Esterases and uses thereof

Patent metadata

Field	Value
Publication number	US-11692181-B2
Application number	US-201917263574-A
Country	US
Kind code	B2
Filing date	Jul 26, 2019
Priority date	Jul 27, 2018
Publication date	Jul 4, 2023
Grant date	Jul 4, 2023

Abstract

The present invention relates to novel esterases, more particularly to esterase variants having improved activity and/or improved thermostability compared to the esterase of SEQ ID NO: 1 and the uses thereof for degrading polyester containing material, such as plastic products. The esterases of the invention are particularly suited to degrade polyethylene terephthalate, and material containing polyethylene terephthalate.

First claim

The invention claimed is:

1. An esterase which (i) comprises an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 1, (ii) with one to fourteen amino acid substitutions selected from the group consisting of A17T, T27S, S48T, L82I, F90L, Y92F, G135A, A140S, N143I, S145T, A149G, S164P, V167Q, S206T, or N213P wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, and (iii) exhibits increased polyester degrading activity and/or increased thermostability compared to an esterase of SEQ ID NO: 1.

2. The esterase according to claim 1 , wherein said esterase comprises one to three amino acid substitutions selected from the group consisting of A17T, T27S, S48T, L82I, F90L, Y92F, G135A, A140S, N143I, S145T, A149G, S164P, V167Q, S206T, or N213P.

3. The esterase according to claim 1 , wherein said esterase exhibits both increased polyester degrading activity and increased thermostability as compared to the esterase of SEQ ID NO: 1.

4. The esterase according to claim 1 , wherein said esterase further comprises the substitution T252S.

5. The esterase according to claim 1 , wherein the esterase further comprises a substitution or combination of substitutions at position corresponding to residues selected from F208, D203+S248, T61, Y92, V170, V177 or E173.

6. The esterase according to claim 5 , wherein the esterase comprises a combination of substitutions selected from D203C+S248C, F208I+D203C+S248C or F208W+D203C+S248C.

7. The esterase according to claim 6 , wherein the esterase further comprises one substitution selected from V170I, V177I, T176N, T61M, S65T, N211D/M or Y92G/P.

8. The esterase according to claim 1 , wherein the esterase comprises a combination of substitutions selected from F208I+D203C+S248C+V170I, F208I+D203C+S248C+Y92G or F208I+D203C+S248C+V170I+Y92G, F208W+D203C+S248C+V170I, F208W+D203C+S248C+Y92G or F208W+D203C+S248C+V170I+Y92G.

9. The esterase according to claim 1 , wherein the esterase has the exact amino acid sequence set forth in SEQ ID No 1 with a combination of substitutions selected from the group consisting of A17T+T27S+S48T+L82I+Y92F+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S, A17T+T27S+S48T+L82I+F90L+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S, A17T+T27S+S48T+L82I+F90L+Y92F+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S, A17T+T27S+S48T+L82I+F90L+Y92F+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+N213P+T252S, T27S+S48T+L82I+F90L+Y92F+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S, A17T+T27S+S48T+L82I+F90L+Y92G+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S, A17T+T27S+S48T+L82I+F90L+Y92F+G135A+A140S+N143I+S145T+A149G+V167Q+S206T+N213P+T252S, A17T+T27S+S48T+L82I+F90L+Y92F+G135A+A140S+S145T+A149G+S164P+V167Q+S206T+N213P+T252S, A17T+T27S+S48T+L82I+F90L+Y92F+G135A+A140S+N143I+S145T+A149G+S164P+S206T+N213P+T252S, A17T+T27S+S48T+L82I+F90L+Y92F+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+T252S, A17T+T27S+S48T+L82I+Y92F+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+D203C+S206T+F208I+N213P+S248C+T252S, A17T+T27S+S48T+L82I+F90L+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+D203C+S206T+F208I+N213P+S248C+T252S, A17T+T27S+S48T+L82I+F90L+Y92G+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+D203C+S206T+F208I+N213P+S248C+T252S, T27S+S48T+L82I+F90L+Y92F+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+D203C+S206T+F208I+N213P+S248C+T252S, A17T+T27S+S48T+L82I+F90L+Y92F+G135A+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S, A17T+T27S+S48T+L82I+F90L+Y92F+G135A+A140S+N143I+S145T+S164P+V167Q+S206T+N213P+T252S or A17T+T27S+S48T+L82I+F90L+Y92F+G135A+A140S+N143I+S145T+A149G+S164P+V167Q+S206T+N213P+T252S+V170I.

10. The esterase according to claim 1 , wherein the esterase has the exact amino acid sequence set forth in SEQ ID No 1 with the combination of substitutions selected from the group consisting of F208I+D203C+S248C+V170I+N213P, F208I+D203C+S248C+Y92G+N213P, F208I+D203C+S248C+V170I+Y92G+N213P, F208I+D203C+S248C+V170I+Y92G+G135A, F208I+D203C+S248C+V170I+Y92G+N213P+G135A, F208I+D203C+S248C+V170I+Y92G+N213P+G135A+N241P, F208I+D203C+S248C+V170I+S212F+N213P, F208I+D203C+S248C+Y92G+S212F+N213P, F208I+D203C+S248C+V170I+Y92G+S212F+N213P, F208I+D203C+S248C+V170I+Y92G+N213P+G135A+R12H, F208I+D203C+S248C+V170I+Y92G+V167Q, F208I+D203C+S248C+V170I+Y92G+N213P+G135A+V167Q, or F208I+D203C+S248C+V170I+Y92G+R12H+V167Q.

11. The esterase according to claim 1 , wherein said esterase further comprises from one to four substitutions at a position selected from T11, R12, A14, W69, R73, A205, N214, A215, A216, I217, F238, V242, D244, P245, A246, L247, D94, R138, D158, Q182, F187, P10, L15, D18, N87, S88, S95, Q99, K159, A174, A125, S218, S13, T16, A62, L67, D91, P93, M131, L202, N204, A209, P210, S212, V219, Y220, Q237, L239, N241, N243, P179, R30, G37, A68, R72, R96, S98, H156, H183, D203+S248, E173, F208, T61, Y92, V177, G53, S65, A121, T157, V170, T176, N211, Y60, D63 or S66.

12. The esterase according to claim 1 , wherein said esterase comprises a single substitution selected from A17T, T27S, S48T, L82I, F90L, Y92F, G135A, A140S, N143I, S145T, A149G, S164P, V167Q, S206T, or N213P, as compared to the amino acid sequence set forth in SEQ ID NO: 1.

13. The esterase according to claim 1 , wherein the esterase further comprises at the N-terminal end an amino acid sequence having at least 55% identity to the full length amino acid sequence set forth in SEQ ID NO: 3.

14. The esterase according to claim 13 , wherein the N-terminal amino acid sequence is selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

15. A nucleic acid encoding an esterase as defined in claim 1 .

16. An expression cassette or vector comprising a nucleic acid of claim 15 .

17. A host cell comprising a nucleic acid encoding an esterase as defined in claim 1 or an expression cassette or vector comprising a nucleic acid encoding said esterase.

18. A composition comprising an esterase as defined in claim 1 , or a host cell expressing said esterase.

19. A method of degrading at least one polyester of a polyester containing material comprising
a) contacting the polyester containing material with an esterase according to claim 1 or a host cell comprising a nucleic acid encoding said esterase; and, optionally
b) recovering monomers and/or oligomers.

20. The method according to claim 19 , wherein the polyester is selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxy alkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), Polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures of these materials.

21. A polyester containing material containing an esterase according to claim 1 or a host cell comprising a nucleic acid encoding said esterase.

22. A detergent composition comprising the esterase according to claim 9 or a host cell comprising a nucleic acid encoding said esterase.

Assignees

• Carbios

Inventors

• Andre Isabelle
• Tournier Vincent
• Gilles Alexandre
• Duquesne Sophie

Classifications

• C08J2367/02

  Polyesters derived from dicarboxylic acids and dihydroxy compounds; (C08J2367/06 takes precedence) · CPC title

• C08J11/105

  by treatment with enzymes · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title

• C12Y301/01074

  Cutinase (3.1.1.74) · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title