Novel esterases and uses thereof

Patent metadata

Field	Value
Publication number	US-2021261931-A9
Application number	US-202016812405-A
Country	US
Kind code	A9
Filing date	Mar 9, 2020
Priority date	Jul 12, 2016
Publication date	Aug 26, 2021
Grant date	—

Abstract

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

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, and (ii) has one or more amino acid modifications as compared to SEQ ID NO: 1 at position(s) selected from D203+S248, E173, L202, N204, A172+A209, V28, S29, R30, L31, S32, V33, S34, G35, F36, G37, G38, G39, A103, L82, G53, L104, L107, L119, A121, L124, I54, M56, L70, L74, A127, V150, L152, L168, V170, P196, V198, V200, V219, Y220, T221, S223, W224, M225, L239, T252, N253, H256, S1, Y4, Q5, R6, N9, S13, T16, S22, T25, Y26, S34, Y43, S48, T50, R72, S98, N105, R108, S113, N122, S145, K147, T160, N162, S181, Q189, N190, S193, T194, S212, N213, N231, T233, R236, Q237, N241, N243, N254, R255 and Q258 or at least one amino acid substitution selected from V177I, Y92G/P, F208I/W, T61M, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

2 . The esterase variant of claim 1 , which has one or more amino acid modifications, as compared to SEQ ID NO: 1, at position(s) selected from D203+S248, E173, L202, N204, V170, V219, S212, N213, N241 and N243 or at least one amino acid substitution selected from V177I, Y92G/P, F208VW, T61M.

3 . The esterase variant of claim 1 , comprising at least the amino acid substitutions D203C+S248C.

4 . The esterase variant of claim 1 , comprising at least the replacement of amino acids V28 to G39 of SEQ ID NO: 1 with an amino acid sequence consisting of E-G-P-S-C or A-G-P-S-C, and the substitution L82A and/or A103C.

5 . The esterase variant of claim 3 , further comprising at least one substitution at a position selected from E173, L202, N204 and F208, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

6 . The esterase variant of claim 5 , wherein the at least one substitution is selected from E173A/R, L202R, N204D and F208W/I.

7 . The esterase variant of claim 1 , comprising the combination of substitutions at positions selected from D203+S248+E173, D203+S248+F208, and D203+S248+E173+N204+L202.

8 . The esterase variant of claim 1 , which comprises at least one substitution or combination of substitutions selected from the group consisting of V177I, Y92G, Y92P, F208W, Y92P+F208W, T61M, V170I+F208W, D203C+S248C, D203C+S248C+E173R, D203C+S248C+E173A, D203C+S248C+F208W, D203C+S248C+F208I, F208W+D203C+S248C+E173A, F208I+D203C+S248C+E173A and D203C+S248C+E173R+N204D+L202R wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

9 . The esterase variant of claim 1 , which is further glycosylated, at a position selected from N9, N143, N162, N204, N231, or combinations thereof.

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

14 . A composition comprising an esterase according to claim 1 and one or several excipients or additives.

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 , thereby degrading the plastic product.

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

17 . The method of claim 15 , wherein the plastic product comprises at least one polyester selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylen terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (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 . The esterase variant of claim 1 which exhibits a polyester degrading activity

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

20 . 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

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

Classifications

• C12Y301/01074

  Cutinase (3.1.1.74) · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C08J11/105

  by treatment with enzymes · CPC title

• Y02W30/62

  Plastics recycling; Rubber recycling · CPC title

• C12Y301/01

  Carboxylic ester hydrolases (3.1.1) · CPC title