WO2001042433A2 - Enzyme - Google Patents

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Publication number
WO2001042433A2
WO2001042433A2 PCT/IB2000/001941 IB0001941W WO0142433A2 WO 2001042433 A2 WO2001042433 A2 WO 2001042433A2 IB 0001941 W IB0001941 W IB 0001941W WO 0142433 A2 WO0142433 A2 WO 0142433A2
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Prior art keywords
xylanase
food
enzyme
feed
xylanase enzyme
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PCT/IB2000/001941
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English (en)
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WO2001042433A3 (fr
Inventor
Troels Nørgaard GRAVESEN
Patrick Maria Franciscus Derkx
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Danisco A/S
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Priority to AU18786/01A priority Critical patent/AU1878601A/en
Publication of WO2001042433A2 publication Critical patent/WO2001042433A2/fr
Publication of WO2001042433A3 publication Critical patent/WO2001042433A3/fr

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    • 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
    • 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
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
    • 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)

Definitions

  • the present invention relates to an enzyme.
  • the present invention also relates to a nucleotide sequence encoding same.
  • the present invention relates to nucleotide sequence encoding a xylanase enzyme.
  • the present invention also relates to the use of the enzyme and nucleotide sequences encoding same in the modification of a food and/or feed supplements comprising a xylan polymer.
  • Plant biomass comprises on average 23% lignin, 40% cellulose and 33% hemicellulose by dry weight (Coughlan & Hazlewood, 1993). Hemicellulose, one of the most abundant of organic substances in existence, accounts for 5-50% of the dry weight of plant materials (Dekker & Lindner, 1979).
  • Xylans constitute the main polymeric component of hemicellulose, being present in the cell walls of all land plants, particularly in tissues that have undergone secondary thickening, where they are known to have important structural functions (Horton & Wolfrom, 1963), Xylans are also present to some extent in the primary walls of growing cells, seeds and bulbs of certain plant species, functioning as reserve polysaccharides.
  • xylans are complex heteropolysaccharides based on a backbone structure of ⁇ -l,4-linked D-xylanopyranose units and, depending on their source and method of extraction, they may be substituted and may be linear or branched (reviewed in Puls & Poutanen, 1989). Substitutions include: acetylation at C-2 or C-3 of the xylose units; ⁇ - 1,2 linked 4-0-methyl- glucuronic acid groups; ⁇ -l,3-linked arabinofuranose units; and ferulic or coumaric acids esterified to C-5 of arabinose (Puls & Poutanen, 1989).
  • the xylans of hardwoods, softwoods and grasses are, on average, comprised of 70-200 D-xylopyranose units joined by ⁇ -l,4-linkages and, depending on their source and method of extraction, they may be substituted and may be linear or branched (Puls & Poutanen, 1989). Very few unsubstituted linear xylans have been isolated, the best known being from the xylans from esparto grass, tobacco stalks and guar seed husk.
  • Xylans having a ⁇ -l,3-linked backbone are found only in marine algae (Dekker & Richards, 1976), where, in certain species, they form highly crystalline fibrillar structures in the absence of cellulose.
  • Mixed link ⁇ -1,3- and 1,4-xylans are found in certain seaweeds such as Palmaria palmata (formerly Rhodymenia palmata) (Barry & Dillon, 1940).
  • Hardwood xylan (10-35% of dry weight) is typically 0-acetyl-4-0- methylglucuronoxylan (Degree of Polymerisation (DP) 200).
  • the xylan of softwoods (10-15% of dry weight) is an arabino-4-0- methylglucuronoxylan (DP >120) and differs from that of hardwoods in not being acetylated.
  • Substituents are 4-0-methylglucuronosyl residues attached to C-2 and L- arabinofuranosyl residues attached to C-3 of the relevant xylose backbone units.
  • the average ratio of the sugar units is 100:20:13 (Xyl:4-0-Me-GlcA:Ara) (Puls & Poutanen,
  • the xylan of grasses and cereals is also an arabino-4-0-methylglucuronoxylan (DP 70), but it has a lower 4-0-methylglucuronic acid content than does hardwood xylan, and a larger proportion of L-arabino-furanosyl side-chains, linked to C-2 or C-3, or both of the xylose main chain residues (Voragen et al, 1992).
  • DP 70 arabino-4-0-methylglucuronoxylan
  • L-arabino-furanosyl side-chains linked to C-2 or C-3, or both of the xylose main chain residues
  • xylan-degrading enzyme system the xylan-degrading enzyme system
  • hydrolytic enzymes include the main chain-cleaving enzymes, the endo-l,4- ⁇ -xylanases (EC 3.2.1.8) and the ⁇ -xylosidases (EC 3.2.1.37) those that liberate side chain substituents, namely ⁇ - glucuronidases, acetylxylanesterases and ⁇ -L-arabinofuranosidases (EC 3.2.1.55).
  • the main-chain enzymes involved are endo- ⁇ -l,4-xylanase ( ⁇ -l,4-D-xylan xylanohydrolase: EC 3.2.1.8), ⁇ -l,4-xylosidases ( ⁇ -l,4-D-xyloside xylohydrolase: EC 3.2.1.37) and possibly exo-xylanases ( ⁇ -l,4-D-xylan xylohydrolase).
  • side-chain cleaving activities necessary include -glucuronidase and acetylxylanesterase (EC 3.1.1.6), as well as the esterases that liberate acetyl, coumaroyl and feruloyl substituents.
  • acetylxylanesterase EC 3.1.1.6
  • esterases that liberate acetyl, coumaroyl and feruloyl substituents have been shown to interact synergistically, e.g. the removal of side-chain substituents to facilitate hydrolysis of the backbone by endoxylanse (Lee & Forsberg, 1987).
  • accessory enzymes will only remove side-chain groups from xylo- oligosaccharides, generated by the prior action of xylanase.
  • the various types of synergistic interactions may be distinguished and described as homeosynergy, heterosynergy, uni- and bi-product synergy and antisynerg
  • Endoxylanase cleaves ⁇ -1,4 linkages in the backbone of xylans, arabinoxylans, glucuronoxylans and related polymers (Coughlan et al 1992). However, depending on the enzyme, such action may require, or be hindered by, side chain substitution of the substrate (for a review see Coughlan et al 1992).
  • ⁇ -xylosidases cleave xylobiose, remove xylose residues from the non-reducing ends of xylo- oligosaccharides, and many can also liberate xylose from artificial substrates such as aryl ⁇ -xylosidases. Generally, ⁇ -xylosidases do not act against 'native' xylans although exceptions have been reported. An example of the latter is the enzyme from Neocallimastix frontalis.
  • Xylanases can also hydrolyse higher xylo-oligosaccharides, the affinity for such substrates increasing with increasing degree of polymerization (DP).
  • DP degree of polymerization
  • ⁇ -xylosidases they do not cleave xylobiose and most such enzymes investigated, including representatives from Aspergillus niger, Aspergillus ochraeus, Bacillus subtilis, Humicola grisea var thermoidea and Streptomyces lividans, do not hydrolyse aryl ⁇ -D-xylosides. Again, however, some exceptions have been noted.
  • the complete enzyme systems or the side-chain cleaving activities may be used in the hydrolysis of xylans to their monomeric constituents for subsequent fermentation to ethanol or xylitol, in the bleaching of pulp, in the preparation of clean cellulose, in silage making and in the preparation of modified hemicelluloses (see for example, Biely, 1985; Wong et al, 1988).
  • Xylanase enxymes have been combined with other protease enzymes and added to animal feeds to enhance the digestion of nutrients (see WO 96/05739).
  • Thermostable xylanases (from, for example, Microtetraspora flesuosa and Thermomonospora fusca) have also been added to animal feeds to enable animals digest the feeds more efficiently (see WO 95/29997).
  • the addition of these xylanase enzymes has not been consistenly effective. It is thought that naturally occuring xylanase inhibitors from cereals, such as wheat, can pose problems especially when xylanase enzymes are added to chicken feed with a high content of ground wheat.
  • some inhibitors from wheat may be resistant to temperatures of from about 80°C to 95°C.
  • variations in temperature during the pelleting process may mean that the naturally occurring xylanase inhibitors are not completely denatured. This may have an adverse effect on the efficacy of added xylanase enzymes.
  • thermostable xylanase enzyme Even if it is an thermostable xylanase enzyme, will not give rise to the effect for which it was added, or will only give rise to such an effect to a limited extent.
  • One possible way of overcoming this problem would be to include significantly greater relative amounts of the thermostable xylanase enzyme to the food and or feed in order to compensate for the deactivation of a certain proportion of the xylanase enzyme.
  • adding such additional amounts of enzyme is disadvantageous from an economic viewpoint.
  • the present invention thus seeks to overcome the problems associated with the preparation of food and/or feed supplements comprising naturally occuring xylanase inhibitors from cereals such as wheat.
  • thermostable xylanase enzyme a thermostable xylanase enzyme.
  • the activity of this xylanase enzyme is substantially independent of any level of a wheat xylanase inhibitor.
  • This thermostable xylanase enzyme can be used in a food and/or feed supplement to modifying a xylan polymer present in that supplement.
  • thermostable xylanase enzyme of the present invention has relatively low specific endo-xylanase activity. Consquently, this xylanase was not an obvious choice for a new food and/or feed xylanase as this enzyme is very different from the xylanase activity of traditional food and/or feed xylanase enzymes which have high specific endo-xylanase activity.
  • thermostable xylanase enzyme of the present invention allows the preparation of food and/or feed supplement at high temperatures without affecting the performance of the xylanase enzyme as the activity of this enzyme is substantially independent of any wheat xylanase inhibitors which may be present in a cereal based food and/or feed supplement.
  • thermostable xylanase enzyme is capable of delivering low molecular weight arabinoxylan fragments in vivo which may have a profound positive influence on intestinal microflora.
  • most of the other known endo-xylanase enzymes produce high molecular weight fragments.
  • the present invention provides a thermostable xylanase enzyme capable of modifying a xylan polymer in a food and/or feed supplement wherein the activity of the xylanase enxyme is substantially independent of any level of a wheat xylanase inhibitor that may be present in the food and/or feed supplement.
  • xylanase refers to a hemicellulase enzyme that cuts through the ⁇ -1,4 bonds within the xylosidic chain of a xylan polymer.
  • Xylanase enzymes can be classified as endo-xylanases or exo-xylanases depending on whether they cleave an internal ⁇ - 1 ,4 linkage or a ⁇ - 1 ,4 linkage at the non-reducing end of the xylan polymer.
  • the xylanase enzyme of the present invention is a thermostable xylanase enzyme which is herein after referred to as a themostable TX-1 xylanase enzyme.
  • thermostable xylanase enzyme of the present invention is derivable from a fungus.
  • thermostable TX-1 xylanase enzyme of the present invention is derivable from Talaromyces emersonii.
  • Talaromyces emersonii produces a complete xylan-degrading enzyme system when grown on the appropriate inducing substrates (Filho et al, 1989: Tuohy & Coughlan, 1992). Hydrolysis experiments with crude preparations provide evidence for the existence of endoxylanase, ⁇ -xylosidase, ⁇ -arabinofuranosidase, ⁇ - glucuronidase, acetylferuloyl-and coumaroyl-esterases. The participation of these enzyms in xylan degradation has been confirmed by independent assays.
  • the physiochemical characteristics of these xylan-degrading enzyme purified thus far include M r values ranging from 30,000- 131 000 and plvalues from pH 3.8-5.3. According to the literature, most of the fungal and bacterial xylanases have M r values in the range 8500-85 000, with pi values ranging from 4.0 to 10.3. Two endoxylanases (one high M r and one low M r ) purified from A. niger filtrates are good examples of this theory (Coughlan et al, 1993). However, exceptions to this rule have been found, for example in Trichoderma pseudokoningii (Baker et al, 1997). Endoxylanases are almost exclusively single subunit proteins. However, Xyl II from T.
  • emersonii differs in being a dimeric protein. This is not the only unusual feature this particular enzyme possesses. Generally, multiple isoenzymic forms of xylanases have been found in the culture filtrates of fungi. Talaromyces emersonii has been shown to be no exception. Dekker and Richards (1976) proposed that the complexity of xylans required the action of multiple xylanases with overlapping yet different specificities to effect extensive hydrolysis. Some of this multiplicity has been demonstrated to be genetically determined. However, post-translational modification, such as differential glycosylation, proteolysis, or aggregation with other polysaccharides may also account for some of this multiplicity.
  • post-translational modification such as differential glycosylation, proteolysis, or aggregation with other polysaccharides may also account for some of this multiplicity.
  • T, emersonii filtrates Other fungi known to produce comparative numbers of xylanase enzymes to those found in T, emersonii filtrates, are Aspergillus niger (15 endoxylanases) Biely et al, 1985) and Trichoderma viride (13 endoxylanases)(Biely et al, 1985).
  • the endoxlanases from T. emersonii are glycoproteins and have been shown to have high temperature optima (67-80°C) and high thermal stabilities. All the isoenzymes have acidic pH optima (pH 2.5-4.7 (Tuohy et al, 1993 a.b). Thermostable enzymes offer particular advantages in many industrial applications.
  • Table 2 shows that Xylobiose (X 2 ) is the major end product of the thermostable TX-1 xylanase of the present invention.
  • X 2 Xylobiose
  • Xyl I, Xyl II and Xyl III in Tuohy M G et al. (1993a).
  • thermostable xylanase enzyme (TX-1) of the present invention is characterised by a molecular weight of 38, 500 Daltons; a pH optimum of 3.0; thermostability at temperatures from about 70°C to about 95°C; a temperature optimum of 80°C; a thoeretical pi of about 4.5; a xylobiose (X ) end product; and a retention of capability to modify a xylan polymer even when naturally occuring wheat xylanase inhibitors are present in the food and/or feed supplement.
  • xylanase inhibitor refers to a protein whose role is to control the depolymerisation of complex carbohydrates, such as arabinoxylan, found in plant cell walls. These xylanase inhibitors are capable of reducing the activity of naturally occuring xylanase enzymes as well as those of fungal or bacterial origin. Although the presence of xylanase inhibitors have been reported in cereal seeds (see for example McLauchlan et al 1999; Rouau and Suget 1998) their impact on the efficacy of xylanase enzymes and in particular thermostable xylanase enzyme which have been added to food and/or feed supplements, has not been extensively examined.
  • the xylanases (and specifically, the endo- ⁇ -l,4-xylanases) which are produced by certain bacteria, fungi and plants and which hydrolyse the ⁇ -1, 4-xylan linkages in the xylan component of plant cell walls have been grouped into two classes, family 10 (also called F) and family 11 (also called G).
  • family 10 also called F
  • family 11 also called G
  • the TX-1 thermostable xylanase enzyme of the present invention is regarded as a member of the family 10 xylanases and is not regarded as a member of the family 1 1 xylanases.
  • McLauchlan et al (1999a) disclose the isolation and characterisation of a protein from wheat that binds to and inhibits two family- 11 xylanases.
  • WO 98/49278 demonstrates the effect of a wheat flour extract on the activity of a group of microbial xylanases all of which are classified as family 11 xylanases.
  • Debyser et al (1999) also disclose that endoxylanases from Aspergillus niger and Bacillus subtilis, which are both members of the family 11 xylanases were inhibited by a wheat xylanase inhibitor called TAXI.
  • the present invention demonstrates the highly surprising finding that the thermostable TX-1 xylanase enzyme of the present invention is effective in modifying a xylan present in a food and/or feed supplement even when naturally occuring xylanase inhibitors are present in a food and/or feed supplement.
  • the present invention demonstrates that the activity of the TX-1 xylanase enzyme is substantially independent of naturally occuring xylanase inhibitors derivable from cereal seeds, particularly naturally occuring xylanase inhibitors derivable from wheat based cereal seeds.
  • the present invention provides a thermostable TX-1 xylanase enzyme capable of modifying a xylan polymer in a food and or feed supplement.
  • modifying refers to a xylan polymer, such as arabinoxylan, which is degraded by a xylanase enzyme to produce low molecular weight (mostly xylobiose, X 2 ) fragments.
  • xylan refers to a polymer of D-xylose residues that are joined through ⁇ -1, 4 linkages and which have various substituent groups. These xylans can include but are not limited to xylans from cereals and grasses such as arabinoxylan and glucuronoxylan. The term “xylan” is often used interchangeably with the term “xylan polymer”.
  • the xylan is a cereal or derivable from a cereal.
  • cereal means any kind of grain used for food and or any grass producing this grain such as but not limited to any one of wheat, milled wheat, barley, maize, sorghum, rye, oats, triticale and rice or combinations thereof.
  • the cereal is a wheat cereal or a legume (such as for example pea or soy legumes).
  • the xylan in the food and/or feed supplement of the present invention is modified by contacting the xylan with the novel thermostable xylanase enzyme (TX-1) of the present invention.
  • contacting includes but is not limited to spraying, coating, impregnating or layering the food and/or feed supplement with the TX-1 enzyme of the present invention.
  • the food and/or feed supplement of the present invention may be prepared by mixing the TX-1 xylanase enzyme directly with a food and/or feed supplement.
  • the TX-1 xylanase enzyme * may be contacted (for example, by spraying) onto a cereal-based food and/or feed supplement such as milled wheat, maize or soya flour.
  • TX-1 xylanase enzyme it is also possible to incorporating the TX-1 xylanase enzyme it into a second (and different) food and/or feed or drinking water which is then added to the food and/or feed supplement of the present invention. Accordingly, it is not essential that the TX-1 xylanase enzyme provided by the present invention is incorporated into the cereal-based food and/or feed supplement itself, although such incorporation forms a particularly preferred aspect of the present invention.
  • the food and/or feed supplement may be combined with other food and/or feed components to produce a cereal-based food and/or feed.
  • Such other food and/or feed components may include one or more other (preferably thermostable) enzyme supplements, vitamin food and/or feed supplements, mineral food and/or feed supplements and amino acid food and/or feed supplements.
  • the resulting (combined) food and/or feed supplement comprising possibly several different types of compounds can then be mixed in an appropriate amount with the other food and/or feed components such as cereal and protein supplements to form a human food and/or an animal feed.
  • the food and/or feed supplement of the present invention can be prepared by mixing different enzymes having the appropriate activities to produce an enzyme mix.
  • a cereal-based food and/or feed supplement formed from e.g. milled wheat or maize may be contacted (eg by spraying) either simultaneously or sequentially with the xylanase enzyme and other enzymes having appropriate activities.
  • enzymes may include but are not limited to any one or more of an amylase, a glucoamylase, a pectinase, a mannanase, an a galactosidase, a phytase, a lipase, a P-glucanase, an-arabinofuranosidase, an amylase, a pectinase and a xylanase.
  • Enzymes having the desired activities may for instance be mixed with the xylanase of the present invention either before contacting these enzymes with a cereal- based food and/or feed supplement or alternatively such enzymes may be contacted simultaneously or sequentially on such a cereal based supplement.
  • the food and/or feed supplement is then in turn mixed with a cereal-based food and/or feed to prepare the final food and/or feed. It is also possible to formulate the food and/or feed supplement as a solution of the individual enzyme activities and then mix this solution with a food and/or feed material prior to processing the food and/or feed supplement into pellets or as a mash.
  • thermostable TX-1 xylanase enzyme can be obtainable from or produced by any suitable source, whether natural or not, or it may be a synthetic thermostable TX-1 xylanase enzyme, a semi-synthetic thermostable TX-1 xylanase enzyme, a mimetic, a derivatised thermostable TX-1 xylanase enzyme, a recombinant thermostable TX-1 xylanase enzyme, a fermentation optimised enzyme, a fusion protein or equivalents, mutants and derivatives thereof as long as it retains the required activity of the thermostable TX-1 xylanase enzyme of the present invention.
  • the term "mimetic" relates to any chemical which may be a peptide, polypeptide, antibody or other organic chemical which has the same activity as the thermostable TX- 1 xylanase enzyme of the present invention.
  • thermostable TX-1 xylanase enzyme includes chemical modification of an thermostable TX-1 xylanase enzyme. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group.
  • the non-native thermostable TX-1 xylanase enzyme includes at least a portion of which has been prepared by recombinant DNA techniques or produced by chemical synthesis techniques or combinations thereof.
  • thermostable TX-1 xylanase enzyme is prepared by the use of chemical synthesis techniques.
  • thermostable TX-1 xylanase enzyme of the present invention or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesize the thermostable TX-1 xylanase enzyme amino acid sequence, in whole or in part.
  • peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
  • thermostable TX-1 xylanase enzyme or variants, homologues, derivatives, fragments or mimetics thereof can be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences obtainable from the thermostable TX-1 xylanase enzyme, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant thermostable TX-1 xylanase enzyme.
  • thermostable TX- 1 xylanase enzyme of the present invention comprises the amino acid sequence set out in SEQ ID No 1 (see Figure 10) which is derivable from T. emersonii.
  • amino acid sequence refers to peptide, polypeptide sequences, protein sequences or portions thereof.
  • thermostable TX-1 xylanase enzyme of the present invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the thermostable TX-1 xylanase enzyme and still be regarded as substantially isolated.
  • the thermostable TX-1 xylanase enzyme of the present invention may also be in a substantially purified form, in which case it will generally comprise the thermostable TX-1 xylanase enzyme in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the thermostable TX-1 xylanase enzyme in the preparation is a peptide comprising SEQ ID No 1 or variants, homologues, derivatives or fragments thereof.
  • Preferred amino acid sequences of the present invention are set out in SEQ ID No 1 or are sequences obtainable from the thermostable TX-1 xylanase enzyme of the present invention but also include homologous sequences obtained from any source, for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof.
  • the present invention covers variants, homologues or derivatives of the amino acid sequences presented herein, as well as variants, homologues or derivatives of the nucleotide sequence coding for those amino acid sequences.
  • a homologous sequence is taken to include an amino acid sequence which is at least 75, 85 or 90% identical, preferably at least 95 or 98%o identical at the amino acid level over at least, for example, the amino acid sequence as set out in SEQ ID No 1 of the sequence listing herein.
  • homology should typically be considered with respect to those regions of the sequence known to be essential for enzyme activity (such as amino acids at positions) rather than non-essential neighbouring sequences.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • variant or derivative in relation to the amino acid sequences of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence has an enzyme activity, preferably having at least the same enzyme activity as the amino acid sequence set out in SEQ ID No 1.
  • SEQ ID No 1 may be modified for use in the present invention. Typically, modifications are made that maintain the enzyme activity of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains the required enzyme activity. Amino acid substitutions may include the use of non-naturally occurring analogues.
  • thermostable TX-1 xylanase enzyme of the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent enzyme. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the enzyme activity of the thermostable TX-1 xylanase enzyme is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
  • thermostable TX-1 xylanase enzyme has been prepared by use of recombinant techniques.
  • thermostable TX-1 xylanase enzyme of the present invention can encode the same thermostable TX-1 xylanase enzyme of the present invention as a result of the degeneracy of the genetic code.
  • skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the thermostable TX-1 xylanase enzyme encoded by the nucleotide sequence of the present invention to reflect the codon usage of any particular host organism in which the thermostable TX-1 xylanase enzyme of the present invention is to be expressed.
  • variant in relation to the nucleotide sequence set out in SEQ ID No 2 (see Figure 11) or SEQ ID No 3 (see Figure 12) of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for a thermostable TX-1 xylanase enzyme having an enzyme activity, preferably having at least the same activity as the nucleotide sequence set out in SEQ ID No 2 or SEQ ID No 3 of the sequence listings.
  • sequence homology preferably there is at least 75%), more preferably at least 85%, more preferably at least 90%) homology to the sequences shown in the sequence listing herein. More preferably there is at least 95%, more preferably at least 98%, homology. Nucleotide homology comparisons may be conducted as described above.
  • a preferred sequence comparison program is the GCG Wisconsin Bestfit program described above.
  • the default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch.
  • the default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.
  • the present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above.
  • Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
  • hybridization shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • Nucleotide sequences of the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 75%), preferably at least 85 or 90% and more preferably at least 95% or 98%) homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
  • Preferred nucleotide sequences of the invention will comprise regions homologous to the nucleotide sequence set out in SEQ ID No 2 or SEQ ID No 3 preferably at least 80 or 90% and more preferably at least 95% homologous to the nucleotide sequence set out in SEQ ID No 2 or SEQ ID No 3.
  • the term "selectively hybridizable" means that the nucleotide sequence used as a probe is used under conditions where a- target nucleotide sequence of the invention is found to hybridize to the probe at a level significantly above background.
  • the background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened.
  • background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
  • Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
  • both strands of the duplex either individually or in combination, are encompassed by the present invention.
  • the nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.
  • Nucleotide sequences which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of sources. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • mammalian cells e.g. rat, mouse, bovine and primate cells
  • Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the nucleotide sequence set out in SEQ I.D. No 2 or SEQ ID No 3 under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the amino acid and/or nucleotide sequences of the present invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences, such as the nucleotide sequence set out in SEQ ID. No 2 or SEQ ID No 3. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the enzyme activity of the thermostable TX-1 xylanase enzyme encoded by the nucleotide sequences.
  • the nucleotide sequences of the present invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the nucleotide sequences may be cloned into vectors.
  • a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the nucleotide sequences may be cloned into vectors.
  • Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term nucleotide sequence of the invention as used herein.
  • nucleotide sequences such as a DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • PCR polymerase chain reaction
  • This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector
  • thermostable TX-1 xylanase enzyme Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used to clone and express the thermostable TX-1 xylanase enzyme. As will be understood by those of skill in the art, it may be advantageous to produce the thermostable TX-1 xylanase enzyme - encoding nucleotide sequences possessing non-naturally occurring codons.
  • Codons preferred by a particular prokaryotic or eukaryotic host can be selected, for example, to increase the rate of the thermostable TX-1 xylanase enzyme expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
  • thermostable TX-1 xylanase enzyme or variants, homologues, derivatives, fragments or mimetics thereof may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
  • the nucleotide sequences of the present invention can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleotide sequence in a compatible host cell.
  • the invention provides a method of making the thermostable TX-1 xylanase enzyme of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell.
  • a nucleotide sequence of present invention which is inserted into a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • Control sequences operably linked to sequences encoding the protein of the invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell in which the expression vector is designed to be used. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
  • operably linked means that the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • the vectors of the present invention may be transformed or transfected into a suitable host cell as described below to provide for expression of an thermostable TX-1 xylanase enzyme of the present invention.
  • This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the thermostable TX-1 xylanase enzyme, and optionally recovering the expressed protein.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • the promoter is typically selected from promoters which are functional in mammalian, cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used.
  • the promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of ⁇ -actin, ⁇ -actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). Tissue-specific promoters specific for may be particularly preferred.
  • Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
  • MMLV LTR Moloney murine leukaemia virus long terminal repeat
  • RSV rous sarcoma virus
  • CMV human cytomegalovirus
  • the promoters may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
  • any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences.
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • thermostable TX-1 xylanase enzyme produced by a host recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing the thermostable TX-1 xylanase enzyme coding sequences can be designed with signal sequences which direct secretion of the thermostable TX-1 xylanase enzyme coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • thermostable TX-1 xylanase enzyme coding sequence may join to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3 -.26328 1), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA).
  • thermostable TX-1 xylanase enzyme is useful to facilitate purification.
  • thermostable TX-1 xylanase enzyme of the invention may also be produced as fusion proteins, for example to aid in extraction and purification.
  • fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences.
  • the fusion protein will not hinder the secondary binding activity of the thermostable TX-1 xylanase enzyme comprising the amino acid sequence of the present invention.
  • the present invention thus provides for fusion proteins comprising the respected target or an enzymatically active fragment or derivative thereof linked to an affinity tag such as glutathione-S-transferase (GST), biotin, His6, myc and hemagglutinin (HA) (as described in Wilson et al (1984 Cell 37 767).
  • GST glutathione-S-transferase
  • HA hemagglutinin
  • the fused recombinant protein comprises an antigenic co-protein such as GST, ⁇ -galactosidase or the lipoprotein D from Haemophilus influenzae which are relatively large co-proteins, which solubilise and facilitate production and purification thereof.
  • the fused protein may comprise a carrier protein such as bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen Inc) and described in Gentz et al (1989 PNAS 86: 821-824).
  • fusion proteins are readily expressable in yeast culture (as described in Mitchell et al 1993 Yeast 5: 715-723) and are easily purified by affinity chromatography.
  • a fusion protein may also be engineered to contain a cleavage site located between the nucleotide sequence encoding the thermostable TX-1 xylanase enzyme and the heterologous protein sequence, so that the thermostable TX-1 xylanase enzyme may be cleaved and purified away from the heterologous moiety.
  • an assay for the target protein may be conducted using the entire, bound fusion protein.
  • the co-protein may act as an adjuvant in the sense of providing a generalised stimulation of the immune system.
  • the co-protein may be attached to either the amino or carboxy terminus of the first protein.
  • thermostable TX-1 xylanase enzyme natural, modified or recombinant sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence to encode a fusion protein.
  • it may be useful to encode a chimeric thermostable TX-1 xylanase enzyme expressing a heterologous epitope that is recognized by a commercially available antibody.
  • thermostable TX-1 xylanase enzyme coding sequence is inserted within a marker gene sequence, recombinant cells containing the thermostable TX-1 xylanase enzyme coding regions may be identified by the absence of marker gene function.
  • a marker gene may be placed in tandem with a the thermostable TX-1 xylanase enzyme coding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the thermostable TX-1 xylanase enzyme as well.
  • Vectors comprising the nucleotide sequences of the present invention may be introduced into host cells for the purpose of replicating the vectors comprising the nucleotide sequences of the present invention and/or expressing the proteins of the invention encoded by the nucleotide sequences of the present invention.
  • Vectors comprising nucleotide sequences of the present invention may introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where the vectors comprising the nucleotide sequences of the present invention are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses, direct injection of nucleic acids and biolistic transformation.
  • retroviruses such as retroviruses, herpes simplex viruses and adenoviruses
  • Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents.
  • transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectamTM and transfectamTM).
  • cationic agents for example calcium phosphate and DEAE-dextran
  • lipofectants for example lipofectamTM and transfectamTM.
  • nucleic acid constructs are mixed with the transfection agent to produce a composition.
  • the vectors comprising nucleotide sequences encoding thermostable TX-1 xylanase enzymes of the present invention for use in affecting viral infections may be administered directly as a naked nucleic acid construct, preferably further comprising flanking sequences homologous to the host cell genome.
  • the amount of nucleic acid administered may typically be in the range of from 1 ⁇ g to 10 mg, preferably from 100 ⁇ g to 1 mg.
  • the nucleotide sequence of the present invention may be inserted into, for example, plasmid pMM68, which is then used to transfect host cells, such as Vibrio sp.60.
  • Host cells comprising nucleotide sequences of the present invention may be used to express the thermostable TX-1 xylanase enzymes of the present invention.
  • the proteins of the invention may be produced using prokaryotic cells as host cells, it is preferred to use eukaryotic cells, for example yeast, insect or mammalian cells, in particular mammalian cells.
  • eukaryotic cells for example yeast, insect or mammalian cells, in particular mammalian cells.
  • Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
  • thermostable TX-1 xylanase enzymes of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
  • thermostable TX-1 xylanase enzyme production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
  • Thermostable TX-1 xylanase enzymes of the present invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.
  • the thermostable TX-1 xylanase enzyme is purified and isolated in a manner known per se. LARGE SCALE APPLICATIONS
  • thermostable TX-1 xylanase enzyme is optimised for large scale production.
  • thermostable TX-1 xylanase enzyme is from about 500 to about 5000 units/kg feed.
  • thermostable TX-1 xylanase enzyme is from about 1000 to about 3000 units/kg feed.
  • thermostable TX-1 xylanase enzyme is advantageous for the preparation of food and/or feed formulations.
  • the cereal-based food and/or feed comprises from about 100 units of thermostable TX-1 xylanase enzyme to about 2000 units of thermostable TX-1 xylanase enzyme per kilo gram (kg) of food.
  • the cereal-based food and/or feed comprises from about 200 units of thermostable TX-1 xylanase enzyme to about 1200 units of thermostable TX-1 xylanase enzyme per kilo gram (kg) of food.
  • the cereal-based food and/or feed comprises from about 300 units of thermostable TX-1 xylanase enzyme to about 600 units of thermostable TX-1 xylanase enzyme per kilo gram (kg) of food.
  • the cereal-based food and/or feed comprises from about 400 units of thermostable TX-1 xylanase enzyme to about 500 units of thermostable TX-1 xylanase enzyme per kilo gram (kg) of food.
  • the cereal-based food and/or feed comprises from about 10%) to about 90% by weight of cereal.
  • the cereal-based food and/or feed comprises from about 20%> to about 70%> by weight of cereal.
  • the cereal-based food and/or feed comprises from about 30% to about 60% by weight of cereal.
  • the cereal-based food and/or feed comprises about 60%) by weight of cereal.
  • the cereal-based food and/or feed comprises from about 10%> to about 90%o by weight of wheat.
  • the cereal-based food and/or feed comprises from about 20%) to about 70%> by weight of wheat.
  • the cereal-based food and/or feed comprises from about 30%> to about 60%) by weight of wheat.
  • the cereal-based food and/or feed comprises about 60% by weight of wheat.
  • the cereal-based feed is prepared in a pellet form.
  • Processing of the food and/or feed supplement into an animal feed may be performed using any of the currently used processing apparatus such as a double-pelleting machine, a steam pelleter, an expander or an extruder.
  • the novel thermostable TX-1 xylanase enzyme of the present invention allows the preparation of food and/or feed supplement at high temperatures without affecting the performance of the enzyme whose activity is substantially independent of any wheat inhibitors which may be present in a cereal based food and/or feed supplement.
  • the preparation of the food and/or feed supplement of the present invention at high temperatures is highly advantageous when preparing cubes and pellets for an animal feed as these high temperatures: (i) improve the quality and durability of the resulting cubes and pellets, (ii) increase the range of ingredients which can be efficiently handled and (iii) also increase the level of liquid ingredients,such as fat and molasses, which can be incorporated into the food and/or feed.
  • the cereal-based feed supplement of the present invention which is prepared in pellet form may be provided to animals such as turkeys, geese, ducks, sheep and cows.
  • the feed is provided to pigs or to poultry.
  • the feed is provided to broiler chickens.
  • the present invention provides a food and/or feed supplement for improving the food/feed conversion ratio (FCR) and/or increasing the digestibility of a cereal-based feed.
  • FCR food/feed conversion ratio
  • the term food feed conversion ratio is the ratio of the amount of food and/or feed consumed relative to the weight gain of a human and/or animal subject.
  • a low FCR indicates that a given amount of food and/or feed results in a growing human and/or animal subject gaining proportionately more weight. This means that the human and/or animal subject is able to utilise the feed more efficiently.
  • One way in which the FCR of a food and/or feed can be improved is to increase its digestibility.
  • the present invention advantageously provides a food and/or feed supplement which can decrease the food and/or feed conversion ratio (FCR) of a cereal-based human food and/or animal feed.
  • thermostable xylanase (TX-1) of the present invention improves the FCR of a food and/or feed without increasing its cost per unit weight.
  • thermostable xylanase (TX-1) of the present invention improves the FCR of a food and/or feed without increasing its cost per unit weight.
  • the inclusion of the above enzyme as a feed supplement in the diet of an animal enables an animal and/or a human subject to digest the diet more efficiently. This increases the proportion of feed protein and energy which an animal and/or human subject can derive from the food and/or feed. This in turn improves the FCR of the feed making it more economical in use.
  • Figure 1 shows a graph
  • Figure 2 shows a photographic representation
  • Figure 3 shows a graph
  • Figure 4 shows a graph
  • Figure 5 shows a graph
  • Figure 6 shows a graph
  • Figure 7 shows a sequence alignment
  • Figure 8a shows a sequence alignment
  • Figure 8b shows a sequence alignment
  • Figure 9 shows a genomic construct
  • Figure 10 shows an amino acid sequence listing (SEQ ID No 1);
  • Figure 11 shows a nucleotide sequence listing (SEQ ID No 2);
  • Figure 12 shows a nucleotide sequence listing (SEQ ID No 3);
  • Figure 13 shows a graph
  • Figure 14 shows a graph
  • Figure 15 shows a graph
  • Figure 16 shows a graph
  • FIG. 1 shows the recovery of the thermostable Talaromyces emersonii (TX-1) enzyme from an ion exchange POROS 10HQ column.
  • Figure 2 shows a silver stained SDS polyacrylamide gel electrophoresis. Mv determination with masss spectrometry (MS) indicated two peaks with a Mv of 38,382 and 38, 987D, indicating micro heterogeneity.
  • MS masss spectrometry
  • FIG. 3 shows the pH optimum determinations which were determined for three different xylanase preparations. These were:
  • thermostable xylanase derived from Thermomyces lanuginosus.
  • TX-1 xylanase derived from Talaromyces emersonii A standard assay for optimum pH determinations was performed in citrate phosphate buffers ranging from pH 2.5 - 6.5.
  • FIG. 4 shows the thermostability of TX-1 which was determined for three different xylanase preparations by exposing a solution of purified enzyme in acetate buffer, pH 5.0, to 40, 50, 60, 70 and 80°C for 15 minutes. After heat treatment the residual activity has been determined.
  • the three different xylanase preparations were: TLX-1, thermostable endo-xylanase derived from Thermomyces lanuginosus. X-l endo-xylanase derived from Aspergillus niger. TX-1 endo-xylanase derived from Talaromyces emersonii.
  • Figure 5 shows the redidual xylanase activity for two enzyme preparations after subjecting the enzymes to a pelleting process.
  • the two enzyme preparations were: X-l endo-xylanase derived from Aspergillus niger and TX-1 endo-xylanase derived from Talaromyces emersonii.
  • FIG. 6 shows the water soluble pentosan (WSP)-activity relative to activity on water insoluble pentosan (WIP) at pH 4 and pH 7.
  • WSP water soluble pentosan
  • WIP water insoluble pentosan
  • thermostable endo-xylanase from Thermomyces lanuginosus.
  • Xy-1 endo-xylanase derived from Aspergillus niger.
  • Figure 7 shows the Primer design for degenerate PCR.
  • Figure 8a shows a BLAST alignment of the TX-1 xylanase amino acid sequence from T. emersonii, with the amino acid sequence from A.bisporus.
  • Figure 8b shows a BLAST alignment of the TX-1 xylanase amino acid sequence from T. emersonii, with the amino acid sequence from H.grisea.
  • Figure 9 shows the genomic organization of txl from T. Emersonii.
  • Figure 10 shows the deduced amino acid sequence for TX-1 (SEQ ID No 1).
  • Figure 11 shows the cDNA sequence (SEQ ID No 2) and the deduced protein sequence for TX-1.
  • Figure 12 shows genomic DNA (SEQ ID No 3).
  • Figure 13 shows the percentage relative activity of the TX-1 xylanase enzyme at pH 3-6 in the presence of a wheat extract.
  • Figure 14 shows the percentage relative activity of family 10 and 1 1 xylanase enzymes in the presence of increasing amounts of a wheat extract.
  • Figure 15 shows the percentage relative activity of TX-1 xylanase enzyme in the presence of xylanase inhibitor from difference cereal sources.
  • Figure 16 shows the temperature optimum for TX-1 enzyme determined in 0.1 acetate buffer, pH 5.0 and during a reaction time of 15 minutes.
  • Talaromyces emersonii, CBS 841.70 was grown on CMA agar medium at 40°C to produce spores for inoculation of shake flask cultures.
  • Shake flask medium containing ground corn cob, wheat bran and ammonium sulphate as a nitrogen source was used. Cultures were incubated at 40°C for 5-8 days at 200 rpm. Typically, 100 mg/liter of xylanase enzyme was routinely recovered.
  • TX-1 xylanase was purified from crude fermentation broth using successively Sephadex G25M for desalting, SOURCE 15Q anion exchanger, SOURCE 15PHE and SOURCE 15ETH for hydrophobic interaction chromatography and Superdex 75 prep grade for gel filtration.
  • Crude culture filtrate was assayed for endo-xylanase activity with Xylazyme tablets, Megazyme, Ireland. Activity was correlated to a standard enzyme preparation and expressed as TXU/gram enzyme.
  • TX-1 xylanase enzyme The purity of the TX-1 xylanase enzyme was checked by (i) running the xylanase on MINI Q and MONO Q HR 5/5 columns (ii) SDS polyacrylamide gel electrophoresis and (iii) mass spectrometry (MS). All material used were from Pharmacia LKB Biotechnology.
  • the dry matter content of a purified TX-1 sample determined by specific absorbance, was calculated to be 3.2 (0.1 %> at 280nm). From the specific absorbance, the specific activity was determined as 2,500 TXU/mg.
  • FIG. 2 shows a silver stained SDS polyacrylamide gel electrophoresis.
  • Mv determination of the staining profile of the TX-1 enzyme using masss spectrometry (MS) showed two peaks with a Mv of 38,382 and 38, 987D, indicating micro heterogeneity.
  • the TX-1 xylanase was deemed to belong to the F family of xylanases.
  • Figure 3 demonstrates the results from pH optimum determinations were carried out for three different xylanase preparations. These were:
  • thermostable endo-xylanase derived from Thermomyces lanuginosus.
  • TX-1 is active within a rather broad pH range, pH 3-8, with the highest activity at pH 3. However, more than 50% relative activity remains at pH 6.
  • Thermostability determinations were carried out for three different xylanase preparations by exposing a solution of purified enzyme in acetate buffer, pH 5.0, to 40, 50, 60, 70 and 80°C for 15 minutes. After heat treatment the residual activity was determined.
  • the three different xylanase preparations were: TLX-1, thermostable endo-xylanase derived from Thermomyces lanuginosus. X- 1 endo-xylanase derived from Aspergillus niger. TX-1 endo-xylanase derived from Talaromyces emersonii.
  • the residual xylanase activity for two enzyme preparations was determined after subjecting the enzymes to a pelleting process.
  • the two enzyme preparations were: X-l endo-xylanase derived from Aspergillus niger and TX-1 endo-xylanase derived from Talaromyces emersonii.
  • the water soluble pentosan (WSP)-activity relative to the activity on water insoluble pentosan (WIP) was determined at pH 4 and pH 7 for three different xylanase preparations.
  • the xylanase preparations used were: TLX-1, thermostable endo-xylanase from Thermomyces lanuginosus.
  • Figure 6 demonstrates that, of the three enzymes studies, only Xyl-1 and TX-1 release WSP degradation product.
  • a comparision of the WSP activities of Xyl-1 and TX-1 indicate that both enzymes have higher activities at pH 4 than at pH 7 but TX-1 has a higher WSP release activity than Xyl-1 at both pHs studied.
  • Table 2 indicates that depolymerisation of arabinoxylan with TX-1 delivers high amounts of xylobiose (X ) and almost no xylotriose (X 3 ) whereas X-l (Aspergillus niger) and TLX-1 (Therinomyces lanuginosus) deliver xylobiose (X ) and xylotriose (X 3 ) in approximately the same proportions.
  • TX-1 xylanase could be characterised as an exo-type xylanase but not a true exo- xylanase, where xylose is cut at the non-reducing end of arabinoxylan.
  • the specific activity of TX-1 compared with other known xylanases is low when procedures for measuring endo-activity are used.
  • the TX-1 enzyme of the present invention has been classified as an endo-xylanase and not an exo-xylanase.
  • this xylanase is used in combination with existing xylanases; the ability to reduce viscosity of water soluble xylan is enhanced and the amount of low molecular AX- fragments is elevated in comparison with reference.
  • Table 3 Activities of xylanases on different substrates
  • WIP-activity determined as the amount of liberated carbohydrate/gram enzyme with reference to standard enzyme
  • the 5' and 3' flanking regions of the nucleotide sequence encoding the TX-1 enzyme were isolated with a genomic walking procedure as described in Siebert et al. 1995. This strategy resulted in the isolation of DNA fragments containing the ORF encoding TX-1.
  • This protein had less than 55%> identity to xylanases from other organisms such as A.bisporus 52%> identical aa (see Figure 8a) and H grisea 41% identical aa (see Figure 8b).
  • the TX-1 protein comprises a predicted signal sequence which is cleaved between aa 23-24 leaving a mature protein of 382 aa (deduced MW 40.8 kDa).
  • a glycosyl hydrolase F10 active site is located at aa 254-264 and a putative cellulose binding domain can be found at the extreme C-terminus of the protein.
  • TX-1 contains also two putative N-linked glycosylation sites. These sites are illustrated in the schematic diagram in Figure 9.
  • the active-enzyme comprises a 330 amino acid sequence.
  • thermostable TX-1 xylanase enzyme of the present invention was assayed in the presence of a wheat derivable extract (200ul) at p ⁇ in the range of 3-6.
  • thermostable TX-1 xylanase enzyme of the present invention is substantially the same at p ⁇ in the range of 3- 6 but with the highest activity at p ⁇ 3.0.
  • TX-1 xylanase derived from Talaromyces emersonii (family 10 xylanase)
  • Figure 14 shows that the percentage relative activity of four different xylanase enzymes when assayed in the present of 1, 100, 200 and 400 ul of a wheat extract.
  • Two of the four enzymes studied (X-3, NN) showed at least a 50% reduction in activity after 30 addition of lOOul of the wheat extract.
  • the X-l enzyme showed at least a 50% reduction in activity after addition of 200 ul of the wheat extract.
  • the TX-1 xylanase enzyme of the present invention showed less than a 10%) reduction in activity after addition of either lOOul or 200ul of the wheat extract.
  • the main objective of this food and/or feeding trial was to study the efficacy of two enzyme preparations (Grindazym GP 5000 (Reference Enzyme) and Test product TS-E 389) in a 60%o wheat diet for broilers.
  • the wheat used was of the Cartaya type, harvested in the Lleida area and its analytical composition is shown in Table 7.
  • Feeds and wheat sample before analysis, all samples of experimental diets and Cartaya wheat were ground through 1.0 mm screen on a cyclone sample mill.
  • the analytical composition of Cartaya wheat was determined, including specific gravity, dry matter, crude protein, crude fat, ash, crude fiber, total and insoluble ⁇ -glucans, total and soluable pentosans. Quality control of the manufactured food and or feed was performed determining dry matter, crude protein, ether extract, crude fiber and ash (Table 8).
  • mice were killed by intravenous injection of sodium pentobarbital and chicks and their pancreas were weighed.
  • Digesta samples from Meckel's diverticulum to 15 cm before ileo-cecal conjunction were taken and stored on ice to measure fresh digesta viscosity.
  • the chyme samples were pooled per intestinal segment per cage (eight samples from two animals per treatments).
  • samples from the last part of ileum (I 5 cm) from treatments T- 1 and T-2 were taken, frozen and freeze-dried to measure ileal amino acid digestibility (eight samples from six animals per treatment).
  • Viscosity was determined using a Brookfield digital viscometer (model LVTDVCP-11, Brookfield Engineefing Laboratories, Stoughton, MA) maintained at 30'C and reading after I min.
  • Values are means of eight replicates of six chicks.
  • thermostable xylanase in an animal food and/or feed in accordance with the present invention enables the crude protein value and/or digestibility and/or the amin ⁇ acid content and/or digestibility coefficients of the food and/or feed to be increased, which permits a reduction in the amounts of alternative protein sources and/or amino acids supplements which have previously had to be included in animal food and/or feeds.
  • protein digestibility coefficient and/or the content of available crude protein of wheat is increased by the addition of the thermostable TX-1 xylanase of the present invention, major savings can be found in the reduced levels of protein and/or energy supplements which have conventionally needed to be added.

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Abstract

L'invention concerne une enzyme xylanase thermostable capable de modifier un polymère xylane dans un aliment et/ou un complément alimentaire. L'activité de l'enzyme xylanase est sensiblement indépendante de toute quantité d'un inhibiteur de xylanase du blé qui peut se trouver dans l'aliment et/ou le complément alimentaire.
PCT/IB2000/001941 1999-12-07 2000-12-06 Enzyme WO2001042433A2 (fr)

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WO2002024926A1 (fr) * 2000-09-21 2002-03-28 Dsm N.V. Talaromyces xylanase
WO2004029084A3 (fr) * 2002-09-27 2004-11-25 Danisco Proteines
WO2007091231A1 (fr) * 2006-02-10 2007-08-16 National University Of Ireland, Galway Systèmes enzymatiques à base de talaromyces emersonii
WO2009079210A2 (fr) * 2007-12-05 2009-06-25 Novozymes A/S Polypeptides ayant une activité de xylanase et polynucléotides codant pour ceux-ci
WO2010081869A1 (fr) 2009-01-16 2010-07-22 Danisco A/S Génération enzymatique de lipides fonctionnels à partir de céréales ou de bi-souches de céréale
WO2010115754A1 (fr) 2009-03-31 2010-10-14 Danisco A/S Prévention du noircissement d'extrait et de la formation de mauvaises odeurs au cours de la solubilisation d'une paroi cellulaire végétale
WO2010126772A1 (fr) * 2009-04-30 2010-11-04 Novozymes, Inc. Polypeptides ayant une activité xylanase et poly-nucléotides codant pour eux
WO2010150213A1 (fr) 2009-06-25 2010-12-29 Danisco A/S Protéine
EP2266405A2 (fr) 2004-03-12 2010-12-29 Danisco A/S Enzymes fongiques lipolytiques
EP2319920A1 (fr) 2005-12-22 2011-05-11 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
WO2011114251A1 (fr) 2010-03-18 2011-09-22 Danisco A/S Produits alimentaires
JP2013099335A (ja) * 2006-02-10 2013-05-23 National Univ Of Ireland Galway タラロマイセス・エマーソニイ酵素系
WO2013068550A3 (fr) * 2011-11-09 2013-07-18 Puratos N.V. Composition alimentaire enrichie d'un xylanase
US8623402B2 (en) 2001-08-20 2014-01-07 Cargill, Incorporated Non-starch-polysaccharides
WO2013182670A3 (fr) * 2012-06-08 2014-03-20 Dsm Ip Assets B.V. Nouvelles enzymes de scytalidium thermophilum de déconstruction de la paroi cellulaire et leurs utilisations
WO2014182990A1 (fr) * 2013-05-10 2014-11-13 Novozymes A/S Polypeptides présentant une activité xylanase et polynucléotides codant pour ceux-ci
US8927038B2 (en) 2008-03-25 2015-01-06 Cargill, Incorporated (Arabino)xylan oligosaccharide preparation
US9012186B2 (en) 2009-04-27 2015-04-21 The Board Of Trustees Of The University Of Illinois Hemicellulose-degrading enzymes
US9061046B2 (en) 2007-09-28 2015-06-23 Cargill, Incorporated Arabinoxylo-oligosaccharides useful against gastrointestinal infections
EP2776561A4 (fr) * 2011-11-11 2015-09-02 Novozymes Inc Polypeptides ayant une activité xylanase et polynucléotides codant pour ceux-ci
WO2015143961A1 (fr) * 2014-03-28 2015-10-01 中国科学院广州能源研究所 Aspergillus niger à haut rendement de production de xylanase et son application
US10202592B2 (en) 2009-11-06 2019-02-12 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2019121930A1 (fr) 2017-12-20 2019-06-27 Dsm Ip Assets B.V. Compositions d'aliments pour animaux et leurs utilisations
CN109997970A (zh) * 2019-03-07 2019-07-12 青岛红樱桃生物技术有限公司 一类酶活和耐热性提高的酸性木聚糖酶突变体及其编码基因和应用
WO2020058228A1 (fr) 2018-09-17 2020-03-26 Dsm Ip Assets B.V. Compositions d'aliments pour animaux et leurs utilisations
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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AU2002339115B2 (en) 2001-05-18 2007-03-15 Dupont Nutrition Biosciences Aps Method of preparing a dough with an enzyme
KR101226156B1 (ko) 2004-07-16 2013-01-24 듀폰 뉴트리션 바이오사이언시즈 에이피에스 효소적 오일-탈검 방법

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WO2002024926A1 (fr) * 2000-09-21 2002-03-28 Dsm N.V. Talaromyces xylanase
US7514110B1 (en) 2000-09-21 2009-04-07 Basf Aktiengesellschaft Talaromyces xylanases
US8623402B2 (en) 2001-08-20 2014-01-07 Cargill, Incorporated Non-starch-polysaccharides
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EP2319920A1 (fr) 2005-12-22 2011-05-11 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
US8409836B2 (en) 2005-12-22 2013-04-02 Roal Oy Treatment of cellulosic material and enzymes useful therein
EP2453014A1 (fr) 2005-12-22 2012-05-16 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
JP2013099335A (ja) * 2006-02-10 2013-05-23 National Univ Of Ireland Galway タラロマイセス・エマーソニイ酵素系
WO2007091231A1 (fr) * 2006-02-10 2007-08-16 National University Of Ireland, Galway Systèmes enzymatiques à base de talaromyces emersonii
US9061046B2 (en) 2007-09-28 2015-06-23 Cargill, Incorporated Arabinoxylo-oligosaccharides useful against gastrointestinal infections
US9771569B2 (en) 2007-12-05 2017-09-26 Novozymes A/S 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
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
US9353360B2 (en) 2007-12-05 2016-05-31 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
US8927038B2 (en) 2008-03-25 2015-01-06 Cargill, Incorporated (Arabino)xylan oligosaccharide preparation
WO2010081869A1 (fr) 2009-01-16 2010-07-22 Danisco A/S Génération enzymatique de lipides fonctionnels à partir de céréales ou de bi-souches de céréale
WO2010115754A1 (fr) 2009-03-31 2010-10-14 Danisco A/S Prévention du noircissement d'extrait et de la formation de mauvaises odeurs au cours de la solubilisation d'une paroi cellulaire végétale
EP3269248A2 (fr) 2009-03-31 2018-01-17 DuPont Nutrition Biosciences ApS Méthode pour la solubilisation d'un matériau de paroi cellulaire végétale
US9012186B2 (en) 2009-04-27 2015-04-21 The Board Of Trustees Of The University Of Illinois Hemicellulose-degrading enzymes
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WO2011114251A1 (fr) 2010-03-18 2011-09-22 Danisco A/S Produits alimentaires
WO2013068550A3 (fr) * 2011-11-09 2013-07-18 Puratos N.V. Composition alimentaire enrichie d'un xylanase
EP2776561A4 (fr) * 2011-11-11 2015-09-02 Novozymes Inc Polypeptides ayant une activité xylanase et polynucléotides codant pour ceux-ci
WO2013182670A3 (fr) * 2012-06-08 2014-03-20 Dsm Ip Assets B.V. Nouvelles enzymes de scytalidium thermophilum de déconstruction de la paroi cellulaire et leurs utilisations
WO2014182990A1 (fr) * 2013-05-10 2014-11-13 Novozymes A/S Polypeptides présentant une activité xylanase et polynucléotides codant pour ceux-ci
US9714417B2 (en) 2013-05-10 2017-07-25 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
US9556465B2 (en) 2013-05-10 2017-01-31 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2015143961A1 (fr) * 2014-03-28 2015-10-01 中国科学院广州能源研究所 Aspergillus niger à haut rendement de production de xylanase et son application
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CN109997970A (zh) * 2019-03-07 2019-07-12 青岛红樱桃生物技术有限公司 一类酶活和耐热性提高的酸性木聚糖酶突变体及其编码基因和应用
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