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WO2003012110A1 - Mannanase thermo-tolerante provenant d'acidothermus cellulolyticus - Google Patents

Mannanase thermo-tolerante provenant d'acidothermus cellulolyticus Download PDF

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Publication number
WO2003012110A1
WO2003012110A1 PCT/US2001/023819 US0123819W WO03012110A1 WO 2003012110 A1 WO2003012110 A1 WO 2003012110A1 US 0123819 W US0123819 W US 0123819W WO 03012110 A1 WO03012110 A1 WO 03012110A1
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Prior art keywords
mannanase
mana
sequence
seq
acid sequence
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PCT/US2001/023819
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English (en)
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Shi-You Ding
William S. Adney
Todd B. Vinzant
Michael E. Himmel
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Midwest Research Institute
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Priority to PCT/US2001/023819 priority Critical patent/WO2003012110A1/fr
Publication of WO2003012110A1 publication Critical patent/WO2003012110A1/fr

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    • 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
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Definitions

  • the invention generally relates to a novel mannanase from Acidothermus cellulolyticus, ManA. More specifically, the invention relates to purified and isolated ManA polypeptides, nucleic acid molecules encoding the polypeptides, and processes for production and use of ManA, as well as variants and derivatives thereof. Background of the Invention
  • Plant biomass is the niost abundant source of carbohydrate in the world due to the lignocellulosic materials comprising the cell walls of all higher plants.
  • Plant cell walls are divided into primary and secondary cell walls.
  • the primary cell wall which provides structure for expanding cells (and hence changes as the cell grows), is composed of three major polysaccharides, cellulose, hemicellulose and pectin, and one group of glycoproteins.
  • the secondary cell wall which is produced after the cell has completed growing, also contains polysaccharides and is strengthened through polymeric lignin covalently cross-linked to hemicellulose.
  • Hemicellulose is a general term used to refer to cell wall polysaccharides that are not celluloses or pectins.
  • Hemicellulose sugar backbones include a variety of compounds, including xylans, xyloglucans, arabinoxylans and mannans.
  • One of the chief constituents of hemicellulose is the aldohexose glucose, mannose, which also may be in the form of a pyranose ring structure, ⁇ -D-mannose. With respect to mannose, the glycosidic linkage is on the 1 -carbon as a ⁇ -bond, having available linkage sites at the 2-, 3-, 4-, and 6-carbons.
  • a particularly rich source of mannans is the hemicellulose content of softwood, and in particular, the waste material from softwood processing in paper manufacturing.
  • One of the more important hemicelluloses in softwood is galactoglucomannan, which is composed of a backbone of ⁇ -(l,4)-linked D- mannopyranose and D-glucopyranose in a ratio of approximately 3:1, respectively (Sjostrom, E. (1992) Wood Chemistry, 2nd Ed., Academic Press: New York, NY, pp 63-70).
  • Other sources of mannans include the endosperm of capra and ivory palm nuts, guar beans, coffee beans, and roots of konjak (Amorphorphallus konjac).
  • mannanases have been identified in Bacillus (Emi et al., (1972) Agr. Biol. Chem. 36:991-1001), Aeromonas (Araki et al., (1983) Agr. Kyushu Univ. 27:89-98), Streptomyces (Takahashi et al., (1984) Biol. Chem. 48:2189-2195) and several fungal species (Hashimoto et al., (1969) J. Nippon Nogeikagaku Kaishi 43:317-322).
  • Mannanases are given an Enzyme Commission (EC) designation according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (Eur. j. Biochem. 264:607-609 and 610-650 (1999)).
  • ⁇ - mannosidase EC 3.2.1.25
  • ⁇ - mannanase EC 3.2.1.78
  • Reese, E.T., (1965) Can. J. Microbiol. 11:167 cleave the ⁇ -mannoside linkages in ⁇ -l,4-D-mannans to yield D-mannose and manno- oligosaccharides, respectively.
  • mannanase activities have been identified, for example, exomannanase (1,4- ⁇ -D-Mannan mannohydrolase) (EC 3.2.1.xx - unassigned) and exomannobiohydrolase (1,4- ⁇ -D-Mannan mannobiohydrolase) (EC 3.2.1.100) (McCleary, B.V., (1988) Methods Enzymol. 160:589-595; Araki et al., (1982) J. Bioche?n. 91:1181).
  • mannanases can be useful in the production of biofuels from plant biomass, where the mannanases participate in the hydrolysis of the hemicellulose fraction to simpler sugars, which are then converted to ethanol via fermentation.
  • thermophile Acidothermus cellulolyticus gen. nov. sp. nov., a bacterium originally isolated from decaying wood in an acidic, thermal pool at Yellowstone National Park (Mohagheghi et al., (1986) Int. J. Systematic Bacteriology 36(3):435-443).
  • One cellulase enzyme produced by this organism, the endoglucanase El is known to display maximal activity at 75°C to 83°C (Tucker et al., Bio/Technology, 7(8):817- 820).
  • El endoglucanase has been described in U.S. Patent 5,275,944. The A.
  • El endoglucanase is an active cellulase; in combination with exocellulase CBH I from Trichoderma reesei, El gives high levels of saccharification and contributes to a degree of synergism. Baker et al., (1994) Appl. Biochem. Biotechnol. 45/46:245-256.
  • the gene encoding El catalytic and cellulose binding domains and linker peptide were described in U.S. Patent 5,536,655.
  • thermal stable mannanases would likely have the desirable characteristic of maximal activity at elevated temperatures, as well as potentially having the thermal tolerant associated properties of resistance to acid inactivation, proteolytic inactivation, and solvent inactivation (Cowan DA in Danson MJ et al. (1992) The Archaebacteria, Biochemistry and Biotechnology at 149-159, University Press, Cambridge, ISBN 1855780100). El has also been expressed as a stable, active enzyme from a wide variety of hosts, including E. coli, Streptomyces lividans, Pichia pastoris, cotton, tobacco, and Arabidopsis (Dai Z, Hooker BS, Anderson DB, Thomas SR. Transgenic Res. 2000 Feb; 9(l):43-54).
  • ManA a novel member of the glycoside hydrolase (GH) family of enzymes, and in particular a thermal tolerant glycoside hydrolase useful in the degradation of mannans.
  • ManA polypeptides of the invention include those having an amino acid sequence shown in SEQ ID NO:l, as well as polypeptides having substantial amino acid sequence identity to the amino acid sequence of SEQ ID NO:l and useful fragments thereof, including, a catalytic domain having significant sequence similarity to the GH5 family, a first carbohydrate binding domain (type H) and a second carbohydrate binding domain (type rn).
  • the invention also provides a polynucleotide molecule encoding ManA polypeptides and fragments of ManA polypeptides, for example catalytic and carbohydrate binding domains.
  • Polynucleotide molecules of the invention include those molecules having a nucleic acid sequence as shown in SEQ ID NO:2; those that hybridize to the nucleic acid sequence of SEQ ID NO:2 under high stringency conditions; and those having substantial nucleic acid identity with the nucleic acid sequence of SEQ ID NO:2.
  • the invention includes variants and derivatives of the ManA polypeptides, including fusion proteins.
  • fusion proteins of the invention include ManA polypeptide fused to a heterologous protein or peptide that confers a desired function.
  • the heterologous protein or peptide can facilitate purification, oligomerization, stabilization, or secretion of the ManA polypeptide, for example.
  • the heterologous polypeptide can provide enhanced activity, including catalytic or binding activity, for ManA polypeptides, where the enhancement is either additive or synergistic.
  • a fusion protein of an embodiment of the invention can be produced, for example, from an expression construct containing a polynucleotide molecule encoding ManA polypeptide in frame with a polynucleotide molecule for the heterologous protein.
  • Embodiments of the invention also comprise vectors, plasmids, expression systems, host cells, and the like, containing a ManA polynucleotide molecule.
  • Genetic engineering methods for the production of ManA polypeptides of embodiments of the invention include expression of a polynucleotide molecule in cell free expression systems and in cellular hosts, according to known methods.
  • the invention further includes compositions containing a substantially purified ManA polypeptide of the invention and a carrier. Such compositions are administered to a biomass containing mannanase for the reduction or degradation of the mannanase or to produce useful oligosaccharides from hemicellulose.
  • the invention also provides reagents, compositions, and methods that are useful for analysis of ManA activity.
  • Tables 4 and 5 includes sequences used in describing embodiments of the present invention.
  • the abbreviations are as follows: CD, catalytic domain; CBD_ ⁇ , carbohydrate binding domain type II; CBD IH, carbohydrate binding domain type HI; and FN-IH, fibronectin domain type m.
  • N* indicates a string of unknown nucleic acid units
  • X* indicates a string of unknown amino acid units, for example about 50 or more.
  • Table 4 includes approximate start and stop information for segments
  • Table 5 includes amino acid sequence data for segments. Table 4. Nucleotide and polypeptide segments.
  • FIG. 1 is a schematic representation of the gene sequence and amino acid segment organization.
  • FIG 2 is a graphic representation of the glycoside hydrolase gene/protein families found in various organisms.
  • amino acid refers to any of the twenty naturally occuring amino acids as well as any modified amino acid sequences. Modifications may include natural processes such as posttranslational processing, or may include chemical modifications which are known in the art. Modifications include but are not limited to: phosphorylation, ubiquitination, acetylation, amidation, glycosylatioin, covalent attachment of flavin, ADP-ribosylation, cross linking, iodination, methylation, and alike.
  • Antibody refers to a Y-shaped molecule having a pair of antigen binding sites, a hinge region and a constant region. Fragments of antibodies, for example an antigen binding fragment (Fab), chimeric antibodies, antibodies having a human constant region coupled to a murine antigen binding region, and fragments thereof, as well as other well known recombinant antibodies are included in the present invention.
  • Antisense refers to polynucleotide sequences that are complementary to target "sense" polynucleotide sequence.
  • Binding activity refers to any activity that can be assayed by characterizing the ability of a polypeptide to bind to substrate.
  • the substrate can be a carbohydrate polymer such as hemicellulose, including mannan, or can be a complex molecule or aggregate of molecules where the entire moiety comprises at least some carbohydrate.
  • Complementary refers to the ability of a polynucleotide in a polynucleotide molecule to form a base pair with another polynucleotide in a second polynucleotide molecule.
  • sequence A- G-T is complementary to the sequence T-C-A.
  • Complementarity may be partial, in which only some of the polynucleotides match according to base pairing, or complete, where all the polynucleotides match according to base pairing.
  • “Expression” refers to transcription and translation occurring within a host cell.
  • the level of expression of a DNA molecule in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of DNA molecule encoded protein produced by the host cell (Sambrook et al., 1989, Molecular cloning: A Laboratory Manual, 18.1-18.88).
  • Fusion protein refers to a first protein having attached a second, heterologous protein.
  • the heterologous protein is fused via recombinant DNA techniques, such that the first and second proteins are expressed in frame.
  • the heterologous protein can confer a desired characteristic to the fusion protein, for example, a detection signal, enhanced stability or stabilization of the protein, facilitated oligomerization of the protein, or facilitated purification of the fusion protein.
  • heterologous proteins useful in the fusion proteins of the invention include molecules having the catalytic domain of ManA, one or more binding domains of ManA, one or more catalytic domains of a glycoside hydrolase other than ManA, one or more binding domains of a glycoside hydrolase other than ManA, or any combination thereof.
  • Fusion proteins are also meant to encompass variants and derivatives of ManA polypeptides that are generated by conventional site-directed mutagenesis and more modern techniques such as directed evolution, discussed infra.
  • Genetically engineered refers to any recombinant DNA or RNA method used to create a prokaryotic or eukaryotic host cell that expresses a protein at elevated levels, at lowered levels, or in a mutated form.
  • the host cell has been transfected, transformed, or transduced with a recombinant polynucleotide molecule, and thereby been altered so as to cause the cell to alter expression of the desired protein.
  • Methods and vectors for genetically engineering host cells are well known in the art; for example various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates). Genetically engineering techniques include but are not limited to expression vectors, targeted homologous recombination and gene activation (see, for example, U.S. Patent No. 5,272,071 to Chappel) and trans activation by engineered transcription factors (see, for example, Segal et al., 1999, Proc Nail Acad Sci USA 96(6):2758-63).
  • glycoside hydrolase family refers to a family of enzymes, which hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety (Henrissat B., (1991) Biochem. J., 280:309-316). Identification of a putative glycoside hydrolase family member is made based on an amino acid sequence comparison and the finding of significant sequence similarity within the putative member's catalytic domain, as compared to the catalytic domains of known family members.
  • Homology refers to a degree of complementarity between polynucleotides, having significant effect on the efficiency and strength of hybridization between polynucleotide molecules. The term also can refer to a degree of similarity between polypeptides.
  • "Host cell” or “host cells” refers to cells expressing a heterologous polynucleotide molecule. Host cells of the present invention express polynucleotides encoding ManA or a fragment thereof. Examples of suitable host cells useful in the present invention include, but are not limited to, prokaryotic and eukaryotic cells.
  • Such cells include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus; fiingi, particularly filamentous fungi such as Trichoderma and Aspergillus, Phanerochaete chrysosporium, and other white rot fungi; also other fungi including Fusaria, molds and yeast including Saccharomyces sp., and Candida sp. and the like; plants e.g.
  • SF9 insect cells (Summers and Smith, 1987, Texas Agriculture Experiment Station Bulletin, 1555), and the like.
  • mammalian cells such as human embyonic kidney cells (293 cells), Chinese hamster ovary (CHO) cells (Puck et al., 1958, Proc. Natl. Acad. Sci.
  • Hybridization refers to the pairing of complementary polynucleotides during an annealing period.
  • Identity refers to a comparison between pairs of nucleic acid or amino acid molecules. Methods for determining sequence identity are known. See, for example, computer programs commonly employed for this purpose, such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wisconsin), that uses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math., 2: 482-489.
  • isolated refers to a polynucleotide or polypeptide that has been separated from at least one contaminant (polynucleotide or polypeptide) with which it is normally associated.
  • an isolated polynucleotide or polypeptide is in a context or in a form that is different from that in which it is found in nature.
  • Mannanase activity refers to any activity that can be assayed by characterizing the enzymatic activity of a mannanase. For example, mannanase activity can be assayed by determining how much reducing sugar is produced during a fixed amount of time for a set amount of enzyme acting on mannan (see Irwin et al., (1998) J.
  • Nucleic acid sequence refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide chain. The deoxyribonucleotide sequence thus codes for the amino acid sequence.
  • Polynucleotide refers to a linear sequence of nucleotides.
  • the nucleotides maybe ribonucleotides, or deoxyribonucleotides, or a mixture of both.
  • Examples of polynucleotides in the context of the present invention include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA.
  • the polynucleotides of the present invention may contain one or more modified nucleotides.
  • Protein “Protein,” “peptide,” and “polypeptide” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.
  • Purify refers to a target protein that is free from at least 5- 10% of contaminating proteins. Purification of a protein from contaminating proteins can be accomplished using known techniques, including ammonium sulfate or ethanol precipitation, acid precipitation, heat precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, size- exclusion chromatography, and lectin chromatography. Various protein purification techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates).
  • Selectable marker refers to a marker that identifies a cell as having undergone a recombinant DNA or RNA event.
  • Selectable markers include, for example, genes that encode antimetabolite resistance such as the DHFR protein that confers resistance to methotrexate (Wigler et al, 1980, Proc Nail Acad Sci USA 77:3567; O'Hare et al., 1981, Proc Natl Acad Sci USA, 78:1527), the GPT protein that confers resistance to mycophenolic acid (Mulligan & Berg, 1981, PNAS USA, 78:2072), the neomycin resistance marker that confers resistance to the aminoglycoside G-418 (Calberre-Garapin et al., 1981, J Mol Biol, 150:1), the Hygro protein that confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147), and the ZeocinTM resistance marker (Invitrogen).
  • herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes can be employed in tk " , hgprf and aprf cells, respectively.
  • Stringency refers to the conditions (temperature, ionic strength, solvents, etc) under which hybridization between polynucleotides occurs.
  • a hybridzation reaction conducted under high stringency conditions is one that will only occur between polynucleotide molecules that have a high degree of complementary base pairing (85% to 100% identity).
  • Conditions for high stringency hybridization may include an overnight incubation at about 42°C for about 2.5 hours in 6 X SSC/0.1% SDS, followed by washing of the filters in 1.0 X SSC at 65°C, 0.1% SDS.
  • a hybridization reaction conducted under moderate stringency conditions is one that will occur between polynucleotide molecules that have an intermediate degree of complementary base pairing (50% to 84% identity).
  • “Substrate targeting moiety” refers to any signal on a substrate, either naturally occurring or genetically engineered, used to target any ManA polypeptide or fragment thereof to a substrate.
  • Such targeting moieties include ligands that bind to a substrate structure. Examples of ligand/receptor pairs include carbohydrate binding domains and mannans. Many such substrate-specific ligands are known and are useful in the present invention to target a ManA polypeptide or fragment thereof to a substrate.
  • a novel example is a ManA carbohydrate binding domain that is used to tether other molecules to a Mannan-containing substrate such as a fabric.
  • Thermal tolerant refers to the property of withstanding partial or complete inactivation by heat and can also be described as thermal resistance or thermal stability. Although some variation exists in the literature, the following definitions can be considered typical for the optimum temperature range of stability and activity for enzymes: psycrophilic (below freezing to IOC); mesophilic (10°C to 50°C); thermophilic (50°C to 75°C); and caldophilic (75°C to above boiling water temperature).
  • psycrophilic below freezing to IOC
  • mesophilic (10°C to 50°C
  • thermophilic 50°C to 75°C
  • caldophilic 75°C to above boiling water temperature
  • thermal tolerance refers to the ability to function in a temperature range of from about 15°C to about 100°C.
  • a preferred range is from about 30°C to about 80°C.
  • a highly preferred range is from about 50°C to about 70°C.
  • a protein that can function at about 45°C is considered in the preferred range even though it may be susceptible to partial or complete inactivation at temperatures in a range above about 45°C and less than about 80°C.
  • the desirable property of thermal tolerance among is often accompanied by other desirable characteristics such as: resistance to extreme pH degradation, resistance to solvent degradation, resistance to proteolytic degradation, resistance to detergent degradation, resistance to oxidizing agent degradation, resistance to chaotropic agent degradation, and resistance to general degradation.
  • 'resistance' is intended to include any partial or complete level of residual activity.
  • Variant means a polynucleotide or polypeptide molecule that differs from a reference molecule. Variants can include nucleotide changes that result in amino acid substitutions, deletions, fusions, or truncations in the resulting variant polypeptide when compared to the reference polypeptide.
  • Vector refers to a first polynucleotide molecule, usually double-stranded, which may have inserted into it a second polynucleotide molecule, for example a foreign or heterologous polynucleotide.
  • the heterologous polynucleotide molecule may or may not be naturally found in the host cell, and may be, for example, one or more additional copy of the heterologous polynucleotide naturally present in the host genome.
  • the vector is adapted for transporting the foreign polynucleotide molecule into a suitable host cell. Once in the host cell, the vector may be capable of integrating into the host cell chromosomes.
  • the vector may optionally contain additional elements for selecting cells containing the integrated polynucleotide molecule as well as elements to promote transcription of mRNA from transfected DNA.
  • additional elements for selecting cells containing the integrated polynucleotide molecule as well as elements to promote transcription of mRNA from transfected DNA.
  • vectors useful in the methods of the present invention include, but are not limited to, plasmids, bacteriophages, cosmids, retroviruses, and artificial chromosomes.
  • Glycoside hydrolases are a large and diverse family of enzymes that hydrolyze the glycosidic bond between two carbohydrate moieties or between a carbohydrate and non-carbohydrate moiety (See FIG. 2).
  • Glycoside hydrolase enzymes are classified into glycoside hydrolase (GH) families based on significant amino acid similarities within their catalytic domains, as compared to the catalytic domain of known family members. Enzymes having related catalytic domains are grouped together within a family (Henrissat et al., (1991) supra; Henrissat et al., (1996), Biochem. J. 316:695-696), where the underlying classification provides a direct relationship between the GH domain amino acid sequence and how the GH domain will fold. This information ultimately provides a common mechanism for how the enzyme will hydrolyze the glycosidic bond within a substrate, i.e., either by a retaining mechanism or inverting mechanism (Henrissat B., (1991) supra).
  • Mannanases belong to the GH family of enzymes. Mannanases are produced by a variety of bacteria and fungi to degrade the ⁇ -1,4 linkages in mannans, glucomannans, galactomannans, and galactoglucomannans and to so produce oligosaccharides. At present, mannanases are found within either the GH5 or GH26 family of glycoside hydrolases (Hilge et al., (1998) Structure 6(11): 1433-1444).
  • Mannanases are characterized by having a multiple domain unit within their overall structure, a GH or catalytic domain is joined to a carbohydrate binding type domain (CBD) by a glycosylated linker peptide (see FIG. 1) (Koivula et al., (1996) Protein Expression and Purification 8:391-400).
  • CBD carbohydrate binding type domain
  • the catalytic domain hydrolysis the mannan, the CBD type domain increases the concentration of the enzyme on the substrate, in this case hemicellulose, and the linker peptide provides flexibility for both larger domains.
  • mannanases Conversion of hemicellulose to oligosaccharides by mannanases elevates the value of the complex substrate, such as in foods, feeds and paper pulp.
  • mannan polymers from the hemicellulose fraction of softwood are known to contaminate the cellulose fibers in paper pulp. Addition of a mannanases to the paper pulp can reduce the amounts of these contaminants and provide a higher quality product.
  • mannanase released oligosaccharides from hemicellulose may be used as bulking agents or stabilizers, for example mannan-oligomers released from palm seed mannan, in food or feeds.
  • mannanases can be useful in the production of biofuels from plant biomass, where the mannanases participate in the hydrolysis of the hemicellulose fraction to oligosaccharides, which can be further reduced to sugars and converted to ethanol via fermentation.
  • ManA can be useful in the production of biofuels from plant biomass, where the mannanases participate in the hydrolysis of the hemicellulose fraction to oligosaccharides, which can be further reduced to sugars and converted to ethanol via fermentation.
  • ManA a novel thermostable mannanase
  • SEQ ID NO:l The predicted amino acid sequence of ManA (SEQ ID NO:l) has an organization characteristic of a mannanase enzyme.
  • ManA contains a GH5 catalytic domain (about amino acid 37 to 41 l)-linker domain-carbohydrate binding type domain (about amino acid 455 to 608) organization, as well as a second carbohydrate binding type domain (about amino acid 662 to 762).
  • significant amino acid similarity of ManA to other mannanases identifies ManA as a mannanase.
  • ManA has a catalytic domain belonging to the GH5 family of glycoside hydrolases.
  • the GH5 domain family includes a number of ⁇ - mannanases, for example, ⁇ -mannanase isolated from Agaricus bisporus, and ⁇ - mannanase isolated from Trichoderma reesei (Hypocreajecorina).
  • the GH5 members degrade substrate using a retaining mechanism. Being a mannanase member of the GH5 family of glycoside hydrolases identifies ManA as having ⁇ -mannanase (EC 3.2.1.78) activity.
  • the predicted amino acid sequence indicates that CBD type II and CBD type m domains are present as characterized by Tomme P. et al. (1995), in Enzymatic Degradation of Insoluble Polysaccharides (Saddler JN & Penner M, eds.), at 142-163, American Chemical Society, Washington. See also Tomme, P. & Claeyssens, M. (1989) FEBS Lett. 243, 239-2431; Gilkes, N.R et al., (1988) J.Biol.Chem. 263, 10401-10407.
  • ManA is also a thermostable mannanase as it is produced by the thermophile Acidothermus cellulolyticus. ManA can have other thermostable associated characteristics - for example, resistance to acidity, resistance to protein degredation, and the like (Cowan D., (1992) supra). Like other members of the mannanase family, and in particular thermostable mannanases, ManA is useful in converting hemicellulose to oligosaccharides and thereby elevating the value of the complex substrate, for example in foods, feeds and paper pulp. Oligosaccharides produced by ManA may also be used as a bulking agent or stabilizer in foods and feeds.
  • ManA is useful in the conversion of biofuels from biomass, and in particular, biofuels from hemicellulose. It is envisioned that ManA could be used alone or in combination with one or more other mannanase or other relevant glycoside hydrolase to perform the uses described herein or known within the relevant art, all of which are within the scope of the present disclosure.
  • ManA Polypeptides :
  • ManA polypeptides of the invention include isolated polypeptides having an amino acid sequence as shown below in Example 1; Table 1 and in SEQ ID NO:l, as well as variants and derivatives, including fragments, having substantial identity to the amino acid sequence of SEQ ID NO:l and that retain any of the functional activities of ManA.
  • ManA polypeptide activity can be determined, for example, by subjecting the variant, derivative, or fragment to a substrate binding assay or a mannanase activity assay such as those described for cellulases in Irwin D et al., J. Bacteriology 180(7): 1709-1714 (April 1998) or U.S. Patent 6,060,299.
  • the isolated ManA polypeptide includes an N-terminal hydrophobic region that functions as a signal peptide, having an amino acid sequence that begins with Ml and extends to approximately A36; a catalytic domain having significant sequence similarity to a GH5 family domain that begins at about A37 and extends to about G411, a carbohydrate binding domain type HI region that begins at about V455 and extends to about T608, and a carbohydrate binding domain type II that begins at about G662 and extends to about S762.
  • ManA polypeptides modified by covalent or aggregative conjugation with other chemical moieties, such as glycosyl groups, polyethylene glycol (PEG) groups, lipids, phosphate, acetyl groups, and the like.
  • PEG polyethylene glycol
  • the amino acid sequence of ManA polypeptides of the invention is preferably at least about 60% identical, more preferably at least about 70% identical, or in some embodiments at least about 90% identical, to the ManA amino acid sequence shown above in Table 1 and SEQ ID NO:l.
  • the percentage identity also termed homology (see definition above) can be readily determined, for example, by comparing the two polypeptide sequences using any of the computer programs commonly employed for this purpose, such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wisconsin), which uses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2: 482-489.
  • Variants and derivatives of the ManA polypeptide may further include, for example, fusion proteins formed of a ManA polypeptide and a heterologous polypeptide.
  • Preferred heterologous polypeptides include those that facilitate purification, oligomerization, stability, or secretion of the ManA polypeptides.
  • Fragments of the ManA polypeptide may include, but are not limited to, the GH5 catalytic domain (SEQ ID NO:3), the carbohydrate binding domain type in (SEQ ID NO:4) and the carbohydrate binding domain type ⁇ (SEQ ID NO:5).
  • ManA polypeptide variants and derivatives can contain conservatively substituted amino acids, meaning that one or more amino acid can be replaced by an amino acid that does not alter the secondary and/or tertiary structure -of the polypeptide.
  • substitutions can include the replacement of an amino acid, by a residue having similar physicochemical properties, such as substituting one aliphatic residue (Tie, Val, Leu, or Ala) for another, or substitutions between basic residues Lys and Arg, acidic residues Glu and Asp, amide residues Gin and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr.
  • the ManA polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • the polypeptides may be recovered and purified from recombinant cell cultures by known methods, including, for example, ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.
  • HPLC high performance liquid chromatography
  • ManA polypeptides are that of recombinant polypeptides as expressed by suitable hosts.
  • the hosts can simultaneously produce other mannanases or glycoside hydrolases such that a mixture is produced comprising a ManA polypeptide and one or more other glycoside hydrolases.
  • Such a mixture can be effective in crude fermentation processing or other industrial processing (see below).
  • ManA polypeptides can be fused to heterologous polypeptides to facilitate purification.
  • Many available heterologous peptides allow selective binding of the fusion protein to a binding partner.
  • Non-limiting examples of peptide tags include 6-His, thioredoxin, hemaglutinin, GST, and the OmpA signal sequence tag.
  • a binding partner that recognizes and binds to the heterologous peptide can be any molecule or compound, including metal ions (for example, metal affinity columns), antibodies, antibody fragments, or any protein or peptide that preferentially binds the heterologous peptide to permit purification of the fusion protein.
  • ManA polypeptides can be modified to facilitate formation of ManA oligomers.
  • ManA polypeptides can be fused to peptide moieties that promote oligomerization, such as leucine zippers and certain antibody fragment polypeptides, for example, Fc polypeptides. Techniques for preparing these fusion proteins are known, and are described, for example, in WO 99/31241 and in Cosman etal., 2001 Immunity 14:123-133. Fusion to an Fc polypeptide offers the additional advantage of facilitating purification by affinity chromatography over Protein A or Protein G columns.
  • LZ leucine-zipper
  • an expanded set of variants and derivatives of ManA polynucleotides and/or polypeptides can be generated to select for useful molecules, where such expansion is achieved not only by conventional methods such as site- directed mutagenesis (SDM) but also by more modern techniques, either independently or in combination.
  • SDM site- directed mutagenesis
  • Site-directed-mutagenesis is considered an informational approach to protein engineering and can rely on high-resolution crystallographic structures of target proteins and some stratagem for specific amino acid changes (Van Den Burg, B.; Vriend, G.; Veltman, O.R.; Venema, G.; Eijsink, V.G.H. Proc. Nat. Acad. Sci. U.S. 1998, 95, 2056-2060).
  • modification of the amino acid sequence of ManA polypeptides can be accomplished as is known in the art, such as by introducing mutations at particular locations by oligonucleotide-directed mutagenesis (Walder et al.,1986, Gene, 42:133; Bauer et al., 1985, Gene 37:73; Craik, 1985, BioTechniques, 12-19; Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press; and U.S. Patent No. 4,518,584 and U.S. Patent No. 4,737,462).
  • SDM technology can also employ the recent advent of computational methods for identifying site-specific changes for a variety of protein engineering objectives (Hellinga, H.W. Nature Structural. Biol. 1998, 5, 525-527).
  • Directed evolution in conjunction with high-throughput screening, allows testing of statistically meaningful variations in protein conformation (Arnold, F.H. Nature Biotechnol. 1998, 16, 617-618).
  • Directed evolution technology can include diversification methods similar to that described by Crameri A. et al. (1998, Nature 391: 288-291), site- saturation mutagenesis, staggered extension process (StEP) (Zhao, H.; Giver, L.; Shao, Z.; Affholter, J.A.; Arnold, F.H. Nature Biotechnol. 1998, 16, 258-262), and DNA synthesis/reassembly (U.S. Patent 5,965,408).
  • ManA polypeptide can be used, for example, to generate specific anti-ManA antibodies. Using known selection techniques, specific epitopes can be selected and used to generate monoclonal or polyclonal antibodies. Such antibodies have utlilty in the assay of ManA activity as well as in purifying recombinant ManA polypeptides from genetically engineered host cells.
  • ManA Polynucleotides :
  • ManA polynucleotide molecules of the invention include polynucleotide molecules having the nucleic acid sequence shown in Table 2 and SEQ ID NO:2, polynucleotide molecules that hybridize to the nucleic acid sequence of Table 2 and SEQ ID NO:2 under high stringency hybridization conditions (for example, 42°, 2.5 hr., 6X SCC, 0.1%SDS); and polynucleotide molecules having substantial nucleic acid sequence identity with the nucleic acid sequence of Table 2 and SEQ ID NO: 2, particularly with those nucleic acids encoding the catalytic domain, GH5 (from amino acid 37 to 411), the carbohydrate binding domain HI (from amino acid 455 to 608) and carbohydrate binding domain II (from amino acid 662 to 762). Table 2. ManA nucleotide sequence.
  • the ManA polynucleotide molecules of the invention are preferably isolated molecules encoding the ManA polypeptide having an amino acid sequence as shown in Table 1 and SEQ ID NO:l, as well as derivatives, variants, and useful fragments of the ManA polynucleotide.
  • the ManA polynucleotide sequence can include deletions, substitutions, or additions to the nucleic acid sequence of Table 2 and SEQ ID NO:2.
  • the ManA polynucleotide molecule of the invention can be cDNA, chemically synthesized DNA, DNA amplified by PCR, RNA, or combinations thereof. Due to the degeneracy of the genetic code, two DNA sequences may differ and yet encode identical amino acid sequences.
  • the present invention thus provides an isolated polynucleotide molecule having a ManA nucleic acid sequence encoding ManA polypeptide, where the nucleic acid sequence encodes a polypeptide having the complete amino acid sequences as shown in Table 1 and SEQ ID NO:l, or variants, derivatives, and fragments thereof.
  • the ManA polynucleotides of the invention have a nucleic acid sequence that is at least about 60% identical to the nucleic acid sequence shown in Table 2 and SEQ ID NO:2, in some embodiments at least about 70% identical to the nucleic acid sequence shown in Table 2 and SEQ ID NO:2, and in other embodiments at least about 90%) identical to the nucleic acid sequence shown in Table 2 and SEQ ID NO:2.
  • Nucleic acid sequence identity is determined by known methods, for example by aligning two sequences in a software program such as the BLAST program (Altschul, S.F et al. (1990) J. Mol. Biol. 215:403-410, from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/).
  • ManA polynucleotide molecules of the invention also include isolated polynucleotide molecules having a nucleic acid sequence that hybridizes under high stringency conditions (as defined above) to a the nucleic acid sequence shown in Table 2 and SEQ ID NO:2.
  • Hybridization of the polynucleotide is to at least about 15 contiguous nucleotides, or at least about 20 contiguous nucleotides, and in other embodiments at least about 30 contiguous nucleotides, and in still other embodiments at least about 100 contiguous nucleotides of the nucleic acid sequence shown in Table 2 and SEQ IDNO:2.
  • Useful fragments of the ManA-encoding polynucleotide molecules described herein include probes and primers.
  • probes and primers can be used, for example, in PCR methods to amplify and detect the presence of ManA polynucleotides in vitro, as well as in Southern and Northern blots for analysis of ManA.
  • Cells expressing the ManA polynucleotide molecules of the invention can also be identified by the use of such probes. Methods for the production and use of such primers and probes are known.
  • 5' and 3' primers corresponding to a region at the termini of the ManA polynucleotide molecule can be employed to isolate and amplify the ManA polynucleotide using conventional techniques.
  • ManA polynucleotides include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence capable of binding to a target ManA mRNA (using a sense strand), or DNA (using an antisense strand) sequence.
  • Antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence capable of binding to a target ManA mRNA (using a sense strand), or DNA (using an antisense strand) sequence.
  • the present invention also provides vectors containing the polynucleotide molecules of the invention, as well as host cells transformed with such vectors.
  • Any of the polynucleotide molecules of the invention may be contained in a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host.
  • the vectors further include suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect genes, operably linked to the ManA polynucleotide molecule. Examples of such regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation.
  • Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the target protein.
  • a promoter nucleotide sequence is operably linked to a ManA DNA sequence if the promoter nucleotide sequence directs the transcription of the ManA sequence.
  • Suitable vectors for the cloning of ManA polynucleotide molecules encoding the target ManA polypeptides of this invention will depend upon the host cell in which the vector will be transformed, and, where applicable, the host cell from which the target polypeptide is to be expressed.
  • Suitable host cells for expression of ManA polypeptides include prokaryotes, yeast, and higher eukaryotic cells, each of which is discussed below.
  • the ManA polypeptides to be expressed in such host cells may also be fusion proteins that include regions from heterologous proteins. As discussed above, such regions may be included to allow, for example, secretion, improved stability, or facilitated purification of the ManA polypeptide.
  • a nucleic acid sequence encoding an appropriate signal peptide can be incorporated into an expression vector.
  • a nucleic acid sequence encoding a signal peptide (secretory leader) may be fused in-frame to the ManA sequence so that ManA is translated as a fusion protein comprising the signal peptide.
  • a signal peptide that is functional in the intended host cell promotes extracellular secretion of the ManA polypeptide.
  • the signal sequence will be cleaved from the ManA polypeptide upon secretion of ManA from the cell.
  • signal sequences that can be used in practicing the invention include the yeast I-factor and the honeybee melatin leader in Sf9 insect cells.
  • Suitable host cells for expression of target polypeptides of the invention include prokaryotes, yeast, and higher eukaryotic cells.
  • Suitable prokaryotic hosts to be used for the expression of these polypeptides include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus. For expression in prokaryotic cells, for example, in E.
  • the polynucleotide molecule encoding ManA polypeptide preferably includes an N-terminal methionine residue to facilitate expression of the recombinant polypeptide.
  • the N-terminal Met may optionally be cleaved from the expressed polypeptide.
  • Expression vectors for use in prokaryotic hosts generally comprise one or more phenotypic selectable marker genes. Such genes encode, for example, a protein that confers antibiotic resistance or that supplies an auxotrophic requirement.
  • phenotypic selectable marker genes encode, for example, a protein that confers antibiotic resistance or that supplies an auxotrophic requirement.
  • a wide variety of such vectors are readily available from commercial sources. Examples include pSPORT vectors, pGEM vectors (Promega, Madison, WI), pPROEX vectors (LTI, Bethesda, MD), Bluescript vectors (Stratagene), and pQE vectors (Qiagen).
  • ManA can also be expressed in yeast host cells from genera including Saccharomyces, Pichia, and Kluveromyces.
  • Preferred yeast hosts are S. cerevisiae and P. pastoris.
  • Yeast vectors will often contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • Vectors replicable in both yeast and E. coli may also be used.
  • a shuttle vector will also include sequences for replication and selection in E. coli. Direct secretion of the target polypeptides expressed in yeast hosts may be accomplished by the inclusion of nucleotide sequence encoding the yeast I-factor leader sequence at the 5' end of the ManA-encoding nucleotide sequence.
  • Insect host cell culture systems can also be used for the expression of ManA polypeptides.
  • the target polypeptides of the invention are preferably expressed using a baculovirus expression system, as described, for example, in the review by Luckow and Summers, 1988 Bio/Technology 6:47.
  • the choice of a suitable expression vector for expression of ManA polypeptides of the invention will depend upon the host cell to be used. Examples of suitable expression vectors for E. coli include pET, pUC, and similar vectors as is known in the art.
  • Preferred vectors for expression of the ManA polypeptides include the shuttle plasmid pU702 for Streptomyces lividans, pGAPalpha-A, B, C and pPICZalpha-A, B, C (Invitorgen) for Pichia pastoris, and pFE-1 and pFE-2 for filamentous fungi and similar vectors as is known in the art.
  • ManA polynucleotide molecule Modification of a ManA polynucleotide molecule to facilitate insertion into a particular vector (for example, by modif ⁇ ying restriction sites), ease of use in a particular expression system or host (for example, using preferred host codons), and the like, are known and are contemplated for use in the invention.
  • Genetic engineering methods for the production of ManA polypeptides include the expression of the polynucleotide molecules in cell free expression systems, in cellular hosts, in tissues, and in animal models, according to known methods.
  • compositions containing a substantially purified ManA polypeptide of the invention and an acceptable carrier are administered to biomass, for example, to degrade the hemicellulose in the biomass into simpler carbohydrate units and ultimately, to sugars. These released sugars from the hemicellulose are converted into ethanol by any number of different catalysts.
  • Such compositions may also be included in detergents for removal, for example, of hemicellulose containing stains within fabrics, or compositions used in the pulp and paper industry to address conditions associated with hemicellulose contamination of the cellulose fraction.
  • Compositions of the present invention can be used in degrading the hemicellulose fraction in the food and feed industry to result in a lower content of hemicellulose in food or feed.
  • compositions of the present invention can also be used to produce oligosaccharide bulking agents and stabilizers from hemicellulose for use in the food and feed industry.
  • Compositions of the present invention that include either the carbohydrate binding domain type in or ⁇ polypeptides can be used as linking agents, for example, to link a target molecule fused to the CBD H or HI to a carbohydrate containing target, including pharmaceutical compositions where the CBD II or UJ is used to target drugs to target carbohydrate expressing cells (see below).
  • the invention provides pharmaceutical compositions containing a substantially purified ManA polypeptide of the invention and if necessary a pharmaceutically acceptable carrier. Such pharmaceutical compositions are administered to cells, tissues, or patients, for example, to aid in delivery or targeting of other pharmaceutical compositions.
  • ManA polypeptides, and in particular the carbohydrate binding domains of ManA may be used where carbohydrate-mediated liposomal interactions are involved with target cells. Vyas SP et al. (2001), J. Pharmacy & Pharmaceutical Sciences May-Aug 4(2): 138-58.
  • compositions of the present invention may also include other known glycoside hydrolases, and preferably, other known thermal tolerant glycoside hydrolases for enhanced treatment of hemicellulose.
  • Antibodies are also included in compositions of the present invention.
  • polypeptides of the present invention may be used to raise polyclonal and monoclonal antibodies that are useful in purifying ManA, or detecting ManA polypeptide expression, as well as a reagent tool for characterizing the molecular actions of the ManA polypeptide.
  • a peptide containing a unique epitope of the ManA polypeptide is used in preparation of antibodies, using conventional techniques. Methods for the selection of peptide epitopes and production of antibodies are known.
  • mannanase activity and binding assays may be performed in a manner similar to those described for cellulases in Irwin et al., J. Bacteriology 180(7): 1709-1714 (April 1998).
  • ManA stimulated activity is determined in the presence and absence of a test agent and then compared.
  • a higher ManA activated test activity in the presence of the test agent than in the absence of the test agent indicates that the test agent has increased the activity of the ManA.
  • Stimulators and inhibitors of ManA may be used to augment, inhibit, or modify ManA mediated activity, and therefore may have potential industrial uses as well as potential use in the further elucidation of ManA's molecular actions.
  • ManA polypeptides of the invention are effective in adding in delivery or targeting of other pharmaceutical compositions within a host.
  • ManA polypeptides and in particular the ManA carbohydrate binding domains, may be used where carbohydrate-mediated liposomal interactions are involved with target cells.
  • ManA polynucleotides and polypeptides, including vectors expressing ManA, of the invention can be formulated as pharmaceutical compositions and administered to a host, preferably mammalian host, including a human patient, in a variety of forms adapted to the chosen route of administration.
  • the compounds are preferably administered in combination with a pharmaceutically acceptable carrier, and may be combined with or conjugated to specific delivery agents, including targeting antibodies and/or cytokines.
  • ManA can be administered by known techniques, such as orally, parentally
  • compositions of the invention can be in the form of suspensions or tablets suitable for oral administration, nasal sprays, creams, sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions or suppositories.
  • compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation.
  • the compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents.
  • the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
  • compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation.
  • the compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons or other solubilizing or dispersing agents known in the art.
  • suitable dispersing or wetting and suspending agents such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • compositions can be prepared by mixing with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.
  • a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.
  • Preferred administration routes include orally, parenterally, as well as intravenous, intramuscular or subcutaneous routes. More preferably, the compounds of the present invention are administered parenterally, i.e., intravenously or intraperitoneally, by infusion or injection.
  • Solutions or suspensions of the compounds can be prepared in water, isotonic saline (PBS) and optionally mixed with a nontoxic surfactant.
  • Dispersions may also be prepared in glycerol, liquid polyethylene, glycols, DNA, vegetable oils, triacetin and mixtures thereof. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage form suitable for injection or infusion use can include sterile, aqueous solutions or dispersions or sterile powders comprising an active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol such as glycerol, propylene glycol, or liquid polyethylene glycols and the like, vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size, in the case of dispersion, or by the use of nontoxic surfactants.
  • the prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, buffers, or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents delaying absorption-- for example, aluminum monosterate hydrogels and gelatin.
  • Sterile injectable solutions are prepared by incorporating the compounds in the required amount in the appropriate solvent with various other ingredients as enumerated above and, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • ManA polypeptides of the invention are effective thermostable mannanases.
  • the mannanase degrading effects of ManA are achieved by treating biomass at a ratio of about 1 to about 50 of ManA:biomass.
  • ManA may be used under extreme conditions, for example, elevated temperatures and acidic pH.
  • Treated biomass is degraded into simpler forms of carbohydrates, which is then used in the formation of ethanol or other industrial chemicals, as is known in the art.
  • Other methods are envisioned to be within the scope of the present invention, including methods for treating fabrics to remove hemicellulose-containing stains and other methods already discussed.
  • ManA polypeptides can be used to degrade the hemicellulose content of a substrate source in the food, feed and paper pulp industries, or alternatively, be used to produce useful oligosaccharides to be added to food and feed as bulking agents or stabilizing agents.
  • ManA polypeptides can be used in any known application currently utilizing a mannanases, all of which are within the scope of the present invention. Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
  • Genomic DNA was isolated from Acidothermus cellulolyticus and purified by banding on cesium chloride gradients. Genomic DNA was partially digested with Sau 3 A and separated on agarose gels. DNA fragments in the range of 9-20 kilobase pairs were isolated from the gels. This purified Sau 3A digested genomic DNA was ligated into the Bam HI acceptor site of purified EMBL3 lambda phage arms (Clontech, San Diego, Calif.). Phage DNA was packaged according to the manufacturer's specifications and plated with E. coli LE392 in top agar which contained the soluble cellulose analog, carboxymethylcellulose (CMC). The plates were incubated overnight (12-24 hours) to allow transfection, bacterial growth, and plaque formation. Plates were stained with Congo Red followed by destaining with 1 M NaCl. Lambda plaques harboring endoglucanase clones showed up as unstained plaques on a red background.
  • CMC carboxymethylcellulose
  • Lambda clones which screened positive on CMC-Congo Red plates were purified by successive rounds of picking, plating and screening. Individual phage isolates were named SL-1, SL-2, SL-3 and SL-4. Subsequent subcloning efforts employed the SL-3 clone which contained an approximately 14.2 kb fragment of A. cellulolyticus genomic DNA.
  • Template DNA was constructed using a 9 kb BamHl fragment obtained from the 14.2 kb lambda clone SL3 prepared from Acidothermus cellulolyticus genomic DNA.
  • the 9-kb BamHI fragment from SL3 was subcloned into pDR540 to generate a plasmid NREL501.
  • NREL501 was first sequenced by the primer walking method as is known in the art.
  • NREL501 was then subcloned into pUC19 using restriction enzymes Pstl and EcoRI and transformed into E. coli XL 1 -blue (Stratagene, La Jolla, California) for the production of template DNA for sequencing. Each subclone was sequenced from both forward and reverse directions.
  • DNA for sequencing was prepared from an overnight growth in 500 mL LB broth using a megaprep DNA purification kit from Promega.
  • the template DNA was PEG precipitated and suspended in de-ionized water and adjusted to a final concentration of 0.25 mg/mL.
  • Custom primers were designed by reading upstream known sequence and selecting segments of an appropriate length to function, as is well known in the art.
  • Primers for cycle sequencing were synthesized at the Macromolecular Resources facility located at Colorado State University in Fort Collins, Colorado. Typically the sequencing primers were 26-30 nucleotides in length, but were sometimes longer or shorter to accommodate a melting temperature appropriate for cycle sequencing.
  • the sequencing primers were diluted in de-ionized water, the concentration measured using UV absorbance at 260 nm, and then adjusted to a final concentration of 5 pmol/ ⁇ L.
  • Templates and sequencing primers were shipped to the Iowa State University DNA Sequencing facility at Ames, Iowa for sequencing using standard chemistries for cycle sequencing. In some cases, regions of the template that sequenced poorly using the standard protocols and dye terminators were repeated with the addition of 2 ⁇ L DMSO and by using nucleotides optimized for the sequencing of high GC content DNA.
  • the high frequency of reoccurring small domains, i.e., CBDs and linkers, with high sequence similarity caused initial difficulties in sequence assignments which were only resolved through extensive review of the data and repeated analysis.
  • Example 2 An ORF of 2289 bp [SEQ ID NO: 2] and deduced amino acid sequence [SEQ ID NO: 1] are shown in Tables 2 and 1, respectively.
  • the amino acid sequence predicted by SEQ ID NO: 1 was determined to have significant homology to known mannanases, as shown below in Example 2 and in Table 3.
  • the amino acid sequence represents a novel member of the family of proteins with mannanase activity. Due to the source of isolation from the thermophilic organism Acidothermus, ManA is a novel member of the mannanase family with properties including thermal tolerance. It is also known that thermal tolerant enzymes may have other properties (see definition above).
  • GH3_Ace Acidothermus cellulolyticus ManA catalytic domain GHS
  • Cel4A_Abi Agaricus bisporus Cel4 (beta-mannanase). GeneBank Ace. # CAA904 23
  • GH5_Ace DVDDVLAKAQAMLSVIRTWGFIDIGS DGSVPTIDGNKNGFYFQY DPSTGAPAYNDGP Cel4a_Abi AMNQAFSDIANAGGTTVRTWGFNEVTSP NGNYYQSWSGAR--PTINTGA Man Tre DVDSTFSHISSSGIiKWRVWGFNDVNTQPSP-- GQIWFQK SATG--STINTGA GH5_Ace TGLQG DYAIASAAAHGLRVIWLTND KEFGGMDQYD-KYG-LPYHDNFYTDPRTQQA
  • Example 3 Mixed Domain GH5, CBD II, CBD III Genes and Hybrid Polypeptides
  • mannanase genes From the putative locations of the domains in the ManA sequence given above and in comparable cloned mannanase sequences from other species, one can separate individual domains and combine them with one or more domains from different sequences.
  • the significant similarity between mannanase genes permit one by recombinant techniques to arrange one or more domains from the Acidothermus cellulolyticus ManA gene with one or more domains from a mannanase gene from one or more other microorganisms.
  • endoglucanase genes include Bacillus polymyxa beta-(l,4) endoglucanase (Baird et al, Journal of Bacteriology, 172: 1576-86 (1992)) Xanthomonas campestris beta-(l,4)- endoglucanase (Gough et al, Gene 89:53-59 (1990)) and Trichoderma harzianum endo-l,3(4)-beta-glucanase (U.S. Patent 6,140,096).
  • the result of the fusion of any two or more domains will, upon expression, be a hybrid polypeptide.
  • Such hybrid polypeptides can have one or more catalytic or binding domains.

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Abstract

L'invention concerne une mannanase thermo-tolérante appartenant à la famille de la glycoside hydrolase. Ladite mannanase est représentée ici par ManA. ManA a été isolée et caractérisée à partir de l'Acidothermus cellulolyticus. L'invention concerne également des formes recombinantes de la ManA identifiée. Des procédés de préparation de polypeptides ManA, y compris des fusions, des variantes, et des dérivés, ainsi que des procédés d'utilisation de la mannanase A, notamment pour le traitement des aliments et pour l'utilisation dans des produits alimentaires en tant qu'agents gonflants et similaires, sont également décrits.
PCT/US2001/023819 2001-07-28 2001-07-28 Mannanase thermo-tolerante provenant d'acidothermus cellulolyticus WO2003012110A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
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EP1881067A1 (fr) * 2006-07-18 2008-01-23 Direvo Biotech AG Mannanases
WO2008009673A2 (fr) 2006-07-18 2008-01-24 Direvo Industrial Biotechnology Gmbh Mannanases
US7846705B2 (en) 2006-07-18 2010-12-07 Direvo Industrial Biotechnology Gmbh Mannanases
WO2011085747A1 (fr) * 2010-01-13 2011-07-21 Direvo Industrial Biotechnology Gmbh Nouveaux variants de la mannanase
WO2013053071A1 (fr) * 2011-10-10 2013-04-18 Huang Daiyong Procédé de production pour la préparation d'un oligosaccharide mannane de grande pureté par enzymolyse d'hémicellulose
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Publication number Priority date Publication date Assignee Title
EP1881067A1 (fr) * 2006-07-18 2008-01-23 Direvo Biotech AG Mannanases
WO2008009673A2 (fr) 2006-07-18 2008-01-24 Direvo Industrial Biotechnology Gmbh Mannanases
WO2008009673A3 (fr) * 2006-07-18 2008-03-06 Direvo Biotech Ag Mannanases
US7846705B2 (en) 2006-07-18 2010-12-07 Direvo Industrial Biotechnology Gmbh Mannanases
WO2011085747A1 (fr) * 2010-01-13 2011-07-21 Direvo Industrial Biotechnology Gmbh Nouveaux variants de la mannanase
EP2857490A1 (fr) * 2010-01-13 2015-04-08 Basf Se Nouveaux variants de la mannanase
US9040277B2 (en) 2010-01-13 2015-05-26 Basf Se Mannanase variants
EA023087B1 (ru) * 2010-01-13 2016-04-29 Басф Се Новые варианты маннаназы
WO2013053071A1 (fr) * 2011-10-10 2013-04-18 Huang Daiyong Procédé de production pour la préparation d'un oligosaccharide mannane de grande pureté par enzymolyse d'hémicellulose
US11889847B2 (en) * 2013-08-15 2024-02-06 Novozymes A/S Method for producing a coffee extract employing enzymes having beta-1,3-galactanase activity

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