WO1997027293A1 - Enzyme possedant une activite de type xylanase - Google Patents

Enzyme possedant une activite de type xylanase Download PDF

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Publication number
WO1997027293A1
WO1997027293A1 PCT/DK1997/000033 DK9700033W WO9727293A1 WO 1997027293 A1 WO1997027293 A1 WO 1997027293A1 DK 9700033 W DK9700033 W DK 9700033W WO 9727293 A1 WO9727293 A1 WO 9727293A1
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Prior art keywords
enzyme
dna sequence
xylanase
dna
strain
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PCT/DK1997/000033
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English (en)
Inventor
Thomas Sandal
Lene Venke Kofod
Markus Sakari Kauppinen
Lene Nonboe Andersen
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Novo Nordisk A/S
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Priority to AU14378/97A priority Critical patent/AU1437897A/en
Publication of WO1997027293A1 publication Critical patent/WO1997027293A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • 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
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes
    • C12C5/006—Beta-glucanase or functionally equivalent enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C7/00—Preparation of wort
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K13/00—Sugars not otherwise provided for in this class
    • C13K13/002—Xylose
    • 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
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes

Definitions

  • TITLE An enzyme with xylanase activity
  • the present invention relates to an enzyme with xylanase activity, a DNA construct encoding the enzyme with xylanase activity, a method of producing the enzyme, an enzyme preparation comprising said enzyme with xylanase activity, and the use of said enzyme and enzyme preparation for a number of 10 industrial applications.
  • Xylan a major component of plant hemicellulose, is a
  • Xylan can be degraded to xylose and xylo-oligomers by acid or enzymatic hydrolysis. Enzymatic hydrolysis of xylan produces free sugars without the by-products formed with acid (e.g. furans) .
  • Enzymes which are capable of degrading xylan and other plant cell wall polysaccharides are important for the feed and food industry.
  • industri xylanases are primarily used as feed enhancers and for processing of feed.
  • xylanases are primarily used for baking, and in fruit
  • WO 92/17573 discloses a substantially pure xylanase derived from the fungal species H. insolens and recombinant DNA encoding said xylanase.
  • the xylanase is stated to be useful as a baking agent, a feed additive, and in the preparation of paper and pulp.
  • WO 92/01793 discloses a xylanase derived from the fungal species Aspergillus tubigensi ⁇ . It is mentioned, but not shown that related xylanases may be derived from other filamentous fungi, examples of which are Aspergillus , Disporotrichum, Penicillium, Neurospora , Fusarium and Trichoderma . The xylanases are stated to be useful in the preparation of bread or animal feed, in breewing and in reducing viscosity or improving filterability of cereal starch.
  • the inventors have now succeeded in isolating and characterizing a DNA sequence, which encodes an enzyme exhibiting xylanase activity, thereby making it possible to prepare a mono-component xylanase preparation. Accordingly, in a first aspect the invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting xylanase activity, which DNA sequence comprises
  • i) is homologous with the DNA sequence defined in a) , or
  • ⁇ i) encodes a polypeptide which is at least 70% homologous with the polypeptide encoded by a DNA sequence comprising the DNA sequence defined in a) , or
  • iv) encodes a polypeptide which is immunologically reac- tive with an antibody raised against the purified xylanase encoded by the DNA sequence defined in a) .
  • the full length DNA sequence SEQ ID No. 1 encoding a xylanase has been derived from a strain of the filamentous fungus Thielavia terrestris and is present in the Escherichia coli strain DSM No. 10363.
  • the xylanase encoding sequence harboured in DSM 10363 is believed to have the same sequence as that identified in SEQ ID NO 1. Accordingly, whenever reference is made to the xylanase encoding part of SEQ ID No. 1 such reference is also intended to include reference to the xylanase encoding DNA sequence present in DSM 10363. Accordingly, the terms "the xylanase encoding part of the DNA sequence SEQ ID No.
  • the invention provides an expression vector harbouring the DNA construct of the invention, a cell comprising said DNA construct or said expression vector and a method of producing an enzyme exhibiting xylanase activity, which method comprises culturing said cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
  • the invention provides an enzyme exhibiting xylanase activity, which enzyme,
  • (b) is produced by the method of the invention; and/or (c) is immunologically reactive with an antibody raised against a purified xylanase encoded by the DNA sequence obtainable from Escherichia coli DSM no. 10363.
  • the present invention provides an enzyme preparation useful for the degradation or modification of plant material or components, said preparation being enriched in an enzyme exhibiting xylanase activity as described above.
  • the present invention relates to the use of an enzyme or an enzyme preparation of the invention for various industrial applications.
  • No. 10363 harbouring a xylanase encoding DNA sequence (the xylanase encoding part of the SEQ ID No. 1) derived from a strain of the filamentous fungus Thielavia terrestris or any mutant of said strain having retained the xylanase encoding capability; and to an isolated substantially pure biological culture of the filamentous fungus Thielavia terrestris NRRL No.
  • DNA Constructs The present invention provides a DNA construct comprising a DNA sequence encoding an enzyme exhibiting xylanase activity, which DNA sequence comprises
  • (iii) encodes a polypeptide which is at least 70% homologous with the polypeptide encoded by a DNA sequence comprising the DNA sequence defined in (a) , or (iv) encodes a polypeptide which is immunologically reactive with an antibody raised against the purified xylanase encoded by the DNA sequence defined in (a) .
  • xylanase encoding part used in connection with a DNA sequence means the region of the DNA sequence which corresponds to the region which is translated into a polypeptide sequence.
  • SEQ ID NO 1 it is the region between the first "ATG” start codon ("AUG” codon in mRNA) and the following stop codon ("TAA”, "TAG” or “TGA”) . In others words this is the translated polypeptide.
  • the translated polypeptide comprises, in addition to the mature sequence exhibiting xylanase activity, an N- ter inal signal sequence.
  • the signal sequence generally guides the secretion of the polypeptide.
  • xylanase encoding part is intended to cover the translated polypeptide and the mature part thereof.
  • a DNA sequence analogous to the xylanase encoding part of the DNA sequence SEQ ID No. 1 is intended to indicate any DNA sequence encoding an enzyme exhibiting xylanase activity, which enzyme has one or more of the properties cited under (i)-(iv) above.
  • the analogous DNA sequence may be isolated from a strain of the filamentous fungus Thielavia terrestris producing the enzyme with xylanase activity, or another or related organism and thus, e . g. be an allelic or species variant of the xylanase encoded by the DNA sequence SEQ ID No. 1.
  • the analogous sequence may be constructed on the basis of the DNA sequence presented as the xylanase encoding part of SEQ ID No. 1, e .g be a sub-sequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the xylanase encoded by the DNA sequence, but which corresponds to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence.
  • amino acid changes are preferably of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding or activity of the protein, small deletions, typically of one to about 30 amino acids; small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • conservative substitutions are within the group of basic amino acids (such as arginine, lysine, histi- dine) , acidic amino acids (such as glutamic acid and aspartic acid) , polar amino acids (such as glutamine and asparagine) , hydrophobic amino acids (such as leucine, isoleucine, valine) , aromatic amino acids (such as phenylalanine, tryptophan, ty ⁇ rosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine) .
  • basic amino acids such as arginine, lysine, histi- dine
  • acidic amino acids such as glutamic acid and aspartic acid
  • polar amino acids such as glutamine and asparagine
  • hydrophobic amino acids such as leucine, isoleucine, valine
  • aromatic amino acids such as phenylalanine, tryptophan, ty ⁇ rosine
  • xylanase activity to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of substrate-enzyme interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (cf. e .g. de Vos et al., (1992), Science 255, 306-312; Smith et al., (1992), J. Mol. Biol. 224, 899-904; Wlodaver et al., (1992), FEBS Lett. 309, 59-64).
  • the xylanase encoded by the DNA sequence of the DNA construct of the invention may comprise a cellulose binding domain (CBD) existing as an integral part of the encoded enzyme, or a CBD from another origin may be introduced into the xylanase enzyme.
  • CBD cellulose binding domain
  • Examples of suitable CBD's are given by Tomme, P. et al. ("Cellulose-Binding Domains: Classification and Properties" in "Enzymatic Degradation of Insoluble Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618, 1996.).
  • W093/21331 discloses a suitable method of introducing a CBD into the xylanase of the invention.
  • the sequence shown as amino acid number 266 to 295 in SEQ ID No 2 is a cellulose binding domain (CBD).
  • the sequence is following: "(266) WGQCGGQGWTGPTCCSQGTCKAQNQWYSQC(295)” and it is presently believed to be a CBD based on the in the art known CBD consensus sequence "WGQCGGXGXXGXXXCXXGXTCXXXNXXYXQC".
  • the homology referred to in i) above is determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
  • the homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package
  • the coding region of the DNA sequence exhibits a degree of identity preferably of at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95% with the xylanase encoding part of the DNA sequence shown in SEQ ID No. 1.
  • the hybridization referred to in (ii) above is intended to indicate that the analogous DNA sequence hybridizes to the same probe as the DNA sequence encoding the xylanase enzyme under certain specified conditions which are described in detail in the Materials and Methods section hereinafter.
  • the probe to be used may conveniently be constructed on the basis of the xylanase encoding part of the DNA sequence SEQ ID No. 1, or a sub-sequence thereof encoding at least 6-7 amino acids of the enzyme. In the latter case the probe is prepared from an amino acid subsequence corresponding to a high number of low degenerated codons.
  • the homology referred to in iii) above is determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
  • the homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453.
  • the polypeptide encoded by an analogous DNA sequence exhibits a degree of identity preferably of at least 70%, more preferably at least 80%, especially at least 90% with the enzyme encoded by a DNA construct comprising the xylanase encoding part of the DNA sequence shown in SEQ ID No. 1.
  • the immunological reactivity may be determined by the method described in the Materials and Methods section below.
  • the DNA sequence encoding a xylanase of the invention can be isolated from the Escherichia coli strain Escherichia coli DSM No. 10363 using standard methods e .g. as described by Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab.; Cold Spring Harbor, NY.
  • DNA sequence encoding an enzyme exhibiting xylanase activity of the invention can also be isolated by any general method involving
  • the DNA sequence encoding the xylanase is derived from a strain of Thielavia, especially a strain of Thielavia terrestris .
  • a DNA sequence encoding an enzyme homologous to the enzyme of the invention may be obtained from other microorganisms.
  • the DNA sequence may be derived by similarly screening a cDNA library of another microorganism, in particular a fungus, such as a strain of an Aspergillus sp. , in particular a strain of A. aculeatus or A . niger, a strain of Trichoderma ⁇ p. , in particular a strain of T. ree ⁇ ei , T. viride , T. longibrachiatum, T. harzianum or T.
  • a strain of a Fu ⁇ arium ⁇ p. in particular a strain of F. oxysporu , or a strain of a Humicola sp. , or a strain of a Neocallimastix ⁇ p. , a Piromyce ⁇ ⁇ p. , a Penicillium ⁇ p. , an AureoJasidium ⁇ p. , a Thermoa ⁇ cu ⁇ sp. , a Paecilomyces ⁇ p. , a Talaromyce ⁇ ⁇ p. , a Magnaporthe ⁇ p. , a Schizophyllum ⁇ p. , a Filiba ⁇ idium ⁇ p. , or a Cryptococcu ⁇ ⁇ p.
  • the expression plasmid pYES 2.0 comprising the full length DNA sequence encoding the xylanase of the invention has been transformed into a strain of the E. coli which was deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutshe Sammlung von Mikroorganismen und Zellkulturen GmbH.
  • the DNA sequence encoding the enzyme exhibiting xylanase activity can for instance be isolated from the above mentioned deposited strains by standard methods.
  • the DNA encoding a xylanase of the invention may, in accordance with well-known procedures, conveniently be isolated from a suitable source, such as any of the above mentioned organisms, by use of synthetic oligonucleotide probes prepared on the basis of a DNA sequence disclosed herein.
  • a suitable oligonucleotide probe may be prepared on the basis of the xylanase encoding part of the nucleotide sequences presented as SEQ ID No. 1 or any suitable subsequence thereof.
  • the invention provides a recombinant expression vector comprising the DNA construct of the invention.
  • the expression vector of the invention may be any expression vector that is conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence encoding the xylanase should be operably connected to a suitable promoter and terminator sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • the procedures used to ligate the DNA sequences coding for the xylanase, the promoter and the terminator, respectively, and to insert them into suitable vectors are well known to persons skilled in the art (cf., for instance, Sambrook et al., (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY) .
  • suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al . , The EMBO J. 4. (1985), 2093 - 2099) or the tpiA promoter.
  • Examples of other useful promoters are those derived from the gene encoding Aspergillu ⁇ oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, Aspergillus niger acid stable a-amylase, Aspergillus niger or Aspergillu ⁇ awamori glucoamylase (gluA) , Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, A ⁇ pergillu ⁇ oryzae triose phosphate isomerase or A ⁇ pergillu ⁇ nidulan ⁇ acetamidase.
  • the invention provides a host cell comprising the DNA construct of the invention and/or the recombinant expression vector of the invention.
  • the host cell of the invention is a eukaryotic cell, in particular a fungal cell such as a yeast or filamentous fungal cell.
  • the cell may belong to a species of Trichoderma, preferably Trichoderma harzianum or
  • Trichoderma ree ⁇ ei or a species of A ⁇ pergillu ⁇ , most preferably A ⁇ pergillu ⁇ oryzae or A ⁇ pergillu ⁇ niger.
  • Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se.
  • the use of A ⁇ pergillu ⁇ as a host microorganism is described in EP 238 023 (Novo Nordisk A/S) , the contents of which are hereby incorporated by ref ⁇ erence.
  • the host cell may also be a yeast cell, e . g .
  • Saccharomyce ⁇ in particular Saccharomyce ⁇ cerevisae, Saccharomyce ⁇ kluyveri or Saccharomyces uvarum, a strain of Schizosaccharomyce ⁇ sp. , such as Schizosaccharomyces pombe , a strain of Han ⁇ enula sp. , Pichia sp. , Yarrowia sp., such as Yarrowia lipolytica , or Kluyveromyce ⁇ sp., such as Kluyveromyces lacti ⁇ .
  • the present invention provides a method of producing an enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question.
  • the expressed xylanase may conveniently be secreted into the culture medium and may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • the present invention relates to an enzyme preparation useful for the degradation of plant cell wall components, said preparation being enriched in an enzyme exhibiting xylanase activity as described above.
  • the enzyme composition having been enriched with an enzyme of the invention may e.g. be an enzyme preparation comprising multiple enzymatic activities, in particular an enzyme preparation comprising multiple plant cell wall degrading enzymes such as Biofeed+®, Energex®, Viscozym®, Pectinex®, Pectinex Ultra SP®, (all available from Novo Nordisk A/S) .
  • the term "enriched" is intended to indicate that the xylanase activity of the enzyme preparation has been increased, e.g. with an enrichment factor of 1.1, conveniently due to addition of an enzyme of the invention prepared by the method described above.
  • the enzyme preparation enriched in an enzyme exhibiting xylanase activity may be one which comprises an enzyme of the invention as the major enzymatic component, e.g. a mono-component enzyme preparation.
  • the enzyme preparation may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry preparation.
  • the enzyme preparation may be in the form of a granulate or a microgranulate.
  • the enzyme to be included in the preparation may be stabilized in accordance with methods known in the art.
  • the enzyme preparation of the invention may, in addition to a xylanase of the invention, contain one or more other enzymes, for instance those with xylanolytic, or pectinolytic activities such as a-arabinosidase, a-glucoronisidase, fo ⁇ xylosidase, xylan acetyl esterase, arabinanase, rhamnogalacturonase, pectin acetylesterase, galactanase, pectin lyase, pectate lyase, glucanase, pectin methylesterase.
  • a-arabinosidase a-glucoronisidase
  • fo ⁇ xylosidase xylan acetyl esterase
  • arabinanase rhamnogalacturonase
  • pectin acetylesterase galactanase
  • the additional enzyme(s) may be producible by means of a microorganism belonging to the genus A ⁇ pergillu ⁇ , preferably A ⁇ pergillu ⁇ niger, A ⁇ pergillu ⁇ aculeatu ⁇ , Aspergillus awamori or Aspergillus oryzae, or Trichoderma, or Humicola insolen ⁇ .
  • the dosage of the enzyme preparation of the invention and other conditions under which the preparation is used may be determined on the basis of methods known in the art. In general terms, the enzyme is to be used in an efficient amount for providing the desired effect.
  • the enzyme preparation according to the invention may be useful for at least one of the following purposes.
  • the enzyme preparation according to the invention is preferably used as an agent for degradation or modification of plant cell walls or any ylan-containing material originating from plant cells walls due to the high plant cell wall degrading activity of the xylanase of the invention.
  • the xylanase of the invention hydrolyse b-1,4 linkages in xylans.
  • Xylans are polysaccharides having a backbone composed of b-1,4 linked xylose.
  • the backbone may have different sidebranches, like arabinose, acetyl, glucuronic acid and 4- methylglucuronic acid sidebranches.
  • the composition and number of sidebranches vary according to the source of the xylan. Arabinose sidebranches dominate in xylans from cereal endosperm, whereas xylans from hard wood contain relatively more acetyl and glucuronic acid substituents (Michael P. Coughlan and Geoffrey P. Hazlewood. Biotechnol.Appl. Biochem. 17 : 259-289 (1993) .
  • Xylan originating from red algae contains a mixture of b-1,4 and b-l,3 linked xylose in the backbone, this type of xylan is degradable by xylanases to varying extent due to the 1,4-links in the backbone.
  • xylan by xylanases The degradation of xylan by xylanases is facilitated by full or partial removal of the sidebranches.
  • Acetyl groups can be removed by alkali, or by xylan acetyl-esterases
  • arabinose sidegroups can be removed by a mild acid treatment or by alpha- arabinosidases and the glucuronic acid sidebranches can be removed by alpha-glucuronisidases.
  • the oligomers with are released by the xylanase or by a combination of xylanases and sidebranch-hydrolysing enzymes as mentioned above can be further degraded to free xylose by beta-xylosidases.
  • the xylanase of the present invention can be used without other xylanolytic enzymes or with limited activity of other xylanolytic enzymes to degrade xylans for production of oligosaccharides.
  • the oligosaccharides may be used as bulking agents, like arabinoxylan oligosaccharides released from cereal cell wall material, or of more or less purified arabinoxylan ⁇ from cereals.
  • the xylanase of the present invention can be used in combination with other xylanolytic enzymes to degrade xylans to xylose and other monosaccharides.
  • the released xylose may be converted to other compounds like furanone flavours.
  • the xylanase of the present invention may be used alone or together with other enzymes like a glucanase to improve the extraction of oil from oil-rich plant material, like corn-oil from corn-embryos.
  • the xylanase of the present invention may be used for separation of components of plant cell materials, in particular of cereal components such as wheat components.
  • cereal components such as wheat components.
  • the separation process may be performed by use of methods known in the art, conveniently a so-called batter process (or wet milling process) performed as a hydroclone or a decanter process.
  • batter process or wet milling process
  • the starting material is a dilute pumpable dispersion of the plant material such as wheat to be subjected to separ ⁇ ation.
  • the dispersion is made normally from wheat flour and water.
  • the xylanase of the invention may also be used in the preparation of fruit or vegetable juice in order to increase yield, and in the enzymatic hydrolysis of various plant cell wall-derived materials or waste materials, e.g. from paper production, or agricultural residues such as wheat-straw, corn cobs, whole corn plants, nut shells, grass, vegetable hulls, bean hulls, spent grains, sugar beet pulp, olive pulp, and the like.
  • various plant cell wall-derived materials or waste materials e.g. from paper production, or agricultural residues such as wheat-straw, corn cobs, whole corn plants, nut shells, grass, vegetable hulls, bean hulls, spent grains, sugar beet pulp, olive pulp, and the like.
  • the plant material may be degraded in order to improve different kinds of processing, facilitate purification or extraction of other component than the xylans like purification of beta-glucan or beta-glucan oligomers from cereals, improve the feed value, decrease the water binding capacity, improve the degradability in waste water plants, improve the conversion of e.g. grass and corn to ensilage, etc.
  • the xylanase of the invention may be used in modifying the viscosity of plant cell wall derived material.
  • the xylanase may be used to reduce the viscosity of feed containing xylan, to promote processing of viscous xylan containing material as in wheat separation, and to reduce viscosity in the brewing process.
  • the xylanase of the present invention may be used in baking so as to improve the development, elasticity and/or stability of dough and/or the volume, crumb structure and/or anti-staling properties of the baked product.
  • the xylanase may be used for the preparation of dough or baked products prepared from any type of flour or meal (e.g. based on rye, barley, oat, or maize) , particularly in the preparation of dough or baked products made from wheat or comprising substantial amounts of wheat.
  • the baked products produced with an xylanase of the invention includes bread, rolls, baquettes and the like.
  • the xylanase of the invention may be used as the only or major enzymatic activity, or may be used in combination with other enzymes such as a lipase, an amylase, an oxidase (e.g. glucose oxidase, peroxidase) , a laccase and/or a protease.
  • a lipase an amylase
  • an oxidase e.g. glucose oxidase, peroxidase
  • laccase e.g., a laccase and/or a protease.
  • the xylanase of the present invention may be used for modification of animal feed and may exert their effect either in vitro (by modifying components of the feed) or in vivo.
  • the xylanase is particularly suited for addition to animal feed compositions containing high amounts of arabinoxylans and glucuronoxylans, e.g. feed containing cereals such as barley, wheat, rye or oats or maize.
  • feed compositions containing high amounts of arabinoxylans and glucuronoxylans e.g. feed containing cereals such as barley, wheat, rye or oats or maize.
  • the xylanase significantly improves the in vivo break-down of plant cell wall material partly due to a reduction of the intestinal viscosity (Bedford et al., 1993), whereby a better utilization of the plant nutrients by the animal is achieved. Thereby, the growth rate and/or feed conversion ratio (i.e. the weight of ing
  • the xylanase of the present invention may be used in the paper and pulp industry, inter alia in the bleaching process to enhance the brightness of bleached pulps whereby the amount of chlorine used in the bleaching stages may be reduced, and to increase the freeness of pulps in the recycled paper process (Eriksson, K.E.L., Wood Science and Technology 24 (1990): 79- 101; Paice, et al., Biotechnol. and Bioeng. 32 (1988): 235-239 and Pommier et al., Tappi Journal (1989): 187-191). Furthermore, the xylanase may be used for treatment of lignocellulosic pulp so as to improve the bleachability thereof.
  • the treament of lignocellulosic pulp may, e.g., be performed as described in WO 93/08275, WO 91/02839 and WO 92/03608. Beer brewing
  • the xylanase of the present invention may be used in beer brewing, in particular to improve the filterability of wort e.g. containing barley and/or sorghum malt.
  • the xylanase may be used in the same manner as pentosanases conventionallly used for brewing, e.g. as described by Vietor et al. , 1993 and EP 227 159.
  • the xylanase may be used for treatment of brewers spent grain, i.e. residuals from beer wort production containing barley or malted barley or other cereals, so as to improve the utilization of the residuals for, e.g., animal feed.
  • Thielavia terrestris NRRL 8126 comprises the xylanase encoding DNA sequence of the invention.
  • Escherichia coli DSM 10363 containing the plasmid comprising the full length DNA sequence, coding for the xylanase of the invention, in the shuttle vector pYES 2.0.
  • Yeast strain The Saccharomyce ⁇ cerevi ⁇ iae strain used was W3124 (MATa; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-1137; prcl::HIS3; prbl:: LEU2; cir+) .
  • the Aspergillus expression vector pHD414 is a derivative of the plasmid p775 (described in EP 238 023) .
  • the construction of pHD414 is further described in WO 93/11249.
  • the xylanase encoding part of the DNA sequence shown in SEQ ID No. 1 coding for the xylanase of the invention can be obtained from the deposited organism Escherichia coli DSM 10363 by extraction of plasmid DNA by methods known in the art (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab. , Cold Spring Harbor, NY) .
  • cDNA synthesis Double-stranded cDNA was synthesized from 5 mg poly(A) + RNA by the RNase H method (Gubler and Hoffman (1983) Gene 25:263-269, Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY) using the hair-pin modification developed by F. S. Hagen (pers. comm.).
  • the poly(A) + RNA (5 mg in 5 ml of DEPC-treated water) was heated at 70°C for 8 min. in a pre- siliconized, RNase-free Eppendorph tube, quenched on ice and combined in a final volume of 50 ml with reverse transcriptase buffer (50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 , 10 mM DTT, Bethesda Research Laboratories) containing 1 mM of dATP, dGTP and dTTP and 0.5 mM 5-methyl-dCTP (Pharmacia), 40 units human placental ribonuclease inhibitor (RNasin, Promega), 1.45 mg of oligo(dT) 18 -Not I primer (Pharmacia) and 1000 units Superscript
  • Second strand cDNA synthesis was performed by incubating the reaction tube at 16°C for 2 hours and additional 15 min. at 25°C The reaction was stopped by addition of EDTA to a final concentration of 20 mM followed by phenol and chloroform extractions.
  • the double-stranded cDNA was precipitated at -20°C for
  • the single- stranded hair-pin DNA was clipped by incubating the reaction at 30°C for 30 min., followed by addition of 70 ml 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction and precipitation with 2 vols of 96% EtOH and 0.1 vol 3 M NaAc, pH 5.2 on ice for 30 min.
  • the double-stranded cDNAs were recovered by centrifugation and blunt-ended in 30 ml T4 DNA polymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing 0.5 mM of each dNTP and 5 units T4 DNA polymerase (New England Biolabs) by incubating the reaction mixture at 16°C for 1 hour. The reaction was stopped by addition of EDTA to a final concentration of 20 mM, followed by phenol and chloroform extractions, and precipitation for 12 hours at -20°C by adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.
  • T4 DNA polymerase buffer 20 mM Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT
  • T4 DNA polymerase New England Biolabs
  • the cDNAs were recovered by centrifugation, washed in 70% EtOH and dried.
  • the cDNA pellet was resuspended in 25 ml ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl 2 , 10 mM DTT, 0.5 mM ATP) containing 2.5 mg non- palindromic BstXl adaptors (Invitrogen) and 30 units T4 ligase (Promega) and incubated at 16°C for 12 hours. The reaction was stopped by heating at 65°C for 20 min. and then cooling on ice for 5 min.
  • the adapted cDNA was digested with Not I restriction enzyme by addition of 20 ml water, 5 ml lOx Not I restriction enzyme buffer (New England Biolabs) and 50 units Not I (New England Biolabs), followed by incubation for 2.5 hours at 37°C The reaction was stopped by heating at 65°C for 10 min.
  • the cDNAs were size-fractionated by gel electrophoresis on a 0.8% SeaPlaque GTG low melting temperature agarose gel (FMC) in lx TBE to separate unligated adaptors and small cDNAs.
  • FMC SeaPlaque GTG low melting temperature agarose gel
  • the cDNA was size-selected with a cut-off at 0.7 kb and rescued from the gel by use of b-Agarase (New England Biolabs) according to the manufacturer's instructions and precipitated for 12 hours at - 20°C by adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.
  • the directional, size-selected cDNA was recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 ml 10 mM Tris-Cl, pH 7.5, 1 mM EDTA.
  • the cDNAs were desalted by gelfiltration through a MicroSpin S-300 HR (Pharmacia) spin column according to the manufacturer's instructions.
  • E. coli DH10B cells (Bethesda research Laboratories) as described (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab. , Cold Spring Harbor, NY) .
  • E. coli DH10B cells
  • Plasmid DNA was isolated from the cells according to the manufacturer's instructions using QIAGEN plasmid kit and stored at -20°C l ml aliquots of purified plasmid DNA (100 ng/ml) from individual pools were transformed into S . cerevisiae W3124 by electroporation (Becker and Guarante (1991) Methods Enzymol. 194:182-187) and the transformants were plated on SC agar containing 2% glucose and incubated at 30°C Identification of positive clones
  • the tranformants was plated on SC agar containing 0.1% AZCL xylan (Megazyme, Australia) and 2% Galactose and incubated for 3-5 days at 30 C Xylanase positive colonies are identified as colonies surrounded by a blue halo.
  • the positive clones were obtained as single colonies, the cDNA inserts were amplified directly from the yeast colony using biotinylated polylinker primers, purified by magnetic beads (Dynabead M-280, Dynal) system and characterized individually by sequencing the 5'-end of each cDNA clone using the chain-termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467) and the Sequenase system (United States Biochemical) .
  • a xylanase-producing yeast colony was inoculated into 20 ml YPD broth in a 50 ml glass test tube. The tube was shaken for
  • DNA was isolated according to WO 94/14953 and dissolved in 50 ml water. The DNA was transformed into E. coli by standard procedures. Plasmid DNA was isolated from E. coli using standard procedures, and analyzed by restriction enzyme analysis. The cDNA insert was excised using appropriate restriction enzymes and ligated into an Aspergillus expression vector.
  • Protoplasts may be prepared as described in WO 95/02043, p. 16, line 21 - page 17, line 12, which is hereby incorporated by reference.
  • Protoplasts are mixed with p3SR2 (an A. nidulan ⁇ amdS gene carrying plasmid) .
  • the mixture is left at room temperature for 25 minutes.
  • 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl 2 and 10 mM Tris-HCl, pH 7.5 is added and carefully mixed (twice) and finally 0.85 ml of the same solution is added and carefully mixed.
  • the mixture is left at room temperature for 25 minutes, spun at 2500 g for 15 minutes and the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one more sedimentation the protoplasts are spread on minimal plates (Cove, Biochem. Biophys. Acta 113 (1966) 51-56) containing 1.0 M sucrose, pH 7.0, 10 mM aceta ide as nitrogen source and 20 mM CsCl to inhibit background growth. After incubation for 4-7 days at 37°C spores are picked and spread for single colonies. This procedure is repeated and spores of a single colony after the second reisolation is stored as a defined transformant.
  • Each of the transformants were inoculated in 10 ml of YPM (cf. below) and propagated. After 2-5 days of incubation at 30°C, the supernatant was removed. The xylanolytic activity was identified by applying 10 ⁇ l supernatant to 4 mm diameter holes punched out in agar plates containing 0.2% AZCLO birch xylan (Megazyme ⁇ , Australia) . Xylanolytic activity is then identified as a blue halo.
  • Suitable hybridization conditions for determining hybridization between an oligonucleotide probe and an "analogous" DNA sequence of the invention may be defined as described below.
  • a suitable oligonucleotide probe to be used in the hybridization may be prepared on the basis of the xylanase encoding part of the DNA sequence shown in SEQ ID No. 1, or any sub-sequence thereof.
  • An example of a suitable probe is the DNA sequence corresponding to the xylanase encoding part of SEQ ID No. 1.
  • the hybridization referred to above is intended to comprise an analogous DNA sequence which hybridizes to the nucleotide probe corresponding to the xylanase encoding part 5 of the DNA sequence shown in SEQ ID NO 1, i.e. nucleotides 1- 894, under at least under at least low stringency conditions and preferably at medium or high stringency conditions as described in detail below.
  • Antibodies to be used in determining immunological cross-reactivity may be prepared by use of a purified xylanase. 35 More specifically, antiserum against the xylanase of the invention may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al. in: A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe, I munochemistry in Practice, Blackwell Scientific Publications, 1982 (more specifically p. 27-31) .
  • Purified immunoglobulins may be obtained from the antisera, for example by salt precipitation ((NH ) 2 S0 4 ) , followed by dialysis and ion exchange chromatography, e .g. on DEAE-Sephadex. Immunochemical characterization of proteins may be done either by Outcherlony double-diffusion analysis (O. Ouchterlony in: Handbook of
  • YPD 10 g yeast extract, 20 g peptone, H 2 0 to 900 ml. Autoclaved, 100 ml 20% glucose (sterile filtered) added.
  • YPM 10 g yeast extract, 20 g peptone, H 2 0 to 900 ml. Autoclaved, 100 ml 20% maltodextrin (sterile filtered) added.
  • 10 x Basal salt 75 g yeast nitrogen base, 113 g succinic acid, 68 g NaOH, H0 ad 1000 ml, sterile filtered.
  • SC-URA 100 ml 10 x Basal salt, 28 ml 20% casamino acids without vitamins, 10 ml 1% tryptophan, H0 ad 900 ml, autoclaved, 3.6 ml 5% threonine and 100 ml 20% glucose or 20% galactose added.
  • SC-agar SC-URA, 20 g/l agar added.
  • SC-variant agar 20 g agar, 20 ml 10 x Basal salt, H 2 0 ad 900 ml, autoclaved
  • Thielavia terrestris NRRL NO. 8126 mRNA Cloning and expression of a xylanase from Thielavia terrestris NRRL NO. 8126 mRNA was isolated from Thielavia terrestri ⁇ , NRRL No. 8126, grown in cellulose-containing fermentation medium with agitation to ensure sufficient aeration. Mycelia were harvested after 3-5 days* growth, immediately frozen in liquid nitrogen and stored at -80°C A library from Thielavia terre ⁇ tri ⁇ , NRRL No. 8126, consisting of approx. 9xl0 5 individual clones was constructed in E. coli as described with a vector background of 1%. Plasmid DNA from some of the pools was transformed into yeast, and 50-100 plates containing 250-400 yeast colonies were obtained from each pool.
  • Xylanase-positive colonies were identified and isolated on SC-agar plates with the AZCL xylan assay.
  • cDNA inserts were amplified directly from the yeast colonies and characterized as described in the Materials and Methods section above.
  • the DNA sequence of the cDNA encoding the xylanase is shown in SEQ ID No. 1.
  • the cDNA is obtainable from the plasmid in DSM 10363.
  • Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E. coli as described above.
  • the DNA was digested with appropriate restriction enzymes, size fractionated on gel, and a fragment corresponding to the xylanase gene was purified.
  • the gene was subsequently ligated to pHD414, digested with appropriate restriction enzymes, resulting in the plasmid pA2X154.
  • the plasmid was transformed into Aspergillu ⁇ oryzae as described above.
  • transformants were tested for enzyme activity as described above. Some of the transformants had xylanase activity which was significantly larger than the Aspergillus oryzae background. This demonstrates efficient expression of the xylanase in Aspergillus oryzae .
  • a homology search with the xylanase of the invention against nucleotide and protein databases was performed.
  • the homology search showed that the most related xylanases were xylanase II from Trichoderma reesei and xylanase A from Aspergillus nidulans.
  • the xylanase from Trichoderma reesei belongs to family 11 of glycosyl hydrolases which indicate that the xylanase of the invention also belongs to family 11 of glycosyl hydrolases (Henrissat, B Biochem. J. 280:309-316, 1991) .
  • the DNA homology of the xylanase of the invention against most prior art xylanases was determined using the computer program GAP.
  • the xylanase of the invention has only 61% DNA homology to the xylanase II from Trichoderma reesei (Torronen, A. et al., Biotechnology (N.Y.) 10 (11), 1461-1465(1992)) and the xylanase of the invention has only 56% DNA homology to xylanase A from Aspergillus nidulans (ACCESSION No. Z49892, Genebank) . This show that the xylanase of the invention indeed is distant from any known xylanases.
  • SEQ ID No. 1 shows the DNA sequence of the full-length DNA sequence comprised in the DNA construct transformed into the deposited Escherichia coli DSM 10363.
  • ORGANISM Thielavia terrestris

Abstract

La présente invention concerne une enzyme possédant une activité de type xylanase, une construction d'ADN qui code l'enzyme possédant cette activité de type xylanase, un procédé de production de cette enzyme, une préparation enzymatique qui contient l'enzyme possédant cette activité de type xylanase, une composition détergente comprenant ladite xylanase, ainsi que l'utilisation de cette enzyme et de ladite préparation enzymatique dans un certain nombre d'applications industrielles.
PCT/DK1997/000033 1996-01-22 1997-01-22 Enzyme possedant une activite de type xylanase WO1997027293A1 (fr)

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WO2004031378A2 (fr) * 2002-10-01 2004-04-15 Novozymes A/S Polypeptides de la famille gh-61
EP1688534A1 (fr) * 2005-02-02 2006-08-09 Wolff Cellulosics GmbH & Co.KG L'utilisation de Arabinoxylanes pour la production de papier
US7273738B2 (en) 2002-10-01 2007-09-25 Novozymes A/S Family GH-61 polypeptides
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EP2106215A2 (fr) 2007-01-16 2009-10-07 Puratos N.V. Pain à teneur augmentée en arabinoxylo-oligosaccharide
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US6692578B2 (en) 2001-02-23 2004-02-17 Battelle Memorial Institute Hydrolysis of biomass material
WO2002067691A1 (fr) * 2001-02-23 2002-09-06 Battelle Memorial Institute Hydrolyse de biomasse
US8623402B2 (en) 2001-08-20 2014-01-07 Cargill, Incorporated Non-starch-polysaccharides
US7803590B2 (en) 2002-10-01 2010-09-28 Novozymes A/S Family GH 61 polypeptides
WO2004031378A2 (fr) * 2002-10-01 2004-04-15 Novozymes A/S Polypeptides de la famille gh-61
WO2004031378A3 (fr) * 2002-10-01 2004-05-06 Novozymes As Polypeptides de la famille gh-61
US7273738B2 (en) 2002-10-01 2007-09-25 Novozymes A/S Family GH-61 polypeptides
EP2302046A1 (fr) * 2002-10-01 2011-03-30 Novozymes A/S Polypeptides de la famille GH-61
EP1862539B1 (fr) * 2003-05-29 2012-01-25 Danisco US Inc. Nouveaux gènes de Trichoderma
WO2006081845A1 (fr) * 2005-02-02 2006-08-10 Wolff Cellulosics Gmbh & Co. Kg Utilisation d'arabinoxylanes en tant qu'additifs dans la fabrication du papier
EP1688534A1 (fr) * 2005-02-02 2006-08-09 Wolff Cellulosics GmbH & Co.KG L'utilisation de Arabinoxylanes pour la production de papier
EP2041277A2 (fr) 2006-06-30 2009-04-01 Katholieke Universiteit Leuven Methode de fabrication d'arabinoxylanes solubles comme co-produit de fermentation de cereales completes
US8034586B2 (en) 2006-06-30 2011-10-11 Fugeia Nv Method for making soluble arabinoxylans as co-product of fermentation of whole-grain cereals
EP2106215A2 (fr) 2007-01-16 2009-10-07 Puratos N.V. Pain à teneur augmentée en arabinoxylo-oligosaccharide
EP2106215B1 (fr) * 2007-01-16 2020-09-02 Puratos N.V. Pain à teneur augmentée en arabinoxylo-oligosaccharide
US9061046B2 (en) 2007-09-28 2015-06-23 Cargill, Incorporated Arabinoxylo-oligosaccharides useful against gastrointestinal infections
WO2009079210A2 (fr) * 2007-12-05 2009-06-25 Novozymes A/S Polypeptides ayant une activité de xylanase et polynucléotides codant pour ceux-ci
US8940515B2 (en) 2007-12-05 2015-01-27 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
WO2009079210A3 (fr) * 2007-12-05 2009-10-29 Novozymes A/S Polypeptides ayant une activité de xylanase et polynucléotides codant pour ceux-ci
US9353360B2 (en) 2007-12-05 2016-05-31 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
US9771569B2 (en) 2007-12-05 2017-09-26 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
US7851193B2 (en) 2007-12-05 2010-12-14 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
US8927038B2 (en) 2008-03-25 2015-01-06 Cargill, Incorporated (Arabino)xylan oligosaccharide preparation

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