+

WO2003012090A2 - Avicelase a tolerance thermique derivee de acidothermus cellulolyticus - Google Patents

Avicelase a tolerance thermique derivee de acidothermus cellulolyticus Download PDF

Info

Publication number
WO2003012090A2
WO2003012090A2 PCT/US2001/023818 US0123818W WO03012090A2 WO 2003012090 A2 WO2003012090 A2 WO 2003012090A2 US 0123818 W US0123818 W US 0123818W WO 03012090 A2 WO03012090 A2 WO 03012090A2
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
avidi
seq
polypeptide
polynucleotide
Prior art date
Application number
PCT/US2001/023818
Other languages
English (en)
Other versions
WO2003012090A3 (fr
Inventor
Shi-You Ding
William S. Adney
Todd B. Vinzant
Michael E. Himmel
Original Assignee
Midwest Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Midwest Research Institute filed Critical Midwest Research Institute
Priority to AU2001277220A priority Critical patent/AU2001277220A1/en
Priority to PCT/US2001/023818 priority patent/WO2003012090A2/fr
Publication of WO2003012090A2 publication Critical patent/WO2003012090A2/fr
Publication of WO2003012090A3 publication Critical patent/WO2003012090A3/fr

Links

Classifications

    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • 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)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • 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)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention generally relates to a novel avicelase from Acidothermus cellulolyticus, Avirn. More specifically, the invention relates to purified and isolated AviHI polypeptides, nucleic acid molecules encoding the polypeptides, and processes for production and use of Aviffl, as well as variants and derivatives thereof. Background of the Invention
  • Plant biomass as a source of energy production can include agricultural and forestry products, associated by-products and waste, municipal solid waste, and industrial waste.
  • Over 50 million acres in the United States are currently available for biomass production, and there are a number of terrestrial and aquatic crops grown solely as a source for biomass (A Wiselogel, et al. Biomass feedstocks resources and composition, hi CE Wyman, ed. Handbook on Bioethanol: Production and Utilization. Washington, DC: Taylor & Francis, 1996, pp 105-118).
  • Biofuels produced from biomass include ethanol, methanol, biodiesel, and additives for reformulated gasoline.
  • Biofuels are desirable because they add little, if any, net carbon dioxide to the atmosphere and because they greatly reduce ozone formation and carbon monoxide emissions as compared to the environmental output of conventional fuels.
  • Plant biomass is the most abundant source of carbohydrate in the world due to the lignocellulosic materials composing the cell walls of all higher plants. Plant cell walls are divided into two sections, the primary and the 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 and one group of glycoproteins.
  • Cellulose is a linear beta-(l,4)-D-glucan and comprises 20% to 30% of the primary cell wall by weight.
  • 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.
  • Carbohydrates, and cellulose in particular can be converted to sugars by well- known methods including acid and enzymatic hydrolysis.
  • Enzymatic hydrolysis of cellulose requires the processing of biomass to reduce size and facilitate subsequent handling. Mild acid treatment is then used to hydrolyze part or all of the hemicellulose content of the feedstock. Finally, cellulose is converted to ethanol through the concerted action of cellulases and saccharolytic fermentation (simultaneous saccharification fermentation (SSF)).
  • SSF solid saccharification fermentation
  • the cost of producing ethanol from biomass can be divided into three areas of expenditure: pretreatment costs, fermentation costs, and other costs.
  • Pretreatment costs include biomass milling, pretreatment reagents, equipment maintenance, power and water, and waste neutralization and disposal.
  • the fermentation costs can include enzymes, nutrient supplements, yeast, maintenance and scale-up, and waste disposal.
  • Other costs include biomass purchase, transportation and storage, plant labor, plant utilities, ethanol distillation, and administration (which may include technology-use licenses).
  • One of the major expenses incurred in SSF is the cost of the enzymes, as about one kilogram of cellulase is required to fully digest 50 kilograms of cellulose.
  • Enzymatic degradation of cellulose requires the coordinate action of at least three different types of cellulases.
  • Such enzymes 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).
  • Endo- beta-(l,4)-glucanases (EC 3.2.1.4) cleave the cellulose strand randomly along its length, thus generating new chain ends.
  • Exo- beta-(l,4)- glucanases (EC 3.2.1.91) are processive enzymes and cleave cellobiosyl units (beta- (l,4)-glucose dimers) from free ends of cellulose strands.
  • beta-D- glucosidases (cellobiases: EC 3.2.1.21) hydrolyze cellobiose to glucose. All three of these general activities are required for efficient and complete hydrolysis of a polymer such as cellulose to a subunit, such as the simple sugar, glucose.
  • thermostable enzymes have been isolated from the cellulolytic thermophile Acidothermus cellulolyticus gen. nov., sp. nov., a bacterium originally isolated from decaying wood in an acidic, thermal pool at Yellowstone National Park. A. 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. M.P. Tucker et al. (1989), 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 the exocellulase CBH I from Trichoderma reesei, El gives a high level of saccharification and contributes to a degree of synergism. Baker JO et al. (1994), Appl. Biochem. Biotechno 45/46: 245-256. The gene coding El catalytic and carbohydrate binding domains and linker peptide were described in U.S. Patent 5,536,655. El has also been expressed as a stable, active enzyme from a wide variety of hosts, including E.
  • heterologous cellulases and in particular novel cellulases with or without any one or more desirable properties such as thermal tolerance and resistance to acid inactivation, proteolytic inactivation, and solvent inactivation.
  • Such expression can occur in filamentous fungi, bacteria, and other hosts.
  • the present invention provides Avii ⁇ , a novel member of the glycoside hydrolase (GH) family of enzymes, and in particular a thermal tolerant glycoside hydrolase useful in the degradation of cellulose.
  • Avii ⁇ 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 GH74 family, a first carbohydrate binding domain (type H.) and a second carbohydrate binding domain (type m). See FIG 1.
  • the invention also provides a polynucleotide molecule encoding Avilll polypeptides and fragments of Aviffl 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 AviDI polypeptides, including fusion proteins.
  • fusion proteins of the invention include AviDI 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 AviDI polypeptide, for example.
  • the heterologous polypeptide can provide enhanced activity, including catalytic or binding activity, for AviDI 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 AviDI 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 AviDI polynucleotide molecule.
  • Genetic engineering methods for the production of AviDI 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 AviDI polypeptide of the invention and a carrier. Such compositions are administered to a biomass containing cellulose for the reduction or degradation of the cellulose.
  • the invention also provides reagents, compositions, and methods that are useful for analysis of AviDI activity.
  • Tables 4 and 5 includes sequences used in describing embodiments of the present invention.
  • the abbreviations are as follows: CD, catalytic domain; CBDJQ. carbohydrate binding domain type D; CBD_DI, carbohydrate binding domain type ID; and FN-DI, fibronectin domain type Dl.
  • 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.
  • 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, glycosylation, 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.
  • Fab antigen binding fragment
  • chimeric antibodies antibodies having a human constant region coupled to a murine antigen binding region, and fragments thereof
  • 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 a substrate.
  • the substrate can be a polymer such as cellulose or can be a complex molecule or aggregate of molecules where the entire moiety comprises at least some cellulose.
  • Cellulase activity refers to any activity that can be assayed by characterizing the enzymatic activity of a cellulase. For example, cellulase activity can be assayed by determining how much reducing sugar is produced during a fixed amount of time for a set amount of enzyme (see Irwin et al., (1998) J. Bacteriology,
  • 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 one or more catalytic domains of AviDI, one or more binding domains of AviDI, one or more catalytic domains of a glycoside hydrolase other than AviDI, one or more binding domains of a glycoside hydrolase other than AviDI, or any combination thereof.
  • Further examples include immunoglobuhn molecules and portions thereof, peptide tags such as histidine tag (6-His), leucine zipper, substrate targeting moieties, signal peptides, and the like. Fusion proteins are also meant to encompass variants and derivatives of AviDI 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 Natl 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 refers to cells expressing a heterologous polynucleotide molecule. Host cells of the present invention express polynucleotides encoding AviDI or a fragment thereof. Examples of suitable host cells useful in the present invention include, but are not limited to, prokaryotic and eukaryotic cells.
  • 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.
  • HELA human cervical carcinoma cells
  • HTB2 human liver cells
  • DLD-1 human colon carcinoma cells
  • Daudi cells ATCC CRL-213
  • murine myeloma cells such as P3/NSI/l-Ag4-l (ATCC TIB-18), P3X63Ag8 (ATCC TD -9), SP2/0-Agl4 (ATCC CRL-1581) and the like.
  • Hybridization refers to the pairing of complementary polynucleotides during an annealing period. The strength of hybridization between two polynucleotide molecules is impacted by the homology between the two molecules, stringency of the conditions involved, the melting temperature of the formed hybrid and the G:C ratio within the polynucleotides.
  • 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.
  • 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 may be 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 Natl 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, JMol 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 “ , hgprt " 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 AviDI polypeptide or fragment thereof to a substrate.
  • targeting moieties include ligands that bind to a substrate structure. Examples of ligand/receptor pairs include carbohydrate binding domains and cellulose.
  • substrate-specific ligands are known and are useful in the present invention to target a AviDI polypeptide or fragment thereof to a substrate.
  • a novel example is a AviDI carbohydrate binding domain that is used to tether other molecules to a cellulose-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 prefe ⁇ ed 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 hydrolyse the glycosidic bond between two carbohydrate moieties or between a carbohydrate and a 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. Enzymes having related catalytic domains are grouped together within a family, (Henrissat et al., (1991) supra, and 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 a GH domain will fold.
  • GH glycoside hydrolase
  • Cellulases belong to the GH family of enzymes. Cellulases are produced by a variety of bacteria and fungi to degrade the ⁇ -1,4 glycosidic bond of cellulose and to so produce successively smaller fragments of cellulose and ultimately produce glucose. At present, cellulases are found within are at least 11 different GH families.
  • exo-acting cellulases which cleave successive disaccharide units from the non-reducing ends of a cellulose chain
  • endo-acting cellulases which randomly cleave successive disaccharide units within the cellulose chain
  • ⁇ -glucosidases which cleave successive disaccharide units to glucose
  • cellulases are characterized by having a multiple domain unit within their overall structure, a GH or catalytic domain is joined to a carbohydrate-binding domain (CBD) by a glycosylated linker peptide (Koivula et al., (1996) Protein Expression and Purification 8:391-400).
  • CBD carbohydrate-binding domain
  • cellulases do not belong to any one family of GH domains, but rather have been identified within at least 11 different GH families to date.
  • the CBD type domain increases the concentration of the enzyme on the substrate, in this case cellulose, and the linker peptide provides flexibility for both larger domains.
  • thermostable cellulases have taken precedent, due to their ability to function at elevated temperatures and under other conditions including pH extremes, solvent presence, detergent presence, proteolysis, etc. (see Cowan DA (1992), supra). Highly thermostable cellulase enzymes are secreted by the cellulolytic themophile Acidothermus cellulolyticus (U.S. Patent Nos. 5,275,944 and 5,110,735).
  • This bacterium was originally isolated from decaying wood in an acidic, thermal pool at Yellowstone National Park and deposited with the American Type Culture Collection (ATCC 43068) (Mohagheghi et al., (1986) Int. J. System. Bacteriol, 36:435-443).
  • thermostable cellulase El endoglucanase
  • Acidothermus cellulolyticus U.S. Patent No. 5,536,655
  • the El endoglucanase has maximal activity between 75 and 83 °C and is active to a pH well below 5.
  • Thermostable cellulase, and El endoglucanase are useful in the conversion of biomass to biofuels, and in particular, are useful in the conversion of cellulose to glucose. Conversion of biomass to biofuel represents an extremely important alternative fuel source that is more environmentally friendly than conventional fuels, and provides a use, in some cases, for waste products.
  • Avii ⁇ As described more fully in the Examples below, AviDI, a novel thermostable cellulase, has now been identified and characterized.
  • the predicted amino acid sequence of Avilll (SEQ ID NO:l) has an organization characteristic of a cellulase enzyme.
  • AviDI contains a carbohydrate binding domain - linker domain - catalytic domain -linker domain- fibronectin domain - linker domain - carbohydrate binding domain unit, i particular, AviDI includes a a GH74 catalytic domain (from about amino acid A37 to about G776), and a carbohydrate binding domain type HI (CBDID) (amino acids from about V859 to about at least Q946).
  • CBDID carbohydrate binding domain type HI
  • AviDI has a catalytic domain, identified as belonging to the GH74 family.
  • the GH74 domain family includes a number of exoglucanases, for example, from Cellulomonas fimi, and exoglucanase E3 isolated from Thermobifida fusca.
  • the GH74 members degrade substrate using an inverting mechanism. Being a member of the GH74 family of proteins identifies AviDI as potentially having cellulase activity.
  • AviDI is also a thermostable cellulase as it is produced by the themophile Acidothermus cellulolyticus. As discussed, AviDI polypeptides can have other desirable characteristics (see Cowan DA (1992), supra). Like other members of the cellulase family, and in particular thermostable cellulases, AviDI polypeptides are useful in the conversion of biomass to biofuels and biofuel additives, and in particular, biofuels from cellulose. It is envisioned that AviDI polypeptides could be used for other purposes, for example in detergents, pulp and paper processing, food and feed processing, and in textile processes. AviDI polypeptides can be used alone or in combination with one or more other cellulases or glycoside hydrolases to perform the uses described herein or known within the relevant art, all of which are within the scope of the present disclosure. Avii ⁇ Polypeptides:
  • AviDI 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 TD NO:l and that retain any of the functional activities of AviDI.
  • AviDI polypeptide activity can be determined, for example, by subjecting the variant, derivative, or fragment to a substrate binding assay or a cellulase activity assay such as those described in Ixwin D et al., J. Bacteriology 180(7): 1709-1714 (April 1998).
  • the isolated AviDI polypeptide includes an N-terminal hydrophobic region that functions as a signal peptide, having an amino acid sequence that begins with Metl and extends to about A36; a catalytic domain having significant sequence similarity to a GH74 family domain that begins with about A37 and extends to about G776, a carbohydrate binding domain having sequence similarity to such type ID domains that begins with about V859 extends to about at least Q946.
  • Variants and derivatives of AviDI include, for example, AviDI 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.
  • other chemical moieties such as glycosyl groups, polyethylene glycol (PEG) groups, lipids, phosphate, acetyl groups, and the like.
  • the amino acid sequence of AviDI 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 AviDI amino acid sequence shown above in Table 1 and SEQ DD 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 AviDI polypeptide may further include, for example, fusion proteins formed of a AviDI polypeptide and a heterologous polypeptide.
  • Preferred heterologous polypeptides include those that facilitate purification, oligomerization, stability, or secretion of the AviDI polypeptides.
  • AviDI 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 ( e, 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.
  • Functional AviDI polypeptide variants include those having amino acid substitutions, deletions, or additions to the amino acid sequence outside functional regions of the protein, for example, outside the catalytic and carbohydrate binding domains. These would include, for example, the various linker sequences that connect functional domains as defined herein.
  • the AviDI 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
  • Another preferred form of AviDI polypeptides is that of recombinant polypeptides as expressed by suitable hosts. Furthermore, the hosts can simultaneously produce other cellulases such that a mixture is produced comprising a
  • AvilU polypeptide and one or more other cellulases can be effective in crude fermentation processing or other industrial processing.
  • AvilD 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.
  • AvilD polypeptides can be modified to facilitate formation of AviDI oligomers.
  • AviDI 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 AviDI 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 AviDI 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).
  • Fragments of the AviDI polypeptide can be used, for example, to generate specific anti-AvilD 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 AviDI activity as well as in purifying recombinant AviDI polypeptides from genetically engineered host cells.
  • the invention also provides polynucleotide molecules encoding the AviDI polypeptides discussed above.
  • AviDI polynucleotide molecules of the invention include polynucleotide molecules having the nucleic acid sequence shown in Table 2 and SEQ DD NO: 2, polynucleotide molecules that hybridize to the nucleic acid sequence of Table 2 and SEQ DD 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 DD NO: 2, particularly with those nucleic acids encoding a catalytic domain, GH74 (from about amino acid A37 to about Gil ), and a carbohydrate binding domain type Dl (from about amino acid V859 to about at least Q946).
  • the AviDI polynucleotide sequence can include deletions, substitutions, or additions to the nucleic acid sequence of Table 2 and SEQ DD NO: 1.
  • the AviDI 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 AviDI nucleic acid sequence encoding AviDI polypeptide, where the nucleic acid sequenc encodes a polypeptide having the complete amino acid sequences as shown in Table 1 and SEQ DD NO: 1 , or variants, derivatives, and fragments thereof.
  • the AviDI 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 DD NO: 2, in some embodiments at least about 70% identical to the nucleic acid sequence shown in Table 2 and SEQ DD NO: 2, and in other embodiments at least about 90% identical to the nucleic acid sequence shown in Table 2 and SEQ DD 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/).
  • the AviDI 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 DD NO: 2. Hybridization of the polynucleotide is to about 15 contiguous nucleotides, or about 20 contiguous nucleotides, and in other embodiments about 30 contiguous nucleotides, and in still other embodiments about 100 contiguous nucleotides of the nucleic acid sequence shown in Table 2 and SEQ DD NO: 2.
  • Useful fragments of the AviDI-encoding polynucleotide molecules described herein, include probes and primers.
  • Such probes and primers can be used, for example, in PCR methods to amplify and detect the presence of AviDI polynucleotides in vitro, as well as in Southern and Northern blots for analysis of AviDI.
  • Cells expressing the AviDI 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 AviDI polynucleotide molecule can be employed to isolate and amplify the AviDI polynucleotide using conventional techniques.
  • 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 AviDI polynucleotide molecule.
  • suitable transcriptional or translational 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 Avim DNA sequence if the promoter nucleotide sequence directs the transcription of the AviDI sequence.
  • Suitable vectors for the cloning of AviDI polynucleotide molecules encoding the target AviDI 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 AviDI polypeptides include prokaryotes, yeast, and higher eukaryotic cells, each of which is discussed below.
  • the AviDI 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 AviDI 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 AviDI sequence so that AviDI 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 AviDI polypeptide.
  • the signal sequence will be cleaved from the AviDI polypeptide upon secretion of AviDI from the cell.
  • Non-limiting examples of 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
  • the polynucleotide molecule encoding AviDI 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.
  • pSPORT vectors examples include pSPORT vectors, pGEM vectors (Promega, Madison, WT), pPROEX vectors (LTI, Bethesda, MD), Bluescript vectors (Stratagene), and pQE vectors (Qiagen).
  • AviDI 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.
  • ARS autonomously replicating sequence
  • shuttle vectors 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 AviDI-encoding nucleotide sequence.
  • Insect host cell culture systems can also be used for the expression of AviDI 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.
  • a suitable expression vector for expression of AviDI polypeptides of the invention will depend upon the host cell to be used.
  • suitable expression vectors for E. coli include pET, pUC, and similar vectors as is known in the art.
  • Preferred vectors for expression of the AviDI polypeptides include the shuttle plasmid pD702 for Streptomyces lividans, pGAPZalpha-A, B, C and pPICZalpha-A, B, C (Invitrogen) for Pichia pastoris, and pFE-1 and pFE-2 for filamentous fungi and similar vectors as is known in the art.
  • AviDI polynucleotide molecule Modification of a AviDI polynucleotide molecule to facilitate insertion into a particular vector (for example, by modifiying restriction sites), ease of use in a particular expression system or host (for example, using prefe ⁇ ed host codons), and the like, are known and are contemplated for use in the invention.
  • Genetic engineering methods for the production of AviDI 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 AviDI polypeptide of the invention and an acceptable carrier are administered to biomass, for example, to degrade the cellulose in the biomass into simpler carbohydrate units and ultimately, to sugars. These released sugars from the cellulose are converted into ethanol by any number of different catalysts.
  • Such compositions may also be included in detergents for removal, for example, of cellulose containing stains within fabrics, or compositions used in the pulp and paper industry, to address conditions associated with cellulose content.
  • Compositions of the present invention can be used in stonewashing jeans such as is well known in the art. Compositions can be used in the biopolishing of cellulosic fabrics, such as cotton, linen, rayon and Lyocell.
  • the invention provides pharmaceutical compositions containing a substantially purified AviDI 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.
  • AviDI polypeptides 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.
  • the invention also provides reagents, compositions, and methods that are useful for analysis of AviDI activity and for the analysis of cellulose breakdown.
  • compositions of the present invention may also include other known cellulases, and preferably, other known thermal tolerant cellulases for enhanced treatment of cellulose.
  • Antibodies are also include other known cellulases, and preferably, other known thermal tolerant cellulases for enhanced treatment of cellulose.
  • the polypeptides of the present invention may be used to raise polyclonal and monoclonal antibodies that are useful in purifying AviDI, or detecting AviDI polypeptide expression, as well as a reagent tool for characterizing the molecular actions of the AviDI polypeptide.
  • a peptide containing a unique epitope of the AviDI polypeptide is used in preparation of antibodies, using conventional techniques. Methods for the selection of peptide epitopes and production of antibodies are known.
  • Agents that modify, for example, increase or decrease, AviDI hydrolysis or degradation of cellulose can be identified, for example, by assay of AviDI cellulase activity and/or analysis of AviDI binding to a cellulose substrate. Incubation of cellulose in the presence of AviDI and in the presence or absence of a test agent and co ⁇ elation of cellulase activity or carbohydrate binding permits screening of such agents.
  • cellulase activity and binding assays may be performed in a manner similar to those described in frwin et al., J. Bacteriology 180(7): 1709-1714 (April 1998).
  • the AviDI stimulated activity is determined in the presence and absence of a test agent and then compared.
  • Stimulators and inhibitors of AviDI may be used to augment, inhibit, or modify AviDI mediated activity, and therefore may have potential industrial uses as well as potential use in the further elucidation of AviDTs molecular actions.
  • the AviDI polypeptides of the invention are effective in adding in delivery or targeting of other pharmaceutical compositions within a host.
  • AviDI polypeptides may be used where carbohydrate-mediated liposomal interactions are involved with target cells.
  • AviDI polynucleotides and polypeptides, including vectors expressing AviDI, 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.
  • AviDI can be administered by known techniques, such as orally, parentally (including subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques), by inhalation spray, topically, by absorption through a mucous membrane, or rectally, in dosage unit formulations containing conventional non- toxic pharmaceutically acceptable carriers, adjuvants or vehicles.
  • Pharmaceutical 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.
  • the 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, ttisintegrants, 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.
  • compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • 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.
  • Prefe ⁇ ed 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 prefe ⁇ ed 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.
  • the AviDI polypeptides of the invention are effective cellulases.
  • the cellulose degrading effects of AviDI are achieved by treating biomass at a ratio of about 1 to about 50, or about 1:40, 1:35, 1:30, 1:25 , 1:20 or even about 1: 70 in some preparations of the AVD of AviDLbiomass.
  • AviDI may be used under extreme conditions, for example, elevated temperatures and acidic pH.
  • Treated biomass is degraded into simpler forms of carbohydrates, and in some cases glucose, 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 cellulose-containing stains and other methods already discussed.
  • AviDI polypeptides can be used in any known application cu ⁇ ently utilizing a cellulase, all of which are within the scope of the present invention.
  • 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 specification 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 kilobase fragment of Acidothermus cellulolyticus genomic DNA.
  • Template DNA was constructed using a 9 kilobase Bam HI fragment obtained from the 14.2 kilobase lambda clone SL-3 prepared from Acidothermus cellulolyticus genomic DNA.
  • the 9 kilobase Bam HI fragment from SL-3 was subcloned into pDR540 to generate a plasmid NREL501.
  • NREL501 was sequenced by the primer walking method as is known in the art.
  • NREL501 was then subcloned into pUC19 using restriction enzymes Pst I and Eco RI and transformed into E. coli XL 1 -blue (Stratagene) for the production of template DNA for sequencing. Each subclone was sequenced from both the 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 templated DNA was PEG precipitated and suspended in de-ionized water and adjusted to a final concentration of 0.25 milligrams/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 to 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 UN absorbance at 260 nm, and then adjusted to a final concentration of 5 pmol/microL.
  • Templates and sequencing primers were shipped to the Iowa State University D ⁇ A 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 microL DMSO and by using nucleotides optimized for the sequencing of high GC content D ⁇ A.
  • An inverse PCR technique known in the art was applied to continue sequencing the genomic D ⁇ A, and a primer walking method was used to sequence the large PCR products. Each PCR fragment was sequenced from both strands, using high fidelity commercial D ⁇ A polymerase.
  • amino acid sequence represents a novel member of the family of proteins with cellulase activity. Due to the source of isolation, from the thermophilic
  • AviDI is a novel member of cellulases with properties including thermal tolerance. It is also known that thermal tolerant enzymes may have other properties (see definition above).
  • Avicelase Dl (endoglucanase) GH74 domain, indicating that the AviDI catalytic domain is a member of the GH74 family (Henrissat et al., (1991) supra).
  • GH74_Ace Acidothermus cellulolyticus AviDI catalytic domain GH74
  • AviHI_Aac Aspergillus aculeatus Avicelase IE (endoglucanase). GeneBank Ace. # BAA ⁇ C ⁇ 1
  • GH74_Ace GNMPGRGMGERLAVDPNNDNILYFGAPSGKGL RSTDSGAT SQ TNFPDVGTYIANPTD Avilll Aac GNMPGRGMGERAVDPNKNSILYFGARSGHG KSTDYGAT SNVTSFT TGTYFQDSSS *************** .._ ****** **;*** . *** ***** .. * . * . . ***: : . : .
  • endoglucanase genes include Bacillus polymyxa beta-(l,4) endoglucanase (Baird et al, Journal of Bacteriology, 172: 1576-86 (1992)) and Xanthomonas campestris beta-(l,4)- endoglucanase A (Gough et al, Gene 89:53-59 (1990)).
  • 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.
  • recombinant techniques may be employed such as the addition of restriction enzyme sites by site-specific mutagenesis. If one is not using one domain of a particular gene, any number of any type of change including complete deletion maybe made in the unused domain for convenience of manipulation.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne une cellulase présentant une tolérance thermique (thermostable) qui est un membre de la famille des glycoside hydrolases. L'invention concerne plus particulièrement la cellulase dénommée AviIII, isolée et caractérisée à partir de Acidothermus cellulolyticus. L'invention concerne en outre des formes recombinées de l'AViIII ainsi identifié, ainsi que des procédés de préparation et d'utilisation des polypeptides d'AviIII, y compris d'hybrides, de variants et de dérivés de ceux-ci.
PCT/US2001/023818 2001-07-28 2001-07-28 Avicelase a tolerance thermique derivee de acidothermus cellulolyticus WO2003012090A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2001277220A AU2001277220A1 (en) 2001-07-28 2001-07-28 Thermal tolerant avicelase from acidothermus cellulolyticus
PCT/US2001/023818 WO2003012090A2 (fr) 2001-07-28 2001-07-28 Avicelase a tolerance thermique derivee de acidothermus cellulolyticus

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2001/023818 WO2003012090A2 (fr) 2001-07-28 2001-07-28 Avicelase a tolerance thermique derivee de acidothermus cellulolyticus

Publications (2)

Publication Number Publication Date
WO2003012090A2 true WO2003012090A2 (fr) 2003-02-13
WO2003012090A3 WO2003012090A3 (fr) 2003-03-27

Family

ID=21742736

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/023818 WO2003012090A2 (fr) 2001-07-28 2001-07-28 Avicelase a tolerance thermique derivee de acidothermus cellulolyticus

Country Status (2)

Country Link
AU (1) AU2001277220A1 (fr)
WO (1) WO2003012090A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009035551A1 (fr) 2007-09-12 2009-03-19 Martek Biosciences Corporation Huiles biologiques et leur production et utilisations
US10344306B2 (en) 2014-07-03 2019-07-09 Sustainable Bioproducts, Inc. Acidophilic fusarium oxysporum strains, methods of their production and methods of their use
US10533155B2 (en) 2016-03-01 2020-01-14 Sustainable Bioproducts, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US11039635B2 (en) 2019-02-27 2021-06-22 The Fynder Group, Inc. Food materials comprising filamentous fungal particles
US11118305B2 (en) 2019-06-18 2021-09-14 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11297866B2 (en) 2017-08-30 2022-04-12 The Fynder Group, Inc. Bioreactor system for the cultivation of filamentous fungal biomass

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536655A (en) * 1989-09-26 1996-07-16 Midwest Research Institute Gene coding for the E1 endoglucanase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536655A (en) * 1989-09-26 1996-07-16 Midwest Research Institute Gene coding for the E1 endoglucanase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] accession number 074170, 1 November 1998 (1998-11-01) M. ARAI ET AL: "Avicelase III from Aspergillus aculeatus" XP002195538 *
M.D. GIBBS ET AL: "Multidomain and multifunctional Glycosyl hydrolases from the extreme Thermophile Caldicellulosiruptor isolate Tok7B.1" CURRENT MICROBIOLOGY, vol. 40, no. 5, 2000, pages 333-340, XP002195536 -& DATABASE EMBL [Online] accession number AAK06388, 11 February 2001 (2001-02-11) M.D. GIBBS ET AL: " Glycosyl hydrolase 5 (caldicellulosiruptor sp. Tok7B.1" XP002195537 *
ROBSON L M ET AL: "ENDO-BETA-1,4-GLUCANASE GENE OF BACILLUS SUBTILIS DLG" JOURNAL OF BACTERIOLOGY, WASHINGTON, DC, US, vol. 169, no. 5, 1 May 1987 (1987-05-01), pages 2017-2025, XP002046715 ISSN: 0021-9193 -& DATABASE SWISS-PROT [Online] GUN1-BACSU, accession number P07983, 1 August 1988 (1988-08-01) L.M. ROBSON ET AL: "Endoglucanase precursor (EC 3.2.1.4) (ENDO-1,4-beta -Glucanase) (Cellulase)" XP002195539 *
TOMME P ET AL: "Characterization and affinity applications of cellulose-binding domains" JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL SCIENCES & APPLICATIONS, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 715, no. 1, 11 September 1998 (1998-09-11), pages 283-296, XP004147002 ISSN: 1570-0232 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9453172B2 (en) 2007-09-12 2016-09-27 Dsm Ip Assets B.V. Biological oils and production and uses thereof
WO2009035551A1 (fr) 2007-09-12 2009-03-19 Martek Biosciences Corporation Huiles biologiques et leur production et utilisations
US10851396B2 (en) 2014-07-03 2020-12-01 The Fynder Group, Inc. Acidophilic fusarium oxysporum strains, methods of their production and methods of their use
US10344306B2 (en) 2014-07-03 2019-07-09 Sustainable Bioproducts, Inc. Acidophilic fusarium oxysporum strains, methods of their production and methods of their use
US11261420B2 (en) 2016-03-01 2022-03-01 The Fynder Group, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US10787638B2 (en) 2016-03-01 2020-09-29 The Fynder Group, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US10577579B2 (en) 2016-03-01 2020-03-03 Sustainable Bioproducts, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US11001801B2 (en) 2016-03-01 2021-05-11 The Fynder Group, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US11015168B2 (en) 2016-03-01 2021-05-25 The Fynder Group, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US12157880B2 (en) 2016-03-01 2024-12-03 The Fynder Group, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US11505779B2 (en) 2016-03-01 2022-11-22 The Fynder Group, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US10533155B2 (en) 2016-03-01 2020-01-14 Sustainable Bioproducts, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US10590379B2 (en) 2016-03-01 2020-03-17 Sustainable Bioproducts, Inc. Filamentous fungal biomats, methods of their production and methods of their use
US11464251B2 (en) 2017-08-30 2022-10-11 The Fynder Group, Inc. Edible foodstuffs and bio reactor design
US11297866B2 (en) 2017-08-30 2022-04-12 The Fynder Group, Inc. Bioreactor system for the cultivation of filamentous fungal biomass
US11478007B2 (en) 2019-02-27 2022-10-25 The Fynder Group, Inc. Food materials comprising filamentous fungal particles and membrane bioreactor design
US11432575B2 (en) 2019-02-27 2022-09-06 The Fynder Group, Inc. Food materials comprising filamentous fungal particles and membrane bioreactor design
US11272726B2 (en) 2019-02-27 2022-03-15 The Fynder Group, Inc. Food materials comprising filamentous fungal particles and membrane bioreactor design
US11039635B2 (en) 2019-02-27 2021-06-22 The Fynder Group, Inc. Food materials comprising filamentous fungal particles
US11427957B2 (en) 2019-06-18 2022-08-30 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11447913B2 (en) 2019-06-18 2022-09-20 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11414815B2 (en) 2019-06-18 2022-08-16 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11118305B2 (en) 2019-06-18 2021-09-14 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11649586B2 (en) 2019-06-18 2023-05-16 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11718954B2 (en) 2019-06-18 2023-08-08 The Fynder Group, Inc. Fungal textile materials and leather analogs

Also Published As

Publication number Publication date
AU2001277220A1 (en) 2003-02-17
WO2003012090A3 (fr) 2003-03-27

Similar Documents

Publication Publication Date Title
US7932054B2 (en) Methods of using thermal tolerant avicelase from Acidothermus cellulolyticus
US7059993B2 (en) Thermal tolerant cellulase from Acidothermus cellulolyticus
Payne et al. Fungal cellulases
US5536655A (en) Gene coding for the E1 endoglucanase
US11530428B2 (en) Nucleic acids encoding fungal cellobiohydrolases for expression in yeast
CN102947442B (zh) 用于糖化纤维素材料的组合物
US7393673B2 (en) Thermal tolerant exoglucanase from Acidothermus cellulolyticus
CN102803482B (zh) 具有改善的表达、活性和/或稳定性的纤维素酶变体，及其用途
US8361752B2 (en) Artificial scaffolding material for protein retention and use of the same
WO2003012109A1 (fr) Cellulase a tolerance thermique a partir d'acidothermus cellulolyticus
JP2012510263A (ja) セルロース分解ポリペプチド、並びに微生物中での溶剤及び燃料の生産に対するその使用
Ferreira et al. The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains
WO2003012095A1 (fr) Exoglucanase thermo-tolerante d'acidothermus cellulolyticus
WO2003012090A2 (fr) Avicelase a tolerance thermique derivee de acidothermus cellulolyticus
US7112429B2 (en) Thermal tolerant mannanase from acidothermus cellulolyticus
US8993276B2 (en) Chimeric enzymes with improved cellulase activities
WO2003012110A1 (fr) Mannanase thermo-tolerante provenant d'acidothermus cellulolyticus
JP2012039967A (ja) 耐熱性セロビオヒドロラーゼ及びその利用
US9080162B2 (en) Cellulase variants
JP5434689B2 (ja) セルラーゼ複合体及びその利用
JP5810489B2 (ja) セルラーゼ活性を有するタンパク質及びその利用
EP2436698A1 (fr) Clostridium acetobutylicum cellulolytique
KR20140146856A (ko) 클로스트리디움 속 미생물 유래의 엔도―베타―1,4―글루칸아제―이, 자일란네이즈―비 및 소형 셀룰로오즈 결합단백질―에이를 이용한 섬유소 분해효소 복합체의 제조방법
JP2012039966A (ja) セルロース結合ドメインが不活性化されたセロビオヒドロラーゼ活性を有するタンパク質
Zverlov et al. The Clostridium thermocellum cellulosome—the paradigm of a multienzyme complex

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GE GH GM HR HU ID IL IN IS JP KE KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX MZ NO NZ PT RO RU SD SE SG SI SK SL TJ TM TT TZ UA UG UZ VN YU ZA

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ CZ DE DE DK DK DM DZ EC EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZW AM AZ BY KG KZ MD TJ TM AT BE CH CY DE DK ES FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载