# US 9,040,277 B2 · Mannanase variants > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,277 B2 - Document ID: US9040277B2 - Status: Granted - Kind: B2 - Application number: 13/522,221 - Filed: 2010-11-24 - Published: 2012-11-08 - Granted: 2015-05-26 - Earliest claimed: 2010-01-13 - Assignees: BASF SE - Applicants: Oliver Kensch - Inventors: Oliver Kensch - Web page: https://patentgrep.com/patent/US9040277B2 ## CPC - C11D 3/38636: {containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase} - C12N 9/2494: {Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase} - C12Y 302/01078: Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase - C09K 8/68: containing organic compounds - C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) - C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE ## Abstract The present disclosure provides novel mannanase variants which have an amino acid sequence that varies from that of the parent/wild type Trichoderma reesei mannanase, and which have one or more advantageous properties like improved thermo stability; temperature/activity profile; pH/activity profile; specific activity; and pH/protease-sensitivity. The novel mannanase variants are useful and used in alcohol fermentations processes and/or productions, for coffee extraction and the processing of coffee waste, as a supplement to food and feed, for enzyme aided bleaching of paper pulps, as bleaching and/or desizing agent in textile industry, for oil and gas well stimulation by hydraulic fracturing, as detergent, as baking ingredients, for removal of biofilms and in delivery systems, for grain processing or for the processing of renewable resources intended for the production of biological fuels, and in the textile, oil drilling, cleaning, laundering, detergent, and cellulose fiber processing industries. ## Claims ### Claim 1 A mannanase variant having mannanase activity and an amino acid sequence that varies from the amino acid sequence of parent/wild type Trichoderma reesei mannanase (SEQ ID NO: 1), wherein the amino acid sequence of the mannanase variant comprises the variations 3R, 201S, 207F and 274L, and variations 66P, 215T and 259R and a sequence identity of at least 90% to SEQ ID NO: 1. ### Claim 2 (depends on 1) The mannanase variant of claim 1, wherein the mannanase variant comprises the variation 181A/H. ### Claim 3 (depends on 1) The mannanase variant of claim 1, wherein the mannanase variant further comprises one or more additional variations at positions 31, 97, 113, 146, 149, 173, 181, 280, 282, 331 or 344. ### Claim 4 (depends on 3) The mannanase variant of claim 3, wherein the variations are 3IY, 97R, 113Y, 146Q, 149K, 173H/T, 181H/A, 280S/L/R, 282D, 331S or 344D. ### Claim 5 (depends on 1) A composition which is suitable for food and feed processing, as supplement to food and feed, for enzyme aided bleaching of paper pulps, for oil and gas well stimulation by hydraulic fracturing, as detergent, for removal of biofilms, or in delivery systems comprising the mannanase variant according to claim 1. ### Claim 6 (depends on 1) A composition comprising the mannanase variant according to claim 1 and one or more enzymes selected from the group consisting of phytases, hemicellulases, alpha-galactosidases, beta-galactosidases, lactases, beta-glucanases, endo-beta-1,4-glucanases, cellulases, xylosidases, xylanases, xyloglucanases, xylan acetylesterases, galactanases, exo-mannanases, pectinases, pectin lyases, pectinesterases, polygalacturonases, arabinases, rhamnogalacturonases, laccases, reductases, oxidases, phenoloxidases, ligninases, proteases, amylases, phosphatases, lipolytic enzymes, and cutinases. ### Claim 7 (depends on 1) A food or feed supplement which comprises the mannanase variant of claim 1. ### Claim 8 (depends on 1) A detergent comprising the mannanase variant of claim 1. ### Claim 9 (depends on 1) A bleaching or desizing agent for textiles comprising the mannanase variant of claim 1. ### Claim 10 (depends on 1) A method for preparing coffee extract, comprising adding the mannanase variant of claim 1 in a liquid coffee extract, and freeze drying the liquid coffee extract. ### Claim 11 (depends on 1) A method of processing coffee waste, the method comprising adding the mannanase variant of claim 1 to spent coffee grounds. ### Claim 12 (depends on 1) A method of bleaching paper pulps, the method comprising adding the mannanase variant of claim 1 to paper pulp under conditions suitable to produce a whitening of the pulp. ### Claim 13 (depends on 1) A method of removing biofilms, the method comprising contacting the biofilm with the mannanase variant of claim 1, resulting in removal of the biofilm. ### Claim 14 A mannanase variant having mannanase activity and an amino acid sequence that varies from the amino acid sequence of parent/wild type Trichoderma reesei mannanase (SEQ ID NO:1, wherein the amino acid sequence of the mannanase variant comprises the variations 3R, 201S, 207F and 274L, and further variations selected from the group consisting of: 1) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280R/N282D/N331S; 2) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/Q280S/N331S; 3) F31Y/S66P/Q97R/N173H/V181H/A215TIQ259R/Q280R/N282D; 4) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280S/N282D/N331S; 5) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280R/N282D; 6) F31Y/S66P/Q97R/Q149K/N173H/V181H/A215T/Q259R/Q280L; 7) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R; 8) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280S/N282D/N331S; 9) S66P/N113Y/N173H/V181H/A215T/Q259R; 10) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181H/A215T/Q259R/Q280L/N28 2D; 11) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/Q280L/N28 2D/ N331S; 12) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181H/A215T/Q259R/Q280L; 13) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/N282D; 14) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/Q280R/N28 2D; 15) S66P/N113Y/V181H/A215T/Q259R; 16) S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280L/N282D; 17) F317/S66P/Q97R/N113YNK146Q/N173H/V181A/A215T/Q259R/Q280L/N28 2D; 18) F31Y/S66P/N173H/V181H/A215T/Q259R/N282D; 19) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280L; 20) S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280R/N282D; 21) F31Y/S66P/Q97R/N113Y/N173T/V181H/A215T/Q259R/Q280R/N282D; 22) F31Y/S66P/Q97R/N173T/V181H/A215T/Q259R/Q280R/N282D; 23) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280S/N282D; 24) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S/N282D; 25) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/Q280S/N282D/N331S; 26) S66P/Q97R/N113Y/V181H/A215T/Q259R/Q280L/N282D; 27) S66P/N113Y/N173H/V181H/A215T/Q259R/N331S; 28) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/Q280R/N28 2D/ N331S; 29) F31Y/S66P/Q97R/N113Y/K146Q/V181H/A215T/Q259R/Q280S/N282D/N33 1S; 30) S66P/Q97R/N113Y/N173T/V181A/A215T/Q259R; 31) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/Q280S/N33 1S; 32) F31Y/S66P/Q97R/N113Y/V181H/A215T/Q259R; 33) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/N331S; 34) F31Y/S66P/Q97R/N113Y/V181H/A215T/Q259R/Q280L; 35) F31Y/S66P/Q97R/N113Y/K146Q/V181H/A215T/Q259R/Q280L; 36) F31Y/S66P/Q97R/K146Q/V181H/A215T/Q259R/Q280R/N282D; 37) S66P/N113Y/V181H/A215T/Q259R/N282D; 38) F31Y/S66P/Q97R/V181H/A215T/Q259R/N282D; 39) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S/N331S; 40) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181H/A215T/Q259R/Q280S/N33 1S; 41) S66P/V181H/A215T/Q259R/N282D; 42) F31Y/S66P/Q97R/N113Y/K146Q/V181H/A215T/Q259R/Q280L/N331S; 43) S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/N282D; 44) S66P/Q97R/N113Y/V181H/A215T/Q259R/N282D; 45) S66P/V181H/A215T/Q259R; 46) S66P/Q97R/N113Y/V181H/A215T/Q259R/Q280R/N282D; 47) F31Y/S66P/N173T/V181H/A215T/Q259R/N282D; 48) F31Y/S66P/N113Y/V181H/A215T/Q259R/Q280R/N344D; 49) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280R/N282D; 50) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280S/N282D/N33 1S; 51) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S; 52) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R; 53) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/Q280S; 54) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S/N282D; 55) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280R/N282D; 56) S66P/N113Y/N173H/V181H/A215T/Q259R; 57) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/N331S; 58) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280S/N282D/N331S; 59) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S/N331S; 60) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/Q280S/N331S; 61) F31Y/S66P/Q97R/Q149K/N173H/V181H/A215T/Q259R/Q280S/N331S; and 62) S66P/A215T/Q259R, and wherein the mannanase variant has a sequence identity of at least 90% to SEQ ID NO: 1.