Esterases and uses thereof

Patent metadata

Field	Value
Publication number	US-11851689-B2
Application number	US-201917263581-A
Country	US
Kind code	B2
Filing date	Jul 26, 2019
Priority date	Jul 27, 2018
Publication date	Dec 26, 2023
Grant date	Dec 26, 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) has at least 75% identity to the full length amino acid sequence set forth in SEQ ID NO: 1 (ii) contains at least four substitutions at positions selected from F208, D203, S248, V170, V177, T176, T61, S65, N211 or Y92 as compared to the amino acid sequence SEQ ID NO: 1 and (iii) exhibits increased polyester degrading activity and/or increased thermostability as compared to the esterase of SEQ ID NO: 1.

2. The esterase according to claim 1 , wherein said esterase exhibits both an increased activity and an increased thermostability.

3. The esterase according to claim 1 , wherein said esterase comprises at least a combination of substitutions at positions F208+D203+S248, and one substitution at position selected from V170, V177, T176, T61, S65, N211 or Y92.

4. The esterase according to claim 1 , wherein said esterase comprises at least a combination of substitutions at positions selected from F208+D203+S248+V170, F208+D203+S248+V177, F208+D203+S248+T61, F208+D203+S248+Y92, F208+D203+S248+T176, F208+D203+S248+S65, F208+D203+S248+N211 or F208+D203+S248+V170+Y92.

5. The esterase according to claim 1 , wherein said esterase comprises at least a combination of substitutions selected from F208I+D203C+S248C or F208W+D203C+S248C and one substitution selected from V170I, V177I, T176N, T61M, S65T, N211D/M or Y92G/P/F.

6. The esterase according to claim 1 , wherein said esterase comprises at least the combination of substitutions selected from F208I+D203C+S248C or F208W+D203C+S248C, and one or two substitutions selected from V170I or Y92G.

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

8. The esterase according to claim 1 , wherein said esterase further comprises at least one amino acid residue selected from S130, D175, or H207, as in the parent esterase.

9. The esterase according to claim 1 , wherein said esterase further comprises at least one substitution at a position corresponding to residues 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, L202, N204, S212, V219, Y220, Q237, L239, N241, N243, A62, L67, D91, P93, M131, P210, A209, P179, R30, G37, R72, S98, A68, R96, H156, H183, A17, T27, S48, F90, L82, G135, A140, N143, S145, A149, S164, V167, S206, N213, T252, E173, G53, A121, T157, N211, Y60, D63 or S66.

10. The esterase according to claim 1 , wherein said esterase further comprises at least one substitution at a position selected from A121, N213, S212, A125, N204, G135, W69, N214, N241, N243, R12, P179, V242, V167 or N211.

11. The esterase according to claim 1 , wherein the esterase further comprises at least one substitution selected from A121S, N213P, S212F/T/I/L, A125G, N204D/I/L/Y/H/F, G135A, W69R, N214D/I/L/F/Y/H, N241P, N243P, R12F/Y/H, P179E, V242Y, V167Q or N211D/M.

12. The esterase according to claim 1 , wherein the esterase has the amino acid sequence set forth in SEQ ID NO: 2 and at least four substitutions at positions selected from F208, D203, S248, V170, V177, T61, T176, S65, N211 or F92, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 2 and exhibits increased polyester degrading activity and/or an increased thermostability as compared to the esterase of SEQ ID NO: 1.

13. The esterase according to claim 1 , wherein the esterase has the amino acid sequence set forth in SEQ ID NO: 2 and at least four substitutions at positions selected from F208, D203, S248, V170, V177, T61, N211 or F92, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 2 and exhibits increased polyester degrading activity and/or an increased thermostability as compared to the esterase of SEQ ID NO: 1.

14. The esterase according to claim 13 , wherein said esterase comprises at least a combination of substitutions at positions F208+D203+S248, and one substitution at position selected from V170, V177, T176, T61, S65, N211 or F92.

15. The esterase according to claim 14 , wherein said esterase comprises at least a combination of substitutions selected from F208I+D203C+S248C or F208W+D203C+S248C and one substitution selected from V170I, V177I, T176N, T61M, S65T, N211D/M or F92G/P.

16. 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.

17. The esterase according to claim 16 , 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.

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

19. An expression cassette or vector comprising a nucleic acid of claim 18 .

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

21. A composition comprising an esterase as defined in claim 1 , or a host cell comprising a nucleic acid encoding said esterase.

22. 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.

23. The method according to claim 22 , 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.

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

25. A detergent composition comprising the esterase according to claim 1 or a host cell comprising a nucleic acid encoding said esterase.

26. The esterase according to claim 1 , wherein said esterase has at least 90% identity with the esterase of SEQ ID NO: 1.

27. The esterase according to claim 1 , wherein said esterase has at least 95% identity with the esterase of SEQ ID NO: 1.

28. The esterase according to claim 1 , wherein said esterase comprises at least four substitutions selected from F208I/W, D203C, S248C, V170I, V177I, T176N, T61M, S65T, N211D/M or Y92G/P/F.

29. The esterase according to claim 1 , wherein said esterase comprises at least one combination of substitutions selected from F208I+D203C+S248C+V170I, F208I+D203C+S248C+Y92G, F208I+D203C+S248C+V170I+Y92G, F208I+D203C+S248C+V177I, F208W+D203C+S248C+Y92G, F208W+D203C+S248C+V177I, F208I+D203C+S248C+N211M, F208W+D203C+S248C+N211M, F208W+D203C+S248C+V170I, F208W+D203C+S248C+T176N, F208I+D203C+S248C+T176N, F208I+D203C+S248C+V170I+A121S, F208I+D203C+S248C+Y92G+A121S,

Assignees

• Carbios

Inventors

• Duquesne Sophie
• Tournier Vincent
• Marty Alain

Classifications

• C12Y301/01074

  Cutinase (3.1.1.74) · CPC title

• C11D3/38636

  containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase · CPC title

• C11D3/38

  Products with no well-defined composition {, e.g. natural products} · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C08J11/105

  by treatment with enzymes · CPC title