# US 9,752,131 B2 · Multi-domain enzymes having cutinase activity, compositions comprising same and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,752,131 B2 - Document ID: US9752131B2 - Status: Granted - Kind: B2 - Application number: 15/044,715 - Filed: 2016-02-16 - Published: 2016-11-03 - Granted: 2017-09-05 - Earliest claimed: 2015-02-16 - Assignees: National Research Council of Canada; Ozymes - Applicants: Ozymes; National Research Council of Canada - Inventors: Kofi Abokitse; Peter C. K. Lau; Stephan Grosse - Web page: https://patentgrep.com/patent/US9752131B2 ## CPC - C12N 9/18: Carboxylic ester hydrolases {(3.1.1)} - A23K 20/189: Enzymes - C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} - C09K 8/582: characterised by the use of bacteria - C11D 3/38636: {containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase} - C11D 3/38681: {Chemically modified or immobilised enzymes} - C11D 3/39: Organic or inorganic per-compounds - C11D 3/43: Solvents - C11D 7/5004: {Organic solvents} - C12N 9/16: acting on ester bonds (3.1) - C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates - C12Y 301/01074: Cutinase (3.1.1.74) - C07K 2319/00: Fusion polypeptide - C07K 2319/036: targeting to the medium outside of the cell, e.g. type III secretion - C07K 2319/20: containing a tag with affinity for a non-protein ligand - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A23K: FODDER - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE - C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL - C12Y: ENZYMES ## 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) The composition of claim 1 comprising a peroxide. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 1) 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. ### Claim 6 (depends on 1) The composition of claim 1 having a pH between 7.5 and 9.5. ### Claim 7 (depends on 1) The composition of claim 1, further comprising: a detergent, a surfactant, a buffer, a chelator, or any combination thereof. ### Claim 8 (depends on 1) The composition of claim 1, further comprising a chelator which is EDTA or GLDA. ### Claim 9 (depends on 1) The composition of claim 1, further comprising a polyol and/or boric acid as a stabilizer. ### Claim 10 (depends on 1) 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. ### Claim 11 (depends on 1) 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. ### Claim 12 (depends on 1) 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. ### Claim 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. ### Claim 14 (depends on 13) 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. ### Claim 15 (depends on 13) 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. ### Claim 16 (depends on 13) 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. ### Claim 17 (depends on 16) The isolated recombinant protein of claim 16, wherein said carrier protein is YebF.