[go: up one dir, main page]

WO2016090473A1 - Nouvelles enzymes de déconstruction de paroi cellulaire issues de rhizomucor miehei, thermoascus thermophilus (dactylomyces thermophilus) et humicola hyalo thermophila et leurs utilisations - Google Patents

Nouvelles enzymes de déconstruction de paroi cellulaire issues de rhizomucor miehei, thermoascus thermophilus (dactylomyces thermophilus) et humicola hyalo thermophila et leurs utilisations Download PDF

Info

Publication number
WO2016090473A1
WO2016090473A1 PCT/CA2015/051284 CA2015051284W WO2016090473A1 WO 2016090473 A1 WO2016090473 A1 WO 2016090473A1 CA 2015051284 W CA2015051284 W CA 2015051284W WO 2016090473 A1 WO2016090473 A1 WO 2016090473A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
present
polypeptides
activity
acid sequence
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CA2015/051284
Other languages
English (en)
Inventor
Adrian Tsang
Justin Powlowski
Gregory Butler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Concordia University
Original Assignee
Concordia University
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 Concordia University filed Critical Concordia University
Publication of WO2016090473A1 publication Critical patent/WO2016090473A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/24—Organic nitrogen compounds
    • A21D2/26—Proteins
    • A21D2/261—Animal proteins
    • A21D2/262—Animal proteins from eggs
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • 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)
    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C1/00—Pretreatment of the finely-divided materials before digesting
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
    • 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
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to novel polypeptides and enzymes having activities relating to biomass processing and/or degradation (e.g., cell wall deconstruction), as well as polynucleotides, vectors, cells, compositions and tools relating to same, or functional variants thereof. More particularly, the present invention relates to secreted enzymes that may be isolated from the fungi Rhizomucor miehei strain CBS 182.67, Thermoascus thermophilus (Dadylomyces thermophilus) strain ATCC 26413, and Humicola hyalothermophila strain CBS 454.80. Uses thereof in various industrial processes such as in biofuels, food preparation, animal feed, pulp and paper, textiles, detergents, waste treatment and others are also disclosed.
  • Biomass-processing enzymes have a number of industrial applications such as in: the biofuel industry (e.g., improving ethanol yield and/or increasing the efficiency and economy of ethanol production); the food industry (e.g., production of cereal-based food products; the feed-enzyme industry (e.g., increasing the digestibility/absorption of nutrients); the pulp and paper industry (e.g., enhancing bleachability of pulp); the textile industry (e.g., treatment of cellulose-based fabrics); the waste treatment industry (e.g., de-colorization of synthetic dyes); the detergent industry (e.g., providing eco-friendly cleaning products); and the rubber industry (e.g., catalyzing the conversion of latex into foam rubber).
  • the biofuel industry e.g., improving ethanol yield and/or increasing the efficiency and economy of ethanol production
  • the food industry e.g., production of cereal-based food products
  • the feed-enzyme industry e.g., increasing the digestibility/absorption of nutrients
  • Conversion of plant biomass to glucose may also be enhanced by supplementing cellulose cocktails with enzymes that degrade the other components of biomass, including hemicelluloses, pectins and lignins, and their linkages, thereby improving the accessibility of cellulose to the cellulase enzymes.
  • Such enzymes include, without being limiting, to: xylanases, mannanases, arabinanases, esterases, glucuronidases, xyloglucanases and arabinofuranosidases for hemicelluloses; lignin peroxidases, manganese-dependent peroxidases, versatile peroxidases, and laccases for lignin; and pectate lyase, pectin lyase, polygalacturonase, pectin acetyl esterase, alpha-arabinofuranosidase, beta-galactosidase, galactanase, arabinanase, rhamnogalacturonase, rhamnogalacturonan lyase, and rhamnogalacturonan acetyl esterase, xylogalacturonosidase, xylogalacturonase, and rham
  • lignin modifiying enzymes may be used to alter the structure of lignin to produce novel materials, and hemicellulases may be employed to produce 5-carbon sugars from hemicelluloses, which may then be further converted to chemical products.
  • Cereal- based food products such as pasta, noodles and bread can be prepared from dough which is usually made from the basic ingredients (cereal) flour, water and optionally salt.
  • Cereal basic ingredients
  • Suitable enzymes include, for example, xylanase, starch degrading enzymes, oxidizing enzymes, fatty material splitting enzymes, protein degrading, and modifying or crosslinking enzymes.
  • Amylases are used for the conversion of plant starches to glucose.
  • Pectin-active enzymes are used in fruit processing, for example to increase the yield of juices, and in fruit juice clarification, as well as in other food processing steps.
  • enzymes are used to make the bleaching process more effective and to reduce the use of oxidative chemicals.
  • enzymatic treatment is often used in place of (or in addition to) a bleaching treatment to achieve a "used" look of jeans, and can also improve the softness/feel of fabrics.
  • enzymes can enhance cleaning ability or act as a softening agent.
  • enzymes play an important role in changing the characteristics of the waste, for example, to become more amenable to further treatment and/or for bio-conversion to value-added products.
  • thermostable enzymes that are "thermostable” in that they retain a level of their function or protein activity at temperatures about 50°C.
  • thermostable enzymes are highly desirable, for example, to be able to perform reactions at elevated temperatures to avoid or reduce contamination by microorganisms (e.g., bacteria).
  • the present invention relates to soluble, secreted proteins relating to biomass processing and/or degradation (e.g., cell wall deconstruction) that may be isolated from the fungi Rhizomucor miehei strain CBS 182.67, Thermoascus thermophilus (Dadylomyces thermophilus) strain ATCC 26413, and Humicola hyalothermophila strain CBS 454.80, as well as polynucleotides, vectors, compositions, cells, antibodies, kits, products and uses associated with same. Briefly, these fungal strains were cultured in vitro and total RNA was isolated therefrom. These nucleic acids were then used to determine/assemble fungal cDNA libraries.
  • biomass processing and/or degradation e.g., cell wall deconstruction
  • Sequences encoding proteins relating to biomass-degradation were identified based on bioinformatics (e.g., the presence of conserved domains). Sequences predicted to encode proteins which are targeted to the mitochondria or bound to the cell wall were removed. cDNA clones comprising full-length sequences predicted to encode soluble, secreted proteins relating to biomass-degradation were fully sequenced and cloned into appropriate expression vectors for protein production and characterization. The full-length coding and polypeptide sequences are disclosed herein, along with corresponding putative (biological) functions and/or protein activities, where available.
  • the soluble, secreted, biomass degradation proteins of the present invention comprise a proteome which is referred to herein as the SSBD proteome of Rhizomucor miehei, Thermoascus thermophilus (Dadylomyces thermophilus), or Humicola hyalothermophila.
  • the present invention relates to an isolated polypeptide which is:
  • polypeptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the polypeptide defined in (a);
  • polypeptide comprising an amino acid sequence encoded by the nucleic acid sequence of any one of SEQ ID NOs: 1-46, 93-132 and 173-297;
  • the above mentioned polypeptide has a corresponding function and/or protein activity according to Tables 1A-1 C.
  • the above mentioned polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 47-92, 133-172, and 298-422.
  • the above mentioned polypeptide is a recombinant polypeptide.
  • the above mentioned polypeptide is obtainable from a fungus.
  • the fungus is from the genus Rhizomucor, Thermoascus (previously Dactylomyces), or Humicola.
  • the fungus is Rhizomucor miehei, Thermoascus thermophilus (Dactylomyces thermophilus), or Humicola hyalothermophila.
  • the present invention relates to an antibody that specifically binds to any one of the above mentioned polypeptides.
  • the present invention relates to an isolated polynucleotide molecule encoding any one of the above mentioned polypeptides.
  • the present invention relates to an isolated polynucleotide molecule which is:
  • a polynucleotide molecule comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleic acid sequence identity to any one of the polynucleotide molecules defined in (a) and (b); or
  • the above mentioned polynucleotide molecule is obtainable from a fungus.
  • the fungus is from the genus Rhizomucor, Thermoascus (previously Dactylomyces), or Humicola.
  • the fungus is Rhizomucor miehei, Thermoascus thermophilus (Dactylomyces thermophilus), or Humicola hyalothermophila.
  • the above mentioned polynucleotide molecule is operably linked to a heterologous promoter.
  • the present invention relates to a vector comprising any one of the above mentioned polynucleotide molecules.
  • the vector comprises a regulatory sequence operatively linked to the polynucleotide molecule for expression of same in a suitable host cell.
  • the suitable host cell is a bacterial cell; a fungal cell; or a filamentous fungal cell.
  • the present invention relates to a recombinant host cell comprising any one of the above mentioned polynucleotide molecules or vectors.
  • the present invention relates to a polypeptide obtainable by expressing the above mentioned polynucleotide or vector in a suitable host cell.
  • the suitable host cell is a bacterial cell; a fungal cell; or a filamentous fungal cell.
  • the present invention relates to a composition
  • a composition comprising any one of the above mentioned polypeptides or the recombinant host cells.
  • the composition further comprises a suitable carrier.
  • the composition further comprises a substrate of the polypeptide.
  • the substrate is biomass.
  • the present invention relates to a method for producing any one of the above mentioned polypeptides, the method comprising: (a) culturing a strain comprising the above mentioned polynucleotide molecule or vector under conditions conducive for the production of the polypeptide; and (b) recovering the polypeptide.
  • the strain is a bacterial strain; a fungal strain; or a filamentous fungal strain.
  • the present invention relates to a method for producing any one of the above mentioned polypeptides, the method comprising: (a) culturing the above mentioned recombinant host cell under conditions conducive for the production of the polypeptide; and (b) recovering the polypeptide.
  • the present invention relates to a method for preparing a food product, the method comprising incorporating any one of the above mentioned polypeptides during preparation of the food product.
  • the food product is a bakery product.
  • the present invention relates to the use of the above mentioned polypeptide for the preparation or processing of a food product.
  • the food product is a bakery product.
  • the present invention relates to the use of any one of the above mentioned polypeptides for the preparation or processing of a food product.
  • the food product is a bakery product.
  • the present invention relates to the above mentioned polypeptide for use in the preparation or processing of a food product.
  • the food product is a bakery product.
  • the present invention relates to the use of any one of the above mentioned polypeptides for the preparation of animal feed. In some aspects the present invention relates to the use of any one of the above mentioned polypeptides for increasing digestion or absorption of animal feed. In some aspects, the present invention relates to any one of the above mentioned polypeptides for use in the preparation of animal feed, or for increasing digestion or absorption of animal feed. In some embodiments, the animal feed is a cereal-based feed.
  • the present invention relates to the use of any one of the above mentioned polypeptides for the production or processing of kraft pulp or paper. In some aspects the present invention relates to any one of the above mentioned polypeptides for the production or processing of kraft pulp or paper. In some embodiments, the processing comprises prebleaching and/or de-inking.
  • the present invention relates to the use of any one of the above mentioned polypeptides for processing lignin. In some aspects the present invention relates to any one of the above mentioned polypeptides for processing lignin.
  • the present invention relates to the use of any one of the above mentioned polypeptides for producing ethanol. In some aspects the present invention relates to any one of the above mentioned polypeptides for producing ethanol.
  • the above mentioned uses are in conjunction with cellulose or a cellulase.
  • the present invention relates to the use of any one of the above mentioned polypeptides for treating textiles or dyed textiles. In some aspects the present invention relates to any one of the above mentioned polypeptides for treating textiles or dyed textiles.
  • the present invention relates to the use of any one of the above mentioned polypeptides for degrading biomass or pretreated biomass. In some aspects the present invention relates to any one of the above mentioned polypeptides for degrading biomass or pretreated biomass.
  • the present invention relates to proteins and/or enzymes that are thermostable.
  • a polypeptide of the present invention retains a level of its function and/or protein activity at about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, or about 95°C.
  • a polypeptide of the present invention retains a level of its function and/or protein activity between about 50°C and about 95°C, between about 50°C and about 90°C, between about 50°C and about 85°C, between about 50°C and about 80°C, between about 50°C and about 75°C, between about 50°C and about 70°C, or between about 50°C and about 65°C.
  • a polypeptide of the present invention has optimal or maximal function and/or protein activity greater than 50°C, greater than 55°C, greater than 60°C, greater than 65°C, or greater than 70°C.
  • a polypeptide of the present invention has optimal or maximal function and/or protein activity between about 50°C and about 95°C, between about 50°C and about 90°C, between about 50°C and about 85°C, between about 50°C and about 80°C, between about 50°C and about 75°C, between about 50°C and about 70°C, or between about 50°C and about 65°C.
  • Thermoascus (Dactylomyces)" , “Thermoascus (previously Dactylomyces)” and “Thermoascus thermophilus (Dactylomyces thermophilus)” , are meant to reflect the transfer of the species Dactylomyces thermophilus to the genus Thermoascus, for example, as noted in Salar et al., Journal of Agricultural Technology (2007), 3(1): 77-107. However, regardless of taxonomic classification, a person of skill in the art would be able to identify the organism used to determine the sequences disclosed herein for example based on the strain's accession number (ATCC 26413).
  • Headings, and other identifiers e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims.
  • the use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.
  • DNA or "RNA” molecule or sequence (as well as sometimes the term “oligonucleotide”) refers to a molecule comprised generally of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C). In “RNA”, T is replaced by uracil (U).
  • rDNA recombinant DNA
  • polynucleotide or “nucleic acid molecule” refers to a polymer of nucleotides and includes DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA), and chimeras thereof.
  • the nucleic acid molecule can be obtained by cloning techniques or synthesized.
  • DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]).
  • nucleic acid molecule and “polynucleotide” as are analogs thereof (e.g., generated using nucleotide analogs, e.g., inosine or phosphorothioate nucleotides). Such nucleotide analogs can be used, for example, to prepare polynucleotides that have altered base-pairing abilities or increased resistance to nucleases.
  • a nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar- phosphodiester linkages, peptide-nucleic acid bonds (referred to as "peptide nucleic acids" (PNA); Hydig-Hielsen et al., PCT Int'l Pub. No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages or combinations thereof.
  • Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy substitutions (containing a 2'-0-methylribofuranosyl moiety; see PCT No.
  • Nitrogenous bases may be conventional bases (A, G, C, T, U), known analogs thereof (e.g., inosine or others; see “The Biochemistry of the Nucleic Acids 5-36", Adams et al., ed., 11 th ed., 1992), or known derivatives of purine or pyrimidine bases (see, Cook, PCT Int'l Pub. No. WO 93/13121 ) or "abasic" residues in which the backbone includes no nitrogenous base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481 ).
  • a nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs).
  • an "isolated nucleic acid molecule” refers to a polymer of nucleotides, and includes, but should not be limited to DNA and RNA.
  • the "isolated” nucleic acid molecule is purified from its natural in vivo state, obtained by cloning or chemically synthesized.
  • gene and “recombinant gene” refer to nucleic acid molecules which may be isolated from chromosomal DNA, and very often include an open reading frame encoding a protein, e.g., polypeptides of the present invention.
  • a gene may include coding sequences, non-coding sequences, introns and regulatory sequences, as well known.
  • Amplification refers to any in vitro procedure for obtaining multiple copies ("amplicons") of a target nucleic acid sequence or its complement or fragments thereof.
  • In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement.
  • In vitro amplification methods include, e.g., transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification and strand-displacement amplification (SDA including multiple strand-displacement amplification method (MSDA)).
  • Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as Q ⁇ -replicase (e.g., Kramer et al., U.S. Pat. No. 4,786,600).
  • PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat. Nos. 4,683, 195, 4,683,202, and 4,800, 159).
  • LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., EP Pat. App. Pub. No. 0320308).
  • SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., Walker et al., U.S. Pat. No. 5,422,252).
  • oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase (e.g., see Kwoh et al., 1990, Am. Biotechnol. Lab. 8: 14 25 and Kwoh et al., 1989, Proc. Nati. Acad. Sci.
  • oligos are designed to bind to a complementary sequence under selected conditions.
  • the terminology "amplification pair” or “primer pair” refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes.
  • hybridizing and “hybridizes” are intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 60%, at least about 70%, at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, more preferably at least 95%, more preferably at least 98% or more preferably at least 99% homologous to each other typically remain hybridized to each other.
  • hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 1X SSC, 0.1 % SDS at 50°C, preferably at 55°C, preferably at 60°C and even more preferably at 65°C.
  • Highly stringent conditions include, for example, hybridizing at 68°C in 5x SSC/5x Denhardt's solution / 1.0% SDS and washing in 0.2x SSC/0.1 % SDS at room temperature. Alternatively, washing may be performed at 42°C. The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions.
  • a polynucleotide which hybridizes only to a poly (A) sequence such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) residues, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • identity and “percent identity” are used interchangeably herein.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are the same length.
  • the term "identical” or “percent identity” in the context of two or more nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95% identity, more preferably at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical.
  • Such a definition also applies to the complement of a test sequence.
  • the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length.
  • Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson et al., Nucl. Acids Res. (1994), 22(22): 4673-4680) or FASTDB (Brutlag et al., Comp. Appl. Biosci. (1990), 6(3): 237-245), as known in the art.
  • the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10.
  • the present invention also relates to nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above-described hybridizing molecule.
  • the term "being degenerate as a result of the genetic code” means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.
  • the present invention also relates to nucleic acid molecules which comprise one or more mutations or deletions, and to nucleic acid molecules which hybridize to one of the herein described nucleic acid molecules, which show (a) mutation(s) or (a) deletion(s).
  • the skilled person will appreciate that all these different algorithms or programs will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • homology refers to a similarity between two polypeptide sequences, but take into account changes between amino acids (whether conservative or not).
  • amino acids can be classified by charge, hydrophobicity, size, etc. It is also well known in the art that amino acid changes can be conservative (e.g., they do not affect significantly, or at all, the function of the protein).
  • homology introduces evolutionistic notions (e.g., pressure from evolution to retain a function of essential or important regions of a sequence, while enabling a certain drift of less important regions).
  • the skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Wo/. Biol. (48): 444-453 (1970)) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity two amino acid or nucleotide sequence is determined using the algorithm of E. Meyers and W.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the Basic Local Alignment Search Tool (BLAST) software package as described by Altschul et al., (1990) J. Wo/. Biol. 215:403-10.
  • BLAST nucleotide searches can be performed with the BLASTN program, to obtain nucleotide sequences homologous to nucleic acid molecules of the invention.
  • BLAST protein searches can be performed with the BLASTP program, to obtain amino acid sequences homologous to protein molecules of the invention.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17): 3389-3402.
  • the default parameters of the respective programs e.g., BLASTP and BLASTN
  • BLASTP and BLASTN can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.
  • corresponding to refers to one or more nucleotide or amino acid positions that are determined to correspond to one another based on sequence and/or structural alignments with a specified reference gene sequence, coding sequence, or protein.
  • a position "corresponding to" an amino acid position of a given protein can be determined empirically by aligning the sequence of amino acids of that given protein with that of a polypeptide of interest that shares a level of sequence identity therewith.
  • Corresponding positions can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, for example, such that identity between the sequences is greater than 95%, 96%, 97%, 98% or 99% or more.
  • Corresponding positions also can be based on structural alignments, for example by using computer simulated alignments of protein structure. Recitation that amino acids of a polypeptide correspond to amino acids in a disclosed sequence refers to amino acids identified upon alignment of the polypeptide with the disclosed sequence to maximize identity or homology (where conserved amino acids are aligned) using a standard alignment algorithm, such as the GAP algorithm.
  • sufficiently complementary is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another sequence by hydrogen bonding between a series of complementary bases.
  • Complementary base sequences may be complementary at each position in sequence by using standard base pairing (e.g., G:C, A:T or A:U pairing) or may contain one or more residues (including abasic residues) that are not complementary by using standard base pairing, but which allow the entire sequence to specifically hybridize with another base sequence in appropriate hybridization conditions.
  • Contiguous bases of an oligomer are preferably at least about 80% (81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% complementary to the sequence to which the oligomer specifically hybridizes.
  • Appropriate hybridization conditions are well known to those skilled in the art, can be predicted readily based on sequence composition and conditions, or can be determined empirically by using routine testing (see Sambrook ef al., Molecular Cloning, A Laboratory Manual, 2 nd ed.
  • the present invention refers to a number of units or percentages that are often listed in sequences. For example, when referring to "at least 80%, at least 85%, at least 90%.", or "at least about 80%, at least about 85%, at least about 90%.", every single unit is not listed, for the sake of brevity. For example, some units (e.g., 81 , 82, 83, 84, 85,... 91 , 92%.%) may not have been specifically recited but are considered encompassed by the present invention. The non-listing of such specific units should thus be considered as within the scope of the present invention.
  • Nucleic acid sequences may be detected by using hybridization with a complementary sequence (e.g., oligonucleotide probes) (see U.S. Patent Nos. 5,503,980 (Cantor), 5,202,231 (Drmanac et al.), 5, 149,625 (Church et al.), 5, 1 12,736 (Caldwell et al.), 5,068, 176 (Vijg et al.), and 5,002,867 (Macevicz)).
  • a complementary sequence e.g., oligonucleotide probes
  • Hybridization detection methods may use an array of probes (e.g., on a DNA chip) to provide sequence information about the target nucleic acid which selectively hybridizes to an exactly complementary probe sequence in a set of four related probe sequences that differ one nucleotide (see U.S. Patent Nos. 5,837,832 and 5,861 ,242 (Chee et al.)).
  • a detection step may use any of a variety of known methods to detect the presence of nucleic acid by hybridization to an oligonucleotide probe.
  • the types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection).
  • Labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds (e.g., protein detection by far western technology: Guichet et al, 1997, Nature 385(6616): 548-552; and Schwartz et al, 2001 , EMBO 20(3): 510- 519).
  • kits containing reagents of the present invention on a dipstick setup and the like include kits containing reagents of the present invention on a dipstick setup and the like.
  • a detection method which is amenable to automation.
  • a non-limiting example thereof includes a chip or other support comprising one or more (e.g., an array) of different probes.
  • a "label” refers to a molecular moiety or compound that can be detected or can lead to a detectable signal.
  • a label is joined, directly or indirectly, to a nucleic acid probe or the nucleic acid to be detected (e.g., an amplified sequence) or to a polypeptide (including for example an antibody) to be detected.
  • Direct labeling can occur through bonds or interactions that link the label to the polynucleotide or polypeptide (e.g., covalent bonds or non-covalent interactions), whereas indirect labeling can occur through the use of a "linker” or bridging moiety, such as additional nucleotides, amino acids or other chemical groups, which are either directly or indirectly labeled.
  • Bridging moieties may amplify a detectable signal.
  • Labels can include any detectable moiety (e.g., a radionuclide, ligand or binding agent such as an antibody (e.g., of an organism which is of a different species than that of the antigen which it recognizes), biotin or (strept)avidin, an enzyme or enzyme substrate, a reactive group, a chromophore such as a dye or colored particle, a precursor of a chromophore, a luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and a fluorescent compound).
  • detectable moiety e.g., a radionuclide, ligand or binding agent such as an antibody (e.g., of an organism which is of a different species than that of the antigen which it recognizes), biotin or (strept)avidin, an enzyme or enzyme substrate, a reactive group, a chromophore
  • a "label" of a nucleic acid or polypeptide as used herein refers to a molecular moiety or compound that is not associated with (e.g., not attached to) the nucleic acid or polypeptide as it occurs in nature, and further does not refer to an inherent property of the nucleic acid or polypeptide as it occurs in nature. I n an embodiment, the label is a synthetic label.
  • expression is meant the process by which a gene or otherwise nucleic acid sequence eventually produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptide(s).
  • peptide and oligopeptide are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context required to indicate a chain of at least two amino acids coupled by peptidyl linkages.
  • polypeptide is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxyl terminus.
  • the one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al., supra. Sequence Listings programs can convert easily this one-letter code of amino acids sequence into a three-letter code.
  • mature polypeptide is defined herein as a polypeptide having biological activity, a polypeptide of the present invention that is in its final form, following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, removal of signal sequences, glycosylation, phosphorylation, etc.
  • polypeptides of the present invention comprise mature of polypeptides of any one of the polypeptides disclosed herein. Mature polypeptides of the present invention can be predicted using programs such as SignalP.
  • mature polypeptide coding sequence is defined herein as a nucleotide sequence that encodes a mature polypeptide as defined above. As well known, some nucleotide sequences are non-coding.
  • the term "purified” or “isolated” refers to a molecule (e.g., polynucleotide or polypeptide) having been separated from a component of the composition in which it was originally present.
  • an "isolated polynucleotide” or “isolated polypeptide” has been purified to a level not found in nature.
  • a “substantially pure” molecule is a molecule that is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
  • the term “crude” means molecules that have not been separated from the components of the original composition in which it was present.
  • the units e.g., 66, 67...81 , 82, 83, 84, 85, ...91 , 92%.
  • an "isolated polynucleotide” or “isolated nucleic acid molecule” is a nucleic acid molecule (DNA or RNA) that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
  • an "isolated polypeptide” or “isolated protein” is intended to include a polypeptide or protein removed from its native environment.
  • recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31 -40 (1988).
  • variant refers herein to a polypeptide, which is substantially similar in structure (e.g., amino acid sequence) to a polypeptide disclosed herein or encoded by a nucleic acid sequence disclosed herein without being identical thereto.
  • a variant can comprise an insertion, substitution, or deletion of one or more amino acids as compared to its corresponding native protein.
  • a variant can comprise additional modifications (e.g., post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc).
  • biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid.
  • Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves.
  • Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste or a combination thereof.
  • biomass examples include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, and animal manure or a combination thereof.
  • Biomass that is useful for the invention may include biomass that has a relatively high carbohydrate value, is relatively dense, and/or is relatively easy to collect, transport, store and/or handle.
  • biomass that is useful includes corn cobs, corn stover, sawdust, and sugar cane bagasse.
  • the terms “cellulosic” or “cellulose-containing material” refers to a composition comprising cellulose.
  • the term “lignocellulosic” refers to a composition comprising both lignin and cellulose.
  • Lignocellulosic material may also comprise hemicellulose.
  • the predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemi-cellulose, and the third is pectin.
  • the secondary cell wall produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose.
  • Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.
  • Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees.
  • the cellulose-containing material can be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues.
  • the cellulose-containing material can be any type of biomass including, but not limited to, wood resources, municipal solid waste, wastepaper, crops, and crop residues (e.g., see Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman. 1994.
  • the cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix.
  • cellulolytic enhancing activity or “cellulolysis-enhancing” is defined herein as a biological activity which enhances the hydrolysis of a cellulose-containing material by proteins having cellulolytic activity.
  • cellulolytic activity is defined herein as a biological activity which hydrolyzes a cellulose- containing material.
  • lignocellulolytic enhancing activity or “Ngnocellulolysis-enhancing” is defined herein as a biological activity which enhances the hydrolysis of a lignocellulose-containing material by proteins having lignocellulolytic activity.
  • lignocellulolytic activity is defined herein as a biological activity which hydrolyzes a lignocellulose-containing material.
  • thermostable refers to an enzyme that retains its function or protein activity at a temperature greater than 50°C; thus, a thermostable cellulose-degrading or cellulase-enhacing enzyme/protein retains the ability to degrade or enhace the degradation of cellulose at this elevated temperature.
  • a protein or enzyme may have more than one enzymatic activity.
  • some polypeptides of the present invention exhibit bifunctional activities such as xylosidase/ arabinosidase activity. Such bifunctional enzymes may exhibit thermostability with regard to one activity, but not another, and still be considered as "thermostable”.
  • Figure 1 is a schematic map of the pGBFIN-49 expression plasmid.
  • Figure 2 shows results of the determination of the temperature optimum of RHIMI_1_02182 using assay protocol CU2 on beta-mannan.
  • Figure 3 shows results of the determination of the temperature optimum of Humhy2p7_013018 using assay protocol CU7.
  • Figure 4 shows results of the determination of the temperature optimum of Humhy2p7_015152 using assay protocol CU2 on xylan.
  • the present invention relates to isolated polypeptides secreted by Rhizomucor miehei, Thermoascus thermophilus (Dactylomyces thermophilus), or Humicola hyalothermophila (e.g., Rhizomucor miehei strain CBS 182.67, Thermoascus thermophilus (Dactylomyces thermophilus) strain ATCC 26413, or Humicola hyalothermophila strain CBS 454.80) having an activity relating to the processing or degradation of biomass (e.g., cell wall deconstruction).
  • Rhizomucor miehei Rhizomucor miehei
  • Humicola hyalothermophila e.g., Rhizomucor miehei strain CBS 182.67, Thermoascus thermophilus (Dactylomyces thermophilus) strain ATCC 26413, or Humicola hyalothermophila strain CBS 454.80
  • the present invention relates to isolated polypeptides comprising the amino acid sequences shown in any one of SEQ ID NOs: 47-92, 133-172, and 298-422.
  • the present invention relates to isolated polypeptides sharing a minimum threshold of amino acid sequence identity with any one of the above-mentioned polypeptides.
  • the present invention relates to isolated polypeptides having at least 60%, 65%, 70%, 71 %, 72, 73%, 74%, 75%, 76%, 77%, 78%, 79, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of the above-mentioned polypeptides.
  • Other specific percentage units that have not been specifically recited here for brevity are nevertheless considered within the scope of the present invention.
  • the present invention relates to a polypeptide encoded by a polynucleotide of the present invention, which includes coding (e.g., SEQ ID NOs: 1-46, 93-132 and 173-297) nucleic acid sequences disclosed herein, polynucleotides hybridizing under medium-high, high, or very high stringency conditions with a full- length complement thereof, as well as polynucleotides sharing a certain degree of nucleic acid sequence identity therewith.
  • coding e.g., SEQ ID NOs: 1-46, 93-132 and 173-297
  • the present invention relates to functional variants of any one of the above- mentioned polypeptides.
  • the term "functional” or “biologically active” relates to the native enzymatic (e.g., catalytic) activity of a polypeptide of the present invention.
  • the present invention relates to a polypeptide comprising a biological activity of any one of the enzymes described below, or a polynucleotide encoding same.
  • Carbohydrase refers to any protein that catalyzes the hydrolysis of carbohydrates.
  • glycoside hydrolase “glycosyl hydrolase” or “glycosidase” refers to a protein that catalyzes the hydrolysis of the glycosidic bonds between carbohydrates or between a carbohydrate and a non-carbohydrate residue.
  • Endoglucanases cellobiohydrolases, beta-glucosidases, alpha-glucosidases, xylanases, beta-xylosidases, alpha-xylosidases, galactanases, alpha-galactosidases, beta-galactosidases, alpha-amylases, glucoamylases, endo-arabinases, arabinofuranosidases, mannanases, beta-mannosidases, pectinases, acetyl xylan esterases, acetyl mannan esterases, ferulic acid esterases, coumaric acid esterases, pectin methyl esterases, and chitosanases are examples of glycosidases.
  • Cellulase refers to a protein that catalyzes the hydrolysis of 1 ,4-D-glycosidic linkages in cellulose (such as bacterial cellulose, cotton, filter paper, phosphoric acid swollen cellulose, Avicel®); cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose); plant lignocellulosic materials, beta-D-glucans or xyloglucans.
  • Cellulose is a linear beta-(1 -4) glucan consisting of anhydrocellobiose units. Endoglucanases, cellobiohydrolases, and beta- glucosidases are examples of cellulases.
  • Endoglucanase refers to a protein that catalyzes the hydrolysis of cellulose to oligosaccharide chains at random locations by means of an endoglucanase activity.
  • Cerabiohydrolase refers to a protein that catalyzes the hydrolysis of cellulose to cellobiose via an exoglucanase activity, sequentially releasing molecules of cellobiose from the reducing or non-reducing ends of cellulose or cello- oligosaccharides.
  • Beta-glucosidase refers to an enzyme that catalyzes the conversion of cellobiose and oligosaccharides to glucose.
  • Hemicellulase refers to a protein that catalyzes the hydrolysis of hemicellulose, such as that found in lignocellulosic materials. Hemicelluloses are complex polymers, and their composition often varies widely from organism to organism, and from one tissue type to another. Hemicelluloses include a variety of compounds, such as xylans, arabinoxylans, xyloglucans, mammans, glucomannans, and galacto(gluco)mannans. Hemicellulose can also contain glucan, which is a general term for beta-linked glucose residues. In general, a main component of hemicellulose is beta-1 ,4-linked xylose, a five carbon sugar.
  • this xylose is often branched as beta-1 ,3 linkages or beta-1 ,2 linkages, and can be substituted with linkages to arabinose, galactose, mannose, glucuronic acid, or by esterification to acetic acid.
  • Hemicellulolytic enzymes i.e., hemicellulases
  • Hemicellulases also include the accessory enzymes, such as acetylesterases, ferulic acid esterases, and coumaric acid esterases.
  • xylanases and acetyl xylan esterases cleave the xylan and acetyl side chains of xylan and the remaining xylo-oligomers are unsubstituted and can thus be hydrolysed with beta-xylosidase only.
  • beta-xylosidase beta-xylosidase
  • several less known side activities have been found in enzyme preparations which hydrolyze hemicellulose. Accordingly, xylanases, acetylesterases and beta- xylosidases are examples of hemicellulases.
  • Xylanase specifically refers to an enzyme that hydrolyzes the beta-1 ,4 bond in the xylan backbone, producing short xylooligosaccharides.
  • Beta-mannanase or "endo-1,4-beta-mannosidase” refers to a protein that hydrolyzes mannan- based hemicelluloses (mannan, glucomannan, galacto(gluco)mannan) and produces short beta-1 ,4- mannooligosaccharides.
  • Mannan endo-1 ,6-alpha-mannosidase refers to a protein that hydrolyzes 1 ,6-alpha-mannosidic linkages in unbranched 1 ,6-mannans.
  • Beta-mannosidase (beta-1 ,4-mannoside mannohydrolase; EC 3.2.1.25) refers to a protein that catalyzes the removal of beta-D-mannose residues from the non-reducing ends of oligosaccharides.
  • Galactanase refers to a protein that catalyzes the hydrolysis of endo-1 ,4-beta-D-galactosidic linkages in arabinogalactans.
  • Glucoamylase refers to a protein that catalyzes the hydrolysis of terminal 1 ,4-linked-D-glucose residues successively from non-reducing ends of the glycosyl chains in starch with the release of beta-D-glucose.
  • Beta-hexosaminidase or “beta-N-acetylglucosaminidase” refers to a protein that catalyzes the hydrolysis of terminal N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosamines.
  • Alpha-L-arabinofuranosidase refers to a protein that hydrolyzes arabinofuranosyl-containing hemicelluloses or pectins. Some of these enzymes remove arabinofuranoside residues from 0-2 or 0-3 single substituted xylose residues, as well as from 0-2 and/or 0-3 double substituted xylose residues. Some of these enzymes remove arabinose residues from arabinan oligomers.
  • Endo-arabinase refers to a protein that catalyzes the hydrolysis of 1 ,5-alpha-arabinofuranosidic linkages in 1 ,5-arabinans.
  • Exo-arabinase refers to a protein that catalyzes the hydrolysis of 1 ,5-alpha-linkages in 1 ,5-arabinans or 1 ,5-alpha-L arabino-oligosaccharides, releasing mainly arabinobiose, although a small amount of arabinotriose can also be liberated.
  • Beta-xylosidase refers to a protein that hydrolyzes short 1 ,4-beta-D-xylooligomers into xylose.
  • Cerellobiose dehydrogenase refers to a protein that oxidizes cellobiose to cellobionolactone.
  • Chitosanase refers to a protein that catalyzes the endohydrolysis of beta-1 ,4-linkages between D- glucosamine residues in acetylated chitosan (i.e., deacetylated chitin).
  • Exo-polygalacturonase refers to a protein that catalyzes the hydrolysis of terminal alpha 1 ,4-linked galacturonic acid residues from non-reducing ends thus converting polygalacturonides to galacturonic acid.
  • Alcohol xylan esterase refers to a protein that catalyzes the removal of the acetyl groups from xylose residues.
  • Alcohol mannan esterase refers to a protein that catalyzes the removal of the acetyl groups from mannose residues
  • ferulic esterase or "ferulic acid esterase” refers to a protein that hydrolyzes the ester bond between the arabinose substituent group and ferulic acid.
  • Coumaric acid esterase refers to a protein that hydrolyzes the ester bond between the arabinose substituent group and coumaric acid.
  • Acetyl xylan esterases, ferulic acid esterases and pectin methyl esterases are examples of carbohydrate esterases.
  • Pectate lyase and pectin lyases refer to proteins that catalyze the cleavage of 1 ,4-alpha-D- galacturonan by beta-elimination acting on polymeric and/or oligosaccharide substrates (pectates and pectins, respectively).
  • Endo-1 ,3-beta-glucanase or “laminarinase” refers to a protein that catalyzes the cleavage of 1 ,3- linkages in beta-D-glucans such as laminarin or lichenin.
  • Laminarin is a linear polysaccharide made up of beta-1 , 3- glucan with beta-1 , 6-linkages.
  • lichenan refers to a protein that catalyzes the hydrolysis of lichenan, a linear, 1 ,3-1 ,4-beta-D glucan.
  • Rhamnogalacturonan is composed of alternating alpha-1 ,4-rhamnose and alpha-1 ,2-linked galacturonic acid, with side chains linked 1 ,4 to rhamnose.
  • the side chains include Type I galactan, which is beta- 1 ,4-linked galactose with alpha-1 ,3-linked arabinose substituents; Type II galactan, which is beta-1 , 3-1 , 6-linked galactoses (very branched) with arabinose substituents; and arabinan, which is alpha-1 , 5-linked arabinose with alpha-1 ,3-linked arabinose branches.
  • the galacturonic acid substituents may be acetylated and/or methylated.
  • Exo-rhamnogalacturonanase refers to a protein that catalyzes the degradation of the rhamnogalacturonan backbone of pectin from the non-reducing end.
  • Rhamnogalacturonan acetylesterase refers to a protein that catalyzes the removal of the acetyl groups ester-linked to the highly branched rhamnogalacturonan (hairy) regions of pectin.
  • Rhamnogalacturonan lyase refers to a protein that catalyzes the degradation of the rhamnogalacturonan backbone of pectin via a beta-elimination mechanism (e.g., see Pages et al., J. Baderioi, 185:4727-4733 (2003)).
  • Alpha-rhamnosidase refers to a protein that catalyzes the hydrolysis of terminal non-reducing alpha- L-rhamnose residues in alpha-L-rhamnosides.
  • Certain proteins of the present invention may be classified as "Family 61 glycosidases" based on homology of the polypeptides to CAZy Family GH61.
  • Family 61 glycosidases may exhibit cellulolytic enhancing activity (e.g., polysaccharide monooxygenase) activity. Additional information on the properties of Family 61 glycosidases may be found in U.S. Patent Application Publication Nos. 2005/0191736, 2006/0005279, 2007/0077630, and in PCT Publication No.. WO 2004/031378.
  • Esterases represent a category of various enzymes including lipases, phospholipases, cutinases, and phytases that catalyze the hydrolysis and synthesis of ester bonds in compounds.
  • EC 3 Hydrolases catalyze the hydrolysis of various bonds
  • EC 4 Lyases cleave various bonds by means other than hydrolysis and oxidation
  • EC 5 Isomerases catalyze isomerization changes within a single molecule
  • EC 6 Ligases join two molecules with covalent bonds.
  • polypeptides/enzymes of the present invention are not meant to be limited to specific enzyme classes as they currently exist.
  • the skilled person would know how to appropriately reclassify (and assign the appropriate functions) to the enzymes of the present invention based on the amino acid sequence information provided herein. Such reclassifications are thus within the scope of the present invention.
  • the present invention relates to a polypeptide comprising a biological activity of any one of the enzymes (or sub-classes thereof), or a polynucleotide encoding same.
  • Cellulose-hydrolyzing enzymes including: endoglucanases (EC 3.2.1.4), which hydrolyze the beta-1 ,4- linkages between glucose units; exoglucanases (also known as cellobiohydrolases 1 and 2) (EC 3.2.1.91), which hydrolyze cellobiose, a glucose disaccharide, from the reducing and non-reducing ends of cellulose; and beta-glucosidases (EC 3.2.1.21 ), which hydrolyze the beta-1 ,4 glycoside bond of cellobiose to glucose;
  • Proteins that enhance or accelerate the action of cellulose-degrading enzymes including: glycoside hydrolase family 61 (GH61), recently reclassified as AA9, proteins (e.g., polysaccharide monooxygenases), which enhance the action of cellulose enzymes on lignocellulose substrates;
  • GH61 glycoside hydrolase family 61
  • AA9 proteins (e.g., polysaccharide monooxygenases), which enhance the action of cellulose enzymes on lignocellulose substrates)
  • Enzymes that degrade or modify xylan and/or xylan-lignin complexes including: xylanases, such as endo- 1,4-beta-xylanase (EC 3.2.1.8), which catalyze the endohydrolysis of 1-4-beta-D-xylosidic linkages in xylans (or xyloglucans); xylosidases, such as xylan 1,4-beta-xylosidases (EC 3.2.1.37), which catalyze hydrolysis of 1 ,4-beta-D-xylans to remove successive D-xylose residues from the non-reducing terminals, and also cleaves xylobiose; arabinosidases, such as alpha-arabinofuranosidases (EC 3.2.1.55), which hydrolyze terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabino
  • Enzymes that degrade or modify mannan including: mannanases, such as mannan endo-1,4-beta- mannosidase (EC 3.2.1.78), which catalyze random hydrolysis of 1 ,4-beta-D-mannosidic linkages in mannans, galactomannans and glucomannans;
  • mannanases such as mannan endo-1,4-beta- mannosidase (EC 3.2.1.78), which catalyze random hydrolysis of 1 ,4-beta-D-mannosidic linkages in mannans, galactomannans and glucomannans
  • alpha-galactosidases (EC 3.2.1.25), which hydrolyze terminal, non-reducing beta-D-mannose residues in beta-D- mannosides; alpha-galactosidases (EC 3.2.1.22), which hydrolyzes terminal, non-reducing alpha-D- galactose residues in alpha-D-galactosides (including galactose oligosaccharides, galactomannans and galactohydrolase); and mannan acetyl esterases;
  • Enzymes that degrade or modify xyloglucans including: xyloglucanases such as xyloglucan-specific endo- beta-1 ,4-glucanase (EC 3.2.1.151 ), which involves endohydrolysis of 1 ,4-beta-D-glucosidic linkages in xyloglucan; and xyloglucan-specific exo-beta-1 ,4-glucanase (EC 3.2.1.155), which catalyzes exohydrolysis of 1 ,4-beta-D-glucosidic linkages in xyloglucan; endoglucanases / cellulases;
  • xyloglucanases such as xyloglucan-specific endo- beta-1 ,4-glucanase (EC 3.2.1.151 ), which involves endohydrolysis of 1 ,4-beta-D-glucosidic linkages in xyloglucan
  • Enzymes that degrade or modify glucans including: Enzymes that degrade beta-1 ,4-glucan, such as endoglucanases; cellobiohydrolases; and beta-glucosidases;
  • Enzymes that degrade beta-1 ,3-1 ,4-glucan such as endo-beta-1 ,3(4)-glucanases (EC 3.2.1.6), which catalyzes endohydrolysis of 1 ,3- or 1 ,4-linkages in beta-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is itself substituted at C-3; endoglucanases (beta-glucanase, cellulase), and beta-glucosidases;
  • galactanases EC 3.2.1.23
  • beta-D-galactose residues in beta-D-galactosides EC 3.2.1.23
  • Enzymes that degrade or modify arabinans including: arabinanases (EC 3.2.1.99), which catalyze endohydrolysis of 1 ,5-alpha-arabinofuranosidic linkages in 1 ,5-arabinans;
  • Enzymes that degrade or modify starch including: amylases, such as alpha-amylases (EC 3.2.1.1 ), which catalyze endohydrolysis of 1 ,4-alpha-D-glucosidic linkages in polysaccharides containing three or more 1 ,4- alpha-linked D-glucose units; and glucosidases, such as alpha-glucosidases (EC 3.2.1.20), which hydrolyze terminal, non-reducing 1 ,4-linked alpha-D-glucose residues with release of alpha-D-glucose; Enzymes that degrade or modify pectin, including: pectate lyases (EC 4.2.2.2), which carry out eliminative cleavage of pectate to give oligosaccharides with 4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non- reducing ends; pectin lyases (EC 4.2.2.10), which
  • Enzymes that degrade or modify lignin including: lignin peroxidases (EC 1.11.1.14), which oxidize lignin and lignin model compounds using hydrogen peroxide; manganese-dependent peroxidases (EC 1.11.1.14), which oxidize lignin and lignin model compounds using hydrogen peroxide; manganese-dependent peroxidases (EC 1.11.1.14), which oxidize lignin and lignin model compounds using hydrogen peroxide; manganese-dependent peroxidases (EC 1.11.1.14), which oxidize lignin and lignin model compounds using hydrogen peroxide; manganese-dependent peroxidases (EC 1.11.1.14), which oxidize lignin and lignin model compounds using hydrogen peroxide; manganese-dependent peroxidases (EC 1.11.1.14), which oxidize lignin and lignin model compounds using hydrogen peroxide; manganese-dependent peroxidases (EC
  • Enzymes acting on chitin including: chitinases (EC 3.2.1.14), which catalyze random hydrolysis of N- acetyl-beta-D-glucosaminide 1 ,4-beta-linkages in chitin and chitodextrins; and hexosaminidases, such as beta-N-acetylhexosaminidase (EC 3.2.1.52), which hydrolyzes terminal non-reducing N-acetyl-D- hexosamine residues in N-acetyl-beta-D-hexosaminides.
  • chitinases EC 3.2.1.14
  • hexosaminidases such as beta-N-acetylhexosaminidase (EC 3.2.1.52), which hydrolyzes terminal non-reducing N-acetyl-D- hexosamine residues in N-acetyl-beta-D
  • the present invention includes the polypeptides and their corresponding activities as defined in Tables 1A-1 C, as well as functional variants thereof.
  • a functional variant as used herein is intended to include a polypeptide which is sufficiently similar in structure and function to any one of the above-mentioned polypeptides (without being identical thereto) to maintain at least one of its native biological activities.
  • a functional variant can comprise an insertion, substitution, or deletion of one or more amino acids as compared to its corresponding native protein.
  • a functional variant can comprise additional modifications (e.g., post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc).
  • functional variants of the present invention can contain one or more conservative substitutions of a polypeptide sequence disclosed herein. Such modifications can be carried out routinely using site-specific mutagenesis.
  • conservative substitution is intended to indicate a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acids having similar side chains are known in the art and include amino acids with basic side chains (e.g., lysine, arginine and hystidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).
  • basic side chains e.g., lysine, arginine and hystidine
  • acidic side chains e.g
  • non-essential amino acid is a residue that can be altered in a polypeptide of the present invention without substantially altering its (biological) function or protein activity.
  • amino acid residues that are conserved among the proteins of the present invention having similar biological activities (and their orthologs) are predicted to be particularly unamenable to alteration.
  • functional variants can include functional fragments (i.e., biologically active fragments) of any one of the polypeptide sequences disclosed herein.
  • Such fragments include fewer amino acids than the full length protein from which they are derived, but exhibit at least one biological activity of the corresponding full-length protein.
  • biologically active fragments comprise a domain or motif with at least one activity of the full-length protein.
  • a biologically active fragment of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the present invention.
  • the present invention includes other functional variants of the polypeptides disclosed herein, which can be identified by techniques known in the art.
  • functional variants can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants), of polypeptides of the present invention for biological activity.
  • a variegated library of variants can be generated by combinatorial mutagenesis at the nucleic acid level.
  • a variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • libraries of fragments of the coding sequence of a polypeptide of the present invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
  • REM Recursive ensemble mutagenesis
  • functional variants of the present invention can encompass orthologs of the genes and polypeptides disclosed herein.
  • Orthologs of the polypeptides disclosed herein include proteins that can be isolated from other strains or species and possess a similar or identical biological activity. Such orthologs can be identified as comprising an amino acid sequence that is substantially homologous (shares a certain degree of amino acid sequence identity) with the polypeptides disclosed herein.
  • substantially homologous refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequences have a common domain.
  • amino acid or nucleotide sequences which contain a common domain having at least 70%, 71 %, 72%, 73% 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 % 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity are defined herein as sufficiently identical.
  • the present invention includes improved proteins derived from the polypeptides of the present invention.
  • Improved proteins are proteins wherein at least one biological activity is improved. Such proteins may be obtained by randomly introducing mutations along all or part of the coding sequences of the polypeptides of the present invention such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity. For instance, the art provides for standard assays for measuring the enzymatic activity of the resulting protein and thus improved proteins may be selected.
  • polypeptides of the present invention may be present alone (e.g., in an isolated or purified form), within a composition (e.g., an enzymatic composition for carrying out an industrial process), or in an appropriate host.
  • polypeptides of the present invention can be recovered and purified from cell cultures (e.g., recombinant cell cultures) by methods known in the art.
  • high performance liquid chromatography HPLC
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending on the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the present invention includes fusion proteins comprising a polypeptide of the present invention or a functional variant thereof, which is operatively linked to one or more unrelated polypeptide (e.g., heterologous amino acid sequences).
  • unrelated polypeptides or “heterologous polypeptides” or “heterologous sequences” refer to polypeptides or sequences which are usually not present close to or fused to one of the polypeptides of the present invention.
  • Such "unrelated polypeptides" or “heterologous polypeptides” having amino acid sequences corresponding to proteins which are not substantially homologous to the polypeptide sequences disclosed herein.
  • fusion protein of the present invention comprises at least two biologically active portions or domains of polypeptide sequences disclosed herein.
  • the term "operatively linked” is intended to indicate that all of the different polypeptides are fused in-frame to each other.
  • an unrelated polypeptide can be fused to the N terminus or C terminus of a polypeptide of the present invention.
  • a polypeptide of the present invention can be fused to a protein which enables or facilitates recombinant protein purification and/or detection.
  • a polypeptide of the present invention can be fused to a protein such as glutathione S-transferase (GST), and the resulting fusion protein can then be purified/detected through the high affinity of GST for glutathione.
  • GST glutathione S-transferase
  • Fusion proteins of the present invention can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences can be ligated together in frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers, which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (e.g., see Current Protocols in Moiecuiar Bioiogy, eds. Ausubel et al., John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a nucleic acid encoding a polypeptide of the present invention can be cloned into such an expression vector so that the fusion moiety is linked in-frame to the polypeptide of interest.
  • a polypeptide of the present invention can be fused to a heterologous signal sequence (e.g., at its N terminus) to facilitate its isolation, expression and/or secretion from certain host cells (e.g., mammalian and yeast host cells).
  • a heterologous signal sequence e.g., at its N terminus
  • host cells e.g., mammalian and yeast host cells.
  • Signal sequences are typically characterized by a core of hydrophobic amino acids, which are generally cleaved from the mature protein during secretion in one or more cleavage events.
  • Such signal peptides may contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.
  • the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).
  • Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California).
  • useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
  • the signal sequence can direct secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved.
  • the protein can then be readily purified from the extracellular medium by known methods.
  • a signal sequence can be linked to a fusion protein of the present invention to facilitate detection, purification, and/or recovery thereof.
  • the sequence encoding a fusion protein of the present invention may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide.
  • the marker sequence can be a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
  • a pQE vector Qiagen, Inc.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the HA tag is another peptide useful for purification, which corresponds to an epitope derived of influenza hemaglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance.
  • nucleic acid sequences of the genes disclosed herein were determined by sequencing cDNA clones or mRNA transcripts obtained from Rhizomucor miehei strain CBS 182.67, Thermoascus thermophilus (Dactylomyces thermophilus) strain ATCC 26413, and Humicola hyalothermophila strain CBS 454.80.
  • the present invention relates to polynucleotides encoding a polypeptide of the present invention, including functional variants thereof.
  • polynucleotides of the present invention comprise the coding nucleic acid sequence of any one of SEQ ID NOs: 1-46, 93-132 and 173-297, or as set forth in Tables 1A-1C.
  • the polynucleotides of the present invention are operably linked to a heterologous promoter.
  • the present invention relates to a polynucleotide comprising a fragment of a cDNA set forth in any one of SEQ ID NOs: 1-46, 93-132 and 173-297, wherein said fragment: (i) comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive residues of the above mentioned cDNA.
  • the present invention relates to isolated polynucleotides sharing a minimum threshold of nucleic acid sequence identity with any one of the above-mentioned polynucleotides.
  • the present invention relates to isolated polynucleotides having at least 60%, 65%, 70%, 71 %, 72, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity to any one of the above-mentioned polynucleotides.
  • Polynucleotides having the aforementioned thresholds of nucleic acid sequence identity can be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences of the present invention such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded polypeptide. Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • the present invention relates to a polynucleotide that hybridizes (or is hybridizable) under medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full- length complement of any one of the polynucleotides defined above.
  • very low stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.
  • low stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
  • medium stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SOS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SOS at 55°C.
  • medium-high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.
  • high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.
  • very high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.
  • a polynucleotide of the present invention can be isolated using the sequence information provided herein in conjunction with standard molecular biology techniques (e.g., as described in Sambrook et al., supra.
  • suitable hybridization oligonucleotides e.g., probes or primers
  • suitable hybridization oligonucleotides can be designed using all or a portion of the nucleic acid sequences disclosed herein and prepared by standard synthetic techniques (e.g., using an automated DNA synthesizer).
  • the oligonucleotides can be employed in hybridization and/or amplification reactions, for example, to amplify a template of cDNA, mRNA or genomic DNA, according to standard PCR techniques.
  • a polynucleotide so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • the present invention relates to polynucleotides encoding functional variants of any one of the polypeptides of the present invention, including a biologically active fragment or domain thereof.
  • the present invention can include nucleic acid molecules (e.g., oligonucleotides) sufficient for use as primers and/or hybridization probes to amplify sequence and/or identify nucleic acid molecules encoding a polypeptide of the present invention or fragments thereof. Designing such polynucleotides/oligonucleotides would be within the grasp of a person of skill in the art in view of the target sequence information disclosed herein and are thus encompassed by the present invention.
  • nucleic acid molecules e.g., oligonucleotides
  • the present invention relates to polynucleotides comprising silent mutations or mutations that do not significantly alter the (biological) function or protein activity of the encoded polypeptide.
  • Guidance concerning how to make phenotypically silent amino acid substitutions is provided for example in Bowie et al., Science 247:1306-1310 (1990) and in the references cited therein.
  • DNA sequence polymorphisms of the genes disclosed herein may exist within a given population, which may differ from the sequences disclosed herein. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Accordingly, in one embodiment, the present invention can include natural allelic variants and homologs of polynucleotides disclosed herein.
  • polynucleotides of the present invention can comprise only a portion or a fragment of the nucleic acid sequences disclosed herein. Although such polynucleotides may not encode a functional polypeptide of the present invention, they are useful for example as probes or primers in hybridization or amplification reactions.
  • Exemplary uses of such polynucleotides include: (1) isolating a gene (as allelic variant thereof) from cDNA library; (2) in situ hybridization (e.g., FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the gene as described in Verma et al., Human Chromosomes: a Manuai of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis for detecting expression of mRNA corresponding to a polypeptide disclosed herein, or a homolog, ortholog or variant thereof, in specific tissues and/or cells; and (4) probes and primers that can be used as a diagnostic tool to analyze the presence of a nucleic acid hybridizable to a polynucleotide disclosed herein in a given biological (e.g., tissue) sample.
  • a given biological e.g., tissue
  • Oligonucleotides typically comprise a region of nucleotide sequence that hybridizes (preferably under highly stringent conditions) to at least 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 37, 39, 40, 50, 60, 70, 80, 90 or 100 contiguous nucleotides of a polynucleotide of the present invention.
  • such oligonucleotides can be used for identifying and/or cloning other family members, as well as orthologs from other species.
  • the oligonucleotide can be attached to a detectable label (e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor).
  • a detectable label e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.
  • Such oligonucleotides can also be used as part of a diagnostic method or kit for identifying cells which express a polypeptide of the present invention.
  • full-length complements of any one of the polynucleotides of the present invention are also encompassed.
  • the full-length complements are antisense molecules with respect to the coding strands of polynucleotides of the present invention, which hybridize (preferably under highly stringent conditions) to at least 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 37, 39, 40, 50, 60, 70, 80, 90 or 100 contiguous nucleotides to a polynucleotide of the present invention.
  • sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.
  • the specific sequences disclosed herein can be readily used to isolate the corresponding complete genes from the organism sequenced herein, which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.
  • nucleotide sequences disclosed herein were determined by sequencing using an automated DNA sequencer, and all amino acid sequences of polypeptides disclosed herein were predicted by translation based on the genetic code. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • vectors e.g., expression vectors
  • a polynucleotide encoding a polypeptide of the present invention e.g., amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino
  • vector includes a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors useful in recombinant DNA techniques are often in the form of plasmids.
  • the terms "plasmid” and “vector” can be used interchangeably herein as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • recombinant expression vectors of the invention can comprise a polynucleotide of the present invention in a form suitable for expression of the polynucleotide in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • operatively linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technoiogy: Methods in Enzymoiogy 185, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the present invention can be introduced into host cells to thereby produce proteins or peptides, encoded by polynucleotides as described herein (e.g., polypeptides of the present invention).
  • recombinant expression vectors of the present invention can be designed for expression of polypeptides of the present invention in prokaryotic or eukaryotic cells.
  • these polypeptides can be expressed in bacterial cells such as E. coii, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel supra).
  • recombinant expression vectors of the present invention can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • expression vectors of the present invention can include chromosomal- episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage
  • a DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • promoters are preferred that are capable of directing a high expression level of biologically active polypeptides of the present invention (e.g., lignocellulose active proteins) from fungi.
  • Such promoters are known in the art.
  • the expression constructs may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipid-mediated transfection or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al., (Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methatrexate.
  • a polynucleotide encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide of the present invention, or on a separate vector. Cells stably transfected with a polynucleotide of the present invention can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • Fusion vectors add a number of amino acids to a protein encoded therein, e.g., to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: (1 ) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Vectors preferred for use in bacteria are for example disclosed in WO-A 1-2004/074468.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Known bacterial promoters suitable for use in the present invention include the promoters disclosed in WO-A1 -2004/074468.
  • the expression vectors will preferably contain selectable markers. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and antibiotic resistance (e.g., tetracyline or ampicillin) for culturing in E. coli and other bacteria.
  • Representative examples of appropriate host include bacterial cells, such as E.
  • coli Streptomyces, Salmonella typhimurium and certain Bacillus species
  • fungal cells such as Aspergillus species, for example A. niger, A. oryzae and A. nidulans
  • yeast cells such as Kluyveromyces, for example K. lactis and/or Pichia, for example P. pastoris
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS and Bowes melanoma
  • Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signal may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • a polypeptide of the present invention may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
  • additional amino acids particularly charged amino acids
  • peptide moieties may be added to the polypeptide to facilitate purification and/or detection.
  • the present invention features cells, e.g., transformed host cells or recombinant host cells that contain a polynucleotide or vector of the present invention.
  • a "transformed cell” or “recombinant cell” is a cell into which (or into an ancestor of which) has been introduced a polynucleotide or vector of the invention by means of recombinant DNA techniques.
  • prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from filamentous fungi, in particular the strain from which the polynucleotide and polypeptide sequences disclosed herein were derived.
  • a cell of the present invention is typically not a wild-type strain or a naturally- occurring cell.
  • Host cells of the present invention can include, but are not limited to: fungi (e.g., Aspergillus niger, Trichoderma reesii, Myceliophthora thermophila and Talaromyces emersonii); yeasts (e.g., Saccharomyces cerevisiae, Yarrowia lipolytica and Pichia pastoris); bacteria (e.g., Escherichia coli and Bacillus sp.); and plants (e.g., Nicotiana benthamiana, Nicotiana tabacum and Medicago sativa).
  • fungi e.g., Aspergillus niger, Trichoderma reesii, Myceliophthora thermophila and Talaromyces emersonii
  • yeasts e.g., Saccharomyces cerevisiae,
  • a polynucleotide (or a polynucleotide which is comprised within a vector) may be homologous or heterologous with respect to the cell into which it is introduced.
  • a polynucleotide is homologous to a cell if the polynucleotide naturally occurs in that cell.
  • a polynucleotide is heterologous to a cell if the polynucleotide does not naturally occur in that cell.
  • the present invention relates to a cell which comprises a heterologous or a homologous sequence corresponding to any one of the polynucleotides or polypeptides disclosed herein.
  • a host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
  • Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skill in the art can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such host cells are well known in the art.
  • host cells can also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.
  • mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.
  • a stably transfected cell line can produce the polypeptides of the present invention.
  • a number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al., (supra).
  • the present invention relates to methods of inhibiting the expression of a polypeptide of the present invention in a host cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule (or a molecule comprising region of double-strandedness), wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention.
  • dsRNA double-stranded RNA
  • the dsRNA is about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more duplex nucleotides in length.
  • the dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA).
  • the dsRNA is small interfering RNA (siRNAs) for inhibiting transcription.
  • the dsRNA is micro RNA (miRNAs) for inhibiting translation.
  • the present invention also relates to such double-stranded RNA (dsRNA) molecules, comprising a portion of the mature polypeptide coding sequence of any one of the coding sequences of the polypeptides disclosed herein of inhibiting expression of that polypeptide in a cell. While the present invention is not limited by any particular mechanism of action, the dsRNA can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs.
  • ssRNA single-stranded RNA
  • RNA interference RNA interference
  • the dsRNAs of the present invention can be used in gene-silencing methods.
  • the invention relates to methods to selectively degrade RNA using the dsRNAi's of the present invention.
  • the process may be practiced in vitro, ex vivo or in vivo.
  • the dsRNA molecules can be used to generate a loss-of-function mutation in a cell, an organ or an oganism. Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art, see, for example, U.S. Patent No.
  • the present invention relates to an isolated binding agent capable of selectively binding to a polypeptide of the present invention.
  • Suitable binding agents may be selected from an antibody, an antigen binding fragment, or a binding partner.
  • the binding agent selectively binds to an amino acid sequence selected from Tables 1A-1C, including to any fragment of any of the above sequences comprising at least one antibody binding epitope.
  • the phrase “selectively binds to” refers to the ability of an antibody, antigen binding fragment or binding partner of the present invention to preferentially bind to specified proteins. More specifically, the phrase “selectively binds” refers to the specific binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, as measured by any standard assay (e.g., an immunoassay), is statistically significantly higher than the background control for the assay.
  • any standard assay e.g., an immunoassay
  • controls when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA, immunoblot assays, etc.).
  • enzyme immunoassays e.g., ELISA, immunoblot assays, etc.
  • Antibodies are characterized in that they comprise immunoglobulin domains and as such, they are members of the immunoglobulin superfamily of proteins.
  • An antibody of the invention includes polyclonal and monoclonal antibodies, divalent and monovalent antibodies, bi- or multi-specific antibodies, serum containing such antibodies, antibodies that have been purified to varying degrees, and any functional equivalents of whole antibodies.
  • Isolated antibodies of the present invention can include serum containing such antibodies, or antibodies that have been purified to varying degrees.
  • Whole antibodies of the present invention can be polyclonal or monoclonal.
  • antibodies such as antigen binding fragments in which one or more antibody domains are truncated or absent (e.g., Fv, Fab, Fab', or F(ab)2 fragments), as well as genetically-engineered antibodies or antigen binding fragments thereof, including single chain antibodies or antibodies that can bind to more than one epitope (e.g., bi-specific antibodies), or antibodies that can bind to one or more different antigens (e.g., bi- or multi-specific antibodies), may also be employed in the invention.
  • Methods for the generation and production of antibodies are well known in the art.
  • Monoclonal antibodies may be produced according to the methodology of Kohler and Milstein (Nature 256:495-497, 1975).
  • Non-antibody polypeptides sometimes referred to as binding partners, may be designed to bind specifically to a protein of the invention. Examples of the design of such polypeptides, which possess a prescribed ligand specificity are given in Beste et al., (Proc. Nat'l Acad. Sci. 96: 1898-1903, 1999).
  • a binding agent of the invention is immobilized on a substrate such as: artificial membranes, organic supports, biopolymer supports and inorganic supports such as for use in a screening assay.
  • antibodies and binding agents specifically binding to polypeptides of the present invention may be produced and used even in absence of knowledge of the precise biological function and/or protein activity of the polypeptide.
  • Such antibodies and binding agent may be useful, for example, as diagnostic, classification, and/or research tools.
  • the antibody is of an organism which is of a different species than that of the antigen to which it selectively binds. Further, in embodiments, the antibody may bear a label as defined herein to facilitate its detection, or may for example be detected via the use of a secondary antibody which itself bears a label.
  • the present invention relates to a composition
  • a composition comprising one or more polypeptides or polynucleotides of the present invention.
  • the compositions are enriched in such a polypeptide.
  • the term "enriched" indicates that the biological activity (e.g., biomass degradation or processing) of the composition has been increased, e.g., with an enrichment factor of at least 1.1.
  • the composition may comprise a polypeptide of the present invention as the major component, e.g., a mono-component composition.
  • the composition may comprise multiple enzymatic activities (e.g., those described herein).
  • the polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the polypeptide composition may be in the form of a granulate or a microgranulate.
  • the polypeptide to be included in the composition may be stabilized in accordance with methods known in the art. Examples are given below of preferred uses of the polypeptide compositions of the present invention.
  • the dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.
  • the present invention relates to the use of the polypeptides (e.g., enzymes) of the present invention a number of industrial and other processes.
  • polypeptides e.g., enzymes
  • these advantages can include aspects such as lower production costs, higher specificity towards the substrate, greater synergies with existing enzymes, less antigenic effect, less undesirable side activities, higher yields when produced in a suitable microorganism, more suitable pH and temperature ranges, better properties of the final product, and food grade or kosher aspects.
  • the present invention seeks to provide one or more of these advantages, or others.
  • the polypeptides of the present invention may be used in new or improved methods for enzymatically degrading or converting plant cell wall polysaccharides from biomass into various useful products.
  • plant cell walls contain associated pectins and lignins, the removal of which by enzymes of the current invention can improve accessibility to cellulases and hemicellulases, or which can themselves be converted to useful products. Therefore the polypeptides of the present invention may be used to degrade biomass or pretreated biomass to sugars. These sugars may be used as such or may be, for example, fermented into ethanol.
  • polypeptides of the present invention may be used in improved methods for the processing of pretreated biomass.
  • Pretreatment technologies may involve chemical, physical, or biological treatments. Examples of pre-treatment technologies include but are not limited to: steam explosion; ammonia; acid hydrolysis; alkaline hydrolysis; solvent extraction; crushing; milling; etc.
  • Bioethanol is usually produced by the fermentation of glucose to ethanol by yeasts such as Saccharomyces cerevisiae: in addition to ethanol, other chemicals may be synthesized starting from glucose.
  • Ethanol, today is produced mostly from sugars or starches, obtained from sugar cane, fruits and grains.
  • cellulosic ethanol is obtained from cellulose, the main component of wood, straw and much of the plants.
  • Sources of biomass for cellulosic ethanol production comprise agricultural residues (e.g., leftover crop materials from stalks, leaves, and husks of corn plants), forestry wastes (e.g., chips and sawdust from lumber mills, dead trees, and tree branches), energy crops (e.g., dedicated fast- growing trees and grasses such as switch grass), municipal solid waste (e.g., household garbage and paper products), food processing and other industrial wastes (e.g., black liquor, paper manufacturing by-products, etc.).
  • agricultural residues e.g., leftover crop materials from stalks, leaves, and husks of corn plants
  • forestry wastes e.g., chips and sawdust from lumber mills, dead trees, and tree branches
  • energy crops e.g., dedicated fast- growing trees and grasses such as switch grass
  • municipal solid waste e.g., household garbage and paper products
  • food processing and other industrial wastes e.g., black liquor, paper manufacturing by-products, etc.
  • Plant biomass is a mixture of plant polysaccharides, including cellulose, hemicelluloses, and pectin, together with the structural polymer, lignin.
  • Glucose is released from cellulose by the action of mixtures of enzymes, including: endoglucanases, exoglucanases (cellobiohydrolases 1 and 2) and beta-glucosidases.
  • Efficient large-scale conversion of cellulosic materials by such mixtures may require the full complement of enzymes, and can be enhanced by the addition of enzymes that attack the other plant cell wall components (e.g., hemicelluloses, pectins, and lignins), as well as chemical linkages between these components.
  • polypeptides of the present invention that are highly expressed, or have high specific activity, stability, or resistance to inhibitors may improve the efficiency of the process, and lower enzyme costs. It would be an advantage to the art to improve the degradation and conversion of plant cell wall polysaccharides by composing cellulase mixtures using cellulase enzymes with such properties. Furthermore, polypeptides of the present invention that are able to function at extremes of pH and temperature are desirable, both since improved enzyme robustness decreases costs, and because enzymes that function at high temperature will allow high processing temperatures under high substrate consistency conditions that decrease viscosity and thus improve yields.
  • Glycoside hydrolases from the family GH61 are known to stimulate the activity of cellulose cocktails on lignocellulosic substrates and are thus considered to exhibit cellulose-enhancing activity (Harris et al., Biochemistry 49, 3305 (2010)). Enhancement of cellulase cocktail efficiency by GH61 proteins of the present invention may contribute to lowering the costs of cellulase enzymes used for the production of glucose from plant cell biomass, as described above.
  • GH61 (glycoside hydrolase family 61 or sometimes referred to as EGIV) proteins are oxygen- dependent polysaccharide monooxygenases (PMO's) according to the latest literature.
  • GH61 was originally classified as an endoglucanase, based on the measurement of very weak endo-1 ,4- -D-glucanase activity in one family member.
  • the term "GH61 " as used herein, is to be understood as a family of enzymes, which share common conserved sequence portions and foldings, originally classified in family 61 (http://www.cazy.org/GH61.html) of the well-established CAZY GH classification system, and now re-classified by CAZY as family AA9 (http://www.cazy.org/AA9.html).
  • GH61 is used herein as being part of the cellulolytic system elaborated by certain fungi to degrade cellulose.
  • Enzymatic hydrolysis of plant hemicellulose yields 5-carbon sugars that either may be fermented to ethanol by some species of yeast, or converted to other types of chemical products. Enzymatic deconstruction of hemicellulose is also known to improve the accessibility of plant cell wall cellulose to cellulase enzymes for the production of glucose from lignocellulosic materials. Hemicellulase enzymes of the present invention that enhance glucose production from lignocellulose would find utility in the bioethanol industry and in other process that rely on glucose or pentose streams from lignocellulose.
  • Lignin is composed of methoxylated phenyl-propane units linked by ether linkages and carbon-carbon bonds.
  • the chemical composition of lignin may, depending on species, include guaiacyl, 4-hydroxyphenyl, and syringyl groups.
  • Enzymatic modification of lignin by the polypeptides of the present invention can be used for the production of structural materials from plant biomass, or alternatively improve the accessibility of plant cellulose and hemicelluloses to cellulase enzymes for the release of glucose from biomass as described above.
  • Enzymes that degrade the lignin component of lignocellulose include lignin peroxidases, manganese-dependent peroxidases, versatile peroxidases, and laccases (Vicuna et al., 2000, Molecular Biotechnology 14: 173-176; Broda et al., 1996, Molecular Microbiology 19: 923-932).
  • polypeptides of the present invention may also, in certain instances, be active in the decolorization of industrial dyes, and thus useful for the treatment and detoxification of chemical wastes.
  • pectin-degrading polypeptides of the present invention can also enhance the action of cellulases on plant biomass by improving the accessibilty of cellulase to the cellulose component of lignocellulose.
  • polypeptides of the present invention may also be useful in other applications for hydrolyzing non-starch polysaccharide (NSP).
  • NSP non-starch polysaccharide
  • esterases of the present invention can be useful in the bioenergy industry such as for the production of biodiesel and hydrolysis of hemicellulose.
  • the present invention relates to methods for degrading or converting a cellulose-containing material, comprising: treating the cellulose-containing material with an effective amount of a cellulolytic enzyme composition in the presence of an effective amount of a polypeptide having cellulolytic enhancing activity of the present invention, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulose-containing material compared to the absence of the polypeptide having cellulolytic enhancing activity.
  • the present invention relates to methods for producing a fermentation product, comprising: (a) saccharifying a cellulose-containing material with an effective amount of a cellulolytic enzyme composition in the presence of an effective amount of a polypeptide having cellulolytic enhancing activity of the present invention, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulose-containing material compared to the absence of the polypeptide having cellulolytic enhancing activity; (b) fermenting the saccharified cellulose-containing material of step (a) with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
  • the present invention relates to methods for preparing a food product comprising incorporating into the food product an effective amount of a polypeptide of the present invention. This can improve one or more properties of the food product relative to a food product in which the polypeptide is not incorporated.
  • the phrase "incorporated into the food product" is defined herein as adding a polypeptide of the present invention to the food product, to any ingredient from which the food product is to be made, and/or to any mixture of food ingredients from which the food product is to be made.
  • a polypeptide of the present invention may be added in any step of the food product preparation and may be added in one, two or more steps.
  • the polypeptide of the present invention is added to the ingredients of a food product which can then be treated by methods including cooking, boiling, drying, frying, steaming or baking as is known in the art.
  • the term "effective amount” is defined herein as an amount of the polypeptide (e.g., enzyme) of the present invention that is sufficient for providing a measurable effect on at least one property of interest of the food product.
  • the term "improved property” is defined herein as any property of a food product which is improved by the action of a polypeptide (e.g., enzyme) of the present invention relative to a food product in which the polypeptide is not incorporated. The improved property may be determined by comparison of a food product prepared with and without addition of a polypeptide of the present invention. Organoleptic qualities may be evaluated using procedures well established in the food industry, and may include, for example, the use of a panel of trained taste-testers.
  • the polypeptides of the present invention may be prepared in any form suitable for the use in question, e.g., in the form of a dry powder, agglomerated powder, or granulate, in particular a non-dusting granulate, liquid, in particular a stabilized liquid, or protected enzyme such as described in WO01/1 1974 and WO02/26044.
  • Granulates and agglomerated powders may be prepared by conventional methods, e.g., by spraying the enzyme according to the invention onto a carrier in a fluid-bed granulator.
  • the carrier may consist of particulate cores having a suitable particle size.
  • the carrier may be soluble or insoluble, e.g., a salt (such as NaCI or sodium sulphate), sugar (such as sucrose or lactose), sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy.
  • a salt such as NaCI or sodium sulphate
  • sugar such as sucrose or lactose
  • sugar alcohol such as sorbitol
  • starch rice, corn grits, or soy.
  • the polypeptide of the present invention (and/or additional polypeptides/enzymes) may be contained in slow-release formulations. Methods for preparing slow-release formulations are well known in the art. Adding nutritionally acceptable stabilizers such as sugar, sugar alcohol, or another polyol, and/or lactic acid or another organic acid according to established methods may for instance, stabilize liquid enzyme preparations.
  • polypeptides of the present invention may also be incorporated in yeast- comprising compositions such as disclosed in EP-A-0619947, EP-A-0659344 and WO02/49441.
  • one or more additional polypeptides/enzymes may be incorporated into a food product of the present invention.
  • the additional enzyme may be of any origin, including mammalian and plant, and preferably of microbial (bacterial, yeast or fungal) origin and may be obtained by techniques conventionally used in the art. Enzymes may conveniently be produced in microorganisms. Microbial enzymes are available from a variety of sources; Bacillus species are a common source of bacterial enzymes, whereas fungal enzymes are commonly produced in Aspergillus species.
  • additional polypeptides/enzymes include starch degrading enzymes, xylanases, oxidizing enzymes, fatty material splitting enzymes, or protein-degrading, modifying or crosslinking enzymes.
  • Starch degrading enzymes include endo-acting enzymes such as alpha-amylase, maltogenic amylase, pullulanase or other debranching enzymes, and exo-acting enzymes that cleave off glucose (amyloglucosidase), maltose (beta-amylase), maltotriose, maltotetraose and higher oligosaccharides.
  • Suitable xylanases are for instance xylanases, pentosanases, hemicellulase, arabinofuranosidase, glucanase, cellulase, cellobiohydrolase, beta- glucosidase, and others.
  • Oxidizing enzymes are for instance glucose oxidase, hexose oxidase, pyranose oxidase, sulfhydryl oxidase, lipoxygenase, laccase, polysaccharide monooxygenase, polyphenol oxidases and others.
  • Fatty material splitting enzymes are for instance triacylglycerol lipases, phospholipases (such as A1 , A2, B, C and D) and galactolipases.
  • Protein degrading, modifying or crosslinking enzymes are for instance endo-acting proteases (serine proteases, metalloproteases, aspartyl proteases, thiol proteases), exo-acting peptidases that cleave off one amino acid, or dipeptide, tripeptide etceteras from the N-terminal (aminopeptidases) or C-terminal (carboxypeptidases) ends of the polypeptide chain, asparagines or glutamine deamidating enzymes such as deamidase and peptidoglutaminase or crosslinking enzymes such as transglutaminase.
  • additional polypeptides/enzymes can include: amylases, such as alpha- amylase (which can be useful for providing sugars that are fermentable by yeast) or beta-amylase; cyclodextrin glucanotransferase; peptidase (e.g., an exopeptidase, which can be useful in flavour enhancement); transglutaminase; lipase, which can be useful for the modification of lipids present in the food or food constituents), phospholipase, cellulase, hemicellulase, protein disulfide isomerase, peroxidase, laccase, or an oxidase (e.g., glucose oxidase, hexose oxidase, aldose oxidase, pyranose oxidase, lipoxygenase or L-amino acid oxidase).
  • amylases such as alpha- amylase (which can be useful for
  • esterases of the present invention have a number of applications in the food industry including, but not limited to, degumming vegetable oils; improving the production of bread (e.g., in situ production of emulsifiers); producing crackers, noodles, and pasta; enhancing flavor development of cheese, butter, and margarine; ripening cheese; removing wax; trans-esterification of flavors and cocoa butter substitutes; synthesizing structured lipids for infant formula and nutraceuticals; improving the polyunsaturated fatty acid content in fish oil; and aiding in digestion and releasing minerals in food processing.
  • degumming vegetable oils e.g., in situ production of emulsifiers
  • producing crackers, noodles, and pasta enhancing flavor development of cheese, butter, and margarine
  • ripening cheese removing wax
  • trans-esterification of flavors and cocoa butter substitutes synthesizing structured lipids for infant formula and nutraceuticals
  • improving the polyunsaturated fatty acid content in fish oil and aiding in digestion and
  • polypeptides of the present invention can be useful in the detergent industry, e.g., for removal of carbohydrate-based stains from soiled laundry.
  • Enzymes are used in detergents in order to improve its efficacy to remove most types of dirt.
  • esterases such as lipases of the present invention are particularly useful for removing fats and lipids.
  • polypeptides of the present invention can be useful in the feed enzyme industry, e.g., for increasing nutritional quality, digestibility and/or absorption of animal feed.
  • Feed enzymes have an important role to play in current farming systems, as they can increase the digestibility of nutrients, leading to greater efficiency in the production of animal products such as meat and eggs. At the same time, they can play a role in minimizing the environmental impact of increased animal production.
  • Non-starch polysaccharides can increase the viscosity of the digesta which can, in turn, decrease nutrient availability and animal performance.
  • Endoxylanases and phytases are the best-known feed-enzyme products.
  • Phytase enzymes hydrolyse phytic acid and release inorganic phosphate, thereby avoiding the need to add inorganic phosphates to the diet and reducing phosphorus excretion.
  • Addition of xylanases to feed has also been shown to have positive effects on animal growth. Adding specific nutrients to feed improves animal digestion and thereby reduces feed costs. A lot of feed additives are being currently used and new concepts are continuously developed.
  • Use of specific enzymes like non-starch carbohydrate degrading enzymes could breakdown fiber, releasing energy as well as increasing the protein digestibility due to better accessibility of the protein when fiber gets broken down. In this way the feed cost could come down, as well as the protein levels in the feed also could be reduced.
  • Non-starch polysaccharides are also present in virtually all feed ingredients of plant origin. NSPs are poorly utilized and can, when solubilized, exert adverse effects on digestion. Exogenous enzymes can contribute to a better utilization of these NSPs and as a consequence reduce any anti-nutritional effects. Accordingly, in a particular embodiment, hemicellulases and other polysaccharide-active polypeptides/enzymes of the present invention can be used for this purpose in cereal-based diets for poultry and, to a lesser extent, for pigs and other species.
  • esterases of the present invention are useful in the feed industry such as for reducing the amount of phosphate in feed.
  • xylanases of the present invention can be useful in the pulp and paper industry, e.g., for prebleaching of kraft pulp.
  • Xylanases have been found to be most effective for that purpose.
  • Xylanases attract increasing scientific and commercial attention due to applications in the pulp and paper industry for removal of hemicellulose from dissolving pulps or for enhancement of the bleachability of pulp and, thus, reduction of the use of environmentally harmful bleaching chemicals.
  • a similar application of xylanases for pulp prebleaching is an already well-established technology and has greatly stimulated research on hemicellulases in the past decade.
  • lignin-active peroxidases of the present invention may also be active in modification of lignin and hence have bleaching properties, such enzymes are generally less attractive for bleaching due to the need to use and recycle expensive redox mediators.
  • polypeptides such as xylanases of the present invention can be used to pre- bleach pulp to reduce the amount of bleaching chemicals to obtain a given brightness. It is suggested that xylanase depolymerises xylan blocks and increases accessibility or helps liberation of residual lignin by releasing xylan- chromophore fragments. In addition to brownstock prior to bleaching, polypeptides such as xylanases of the present invention can save on bleaching chemicals. The enzymes hydrolyze surface xylans and are able to break linkages between hemicellulose and lignin. Other polypeptides (e.g., hemicellulase active enzymes) of the present invention which can break these linkages can function effectively in bleaching or pre-bleaching of pulp, and thus such uses are also within the scope of the present invention.
  • hemicellulase active enzymes e.g., hemicellulase active enzymes
  • esterases of the present invention are useful for the removal of triglycerides, steryl esters, resin acids, free fatty acids, and sterols (e.g., lipophilic wood extractives).
  • polypeptides such as xylanases of the present invention can be used in antibacterial formulations, as well as in pharmaceutical products such as throat lozenges, toothpastes, and mouthwash.
  • Chitin is a beta-(1 ,4)-linked polymer of N-acetyl D-glucosamine (GlcNAc), found as a structural polysaccharide in fungal cell walls as well as in the exoskeleton of arthropods and the outer shell of crustaceans. Approximately 75% the total weight of shellfish, is considered waste, and a large proportion of the material making up the waste is chitin.
  • GlcNAc N-acetyl D-glucosamine
  • polypeptides such as chitin-degrading enzymes of the present invention are useful in the modification and degradation of chitin, allowing the production of chitin-derived material, such as chitooligosaccharides and N-acetyl D-glucosamine, from chitin waste.
  • polypeptides such as chitinase enzymes of the present invention can be useful as antifungal agents.
  • polypeptides of the present invention can be used in the textile industry (e.g., for the treatment of textile substrates).
  • cellulases e.g., endo-, exocellulases and cellobiohydrolases
  • enzymatic treatment by a polypeptide of the present invention is can be used in place of (or in addition to) a bleaching treatment to achieve a "used" look of jeans or other suitable fabrics.
  • Polypeptides of the present invention can also improve the softness/feel of such fabrics.
  • enzymes of the present invention can enhance cleaning ability or act as a softening agent.
  • polypeptides such as cellulases of the present invention can be used in combination with polymeric agents in processes for providing a localized variation in the color density of fibers.
  • polypeptides of the present invention can be used in the waste treatment industry (e.g., for changing the characteristics of the waste to become more amenable to further treatment and/or for bio-conversion to value-added products).
  • Polypeptides such as lipases, cellulases, amylases, and proteases of the present invention can be used in addition to microorganisms to break down polymeric substances like proteins, polysaccharides and lipids, thereby facilitating this process.
  • polypeptides of the present invention can be used in industries such as biocatalysis; sewage treatment; cleaning up oil pollution; the synthesis of fragrances; and enhancing the recovery of oil (e.g., during drilling).
  • the polynucleotides, polypeptides and antibodies of the present invention can be useful for diagnostic and classification tools.
  • designing hybridization probes or primers that are specific for a particular genus, species or strain e.g., the genus, species, or strain from which the sequences disclosed herein were derived
  • a skilled person would be able to select an epitope of a polypeptide of the present invention which is specific for a particular genus, species or strain (e.g., the genus, species, or strain from which the sequences disclosed herein were derived) and generate an antibody or binding agent that binds specifically thereto.
  • Such tools are useful, for example, in diagnostic methods for detecting the presence or absence of a particular organism (e.g., the organism from which the sequences disclosed herein were derived) in a sample; as research tools (e.g., for designing and producing microarrays for studying fungal gene expression); for rapidly classifying an organism of interest based the detection of a sequence or polypeptide specific for that organism.
  • a particular organism e.g., the organism from which the sequences disclosed herein were derived
  • research tools e.g., for designing and producing microarrays for studying fungal gene expression
  • for rapidly classifying an organism of interest based the detection of a sequence or polypeptide specific for that organism.
  • the skilled person would recognize that knowledge of the precise (biological) function or protein activity of a polypeptide of the present invention is not absolutely necessary for the aforementioned tools to be useful for diagnostic, research, or classification purposes.
  • Sequences that are particularly useful in this regard are the coding and amino acid sequences corresponding to the polypeptides of the present invention annotated as "unknown" in Tables 1A- 1 C. These sequences show little sequence identity with those in the art and thus can be useful as markers for identifying the organisms from which the sequences of the present invention were derived. The skilled person would know how to search various sequence databases to design specific hybridization oligonucleotides (e.g., probes and primers), as well as produce antibodies specifically binds to the aforementioned sequences.
  • the present invention relates to a method for identifying and/or classifying an organism (e.g., a fungal species) based on a biological sample, the method comprising detecting the presence or absence of any one of the polynucleotides or polypeptides of the present invention (e.g., those recited in the preceding paragraph) and determining that said organism is present or classifying said organism based on the presence of the polynucleotide or polypeptide.
  • the detecting step can be carried out using one or more oligonucleotides or antibodies of the present invention.
  • the detecting step can be carried out by performing an amplification and/or hybridization reaction.
  • polypeptide of the present invention may not be known per se (e.g., in the case of proteins of the present invention labelled as "unknown" in Tables 1A-1 C), the polypeptide may be nevertheless useful for carrying out an industrial process (e.g., cellulase-enhancing, cellulose-degrading, hemicellulose-degrading, cellulolysis-enhancing, lignocellulolysis-enhancing, and other biological functions listed in Tables 1A-1C).
  • an industrial process e.g., cellulase-enhancing, cellulose-degrading, hemicellulose-degrading, cellulolysis-enhancing, lignocellulolysis-enhancing, and other biological functions listed in Tables 1A-1C).
  • proteins labelled herein as "unknown” comprise proteins whose precise enzymatic activities may not be deduceable from sequence comparisons, but that are nevertheless indentified as interesting targets for industrial applications for other reasons (e.g., their expression is induced by growth under certain conditions such as in the presence of cellulostic and/or lignocellulostic biomass).
  • Example 1 Fermentation of the organisms
  • starter mycelium was grown in rich medium (either mycological broth or yeast malt broth (the latter being indicated with YM)) and then washed with water. The starter was then used to inoculate different liquid media or solid substrate and the resulting mycelium was used for RNA extraction and library construction.
  • rich medium either mycological broth or yeast malt broth (the latter being indicated with YM)
  • Trace Element Solution contains 2 mM Iron(ll) sulphate heptahydrate (FeSC hW), 1 mM Copper (II) sulphate pentahydrate (CuSCvShbO), 5 mM Zinc sulphate heptahydrate (ZnSC hbO), 10 mM Manganese sulphate monohydrate (MnSCrhbO), 5 mM Cobalt(ll) chloride hexahydrate (CoCWhbO), 0.5 mM Ammonium molybdate tetrahydrate ((NhUJeMozC ⁇ h O), and 95 mM Hydrochloric acid (HCI) dissolved in double-distilled water.
  • FeSC hW Iron(ll) sulphate heptahydrate
  • II Copper
  • CuSCvShbO Copper
  • ZnSC hbO Zinc sulphate heptahydrate
  • MnSCrhbO Manganese sulphate monohydrate
  • TDM Trametes Defined Medium
  • TDM-1 Medium was prepared as in basic recipe described above.
  • TDM-2 Quantity of asparagine monohydrate was reduced to 0.15 g.
  • the quantity of manganese sulphate monohydrate was raised to 0.2 mM final concentration in the
  • TDM-5 The quantity of copper (II) sulphate pentahydrate was raised to 20 ⁇ .
  • TDM-6 Glucose was replaced with 10 g per liter of cellulose (Solka-Floc, 200FCC)
  • TDM-7 Glucose was replaced with 10 g per liter of xylan from birchwood (Sigma Cat. # X-0502)
  • TDM-8 Glucose was replaced with 10 g per liter of wheat bran 1 .
  • TDM-9 Glucose was replaced with 10 g per liter of citrus pectin (Sigma Cat. # P-9135).
  • TDM-10 TweenTM 80 was omitted from the medium.
  • TDM-12 The double-distilled water was replaced with Whitewater 2 collected from newsprint manufacture.
  • TDM-13 Glucose was replaced with 5 g per liter of ground hardwood kraft pulp 3 .
  • TDM-14 The medium's pH was raised to 7.5.
  • TDM-15 The strain was incubated at 5°C above its optimum growth temperature.
  • TDM-16 The strain was incubated at 10°C below its optimum growth temperature.
  • Potassium phosphate monobasic was replaced with 5 mM phytic acid from rice (Sigma Cat. #
  • TDM-19 Asparagine monohydrate was increased to 4 g per liter.
  • Asparagine monohydrate was increased to 4g per liter and glucose was replaced with 2% fructose.
  • Asparagine monohydrate was increased to 4 g per liter; 100 mL of double-distilled water was
  • Asparagine monohydrate was increased to 4 g per liter; 100 mL of double-distilled water was
  • Asparagine monohydrate was increased to 4 g per liter; one half of the double-distilled water was
  • TDM-23 replaced with 25% Whitewater from newsprint manufacture plus 25% white water from peroxide bleaching. Glucose was omitted.
  • Asparagine monohydrate was increased to 4 g per liter and manganese sulphate monohydrate was
  • TDM-26 Asparagine monohydrate was increased to 4 g per liter; and potassium phosphate monobasic was replaced with 5mM phytic acid from rice (Sigma Cat. # P3168).
  • TDM-27 Glucose was replaced with 10g per liter of olive oil (Sigma cat. # 01514)
  • Glucose was omitted. TDM-29 Glucose was replaced with 10 g per liter of tallow.
  • TDM-30 Glucose was replaced with 10 g per liter of yellow grease.
  • TDM-31 Glucose was replaced with 10 g per liter of defined lipid (Sigma cat. # L0288).
  • TDM-32 Glucose was replaced with 50 g per liter of D-xylose.
  • TDM-33 Glucose was replaced with 20 g per liter of glycerol and 20ml per liter of ethanol.
  • TDM-34 Glucose was reduced to 1 g per liter and 10 g per liter of bran was added.
  • TDM-35 Glucose was reduced to 1 g per liter and 10 g per liter of pectin (Sigma Cat. # P-9135) was added.
  • TDM-36 Glucose was replaced with 10 g per liter of biodiesel.
  • TDM-37 Glucose was replaced with 10 g per liter of soy feedstock.
  • TDM-38 Glucose was replaced with 10g per liter of locust bean gum (Sigma cat # G0753).
  • TDM-40 The medium's pH was raised to 8.5.
  • TDM-42 Glucose was replaced with 5 g per liter of yellow grease and 5 g per liter of soy feedstock
  • TDM-43 Glucose was replaced with 20g per liter of fructose.
  • Glucose was replaced with 10 g per liter of cellulose (Solka-Floc, 200FCC) plus 1 g per liter of
  • TDM-45 The medium's pH was raised to 8.84.
  • Kerosene was sourced from a general hardware store.
  • Rhizomucor miehei Thermoascus thermophilus (Dadylomyces thermophilus), and Humicola hyalothermophila strains were grown according to the methods described above under the following growth conditions: TDM-1 , -2, -3, -4, -5, -6, -7, -8, -9, -10, -13, -14, -15, -39; YM, whereby the following optimal growth temperature was used: 25°C.
  • strains carrying the recombinant genes were grown according to the methods described above under the following growth conditions: minimal medium as described in Kafer et al., (1977, Adv. Genet. 19:33- 131) except that the salt concentrations were raised ten-fold and the glucose concentration was 150 grams per liter, at 30°C.
  • the mycelia were collected by filtration through Miracloth and washed with water by filtration. The mycelia were padded dry using paper towels, and frozen in liquid nitrogen and stored at -80°C.
  • To extract total RNA the frozen mycelia or cells were ground to a fine powder in liquid nitrogen using pestle and mortar. Approximately 1 -1.5 gram of frozen fungal powder was dissolved in 10 mL of TRIzol® reagent and RNA was extracted according to the manufacturer's protocol (Invitrogen Life Sciences, Cat. #15596-018). Following extraction, the RNA was dissolved at 1 -1.5 mg/ml of DEPC-treated water.
  • the PolyATtract® mRNA Isolation Systems (Promega, Cat. #Z5300) was used to isolate poly(A)+RNA. In general, equal amounts of total RNA extracted from up to ten culture conditions were pooled. One milligram of total RNA was used for isolation of poly(A)+RNA according to the protocol provided by the manufacturer. The purified poly(A)+RNA was dissolved at 200-500 pg/mL of DEPC-treated water.
  • Double-stranded cDNA was synthesized using the ZAP-cDNA® Synthesis Kit (Stratagene, Cat. #200400) according to the manufacturer's protocol with the following modifications.
  • An anchored oligo(dT) linker-primer was used in the first-strand synthesis reaction to force the primer to anneal to the beginning of the poly(A) tail of the mRNA.
  • the anchored oligo(dT) linker-primer has the sequence:
  • a second modification was made by adding trehalose at a final concentration of 0.6 M and betaine at a final concentration of 2 M in the buffer of the first-strand synthesis reaction to promote full-length synthesis. Following synthesis and size fractionation, fractions of double-stranded cDNA with sizes longer than 600 bp were pooled.
  • the pooled cDNA was cloned directionally into the plasmid vector BlueScript KS+® (Stratagene) or a modified BlueScript KS+ vector that contained Gateway® (Invitrogen) recombination sites.
  • the cDNA library was transformed into E. coli strain XL10-Gold ultracompetent cells (Stratagene, Cat. #Z00315) for propagation.
  • Bacterial cells carrying cDNA clones were grown on LB agar containing the antibiotic ampicillin for selection of plasmid-borne bacteria and X-gal and IPTG to use the blue/white system to screen for the presence cDNA inserts.
  • RNA-seq data was assembled into contigs using the Velvet assembler (D. R. Zerbino and E. Birney. Genome Research 18:821-829). Potential full length transcripts were identified using blastx to the NCBI non redundant database.
  • Proteins targeted to the extracellular space by the classical secretory pathway possess an N- terminal signal peptide, composed of a central hydrophobic core surrounded by N- and C- terminal hydrophilic regions.
  • Phobius available at http://phobius.cgb.ki.se
  • SignalP® version 3 available at http://www.cbs.dtu.dk/services/SignalP
  • TargetP® available at http://www.cbs.dtu.dk/services/TargetP
  • Big-PI Fungal Predictor available at http://mendel.imp.ac.at/gpi/fungi_server.html
  • the PCR amplified products were cloned into an appropriate expression vector for protein production in host cells.
  • the coding and polypeptide sequences were assigned SEQ ID NOs, annotations, general functions, protein activities, CAZy family classifications, as summarized in Tables 1A-1C. Where appropriate, carbohydrate-binding modules (CBMs) of particular interest for the degradation of biomass were also listed in Tables 1A-1 C.
  • CBMs carbohydrate-binding modules
  • Polypeptides of the present invention may be additionally cloned into an expression vector, expressed and characterized (e.g., in sugar release assays) for activity relating to their ability to breakdown and/or process biomass as described in WO/2012/92676, WO/2012/130950, WO/2012/130964, WO/2013/181760, and WO/2014/110675 using appropriate substrates (e.g., acid pre-treated corn stover (aCS), hot water treated washed wheat straw, or hot water treated washed corn fiber substrate). Soluble sugars that are released can be analyzed for example by proton NMR.
  • aCS acid pre-treated corn stover
  • Soluble sugars that are released can be analyzed for example by proton NMR.
  • a number of assays may be used to characterize the polypeptides of the present invention. Selected non-limiting examples of such assays are described and/or referenced below. Of course, other assays not explicitly mentioned or referenced here may also be used, and the expression "can be” used below is intended to reflect this possibility. Furthermore, a person of skill in the art would be able to modify or adapt these and other assays, as necessary, to characterize a particular polypeptide.
  • polypeptides of the present invention having this activity catalyze the random, internal hydrolysis of beta-1 ,4-linkages between glucosamine residues in chitin and chitosan, and can be characterized for example as described in Takaya et al., Microbiology (Reading, Engl.) 1998 Sep; 144 (Pt 9):2647-54.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Erfle and Teather, Appl Environ Microbiol. (1991 ), 57(1 ): 122-129.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Chanzy et al., FEBS Letters (1983), 153(1 ): 113-118; Gielkens et al., Appl. Environ. Microbiol. (1999), 65: 4340-4345; Hanif et al, Bioresour. Technol. (2004), 94:31 1-319; Liu et al, Biosci. Biotechnol. Biochem. (2009), 73(6): 1432-4; or Yoshida et al, Biosci. Biotechnol. Biochem. (2009), 73(1 ): 67-73.
  • 11-beta-hydroxysteroid dehydrogenase 1B Polypeptides of the present invention having this activity can be characterized for example as described in Blum et al. Biochemistry (2003), 42(14):4108-17.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Sliwa et al, Biochemistry ⁇ 2m), 49(16):3487-98.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Tenkanen and Siika-aho, J. Biotechnol. (2000), 78(2): 149-61 ; or Chong et al., Appl. Microbiol. Biotechnol. (2011 ), 90(4): 1323-32.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Tubeleviciute et al, Appl Microbiol Biotechnol. (2014) 98(12): 5471 - 85.
  • 2-hydroxyacid dehydrogenase (EC 1.1.99.6).
  • Polypeptides of the present invention having this activity can be characterized for example as described in Cammack, Biochem. J. (1970), 1 18(3): 405-408.
  • 3-hydroxybenzoate 6-hydroxylase Polypeptides of the present invention having this activity can be characterized for example as described in Sucharitakul et al, J Biol Chem. (2013), 288(49): 35210-21 ; Montersino et al, J Biol Chem. (2013), 288(36): 26235-45; or Montersino and van Berkel, Biochim Biophys Acta. (2012), 1824(3): 433-42.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Kim et al, Biochem Biophys Res Commun. (2014), 443(3): 783-8; Madan et al, Eur. J. Biochem. (1973), 32(1 ): 51-6.
  • Polypeptides of the present invention having this activity can be characterized as described in Liu et al, Biosci. Biotechnol. Biochem. (2009), 73(6): 1432-4; Yoshida et al, Biosci. Biotechnol. Biochem. (2009), 73(1): 67-73.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Li et al, FEBS Lett. (2007), 581 (21): 4029-35; Spanikova and Biely, FEBS Lett. (2006), 580(19): 4597-601.
  • Acetylxylan esterase Polypeptides of the present invention having this activity can be characterized as described in Water et al, Appl Environ Microbiol. (2012), 78(10): 3759-62; Yang et al. International Journal of Molecular Sciences (2010), 1 1 (12): 5143-5151 ; or in US patent No. 8,129,590.
  • Adhesin protein Mad1 Adhesin protein Mad1.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Wang and St Leger, Eukaryot. Cell (2007), 6(5): 808-816.
  • Adhesin Polypeptides of the present invention having this activity (reviewed in Dranginis et al. Microbiology and Molecular Biology Reviews (2007), 71 (2): 282-294) can be characterized using techniques well known in the art (e.g. adhesion assays).
  • Polypeptides of the present invention having this activity can be characterized for example as described in Krog et al, PLoS One (2013) 8(3):e59188.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Beeson et al, Appl Environ Microbiol. (201 1 ), 77(2): 650-6; Ishikawa et al, J Biol Chem. (2008), 283(45): 31 133-41.
  • Aldose 1-epimerase mutarotase, aldose mutarotase.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Timson and Reece, FEBS Letters (2003), 543(1 -3):21 -24; and Villalobo et al, Exp. Parasitol. (2005) 110(3): 298-302.
  • Alkaline protease 2 Alkaline protease 2.
  • Polypeptides of the present invention having this activity can be characterizedfor example as described in Gomaa, Braz J Microbiol. (2013) 44(2):529-37; or Yao et al, J Food Sci Technol. (2012), 49(5):626-31.
  • Polypeptides of the present invention having this activity can be characterizedfor example as described in Bowyer et al. Medical Mycology (2007), 45(1 ): 17-26.
  • Alpha-arabinofuranosidase Polypeptides of the present invention having this activity can be characterized for example as described in Poutanen et al. ⁇ Appl. Microbiol. Biotechnol. 1988, 28, 425-432) using 5 mM p-nitrophenyl alpha-L-arabinofuranoside as substrates.
  • the reactions may be carried out in 50 mM citrate buffer at pH 6.0, 40°C with a total reaction time of 30 min.
  • the reaction is stopped by adding 0.5 ml of 1 M sodium carbonate and the liberated p-nitrophenol is measured at 405 nm. Activity is expressed in U/ml.
  • arabionofuranosidases may also be useful in animal feed compositions to increase digestibility.
  • Corn arabinoxylan is heavily di-substituted with arabinose. In order to facilitate the xylan degradation it is advantageous to remove as many as possible of the arabinose substituents.
  • the in vitro degradation of arabinoxylans in a corn based diet supplemented with a polypeptide of the present invention having alpha-arabinofuranosidase activity and a commercial xylanase is studied in an in vitro digestion system, as described in WO/2006/114094.
  • Alpha-fucosidase Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,637,490; in Zielke et al., J. Lab. Clin. Med. (1972), 79: 164; or using commercially available kits (e.g., Alpha-L-Fucosidase (AFU) Assay Kit, Cat. No. BQ082A-EALD, BioSupplyUK).
  • Alpha-galactosidase Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2010/0273235 A1. Briefly, a synthetic substrate, 4- Nitrophenyl-a-D-galactoside is used and the release of p-Nitro-phenol is followed at a wavelength of 405 nm in a reaction buffer containing 100 mM sodium phosphate, 50 mM sodium chloride, pH 6.8 at 26°C.
  • Alpha-glucosidase Polypeptides of the present invention having this activity can be characterized for example as described in Yamamoto et al., Eur. J. Biochem. (2004), 271 (16): 3414-3420; or for example using commercial kits (e.g., available from Sigma-Aldrich).
  • Alpha-glucuronidase GH67 Polypeptides of the present invention having this activity can be characterized for example as described in Lee et al., J Ind Microbiol Biotechnoi. (2012), 39(8): 1245-51 , or Nagy et al., J. Bacte ol. (2002), 184: 4925-4929.
  • Alpha-L-arabinofuranosidase Polypeptides of the present invention having this activity can be characterized for example as described in Giacobbe et al, N Biotechnoi. (2014), 31 (3):230-6; Shi et al, Biotechnoi Lett. (2014), 36(6): 1321 -8; Maehara et al, J Biol Chem. (2014), 289(1 1)7962-72.
  • Alpha-L-rhamnosidase Polypeptides of the present invention having this activity can be characterized for example as described in Rosenfeld and Wiederschein, Bull. Soc. Chim. Biol. (1965), 47 (7): 1433-1440, PMID 5855461 ; Fujimoto et al, J Biol Chem. (2013), 288(17): 12376-85.
  • Alpha-N-arabinofuranosidase Polypeptides of the present invention having this activity can be characterized for example as described in Kaji and Tagawa, Biochim. Biophys. Acta (1970), 207: 456-464; Kaji and Yoshihara, Biochim. Biophys. Acta (1971 ), 250: 367-371 ; Tagawa and Kaji, Carbohydr. Res. (1969), 1 1 : 293-301.
  • Alpha-rhamnosidase Polypeptides of the present invention having this activity can be characterized for example as described in Fujimoto et al, J Biol Chem. (2013), 288(17): 12376-85; Rodriguez et al, J AppI Microbiol. (2010), 109(6): 2206-13; Grandits et al, J Mol Catal B Enzym. (2013), 92(100): 34-43.
  • Aminopeptidase Y Polypeptides of the present invention having this activity can be characterized for example as described in Yasuhara et al, J. Biol. Chem. (1994) 269(18) : 13644-50.
  • Arabinan endo-1,5-alpha-L-arabinosidase A Polypeptides of the present invention having this activity can be characterized for example as described in Flipphi et al, AppI. Microbiol. Biotechnoi. (1993), 40: 318-326; and Leal and de Sa-Nogueira, FEMS Microbiol. Lett. (2004), 241 : 41-48.
  • Arabinogalactan endo-1 ,4-beta-galactosidase Polypeptides of the present invention having this activity can be characterized for example as described in Emi and Yamamoto, Agric. Biol. Chem. (1972), 36: 1945-1954; Labavitch et al, J. Biol. Chem. (1976), 251 : 5904-5910; or Shipkowski and Brenchley, AppI. Environ. Microbiol. (2006), 72: 7730-7738.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Yamamoto and Emi, Methods in Enzymology (1988), 160: 719-725.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Pei and Shao, AppI Microbiol Biotechnoi. (2008), 78(1): 1 15-21 ; Xiong et al. Journal of Experimental Botany (2007), 58(1 1 ): 2799-2810.
  • Arabinoxylan arabinofuranohydrolase (AXH) GH43 Polypeptides of the present invention having this activity can be characterized for example as described in Yoshida et al. Journal of Bacteriology (2010), 192(20): 5424- 5436.
  • Arabinoxylan arabinofuranosidase GH62 Polypeptides of the present invention having this activity can be characterized for example as described in Sakamoto et al. Applied Microbiology and Biotechnology (201 1 ), 90(1 ): 137-146. Aryl-alcohol oxidase. Polypeptides of the present invention having this activity can be characterized for example as described in Hernandez-Ortega et al., Biochemistry (2012), 51 (33): 6595-608; Hernandez-Ortega et al., Appi Microbiol Biotechnol. (2012), 93(4): 1395-410.
  • Aspartate-semialdehyde dehydrogenase (EC 1.2.1.1 1 ).
  • Polypeptides of the present invention having this activity can be characterized for example as described in Black et al., J. Bioi. Chem. (1955) 213:39-50; US Patent No. 7,723,097.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Tacco et al., Wed. Mycoi. (2009), 47(8): 845-854; or in Hu et al., Joumai of Biomedicine and Biotechnology (2012), 2012:728975.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Tjalsma et al., J. Biol. Chem. (1999), 274: 28191 -28197.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Huang et al., Joumai of Biological Chemistry (2000), 275(34): 26607-14.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Kwak et al., Phytopathology (2010), 100(5): 404-14; or in Bowyer et al., Science (1995), 267(5196): 371 -4.
  • Beta-galactosidase Polypeptides of the present invention having this activity can be characterized for example using commercially available kits (e.g., ⁇ -Galactosidase Enzyme Assay System with Reporter Lysis Buffer, Cat. No. E2000, Promega).
  • Beta-glucanase Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication number US 2012/0023626 A1 ; or in US patent No. 8,309,338.
  • Beta-glucosidase Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO/2007/019442; or by using a commercially available kit (e.g., Beta- Glucosidase Assay Kit, Cat. No. KA161 1 , Abnova Corp).
  • Beta-glucuronidase Polypeptides of the present invention having this activity can be characterized for example as described in Eudes et al., Plant Cell Physiology (2008), 49(9): 1331 -41 ;or Michikawa et al., Journal of Biological Chemistry (2012), 287: 14069-14077.
  • Beta-hexosaminidase Polypeptides of the present invention having this activity can be characterized for example as described in Wendeler and Sandhoff, Glycoconj J. (2009), (8):945-52.
  • Beta-mannanase Polypeptides of the present invention having this activity can be characterized for example as described in European patent application No. EP 2261359 A1 ; or in PCT application publication No. WO2008009673A2.
  • Beta-mannosidase Polypeptides of the present invention having this activity can be characterized for example as described in Park et al., N. Biotechnol. (2011 ), 28(6): 639-48; Duffaud et al., Appl Environ Microbiol. (1997), 63(1 ): 169-77; or in Fliedrova et al., Protein Expr Purif. (2012), 85(2): 159-64.
  • Beta-xylosidase xylan 1 ,4-beta xylosidase.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Wagschal et al., Applied and Environmental Microbiology (2005), 71 (9): 5318-5323; or Shao et al., Appl Environ Microbiol. (201 1), 77(3): 719-726.
  • Bifunctional alpha-arabinofuranosidase/beta-xylosidase GH43 Polypeptides of the present invention having this activity can be characterized for example as described in Viborg et al., AMB Express. (2013), 3(1 ):56; Shi et al., Biotechnol Biofuels (2013), 6(1):27; or Kim and Yoon, J Microbiol Biotechnol. (2010), (12): 171 1 -6.
  • Bifunctional solanapyrone synthase Polypeptides of the present invention having this activity can be characterized for example as described in Kasahara et al., ChemBioChem (2010), 11 : 1245-1252; Katayama et al., Biosci. Biotechnol. Biochem. (2008), 72: 604-607; or Katayama et al., Biochim. Biophys. Acta (1998), 1384: 387- 395.
  • Bifunctional xylanase/deacetylase Polypeptides of the present invention having this activity can be characterized for example as described in Cepeljnik et al., Folia Microbiol. (2006), 51 (4): 263-267; US patent application publication No.
  • Carbohydrate-binding cytochrome Carbohydrate-binding cytochrome.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Yoshida et al., Appl Environ Microbiol. (2005) 71 (8): 4548-4555.
  • Carboxylesterase Polypeptides of the present invention having this activity can be characterized for example using a commercially available kit such as the Carboxylesterase 1 (CES1 ) Specific Activity Assay Kit (ab109717) (Abeam, Cambridge, MA, USA).
  • CES1 Carboxylesterase 1
  • ab109717 Specific Activity Assay Kit
  • Carboxypeptidase Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2007/016071 1 A1 ; or in PCT application publication No. WO 1998/014599A1.
  • Cellobiohydrolase GH6 Cellobiohydrolase GH6.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Takahashi et al., Applied and Environmental Microbiology (2010), 76(19): 6583-6590.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Segato et al., Biotechnology for Biofuels (2012), 5:21 ; or Baumann et al., Biotechnol. for Biofuels (2011 ), 4:45; or Naran et al., Plant J. (2007), 50(1 ):95-107.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Schou et al., Biochem. J. (1998), 330: 565-571 ; or Baminger et al., J. Microbiol Methods. (1999), 35(3): 253-9.
  • Polypeptides of the present invention having this activity can be characterized for example as described in European patent application No. EP 0610320 B1.
  • Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 7,087,810.
  • Chitinase-3-like protein 1 Polypeptides of the present invention having this activity can be characterized for example as described in Dela Cruz et al., Cell Host Microbe (2012), 12(1):34-46.
  • Polypeptides of the present invention having this activity can be characterized for example as described in John et al., Proc Natl Acad Sci USA (1993), 90(2): 625-9.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Boucher et al., J. Biol. Chem. (1995), 270(52): 31077-82; WO2012/100345.
  • Chitotriosidase-1 Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 6,057, 142.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Gadda and McAllister-Wilkins, Appl. Environ. Microbiol. (2003) 69(4): 2126-32; or Takabe et al., J. Biol. Chem. (2003), 278 (7): 4932-42.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Abass Askar et al., Canadian Journal Veterinary Research (2011 ), 75(4): 261-270; or Catia et al., PLoS One (2012), 7(3): e33975.
  • Cis-2,3-dihydrobiphenyl-2,3-diol dehydrogenase Polypeptides of the present invention having this activity can be characterized for example as described in Sylvestre et al., Appl. Environ. Microbiol. (1996), 62(8): 2710-5; Takagi and Yano, Biochem. Biophys. Res. (1994), Commun. 202 (2): 850-6.; or Hofer et al., Gene. (1993), 130(1 ): 47-55.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Joshi et al., FEMS Microbiol Lett. (1995), 125(2-3): 21 1-7; Sheng et al., Curr Microbiol. (2006), 53(2): 124-8.
  • Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2012/0028318 A1 ; or in Chen et al., J. Biol Chem. (2008), 283(38): 25854-62. Cys-Gly metallodipeptidase. Polypeptides of the present invention having this activity can be characterized for example as described in Ganguli et al. Genetics (2007), 175: 1 137-1151 ; Kaur et al, J. Biol. Chem. (2009), 284: 14493-14502.
  • Cytochrome P450 Polypeptides of the present invention having this activity can be characterized for example as using commercially available kits (e.g., P450-GloTM Assays, Promega); or as described in Walsky and Obach, Drug Metab Dispos. (2004), 32(6): 647-60.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Mayer and Arnold, J. Biomol. Screen. (2002), 7(2): 135-140.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Ko and Khouw, Can. J. Biochem. (1970), 48(3): 225-227; or Zohra et al, Cabohydrate Polymers (2013), 92: 2149-2153.
  • Dipeptidyl peptidase Dipeptidyl peptidase.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Ohara-Nemoto et al, J Biol Chem. (2014), 289(9) :5436-48.
  • Endo-1 ,3(4)-beta-glucanase (laminarinase).
  • Polypeptides of the present invention having this activity can be characterized for example as described in Akiyama et al, J Plant Physiol. (2009), 166(16): 1814-25; or Hua et al, Biosci Biotechnol Biochem. (201 1), 75(9): 1807-12.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Song et al. Enzyme and Microbial Technology (2013). 52(3): 170-176.
  • Endo-1 ,5-alpha-arabinanase Polypeptides of the present invention having this activity can be characterized for example as described in US patent publication No. US 2012/0270263. More particularly, this assay of arabinase activity is based on colorimetrically determination by measuring the resulting increase in reducing groups using a 3,5-dinitrosalicylic acid reagent. Enzyme activity can be calculated from the relationship between the concentration of reducing groups, as arabinose equivalents, and absorbance at 540 nm. The assay is generally carried out at pH 3.5, but it can be performed at different pH values for the additional characterization and specification of enzymes. Polypeptides of the present invention having this activity can also be characterized for example as described in Hong et al, Biotechnol Lett. (2009), 31 (9): 1439-43.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Bryant et al. Fungal Genet Biol. (2007), 44(8): 808-17; or in Oyama et al, Biosci Biotechnol Biochem. (2006), 70(7): 1773-5.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Takao et al, Biosci Biotechnol Biochem. (2002), 66(2): 430-3; Takao et al, AppI Environ Microbiol. (2002), 68(4): 1639-46.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Kotake et al, J Biol Chem. (2011 ), 286(31 ): 27848-54. Ichinose et al, AppI Environ Microbiol. (2006), 72(5): 3515-3523.
  • Endo-beta-1 ,3(4)-glucanase (EC 3.2.1.6).
  • Polypeptides of the present invention having this activity can be characterized for example as described in WO 1995031533 and WO2013037933.
  • Endo-beta-1,4-glucanase celB Polypeptides of the present invention having this activity can be characterized for example as described in Baird et al, J Bacteriol. (1990), 172(3): 1576-1586; Jorgensen and Hansen, Gene. (1990), 93(1 ):55-60; Jagtap et al, "Characterization of a novel endo- -1 ,4-glucanase from Armillaria gemina and its application in biomass hydrolysis", AppI Microbiol Biotechnol. (2013).
  • Polypeptides of the present invention having this activity can be characterized for example as described in Ichinose et al, AppI Environ Microbiol. (2008), 74(8): 2379-83.
  • Endochitinase Polypeptides of the present invention having this activity can be characterized for example as described in Wen et al, Biotechnol. Applied Biochem. (2002), 35: 213-219. Endoglucanase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 8,063,267; Couturier et al, Microb Cell Fact. (2011 ), 10: 103; Badieyan et al, Biotechnol Bioeng. (2012), 109(1 ): 31 -44; Pereira et al, J Struct Biol.
  • thermostable GH7 endoglucanase from Myceliophthora thermophila capable of high-consistency enzymatic liquefaction
  • Appl Environ Microbiol. (2013), 79(14): 4220-9 Appl Environ Microbiol. (2013), 79(14): 4220-9.
  • Endoglycoceramidase Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,795,765; or US patent application publication No. US 2009/0170155 A1.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Vandamme et al, FEBS Open Bio. (2013), 3: 467-472; US patent No. 8,309,079.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Stals et al, PLoS One. (2012), 7(7): e40854; Stals et al, FEMS Microbiol Lett. (2010), 303(1 ): 9-17.
  • Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO 1994/014952 A1 ; or in European patent application publication Nos. EP1614748 A1 and EP1 1 14165 A1.
  • Endo-rhamnogalacturonase GH28 Polypeptides of the present invention having this activity can be characterized for example as described in Sprockett et al. Gene (201 1), 479(1 -2): 29-36; or An et al. Carbohydrate Research (1994), 264(1): 83-96.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Choi et al., Gene(1993), 125(2): 135-41 ; Williams et al. Arch. Biochem. Biophys. (1972), 149: 52-61.
  • Exo-1 ,3-beta-galactanase GH43 Polypeptides of the present invention having this activity can be characterized for example as described in lchinose et al, Appl Environ Microbiol. (2006), 72(5): 3515-3523.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Wojtkowiak et al. Acta Crystallogr D Biol Crystallogr. (2013) 69(Pt 1 ):52-62; Tao et al. Gene (2013), 527(1 ): 154-60.
  • Polypeptides of the present invention having this activity can be characterized for example as described in O'Connell et al, Appl Microbiol Biotechnol. (201 1 ), 89(3): 685-96; or Santos et al, J Bacterid. (1979), 139(2): 333-338.
  • Exo-1 ,4-beta-xylosidase Polypeptides of the present invention having this activity can be characterized for example as described in La Grange et al. Applied and Environmental Microbiology (2001), 67(12): 5512-5519.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Tatsuji Sakamoto and Thibault, Appl Environ Microbiol. (2001), 67(7): 3319-3321.
  • Exo-beta-D-glucosaminidase Polypeptides of the present invention having this activity can be characterized for example as described in Honda et al, Glycobiology. (201 1), 21 (4): 503-11 ; Li et al, Carbohydr Res. (2009), 344(8): 1046-9; Fukamizo et al, Glycobiology. (2006), 16(1 1): 1064-72; Nogawa et al, Appl Environ Microbiol. (1998), 64(3): 890-5; Jung et al. Protein Expr Purif. (2006), 45(1): 125-31.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Creuzet et al, FEMS Microbiology Letters (1983), 20(3): 347-350; or Kruus et al. Journal of Bacteriology (1995), 177(6): 1641 -1644.
  • Exo-glucosaminidase GH2 Exo-glucosaminidase GH2.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Tanaka et al. Journal of Bacteriology (2003), 185(17): 5175-5181.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Kulminskaya et al, Biochimica et Biophysica Acta (2003), 1650(1-2):22-9; Pessoni et al, Mycologia. (2007), 99(4):493-503.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Dong and Wang, BMC Biochem. (201 1), 12: 51. Exo-rhamnogalacturonase GH28. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,81 1 ,291.
  • Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO 2005/030965 A2; or in US patent No. 7,001 ,743.
  • Expansin-like protein 1 Polypeptides of the present invention having this activity can be characterized for example as described in Lee et al., Molecules and Cells (2010), 29(4): 379-85.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Inacio and de Sa-Nogueira, J Bacteriol. (2008), 190(12):4272-80.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Matsuo et al., Biochem. J. (2000), 346:9-15.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Almeida et al., Parasitol Res. (2003), 89(4); Shibata et al., J Biol Chem. (2000), 275(12):8349-54; Kim and Kim, Can J Microbiol. (1994), 40(2): 120-6.
  • Feruloyl esterase Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO 2009/076122 A1.
  • Fumarate reductase Fumarate reductase.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Iverson et al. Science (1999), 284(5422): 1961 -6; Lancaster et al. Nature (1999), 402(6760): 377- 85; or Maklashina et al, J Biol Chem. (2006), 281 (16): 11357-65.
  • Polypeptides of the present invention having this activity can be characterized for example as described in lchinose et al. Applied and Environmental Microbiology (2008), 74(8): 2379-2383.
  • Galacturan 1 ,4-alpha-galacturonidase C Polypeptides of the present invention having this activity can be characterized for example as described in Dong and Wang, BMC Biochem. (201 1), 12:51.
  • GTT Gamma-glutamyltransferase
  • Glucan 1 ,3-beta-glucosidase Polypeptides of the present invention having this activity can be characterized for example as described in Boonvitthya et al, Biotechnol Lett (2012), 34(10): 1937-43.
  • Glucan endo-1 ,3-beta-glucosidase Polypeptides of the present invention having this activity can be characterized for example as described in Sperisen et al, Proc Natl Acad Sci USA (1991), 88(5): 1820-4.
  • Glucan endo-1 ,6-beta-glucosidase B Polypeptides of the present invention having this activity can be characterized for example as described in Fayad et al, Appl Microbiol Biotechnol. (2001), 57(1 -2): 117-23.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Kondo et al, Proc Natl Acad Sci USA (2006), 103(15):5723-8; and Tarighi et al. Microbiology (2008), 154(Pt 10):2979-90.
  • Polypeptides of the present invention having this activity can be characterized using a commercially available kit such as Amplex® Red Glucose/Glucose Oxidase Assay Kit (Cat. No. A22189, Life Technologies).
  • Glucose-6-phosphate 1-epimerase Polypeptides of the present invention having this activity can be characterized for example as described in Wurster and Hess, Methods Enzymol. (1975), 41 : 488-93.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Vaccaro et al, Eur J Biochem. (1985), 146(2): 315-21 ; Vaccaro et al. Enzyme. (1989), 42(2): 87- 97; Vaccaro et al, Clin Chim Acta. (1982), 1 18(1): 1-7.
  • Glycerol-3-phosphate dehydrogenase Polypeptides of the present invention having this activity can be characterized for example as described in Albertyn et al, FEBS Lett. (1992) 308: 130-132.
  • Glycosidase Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 8, 1 19,383. Glyoxylate reductase. Polypeptides of the present invention having this activity can be characterized for example as described in Duan et al., Enzyme Microb Technol. (2014), 60: 72-9.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Hoover et al., Biochim Biophys Acta. (2013), 1834(12): 2663-71.
  • Hephaestin-like protein 1 Polypeptides of the present invention having this activity can be characterized for example as described for oxioreductases.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Wendeler and Sandhoff, Glycoconj J. (2009), 26(8):945-952.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Bettini et al., Canadian Journal of Microbiology (2012), 58(8): 965-972; or Niu et al., Amino Acids. (2012), 43(2)763-71.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Bacon, J.S.D., Methods in Enzymology (1955), Volume I, 258-262; Lever, M. Analytical Biochemistry (1972), Volume 47, 273-279; Us patent No. US 5,665,579.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Askwith et al., Cell (1994), 76: 403-10; or De Silva et al., J. Biol. Chem. (1995) 270: 1098-1 101.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Dedeyan et al., Appl Environ Microbiol. (2000), 66(3): 925-929.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Khersonsky and Tawfik, ChemBioChem (2006), 7(1 ): 49-53; or Chow et al., J Biol Chem. (2010) 285(52):4091 1 -20.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Ishida et al., J Biol Chem. (2009), 284(15): 10100-10109; or Kawai et al., Biotechnol Lett. (2006), 28(6): 365-71.
  • Polypeptides of the present invention having this activity can be characterized for example as described in US patent Nos. 5,612,208 and 5,180,672.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Aurich et al., Biochim Biophys Acta. (1967), 139(2): 505-7; or US patent No. 5, 156,966.
  • Leucine aminopeptidase 1 Polypeptides of the present invention having this activity can be characterized for example as described in Beattie et al., Biochem. J. (1987), 242: 281 -283.
  • Licheninase (beta-D-glucan 4-glucanohydrolase).
  • Polypeptides of the present invention having this activity can be characterized for example as described in Tang et al., J Agric Food Chem. (2012), 60(9): 2354-61.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Arora and Gill, Enzyme and Microbial Technology (2001 ), 28(7-8): 602-605.
  • Polypeptides of the present invention having this activity can be characterized for example as described in US patent Nos. 7,662,602 and 7,893,232.
  • L-lactate dehydrogenase A Polypeptides of the present invention having this activity can be characterized for example as described in Yeswanth et al., Anaerobe (2013), 24:43-8; Xia et al., Mol Biol Rep. (201 1), 38(3): 1853-60.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Quiroz-Castafieda et al., Microbial Cell Factories (2011 ), 10:8.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Shinjoh et al., Applied and Environment Microbiology (1995), 61 (2): 413-420.
  • Lysophospholipase Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,965,422. Lysozyme. Polypeptides of the present invention having this activity can be characterized for example as described in EnzChek® Lysozyme Assay Kit (cat. No. E22013, Life Technologies); Shugar, D. Biochimica et Biophysica Acta (1952), 8: 302-309.
  • Mannan endo-1,4-beta-mannosidase Polypeptides of the present invention having this activity can be characterized for example as described in Zhao et al., Bioresour Techno!. (201 1), 102(16): 7538-47; Songsiriritthigul et al, Microb Cell Fact. (2010), 9:20; or Do et al, Microb Cell Fact (2009), 8: 59.
  • Metallocarboxypeptidase Polypeptides of the present invention having this activity can be characterized for example as described in Tayyab et al, J Biosci Bioeng. (201 1 ), 11 1 (3): 259-65; or Song et al, J Biol Chem. (1997), 272(16): 10543-50.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Wohlfarth et al, J Bacteriol. (1991), 173(4): 1414-1419.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Clark et al, Carbohydr Res. (1978), 61 : 457-477.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Haghbeen and Tan, Anal Biochem. (2003), 312(1 ): 23-32.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Baudy et al, FEBS Lett. (1988), 235: 271-274.
  • Multicopper oxidase Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2012/0094335 A1.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Pleszczynska, Biotechnol Lett. (2010), 32(11 ): 1699-1704; or WO 1998/000528 A1.
  • N-acetylglucosaminidase GH18 N-acetylglucosaminidase GH18.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Murakami et al, Glycobiology (2013), 23(6):736-44; or in US patent application publication No. US20120258089 A1.
  • N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Guo et al, J Lipid Res. (2013), 54(1 1 ):3151 -7.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Fujisawa et al, Biochim Biophys Acta. (2003), 1645(1 ): 89-94.
  • NADPH-cytochrome P450 reductase NADPH-cytochrome P450 reductase.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Guengerich et al, Nat Protoc. (2009), 4(9): 1245-51.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Colrat et al. Plant Physiol. (1999), 1 19(2): 621-6.
  • NADPH-dependent methylglyoxal reductase GRE2 NADPH-dependent methylglyoxal reductase GRE2.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Murata et al, Eur. J. Biochem. (1985), 151 (3): 631-636; Johnston et al. Yeast. (2003), 20 (6): 545-554.
  • Non-hemolytic phospholipase C Polypeptides of the present invention having this activity can be characterized for example as described in Weingart and Hooke, Curr Microbiol. (1999), 38(4): 233-8; Korbsrisate et al, J Clin Microbiol. (1999), 37(1 1): 3742-5.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Gengan et al. Prep Biochem Biotechnol. (2006), 36(4): 297-306.
  • Polypeptides of the present invention having this activity can be characterized for example using a number of commercially available kits [e.g., Amplex® Red Galactose/Galactose Oxidase Kit (A22179) and Amplex® Red Glucose/Glucose Oxidase Assay Kit (Molecular Probes/lnvitrogen); Cytochrome C Oxidase Assay Kit (Cat. No.
  • Oxidoreductase Polypeptides of the present invention having this activity can be characterized for example as described in Hommes et al., Anal Chem. (2013), 85(1 ): 283-291.
  • Oxygen-dependent choline dehydrogenase Polypeptides of the present invention having this activity can be characterized for example as described in Gadda et al., Appl Environ Microbiol. (2003), 69(4): 2126-32.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Moore and Arnold, Nat Biotechnol. (1996), 14(4): 458-67.
  • Pectate lyase Polypeptides of the present invention having this activity can be characterized for example as described in Wang et al., BMC Biotechnology (2011 ), 1 1 : 32.
  • Pectin lyase Polypeptides of the present invention having this activity can be characterized for example as described in Yadav et al., J Basic Microbiol. (2014), 54 Suppl 1 :S161 -9; Perez-Fuentes et al., Fungal Biol. (2014), 1 18(5-6): 507-15.
  • Pectin methylesterase Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO 1997/031102 A1.
  • Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,053,232.
  • Penicillopepsin Polypeptides of the present invention having this activity can be characterized for example as described in Cao et al., Protein Sci. (2000), 9(5): 991-1001 ; or Hofmann et al., Biochemistry. (1984), 14;23(4): 635- 43.
  • Polypeptides of the present invention having this activity can be characterized for example using a commercially available kit such as the EnzChek® Peptidase/Protease Assay Kit (Life technologies, Cat. No. E33758).
  • Polypeptides of the present invention having this activity can be characterized for example using a number of commercially available kits [e.g., Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes/lnvitrogen); Peroxidase Activity Assay Kit (Cat. No. K772-100; BioVision); QuantiChromTM Peroxidase Assay Kit (Cat. No. DPOD-100, BioAssay Systems].
  • Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit Molecular Probes/lnvitrogen
  • Peroxidase Activity Assay Kit Cat. No. K772-100; BioVision
  • QuantiChromTM Peroxidase Assay Kit Cat. No. DPOD-100, BioAssay Systems.
  • Peroxisomal hydratase-dehydrogenase-epimerase Polypeptides of the present invention having this activity can be characterized for example as described in Nuttley et al., Gene. (1988), 69(2): 171 -80.
  • Phenol 2-monooxygenase (phenol hydroxylase).
  • Polypeptides of the present invention having this activity can be characterized for example as described in Nakagawa and Takeda, Biochim. Biophys. Acta. (1962), 62 (2): 423- 6; Neujahr and Gaal, Eur. J. Biochem. (1973), 35(2): 386-400; Neujahr and Gaal, Eur. J. Biochem. (1975), 58(2): 351-7; Kirchner et al., J Biol Chem. (2003), 278(48): 47545-53.
  • Polypeptides of the present invention having this activity can be characterized for example using commercially available kits (Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit, Molecular Probes/lnvitrogen).
  • Polypeptides of the present invention having this activity can be characterized for example as described in Paoletti et al., Gene (1998), 210(1): 45-52; Ortega et al., J Basic Microbiol. (2014), 54 Suppl 1 :S170-7; or Chavez-Sanchez et al., J Food Sci Technol. (2013), 50(1 ): 101 -7.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Ortiz et al., Anal Biochem. (2014), 454: 33-5; or Chen et al., Int J Mol Sci. (2014), 15(4): 5717-29.
  • Polyphenol oxidase 1 Polyphenol oxidase 1.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Tao et al., J Agric Food Chem. (2013), 61 (51): 12662-9; Dawson and Magee, Methods in Enzymology ll (1955), 817-821 ; Marumo and Waite, Biochim. Biophys. Acta (1986), 872: 98-103.
  • Polysaccharide deacetylase Polypeptides of the present invention having this activity can be characterized for example as described in Kobayashi et al., J. Biol. Chem. (2012) 287(13): 9765-76. Polysaccharide lyase. Polypeptides of the present invention having this activity can be characterized for example as described in Macdonald and Berger, "A polysaccharide lyase from Stenotrophomonas maltophilia with unique, pH-regulated substrate specificity.”, J. Biol Chem.
  • Polysaccharide monooxygenase Polypeptides of the present invention having this activity can be characterized for example as described in Kittl et al, Biotechnol Biofuels. (2012), 5(1 ):79, Phillips et al, ACS Chem Biol (2011), 6(12): 1399-1406, Wu et al., J. Biol. Chem (2013), 288(18): 12828-39.
  • Polysaccharide monooxygenases sometimes referred to functionally as "cellulase-enhancing proteins" generally belong the enzyme class GH61 and are reported to cleave polysaccharides with the insertion of oxygen.
  • Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2005/0010037 A1 ; using the EnzChek® Peptidase/Protease Assay Kit (Cat. No. E33758, Life Technologies).
  • Polypeptides of the present invention having this activity can be characterized for example as described in Staudigl et al, Biomolecules (2013), 3: 535-552; Tan et al, PLoS One. (2013), 8(1 ):e53567; Kujawa et al, FEBS J. (2007), 274(3): 879-94.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Molgaard et al, Structure(2000), 8(4):373-83; or Kauppinen et al, J Biol Chem. (1995), 270(45):27172-8.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Azadi et al, Glycobiology. (1995), 5(8): 783-9.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Mutter et al. Plant Physiol. (1998), 117: 153-163; or de Vries, AppI. Microbiol Biotechnol. (2003), 61 : 10-20.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Paynel et al. Plant Physiol Biochem. (2013), 62: 54-62; Normand et al, AppI Microbiol Biotechnol. (2012), 94(6): 1543-52.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Jensen et al, J Mol Biol. (2010), 404(1): 100-11.
  • Rhizopuspepsin-3 Polypeptides of the present invention having this activity can be characterized for example as described in Chen et al, J Agric Food Chem. (2009), 57(15):6742-7; Flentke et al. Protein Expr Purif. (1999), 16(2):213-20.
  • Rodlet protein Polypeptides of the present invention having this activity can be characterized for example as described in Yang et al, Biopolymers (2013), 99(1 ): 84-94.
  • Saccharopine dehydrogenase [NADP(+), L-glutamate-forming]
  • Polypeptides of the present invention having this activity can be characterized for example as described in Kumar et al. Arch Biochem Biophys. (2012), 522(1):57-61 ; Ekanayake et al. Arch Biochem Biophys. (201 1 ), 514(1-2):8-15.
  • Serine-type carboxypeptidase F Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 6,379,913.
  • Short-chain dehydrogenase reductase 2a Polypeptides of the present invention having this activity can be characterized for example as described in Bijtenhoorn et al, PLoS One. (201 1), 6(10):e26278; Polizzi et al, Chem Commun (Camb). (2007), 18:1843-5.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Bautz et al, G3 (Bethesda). (2013), 3(10): 1819-25. Subtilisin-like protease. Polypeptides of the present invention having this activity can be characterized for example as described in Dang et al., J I nvertebr Pathol. (2013), 112(2): 166-74; Acevedo et al., J Appl Microbiol. (2013), 114(2):352-63.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Jager et al., Biotechnol Biofuels. (201 1 ), 4: 33; or Saloheimo et al., Eur J Biochem. (2002), 269(17): 4202-1 1.
  • Trihydrophobin Polypeptides of the present invention having this activity can be characterized for example as described in Cheng et al., J. Biol. Chem. (2009), 284(13):8786-96.
  • Tripeptidyl-peptidase sedl Tripeptidyl-peptidase sedl .
  • Polypeptides of the present invention having this activity can be characterized for example as described in Du et al., Biol Chem. (2001 ), 382(12): 1715-25; Hilbi et al, Biochim Biophys Acta. (2002), 1601 (2): 149-54; Renn et al, J Biol Chem. (1998), 273(30): 19173-82.
  • Tyrosinase Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 201 1/0311693 A1 ; Duckworth and Coleman, J. Biol. Chem. (1970) , 245: 1613-1625; Park et al, J Protein Chem. (2003), 22(5): 473-80.
  • Unsaturated rhamnogalacturonyl hydrolase Polypeptides of the present invention having this activity can be characterized for example as described in Itoh et al, Biochem Biophys Res Commun. (2006), 347(4): 1021-9; or Itoh et al, J Mol Biol. (2006), 360(3): 573-85.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Lankinen et al, Appl Microbiol Biotechnol. (2005), 66(4): 401 -7; Banci et al, J Biol Inorg Chem. (2003), 8(7): 751-60; Perez-Boada et al, J Mol Biol. (2005), 354(2): 385-402.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Ishihara, M. and Shimizu, K, "alpha-(1 ->2)-Glucuronidase in the enzymatic saccharification of hardwood xylan: Screening of alpha-glucuronidase producing fungi.” Journal Mokuzai Gakkaishi, (1988) 34: 58-64.
  • Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2012/0028306 A1 ; US patent No. 7,759, 102; PCT application publication No. WO 2006/078256 A2; Chen et al, Agric. Biol. Chem. (1986), 50: 1183-1 194; Lever, M, Analytical Biochemistry (1972), 47: 273-279.
  • Polypeptides of the present invention having this activity can be characterized for example as described in van der Vlugt-Bergmans et al. Applied and Environmental Microbiology (2000), 66(1): 36-41.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Master et al, Biochem. (2008), 41 1 (1 ): 161-170; Ariza et al, J Biol Chem. (201 1), 286(39): 33890-900; Qi et al., Biochemistry (Mosc). (2013), 78(4): 424-30; US patent No. 6,815, 192.
  • Polypeptides of the present invention having this activity can be characterized for example as described in European patent application publication No. EP0972016 B1 ; in US patent No. 6,077,702; Damasio et al, Biochim Biophys Acta. (2012), 1824(3): 461 -7; or Wong et al, Appl Microbiol Biotechnol. (2010), 86(5): 1463-71.
  • Polypeptides of the present invention having this activity can be characterized for example as described in Whitehead and Cotta, Curr Microbiol. (2001 ), 43(4): 293-8; or Xiong et al. Journal of Experimental Botany (2007), 58(1 1): 2799-2810.
  • Standard molecular cloning techniques such as DNA isolation, gel electrophoresis, enzymatic restriction modifications of nucleic acids, E. coli transformation, etc, were performed as described by Sambrook et al., 1989, (Molecular cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York and Innes et al. (1990) PCR protocols, a guide to methods and applications, Academic Press, San Diego, edited by Michael A. Innis et al). Primers were prepared by IDT (Integrated DNA Technologies). Sanger DNA sequencing was performed using an Applied Biosystem's 3730x1 DNA Analyzer technology at the Innovation Centre (Genome Quebec), McGill University in Montreal.
  • pGBFIN-49 Genes of interest were cloned into the expression vector pGBFIN-49.
  • This vector is a derivative of pGBFIN-41 that contains the A. niger glaA promoter, A. niger TrpC terminator, A. nidulans gpdA promoter, gene encoding the pheomycin resistance gene, A. niger glaA terminator and an E. coli backbone.
  • Figure 1 represents a schematic map of pGBFIN-49 and the complete nucleotide sequence is presented as SEQ ID NO: 434. Details of the construction of pGBFIN-49 are as follows:
  • TtrpC terminator was PCR amplified using purified pGBFIN33 plasmid as a template. The following primers and PCR program were used:
  • Primer-3 5 ' -GTCCGTCGCCGTCCTTCAccgccggtccgacg-3 ' (SEQ ID NO: 424)
  • Primer-4 5 ' -GCGGCCGGCGTATTGGGTGttacggagc-3 ' (SEQ ID NO: 425)
  • Primer-4 is entirely specific to the TtrpC 3' end.
  • Primer-3 was designed to suit the LIC cloning strategy but also to keep the TtrpC sequence as close to the original sequence. To do so, five adenines were replaced by thymines (underlined).
  • PCR program 1 x 98°C, 2 min; 25 x (98°C, 30 sec; 68°C, 30 sec; 72°C, 1 min); 72°C, 7 min.
  • Reaction conditions 5 [il of the PCR reaction was separated by electrophoresis on 1.0% agarose remaining was purified using QIAEX IITM gel Extraction kit (QIAGEN) and resuspended in nuclease-
  • Vector backbone was PCR amplified using pGBFIN41 as a template. Primers were designed outside of the ccdA region (not included in pGBFIN49). The following primers and PCR program were used: Primer-2: 5 ' -CACCCAATACGCCGGCCGCgcttccagacagctc-3 ' (SEQ ID NO: 426)
  • Primer-lC 5 ' -GGTGTTTTGTTGCTGGGGAtgaagctcaggctctcagttgcgtc-3 ' (SEQ ID NO: 427)
  • Primer-2 contains a pgpdA-specific region and an extra sequence specific to TtrpC 3' end (also included in Primer-4).
  • Primer-1C was designed to suit the LIC cloning strategy but also to keep PgalA region as close to the original sequence. To do so, three thymines were replaced by adenines (underlined).
  • PCR program 1 x 98°C, 3 min; 10x (98°C, 30 sec ; 68°C, 30 sec, 72°C, 5 min); 20 x (98°C, 30 sec, 68°C, 30 sec, 72°C, 5 min + 10 sec/cycle); 72°C, 10 min.
  • Reaction conditions 5 ⁇ _ of the PCR reaction was separated on a 0.5% agarose gel and remaining was purified using QIAEX IITM gel Extraction kit (QIAGEN) and resuspended in nuclease-free water.
  • Overlap-extension / Long range PCR was performed to: a) fuse the two PCR pieces together; b) add an Sfol restriction site to re-circularize the vector. No primers were used in the overlap-extension stage. Primer-11 and Primer-12 were used for the long range PCR reaction.
  • Primer-ll 5 ' -CACCGGCGCCGTCCGTCGCCGTCCTTC -3' (SEQ ID NO: 428)
  • Primer-12 5 ' -ACGGCGCCGGTGTTTTGTTGCTGGGGATG -3' (SEQ ID NO: 429)
  • Primer-1 1 is specific to the LIC tag located on the TtrpC terminator, while Primer-12 is specific to the LIC tag located on the PglaA region.
  • the Sfol restriction site sequence is underlined above.
  • a standard PCR master mix was prepared to perform overlap-extension PCR using pGBFIN41 and TtrpC purified PCR products as templates. No primers were added.
  • PCR program - overlap (no primers): 1x 98°C, 2 min; 5x (98°C, 15 sec; 58°C, 30 sec; 72°C, 5 min), 5x (98°C, 15 sec; 63°C, 30 sec; 72°C, 5 min), 5x (98°C, 15 sec; 68°C, 30 sec; 72°C, 5 min); 72°C, 10 min.
  • Reaction conditions 5 ⁇ _ of the PCR reaction was separated on 0.5% agarose gel and remaining was purified using QIAEX IITM gel Extraction kit and resuspended in nuclease-free water. Then, Sfol digestion was performed and digested product was purified using QIAEX II gel extraction kit follow the procedure as described by the manufacturer.
  • Example 8 Cloning of Rhizomucor miehei, Thermoascus thermophilus (Dactylomyces thermophilus), and Humicola hyalothermophila genes in E. coii
  • Cloning genes of interest in the pGBFIN-49 expression vector was performed using the Ligation- independent cloning (LIC) method according to Aslanidis, C, de Jong, P. (1990) Nucleic Acids Research Vol. 18 No. 20, 6069-6074.
  • LIC Ligation- independent cloning
  • Coding sequences from genes of interest were amplified by PCR using primers containing LIC tags, which are homologous to Pg/a and TrpC sequences in the pGBFIN-49 cloning vector fused to sequences homologous to the coding sequences of the gene of interest, using cDNA as template.
  • Primers have the following sequences: Forward primer: 5 -CCCCAGCAACAAAACACCTCAGCAATG ...15-20 nucleotides specific to each gene to be cloned (portion of primer for which sequence is shown is set forth in SEQ ID NO: 430)
  • PCR mix consists of following components:
  • Phusion DNA Polymerase (FinnzymesTM) 0.5 ⁇
  • Expression vector pGBFIN-49 was PCR amplified using primers with following sequences:
  • PCR mix consists of following components:
  • Obtained PCR fragments were treated with T4 DNA polymerase in the presence of dTTP to create single stranded tails at the ends of the PCR fragments.
  • the single stranded tails of the PCR fragment are complementary to those of the vector, thus permitting non-covalent bi-molecular associations, e.g., circularization between molecules.
  • Example 9 Transformation of Rhizomucor miehei, Thermoascus thermophilus (Dactylomyces thermophilus), and Humicola hyalothermophila gene expression cassettes into A. niger
  • A. niger GBA307 As host strain for enzyme production, A. niger GBA307 was used. Construction of A. niger GBA307 is described in WO 2011/009700.
  • Transformation of A. niger was performed essentially according to the method described by Tilburn, J. et al. (1983) Gene 26, 205-221 and Kelly, J & Hynes, M. (1985) EMBO J., 4, 475-479 with the following modifications:
  • Aspergillus minimal medium contains per liter: 6 g NaNOa; 0.52 g KCI; 1.52 g KH2PO4; 1.12 ml 4 M KOH; 0.52 g MgS0 4 -7H 2 0; 10 g glucose; 1 g casamino acids; 22 mg ZnS0 4 -7H 2 0; 11 mg H3BO3; 5 mg FeS0 4 » 7H 2 0; 1.7 mg CoCI 2 -6H 2 0; 1.6 mg CuS0 4 -5H 2 0; 5 mg MnCI 2 -2H 2 0; 1.5 mg Na 2 Mo0 4 *2 H 2 0; 50 mg EDTA; 2 mg riboflavin; 2 mg thiamine-HCI; 2 mg nicotinamide; 1 mg pyridoxine-HCI; 0.2 mg panthotenic acid; 4 g biot
  • Glucanex 200G (Novozymes) was used for the preparation of protoplasts
  • the protoplasts were resuspended gently in 1 mL 1.2 M sorbitol and plated onto selective regeneration medium consisting of Aspergillus minimal medium without riboflavin, thiamine. HCI, nicotinamide, pyridoxine, panthotenic acid, biotin, casamino acids and glucose, supplemented with 150 pg/mL Phleomycin (Invitrogen), 0.07 M NaN03, 1 M sucrose, solidified with 2% bacteriological agar #1 (Oxoid, England).
  • Example 10 Aspergillus niger microtiter plate fermentation
  • 96 wells microtiter plates (MTP) with sporulated Aspergillus niger strains were used to harvest spores for MTP fermentations.
  • 100 ⁇ _ water was added to each well and after resuspending the mixture, 40 ⁇ _ of spore suspension was used to inoculate 2 mL A.niger medium (70 g/L glucose*H 2 0, 10 g/L yeast extract, 10 g/L (NH 4 ) 2 S0 4 , 2 g/L K 2 S0 4 , 2 g/L KH 2 P0 4 , 0.5 g/L MgS0 4 -7H 2 0, 0.5 g/L ZnS0 4 -7H 2 0, 0.2 g/L CaCI 2 , 0.01 g/L MnS0 4 -7H 2 0, 0.05 g/L FeS0 4 -7H 2 0, 0.002 Na 2 Mo0 4 -2H 2 0, 0.25 g/L TweenTM-80
  • Example 11 Aspergillus niger shake flask fermentation
  • Example 12 Shake flask concentration and protein concentration determination with TCA-biuret method [00280]
  • the fermentation supernatants obtained as described above volume between 75 and 100 mL
  • the protein concentration in the concentrated supernatant was determined via a TCA-biuret method.
  • A. niger strains expressing Rhizomucor miehei, Thermoascus thermophilus (Dactylomyces thermophilus), and Humicola hyalothermophila clones were grown in shake flask, as described above, in order to obtain greater amounts of material for further testing.
  • the fermentation supernatants (volume between 40 and 80ml) were concentrated using a 10 kDa spin filter to a volume of approximately 5 ml. Subsequently, the protein concentration in the concentrated supernatant was determined via a TCA-biuret method, as described above.
  • pH optima are determined by first determining the range of enzyme concentration that reproducibly displays initial velocity kinetics at standard pH and temperature for the appropriate assay. Enzyme is then diluted to an amount within this range, divided into aliquots, and each aliquot is assayed simultaneously at the different pHs.
  • Temperature optima are determined by first determining the range of enzyme concentration that reproducibly displays initial velocity kinetics at 40°C and at the enzyme's optimal pH (see Example 13.1) in the appropriate assay. Enzyme is then diluted to an amount within this range, divided into aliquots, and, where possible, each aliquot is assayed simultaneously at the different temperatures (e.g., when reaction is incubated in a dry bath heater, then transferred to a plate reader for endpoint measurement). Where simultaneous measurements at different temperatures are impossible (e.g., when reaction is incubated in a plate reader for continuous measurement) activities are measured in sequence at different temperatures. 13.3 Assay Procedure CU1 : Colorimetric assay for glycosidase or esterase activity, measuring release of 4-nitrophenol
  • Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in water, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L.
  • 10 L of diluted enzyme sample is added to 30 ⁇ _ of 50 mM acetate-phosphate-borate reaction buffer at appropriate pH (made by dissolving 2.88 mL 99.7% glacial acetic acid, 3.42 mL 85% phosphoric acid, and 3.10 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) in a PCR plate and preheated to appropriate temperature in a dry bath heater, and reaction is started by addition of 10 ⁇ _ of preheated 5 mM substrate in water (see Table CU-1.1) to buffer and sample.
  • Standards contain 10 ⁇ _ of 4-nitrophenol (from 0 to 3 mM; 3 mM solution is made by dissolving 139 mg 4- nitrophenol in isopropyl alcohol and diluting 300 ⁇ _ of resulting 100 mM solution to 10 mL in water) and 40 il of reaction buffer.
  • Sample blank contains 10 il of enzyme sample and 40 il of reaction buffer.
  • Substrate blank contains 10 il of substrate (see Table CU-1.1) and 40 il of reaction buffer.
  • 50 ⁇ il of [1 ] for 4-nitrophenyl acetate, 1 M HEPES buffer pH 8 in water; [2] for all other substrates, 1 M Na 2 C0 3 in water is added.
  • Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in water, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L.
  • 10 il of diluted sample is added to 30 il of either [1] 50 mM acetate-phosphate-borate reaction buffer at appropriate pH (made by dissolving 2.88 mL 99.7% glacial acetic acid, 3.42 mL 85% phosphoric acid, and 3.10 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) or [2] for enzymes that utilize calcium, 50 mM acetate-MOPS-borate reaction buffer at appropriate pH (made by dissolving 2.88 mL 99.7% glacial acetic acid, 10.45 g MOPS, and 3.10 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) in a PCR plate and preheated to appropriate temperature in
  • the reaction is started by addition of 10 L of preheated substrate in water (see Table CU-2.1) to buffer and sample.
  • Standards contain 10 il of 0 to 7.5 mM monosaccharide solution (see Table CU-2.1) in water and 40 ⁇ _ of reaction buffer.
  • Enzyme sample blank contains 10 ⁇ _ of sample and 40 ⁇ _ of reaction buffer.
  • Substrate blank contains 10 ⁇ _ of substrate (see Table CU-2.1) and 40 ⁇ _ of reaction buffer.
  • PCR plate After appropriate incubation time, 10 is removed and added to another PCR plate containing 95 ⁇ _ of BCA Reagent A (made by dissolving 0.543 g Na2C03, 0.242 g NaHC03 and 19 mg disodium 2,2'-bicinchoninate in water and diluting to 1 L) and 95 ⁇ _ of BCA Reagent B (made by dissolving 12 mg CuSC and 13 mg L-Serine in water and diluting to 1 L), sealed and incubated in a dry bath heater for 25 minutes at 80°C. PCR plate is put on ice for 5 minutes, then 160 ⁇ _ is transferred to a clear microtiter flat-bottomed plate, absorbance is read at 562 nm and compared to the standard curve.
  • BCA Reagent A made by dissolving 0.543 g Na2C03, 0.242 g NaHC03 and 19 mg disodium 2,2'-bicinchoninate in water and diluting to 1 L
  • BCA Reagent B
  • One unit is defined as the amount of enzyme that releases one micromole of monosaccharide-equivalent reducing ends per minute at the specified pH and temperature.
  • Colloidal chitin is prepared by mixing 10 g chitin from crab shell in 100 mL concentrated hydrochloric acid, stirring overnight at room temperature, then adding 1 L cold distilled water, filtering resulting suspension through Whatman No. 1 paper washing retentate with distilled water until pH is greater than 4, determining dry weight by gravimetry and diluting to 1 % solution with distilled water. (Adapted from Shimahara et al. (1988) Methods in Enzymology 161 , 417-423).
  • Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in dH 2 0, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L.
  • 20 ⁇ _ of diluted sample is added to 20 ⁇ _ of 300 mM acetate-phosphate-borate reaction buffer at appropriate pH (made by dissolving 17.28 mL 99.7% glacial acetic acid, 20.52 mL 85% phosphoric acid, and 18.6 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) in a clear microtiter plate and preheated to appropriate temperature in the plate reader.
  • the reaction is started by addition of 160 il 0.5 mM alpha-naphthyl acetate substrate solution in water (prepared by diluting 46.55 mg of a-Naphthyl acetate in 1 mL of acetone and then transferring to 499 ml of water), preheated to assay temperature in a dry block heater, to the buffer and enzyme sample.
  • Standards contain 180 ⁇ _ of 0 to 0.1 mM alpha-naphthol in water and 20 ⁇ _ of reaction buffer.
  • Blank contains 20 ⁇ _ of reaction buffer, 20 ⁇ _ of water and 160 ⁇ _ of substrate solution.
  • Absorbance is continuously monitored at 303 nm and compared to that of the standards.
  • One unit is the amount of enzyme that produces one micromole of alpha-naphthol per minute under the specified conditions. (Adapted from Yuorno et al. (1981 ), Anal. Biochem. 1 15, 188-193).
  • Enzyme sample is diluted in 50 mM acetate-MOPS-borate reaction buffer at appropriate pH (made by dissolving 2.88 mL 99.7% glacial acetic acid, 10.45 g MOPS, 3.10 g boric acid and 1.1 1 g calcium chloride in water, adjusting pH with 10 M NaOH and diluting to 1 L) and left to equilibrate for 30 minutes at room temperature.
  • Reaction buffer is mixed in a 1 : 1 ratio with substrate solution (1 % polygalacturonic acid in water or 0.75% Rhamnogalacturonan I from potato in water) and preheated to reaction temperature in a dry bath heater (if reaction temperature is greater than plate reader maximum temperature) or in a microtiter plate in plate reader.
  • Reaction is started by addition of 10 L of diluted enzyme sample to 240 ⁇ _ of reaction buffer/substrate in UV- transparent microtiter flat-bottomed plate.
  • Blank contains 10 ⁇ _ of reaction buffer added to 240 ⁇ _ of reaction buffer/substrate solution.
  • Absorbance at 235 nm is continuously monitored, and the molar absorptivity coefficient of unsaturated galacturonic acid is used to determine activity.
  • One unit is the amount of enzyme that releases one micromole of unsaturated galacturonic acid equivalents per minute under the specified conditions. Adapted from Hansen et al. (2001) J. AOAC International, 84, 1851 -1854).
  • Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in water, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L.
  • 10 ⁇ _ of diluted sample is added to 30 ⁇ _ of 50 mM acetate-phosphate-borate reaction buffer at appropriate pH (made by dissolving 2.88 mL 99.7% glacial acetic acid, 3.42 mL 85% phosphoric acid, and 3.10 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) in a PCR plate and preheated to appropriate temperature in a dry bath heater.
  • the reaction is started by addition of 10 il of preheated 1 mM substrate in water (made by diluting 5.0 mg of 4- methylumbelliferyl cellobioside or 4-methylumbelliferyl lactoside in 10 mL water) to buffer and sample.
  • Standards contain 10 il of 4-methylumbelliferone (from 0 to 50 uM; 19.8 mg of 4-methylumbelliferone sodium salt is dissolved in 100 mL methanol and resulting solution is diluted 20X in water) and 40 il of reaction buffer.
  • Enzyme sample blank contains 10 il of enzyme sample and 40 il of reaction buffer.
  • Substrate blank contains 10 il of substrate and 40 il of reaction buffer.
  • Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in dhbO, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L. 40 ⁇ _ of 1 % acetylated xylan from birchwood (prepared by Swern oxidation in a fume hood according to Johnson et al.
  • Sample blank contains 20 ⁇ _ of diluted enzyme sample, 40 ⁇ _ of reaction buffer and 40 ul of water.
  • Substrate blank contains 20 ⁇ _ of substrate 40 ⁇ _ of reaction buffer and 40 ⁇ _ of water.
  • the plate is heated to 90°C for 5 minutes and centrifuged 10 minutes at 1500 X g.
  • the amount of acetic acid in the supernatant is then determined with the K-ACETAKTM kit by Megazyme; one unit is defined as the amount of enzyme required to release one micromole of acetic acid per minute under the specified conditions. (Adapted from Johnson et al. (1988), Methods in Enzymoiogy 160, 551 - 560 and K-ACETAK assay kit procedure by Megazyme (Ireland)).
  • Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in water, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L.
  • the reaction buffer used is 50 mM acetate-phosphate-borate reaction buffer at appropriate pH (made by dissolving 2.88 mL 99.7% glacial acetic acid, 3.42 mL 85% phosphoric acid, and 3.10 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L). (Adapted from K-AGLUATM assay kit procedure by Megazyme, Ireland).
  • a niger strains expressing Rhizomucor miehei, Thermoascus thermophiius (Dactyiomyces thermophiius), and Humicoia hyaiothermophiia clones were grown in shake flask, as described above, in order to obtain greater amounts of material for further testing.
  • the fermentation supernatants (volume between 40 and 80ml) were concentrated using a 10 kDa spin filter to a volume of approximately 5 ml. Subsequently, the protein concentration in the concentrated supernatant was determined via a TCA-biuret method, as described above.
  • the proteins were spiked based on dosage in a total volume of 20 ml at a feedstock concentration of 10% aCS (w/w) in a 40-ml centrifuge bottle (Nalgene Oakridge). All experiments were performed at least in duplicate and were incubated for 72 hours at 65°C in an oven incubator (Techne HB-1 D hybridization oven) while rotating at set-point 3.
  • the (hemi)cellulose activity of the proteins can also be tested on 5.5% (w/w) washed acid pretreated corn stover.
  • the feedstock solution was prepared via the dilution of a washed (with MQ until pH is neutral and no soluble sugars are detected anymore in the liquid) concentrated feedstock solution with water.
  • the sugar content of the samples after enzymatic hydrolysis were analyzed using a High- Performance Liquid Chromatography System (Agilent 1100) equipped with a refection index detector (Agilent 1260 Infinity).
  • the separation of the sugars was achieved by using a 300 X 7.8 mm Aminex HPX-87P (Bio rad cat no 125-0098) column; Pre-column: Micro guard Carbo-P (Bio Rad cat. no. 125-0119); mobile phase was HPLC grade water; flow rate of 0.6 ml/min and a column temperature of 85 °C.
  • the injection volume was 10 ⁇ .
  • the samples were diluted with HPLC grade water to a maximum of 10 g/l glucose and filtered by using 0.2 ⁇ filter (Afridisc LC25mm syringe filter PVDF membrane).
  • the glucose was identified and quantified according to the retention time, which was compared to the external glucose standard (D-(+)-Glucose Sigma cat no: G7528) ranging from 0.2; 0.4; 1.0; 2.0 g/l.
  • the analysis can also be performed on a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (2 mm ID x 50 mm) and a Dionex PAD-detector (Dionex Co. Sunnyvale).
  • a flow rate of 0.3 mL/min is used and elution from 0-15 min with 22 mM NaOH, a gradient from 15.0-15.1 min; 22-100mM NaOH, 15.1 -18.0 min; 100 mM NaOH, followed by a gradient of sodium acetate in 0.1 M NaOH: 18-28 min, 0-200 mM.
  • Each elution is followed by a washing step of 3 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 10 min at 100 mM NaOH and 20 min 22 mM NaOH.
  • Standards of glucose, xylose, cellobiose and xylobiose (Sigma) are used to identify and quantify the mono- and oligomers released by the action of the enzyme.
  • This assay measures the ability of a-arabino(furano)sidases to remove the a-L-arabinofuranosyl residues from substituted xylose residues.
  • Single and double substituted oligosaccharides are prepared by incubating wheat arabinoxylan (WAX medium viscosity; 2 mg/mL; Megazyme, Bray, Ireland) in 50 mM acetate buffer pH 4.5 with an appropriate amount of endo-xylanase (Aspergillus Awamori, FJM, Kormelink, Carbohydrate Research, 249 (1993) 355-367) for 48 hours at 50°C to produce a sufficient amount of arabinoxylo-oligosaccharides. The reaction is stopped by heating the samples at 100°C for 10 minutes. The samples are centrifuged for 5 minutes at 10.000 x g. The supernatant is used for further experiments.
  • WAX medium viscosity 2 mg/mL; Megazyme, Bray, Ireland
  • HPAEC High Performance Anion Exchange Chromatography
  • the analysis is performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (2 mm ID x 50 mm) and a Dionex PAD-detector (Dionex Co. Sunnyvale).
  • a flow rate of 0.3 mL/min is used with the following gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM.
  • Each elution is followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.
  • Arabinose release is quantified by an arabinose standard (Sigma) and compared to a sample where no enzyme was added.
  • Example 15 Improvement of thermophilic cellulase mixture by Rhizomucor miehei proteins in an activity assay.
  • Rhizomucor miehei proteins were cloned and expressed in A niger as described above. Concentrated supernatants from shake flask fermentations were used in sugar release activity assays as described above, using 10% aCS NREL as feedstock. In one set of experiments, supernatant of a Rhizomucor miehei protein was spiked in a dosage of 2 mg/gDM op top of a TEC-210 base mix in a dosage of 2.5 mg/gDM, as described above. The protein showed increased sugar release as shown below in Table 4.
  • Table 4 Effect of a Rhizomucor miehei protein spiked on top of TEC-210 using 10%DM aCS substrate.
  • Example 16 Improvement of a thermophilic cellulase mixture composed of three enzymes by a Humicoia hyalothermophilia protein in an activity assay.
  • Humicoia hyaiothermophiiia cellobiohydrolase I (CBHI) protein (Humhy2p7_013018) was further analysed.
  • niger expressing Humhy2p7_013018 shake flask fermentation was concentrated and spiked in a dosage of 1.25 mg/gDM on top of a base activity of a three enzyme base mix (3.75 mg/gDM composed of: beta-glucosidase (BG) at 0.45 g/gDM, cellobiohydrolase II (CBHII) at 1.5 mg/gDM and GH61 at 1.8 mg/gDM) at a feedstock concentration of 10% (w/w) aCS, as described above.
  • BG beta-glucosidase
  • CBHII cellobiohydrolase II
  • GH61 GH61 at 1.8 mg/gDM
  • the 3 enzyme base mix was also tested. The experiments were performed at least in duplicate and were incubated for 72 hours at 62°C in an oven incubator (Techne HB-1 D hybridization oven) while rotating at set- point 3. After incubation, the samples were centrifuged and soluble sugars were analysed by HPLC as described above.
  • Table 5 Effect of CBHI Humhy2p7_013018 protein spiked on top of a 3E mix using aCS substrate substrate.
  • Example 17 Determination of temperature optima of Rhizomucor miehei and Humicola hyalothermophila proteins
  • SEQ ID NOs: 1-92 relate to sequences from Rhizomucor miehei (RHIMY);
  • SEQ ID NOs: 93-172 relate to sequences from Thermoascus thermophilus (Dactylomyces thermophilus) (THETH);
  • SEQ ID NOs: 173-422 relate to sequences from Humicola hyalothermophila (HUMHY).
  • thermophilus

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Husbandry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne de nouveaux polypeptides et de nouvelles enzymes (p. ex. des protéines et des enzymes thermostables) ayant des activités associées au traitement et/ou à la dégradation d'une biomasse (p. ex. la déconstruction d'une paroi cellulaire), ainsi que des polynucléotides, des vecteurs, des cellules, des compositions et des outils associés ou leurs variantes fonctionnelles. Plus particulièrement, la présente invention concerne des enzymes secrétées pouvant être isolées à partir des champignons suivants : souche de Rhizomucor miehei CBS 182.67, souche de Thermoascus thermophilus (Dactylomyces thermophilus) ATCC 26413 et souche de Humicola hyalothermophila CBS 454.80. L'invention concerne également leurs utilisations dans différents processus industriels tels que dans des biocarburants, des préparations alimentaires, de la nourriture pour animaux, de la pâte et du papier, des textiles, des détergents, le traitement de déchets et autres.
PCT/CA2015/051284 2014-12-09 2015-12-08 Nouvelles enzymes de déconstruction de paroi cellulaire issues de rhizomucor miehei, thermoascus thermophilus (dactylomyces thermophilus) et humicola hyalo thermophila et leurs utilisations Ceased WO2016090473A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US201462089380P 2014-12-09 2014-12-09
US201462089383P 2014-12-09 2014-12-09
US201462089371P 2014-12-09 2014-12-09
US62/089,380 2014-12-09
US62/089,371 2014-12-09
US62/089,383 2014-12-09

Publications (1)

Publication Number Publication Date
WO2016090473A1 true WO2016090473A1 (fr) 2016-06-16

Family

ID=56106356

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2015/051284 Ceased WO2016090473A1 (fr) 2014-12-09 2015-12-08 Nouvelles enzymes de déconstruction de paroi cellulaire issues de rhizomucor miehei, thermoascus thermophilus (dactylomyces thermophilus) et humicola hyalo thermophila et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2016090473A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3385362A1 (fr) * 2017-04-05 2018-10-10 Henkel AG & Co. KGaA Compositions détergentes comprenant des mannanases fongiques
EP3434763A1 (fr) * 2017-07-25 2019-01-30 DirectSens GmbH Déshydrogénase de cellobiose mutée dotée d'une spécificité de substrat modifiée
WO2020014407A1 (fr) * 2018-07-11 2020-01-16 Novozymes A/S Procédés de production de produits de fermentation
WO2020058228A1 (fr) * 2018-09-17 2020-03-26 Dsm Ip Assets B.V. Compositions d'aliments pour animaux et leurs utilisations
WO2021055395A1 (fr) 2019-09-16 2021-03-25 Novozymes A/S Polypeptides dotés d'une activité bêta-glucanase et polynucléotides codant pour ces polypeptides
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes
CN118931881A (zh) * 2024-09-26 2024-11-12 山东福瑞达医药集团有限公司 一种蜘蛛毒液来源的重组透明质酸酶、酵母工程菌及构建方法与应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013181760A1 (fr) * 2012-06-08 2013-12-12 Concordia University Nouvelles enzymes de déconstruction de la paroi cellulaire de scytalidium thermophilum, myriococcum thermophilum et aureobasidium pullulans, et leurs utilisations
WO2014138983A1 (fr) * 2013-03-14 2014-09-18 Concordia University Nouvelles enzymes de dégradation de paroi cellulaire de malbranchea cinnamomea, thielavia australiensis et paecilomyces byssochlamydoides et utilisations associées

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013181760A1 (fr) * 2012-06-08 2013-12-12 Concordia University Nouvelles enzymes de déconstruction de la paroi cellulaire de scytalidium thermophilum, myriococcum thermophilum et aureobasidium pullulans, et leurs utilisations
WO2014138983A1 (fr) * 2013-03-14 2014-09-18 Concordia University Nouvelles enzymes de dégradation de paroi cellulaire de malbranchea cinnamomea, thielavia australiensis et paecilomyces byssochlamydoides et utilisations associées

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3385362A1 (fr) * 2017-04-05 2018-10-10 Henkel AG & Co. KGaA Compositions détergentes comprenant des mannanases fongiques
EP3434763A1 (fr) * 2017-07-25 2019-01-30 DirectSens GmbH Déshydrogénase de cellobiose mutée dotée d'une spécificité de substrat modifiée
WO2019020673A1 (fr) * 2017-07-25 2019-01-31 Directsens Gmbh Cellobiose déshydrogénase mutée à spécificité de substrat modifiée
WO2020014407A1 (fr) * 2018-07-11 2020-01-16 Novozymes A/S Procédés de production de produits de fermentation
CN112368393A (zh) * 2018-07-11 2021-02-12 诺维信公司 用于生产发酵产物的方法
WO2020058228A1 (fr) * 2018-09-17 2020-03-26 Dsm Ip Assets B.V. Compositions d'aliments pour animaux et leurs utilisations
WO2021055395A1 (fr) 2019-09-16 2021-03-25 Novozymes A/S Polypeptides dotés d'une activité bêta-glucanase et polynucléotides codant pour ces polypeptides
US12275967B2 (en) 2019-09-16 2025-04-15 Novozymes A/S Processes for producing fermentation products and compositions used therein
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes
CN118931881A (zh) * 2024-09-26 2024-11-12 山东福瑞达医药集团有限公司 一种蜘蛛毒液来源的重组透明质酸酶、酵母工程菌及构建方法与应用

Similar Documents

Publication Publication Date Title
US20150175980A1 (en) Novel cell wall deconstruction enzymes of scytalidium thermophilum, myriococcum thermophilum, and aureobasidium pullulans, and uses thereof
WO2014138983A1 (fr) Nouvelles enzymes de dégradation de paroi cellulaire de malbranchea cinnamomea, thielavia australiensis et paecilomyces byssochlamydoides et utilisations associées
WO2015109405A1 (fr) Nouvelles enzymes de déconstruction de la paroi cellulaire de chaetomium thermophilum, thermomyces stellatus et corynascus sepedonium, et utilisations desdites enzymes
WO2014059541A1 (fr) Nouvelles enzymes de déconstruction de la paroi cellulaire de thermoascus aurantiacus, myceliophthora fergusii (corynascus thermophilus) et pseudocercosporella herpotrichoides et leurs utilisations
WO2016090474A1 (fr) Nouvelles enzymes de déconstruction de paroi cellulaire issues de chaetomium olivicolor, acremonium thermophilum, et myceliophthora hinnulea et leurs utilisations
DK2519630T3 (en) METHOD OF TREATING CELLULOS MATERIAL AND CBHII / CEL6A ENZYMES THAT CAN BE USED THEREOF
WO2014110675A1 (fr) Nouvelles enzymes de déconstruction de paroi cellulaire issues d'amorphotheca resinae, rhizomucor pusillus et calcarisporiella thermophila et leurs utilisations
WO2012130950A1 (fr) Nouvelles enzymes de déconstruction de la paroi des cellules de talaromyces thermophilus et utilisations de celles-ci
WO2016090472A1 (fr) Nouvelles enzymes de déconstruction de paroi cellulaire issues de remersonia thermophila (stilbella thermophila), melanocarpus albomyces et lentinula edodes et leurs utilisations
WO2012130964A1 (fr) Nouvelles enzymes de déconstruction des parois cellulaires de thermomyces lanuginosus et leurs utilisations
WO2016090473A1 (fr) Nouvelles enzymes de déconstruction de paroi cellulaire issues de rhizomucor miehei, thermoascus thermophilus (dactylomyces thermophilus) et humicola hyalo thermophila et leurs utilisations
WO2012092676A1 (fr) Nouvelles enzymes de déconstruction de parois cellulaires et leurs utilisations
FI124247B (en) Novel esterases in the treatment of cellulose and lignocellulosic materials
WO2012093149A2 (fr) Nouvelles enzymes de déconstruction des parois cellulaires et leurs utilisations
WO2014140165A1 (fr) Enzymes de déconstruction de la paroi cellulaire de paecilomyces byssochlamydoides et utilisations de celles-ci
US20190345461A1 (en) Polypeptides having cellulolytic enhancing activity and uses thereof
WO2013182669A2 (fr) Nouvelles enzymes de myriococcum thermophilum de déconstruction de la paroi cellulaire et leurs utilisations
EP3387139B1 (fr) Bêta-glucosidase et leurs utilisations
US10815471B2 (en) Method for producing reducing sugar from lignocellulosic substrates
WO2014060379A1 (fr) Enzymes de dégradation de la paroi cellulaire tirées de myceliophthora fergusii (corynascus thermophilus) et leurs utilisations
CN108884481A (zh) β-葡糖苷酶及其用途
US20190002859A1 (en) Beta-glucosidase and uses thereof
WO2014140167A1 (fr) Enzymes de décomposition de la paroi cellulaire issues de malbranchea cinnamomea et leurs utilisations
WO2013182670A2 (fr) Nouvelles enzymes de scytalidium thermophilum de déconstruction de la paroi cellulaire et leurs utilisations
WO2014060380A1 (fr) Enzymes de dégradation de la paroi cellulaire tirées de thermoascus aurantiacus et leurs utilisations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15867063

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15867063

Country of ref document: EP

Kind code of ref document: A1