Esterases and uses thereof

Patent metadata

Field	Value
Publication number	US-11072784-B2
Application number	US-202016819222-A
Country	US
Kind code	B2
Filing date	Mar 16, 2020
Priority date	Jul 12, 2016
Publication date	Jul 27, 2021
Grant date	Jul 27, 2021

Abstract

The present invention relates to novel esterase, more particularly to esterase variants having improved activity 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

We claim:

1. An esterase variant which (i) has at least 75% identity to the full length amino acid sequence set forth in SEQ ID NO: 1, (ii) has at least one amino acid substitution at a position selected from F208, T157, T176, S65, P58, A62, A64, L67, A68, N85, T86, R89, D91, P93, R96, G128, M131, G133, G134, L152, T153, P154, H156, A178, P179, H183, S206, A209, P210, N211, W155, Y60, T61, D63, S66, F90, Y92, H129, V177 wherein the substitutions are different from Y60A/F, T61A/G, A62G/S, D63T/R, S66A, F90A/R/Y, Y92A, H129W and V177A or at least one amino acid substitution selected from G53L, A121R/W and V170I, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1 and (iii) exhibits a polyester degrading activity.

2. The esterase variant according to claim 1 , wherein said esterase comprises at least one amino acid substitution at a position selected from the group consisting of Y60, T61, A62, D63, S66, F90, Y92, F208, T157, T176, S65, N211, N85, T86, R89, A178, P179 and 5206 wherein the substitutions are different from Y60A/F, T61A/G, A62G/S, D63T/R, S66A, F90A/R/Y and Y92A or at least one amino acid substitution selected from G53L, A121R/W and V170I, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

3. The esterase variant according to claim 1 , wherein said esterase comprises at least one amino acid substitution at a position selected from the group consisting of Y60, T61, D63, S66, F90, Y92, F208, T157, T176, S65 and N211, wherein the substitutions are different from Y60A/F, T61A/G, D63T/R, S66A, F90A/R/Y and Y92A or at least one amino acid substitution selected from G53L, A121R/W and V170I, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

4. The esterase variant according to claim 1 , wherein said esterase comprises at least one substitution selected from the group consisting of Y60M, T61M/V, D63N/Q, S66H, F90W, Y92G/N/P/Q/T, F208I/L/W/G/S/N/A/R/T, T157E/QN/G, T176H/N/Q, S65T, G53L, A121R/W, V170I and N211Q, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

5. The esterase variant according to claim 1 , which has at least two substitutions at positions selected from G53, P58, G59, Y60, T61, A62, D63, A64, S65, S66, L67, A68, N85, T86, R89, F90, D91, Y92, P93, R96, A121, G128, H129, M131, G132, G133, G134, L152, T153, P154, W155, H156, T157, V170, T176, V177, A178, H183, 5206, F208, A209, P210, S223 and N211, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

6. The esterase variant according to claim 5 , wherein said esterase comprises at least two substitutions at positions selected from Y60, T61, A62, D63, A64, S66, A68, N85, R89, F90, Y92, H129, W155, T176, V177, S206, F208 and N211, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

7. The esterase variant according to claim 5 , wherein said esterase comprises at least two substitutions at positions selected from S65, Y92, A121, T157, V170, T176, F208 and S223, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

8. The esterase variant according to claim 1 , wherein said esterase further comprises the combination of substitutions consisting in D203C+S248C, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

9. The esterase variant according to claim 1 , which comprises at least one substitution or combination of substitutions selected from the group consisting of G53L, A121R/W, V170I Y60M, T61M/V, D63N/Q, S66H, F90W, Y92G/N/P/Q/T, S65T, T157E/Q/N/G, T176H/N/Q, N211Q, F208I/L/W/G/SN/A/R/T, F208W+V170I, Y92P+F208L, Y92P+F208W, T176H+F208W, V170I+A121S, V170I+A121S+S223A, F208W+T157Q, F208W+T157N, F208W+T157S, F208W+S65T, F208W+T157E, F208W+D203C+S248C and F208I+D203C+S248C, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

10. A nucleic acid encoding an esterase variant according to claim 1 .

11. An expression cassette or vector comprising the nucleic acid of claim 10 .

12. A host cell comprising the nucleic acid of claim 10 .

13. A composition comprising an esterase variant according to claim 1 .

14. A method of producing an esterase comprising:
(a) culturing the host cell according to claim 12 under conditions suitable to express the nucleic acid encoding said esterase; and
(b) recovering said esterase from the cell culture.

15. A method of degrading a plastic product containing at least one polyester comprising (a) contacting the plastic product with an esterase according to claim 1 and thereby degrading the at least one polyester.

16. The method according to claim 15 , further comprising (b) recovering monomers and/or oligomers resulting from the degradation of the at least one polyester.

17. The method according to claim 15 , wherein the plastic product comprises at least one polyester 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.

18. A polyester containing material comprising an esterase variant according to claim 1 and/or a host expressing said esterase variant.

19. A plastic compound comprising at least one polyester and an esterase variant according to claim 1 and/or a host cell expressing said esterase variant.

Assignees

• Carbios

Inventors

• Tournier Vincent
• Texier Hélène
• Desrousseaux Marie-Laure
• Topham Christopher
• Andre Isabelle
• Barbe Sophie
• Duquesne Sophie
• Marty Alain

Classifications

• C12Y301/01074

  Cutinase (3.1.1.74) · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C08J2367/02

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

• C08J11/105

  by treatment with enzymes · CPC title

• Y02W30/62

  Plastics recycling; Rubber recycling · CPC title