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WO2016005519A1 - Composition d'oligosaccharide et son procédé de préparation - Google Patents

Composition d'oligosaccharide et son procédé de préparation Download PDF

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Publication number
WO2016005519A1
WO2016005519A1 PCT/EP2015/065747 EP2015065747W WO2016005519A1 WO 2016005519 A1 WO2016005519 A1 WO 2016005519A1 EP 2015065747 W EP2015065747 W EP 2015065747W WO 2016005519 A1 WO2016005519 A1 WO 2016005519A1
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WIPO (PCT)
Prior art keywords
seq
xylanase
set forth
amino acids
amino acid
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PCT/EP2015/065747
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English (en)
Inventor
Jens EKLÖF
Fiona Becker
Jeppe Wegener Tams
Kristian Bertel Rømer M. KROGH
Martin Simon Borchert
Mary Ann Stringer
Sven Pedersen
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Novozymes A/S
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Publication of WO2016005519A1 publication Critical patent/WO2016005519A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/262—Cellulose; Derivatives thereof, e.g. ethers

Definitions

  • the present invention relates to oligosaccharide compositions suitable for use as bulking agents and to processes for producing such compositions from highly branched xylan.
  • Oligosaccharide compositions useful in food products e.g. as a bulking agent, preferably have low viscosity and low sugar content.
  • mono or dimeric carbohydrates DP1 -2, where DP is degree of polymerization
  • Xylans are hemicelluloses found in all land plants (Popper and Tuohy, Plant Physiology,
  • glucurono arabinoxylans are the main hemicellulose and are present as a soluble or insoluble dietary fibre in many grass-derived food- and feed products.
  • Plant xylans have a ⁇ -1 ,4-linked xylopyranose backbone that can be substituted at the 02 or 03 position with arabinose, glucuronic acid and acetic acid in a species and tissue specific manner.
  • the starch-rich seeds of the Panicoideae with economically important species such as corn and sorghum have special types of highly substituted xylans in their cell walls.
  • wheat flour that has above 60% of the xylosyl units in the arabinoxylan backbone un- substituted the corresponding percentage of unsubstituted backbone xylosyls in corn kernel xy- Ian is 20-30% and 35-40% in sorghum (Huismann et al.
  • the known enzymes responsible for the hydrolysis of the xylan backbone are classified into enzyme families based on sequence similarity (www.cazy.org).
  • the enzymes with mainly enc/o-xylanase activity have previously been described in Glycoside hydrolase family (GH) 5, 8, 10, 1 1 and 30.
  • the enzymes within a family share some characteristics such as 3D fold and they usually share the same reaction mechanism.
  • Some GH families have narrow or monospecific substrate specificities while other families have broad substrate specificities.
  • the xy- lanases described in this invention belongs to GH5, a GH family with a wide array of substrate specificities.
  • the relationship between sequences within GH5 has been clarified by defining subfamilies of related sequences (Aspeborg et al.
  • GH5 xylanases belonging to subfamilies GH5_21 , GH5_34 and GH5_34 all have the capacity to access and degrade highly substituted xylans (meaning that more than 50% of xylosyl units in the arabinoxylan backbone are substituted) that are resistant to xylanase degradation by xylanases of other GH families resulting in an oligosaccharide composition having an average molecular weight and a viscosity suitable for use as a bulking agent.
  • the invention therefore provides in a first aspect an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 50.000 Da, the weight average molecular weight, Mw, is less than 100.000.
  • the present invention provides a food product comprising the oligosaccharide composition according to the invention.
  • the present invention relates to a one step process for making an oligosaccharide composition, wherein the peak value, Mp, expressed as peak molecular weight, is less than 50.000 Da, the weight average molecular weight, Mw, is less than 100.000, comprising hydrolyzing a substrate containing highly branched xylan with a GH5 xylanase.
  • the present invention relates to a use of the composition according to the invention in a food product.
  • the present invention relates to a use of the composition according to the invention as a fat and/or sugar replacement in a food product.
  • the present invention relates to use of the oligosaccharide composition according to any of the claims 1 -28 as caloric reduction of a food product.
  • the present invention relates to use of the oligosaccharide composition according to any of the claims 1 -28 as fiber enhancement of a food product.
  • SEQ ID NO: 1 Codon optimized gene encoding a GH5_35 xylanase from Paenibacillus illinoisensis.
  • SEQ ID NO: 2 GH5_35 xylanase from Paenibacillus illinoisensis including a signal peptide from Bacillus clausii and a His-tag.
  • SEQ ID NO: 3 Amino acid sequence of mature GH5_35 xylanase from Paenibacillus illinoisensis.
  • SEQ ID NO: 4 Codon optimized gene encoding a GH5_35 xylanase from Paenibacillus sp 18054.
  • SEQ ID NO: 5 (previously 4): GH5_35 xylanase from Paenibacillus sp. including a signal peptide from Bacillus clausii and a His-tag.
  • SEQ ID NO: 6 Amino acid sequence of mature GH5_35 xylanase from Paenibacillus sp.
  • SEQ ID NO: 7 Codon optimized gene encoding GH5_21 xylanase from unknown organ- ism
  • SEQ ID NO: 8 GH5_21 xylanase including a signal peptide from Bacillus clausii and a
  • SEQ ID NO: 13 is the amino acid sequence as deduced from SEQ ID NO: 12.
  • SEQ ID NO: 14 is the amino acid sequence of the mature GH5 xylanase from Chryseo- bacterium sp-10696.
  • SEQ ID NO: 15 is the DNA sequence of the recombinant expressed DNA sequence from
  • SEQ ID NO: 16 is the amino acid sequence as deduced from SEQ ID NO: 15.
  • SEQ ID NO: 17 is the amino acid sequence of the mature GH5 xylanase obtained from SEQ ID NO. 16.
  • SEQ ID NO: 18 is the cDNA sequence of GH5 xylanase as isolated from elephant dung metagenome.
  • SEQ ID NO: 19 is the amino acid sequence as deduced from SEQ ID NO: 18.
  • SEQ ID NO: 20 is the amino acid sequence of the mature GH5 xylanase from elephant dung metagenome.
  • SEQ ID NO: 21 is the DNA sequence of the recombinant expressed DNA sequence from
  • SEQ ID NO: 22 is the amino acid sequence as deduced from SEQ ID NO: 21 .
  • SEQ I D NO: 23 is the amino acid sequence of the mature GH5 xylanase obtained from SEQ ID NO. 21 .
  • SEQ ID NO: 24 is the cDNA sequence of GH5 xylanase as isolated from elephant dung metagenome.
  • SEQ ID NO: 25 is the amino acid sequence as deduced from SEQ ID NO: 24.
  • SEQ I D NO: 26 is the amino acid sequence of the mature GH5 xylanase from elephant dung metagenome.
  • SEQ ID NO: 27 is the DNA sequence of the recombinant expressed DNA sequence from SEQ ID NO: 23 with HQ-tag and Savinase signal peptide.
  • SEQ ID NO: 28 is the amino acid sequence as deduced from SEQ ID NO: 27.
  • SEQ I D NO: 29 is the amino acid sequence of the mature GH5 xylanase obtained from SEQ ID NO. 27.
  • SEQ ID NO: 30 is the cDNA sequence of GH5 xylanase as isolated from elephant dung metagenome.
  • SEQ ID NO: 31 is the amino acid sequence as deduced from SEQ ID NO: 30.
  • SEQ I D NO: 32 is the amino acid sequence of the mature GH5 xylanase from elephant dung metagenome.
  • SEQ ID NO: 33 is the DNA sequence of the recombinant expressed DNA sequence from SEQ ID NO: 30 with HQ-tag and Savinase signal peptide.
  • SEQ ID NO: 34 is the amino acid sequence as deduced from SEQ ID NO: 33.
  • SEQ ID NO: 35 is the amino acid sequence of the mature GH5 xylanase obtained from SEQ ID NO. 33.
  • SEQ ID NO: 36 is a putative GH5 xylanase peptide sequence from the draft genome sequence of Gonapodya prolifera.
  • SEQ ID NO: 37 is a Gonapodya prolifera GH5 xylanase coding sequence.
  • SEQ ID NO: 38 is a truncated GH5 xylanase peptide sequence from the draft genome sequence of Gonapodya prolifera excluding the proline rich region.
  • Highly branched in the context of the present invention means that more than 50% of xylosyl units in the arabinoxylan backbone are substituted.
  • Mature polypeptide means a polypeptide in its final form fol- lowing translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
  • the mature polypeptide is amino acids 37 to 573 of SEQ ID NO: 2.
  • Amino acids 1 to 27 of SEQ ID NO: 2 are a signal peptide.
  • Amino acids 28 to 36 of SEQ I D NO: 2 are a His-tag.
  • the mature polypeptide with the His-tag is set forth in SEQ ID NO: 3.
  • the mature polypeptide is amino acids 36 to 582 of SEQ ID NO: 5.
  • Amino acids 1 to 27 of SEQ ID NO: 5 are a signal peptide. Amino acids 28 to 35 of SEQ ID NO: 5 are a his-tag. The mature polypeptide with the His-tag is set forth in SEQ ID NO: 6.
  • the mature polypeptide is amino acids 36 to 633 of SEQ ID NO: 8.
  • Amino acids 1 to 27 of SEQ ID NO: 8 are a signal peptide.
  • Amino acids 28 to 35 of SEQ ID NO: 8 are a his-tag.
  • the mature polypeptide with the His-tag is set forth in SEQ ID NO: 9.
  • the mature polypeptide is amino acids 1 to 620 of SEQ ID NO: 10. In another aspect the mature polypeptide is amino acids 1 to 865 of SEQ ID NO: 1 1 .
  • the mature polypeptide is a mature polypeptide of SEQ ID NO: 13, such as amino acids 1 to 550 of SEQ ID NO: 13, corresponding to amino acids to the sequence set forth in SEQ ID NO: 14.
  • amino acids -23 to -1 are a signal peptide.
  • the mature polypeptide is amino acids 1 to 558 of SEQ ID NO: 16, corresponding to the amino acid sequence set forth in SEQ ID NO: 17.
  • amino acids -27 to -1 are a signal peptide.
  • the mature polypeptide is is a mature polypeptide of SEQ I D NO: 19, such as amino acids 1 to 631 of SEQ ID NO: 19. corresponding to the amino acid sequence set forth in SEQ ID NO: 20.
  • amino acids -25 to -1 are a signal peptide.
  • the mature polypeptide is amino acids 1 to 639 of SEQ ID NO: 22. corresponding to the amino acid sequence set forth in SEQ ID NO: 23.
  • amino acids -27 to -1 are a signal peptide.
  • the mature polypeptide is a mature polypeptide of SEQ ID NO: 25, such as amino acids 1 to 828 of SEQ ID NO: 25. corresponding to the amino acid sequence set forth in SEQ ID NO: 26.
  • amino acids -18 to -1 are a signal peptide.
  • the mature polypeptide is amino acids 1 to 836 of SEQ ID NO: 28. corresponding to the amino acid sequence set forth in SEQ ID NO: 29.
  • ami- no acids -27 to -1 are a signal peptide.
  • the mature polypeptide is a mature polypeptide of SEQ ID NO: 31 , such as amino acids 1 to 577 of SEQ ID NO: 31 . corresponding to the amino acid sequence set forth in SEQ ID NO: 32.
  • amino acids -27 to -1 are a signal peptide.
  • the mature polypeptide is amino acids 1 to 585 of SEQ ID NO: 34, corresponding to the amino acid sequence set forth in SEQ ID NO: 35.
  • amino acids -27 to -1 are a signal peptide.
  • the mature polypeptide is amino acids 24-443 of SEQ ID NO: 36 or amino acids 24-337 of SEQ ID NO: 38. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.
  • Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having xylanase activity.
  • the mature polypeptide coding sequence is nucleotides 109 to 1719 of SEQ ID NO: 1.
  • Nu- cleotides 1 to 81 of SEQ ID NO: 1 encode a signal peptide.
  • Nucleotides 82 to 108 of SEQ ID NO: 1 encode a his-tag.
  • the mature polypeptide coding sequence is nucleotides 106 to 1746 of SEQ ID NO: 4.
  • Nucleotides 1 to 81 of SEQ ID NO: 4 encode a signal peptide.
  • Nucleotides 82 to 105 of SEQ ID NO: 4 encode a his-tag.
  • the mature polypeptide coding sequence is nucleotides 106 to 1899 of SEQ ID NO: 7.
  • Nucleotides 1 to 81 of SEQ ID NO: 7 encode a signal peptide.
  • Nucleotides 82 to 105 of SEQ ID NO: 7 encode a his-tag.
  • the mature polypeptide coding sequence is nucleotides 70 to 17229 of SEQ ID NO: 12. Nucleotides 1 to 69 of SEQ ID NO: 12 encode a signal peptide. In one aspect, the mature polypeptide coding sequence is nucleotides 82 to 1755 of SEQ ID NO: 15. Nucleotides 1 to 81 of SEQ I D NO: 15 encode a signal peptide. In one aspect, the mature polypeptide coding sequence is nucleotides 76 to 1968 of SEQ ID NO: 18. Nucleotides 1 to 75 of SEQ ID NO: 18 encode a signal peptide. In one aspect, the mature polypeptide coding sequence is nucleotides 82 to 1998 of SEQ ID NO: 21.
  • Nucleotides 1 to 81 of SEQ ID NO: 21 encode a signal peptide.
  • the mature polypeptide coding sequence is nucleotides 55 to 2538 of SEQ ID NO: 24.
  • Nucleotides 1 to 54 of SEQ ID NO: 24 encode a signal peptide.
  • the mature polypeptide coding sequence is nucleotides 82 to 2589 of SEQ ID NO: 27.
  • Nucleotides 1 to 81 of SEQ I D NO: 27 encode a signal peptide.
  • the mature polypeptide coding sequence is nucleotides 64 to 1791 of SEQ ID NO: 30.
  • Nucleotides 1 to 63 of SEQ ID NO: 30 encode a signal peptide.
  • the mature polypeptide coding sequence is nucleotides 82 to 1836 of SEQ ID NO:33. Nucleotides 1 to 81 of SEQ ID NO: 33 encode a signal peptide. In another aspect, the mature polypeptide coding sequence is nucleotides 70-101 1 of SEQ ID NO: 37. Nucleotides 1 to 69 of SEQ ID NO: 37 encode a signal peptide.
  • Oligosaccharide composition means oligo- and poly saccharides but does not include mono- and disaccharides (DP1 and DP2).
  • Sequence identity The relatedness between two amino acid sequences or between two nu- cleotide sequences is described by the parameter "sequence identity".
  • sequence identity For purposes of the pre- sent invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • Needle labeled "longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
  • sequence identity between two deoxyribonu- cleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxynbonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
  • Xylanase means a 1 ,4-beta-D-xylan-xylohydrolase (E.C.
  • Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C.
  • One unit of xylanase activity is defined as 1 .0 ⁇ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodi- urn phosphate pH 6.
  • Viscosity refers to the resistance of a fluid to flow. The viscosity of a syrup is typically affected by temperature and dry solids (DS) concentration. Viscosity is expressed in terms of poise (P) or centipoise (cps) at a given temperature and a given % DS.
  • Wild-type strain means a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.
  • the solutions provided according to the present invention are the oligosaccharide compositions according to the invention obtainable by hydrolysing a highly branched xylan substrate with a GH5 xylanase.
  • compositions according to the invention are highly desirable due to the low sugar content (almost no DP1 -2 sugars are present in the hydrolysate after hydrolysis of the substrate with a GH5 xylanase), and the low viscosity, which makes the oligosaccharide compositions particularly suitable for use in a food product, e.g. as a bulking agent.
  • the present invention therefore relates to an oligosaccharide composition, wherein the peak value, Mp, expressed as peak molecular weight, is less than 50.000 Da, the weight average molecular weight, Mw, is less than 100.000.
  • Oligosaccharide compositions according to the invention are suitable for use in a food product, e.g., for use as a bulking agent. It is desirable that the viscosity is low. Viscosity as used herein refers to the resistance of a fluid to flow. The viscosity of a syrup is typically affected by temperature and dry solids (DS) concentration. Viscosity is expressed in terms of poise (P) or centipoise (cps) at a given temperature and a given % DS.
  • the oligosaccharide composition according to the invention may be a blend of two or more hydrolysed branched xylan substrates.
  • the oligosaccharide composition may be a blend of two or more hydrolysed branched xylan substrates, which are different from each other; e.g. having different peak molecular weights and/or viscosities.
  • the oligosaccharide composition according to the invention preferably comprises at least one corn fibre xylan hydrolysate.
  • the oligosaccharide composition has a viscosity profile at 24°C and a dry solid (DS) content of 10% in the range from 0.1 - 5.0 cps (centi poise); particularly 0.5 - 4.0 cps; particularly 1 .0 - 3.0 cps; such as about 2.5 cps.
  • DS dry solid
  • the oligosaccharide composition has a viscosity profile at 24°C and a dry solid (DS) content of 10% of less than 5 cps, particularly less than 4 cps, particularly less than 3 cps.
  • DS dry solid
  • the oligosaccharide composition according to the invention is obtainable by hydrolyzing a highly branched xylan substrate with a GH5 xylanase.
  • the xylan containing substrates to be used according to the invention are preferable highly branched xylan substrates.
  • "Highly branched" in the context of the present invention means that more than 50% of xylosyl units in the arabinoxylan backbone are substituted.
  • the oligosaccharide compositions of the invention are intended for use in food products and thus presence of DP1 -2 content saccharides in the hydrolyzate is undesirable.
  • An advantageous effect of using GH5 xylanases for producing the oligo-saccharide composition of the in- vention is that very low levels of DP1 and DP2 are generated during hydrolysis. There is thus no need for further purification.
  • the DP1 , DP2 fraction present in the hydrolysate is less than 10%, particularly less than 5%, more particularly less than 3%, more particularly less than 1 %, more particularly less than 0.5%.
  • the GH5 xylanases are in one embodiment selected from the group consisting of subfamilies 21 , 34, or 35.
  • the subfamily 21 GH5 xylanase is selected from the xy- lanase shown as amino acids 36 to 633 of SEQ ID NO: 8, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 36 to 633 of SEQ ID NO: 8.
  • the subfamily 21 GH5 xylanase is selected from the His-taggged xylanase set forth in SEQ ID NO: 9, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 9.
  • the subfamily 34 GH5 xylanase is selected from the xylanase shown as SEQ ID NO: 10, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO: 10.
  • the subfamily 34 GH5 xylanase is selected from the xylanase shown as SEQ ID NO: 1 1 , or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO: 1 1 .
  • the subfamily34 GH5 xylanase is selected from the xylanases shown as amino acids 24-443 of SEQ ID NO: 36 and amino acids 24-337 of SEQ ID NO: 38 or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 24-443 of SEQ ID NO: 36 and amino acids 24-337 of SEQ ID NO: 38.
  • the subfamily 35 GH5 xylanase is selected from the xylanase shown as amino acids 37 to 573 of SEQ ID NO: 2, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 37 to 573 of SEQ ID NO: 2.
  • the subfamily 35 GH5 xylanase is selected from the His-tagged xylanase set forth in SEQ ID NO: 3, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the His-tagged xylanase set forth in SEQ ID NO: 3.
  • the subfamily 35 GH5 xylanase is selected from the xylanase shown as amino acids 36 to 582 of SEQ ID NO: 5, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 36 to 582 of SEQ ID NO: 5.
  • the subfamily 35 GH5 xylanase is selected from the His-tagged xylanase set forth in SEQ ID NO: 6, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the His-tagged xylanase set forth in SEQ ID NO: 6.
  • the subfamily 21 GH5 xylanase is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:13 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature polypeptide of the amino acid set forth in SEQ ID NO:13.
  • the subfamily 21 GH5 xylanase is selected from the xylanase shown as amino acids 1 to 558 of SEQ I D NO: 16 (corresponding to the amino acid se- quence set forth in SEQ ID NO: 17), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 558 of SEQ ID NO: 16 (corresponding to the amino acid sequence set forth in SEQ ID NO: 17).
  • the subfamily 21 GH5 xylanase is selected from the ma- ture polypeptide of the amino acid set forth in SEQ ID NO:19 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature polypeptide of the amino acid set forth in SEQ ID NO:19.
  • the subfamily 21 GH5 xylanase is selected from the xy- lanase shown as amino acids 1 to 639 of SEQ ID NO: 22 (corresponding to the amino acid sequence set forth in SEQ ID NO: 23), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 639 of SEQ ID NO: 22 (corresponding to the amino acid sequence set forth in SEQ ID NO: 23).
  • the subfamily 21 GH5 xylanase is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:25 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature polypeptide of the amino acid set forth in SEQ ID NO:13.
  • the subfamily 21 GH5 xylanase is selected from the xylanase shown as amino acids 1 to 836 of SEQ ID NO: 28 (corresponding to the amino acid sequence set forth in SEQ ID NO: 29), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 836 of SEQ ID NO: 28 (corresponding to the amino acid sequence set forth in SEQ ID NO: 29).
  • the subfamily 21 GH5 xylanase is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:31 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature polypeptide of the amino acid set forth in SEQ ID NO:31.
  • the subfamily 21 GH5 xylanase is selected from the xylanase shown as amino acids 1 to 585 of SEQ I D NO: 34 (corresponding to the amino acid se- quence set forth in SEQ ID NO: 34), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 585 of SEQ ID NO: 34 (corresponding to the amino acid sequence set forth in SEQ ID NO: 35).
  • the xylan containing substrate is obtained from plant based material from sub-family Panicoideae.
  • the plant based material from the sub-family Panicoideae is from the tribe Andropogoneae such as the rank Andropogon or Andropterum or Apluda or Apocopis or Arthraxon or Bothriochloa or Capillipedium or Chionachne or Chrysopogon or Coelorachis or Coix or Cymbopogon or Dichanthium or Diheteropogon or Dimeria or Elionurus or Eremochloa or Euclasta or Eulalia or Germainia or Hemarthria or Heteropholis or Heteropogon or Hyparrhe- nia or Hyperthelia or Imperata or Ischaemum or Iseilema or Kerriochloa or Microstegium or Ms- canthidium or Miscanthus or Mnesithea or Ophiuros or Oxyrhachis or Phacelurus or Pholiurus or Pogonatherum or Polyto
  • the plant based material from the sub-family Panicoideae is from the rank Zea, such as the species Zea diploperennis, Zea luxurians, Zea mays, Zea nica- raguensis or Zea perennis
  • the plant based material from the sub-family Panicoideae is from the rank Sorghum, such as the species Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum australiense, Sorghum bicolor, Sorghum brachypodum, Sorghum bulbosum, Sorghum ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum hybrid cultivar, Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum nitidum, Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum stipoideum, Sorghum sudanense, Sorghum timorense, Sorghum versicolor, Sorghum x almum, Sorghum x drum-
  • the plant based material from the sub-family Panicoideae is from the tribe Paniceae such as the rank Acritochaete, Acroceras, Alexfloydia, Alloteropsis, Amphicarpum, Ancistrachne, Anthephora, Brachiaria, Calyptochloa, Cenchrus, Chaetium, Chaetopoa, Chamaeraphis, Chlorocalymma, Cleistochloa, Cypochlaena, Cyrtococcum, Dichanthelium, Digitaria, Dissochondrus, Echinochloa, Entolasia, Eriochloa, Homopholis, Hy- grochloa, Hylebates, Ixophorus, Lasiacis, Leucophrys, Louisiella, Megaloprotachne, Megathyr- sus, Melinis, Microcalamus, Moorochloa, Neurachne, Odonte
  • the plant based material from the sub-family Panicoideae is from the rank Panicum, such as the species Panicum adenophorum, Panicum aff. aquaticum JKT-2012, Panicum amarum, Panicum antidotale, Panicum aquaticum, Panicum arctum, Panicum arundinariae, Panicum atrosanguineum, Panicum auricomum, Panicum auritum, Panicum bartlettii, Panicum bergii, Panicum bisulcatum, Panicum boliviense, Panicum brazzavillense, Panicum brevifolium, Panicum caaguazuense, Panicum campestre, Panicum capillare, Panicum cayennense, Panicum cayoense, Panicum cervicatum, Panicum chloroleucum, Panicum clayto- nii, Panicum coloratum, Panicum cyan
  • the plant based material from the sub-family Panicoideae is maize ⁇ Zea), corn ⁇ Zea), sorghum (Sorghum), switchgrass (Panicum virgatum) or millet (Panicum miliaceum), or in a processed form such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled pearl and foxtail millet, or any combination thereof.
  • the xylan containing substrate is derived from corn or sorghum. More particularly the substrate is corn fiber gum.
  • the xylan containing substrate is obtained from plant based material from the sub-family Ehrhartoideae; in particular from one or more members of the tribe Oryzeae, such as one or more of the species Oryza australiensis, Oryza barthii, Oryza brachyantha, Oryza coarctata, Oryza eichingeri, Oryza glaberrima, Oryza grandiglumis, Oryza latifolia, Oryza longiglumis, Oryza longistaminata, Oryza meyeriana, Oryza minuta, Oryza neo- caledonica, Oryza officinalis, Oryza punctate, Oryza ridleyi, Oryza rufipogon, Oryza sativa and Oryza Aveeri. It will be understood that the xylan containing substrate may also be obtained form any processed form of said plants.
  • the xylan containing substrate is obtained from plant based material from Oryza sativa.
  • the xylan containing substrate may also be obtained form any processed form of said plants.
  • the oligosaccharide composition preferably has a peak value, Mp, expressed as molecular weight after complete xylan hydrolysis is less than 50.000 Da, particularly less than 40.000 Da, more particularly less than 30.000 Da, more particularly less than 20.000 Da, and more par- ticularly less than 10.000 Da.
  • the oligosaccharide composition according to the invention preferably has a weight average molecular weight, Mw, after complete xylan hydrolysis of less than 100.000 Da, particularly less than 75.000 Da, particularly less than 50.000 Da, particularly less than 40.000 Da, more particularly less than 30.000 Da, more particularly less than 20.000 Da, more particularly less than 10.000 Da.
  • oligosaccharide composition for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000.
  • the oligosaccharide composition according to the invention may in particular be one in which the amount of monosaccharides (DP1 ) is less than 10% (w/w) of the amount of oligosaccharides (DP2 or more), such as less than 8% (w/w), less than 6% (w/w), less than 4% (w/w) or such as less than 2% (w/w).
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 21 GH5 xylanase, which is selected from the xylanase shown as amino acids 36 to 633 of SEQ ID NO: 8, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 36 to 633 of SEQ ID NO: 8.
  • subfamily 21 GH5 xylanase which is selected from the xylanase shown as amino acids 36 to 633 of SEQ ID NO: 8, or a GH5 xylanase having at least 75%, at least 80%, at least
  • the subfamily 21 GH5 xylanase may be selected from the His-taggged xylanase set forth in SEQ ID NO: 9, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 9.
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 34 GH5 xylanase, which is selected from the xylanase shown as SEQ ID NO: 10, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO: 10.
  • subfamily 34 GH5 xylanase which is selected from the xylanase shown as SEQ ID NO: 10
  • a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 9
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 34 GH5 xylanase, which is selected from the xylanase shown as SEQ ID NO: 1 1 , or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO: 1 1.
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 35 GH5 xylanase, which is selected from the xylanase shown as amino acids 37 to 573 of SEQ ID NO: 2, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 37 to 573 of SEQ ID NO: 2.
  • the 35 GH5 xylanase may be selected from the His-tagged xylanase set forth in SEQ ID NO: 3, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the His-tagged xylanase set forth in SEQ ID NO: 3.
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 35 GH5 xylanase, wihch is selected from the xylanase shown as amino acids 36 to 582 of SEQ ID NO: 5, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 36 to 582 of SEQ ID NO: 5
  • the 35 GH5 xylanase may be selected from the His-tagged xylanase set forth in SEQ ID NO: 6, or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the His-tagged xylanase set forth in SEQ ID NO: 6.
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 21 GH5 xylanase, which is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:13 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature polypeptide of the amino acid set forth in SEQ ID NO:13.
  • subfamily 21 GH5 xylanase which is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:13 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 21 GH5 xylanase, which is selected from the xylanase shown as amino acids 1 to 558 of SEQ ID NO: 16 (corresponding to the amino acid sequence set forth in SEQ ID NO: 17), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 558 of SEQ ID NO: 16 (corresponding to the amino acid se- quence set forth in SEQ ID NO: 17).
  • subfamily 21 GH5 xylanase which is selected from the xylanase shown as amino acids
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 21 GH5 xylanase, which is selected from
  • the subfamily 21 GH5 xylanase is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:19 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature polypeptide of the amino acid set forth in SEQ ID NO:19.
  • the subfamily 21 GH5 xylanase may be selected from the xylanase shown as amino acids 1 to 631 of SEQ ID NO: 19 (corresponding to the amino acid sequence set forth in SEQ ID NO: 20), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 631 of SEQ ID NO: 19 (corresponding to the amino acid sequence set forth in SEQ ID NO: 20).
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 21 GH5 xylanase, which is selected fromln another specific embodiment the subfamily 21 GH5 xylanase is selected from the xylanase shown as amino acids 1 to 639 of SEQ I D NO: 22 (corresponding to the amino acid sequence set forth in SEQ ID NO: 23), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 639 of SEQ ID NO: 22 (corresponding to the amino acid sequence set forth in SEQ ID NO: 23).
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 21 GH5 xylanase, which is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:25 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature polypeptide of the amino acid set forth in SEQ ID NO:13.
  • subfamily 21 GH5 xylanase which is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:25 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at
  • the subfamily 21 GH5 xylanase may be selected from the xylanase shown as amino acids 1 to 828 of SEQ ID NO: 25 (corresponding to the amino acid sequence set forth in SEQ ID NO: 26), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 828 of SEQ ID NO: 25 (corresponding to the amino ac- id sequence set forth in SEQ ID NO: 26).
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 21 GH5 xylanase, which is selected fromln another specific embodiment the subfamily 21 GH5 xylanase is selected from the xylanase shown as amino acids 1 to 836 of SEQ I D NO: 28 (corresponding to the amino acid sequence set forth in SEQ ID NO: 29), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 836 of SEQ ID NO: 28 (corresponding to the amino acid sequence set forth in SEQ ID NO: 29).
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 21 GH5 xylanase, which is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:31 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature polypeptide of the amino acid set forth in SEQ ID NO:31.
  • subfamily 21 GH5 xylanase which is selected from the mature polypeptide of the amino acid set forth in SEQ ID NO:31 and a GH5 xylanase having at least 75%, at least 80%, at least 85%, at
  • the subfamily 21 GH5 xylanase may be selected from the xylanase shown as amino acids 1 to 576 of SEQ ID NO: 31 (corresponding to the amino acid sequence set forth in SEQ ID NO: 32), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 576 of SEQ ID NO: 31 (corresponding to the amino acid sequence set forth in SEQ ID NO: 32).
  • the present invention relates to an oligosaccharide composition, for a bulking agent, wherein the peak value, Mp, expressed as peak molecular weight is less than 10.000 Da, the weight average molecular weight, Mw, is less than 30.000, obtainable by hydrolyzing corn fiber gum with subfamily 21 GH5 xylanase, which is selected fromln another specific embodiment the subfamily 21 GH5 xylanase is selected from the xy- lanase shown as amino acids 1 to 585 of SEQ I D NO: 34 (corresponding to the amino acid sequence set forth in SEQ ID NO: 34), or a GH5 xylanase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acids 1 to 585 of SEQ ID NO: 34 (corresponding to the amino acid sequence set forth in SEQ ID NO: 35).
  • the present invention relates to food product comprising the oligosaccharide composition according to the invention.
  • the oligosaccharide composition is useful as a bulking agent.
  • the present invention further relates to a process for producing the oligosaccharide composition of the invention.
  • the process can be performed as a one step process.
  • Another aspect of the present invention therefore relates to a one step process for making an oligosaccharide composition, wherein the peak value, Mp, expressed as peak molecular weight, is less than 50.000 Da, the weight average molecular weight, Mw, is less than 100.000, comprising hydrolyzing a substrate containing highly branched xylan with a GH5 xylanase.
  • Hy- drolysis is in a particular embodiment performed until completion. However, hydrolysis times may be varied according to the desired product composition.
  • oligosaccharide compositions of the invention relate to specific uses for the oligosaccharide compositions of the invention.
  • One specific embodiment relates to a use of the oligo-saccharide composition according to the invention in a food product.
  • Another specific embodiment retests to a use of the oligo-saccharide composition according to the invention as a fat replacement in a food product.
  • Another specific embodiment relates to a use of the oligo-saccharide composition according to the invention as a sugar replacement in a food product.
  • a further specific embodiment of theinvention relates to use of the oligosaccharide composition according to the invention for fiber enhancement of a food product.
  • an embodiment of the invention provide use of the oligosaccharide composition according to the invention for caloric reduction of a food product.
  • the xylanase from Acetivibrio cellulolyticus CD2 was identified in part of its public genome sequence as originally published under the accession number UniProt: E1 KC96 (Lucas S., Copeland A., Lapidus A., Cheng J.-F., Bruce D., Goodwin L.,Pitluck S., Land M.L., Hauser L., Chang Y.-J., Jeffries C, Mouttaki H., He Z., Zhou J., Hemme C.L., Woyke T.J.; "The draft genome of Acetivibrio cellulolyticus CD2.”; Submitted (AUG-2010) to the EMBL/GenBank DDBJ databases).
  • the draft genome of Gonapodya prolifera (Chang, Y. et al., 2015) contained a putative peptide sequence (SEQ ID NO: 36) including a predicted signal peptide a GH5_34 domain and a proline rich region C-terminal to the catalytic domain.
  • the xylanase from the strains Paenibacillus sp. 18054 (SEQ ID NO: 4 and 5) U2AGD and Paenibacillus illinoisensis (SEQ ID NO: 1 and 2) were identified by shotgun genome sequencing. Both xylanases belong to family GH5_35. The strains were isolated from a thermal sample from New Zealand in 1991. P. illionensis is deposited as DSMZ under accession number DSM16232.
  • Chryseobacterium sp-10696 was obtained from NCIMB Ltd, Aberdeen, Scotland as NCIMB1314 deposited under the name Flavobacterium sp. According to NCIMB Ltd, the sample was isolated from a suspension of minced fish muscle in water which was isolated on or before 1965.
  • the GH5 xylanase of SEQ ID NO: 12 was identified by shotgun genome sequencing
  • the elephant dung metagenome xylanase belongs to GH5_21 family was obtained by deep sequencing of a metagenome extract.
  • the DNA isolation was performed with the QIAamp DNA Stool kit from Qiagen (Hil- den, Germany) as described in the manufacturer's protocol.
  • Genome sequencing the subsequent assembly of reads and the gene discovery (i.e. annotation of gene functions) is known to the person skilled in the art and the service can be purchased commercially.
  • the all xylanases except SEQ ID NO: 38 were cloned into a Bacillus expression vector as described in WO 12/025577.
  • the xylanases were expressed with a Bacillus clausii secretion signal (BcSP; with the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA, originating from the protease AprH of B. clausii).
  • BcSP replaced all native secretion signals respectively in all genes.
  • an affinity tag sequence was introduced downstream of the BcSP sequence to ease the purification process (Histag; with the following amino acid sequence: HHHHHHPR for the xylanase from elephant dung metagenome, Chryseobacterium sp-10696, Paenibacillus sp. 18054 and Acetivibrio cellulolyticus and HQHQHQHPR for the Paenibacillus illinoisensis xylanase).
  • the gene that was expressed therefore comprised the BcSP sequence followed by the Histag sequence followed by the mature wild type xylanase sequence
  • the final expression plasmids (BcSP-Histag-xylanase) were individually transformed into a Bacillus subtilis expression host.
  • the xylanase BcSP-fusion genes were integrated by homologous recombination into the Bacillus subtilis host cell genome upon transformation.
  • the gene construct was expressed under the control of a triple promoter system (as described in WO 99/43835).
  • the gene coding for chloramphenicol acetyltransferase was used as maker (as described in (Diderichsen et al., 1993, Plasmid 30: 312-315).
  • Transformants were selected on LB media agar supplemented with 6 microgram of chloramphenicol per ml.
  • One recombinant Bacillus subtilis clone containing the respective xylanase expression construct was selected and was cultivated on a rotary shaking table in 500 ml baffled Erlenmeyer flasks each containing 100 ml yeast extract-based media. After 3-5 days cultivation time at 30 °C to 37°C, enzyme containing supernatants were harvested by centrifugation and the enzymes were purified by Histag purification.
  • the synthetic construct was subcloned into the Aspergillus expression vector pMStr57 (WO 2004032648), and the resulting expression construct was transformed into the Aspergillus oryzae strain MT3568 (WO 201 1057140).
  • Transformants were selected during regeneration from protoplasts based on the ability to utilize acetamide as a nitrogen source conferred by the vector borne selectable marker, and subsequently re-isolated under selection (Christensen et al., 1988 and WO 2004032648).
  • a single Aspergillus oryzae trans- formant was cultured in two 500ml baffled flasks each containing 150ml of DAP-4C-1 medium (WO 2012103350). The cultures were shaken on a rotary table at 150 RPM at 30°C for 4 days. The culture broth subsequently was separated from cellular material by passage through a 0.22 urn filter and purified by HIC chromatography.
  • Termamyl 120 L is a Bacillus licheniformis alpha amylase, however, other acid alpha amylases showing good temperature stability may also be used. Examples include, a Bacillus stearothermophilus alpha-amylases, which have a double deletion corresponding to delta(181 - 182) and further comprise a N193F substitution (also denoted 1181 * + G182 * + N193F) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO: 4 disclosed in WO 99/19467.
  • a 2nd extraction (CFG2) was made by dissolving the pellet in 200 mL 1 M NaOH. The mixture was kept for 1 hour at 96 C and then centrifuged for 20 min at 6000 g and the supernatant adjusted to pH 6 with 4 M HCI.
  • the purified enzymes used for the hydrolysis was SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1 (pentamodular GH5_34 from Clostridium thermocel- lum NzyTech (Catalogue Number (SKU): CR0061 , Correia et al. J. Biol. Chem. 201 1 , 286:22510-22520)), SEQ ID NO: fGH5_34_2, Pentopan mono, Pulpzyme, and Shearzyme.
  • SKU Clostridium thermocel- lum NzyTech
  • the columns used were a PWXL guard column and a PWXL-3000 and -5000 (Tosoh) connected in series.
  • the el- uent was 50 mM NaOAc pH 5 and the flow rate 0.5 ml/min. retention times were compared to a pullulan molecular weight standard between 803-1 .3 kDa and glucose, maltotriose and malto- pentaose.
  • the corn fiber gum hydrolysate data clearly showed that all the tested GH5 xylanases from subfamilies GH5_21 , GH5_34 and GH5_35 have the capacity to degrade this highly substituted xylan.
  • the DP1 -DP2 fraction was below 1 % under all tested conditions.
  • Substrate DDGS from a European corn-based fuel ethanol plant was used as substrate.
  • the DDGS was grinded in a coffee blender and washed extensively (five times, each wash followed by centrifugation) to remove soluble dry matter.
  • Enzymes were inactivated by boiling for 10 minutes.
  • the soluble dry substance (Brix) was measured after the sample was filtered (0,2 ⁇ filter) (Mettler Toledo Brix).
  • Adsorption at 320 nm was measured after filtration (0,2 ⁇ filter) after 50x dilution. Adsorption at 320 nm is a measure of the soluble fragments containing ferulic acid. The assay is calibrated with a ferulic acid standard; see standard curve in Figure 1.
  • Table 1 1 Analysis of supernatants after GH5 xylanase treatment. Dry matter is measured as Brix and the xylo oligos are quantified by the amount of ferulic acids. Free xylose was also measured.
  • HPLC as well as the Brix-measurements show that about 1 % or 15 g/l goes into solution by the xylanase treatment. Most of this ( ⁇ 80%) are soluble oligosaccharides with DP>3.
  • oligosaccharides are branched arabinoxylans that are useful as soluble bulking agents.

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Abstract

L'invention concerne, dans un premier aspect, une composition d'oligosaccharide pour un agent gonflant, la valeur de crête, Mp, exprimée en masse moléculaire de crête étant inférieure à 50 000 Da, la masse moléculaire moyenne en poids, Mw, étant inférieure à 100 000, pouvant être obtenue par hydrolyse d'un substrat de xylane hautement ramifié avec un xylanase GH5.
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WO2017044039A1 (fr) * 2015-09-11 2017-03-16 Carbiotix Ab Arabinoxylanes de bas poids moléculaire avec des oligosaccharides ramifiés
US11406120B2 (en) 2015-09-11 2022-08-09 Carbiotix Ab Low molecular weight arabinoxylans with branched oligosaccharides
US12005456B2 (en) 2016-09-16 2024-06-11 Novozymes A/S Fiber washing method and system
WO2019023222A1 (fr) * 2017-07-24 2019-01-31 Novozymes A/S Gh5 et gh30 dans le broyage humide
CN110770283A (zh) * 2017-07-24 2020-02-07 诺维信公司 湿磨中的gh5和gh30
US11746366B2 (en) 2017-07-24 2023-09-05 Novozymes A/S GH5 and GH30 in wet milling
EP3658610B1 (fr) 2017-07-24 2023-10-25 Novozymes A/S Gh5 et gh30 dans le broyage humide
US11525016B2 (en) 2018-05-10 2022-12-13 Comet Biorefining Inc. Compositions comprising glucose and hemicellulose and their use
US12269903B2 (en) 2018-05-10 2025-04-08 Comet Biorefining Inc. Compositions comprising glucose and hemicellulose and their use
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