Esterases and uses thereof
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.
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+5248, E173, L202, N204, F208, A172+A209, 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, N204, S212, N213, N231, T233, R236, Q237, N241, N243, N254, R255 and Q258, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, (iii) has a polyester degrading activity and (iv) exhibits increased thermostability as compared to the esterase of 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, N204, L202, F208, and V170.
3. The esterase variant of claim 1 , comprising, by reference to the esterase of SEQ ID NO: 1:
at least one additional disulphide bridge; and/or
at least one additional salt bridge; and/or
at least one mutation of an amino acid residue located in a void solvent-excluded cavity of the esterase; and/or
a suppression of at least one N- and/or C-terminal amino acid residue.
4. The esterase variant of claim 1 , comprising:
amino acid substitutions D203C+S248C; and/or
amino acid substitutions G39C+A103C+L82A; and 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.
5. The esterase variant of claim 1 , 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.
7. The esterase variant of claim 1 , comprising one or more amino acid substitution(s) as compared to SEQ ID NO: 1 at a position selected from T61, Y92 and V177, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, and wherein the substitutions are different from T61A/G, Y92A and V177A.
8. The esterase variant of claim 7 , wherein the substitutions are selected from V177I, Y92G, Y92P, Y92P+F208W and T61M.
9. The esterase variant of claim 1 , comprising the substitutions at positions selected from D203+S248+E173, D203+S248+F208, and D203+S248+E173+N204+L202.
10. The esterase variant of claim 1 , comprising substitutions selected from D203C+S248C+E173R, D203C+S248C+E173A, D203C+S248C+F208W, D203C+S248C+F208I, F208W+D203C+S248C+E173A, F2081+D203C+S248C+E173A and D203C+S248C+E173R+N204D+L202R.
11. The esterase variant of claim 1 , which is glycosylated, at a position selected from N9, N143, N162, N204, N231, or combinations thereof.
12. A nucleic acid encoding an esterase as defined in claim 1 .
13. An expression cassette or vector comprising the nucleic acid of claim 12 .
14. A host cell comprising the nucleic acid of claim 12 .
15. A method of producing an esterase comprising:
(a) culturing the host cell according to claim 14 under conditions suitable to express the nucleic acid encoding the esterase; and
(b) recovering said esterase from the cell culture.
16. A composition comprising an esterase according to claim 1 and one or several excipients or additives.
17. 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.
18. The method of claim 17 , further comprising (b) recovering monomers and/or oligomers resulting from the degradation of the at least one polyester.
19. The method of claim 17 , 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.
20. The method of claim 17 , wherein step (a) is conducted at a temperature comprised between 50° C. and 90° C.