Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2477—Hemicellulases not provided in a preceding group
  • C12N9/2488—Mannanases
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2477—Hemicellulases not provided in a preceding group
  • C12N9/2488—Mannanases
  • C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
  • C11D2111/10—Objects to be cleaned
  • C11D2111/12—Soft surfaces, e.g. textile
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
  • C11D2111/10—Objects to be cleaned
  • C11D2111/14—Hard surfaces

Definitions

• Mannanase enzymes including endo-P-mannanases, have been employed in detergent cleaning compositions for the removal of gum stains by hydrolyzing mannans.
• a variety of mannans are found in nature, such as, for example, linear mannan, glucomannan, galactomannan, and glucogalactomannan.
• Each such mannan is comprised of polysaccharides that contain a P-l,4-linked backbone of mannose residues that may be substituted up to 33% with glucose residues (Yeoman et al., Adv Appl Microbiol, 70: 1, 2010, Elsevier).
• galactomannans or glucogalactomannnans galactose residues are linked in alpha- 1,6-linkages to the mannan backbone (Moreira and Filho, Appl Microbiol Biotechnol, 79: 165, 2008).
• hydrolysis of mannan to its component sugars requires endo-l,4-P-mannanases that hydrolyze the backbone linkages to generate short chain manno-oligosaccharides that are further degraded to monosaccharides by 1,4-P-mannosidases.
• mannanases such as, for example, endo-P-mannanases have been known in the art of industrial enzymes, there remains a need for improved mannanase variants.
• Variants, compositions and methods disclosed herein relate to a recombinant mannanase, or a recombinant polypeptide generated through conventional molecular biology techniques (see, e.g., Sambrook et al, Molecular Cloning: Cold Spring Harbor Laboratory Press).
• mannanase variants are provided, where the mannanase variants comprise one or more amino acid substitutions at one or more positions selected from 32, 72, 161 and 172 wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
• the mannanase variant is a variant comprising an amino acid substitution selected from the group consisting of 19D, 32Y, 34D, 72V, 93Q, 13 IS, 136P, 139R, 161G, 172F, 225N/Q, 259D, 261DZE and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
• the mannanase variant is a variant comprising two or more amino acid substitutions selected from the group consisting of 19D, 32Y, 72V, 93Q, 13 IS, 136P, 139R, 161G, 168T, 172F, 225N/Q, 259DZE, 261DZE and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
• the mannanase variant is a variant comprising amino acid substitutions selected from the group consisting of 19D-276W, 32Y-259D, 93Q-276W, 131S- 276W, 136P-276W, 139R-276W, 161G-276W, 225N/Q-276W, 259D/E-276W, and 261D/E- 276W.
• the mannanase variant is a variant comprising amino acid substitutions selected from the group consisting of 19D-131S-276W, 32Y-261D-276W, 32Y- 259D-276W, 32Y-172F-259D, 168T-259D-276W, 259D-261E-276W, and 259Q-261E-276W.
• the mannanase variant is a variant comprising amino acid substitutions selected from the group consisting of Y061W-G259D-R261E-F276W, Y061W- T062E-G259D-R261E-F276W, Y061W-V228T-G259D-R261E-F276W, V228T-G259D- R261E-F276W, F032Y-G259D-R261E-F276W, F032Y-Y061W-Y167F-P168S-G259D-R261E- F276W, F032Y-Y061W-G259D-R261E-F276W, F032Y-Y061W-T062E-G259D-R261E- F276W, F032Y-T062E-R261D-F276W, F032Y-T062
• N010T-F032Y-V059S-Q060L-T062E-Y167F-P168S-V228T-G259D-F276W-T284E N010T-F032Y-V059S-Q060L-T062E-I072V-V228T-G259D-F276W-T284E
• Still further embodiments are directed to methods of producing a mannanase variant described herein comprising: stably transforming a host cell with an expression vector comprising a polynucleotide encoding the mannanase variant; culturing the transformed host cell under suitable conditions to produce the mannanase variant; and recovering the mannanase variant.
• the nomenclature of the amino acid substitutions of the one or more mannanase variants described herein uses one or more of the following: position; positiomamino acid substitution(s); or starting amino acid(s):position:amino acid substitution(s).
• Reference to a “position” e.g. 5, 8, 17, 22, etc) encompasses any starting amino acid that may be present at such position, and any substitution that may be present at such position.
• Reference to a “position: amino acid substitution(s)” e.g. 1S/T/G, 3G, 17T, etc) encompasses any starting amino acid that may be present at such position and the one or more amino acid(s) with which such starting amino acid may be substituted.
• the position of an amino acid residue in a given amino acid sequence is numbered by correspondence with the amino acid sequence of SEQ ID NO: 1. That is, the amino acid sequence of SEQ ID NO: 1 serves as a reference sequence for numbering of positions of an amino acid residue.
• the amino acid sequence of one or more mannanase variant described herein is aligned with the amino acid sequence of SEQ ID NO: 1 using an alignment algorithm as described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with an amino acid residue in SEQ ID NO: 1 is conveniently numbered by reference to the numerical position of that corresponding amino acid residue.
• Sequence alignment algorithms such as, for example, described herein will identify the location or locations where insertions or deletions occur in a subject sequence when compared to a query sequence (also sometimes referred to as a “reference sequence”).
• mannan endo-l,4-P-mannosidase refers to an enzyme capable of the random hydrolysis of 1,4-P-D-mannosidic linkages in mannans, galactomannans and glucomannans.
• Endo-l,4-P-mannanases are members of several families of glycosyl hydrolases, including GH26 and GH5.
• endo-P-mannanases constitute a group of poly saccharases that degrade mannans and denote enzymes that are capable of cleaving polyose chains containing mannose units ( . e. , are capable of cleaving glycosidic bonds in mannans, glucomannans, galactomannans and galactogluco-mannans).
• endo-P- mannanases may possess additional enzymatic activities (e.g., endo-l,4-P- glucanase, 1,4- p -mannosidase, and cellodextrinase activities).
• mannanase refers to an enzyme, polypeptide, or protein that can degrade mannan.
• the mannanase enzyme may, for example, be an endo-P- mannanase, an exo-P-mannanase, or a glycosyl hydrolase.
• mannanase activity may be determined according to any procedure known in the art (See, e.g., Lever, Anal. Biochem, 47:273, 1972; Eriksson and Winell, Acta Chem. Scand., (1968), 22: 1924; US 6,602,842; and WO 95/35362A1).
• mannans are polysaccharides having a backbone composed of P-1, 4- linked mannose
• glucomannans are polysaccharides having a backbone of more or less regularly alternating P-1,4 linked mannose and glucose
• galactomannans and galactoglucomannans are mannans and glucomannans with alpha- 1,6 linked galactose side- branches. These compounds may be acetylated. The degradation of galactomannans and galactoglucomannans is facilitated by full or partial removal of the galactose side-branches.
• acetylated mannans, glucomannans, galactomannans and galactoglucomannans is facilitated by full or partial deacetylation.
• Acetyl groups can be removed by alkali or by mannan acetylesterases.
• the oligomers that are released from the mannanases or by a combination of mannanases and alpha-galactosidase and/or mannan acetyl esterases can be further degraded to release free maltose by P-mannosidase and/or P-glucosidase.
• protease refers to an enzyme that has the ability to break down proteins and peptides.
• a protease has the ability to conduct “proteolysis,” by hydrolysis of peptide bonds that link amino acids together in a peptide or polypeptide chain forming the protein.
• proteolytic activity This activity of a protease as a protein-digesting enzyme is referred to as “proteolytic activity.”
• proteolytic activity may be ascertained by comparative assays that analyze the respective protease’s ability to hydrolyze a suitable substrate.
• Exemplary substrates useful in the analysis of protease or proteolytic activity include, but are not limited to, di-methyl casein (Sigma C- 9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and Keratin Azure (Sigma-Aldrich K8500). Colorimetric assays utilizing these substrates are well known in the art (See e.g, WO99/34011 and US 6,376,450).
• modification refers to any change or alteration in an amino acid sequence, including the substitution of an amino acid at the identified position of the amino acid sequence of interest with an amino acid that is different from the starting amino acid, deletion of an amino acid at the identified position of the amino acid sequence of interest, insertion of an amino acid at the identified position of the amino acid sequence of interest, replacement of an amino acid side chain in the amino acid sequence of interest, and or chemical modification of the amino acid sequence of interest.
• catalytic activity or “activity” describes quantitatively the conversion of a given substrate under defined reaction conditions.
• residual activity is defined as the ratio of the catalytic activity of the enzyme under a certain set of conditions to the catalytic activity under a different set of conditions.
• specific activity describes quantitatively the catalytic activity per amount of enzyme under defined reaction conditions.
• pH-stability describes the ability of a protein to withstand a limited exposure to pH-values significantly deviating from the pH where its stability is optimal (e.g., more than one pH-unit above or below the pH-optimum), without losing its activity under conditions where its activity is measurable.
• detergent stability refers to the stability of a specified detergent composition component (such as a hydrolytic enzyme) in a detergent composition mixture.
• perhydrolase refers to an enzyme capable of catalyzing a reaction that results in the formation of a peracid suitable for applications such as cleaning, bleaching, and disinfecting.
• aqueous refers to a composition that is made up of at least 50% water.
• An aqueous composition may contain at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% water.
• surfactant refers to any compound generally recognized in the art as having surface active qualities. Surfactants generally include anionic, cationic, nonionic, and zwitterionic compounds, which are further described, herein.
• chelator stability refers to mannanase variants of the present disclosure that retain a specified amount of enzymatic activity over a given period of time under conditions prevailing during the mannosidic, hydrolyzing, cleaning, or other process disclosed herein, for example while exposed to or contacted with chelating agents.
• the mannanase variant retains at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% mannanase activity after contact with a chelating agent over a given time period, for example, at least about 10 minutes, about 20 minutes, about 40 minutes, about 60 minutes, about 100 minutes, etc.
• thermo stability and “thermostable” refer to mannanase variants that retain a specified amount of enzymatic activity after exposure to elevated temperatures over a given period of time under conditions prevailing during the mannosidic, hydrolyzing, cleaning, or other process, for example, while exposed to elevated temperatures.
• cleaning activity refers to the cleaning performance achieved by a mannanase variant under conditions prevailing during the mannosidic, hydrolyzing, cleaning, or other process disclosed herein.
• cleaning performance is determined by the application of various cleaning assays concerning enzyme sensitive stains arising from food products, household agents or personal care products.
• Some of these stains include, for example, ice cream, ketchup, BBQ sauce, mayonnaise, soups, chocolate milk, chocolate pudding, frozen desserts, shampoo, body lotion, sun protection products, toothpaste, locust bean gum, or guar gum as determined by various chromatographic, spectrophotometric or other quantitative methodologies after subjection of the stains to standard wash conditions.
• the term “effective amount” when used in conjunction with a mannanase variant refers to the quantity of mannanase variant needed to achieve the desired level of enzymatic activity in the specified cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular mannanase variant that is used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar, powder, solid, liquid, tablet, gel, paste, foam, sheet, or unit dose) composition is required.
• a liquid or dry composition e.g., granular, bar, powder, solid, liquid, tablet, gel, paste, foam, sheet, or unit dose
• adjunct ingredient when used in conjunction with a cleaning composition means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, powder, bar, paste, spray, tablet, gel, unit dose, sheet, or foam composition), which materials are also preferably compatible with the mannanase variant used in the composition.
• granular compositions are in “compact” form, while in other embodiments, the liquid compositions are in a “concentrated” form.
• cleaning compositions and “cleaning formulations” refer to admixtures of chemical ingredients that find use in the removal of undesired compounds (e.g., soil or stains) from items or surfaces to be cleaned, such as, for example, fabric, dishes, contact lenses, solid surfaces, hair, skin, and teeth.
• the compositions or formulations may be in the form of a liquid, gel, granule, powder, bar, paste, spray tablet, gel, unit dose, sheet, or foam, depending on the surface or item to be cleaned and the desired form of the composition or formulation.
• Detergent composition and “detergent formulation” refer to mixtures of chemical and/or biological ingredients intended for use in a wash medium for the cleaning of soiled objects.
• Detergent compositions/formulations generally include at least one surfactant, and may optionally include hydrolytic enzymes, oxido-reductases, builders, bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, solubilizers, and one or more microorganisms or microbes or microbial extracts.
• Microorganisms may be used as the only biologically active ingredient, but they may also be used in conjunction with one or more of the enzymes described herein.
• the term “dishwashing composition” refers to all forms of compositions including, for example, granular, unit-dose, and liquid forms for cleaning dishware and cutlery.
• the dishwashing composition is an “automatic dishwashing” composition that finds use in automatic dishwashing machines.
• the term “dishware” refers to dishes e.g., plates, cups, glasses, bowls, and containers) and cutlery (e.g., utensils including, but not limited to spoons, knives, and forks) of any material, including but not limited to ceramics, plastics, metals, china, glass, and acrylics.
• bleaching refers to the treatment of a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient length of time and under appropriate pH and temperature conditions to effect a brightening (i.e., whitening) and/or cleaning of the material.
• chemicals suitable for bleaching include but are not limited to CIO2, H2O2, peracids, and NO2.
• wash performance of a mannanase variant refers to the contribution of the variant to washing that provides additional cleaning performance to the detergent composition. Wash performance is compared under relevant washing conditions.
• relevant washing conditions is used herein to indicate the conditions, particularly washing temperature, time, washing mechanics, suds concentration, type of detergent, and water hardness, actually used in households in a dish or laundry detergent market segment.
• the term “disinfecting” refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items.
• the “compact” form of the cleaning compositions herein is best reflected by density and, in terms of composition, by the amount of inorganic filler salt.
• Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, the filler salts are present in substantial amounts, typically about 17 to about 35% by weight of the total composition. In contrast, in compact compositions, the filler salt is present in amounts not exceeding about 15% of the total composition. In some embodiments, the filler salt is present in amounts that do not exceed about 10%, or more preferably, about 5%, by weight of the composition.
• the inorganic filler salts are selected from the alkali and alkaline-earth-metal salts of sulfates and chlorides. In some embodiments, a preferred filler salt is sodium sulfate.
• fabric refers to, for example, woven, knit, and non-woven material, as well as staple fibers and filaments that can be converted to, for example, yams and woven, knit, and non-woven fabrics.
• the term encompasses material made from natural, as well as synthetic (e.g., manufactured) fibers.
• a nucleic acid or polynucleotide is “isolated” when it is at least partially or completely separated from other components, including but not limited to, for example, other proteins, nucleic acids, and cells.
• a polypeptide, protein or peptide is “isolated” when it is at least partially or completely separated from other components, including but not limited to, for example, other proteins, nucleic acids, and cells.
• an isolated species is more abundant than are other species in a composition.
• an isolated species may comprise at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (on a molar basis) of all macromolecular species present.
• the species of interest is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods).
• Purity and homogeneity can be determined using a number of techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of a nucleic acid or a protein sample, respectively, followed by visualization upon staining.
• a high-resolution technique such as high performance liquid chromatography (HPLC) or a similar means can be utilized for purification of the material.
• nucleic acids or polypeptides generally denotes a nucleic acid or polypeptide that is essentially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation).
• a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is “purified.”
• a purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more pure (e.g., percent by weight on a molar basis).
• a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique.
• the term “enriched” refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than in a starting composition.
• wild-type and “native” refer to polypeptides or polynucleotides that are found in nature.
• a polynucleotide encoding a wild-type or parental polypeptide is not limited to a naturally- occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type or parental polypeptide.
• the term “reference”, with respect to a polynucleotide refers to a naturally-occurring polynucleotide that does not include a man-made substitution, insertion, or deletion of one or more nucleosides, as well as a polynucleotide that includes one or more man-made substitutions, insertions, or deletions at one or more nucleosides.
• a polynucleotide encoding a wild-type or parental polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type or parental polypeptide.
• the one letter code “Z” identifies an insertion or deletion in a parent or reference amino acid sequence.
• the one letter code “Z” is on the left side of the position number and further includes a number (e.g., .01) before each amino acid being inserted therein to indicate the order of the insertions.
• the insertion of one amino acid, glutamine (Q), at position 298 would be depicted as “Z298.01Q”; the insertion of one amino acid, X (where X can be any amino acid) at position 298 would be depicted as “Z298.01X”; and the insertion of three amino acids alanine (A), serine (S) and tyrosine (Y) between position 87 and 88 would be depicted as “Z87.01A/Z87.02S/Z87.03Y”.
• the one letter code "Z" is on the right side of the position number.
• the deletion of an alanine (A) from position 100 would be depicted as A100Z.
• a combination of some of the above insertions and deletions would be depicted as: “G87S/Z87.01A/Z87.02S/Z87.03Y/A100Z”.
• a mannanase variant has at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with a reference polypeptide.
• a variant polynucleotide has at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity with a parent polynucleotide.
• derived from encompasses the terms “originated from,” “obtained from,” “obtainable from,” “isolated from,” and “created from” and generally indicates that one specified material find its origin in another specified material or has features that can be described with reference to the another specified material.
• hybridization refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing, as known in the art.
• hybridization conditions refers to the conditions under which hybridization reactions are conducted. These conditions are typically classified by degree of “stringency” of the conditions under which hybridization is measured.
• the degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe.
• Tm melting temperature
• maximum stringency typically occurs at about T m -5°C (5°C below the Tm of the probe); “high stringency” at about 5-10°C below the T m ; “intermediate stringency” at about 10- 20°C below the Tm of the probe; and “low stringency” at about 20-25°C below the Tm.
• maximum stringency conditions may be used to identify nucleic acid sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify nucleic acid sequences having about 80% or more sequence identity with the probe.
• it is typically desirable to use relatively stringent conditions to form the hybrids e.g., relatively low salt and/or high temperature conditions are used).
• substantially similar and “substantially identical” in the context of at least two nucleic acids or polypeptides means that a polynucleotide or polypeptide comprises either a sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a parent or reference sequence, or a sequence that includes amino acid substitutions, insertions, deletions, or modifications made only to circumvent the present description without adding functionality.
• expression vector refers to a DNA construct containing a DNA sequence that encodes the specified polypeptide and is operably linked to a suitable control sequence capable of effecting the expression of the polypeptides in a suitable host.
• control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
• the vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
• the term “recombinant” refers to genetic material (z.e., nucleic acids, the polypeptides they encode, and vectors and cells comprising such polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at a decreased or elevated levels, expressing a gene conditionally or constitutively in manner different from its natural expression profile, and the like.
• signal sequence refers to a sequence of amino acids bound to the N-terminal portion of a polypeptide, and which facilitates the secretion of the mature form of the protein from the cell.
• the mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process.
• selectable marker refers to a gene capable of expression in a host cell that allows for ease of selection of those hosts containing an introduced nucleic acid or vector.
• selectable markers include but are not limited to antimicrobial substances (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage, on the host cell.
• selectable gene product refers to a gene that encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.
• regulatory element refers to a genetic element that controls some aspect of the expression of nucleic acid sequences.
• a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Additional regulatory elements include splicing signals, polyadenylation signals and termination signals.
• host cells generally refers to prokaryotic or eukaryotic hosts which are transformed or transfected with vectors constructed using recombinant DNA techniques known in the art. Transformed host cells are capable of either replicating vectors encoding the protein variants or expressing the desired protein variant. In the case of vectors which encode the pre- or pro-form of the protein variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium.
• the term “introduced” in the context of inserting a nucleic acid sequence into a cell means transformation, transduction, or transfection.
• Means of transformation include protoplast transformation, calcium chloride precipitation, electroporation, naked DNA, and the like as known in the art. (See, Chang and Cohen [1979] Mol. Gen. Genet. 168:111-115; Smith et al. [1986] Appl. Env. Microbiol. 51 :634; and the review article by Ferrari et al., in Harwood, Bacillus ⁇ Plenum Publishing Corporation, pp. 57-72, 1989).
• variants, compositions and methods disclosed herein relate to a recombinant mannanase, comprising one or more modifications, wherein such variants are generated through conventional molecular biology techniques (see, e.g., Sambrook et al, Molecular Cloning: Cold Spring Harbor Laboratory Press).
• the variant mannanase comprises one or more modifications selected from at least one substitution, at least one deletion, and at least one insertion.
• the modification comprises a combination of mutations, such as, for example, a combination of at least one substitution and at least one deletion, at least one deletion and at least one insertion, at least one insertion and at least one substitution, or at least one substitution, at least one deletion, and at least one insertion.
• mannanase variants are provided, where the mannanase variants comprise one or more amino acid substitutions at one or more positions selected from 32, 72, 161 and 172 wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
• the mannanase variant is a mannanase variant comprising an amino acid substitution selected from the group consisting of 19D, 32Y, 34D, 72V, 93Q, 13 IS, 136P, 139R, 161G, 172F, 225N/Q, 259D, 261D/E and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
• the mannanase variant is a mannanase variant described herein, wherein said variant is derived from a parent or reference polypeptide with 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 1.
• the mannanase variants or recombinant polypeptides are substantially identical to SEQ ID NO:1 or 2, meaning that they can contain amino acid substitutions, insertions, or deletions that do not significantly affect the structure, function, or expression of the variant or polypeptide .
• Such mannanase variants or recombinant polypeptides include those designed only to circumvent the present description.
• the mannanase variants have 1,4-P-D-mannosidic hydrolase activity, which includes mannanase, endo-l,4-P-D-mannanase, exo-l,4-P-D-mannanase galactomannanase, and/or glucomannanase activity.
• 1,4-P-D-mannosidic hydrolase activity can be determined and measured using the assays described herein, or by other assays known in the art.
• a polypeptide of the present invention has activity in the presence of a detergent composition.
• the mannanase variants described herein are produced as an N- and/or C-terminal fusion protein, for example, to aid in extraction, detection and/or purification and/or to add functional properties to the variant or recombinant polypeptides or active fragments thereof.
• fusion protein partners include, but are not limited to, glutathione-S-transferase (GST), 6XHis, GAL4 (DNA binding and/or transcriptional activation domains), FLAG, MYC, BCE103 (WO 2010/044786), or other tags well known to anyone skilled in the art.
• a proteolytic cleavage site is provided between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences.
• the fusion protein does not hinder the activity of the mannanase variants or recombinant polypeptides described herein.
• the mannanase variants or recombinant polypeptides described herein are fused to a functional domain including a leader peptide, propeptide, one or more binding domain (modules) and/or a catalytic domain.
• Suitable binding domains include, but are not limited to, carbohydrate-binding modules (CBM) of various specificities, providing increased affinity to carbohydrate components present during the application of the mannanase variants or recombinant polypeptides described herein.
• CBM carbohydrate-binding modules
• the CBM and catalytic domain of a polypeptide of the present invention are operably linked.
• a CBM is defined as a contiguous amino acid sequence within a carbohydrateactive enzyme with a discreet fold having carbohydrate-binding activity.
• CBMs in cellulosomal scaffold in proteins and rare instances of independent putative CBMs.
• the requirement of CBMs existing as modules within larger enzymes sets this class of carbohydrate-binding proteins apart from other non-catalytic sugar binding proteins such as lectins and sugar transport proteins.
• CBMs were previously classified as cellulose-binding domains (CBDs) based on the initial discovery of several modules that bound cellulose (Tomme et al., Eur J Biochem, 170:575-581, 1988; and Gilkes et al., J Biol Chem, 263: 10401-10407, 1988).
• CBDs cellulose-binding domains
• additional modules in carbohydrate-active enzymes are continually being found that bind carbohydrates other than cellulose, yet otherwise meet the CBM criteria, hence the need to reclassify these polypeptides using more inclusive terminology.
• Previous classification of cellulose-binding domains was based on amino acid similarity. Groupings of CBDs were called "Types" and numbered with Roman numerals (e.g. Type I or Type II CBDs).
• Families 1 to 13 are the same as Types I to XIII (Tomme et al., in Enzymatic Degradation of Insoluble Polysaccharides (Saddler, J.N. & Penner, M., eds.), Cellulose-binding domains: classification and properties, pp. 142-163, American Chemical Society, Washington, 1995).
• a detailed review on the structure and binding modes of CBMs can be found in Boraston et al., Biochem J, 382:769-81, 2004.
• CBMs The family classification of CBMs is expected to aid in the identification of CBMs, predict binding specificity, aid in identifying functional residues, reveal evolutionary relationships, and possibly be predictive of polypeptide folds. Because the fold of proteins is better conserved than their sequences, some of the CBM families can be grouped into superfamilies or clans. The current CBM families are 1- 63. CBDs are found at the N-and C-termini of proteins or are internal.
• Enzyme hybrids are known in the art (See e.g., W090/00609 and WO95/16782) and may be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the cellulose-binding domain ligated, with or without a linker, to a DNA sequence encoding a mannanase variant described herein and growing the host cell to express the fused gene.
• Enzyme hybrids may be described by the following formula: CBM-MR-X or X-MR-CBM, wherein CBM is the N-terminal or the C-terminal region of an amino acid sequence corresponding to at least the carbohydrate-binding module; MR is the middle region (the linker), and may be a bond, or a short linking group of from about 2 to about 100 carbon atoms, from about 2 to about 40 carbon atoms, from about 2 to about 100 amino acids, or from about 2 to about 40 amino acids; and X is an N-terminal or C-terminal region of a mannanase variant described herein that has mannanase catalytic activity.
• a mannanase may contain more than one CBM or other module(s)/domain(s) of non-glycolytic function.
• module and “domain” are used interchangeably in the present disclosure.
• catalytic domains include: cellulases; hemicellulases, such as xylanase; exo-mannanases; glucanases; arabinases; galactosidases; pectinases; and/or other activities such as proteases, lipases, acid phosphatases and/or others or functional fragments thereof.
• Fusion proteins are optionally linked to a mannanase variant described herein through a linker sequence that simply joins the mannanase variant and the fusion domain without significantly affecting the properties of either component, or the linker optionally has a functional importance for the intended application.
• the enzymes are mannanse variants as provided herein in combination with one or more additional enzymes selected from the group consisting of acyl transferases, amylases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinases, arabinosidases, aryl esterases, beta-galactosidases, beta-glucanases, carrageenases, catalases, cellulases, chondroitinases, cutinases, dispersins, DNAses (also known as nucleases or dispersins), endo-glucanases, endo-beta-mannanases, exo-beta-mannanases, esterases, exo- mannanases, galactanases, glucoamylases, hemicellulases, hexosaminidase i hyaluronidases, ker
• a mannanase variant described herein is fused to a signal peptide for directing the extracellular secretion of the variant or polypeptide .
• the signal peptide is the native signal peptide of the mannanase variant described herein.
• the signal peptide is a non-native signal peptide such as the B. subtilis AprE signal peptide.
• a polypeptide of the present invention is expressed in a heterologous organism, i.e., an organism other than Paenibacillus spp.
• exemplary heterologous organisms are Gram(+) bacteria such as B. subtilis, B. Ucheniformis, B. lentus, B. brevis, Geobacillus (formerly Bacillus') stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium, B. thuringiensis, S. lividans, or S. murinus,' Gram(-) bacteria such as E.
• yeast such as Saccharomyces spp. or Schizosaccharomyces spp., e.g. S. cerevisiae and filamentous fungi such as Aspergillus spp., e.g., A. oryzae or A. niger, and T. reesei.
• filamentous fungi such as Aspergillus spp., e.g., A. oryzae or A. niger, and T. reesei.
• a mannanase variant described herein is expressed in a heterologous organism as a secreted polypeptide, in which case, the compositions and method encompass a method for expressing the variant as a secreted polypeptide in a heterologous organism.
• Yet another embodiment is directed to a polynucleotide that encodes a mannanase variant described herein.
• the polynucleotide is contained in an expression vector contained in a heterologous organism, such as those identified, herein.
• the polynucleotide may be operably-linked to regulatory elements (e.g., a promoter, terminator, enhancer, and the like) to assist in expressing the encoded variants or recombinant polypeptides described herein.
• Some embodiments are directed to a polynucleotide that encodes a mannanase variant having at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1-6.
• the polynucleotide is codon-optimized for expression in a different host, mutated to introduce cloning sites, or otherwise altered to add functionality.
• the polynucleotide that encodes a mannanase described herein is fused downstream of a coding sequence of a signal peptide that directs the extracellular secretion of variant .
• Expression vectors may be provided in a heterologous host cell suitable for expressing a variant described herein, or suitable for propagating the expression vector prior to introducing it into a suitable host cell.
• DNA that encodes a mannanase variant described herein can be chemically synthesized from published sequences or obtained directly from host cells harboring the gene (e.g., by cDNA library screening or PCR amplification).
• a polynucleotide is included in an expression cassette and/or cloned into a suitable expression vector by standard molecular cloning techniques.
• Such expression cassettes or vectors contain sequences that assist initiation and termination of transcription (e.g., promoters and terminators), and generally contain a selectable marker.
• the expression cassette or vector is introduced into a suitable expression host cell, which then expresses the corresponding mannanase variant described herein.
• suitable expression hosts are bacterial expression host genera including Escherichia (e.g., E. coli), Pseudomonas (e.g., P. fluorescens or P. stutzerei), Proteus (e.g., P. mirabilis), Ralstonia (e.g., R. eutropha), Streptomyces, Staphylococcus (e.g., S. carnosus), Lactococcus (e.g., L.
• Escherichia e.g., E. coli
• Pseudomonas e.g., P. fluorescens or P. stutzerei
• Proteus e.g., P. mirabilis
• Ralstonia e.g., R. eutropha
• Streptomyces
• yeast expression hosts such as S. cerevisiae, S. pombe, Y. lipolytica, H. polymorpha, K. lactis or P. pastoris.
• fungal expression hosts such as C. lucknow ense, Aspergillus (e.g., A. oryzae, A. niger, A. nidulans, etc.) or T. reesei.
• mammalian expression hosts such as mouse (e.g, NS0), Chinese Hamster Ovary (CHO) or Baby Hamster Kidney (BHK) cell lines.
• eukaryotic hosts such as insect cells or viral expression systems (e.g, bacteriophages such as M13, T7 phage or Lambda, or viruses such as Baculovirus) are also suitable for producing a mannanase variant described herein.
• Promoters and/or signal sequences associated with secreted proteins in a particular host of interest are candidates for use in the heterologous production and secretion of mannanases in that host or in other hosts.
• the promoters that drive the genes for cellobiohydrolase I (cbhl), glucoamylase A (glaA), TAKA- amylase (amyA), xylanase (exlA), the gpd-promoter cbhl, cbhll, endoglucanase genes EGI- EGV, Cel61B, Cel74A, egll-egl5, gpd promoter, Pgkl, pkil, EF-lalpha, tefl, cDNAl and hexl are particularly suitable and can be derived from a number of different organisms (e.g., A.
• the polynucleotide is recombinantly associated with a polynucleotide encoding a suitable homologous or heterologous signal sequence that leads to secretion of a mannanase variant described herein into the extracellular (or periplasmic) space, thereby allowing direct detection of enzyme activity in the cell supernatant (or periplasmic space or lysate).
• a suitable homologous or heterologous signal sequence that leads to secretion of a mannanase variant described herein into the extracellular (or periplasmic) space, thereby allowing direct detection of enzyme activity in the cell supernatant (or periplasmic space or lysate).
• Particularly suitable signal sequences for A are particularly suitable signal sequences for A.

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