IP Library › Granted Patent US 11,345,906
Granted Patent B2
US 11,345,906 · App. 17/037,775 · Granted May 31, 2022

Plastic degrading proteases

Inventors: Alain Marty (Toulouse, FR); Sophie Duquesne (Toulouse, FR); Marie Guicherd (Toulouse, FR); Marlene Vuillemin (Søborg, DK); Maher Ben Khaled (Toulouse, FR)
Assignee: CARBIOS
C12N9/6424C08J11/105C12N15/52C12N15/74C12P21/02C08L2201/06
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Quick Facts
Patent No.
US 11,345,906
App. No.
17/037,775
Granted
May 31, 2022
Kind
B2
Abstract

The present invention relates to novel proteases, more particularly to protease variants having improved activity compared to the protease of SEQ ID N° 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.

Claims (29)

1. A protease 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 S104L, N102S/F, S106T, N107T/I, G132I, G134K, D160E or Y167R, 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 selected from S104L, N102S/F, S106T, N107T/I, G132I, G134K or Y167R.

3. The protease variant according to claim 1 , wherein said protease comprises at least one amino acid substitution selected from Y167R, N102F, S104L, S106T and N107I.

4. The protease variant according to claim 1 , wherein said protease comprises at least one combination of substitutions selected from the group consisting of D160E+Y167R, N102S+Y167R, S106T+Y167R, G134K+Y167R, S104L+G134K, N107T+Y167R, S104L+Y167R, S104L+N107I, N102S+S106T+Y167R, N102S+N107T+Y167R, N102S+D160E+Y167R, S106T+N107T+Y167R, S106T+D160E+Y167R, N107T+D160E+Y167R, N102F+S104L+Y167R, N102F+S104L+N107I, S104L+G134K+Y167R, G134K+D160E+Y167R, S104L+N107I+G134K, N102F+N107I+Y167R, S104L+N107I+Y167R, S104L+G132I+Y167R, N102S+S106T+N107T+Y167R, N102S+S106T+D160E+Y167R, N102S+N107T+D160E+Y167R, S106T+N107T+D160E+Y167R, N102F+S104L+N107I+G134K, S104L+N107I+G134K+Y167R, N102F+S104L+D160E+Y167R, N102F+S104L+S106T+N107I N102S+S106T+N107T+D160E+Y167R, N102F+S106T+N107T+D160E+Y167R, N102F+S104L+N107I+D160E+Y167R, N102F+S104L+N107I+G134K+Y167R, N102F+S104L+S106T+N107I+G134K, S104L+S106T+N107I+G134K+Y167R, N102F+S104L+S106T+N107I+Y167R, N102F+S104L+S106T+N107I+G132I, N102F+S104L+S106T+N107I+D160E+Y167R, N102F+S104L+S106T+N107I+G134K+Y167R, N102F+S104L+S106T+N107I+G132I+Y167R, N102F+S104L+S106T+N107I+G132I+D160E+Y167R or N102F+S104L+S106T+N107I+G134K+D160E+Y167R, N102F+S104L+S106T+N107I+G132I+D160E+Y167R and N102F+S104L+S106T+N107I+G132I+G134K+D160E+Y167R.

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 1 , wherein said protease comprises an additional sequence at the N-terminal end which has at least 85% identity to the full length amino acid sequence set forth in SEQ ID NO:42.

8. The protease variant according to claim 7 , wherein the additional sequence has 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.

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

10. A host cell comprising an expression cassette or vector of claim 9 .

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

12. A method of producing a protease comprising:

(a) culturing the 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.

13. The method of producing a protease according to claim 12 , further comprising: (b) recovering said protease from the cell culture.

14. 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 in order to degrade said at least one polyester up to monomers and oligomers.

15. The method of degrading a plastic product according to claim 14 , further comprising: (b) recovering monomers and/or oligomers.

16. The method according to claim 14 , 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.

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

18. The plastic compound of claim 17 , comprising at least polylactic acid.

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

20. The protease variant according to claim 1 , wherein said protease has at least 80% identity to the full length amino acids sequence set forth in SEQ ID NO:1.

21. The protease variant according to claim 1 , which exhibits an increased polyester degrading activity as compared to the protease of SEQ ID NO:1.

22. The protease variant according to claim 1 , wherein said protease comprises at least one substitution selected from S104L, N102F, N107I, G132I, G134K or Y167R.

23. The protease variant according to claim 1 , wherein said protease comprises at least one substitution selected from S104L, N102F, N107I, G132I, G134K or Y167R or at least one combination of substitutions selected from 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.

24. The protease variant according to claim 23 , which exhibits an increased polyester degrading activity as compared to the protease of SEQ ID NO:1.

25. The masterbatch composition of claim 19 , wherein said masterbatch composition comprises at least polylactic acid (PLA) or polycaprolactone (PCL) and a protease variant wherein said protease comprises at least one substitution selected from S104L, N102F, N107I, G132I, G134K or Y167R or at least one combination of substitutions selected from 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.

26. The method according to claim 14 , wherein the plastic product comprises at least polylactic acid (PLA) and is contacted with a protease wherein said protease comprises at least one substitution selected from S104L, N102F, N107I, G132I, G134K or Y167R or at least one combination of substitutions selected from 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.

27. The plastic compound according to claim 17 , wherein said plastic compound comprises at least polylactic acid (PLA) and a protease variant wherein said protease comprises at least one substitution selected from S104L, N102F, N107I, G132I, G134K or Y167R or at least one combination of substitutions selected from 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.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 30, 2020
From: MARTY, ALAIN; DUQUESNE, SOPHIE; GUICHERD, MARIE; VUILLEMIN, MARLÈNE; BEN KHALED, MAHER
To: CARBIOS
Reel/Frame 053927/0649 →
Priority Claims (1)
EP 16306702 · Dec 16, 2016 · regional
Continuity (2)
Continuation 16470295
Related Publication 20210009980A1 · Jan 14, 2021
Cited By (6)
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