Multi-domain enzymes having cutinase activity, compositions comprising same and uses thereof

Patent metadata

Field	Value
Publication number	US-9752131-B2
Application number	US-201615044715-A
Country	US
Kind code	B2
Filing date	Feb 16, 2016
Priority date	Feb 16, 2015
Publication date	Sep 5, 2017
Grant date	Sep 5, 2017

Abstract

Multi-domain recombinant proteins have a cutinase catalytic domain. The cutinase catalytic domain is a modular domain, which may be combined with other protein domains that also function in a modular fashion. For example, the cutinase catalytic domain can be operably linked to a polymer binding domain via a linker domain (e.g., a threonine/proline-rich linker polypeptide). The cutinase catalytic domain can be from an endogenous cutinase having a multi-domain modular organization, such as from the actinobacterium Kineococcus radiotolerans . The recombinant proteins are used in compositions, methods and uses, such as for use in industrial applications.

First claim

What is claimed is:

1. A composition comprising:
(i) an isolated recombinant protein having cutinase activity, said protein comprising:

(a) a cutinase catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO: 2;
(b) a proline/threonine-rich linker domain having at least 85% amino acid sequence identity to SEQ ID NO: 3, which is positioned C-terminal of domain (a); and
(c) a polymer binding domain having at least 85% amino acid sequence identity to SEQ ID NO: 4, which is positioned C-terminal of domain (b); and
(ii) a bleaching agent and/or an organic solvent,

wherein said recombinant protein retains said cutinase activity when present in said composition, and
wherein the composition is: a cleaning product, a degreaser, a disinfectant, a bleaching product, a product for controlling biofilm, or any combination thereof; feed or a feed additive; a product for inactivating mycotoxin; a product for bioscouring or other treatment of fabrics or textiles; a product for catalyzing trans-esterification reactions; a product for vegetable oil extraction; or a product for enhancing oil recovery.

2. The composition of claim 1 comprising a bleaching agent which is: hydrogen peroxide, a peroxide other than hydrogen peroxide, a non-peroxide oxidizing agent, ozone, sodium percarbonate, sodium perborate, and any combination thereof.

3. The composition of claim 1 comprising a peroxide.

4. The composition of claim 1 comprising a bleaching agent which is: sodium dithionite, sodium dithionite, sulfur dioxide, a sulfite, a bisulfite, a sodium borohydride, or any combination thereof.

5. The composition of claim 1 comprising an organic solvent which is: methanol, 2-propanol, or an alcohol other than methanol or 2-propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or acetone.

6. The composition of claim 1 having a pH between 7.5 and 9.5.

7. The composition of claim 1 , further comprising: a detergent, a surfactant, a buffer, a chelator, or any combination thereof.

8. The composition of claim 1 , further comprising a chelator which is EDTA or GLDA.

9. The composition of claim 1 , further comprising a polyol and/or boric acid as a stabilizer.

10. The composition of claim 1 , further comprising an additional recombinant enzyme which is: a lipase, an esterase, a pectate lyase, a pectinase, a cutinase, a cellulase, a hemicellulose, an amylase, or any combination thereof.

11. The composition of claim 1 , wherein the isolated recombinant protein having cutinase activity comprises:
(a) a cutinase catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO: 2;
(b) a proline/threonine-rich linker domain having at least 90% amino acid sequence identity to SEQ ID NO: 3; and
(c) a polymer binding domain having at least 90% amino acid sequence identity to SEQ ID NO: 4.

12. The composition of claim 1 , wherein the isolated recombinant protein having cutinase activity comprises:
(a) a cutinase catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO: 2;
(b) a proline/threonine-rich linker domain having at least 95% amino acid sequence identity to SEQ ID NO: 3; and
(c) a polymer binding domain having at least 95% amino acid sequence identity to SEQ ID NO: 4.

13. An isolated recombinant protein having cutinase activity, said protein comprising:
(a) a cutinase catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO: 2;
(b) a proline/threonine-rich linker domain having at least 85% amino acid sequence identity to SEQ ID NO: 3, which is positioned C-terminal of domain (a); and
(c) a polymer binding domain having at least 85% amino acid sequence identity to SEQ ID NO: 4, which is positioned C-terminal of domain (b),
wherein the recombinant protein does not comprise an N-terminal signal sequence which is the polypeptide of 1-33 of SEQ ID NO: 1.

14. The isolated recombinant protein of claim 13 , wherein:
(a) the cutinase catalytic domain has at least 90% amino acid sequence identity to SEQ ID NO: 2;
(b) the proline/threonine-rich linker domain has at least 90% amino acid sequence identity to SEQ ID NO: 3; and
(c) the polymer binding domain has at least 90% amino acid sequence identity to SEQ ID NO: 4.

15. The isolated recombinant protein of claim 13 , wherein:
(a) the cutinase catalytic domain has at least 95% amino acid sequence identity to SEQ ID NO: 2;
(b) the proline/threonine-rich linker domain has at least 95% amino acid sequence identity to SEQ ID NO: 3; and
(c) the polymer binding domain has at least 95% amino acid sequence identity to SEQ ID NO: 4.

16. The isolated recombinant protein of claim 13 , which is recombinantly produced fused to a heterologous carrier protein or heterologous tag that enables secretion of said recombinant protein, and/or facilitates purification or detection of said recombinant protein.

17. The isolated recombinant protein of claim 16 , wherein said carrier protein is YebF.

Assignees

• Ozymes
• Nat Res Council Canada

Inventors

• Abokitse Kofi
• Lau Peter C K
• Grosse Stephan

Classifications

• C07K2319/20

  containing a tag with affinity for a non-protein ligand · CPC title

• C07K2319/036

  targeting to the medium outside of the cell, e.g. type III secretion · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12Y301/01074

  Cutinase (3.1.1.74) · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title