# US 10,501,730 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 10,501,730 B2 - Document ID: US10501730B2 - Status: Granted - Kind: B2 - Application number: 15/608,179 - Filed: 2017-05-30 - Published: 2018-12-06 - Granted: 2019-12-10 - Earliest claimed: 2017-05-30 - Assignees: AB Enzymes Oy - Applicants: AB Enzymes Oy - Inventors: Taija Leinonen; Leena Valtakari; Michael Seefried; Kari Juntunen; Terhi Puranen - Web page: https://patentgrep.com/patent/US10501730B2 ## CPC - C12N 9/2494: {Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase} - A23C 11/106: {Addition of, or treatment with, microorganisms (A23C20/025 takes precedence)} - A23K 20/147: Polymeric derivatives, e.g. peptides or proteins - A23K 20/189: Enzymes - A23L 2/84: using microorganisms or biological material, e.g. enzymes - A23L 11/65: Soy drinks - C09K 8/035: Organic additives - C09K 8/62: Compositions for forming crevices or fractures - C09K 8/68: containing organic compounds - C09K 8/86: containing organic compounds - C11D 3/38636: {containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase} - C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60) - C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} - C12Y 302/01078: Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase - C09K 2208/24: Bacteria or enzyme containing gel breakers - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A23C: DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF (preservation thereof A23B11/00) - A23K: FODDER - A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B) - 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 - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12Y: ENZYMES ## Abstract Variants of mannanase, compositions including the variants, to methods for their production and to methods of using the variants to degrade and modify mannan containing material. ## Claims ### Claim 1 A variant of mannanase comprising at least one substitution of an amino acid at a position corresponding to the position 123, 158, 180, 272, 307, or 316 wherein the variant has mannanase activity and is selected from the group consisting of 1) a polypeptide having at least 85% sequence identity to residues 27-331 of SEQ ID NO: 2; 2) a variant encoded by a polynucleotide that hybridizes under high stringency conditions with a) nucleotides 79-993 of SEQ ID NO: 1 (man 7) or b) the full-length complement of a); and 3) a variant encoded by a polynucleotide having at least 95% sequence identity to the SEQ ID NO: 1; and wherein the amino acid numbering corresponds to the amino acid numbering of SEQ ID NO: 2 full length amino acid sequence containing a signal sequence; and wherein the variant comprises at least one additional substitution selected from: a substitution which prevents glycosylation of N283;or a substitution of 283 or 285; or a substitution of N283, T285, or S285; or a substitution of T285, or S285 to a residue other than T or S; or a substitution of T285, or S285 to the residue A. ### Claim 2 (depends on 1) The variant of claim 1 comprising at least one further substitution at the position M123, A158, F180, G272, T307, or L316, or a combination thereof. ### Claim 3 (depends on 1) The variant of claim 1 comprising at least one set of substitutions at the position(s): M123 and G272; or A158 and T307; or L316; or T307; or M123, A158 and T307; or M123 and L316; or A158, T307, and L316; or F180 and L316; or M123, A158, G272; T307, and L316; or a combination thereof. ### Claim 4 (depends on 1) The variant of claim 1 wherein the substitution results into an improved stability of the variant. ### Claim 5 (depends on 1) The variant of claim 1 wherein the additional substitution results into an altered glycosylation of the variant when produced in a host cell capable of N-linked glycosylation. ### Claim 6 (depends on 1) An enzyme composition comprising the variant of claim 1 and a. at least one preservative selected from organic acid, citric acid, ascorbic acid, benzoic acid and their salts and derivatives, sodium benzoate, benzoate, hydroxybenzoate and derivatives, sorbic acid, sodium sorbate, sorbate, salts, sodium chloride, potassium chloride, 1,2-Benzisothiazolin-3-one (BIT) or a combination thereof; b. optionally at least one stabilizer selected from polyol, propylene glycol, polyethylene glycol, hexylene glycol, glycerol, a sugar, sugar alcohol, polysaccharide, lactic acid, boric acid, boric acid derivative, aromatic borate ester, 4-formylphenyl boronic acid, phenyl boronic acid derivative, peptide, surfactant or a combination thereof; c. optionally at least one enzyme selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, phytases, DNAses, pectinases, pectinolytic enzymes, pectate lyases, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases and oxidases with or without a mediator, or a combination thereof; and d. optionally at least one filler selected from maltodextrin, flour, sodium chloride, sulfate, sodium sulfate, or a combination thereof. ### Claim 7 (depends on 6) The enzyme composition of claim 6 in the form of a liquid composition, solid composition, solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal, crystal slurry, gel, or pellet. ### Claim 8 (depends on 1) A detergent composition comprising the variant of mannanase of claim 1. ### Claim 9 (depends on 8) The detergent composition of claim 8 in the form of a liquid detergent, solid detergent, a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a granulate, a paste, a gel, or a regular, compact or concentrated liquid. ### Claim 10 (depends on 8) The detergent composition of claim 8 further comprising one or more additional enzymes selected from the group consisting of protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, pectatelyase, pectinolytic enzyme, esterase, mannanase, arabinase, galactanase, xylanase, oxidase, xanthanase, xyloglucanase, laccase, DNAse and/or peroxidase. ### Claim 11 (depends on 6) A method for degrading or modifying mannan containing material comprising treating said mannan containing material with an effective amount of the enzyme composition of claim 6. ### Claim 12 (depends on 11) The method of claim 11 wherein the mannan containing material is plant based material, textile, waste water, sewage, oil, or a combination thereof. ### Claim 13 (depends on 6) An animal feed comprising the enzyme composition of claim 6, and at least one protein source of plant origin or a mannan containing product or by-product, and a. Optionally at least one enzyme selected from protease, amylase, phytase, xylanase, endoglucanase, beta-glucanase, or a combination thereof; and b. Optionally at least one filler selected from maltodextrin, flour, salt, sodium chloride, sulfate, sodium sulfate, or a combination thereof. ### Claim 14 (depends on 6) A feed supplement comprising the enzyme composition of claim 6; and a. Optionally at least one enzyme selected from protease, amylase, phytase, xylanase, endoglucanase, beta-glucanase, or a combination thereof; and b. Optionally at least one filler selected from maltodextrin, flour, salt, sodium chloride, sulfate, sodium sulfate or a combination thereof. ### Claim 15 (depends on 1) A recombinant host cell comprising genetic elements that allow producing at least one recombinant polypeptide comprising the variant of claim 1. ### Claim 16 The recombinant host cell of 15, wherein the host cell is selected from the group consisting of: fungal cells, filamentous fungal cells from Division Ascomycota, Subdivision Pezizomycotina; from the group consisting of members of the Class Sordariomycetes, Subclass Hypocreomycetidae, Orders Hypocreales and Microascales and Aspergillus, Chrysosporium, Myceliophthora and Humicola; Families Hypocreacea, Nectriaceae, Clavicipitaceae, Microascaceae, and Genera Trichoderma (anamorph of Hypocrea), Fusarium, Gibberella, Nectria, Stachybotrys, Claviceps, Metarhizium, Villosiclava, Ophiocordyceps, Cephalosporium, and Scedosporium; Trichoderma reesei (Hypocrea jecorina), T. citrinoviridae, T. longibrachiatum, T. virens, T. harzianum, T. asperellum, T. atroviridae, T. parareesei, Fusarium oxysporum, F. gramineanum, F. pseudograminearum, F. venenatum, Gibberella fujikuroi, G. monihformis, G. zeaea, Nectria (Haematonectria) haematococca, Stachybotrys chartarum, S. chlorohalonata, Claviceps purpurea, Metarhizium acridum, M. anisopliae, Villosiclava virens, Ophiocordyceps sinensis, Acremonium (Cephalosporium) chrysogenum, and Scedosporium apiospermum, and Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Chrysosporium lucknowense, Mycellophthora thermophila, Humicola insolens, and Humicola grisea, bacterial cells, gram positive Bacilli B. subtilis, B. licheniformis, B. megaterium, B. amyloliquefaciens, B. pumilus, gram negative bacteria, Escherichia coli, actinomycetales, Streptomyces sp., and yeasts, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Trichoderma reesei or Bacillus. ### Claim 17 (depends on 15) The recombinant host cell of claims 15, wherein the recombinant polypeptide is a fusion protein which, in addition to having the amino acid sequence having mannanase activity, comprises at least one of: an amino acid sequence providing a secretory signal sequence; an amino acid sequence which facilitates purification, or is an affinity tag or His-tag; an amino acid sequence which is a carrier or CBM; an amino acid sequence having an enzyme activity; and an amino acid sequence providing for the fusion protein with binding affinity, or a carbohydrate binding moiety. ### Claim 18 (depends on 15) A method for producing a recombinant polypeptide comprising a variant of mannanase comprising: a. cultivating a recombinant host cell of claim 15, wherein the genetic elements comprise at least one control sequence which controls the production of the recombinant polypeptide in the recombinant host cell; the genetic elements optionally comprise at least one sequence encoding a signal sequence for transporting the recombinant polypeptide outside the host cell; and cultivating is carried out in conditions allowing production of the recombinant polypeptide; and b. recovering the recombinant polypeptide. ### Claim 19 (depends on 1) A process of controlling viscosity of a drilling fluid in oil drilling or hydro-fracturing, comprising providing a composition comprising the variant of claim 1 in contact with the drilling fluid. ### Claim 20 (depends on 1) A process of controlling viscosity in manufacturing of coffee extract, fruit juice, pineapple juice, or soya milk, comprising providing a composition comprising the variant of claim 1 in contact with the coffee extract, fruit milk or soya milk.