Esterases and uses thereof

Patent metadata

Field	Value
Publication number	US-11535832-B2
Application number	US-201917263569-A
Country	US
Kind code	B2
Filing date	Jul 26, 2019
Priority date	Jul 27, 2018
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

The present invention relates to esterases, more particularly to esterase variants having improved activity and/or improved themostability 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, and (ii) has at least one amino acid substitution at a position corresponding to residues selected from Q182, T11, R12, A14, W69, R73, A205, N214, A215, A216, I217, F238, V242, D244, P245, A246, L247, D94, R138, F187, P10, L15, D18, N87, S88, S95, Q99, K159, A125, and S218 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 an increased thermostability compared to the esterase of SEQ ID NO: 1.

2. The esterase according to claim 1 , wherein said esterase comprises at least one amino acid substitution at a position corresponding to residues selected from T11, R12, A14, W69, R73, A205, N214, A215, A216, I217, F238, V242, D244, P245, A246 and L247.

3. The esterase according to claim 1 , wherein said esterase comprises at least one substitution selected from T11M/E/I/S/N/D/Q, R12Q/D/N/G/P/F/V/E/L/Y, R12H, A14E/D, W69D/M/E/R, R73I/G/M/D/E/S/C/Q/F/N/V, A205D, N214D/E/C, N214I/L/F/Y/H, A215N, A216Q, F238E, V242P/Y, D244E/C, P245D/Y/E, A246S/D/H/E or L247T.

4. The esterase according to claim 1 , wherein said esterase comprises at least one amino acid substitution at a position corresponding to residues selected from D94, R138, Q182 or F187.

5. The esterase according to claim 4 , wherein said esterase comprises at least one amino acid substitution selected from Q182D/E or F187Y/I.

6. The esterase according to claim 1 , wherein said esterase comprises at least one amino acid substitution selected from Q182D/E, T11M/E/I/S/N/D/Q, R12Q/D/N/G/P/F/V/E/L/Y/H, A14E/D, W69D/M/E/R, R73I/G/M/D/E/S/C/Q/F/N/V, A205D, N214D/E/C, N214I/L/F/Y/H A215N, A216Q, F238E, V242P/Y, D244E/C, P245D/Y/E, A246S/D/H/E, L247T, F187Y/I, A125G or S218A/E.

7. The esterase according to claim 1 , wherein said esterase comprises at least one amino acid residue selected from S130, D175, or H207 wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

8. The esterase according to claim 1 , wherein said esterase further comprises at least one substitution at a position corresponding to residues selected from 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, A17, T27, S48, L82, F90, Y92, G135, A140, N143, S145, A149, S164, V167, S206, N213, T252, D203+S248, E173, F208, T61, Y92, V177, G53, S65, A121, T157, V170, T176, N211, Y60, T61, D63 or S66.

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

10. The esterase according to claim 1 , wherein the esterase has the amino acid sequence set forth in SEQ ID NO: 2, and at least one amino acid substitution as compared to SEQ ID NO: 2 at a position corresponding to residues selected from Q182, T11, R12, A14, W69, R73, A205, N214, A215, A216, I217, F238, V242, D244, P245, A246, L247, D94, R138, F187, P10, L15, D18, N87, S88, S95, Q99, K159, A125 or S218, 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.

11. The esterase according to claim 1 , wherein the esterase further comprises at least one combination of substitution selected from D203C+S248C, F208I+D203C+S248C or F208W+D203C+S248C.

12. 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.

13. The esterase according to claim 12 , 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.

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

15. An expression cassette or vector comprising a nucleic acid of claim 14 .

16. A host cell comprising a nucleic acid according to claim 14 or an expression cassette or vector comprising said nucleic acid.

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

18. 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 expressing said esterase.

19. The method according to claim 18 , 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.

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

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

Assignees

• Carbios

Inventors

• David Benoît
• Andre Isabelle
• Ben Khaled Maher
• Duquesne Sophie
• Marty Alain

Classifications

• C12Y301/01074

  Cutinase (3.1.1.74) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C08J11/105

  by treatment with enzymes · CPC title

• C08J11/10

  by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation (depolymerisation to the original monomer C07) · CPC title