the amino acid substitution is A184C. In some embodiments, the amino acid substitution is A184G. It is 184S.

[0157] In some embodiments, the variant Bhr-PETase has a sequence similar to that of position 185 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, asparagine, Lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine leucine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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. 1185Q. In some embodiments, the amino acid substitution is I185R. In some embodiments, the amino acid substitution is I185S. It's 5Y.

[0158] In some embodiments, the variant Bhr-PETase comprises the nucleotide sequence at position 189 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids of glutamine. Naturally occurring amino acids, namely, serine, threonine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is Q189I. is Q189L. In some embodiments, the amino acid substitution is Q189V.

[0159] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 190 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is N190S.

[0160] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 193 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is at S193 In some embodiments, the amino acid substitution is S193F. 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. 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 a sequence similar to that at position 194 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is T194G. is the T194S.

[0162] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 195 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is T195F.

[0163] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase at position 198 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan In some embodiments, cysteine ​​(in disulfide form) is used. (due to the possibility of synthesis) and proline (due to steric effects). In one embodiment, the amino acid substitution is V198A.

[0164] In some embodiments, the variant Bhr-PETase comprises the Bhr-PETase at position 200 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan In some embodiments, cysteine ​​(in disulfide form) is used. (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is V200L.

[0165] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 203 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. of the naturally occurring amino acids, namely, serine, threonine, glutamine, and asparagine , lysine, arginine, histidine, glutamic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is D203N. is D203R. In some embodiments, the amino acid substitution is D203V.

[0166] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 204 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 an embodiment, the amino acid substitution is N204S.

[0167] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 206 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is T206. In some embodiments, the amino acid substitution is T206K. 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 a nucleotide sequence similar to that of position 208 of SEQ ID NO:1. In some embodiments, the substitution is any other 19 amino acid sequence. Naturally occurring amino acids of the species, namely, serine, threonine, glutamine, asparagine lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, Lysine, proline, alanine, isoleucine, leucine, methionine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is F208G. In some embodiments, the amino acid substitution is F208R. In an embodiment, the amino acid substitution is F208T.

[0169] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 209 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In one embodiment, the amino acid substitution is A209V.

[0170] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 211 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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. It is 211V.

[0171] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 212 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is S212F. is S212L. In some embodiments, the amino acid substitution is S212M.

[0172] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase having a sequence similar to that at position 213 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is P213 In some embodiments, the amino acid substitution is P213R.

[0173] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 216 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is A216. In some embodiments, the amino acid substitution is A216P. 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 a sequence similar to that of position 217 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, asparagine, Lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine leucine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is I217S.

[0175] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 218 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is S218A.

[0176] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase at position 219 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan In some embodiments, cysteine ​​(in disulfide form) is used. (due to the possibility of synthesis) 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. It is 219R.

[0177] In some embodiments, the variant Bhr-PETase has a sequence similar to that at position 221 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, glutamine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is T221S.

[0178] In some embodiments, the variant Bhr-PETase has a nucleotide sequence similar to that of position 222 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, asparagine, Lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine leucine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is I222L.

[0179] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 223 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, Valine and tyrosine are used, and some embodiments use proline (due to steric effects). In some embodiments, the amino acid substitution is S223A. In an embodiment, the amino acid substitution is S223C.

[0180] In some embodiments, the variant Bhr-PETase has a nucleotide sequence similar to that at position 225 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine , proline, alanine, isoleucine, leucine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In one embodiment, the amino acid substitution is M225L.

[0181] In some embodiments, the variant Bhr-PETase has a locus at position 227 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is L227R.

[0182] In some embodiments, the variant Bhr-PETase comprises a nucleotide sequence similar to that at position 228 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. of the naturally occurring amino acids, namely, serine, threonine, glutamine, and asparagine , lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, glycolipid Leucine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is W228F.

[0183] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase at position 229 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan In some embodiments, proline (due to steric effects) is used. In some embodiments, the amino acid substitution is V229C. In some embodiments, the amino acid substitution is V229I. It is 229L.

[0184] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 231 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is N231L. is N231Q. In some embodiments, the amino acid substitution is N231S.

[0185] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 236 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, Valine and tyrosine are used, and some embodiments use proline (due to steric effects). In some embodiments, the amino acid substitution is R236C. In some embodiments, the amino acid substitution is R236E. 236H. In some embodiments, the amino acid substitution is R236K. In this embodiment, the amino acid substitution is R236Q.

[0186] In some embodiments, the variant Bhr-PETase comprises the nucleotide sequence at position 237 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids of glutamine. Naturally occurring amino acids, namely, serine, threonine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is Q237R.

[0187] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 241 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is N241. It's P.

[0188] In some embodiments, the variant Bhr-PETase comprises a Bhr-PETase at position 242 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Leucine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan In some embodiments, cysteine ​​(in disulfide form) is used. (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is V242T.

[0189] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 243 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is N243. It's P.

[0190] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 246 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present in arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine, Proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 an embodiment, the amino acid substitution is A246T.

[0191] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 249 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. of the naturally occurring amino acids, namely, serine, threonine, glutamine, and asparagine , lysine, arginine, histidine, glutamic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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. It is 249T.

[0192] In some embodiments, the variant Bhr-PETase has a nucleotide sequence similar to that at position 250 of SEQ ID NO:1. In some embodiments, the substitution is any other 19 amino acid sequence. Naturally occurring amino acids of the species, namely, serine, threonine, glutamine, asparagine lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine, Lysine, proline, alanine, isoleucine, leucine, methionine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 an embodiment, the amino acid substitution is F250Y.

[0193] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 251 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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. 251L. In some embodiments, the amino acid substitution is R251Q. In some embodiments, the amino acid substitution is R251T. It is 1V.

[0194] In some embodiments, the variant Bhr-PETase comprises a nucleotide sequence similar to that at position 252 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 naturally occurring amino acids. The amino acids present, namely threonine, glutamine, asparagine, lysine, arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is S252T.

[0195] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 253 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is N253S. is N253Y.

[0196] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 254 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely, serine, threonine, glutamine, lysine, and arginine Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is N254R.

[0197] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 255 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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 R255V. In some embodiments, the amino acid substitution is R255W. It's 5Y.

[0198] In some embodiments, the variant Bhr-PETase comprises the nucleotide sequence at position 258 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids of glutamine. Naturally occurring amino acids, namely, serine, threonine, asparagine, lysine, and arginine, Nin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is Q258. It's P.

[0199] In some embodiments, the variant Bhr-PETase is an arginine-binding domain of SEQ ID NO: 1 at position 8. In some embodiments, the substitutions are with any of the other 19 naturally occurring amino acids. The amino acids present are serine, threonine, glutamine, asparagine, and lysine. , histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, ara Nine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan, and tyrosine, and some embodiments use cysteine ​​(for disulfide formation). In some embodiments, the amino acid substitution is P8T. .

[0200] In some embodiments, the variant Bhr-PETase has an amino acid sequence similar to that of position 31 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids. The amino acids present in histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In an embodiment, the amino acid substitution is L31M.

[0201] In some embodiments, the variant Bhr-PETase has an amino acid sequence similar to that of position 38 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids. The amino acids present in histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In embodiments, the amino acid substitution is G38D.

[0202] In some embodiments, the variant Bhr-PETase has an amino acid sequence similar to that of position 95 of SEQ ID NO:1. In some embodiments, the substitution is with any of the other 19 naturally occurring amino acids. The amino acids present in histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Leucine, isoleucine, leucine, methionine, phenylalanine, tryptophan, with phosphorus and tyrosine, and some embodiments with cysteine ​​(disulfide forming (due to possible side effects) and proline (due to steric effects). In an embodiment, the amino acid substitution is S95N.

[0203] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 126 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is V126I.

[0204] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 137 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In this embodiment, the amino acid substitution is L137M.

[0205] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 165 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is V165I.

[0206] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 169 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, Valine and tyrosine are used, and some embodiments use proline (due to steric effects). In some embodiments, the amino acid substitution is I169C. In some embodiments, the amino acid substitution is I169L. It is 169V.

[0207] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 172 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In an embodiment, the amino acid substitution is A172T.

[0208] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 191 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) and proline (due to steric effects). In some embodiments, the amino acid substitution is L191F. is L191V.

[0209] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 192 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form In some embodiments, the amino acid substitution is P192 It's A.

[0210] In some embodiments, the variant Bhr-PETase comprises the amino acid sequence at position 197 of SEQ ID NO:1. In some embodiments, the substitution is any of the other 19 amino acids. Naturally occurring amino acids, namely serine, threonine, glutamine, asparagine, gin, histidine, glutamic acid, aspartic acid, cysteine, glycine, proline, Alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, With valine and tyrosine, and some embodiments with cysteine ​​(in disulfide form (due to the possibility of synthesis) 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. It is 197Y.

[0211] In some embodiments, the variant Bhr-PETase enzyme has the sequence Number 1 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, 2 It has one or more amino acid substitutions at 9 or 30 positions.

[0212] D. Nucleic Acids of the Invention The present invention additionally provides nucleic acids encoding the variant Bhr-PETases of the present invention. As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, all A large number of nucleic acids encoding bright variant Bhr-PETases can be generated. Once a particular amino acid sequence is identified, one skilled in the art can easily identify the amino acid sequence of the protein. Many different sequences can be generated by simply altering the sequence of one or more codons so that the sequence remains unchanged. Thus, providing the amino acid sequence allows the production of a protein. This allows for the generation of a large number of different nucleic acid sequences encoding

[0213] In some embodiments, the specific variant Bhr-PETase is selected from the group consisting of SEQ ID NOs: 2 and 3. In some embodiments, the specific barrier The Bhr-PETase has at least about 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, 3, 84, 85, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 , 96, 97, 98, 99 or 100% identity to a nucleic acid sequence .

[0214] As is well known in the art, nucleic acids encoding the components of the present invention can be prepared using methods known in the art. The host cells used to produce the heterodimeric antibodies of the present invention are well known in the art. Generally, the nucleic acid may be incorporated into an expression vector according to the invention. motors, replication origins, selectable markers, ribosome binding sites, inducers, etc. The expression vector may be an extrachromosomal or an integrating vector.

[0215] The nucleic acids and / or expression vectors of the invention can then be expressed in mammalian, bacterial, yeast, insect and Many different types of host cells are known in the art, including bacteria and / or fungi. Cells, bacteria, yeast and fungi find use in many embodiments and can be transformed. .

[0216] 1. Variant Preparation Nucleic acids encoding the variant Bhr-PETases of the present invention are well known in the art. As is known, methods known in the art, such as site-directed mutagenesis and synthetic gene construction, can be used to It may be prepared using any mutagenesis procedure.

[0217] Synthetic gene construction involves the synthesis of a polynucleotide designed to encode a polypeptide of interest. Gene synthesis requires in vitro synthesis of the gene molecule. Multiplex mycobacterium erythroblastoma (Nature 432:1050-1054) The technology is based on a microchip, and oligonucleotides are synthesized and photoprogrammable ( similar to those assembled on a photo-programmable microfluidic chip. This can be implemented using a number of techniques, such as the technique of GenScript ( is a registered trademark).

[0218] 2. Control arrays The invention provides for the expression of a coding sequence in a suitable host cell under conditions compatible with the regulatory sequences. The variant of the present invention is operably linked to one or more regulatory sequences that direct the The present invention also relates to nucleic acid constructs comprising the polynucleotides encoding the nucleic acid sequences.

[0219] Polynucleotides may be manipulated in a variety of ways to result in the expression of variants. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable depending on the expression vector. It may be necessary or desirable to use recombinant DNA techniques to modify the polynucleotide. Techniques for this are known in the art.

[0220] The regulatory sequence may be a promoter, a sequence recognized by a host cell for expression of the polynucleotide. A promoter can be a polynucleotide that mediates the expression of a variant. Promoters include mutant, truncated and hybrid promoters. The polynucleotide may be any polynucleotide that exhibits transcriptional activity in a host cell, including the host cell. Genes encoding extracellular or intracellular polypeptides, either congener or heterologous It can be obtained from children.

[0221] Promoters for bacteria, yeast, and fungi are known in the art. As an exemplary operon for expression in bacteria, The lactose operon or Ll of S. thermophilus is well used for actic lac ABCDFEGX operon (e.g., Simons et al. , 1993, J. Bact. 175:5186~5175; Mollet et al., 1993, (See J. Bact. 175:4315-4324.) Constitutive processes for bacteria Additional non-limiting examples of promoters include the lac promoter, the trp promoter, and the t ac promoter, T7 promoter, erm promoter, tip promoter, ni Exemplary promoters for yeast include the t promoter and the Sp6 promoter. Examples include, but are not limited to, the AOX1 promoter, the ADH promoter, and PH0 5 promoter, gal10 promoter, PKG promoter and GAP promoter Other useful promoters for yeast host cells include Romanoset et al., 1992, Yeast 8:423-488. Examples of useful promoters for vectors include, but are not limited to, Aspergillus oryzae. illus niger GLA promoter, Aspergillus nidula Aspergillus ni promoters such as ns GPD promoter and adh3 promoter dulans glycolytic pathway genes (McKnight et al., EMBO J. 4:209 3~2099,1985).

[0222] 3. Codon optimization The wild-type coding sequence for Bhr-PETase is shown in SEQ ID NO: 1, however, those skilled in the art will recognize heterologous Bhr-PETase. Codon optimization may be used to increase expression in any particular host organism for genomic expression. It will be appreciated that codon optimization may be performed to optimize expression in the host cell used. To optimize expression, any variant Bhr-PETase polypeptide of the invention may be Such methods are known in the art and can be used, for example, with WO2 In heterologous expression systems, the optimization step is carried out to optimize the desired The ability of the host to produce the variant Bhr-PETase polypeptide can be improved. Protein expression affects transcription, mRNA processing, and translation stability and initiation. The polynucleotide optimization step is governed by host factors, including those that can be Steps for improving the host's ability to produce the desired protein and for efficiently expressing the expression construct. The optimization strategy may include, for example, assisting researchers in systematically designing a translation initiation region. This may include altering the region, altering mRNA structural elements, and using different codon bias. The following paragraphs describe potential issues that may result in reduced heterologous protein expression, and how these issues may be addressed. We will consider technologies that can overcome this.

[0223] In some embodiments, the reduction of heterologous protein expression is achieved by rare codon-induced translational pausing (ra re codon-induced translational pause Rare codon-induced translation pausing occurs when codons that are rarely used in the host organism are deleted. their presence in the available tRNA pool, including their presence in the target polynucleotide deficiency can have a negative effect on protein translation. One way to achieve this is to modify rare host codons in the synthetic polynucleotide sequence. This includes performing possible codon optimization.

[0224] In some embodiments, the reduction in heterologous protein expression results from alternate translation initiation. Each translation initiation site contains a motif that can function as a ribosome binding site (RBS). These sites may contain synthetic polynucleotide sequences that are inadvertently included in the gene. This may result in the initiation of translation of truncated proteins from internal sites. One way to reduce the likelihood of producing truncated proteins, which can be difficult, is to This involves modifying the predicted internal RBS sequence from the engineered polynucleotide sequence.

[0225] In some embodiments, the reduction in heterologous protein expression is due to a repeat-induced polymerase chain reaction. Repeat-induced polymerase slippage occurs through frameshift mutations. This can cause slippage or stuttering of DNA polymerase, which can lead to abnormalities. Such repeat sequences are involved in the synthesis of RNA polymerase In organisms with a high G+C content bias, slippage of G or C nucleotides can also occur. There may be higher degrees of repetition consisting of nucleotide repeat sequences. Therefore, one way to reduce the possibility of inducing RNA polymerase slippage is to or C nucleotide repeat stretches.

[0226] In some embodiments, the reduction of heterologous protein expression is achieved through disrupting secondary structure. The secondary structure may separate the RBS sequence or the start codon, resulting in the formation of a protein. Stem-loop structures may also be involved in pausing and attenuating transcription. The optimized polynucleotide sequence is then used to synthesize the nucleoside analogue to allow for improved transcription and translation. The RBS and gene coding regions of the peptide sequence may contain minimal secondary structure.

[0227] In some embodiments, the restriction site may affect heterologous protein expression. By modifying restriction sites that may prevent subsequent subcloning into a host expression vector Thus, the polynucleotide sequence can be optimized.

[0228] Optimizing the DNA sequence can negatively or positively affect gene expression or protein production. For example, changing a less common codon to a more common codon can affect the This can affect the half-life of the mRNA or interfere with translation of the message. The structure can be modified by introducing a secondary structure. , it may be necessary to modify the optimized message.

[0229] All or part of the gene may be optimized. In some embodiments, the desired expression Regulation is achieved by essentially optimizing the entire gene. The desired regulation is achieved by optimizing some but not all of the genes.

[0230] The codon usage of any coding sequence can be varied to achieve desirable properties, e.g., high levels in a particular cell type. The starting point for such optimization is 100%. Coding sequences using common codons or a mixture of common and uncommon codons The coding sequence may contain a combination of the two.

[0231] Two or more candidate sequences that differ in codon usage can be generated and compared to determine whether they have the desired properties. Candidate sequences can be tested to determine whether they act as silencers or enhancers. to investigate the presence of regulatory elements such as sar and changes in codon usage Search for the presence of regions of the coding sequence that can be converted into such regulatory elements by Additional criteria include the use of computers to assess the The abundance of specific nucleotides, e.g., A, C, G, or U, and the abundance of specific amino acids codon bias in the mRNA, or the presence or absence of specific mRNA secondary or tertiary structures. Adjustments to the candidate sequences can be made based on a number of such criteria. .

[0232] Promising candidate sequences are constructed and then experimentally evaluated. Alternatively, the process may use the most promising candidates as a new starting point and, if or by combining two or more candidate regions to produce a novel hybrid. Any of these may be iterative and may include further rounds of modification and evaluation.

[0233] Altering the codon usage of a candidate sequence can result in the creation of either positive or negative elements. Generally, a positive element is a candidate element that can be modified or removed from the candidate sequence. may result in a decrease in expression of a therapeutic protein, or its creation may result in a decrease in expression of a therapeutic protein. For example, enhancers are positive elements. -, promoter, downstream promoter element, positive regulator (e.g., transcriptional activator) DNA binding sites for the target molecule, or to provide or modify mRNA secondary or tertiary structure Negative elements include sequences that are the cause of the change from the candidate sequence or The removal may result in increased expression of the therapeutic protein, or its creation may result in increased expression of the therapeutic protein. Negative elements include silencers, DNA binding sites for negative regulators (e.g., transcriptional repressors), transcriptional pause sites, or These include sequences that are responsible for imparting or modifying mRNA secondary or tertiary structure. In general, negative elements occur more frequently than positive elements. Any change in codon usage that results in an increase in expression is more likely to result in the creation of a positive element. It is more likely that the alteration of the candidate sequence is due to the disruption of a negative element. , are more likely to destroy positive elements than to create them. In this embodiment, candidate sequences are selected and modified to increase production of the therapeutic protein. The candidate sequence is varied, for example, by sequentially varying the codons or by The modified candidate can then be The sequences can be analyzed by measuring the level of expression of the resulting therapeutic protein or by another protein. parameter, e.g., by assessing a parameter that correlates with the level of expression Candidate sequences that result in increased levels of a therapeutic protein compared to the unmodified candidate sequence. is selected.

[0234] In some embodiments, a codon or group of codons may be used to encode, for example, a protein or message. Alternatively, one or more codons may be modified and tested without reference to the codon structure. is a characteristic of the message level, e.g., location in a predetermined region, e.g., high if Locations in regions with low GC content, enhancers, or silencers In regions that can be modified to introduce structures such as enhancers or silencers, The present invention relates to a method for producing a nucleic acid sequence having or predicted to have secondary or tertiary structure, e.g., intrastrand pairing, interstrand pairing, etc. the location in the region where the secondary or tertiary structure is lacking, e.g., intra- or inter-strand pairings The specific modified gene may be selected based on its location in the region predicted to be missing or missing. The region is selected if it produces the desired result.

[0235] Methods for systematically generating candidate sequences are useful. For example, A codon or group of codons, e.g., a contiguous block of codons, is a common codon (or using uncommon codons, e.g., if the starting sequence is optimized ) may be modified and the resulting sequences may be evaluated. Candidates may be sequenced to generate the first candidate. Optimize (or deoptimize) a given "window" of codons in the string, then Move the mouse to a new position in the sequence and click the new mouse under the window to provide a second candidate. These sequences can be generated by optimizing (or deoptimizing) codons at new positions. Candidates can be identified by determining the level of expression they produce or by another parameter. -, for example, by assessing a parameter that correlates with the level of expression Some parameters can be assessed by survey or computationally, e.g. sequence elements such as high or low GC content; enhancers or silencers; secondary or tertiary structure, e.g., intrastrand or interstrand pairing; g) Possession or lack thereof.

[0236] In some embodiments, the optimized nucleic acid sequence is a variant Bhr-PETa of the invention. se polypeptide at least at the level expressed by a non-optimized nucleic acid sequence. Also about 110%, 150%, 200%, 500%, 1,000%, 5,000% or more can be expressed at a level of 10,000%.

[0237] Starting from the amino acid sequence of the variant Bhr-PETase, candidate DNA sequences were designed. When designing a synthetic DNA sequence, the frequency of codon usage should be determined based on the codon usage of the host expression organism. The synthetic candidate may be compared to the original and rare host codons may be modified in the synthetic sequence. The coDNA sequence may be modified to remove undesired enzyme restriction sites and include any desired signal sequence, linker sequence, or both. The DNA sequence may be modified to add or change anchors or untranslated regions. The translation process is disrupted by secondary structures such as G / C repeats and stem-loop structures. Before candidate DNA sequences are synthesized, optimized sequence designs can be analyzed for the presence of , the sequence may be checked to verify that it correctly encodes the desired amino acid sequence. Finally, candidate DNA sequences can be synthesized using DNA synthesis techniques such as those well known in the art. It can be synthesized using

[0238] In some embodiments, a common phenotype in a host organism, such as any of those described herein. Suitable codon usage is used to optimize expression of heterologous polynucleotide sequences in the host organism. It is unlikely that there will be a preference for a particular amino acid in the host expression system. The percentage and distribution of unused codons can be estimated. Values ​​for 5% and 10% usage are used to estimate the percentage of rare codons. It can be used as a cutoff value for determining the codon.

[0239] 4. Host Cells and Production Strains In one aspect, the disclosure provides a gene encoding a variant Bhr-PETase as described herein. In another aspect, the disclosure relates to an expression vector comprising the nucleic acid. 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. The host cells are bacteria, including but not limited to E. coli and Bacillus species. In some embodiments, the host cell may be, but is not limited to, Saccharomyces cerevisiae. The yeast may be a yeast such as myces cerevisiae or Pichia. In this embodiment, the host cell may be any of a variety of cells, including but not limited to A. niger, T. reseei, or Myceliophthora thermophila obtain.

[0240] The expression vector may be integrated into the genome or may be expressed autonomously in the selected host. It can be any integrating vector that is a replicating plasmid. In one embodiment, the vector The variant Bhr-PETase gene is supported in a suitable state for expression. The expression vector can be stably maintained in the cell into which it is introduced. The vectors can be selected to be suitable for the particular host cell in which they are intended to be used. Specific examples include, but are not limited to, Escherichia coli as a host. pBR322, pACYC184, pUC18, pKK22 when E. coli is used 3-2, pHSG398 (Takara Bio Inc.), pTrcHis (Inv pET11a (Stratagene Corporation) and pET11a (Stratagene pBBR122 (Mobio Corporation) for other Gram-negative bacteria tech) and pBHR1 (Mobiotech); pH for Bacillus spp. W1520 (Mobiotech) and pHY300PLK (Takara Bio Inc.); pSH19 for actinomycetes (Herai et al., Proc. Natl. Acad. Sci. .Sci., 101, 14031~14035, 2004), pIJ702 (John Innes Centre), pIJ943(John Innes Centre), pIJ8600 (John Innes Centre), pIJ602 (John I Nnes Centre), 1, pTip-vector (Nakashima et al., Appl i.Environ.Microbiol.,70,5557~5568,2004), pTYM19 (Onaka et al., J. Antibiot., 56,950-956,200 3); pPICZα and pPIC9 (Thermo Fish) for Pichia spp. pAO815 (Invitrogen Scientific) for fungi n Corporation), pAUR101 (Takara Bio Inc.), pAUR123 (Takara Bio Inc.) and pAUR316 (Takar a Bio Inc.

[0241] In another aspect, the present disclosure provides a method for expressing a variant Bhr-PETase in a host cell. In another aspect, the present disclosure relates to a method for producing a variant Bhr-PETase. and recovering said variant Bhr-PETase. The present invention also relates to a method for producing said variant Bhr-PETase, comprising:

[0242] Cultivation of transformed organisms affected the transformation of variant Bhr-PETase. The method may be carried out in a medium that may be the nutrient medium of the transformed host cells without any modification. Such a medium contains appropriate carbon sources, nitrogen sources, inorganic salts, natural organic nutrients, etc. As sources, glucose, fructose, glycerin, sorbitol, and organic acids are available individually or The concentration of the carbon source is not particularly limited and may be 1 to 10%. Nitrogen sources include ammonium, urea, ammonium sulfate, and ammonium nitrate. , ammonium acetate, etc. may be used individually or in combination of two or more of these members. Inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, Salts such as manganese sulfate and iron sulfate may be used. In addition, they may promote the growth of the bacteria used. Peptone, meat extract, yeast extract, cornstarch, and other organic nutrients that promote muscle growth are also used. Corn steep liquor and casamino acids may be used, as well Additionally, small amounts of vitamins and nucleic acids may be included in the medium.

[0243] 5. PETase Preparation and Use As will be appreciated by those skilled in the art, formulations of the variant Bhr-PETase of the present invention may be The Bhr-PETas of the present invention are also useful for the production of PETs containing PEG-14. Suitable formulations of e include liquids, dried formulations (including spray-dried formulations), powders, These include granules, pellets, and granules. Bhr-PETase is a naturally occurring decomposer. It may also be formulated "embedded in PET particles" for

[0244] In some embodiments, the enzyme compositions (i.e., polypeptide compositions) of the present invention may be prepared using, for example, For example, crude fermentation broth with or without cell removal, if containing cellular debris, as a source of enzymes, or as a cell lysate, semi-purified or purified enzyme composition, or enzymes containing or not containing It may be in any form suitable for use, such as a host cell.

[0245] In some embodiments, the enzyme composition is a dry powder or granule, non-dusty granule. dusting granulate), liquid, stabilized liquid or stabilized protected enzyme (sta The liquid enzyme composition may be a liquid enzyme composition containing a soluble protected enzyme. For example, sugar, sugar alcohol or another polyol, and / or lactic acid or The solubility may be stabilized by adding a stabilizer such as another organic acid according to the process described above.

[0246] In some embodiments, dosages and compositions of the polypeptide compositions of the invention are used Other conditions can be determined based on methods well known in the art.

[0247] The above composition is suitable for PET biodegradation, PET biocycling, and PET upcycling. and / or suitable for use in 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 Pretreatments are broadly classified into a) mechanical pretreatment, b) thermomechanical pretreatment, and c) chemical pretreatment. The mechanical process can be used to pulverize the PET film into particles less than about 500 μm after sieving. This process involves crushing the PET film in liquid nitrogen, primarily to facilitate the crushing process. The advantage of this type of mechanical pretreatment is the reduction of particle size. In thermomechanical degradation, the main chain scission reaction occurs in the cyclic oligomers. During thermomechanical pretreatment, the highly crystalline PET flakes An extruder equipped with a melt pump is used to extrude at very high temperatures (>2 60°C) and then micronized to a particle size of less than 500 μm. An example of a commercial method for pretreating PET is extrusion molding. st) process, i.e., melted PET on a chilled roll to solidify. Thermomechanical pretreatment improves the crystallinity and toughness of industrial grade PET. and particle size, making it accessible to enzymes for depolymerization. In the case of chemical pretreatment, ionic solutions, strong acids, bases, solvents, etc., are used to further depolymerize the polymer into monomers. To facilitate the access of enzymes that cause oxidation, to reduce the crystallinity of PET, or to improve the surface structure It can be used to change the structure.

[0249] In recycled plastics, regardless of whether chemical or biological methods are used Two key obstacles are material variability and the difficulty of converting waste plastic into acceptable grades. The costs associated with identifying and separating different types of plastics and The grades vary in density and molecular weight. The advantage of knowing the properties of a substance is that it can be used to improve its value and However, the sorting process is resource-intensive, time-consuming, and time-consuming. This is a major obstacle in terms of time management and cost-effectiveness. When looking at biological mitigation to overcome other related obstacles, it is important to have broad substrate specificity. Depolymerization of mixed plastics using robust enzymes is becoming a high priority. Tick ​​sources all plastic packaging, not just bottles, from the domestic waste stream. It is a term that encompasses various polymer types and materials typically found in household trash cans. Includes rigid and flexible plastic products in a variety of colors and colours. Plastic refers to a mixture of various plastics. The different polymer types are PET and and / or biologically or chemically derived analogues of PET, PET-like substances or PE Examples of PET analogs, PET-like materials or PET substitutes include Including but not limited to polybutylene terephthalate (PBT), polycarbonate (PC) , polycaprolactone (PCL), polyethylene furanoate (PEF) and high density polyethylene Bhr-PETase can be used alone or in combination with other enzymes. In conjunction with accessory enzymes, it is possible to innovate biological methods for the depolymerization of mixed plastics. The process of enzymatic depolymerization of plastics is environmentally safe, in contrast to chemical methodologies. Such biological treatment may also be beneficial in terms of reducing the cost of wastewater treatment and preserving the market value of secondary use applications. It has also been combined with mild chemical or thermomechanical pretreatment to achieve high depolymerization efficiency. This may also be done.

[0250] After PET pre-treatment and enzymatic degradation, the products can be reused to produce other valuable chemicals In one embodiment, TPA (terephthalic acid) can be purified using an industrially suitable process. The process is carried out using chemicals commonly used in the surfactant, paper and glass industries. In another embodiment, the recycled TPA is added to the unused P It is used as a starting material for synthesizing PET. PET synthesized from recycled TPA is The average molecular weight and intrinsic viscosity are the same as those of PET synthesized using the petrochemical product TPA. In one embodiment, bottles made from recycled PET exhibit similar properties to regular PET bottles. It exhibits similar mechanical properties to tungsten but better brightness.

[0251] In some embodiments, the present invention provides a method for the efficient turnover of pre-treated PET by PET degradation. To produce an enzyme cocktail, the variant Bhr-PETase described herein can be used in combination with other Prepare an enzyme cocktail containing PET along with accessory enzymes and downstream MHETase This provides a method for [Example]

[0252] PET hydrolase gene selection, synthesis, and cloning Multiple novel PET hydrolases were selected based on bioinformatic analysis and analyzed using GeneWiz (https: / / www.genewiz.com / en / ) The gene was transfected into the pET28b(+) vector (Millipore Inc., catalog no. 6 Three hydrolases (Lcc-PETase, Bhr- PETase, Is-PETase) was selected for further purification and characterization. [Example]

[0253] Produced by Escherichia coli in 250 ml shake flasks Preparation of selected PET hydrolases Chemical colonies of BL21(DE3) containing recombinant hydrolase-encoding genes were isolated from single colonies. Infectious E. coli (ThermoFisher Scientific, USA) (Cat. No. C600003) with 1% glucose and 50 μg / mL kanamycin The cultures were inoculated into individual culture tubes containing 5 mL of lysogeny broth (LB broth) containing 1000 kJ / ml of lysogeny broth. Grow overnight at 30°C, 200 rpm and 85% humidity. Add 1 mL of overnight culture to 50 μL of PBS. Shake 50 mL of Terrific Broth (TB broth) containing 100 μg / mL kanamycin. The flasks were then incubated at 37°C, 250 rpm and humidity for 2-2.5 hours. The incubation was carried out at 85%. The OD before induction was measured at 2 to 2.5 hours. When it reaches approximately 0.6-0.8, adjust the pH to obtain a final concentration of 0.5 mM IPTG in the flask. Perform induction using IPTG (isopropyl β-D-1-thiogalactopyranoside) All flasks were then grown at 16°C, except for Is-PETase. The mixture was incubated for 18-24 hours at 30°C, 250 rpm, and 85% humidity. The lysate was transferred to a 50 mL centrifuge tube and stored at -20°C prior to activity assay. Three candidates (Lcc-PETase, Bhr-PETase, Is-PETase) were identified. The cells were grown according to the procedures described. [Example]

[0254] Ni column purification of His-tagged PET hydrolase produced in shake flasks Each sample was analyzed using Thermo Scientific HisPur™ Ni-NTA S The solution was concentrated to 15-20 ml before use on a pin column (Cat. No. 88226). Three PET hydrolases (Lcc-PETase, Bhr-PETase, Is-PETase) The enzyme was purified from cell cultures according to the manufacturer's guidelines. Elution was performed in 250 mM imidazole and proteins from the eluate in various fractions were collected. Desalt the combined fractions stock solution based on band intensity (measured using gel quantitation) Used for steps. [Example]

[0255] Desalting of purified PET hydrolase produced in shake flasks Disposable Thermo Scientific Zeba™ Spin Desal A 7K MWCO Column (Cat. No. 89892) was used to separate imidazole and Ni and NaCl were removed from the purified enzyme. The desalting protocol was performed according to the manufacturer's guidelines. The desalted enzyme was then quantified to determine the protein concentration (g / L). [Example]

[0256] Es using BHET (bis(2-hydroxyethyl) terephthalate) as a substrate Thermostability study of PET hydrolase produced by Cherichia coli. The normalized PET hydrolase protein of each candidate was incubated at 30°C, 50°C, or 70°C. The enzyme was kept on ice for the same time period and used as the untreated condition. After treatment, 250 μL of 10 mM B HET (bis(2-hydroxyethyl) terephthalate), 62.5 μL untreated and The treated enzyme and 170 μL of 0.1 M sodium phosphate buffer, pH 8, were added. The plates were incubated at 50°C for 2 hours. After 2 hours, the samples were rotated at 4,000 rpm for 2 To determine the amount of TPA produced in our reaction, The supernatant was analyzed using high performance liquid chromatography (HPLC). Eclipase Plus C18(Rapid Resolution HD 2. 1 x 50mm (1.8 micron) column, part number 959757-902 as guard column , used with part number 82175-901. Flow rate: 0.6 mL / min, column temperature: 35°C The sample was set up for running on HPLC. Mobile phase 1 consisted of acetonitrile containing 0.1% trifluoroacetic acid, and mobile phase 2 consisted of acetonitrile containing 0.1% trifluoroacetic acid. A gradient method was used to detect terephthalic acid (TPA) using each P The percentage of ET hydrolase remaining was calculated from the following formula: (MHET produced under treatment conditions) +TPA) / (MHET produced in untreated conditions +TPA)*100.

[0257] Three candidates (Lcc-PETase, Bhr-PETase, Is-PETase) The thermal stability results are shown in Figure 4. Under the experimental conditions, Is-PETase showed a thermal stability of 9 at 50°C. Bhr-PETase and Lcc-PETase were the least stable candidates, losing 0% of their activity. PETase retains ≥80% residual activity at all temperatures tested. [Example]

[0258] Large-scale P for evaluating Escherichia coli-produced PET hydrolases ET film assay These experiments were carried out in 250 ml glass bottles. Two types of PET substrates were used. A) Amorphous PET film was used in the experiments of Go et al. PET film was obtained from Woodfellow (Cat. No. ES301445). Use a paring cutter to cut into 1 x 30 cm pieces, then cut again into approximately 1 x 0.25 cm pieces. Pieces of PET film were ground to a fine powder using a mechanical grinder. B) >40% Highly crystalline PET powder was obtained from Goodfellow (catalog number ES306 2-10 g / L PET powder was dissolved in 0.1 M sodium phosphate buffer. The three candidate normalized protein species were loaded in a final volume of 50 ml at pH 8.0. Different loads were used for evaluation at either 65°C or 72°C with 200 rpm shaking. The reaction was continued for a period ranging from 6 to 144 hours. At various time points, the bottles were sampled and the To determine the amount of TPA produced in our reaction, the reaction was analyzed by high performance liquid chromatography. The enzymes were analyzed using HPLC. Plus C18(Rapid Resolution HD 2.1×50mm 1. 8 micron) column, part number 959757-902 and guard column, part number 8217 The flow rate was 0.6 mL / min and the column temperature was 35°C. The run was set up as follows: Mobile phase 1 consisted of water with 0.1% trifluoroacetic acid; Mobile phase 2 consisted of acetonitrile containing 0.1% trifluoroacetic acid. The 'Cent' method was used to detect terephthalic acid (TPA).

[0259] The results for the PET assay are summarized in Figures 5 and 6. Amorphous and >40 In both % crystalline PET, Bhr-PETase was superior to Lcc-PETase. The results showed that Lcc-PETase outperformed Bhr-PETase regardless of the type of substrate. This further suggests that the amorphous PE is less thermally stable than e. T can be depolymerized at both 65°C and 72°C with an enzyme dosage of 2.4 mg / g PET. Compared with hr-PETase (Figure 5), Lcc-PETase deactivates ATP at higher inputs. The polymerization activity was significantly lower (data not shown). Figure 6 shows that Bhr-PETase is highly crystalline. cannot completely degrade PET and requires additional pre-treatment or accessory enzymes to do so. This further suggests that the element may be required.

[0260] Overall, Bhr-PETase was the best PET hydrolase in all the evaluated studies. Therefore, Bhr-PETase was identified as a target for further improvement. was selected as the target. [Example]

[0261] Design and construction of the Bhr-PETase collection To further improve the activity and thermostability of Bhr-PETase, multiple G1 barrier enzymes were used. A collection of ants was designed based on sequence and structural analysis. The design consisted of one per variant. The G1 variant collection was then subjected to standard site-directed mutagenesis. Bhr-PETase G1P (wild-type Bhr-PETase, generation 1 parent) was isolated using the infusion method. ) and then used as a vector for production in Escherichia coli. ET28b(+) vector (Millipore Inc., catalog number 69865) was cloned into

[0262] To further improve the activity and thermostability of Bhr-PETase, multiple G2 barriers were used. A collection of ants was designed based on sequence and structural analysis. The design consisted of one per variant. The G2 variant collection contains multiple specific mutations from We used Bhr-PETase G2P (generation 2 parent, i.e., with the amino acid substitution S27L) G1P) and then transfected into a suitable vector for production in the desired host. was cloned into [Example]

[0263] HTP proliferation: Bhr-PETase G1P, G1 in microtiter plates Preparation of variants, G2P and G2 variants BL21(DE3) containing the recombinant Bhr-PETase-encoding gene from a single colony Chemically competent E. coli, (ThermoFisher Scientific (USA: Cat. No. C600003) with 1% glucose and 50 μg / mL calcium of a 96-well plate containing 180 μl lysogeny broth (LB broth) containing namycin. Individual wells were inoculated. Cultures were grown overnight at 30°C, 200 rpm and 85% humidity. 20 μL of the overnight culture was added to 380 μL of Terifi containing 50 μg / mL kanamycin. The cells were then transferred from each well to a 96-well plate containing TB broth. The plates were incubated for 2-2.5 hours at 37°C, 250 rpm and 85% humidity. Measure the OD before induction at 2-2.5 hours, and when the OD600 reaches approximately 0.6-0.8, Add IPTG (isopropyl β-D) to obtain a final concentration of 0.5 mM IPTG in the wells. Induction with 1-thiogalactopyranoside was then performed. The plates were then incubated for 18-24 hours. The supernatant was transferred to a round-bottom plate and incubated at 30°C, 250 rpm and 85% humidity for 1 hour. and stored at −20° C. prior to activity assay.

[0264] The recombinant Bhr-PETase-encoding gene-containing host from a single colony was cultured in an appropriate medium. The cultures were then incubated overnight for 30 min at 4°C for 1 hour. Overnight cultures were grown at 200°C, 200 rpm and 85% humidity. The plates were then incubated at 30°C, 200 rpm, and humidified. The cultures were incubated at 85% RH with each inducer every 24 hours. The induction was continued for up to 120 hours. The supernatant was transferred to a round-bottom plate at the desired harvest time and used for activity assays. Previously stored at -20°C. [Example]

[0265] Bhr-PETase G1P, G1 variant, G2P and G2 variant activity / HTP PET film assay to evaluate thermal stability For total activity testing, amorphous PET films were prepared using Goodfe's 0.25 mm thick film. The PET film was obtained from Illow (catalog number ES301445). The slices were cut into 1 x 30 cm pieces using a knife, and then recut into approximately 1 x 0.25 cm pieces. Pieces of ET film were ground to a fine powder using a mechanical grinder. The PET powder was mixed with a resin Approximately 8-9 mg of PET powder was loaded into a 96-well plate using a 96-well reader. Dispense into each of the 6 coaster deep wells. To the well, 1.4 mL of 0.1 M sodium phosphate buffer, pH 8.0, was added. Dispense 1 μL of enzyme into the COSTER deep wells, seal the plate, and incubate at 65°C for 7 After 72 hours, the plate was centrifuged at 4,000 rpm for 2 minutes. Transfer 180 μL of the reaction mixture to a Costar round-bottom plate and add 20 μL of The Is-MHETase prepared in step 1 was added to the plate. The plate was then incubated at 50°C for 30 minutes. After 30 minutes, the plate was centrifuged at 4,000 rpm for 2 minutes. for linear assay detection 50 μL of sodium phosphate buffer, pH 7.2, was added to Costar Deep The reaction was diluted with 10 mM EDTA and 50 μL of 10 mM FeSO4. Incubate in the dark for 10 min and then transfer 200 μL of the reaction to a black, clear-bottom fluorometric plate. Centrifuge the plate at 4,000 rpm for 2 minutes before transferring to the end point. The reading was taken after 10 minutes for TPA (terephthalic acid) activity at excitation 328 nm and emission Measured at 421.

[0266] For thermostability testing, 150 μL of enzyme was transferred to a PCR plate. After 3 hours, 100 μL of enzyme was added to 1.4 mL of 0.1 M sodium phosphate buffer, pH 8.0 and 8–9 mg / well of crushed PET powder The plates were 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 plate, and 20 μL of Is-MHETase was added. The plates were incubated at 50°C for 30 minutes. After 30 minutes, the plates were The mixture was centrifuged at 0 rpm for 2 minutes. The amount of TPA produced in our reaction was determined. The reaction was analyzed using high performance liquid chromatography (HPLC) to determine . Zorbax Eclipase Plus C18(Rapid Resoluti on HD 2.1 x 50 mm (1.8 micron) column, part number 959757-90 2 was used with a guard column, part number 82175-901. Flow rate: 0.6 mL / min. The column temperature was set at 35°C for the HPLC run. Mobile phase 1 consisted of water with acid and acetonitrile with 0.1% trifluoroacetic acid A gradient method was used to detect terephthalic acid (TPA) using mobile phase 2 consisting of Used for.

[0267] The results are summarized in Figures 7, 8, and 9. All G1 variants were Bhr-PETas e Improved total activity and / or thermostability compared to G1P (wild-type Bhr-PETase) The Bhr-PETase G1 variant with the amino acid substitution S27L exhibited 1.80-fold improvement in total activity and 3.36-fold improvement in total activity compared to Bhr-PETase G1P It showed improved thermostability and was therefore selected as Bhr-PETase G2P. All G2 variants showed further improvements in total activity compared to Bhr-PETase G2P. showed.

Claims

1. 1. A composition comprising a variant Bhr-PETase, The variant Bhr-PETase is (i) has at least 90% identity to the full-length amino acid sequence of SEQ ID NO: 1; (ii) at least one amino acid substitution selected from the group consisting of L90Y, D203R, and F92L, wherein the amino acid positions are numbered with reference to the amino acid sequence of SEQ ID NO: 1; (iii) exhibits increased PETase activity compared to the enzyme of SEQ ID NO: 1; composition.

2. The variant Bhr-PETases were L90F, D203V, F92G, F92Y, R12K, P20T, V23T, V23L, A24V, A24T, A174R, A174K, I222L, V229I, V229C, N2R, A14K, T17A, T17C, T17G, T17H, T17L, T17Q, T17S, D18R, F21W, S32K, S32M, S32Q, Y60H, N87F, N87H, N87I, N87L, N87M, and 2. The composition of claim 1, further comprising one or more amino acid substitutions selected from the group consisting of S101A, S101H, S101K, S101M, S101N, S101Q, S101R, S101W, T109K, T109L, T109R, S110R, A114K, A114V, A117N, A117Q, A125S, T136A, T136V, Q142L, Q142W, D158E, D158I, D158L, A216P and R236H.

3. The amino acid substitution is P20T / L90Y / F92L / S101M / D158E / A174K / I222L, 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, S32K / L90F / F92L / T109K / A114K / Q142W, V23T / S32Q / N87I / L90F / F92Y / S101N / A114V / Q142W / A174R / D203R / I222L / V229I, 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, T17S / D18R / L90Y / F92G / A114V / A117Q / D203R, L90Y / F92L / S101A / S110R / A125S / Q142W / A174R / A216P, 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, T17C / L90Y / F92L / S101N / T109L / S110R / A125S / A174K / D203R, T17C / L90Y / F92L / A125S / T136V / D158E / A174K, L90Y / F92L / S110R / A125S / D158E / D203V, T17Q / L90Y / F92L / T109L / T136A / D158E / A174R / D203V / V229C, R12K / V23T / A24V / S32Q / F92G / D158L / A174R / D203R, 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, R12K / A14K / F92G / S110R / A117N / A125S / A174R / D203R, T17Q / A24V / N87M / F92G / A114K / A117N / D158E / D203R / I222L, R12K / P20T / D158E / D203R / I222L, S32K / L90Y / F92Y / S101A, V23T / N87I / F92G / A125S / T136A / D158L / D203R, A24T / L90F / F92L / A125S / T136A / D203V, R12K / A14K / F92G / T109R / T136A / Q142L / D158E / A174K / D203R / I222L, D18R / S32Q / N87H / L90Y / F92G / S101K / Q142L / D158I / A174R, T17A / F21W / S32K / N87F / L90F / F92Y / S101Q / T136A / D203R, T17Q / L90Y / F92G / S101N / T109L / S110R / T136V / Q142L / D203V / A216P, S32Q / L90Y / F92L / T109K / A125S / T136V, P20T / L90Y / T109K / T136A / D158I / D203R, N2R / S32Q / L90Y / F92Y / T109K / D203V, T17H / A24V / S32Q / L90Y / F92L / S101H / A114K / D158I / A174R / D203V, 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, 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, and F21W / S32K / L90Y / A125S / D158E / A216P / V229I 10. The composition of claim 1, selected from the group consisting of:

4. A nucleic acid encoding the variant Bhr-PETase of any one of claims 1-3.

5. An expression vector comprising the nucleic acid of claim 4.

6. A host cell comprising the expression vector of claim 5.

7. The host cell of claim 6 , which is a bacterium, yeast or fungus.

8. A method for producing the variant Bhr-PETase of any one of claims 1 to 3, comprising culturing the host cell of claim 6 under conditions in which the variant Bhr-PETase is produced, and recovering the variant Bhr-PETase.

9. A method for degrading PET, comprising contacting PET with the variant Bhr-PETase of any one of claims 1-3.

10. 10. The method of claim 9, 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.

11. The method of claim 10, wherein the mechanical pretreatment comprises grinding the PET into particles.

12. 11. The method of claim 10, wherein the thermomechanical pretreatment comprises extruding the PET at a temperature set to amorphize the PET and reduce its crystallinity.

13. 11. The method of claim 10, 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.

14. 10. The method of claim 9, wherein PET in a mixed plastic composition is degraded.

15. 15. The method of claim 14, wherein the mixed plastic composition comprises a biologically or chemically derived PET analog, PET-like material, or PET substitute.

16. 15. The method of claim 14, 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).

17. 10. The method of claim 9, which does not include separating plastics to select PET from a mixture of plastics.

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