Plastic degrading proteases

Patent metadata

Field	Value
Publication number	US-10829754-B2
Application number	US-201716470295-A
Country	US
Kind code	B2
Filing date	Dec 15, 2017
Priority date	Dec 16, 2016
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

The present invention relates to novel proteases, more particularly to protease variants having improved activity compared to the protease of SEQ ID NO:1 and the uses thereof for degrading polyester containing material, such as plastic products. The proteases of the invention are particularly suited to degrade polylactic acid, and material containing polylactic acid.

First claim

The invention claimed is:

1. A protease variant which (i) has at least 85% 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 S104, N102, S106, N107, G132, G134, D160, or Y167, 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 protease variant according to claim 1 , wherein said protease comprises at least one amino acid substitution at position S104, wherein the position is numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

3. The protease variant according to claim 1 , wherein said protease comprises at least one amino acid substitution selected from N102S/F, S104L, S106T, N107T/I, G132I, G134K, D160E and Y167R.

4. The protease variant according to claim 3 , wherein said protease comprises at least one amino acid substitution selected from S106T and S104L.

5. The protease variant according to claim 1 , wherein said protease comprises at least one combination of substitutions selected from the group consisting of S104L+N107I, S104L+Y167R, D160E+Y167R, N102S+D160E+Y167R, S106T+D160E+Y167R, N107T+D160E+Y167R, N102S+S106T+D160E+Y167R, S106T+N107T+D160E+Y167R, N102S+N107T+D160E+Y167R, N102S+S106T+N107T+Y167R, N102F+S104L+S106T+N107I, N102F+S104L+D160E+Y167R, N102F+S104L+N107I+D160E+Y167R, N102S+S106T+N107T+D160E+Y167R, N102F+S104L+S106T+N107I+Y167R, N102F+S104L+S106T+N107I+G132I, N102F+S106T+N107T+D160E+Y167R, N102F+S104L+S106T+N107I+D160E+Y167R, N102F+S104L+S106T+N107I+G132I+Y167R, and N102F+S104L+S106T+N107I+G132I+D160E+Y167R, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

6. The protease variant according to claim 1 , wherein said protease comprises a single amino acid substitution selected from N102S/F, S104L, S106T, N107T/I, G132I, G134K, D160E, and Y167R.

7. The protease variant according to claim 6 , wherein said protease comprises a single amino acid substitution selected from S104L and Y167R.

8. The protease variant according to claim 1 , wherein said protease comprises an additional sequence at the N-terminal end an amino acid sequence which has 100% identity to the full length amino acid sequence set forth in SEQ ID NO: 42.

9. The protease variant according to claim 1 , wherein said protease comprise an additional amino sequence at the N-terminal end, said additional amino acid sequence having at least 85% identity to the full length amino acid sequence set forth in SEQ ID NO: 42 and at least one amino acid substitution at a position selected from R24, Y75, D106, Q107, E108, V109, R110, A111, and F112, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 42.

10. An expression cassette or vector comprising a nucleic acid encoding a protease variant as defined in claim 1 .

11. A host cell comprising an expression cassette or vector of claim 10 .

12. A composition comprising the protease variant as defined in claim 1 .

13. A method of producing a protease according to claim 1 comprising:
(a) culturing a host cell comprising a nucleic acid encoding a protease variant as defined in claim 1 under conditions suitable to express the nucleic acid encoding said protease.

14. The method of producing a protease according to claim 13 , further comprising:
(b) recovering said protease from the cell culture.

15. A method of degrading a plastic product containing at least one polyester comprising:
(a) contacting the plastic product with a protease according to claim 1 , suitable for degrading said at least one polyester in order to degrade said at least one polyester up to monomers and oligomers.

16. The method according to claim 15 further comprising the step of:
(b) recovering monomers and/or oligomers.

17. The method according to claim 15 , wherein the plastic product comprises at least one polyester selected from polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET) polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA) and blends/mixtures of these materials.

18. A plastic compound containing a protease of claim 1 and at least one polyester.

19. The plastic compound according to claim 18 , comprising at least polylactic acid.

20. A masterbatch composition, containing a protease of claim 1 and at least one polyester.

Assignees

• Carbios

Inventors

• Marty Alain
• Duquesne Sophie
• Guicherd Marie
• Vuillemin Marlene
• Ben Khaled Maher

Classifications

• Y02W30/62

  Plastics recycling; Rubber recycling · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C08J11/105

  by treatment with enzymes · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C12N9/6424 Primary

  Serine endopeptidases (3.4.21) · CPC title