U.S. patent application number 16/839897 was filed with the patent office on 2020-10-08 for xylanases for solubilising arabinoxylan-containing material.
The applicant listed for this patent is DUPONT NUTRITION BIOSCIENCES APS. Invention is credited to SOREN DALSGAARD, ELIJAH G. KIARIE, BART C KOOPS, FLOOR K KOOY, ROSALYN LAU, BRIAN SOEGAARD LAURSEN, SUSAN ARENT LUND, MADS BROEGGER PEDERSEN, LUIS FERNANDO ROMERO MILLAN, JAN HENDRIK A VAN TUIJL, ZHEYONG YU, ZHENGHONG ZHANG.
Application Number | 20200318092 16/839897 |
Document ID | / |
Family ID | 1000004914875 |
Filed Date | 2020-10-08 |
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United States Patent
Application |
20200318092 |
Kind Code |
A1 |
ROMERO MILLAN; LUIS FERNANDO ;
et al. |
October 8, 2020 |
XYLANASES FOR SOLUBILISING ARABINOXYLAN-CONTAINING MATERIAL
Abstract
The present invention relates to a method for solubilising
arabinoxylan-containing material (particularly insoluble
arabinoxylan-containing material), comprising admixing a
xylan-containing material with a xylanase comprising a polypeptide
sequence shown herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1,
SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a
variant, homologue, fragment or derivative thereof having at least
75% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1 or
SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or
a polypeptide sequence which comprises SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity with SEQ ID No. 6, SEQ ID
No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID
No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code, or a xylanase obtainable (or obtained) from
Fusarium verticilloides. The present invention also relates to a
novel xylanase comprising (or consisting of) a polypeptide sequence
shown herein as SEQ ID No. 3, SEQ ID No. 2 or SEQ ID No. 1, or a
variant, homologue, fragment or derivative thereof having at least
99% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1; or
a xylanase which is encoded by a nucleotide sequence shown herein
as SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4, or a nucleotide
sequence which can hybridize to SEQ ID No. 4 or SEQ ID No. 5 under
high stringency conditions, or a nucleotide sequence which has at
least 97.7% identity (preferably 98% identity) with SEQ ID No. 6,
SEQ ID No. 5 or SEQ ID No. 4. The present invention yet further
relates to methods relating to feedstuffs, malting and brewing,
processing of grain-based materials such as during the production
of bioethanol or biochemical (e.g. bio-based isopropanol), or wheat
gluten-starch separation processes and the like.
Inventors: |
ROMERO MILLAN; LUIS FERNANDO;
(WILSHIRE, GB) ; LUND; SUSAN ARENT; (BRABRAND,
DK) ; LAURSEN; BRIAN SOEGAARD; (KALUNDBORG, DK)
; KIARIE; ELIJAH G.; (WUAKESHA, WI) ; ZHANG;
ZHENGHONG; (SHANGHAI, CN) ; LAU; ROSALYN;
(SHANGHAI, CN) ; YU; ZHEYONG; (SHANGHAI, CN)
; KOOY; FLOOR K; (LEIDEN, NL) ; VAN TUIJL; JAN
HENDRIK A; (ZOETERMEER, NL) ; KOOPS; BART C;
(PIJNACKER, NL) ; PEDERSEN; MADS BROEGGER;
(Frederiksberg, DK) ; DALSGAARD; SOREN;
(SILKEBORG, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DUPONT NUTRITION BIOSCIENCES APS |
Copenhagen |
|
DK |
|
|
Family ID: |
1000004914875 |
Appl. No.: |
16/839897 |
Filed: |
April 3, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15843023 |
Dec 15, 2017 |
10612014 |
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16839897 |
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14418900 |
Jan 30, 2015 |
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PCT/EP2013/066255 |
Aug 2, 2013 |
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15843023 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 7/10 20130101; A23K
50/75 20160501; A23K 10/38 20160501; C12P 5/007 20130101; C12Y
302/01008 20130101; A23K 50/30 20160501; C12N 9/2482 20130101; C13K
1/06 20130101; C12C 5/004 20130101; Y02P 60/87 20151101; A23K 10/10
20160501; C12P 19/14 20130101; Y02E 50/10 20130101; C13K 1/02
20130101; A23K 10/14 20160501 |
International
Class: |
C12N 9/24 20060101
C12N009/24; C12P 19/14 20060101 C12P019/14; C12P 7/10 20060101
C12P007/10; C12C 5/00 20060101 C12C005/00; C13K 1/02 20060101
C13K001/02; C13K 1/06 20060101 C13K001/06; A23K 10/10 20060101
A23K010/10; A23K 10/38 20060101 A23K010/38; A23K 10/14 20060101
A23K010/14; A23K 50/75 20060101 A23K050/75; A23K 50/30 20060101
A23K050/30; C12P 5/00 20060101 C12P005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2012 |
CN |
PCT/CN2012/079650 |
Jul 23, 2013 |
CN |
201310311358.1 |
Claims
1-17. (canceled)
18. A recombinant host cell comprising a nucleic acid which encodes
a polypeptide sharing 85% sequence identity to the amino acid
sequence of SEQ ID NO:3.
19-26. (canceled)
27. The host cell of claim 18, wherein the host cell is a
bacterial, fungal, or yeast cell.
28. The host cell of claim 27, wherein the bacterial cell is a
Streptomyces spp. or a Bacillus spp.
29. The host cell of claim 27, wherein the fungal cell is a
Fusarium spp., an Aspergillus spp., a Thermomyces spp., an
Acremonium spp., a Penicillium spp., a Mucor spp., a Neurospora
spp., or a Trichoderma spp.
30. A method for degrading for degrading arabinoxylan-containing
material in a xylan-containing material comprising contacting the
material with the polypeptide produced by the host cell of claim
18.
31. The method of claim 30, wherein the xylan-containing material
is selected from one or more of the group consisting of: a feed or
feedstuff, a feed component, a grain-based material, a mash, a
wort, a malt, malted barely, an adjunct, a barley mash, and a
cereal flour.
32. The method of claim 31, wherein the feed or feedstuff or feed
component comprises or consists of corn, Distillers Dried Grains
with Solubles (DDGS), corn Distillers Dried Grain Solubles (cDDGS),
wheat, wheat bran or a combination thereof.
33. The method according to claim 31 wherein the feed or feedstuff
is a corn-based feedstuff.
34. The method of claim 30, wherein the polypeptide is used in
combination with one or more of the enzymes selected from the group
consisting of a protease, an amylase, and a phytase.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of xylanases having
unusual properties in applications, including in feedstuffs, in
brewing or malting, in the treatment of arabinoxylan containing raw
materials like grain-based materials, e.g. in the production of
biofuel or other fermentation products, including biochemicals
(e.g. bio-based isoprene), and/or in the wheat gluten-starch
separation industry, and methods using these xylanases, as well as
compositions (such as feed additive compositions) comprising said
xylanases. The present invention also relates to a new xylanase
with unusual properties that renders it useful in applications,
including in feedstuffs, in brewing or malting, in the treatment of
arabinoxylan containing raw materials like grain-based materials,
e.g. in the production of biofuel or biochemicals (e.g. bio-based
isoprene), and/or in the wheat gluten-starch separation
industry.
BACKGROUND OF THE INVENTION
[0002] For many years, endo-.beta.-1,4-xylanases (EC 3.2.1.8)
(referred to herein as xylanases) have been used for the
modification of complex carbohydrates derived from plant cell wall
material. It is well known in the art that the functionality of
different xylanases (derived from different microorganisms or
plants) differs enormously. Xylanase is the name given to a class
of enzymes which degrade the linear polysaccharide beta-1,4-xylan
into xylooligosaccharides or xylose, thus breaking down
hemicellulose, one of the major components of plant cell walls.
[0003] Based on structural and genetic information, xylanases have
been classified into different Glycoside Hydrolase (GH) families
(Henrissat, (1991) Biochem. J. 280, 309-316).
[0004] Initially all known and characterized xylanases belonged to
the families GH10 or GH11. Further work then identified numerous
other types of xylanases belonging to the families GH5, GH7, GH8
and GH43 (Collins et al (2005) FEMS Microbiol Rev., 29 (1),
3-23).
[0005] Until now the GH11 family differs from all other GH's, being
the only family solely consisting of xylan specific xylanases. The
structure of the GH11 xylanases can be described as a .beta.-Jelly
roll structure or an all .beta.-strand sandwich fold structure
(Himmel et al 1997 Appl. Biochem. Biotechnol. 63-65, 315-325). GH11
enzymes have a catalytic domain of around 20 kDa.
[0006] GH10 xylanases have a catalytic domain with molecular
weights in the range of 32-39 kDa. The structure of the catalytic
domain of GH10 xylanases consists of an eightfold p/a barrel
(Harris et al 1996--Acta. Crystallog. Sec. D 52, 393-401).
[0007] Three-dimensional structures are available for a large
number of Family GH10 enzymes, the first solved being those of the
Streptomyces lividans xylanase A (Derewenda et al J Biol Chem 1994
Aug. 19; 269(33) 20811-4), the C. firmi endo-glycanase Cex (White
et al Biochemistry 1994 Oct. 25; 33(42) 12546-52), and the
Cellvibrio japonicus Xyn10A (previously Pseudomonas fluorescens
subsp. xylanase A) (Harris et al Structure 1994 Nov. 15; 2(11)
1107-16.). As members of Clan GHA they have a classical
(.alpha./.beta.).sub.8 TIM barrel fold with the two key active site
glutamic acids located at the C-terminal ends of beta-strands 4
(acid/base) and 7 (nucleophile) (Henrissat et al Proc Natl Acad Sci
USA 1995 Jul. 18; 92(15) 7090-4).
[0008] Comprehensive studies characterising the functionality of
xylanases have been done on well characterised and pure substrates
(Kormelink et al., 1992 Characterisation and mode of action of
xylanases and some accessory enzymes. Ph.D. Thesis, Agricultural
University Wageningen, Holland (175 pp., English and Dutch
summaries)). These studies show that different xylanases have
different specific requirements with respect to substitution of the
xylose backbone of the arabinoxylan (AX). Some xylanases require
three un-substituted xylose residues to hydrolyse the xylose
backbone; others require only one or two. The reasons for these
differences in specificity are thought to be due to the three
dimensional structure within the catalytic domains, which in turn
is dependent on the primary structure of the xylanase, i.e. the
amino acid sequence. However, the translation of these differences
in the amino acid sequences into differences in the functionality
of the xylanases, has up until now not been documented when the
xylanase acts in a complex environment, such as a plant material,
e.g. in a feedstuff.
[0009] The xylanase substrates in plant material, e.g. in wheat,
have traditionally been divided into two fractions: The water
un-extractable AX (WU-AX) and the water extractable AX (WE-AX).
There have been numerous explanations as to why there are two
different fractions of AX. The older literature (D'Appolonia and
MacArthur--(1976, Cereal Chem. 53. 711-718) and Montgomery and
Smith (1955, J. Am. Chem. Soc. 77. 3325-332) describes quite high
differences in the substitution degree between WE-AX and WU-AX. The
highest degree of substitution was found in WE-AX. This was used to
explain why some of the AX was extractable. The high degree of
substitution made the polymer soluble, compared to a lower
substitution degree, which would cause hydrogen bonding between
polymers and consequently precipitation.
[0010] The difference between the functionality of different
xylanases has been thought to be due to differences in xylanase
specificity and thereby their preference for the WU-AX or the WE-AX
substrates.
[0011] Xylanase enzymes have been reported from nearly 100
different organisms, including plants, fungi and bacteria. The
xylanase enzymes are classified into several of the more than 40
families of glycosyl hydrolase enzymes. The glycosyl hydrolase
enzymes, which include xylanases, mannanases, amylases,
.beta.-glucanases, cellulases, and other carbohydrases, are
classified based on such properties as the sequence of amino acids,
their three dimensional structure and the geometry of their
catalytic site (Gilkes, et al., 1991, Microbiol. Reviews 55:
303-315).
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a polypeptide sequence (SEQ ID No. 1) of a
xylanase of the present invention (FveXyn4). This is the
pre-pro-protein. Underlined (lower case) portion of the sequence
reflects an N terminal signal peptide can be cleaved before the
enzyme is matured. The amino acids shown in bold and italicized may
also be cleaved by post-translational modification before the
enzyme is fully matured.
[0013] FIG. 2 shows a polypeptide sequence (SEQ ID No. 2) of a
xylanase of the present invention (FveXyn4). This is the
pro-protein. The amino acids shown in bold and italicized may also
be cleaved by post-translational modification before the enzyme is
fully matured.
[0014] FIG. 3 shows a polypeptide sequence (SEQ ID No. 3) of a
xylanase of the present invention (FveXyn4). This is the active
form of the enzyme. This may be referred to herein as the mature
form of the enzyme.
[0015] FIG. 4 shows a nucleotide sequence (SEQ ID No. 4) encoding a
xylanase of the present invention (FveXyn4). The lower case
nucleotides which are in bold show the intron sequence. The signal
sequence is shown bold (upper case).
[0016] FIG. 5 shows a nucleotide sequence (SEQ ID No. 5) encoding a
xylanase of the present invention (FveXyn4). The signal sequence is
shown bold (upper case).
[0017] FIG. 6 shows a nucleotide sequence (SEQ ID No. 6) encoding a
xylanase of the present invention (FveXyn4).
[0018] FIG. 7 shows a scheme of an auto-analyzer for the
determination of pentosan by an automated phloroglucinol method:
(a) Acetic acid mixed with HCl; (b) air bubbling; (c)
phloroglucinol in ethanol; (d) sample; (e) sample accelerator; (f)
flow cell way-out; (g) peristaltic pump; (h) glass coil; (i)
thermostat (96.degree. C.); (j) multiple wavelength
spectrophotometer (410, 510, 550, and 620 nm); (k) waste; (1)
computer (Rouau & Surget, 1994 Carbohydrate Polymers, 24,
123-32).
[0019] FIG. 8 shows pentosan (C-5 sugar) release (solubilisation of
pentosans) from wheat bran as a function of xylanase dosage. The
xylanases used were the xylanase of the present invention (FveXyn4)
compared with abenchmark xylanase namely Econase.RTM. XT.
[0020] FIG. 9 shows solubilisation of pentosans from cDDGS as a
function of xylanase dosage. The xylanases used were the xylanase
of the present invention (FveXyn4) compared with abenchmark
xylanase namely Econase.RTM. XT.
[0021] FIG. 10 shows a plasmid map of pZZH254.
[0022] FIG. 11 shows the pH activity profile of FveXyn4.
[0023] FIG. 12 shows the temperature profile of FveXyn4.
[0024] FIG. 13 shows viscosity reduction on high viscosity wheat by
FveXyn4 in an in vitro animal model (where pH was maintained at
6.0).
[0025] FIG. 14 shows RVA viscosity profiles in whole ground wheat
starting at pH5.2 for the xylanase in accordance with the present
invention namely FveXyn4 and the benchmark enzymes
Xylathin.TM..
[0026] FIG. 15 shows a polypeptide sequence (SEQ ID No. 9) of a
xylanase of the present invention (FoxXyn2). This is the
pre-pro-protein. Underlined (lower case) portion of the sequence
may reflect an N terminal signal peptide which can be cleaved
before the enzyme is matured. The amino acids shown in bold and
italicized may also be cleaved by post-translational modification
before the enzyme is fully matured.
[0027] FIG. 16 shows a polypeptide sequence (SEQ ID No. 10) of a
xylanase of the present invention (FoxXyn2). This is the
pro-protein. The amino acids shown in bold and italicized may also
be cleaved by post-translational modification before the enzyme is
fully matured.
[0028] This sequence may be an active form of the protein and may
be one active form of the protein. This may be referred to herein
as the mature form of the enzyme.
[0029] FIG. 17 shows a polypeptide sequence (SEQ ID No. 11) of a
xylanase of the present invention (FoxXyn2). This is another active
form of the enzyme. In some embodiments, this may be referred to
herein as the mature form of the enzyme.
[0030] FIG. 18 shows RVA viscosity profiles in whole ground wheat
starting at pH5.2 for the xylanase in accordance with the present
invention namely FoxXyn2 and the benchmark enzymes
Xylathin.TM..
[0031] FIG. 19 shows a nucleotide sequence (SEQ ID No. 12) encoding
a xylanase of the present invention (FoxXyn2). The lower case
nucleotides which are in bold show the intron sequence. The signal
sequence is shown bold (upper case).
[0032] FIG. 20 shows a nucleotide sequence (SEQ ID No. 13) encoding
a xylanase of the present invention (FoxXyn2). The signal sequence
is shown bold (upper case).
[0033] FIG. 21 shows a nucleotide sequence (SEQ ID No. 14) encoding
a xylanase of the present invention (FoxXyn2).
[0034] FIG. 22 shows a plasmid map of pZZH135.
[0035] FIG. 23 shows the pH profile of FoxXyn2.
[0036] FIG. 24 shows the temperature profile of FoxXyn2.
[0037] FIG. 25 shows a polypeptide sequence (SEQ ID No. 15) of a
xylanase of the present invention (from Fusarium)--Fusarium
Comparative Sequencing Project, Broad Institute of Harvard and MIT
(http://www.broadinstitute.or/)). In some embodiments, this may be
referred to herein as the mature form of the enzyme.
[0038] FIG. 26 shows an alignment of the mature proteins for
FveXyn4 (SEQ ID No. 3), FoxXyn2 (SEQ ID No. 11) and the xylanase
shown herein as SEQ ID No. 15.
[0039] FIG. 27 shows the effect of the xylanase and protease
treatments alone and in combination on the solubilization of
pentosan and protein from insoluble corn DDGS. Letters a-d are
significant different according to on-way ANOVA and Holm-Sidak
comparisons with overall significance level at P=0.05. Error bars
indicate S.D.
[0040] FIG. 28 shows the effect of the xylanase and protease
treatments alone and in combination on the solubilization of
pentosan and protein from insoluble wheat DDGS. Letters a-d are
significant different according to on-way ANOVA and Holm-Sidak
comparisons with overall significance level at P=0.05. Error bars
indicate S.D.
[0041] FIG. 29 shows solubilisation of pentosans from cDDGS as a
function of xylanase dosage. The xylanases used were the xylanases
of the present invention (FveXyn4 and FoxXyn2).
[0042] FIG. 30 shows a nucleotide sequence (SEQ ID No. 16) encoding
a xylanase for use in the present invention from Fusarium--obtained
from Fusarium Comparative Sequencing Project, Broad Institute of
Harvard and MIT (http://www.broadinstitute.org/)). The lower case
nucleotides which are in bold show the intron sequence. The signal
sequence is shown bold (upper case). Changes compared with SEQ ID
No. 4 are underlined.
[0043] FIG. 31 shows a nucleotide sequence (SEQ ID No. 17) encoding
a xylanase for use in the present invention from Fusarium--obtained
from Fusarium Comparative Sequencing Project, Broad Institute of
Harvard and MIT (http://www.broadinstitute.org/)). The signal
sequence is shown bold (upper case). Changes compared with SEQ ID
No. 5 are underlined.
[0044] FIG. 32 shows a nucleotide sequence (SEQ ID No. 18) encoding
a xylanase for use in the present invention from Fusarium--obtained
from Fusarium Comparative Sequencing Project, Broad Institute of
Harvard and MIT (http://www.broadinstitute.or/)). Changes compared
with SEQ ID No. 6 are underlined.
SUMMARY OF THE INVENTION
[0045] A seminal finding of the present invention is a novel
xylanase isolated from Fusarium verticilloides, which enzyme has
surprising and unexpected properties. In particular it is
unexpectedly good at breaking down (solubilising) insoluble
arabinoxylans (AXinsol).
[0046] Surprisingly the enzyme has been found to efficiently
breakdown (solubilise) AXinsol from a wide range of substrates,
including corn, wheat, DDGS, etc, in particular corn and corn based
substrates, in particular both wheat (including wheat-based)
products and corn (including corn-based products). This contrasts
with prior-known enzymes, which are often inferior at solubilising
AXinsol in corn or corn-based substrates or which are not efficient
in both wheat- and corn-based substrates.
[0047] In addition, the enzyme of the present invention is
particularly good at not only breaking down (solubilising) AXinsol,
but also breaking down (or degrading) the solubilized polymers
efficiently. By being able to efficiently (quickly) breakdown
(degrade) the solubilized polymers (obtained from dissolving
AXinsol), a (fast) reduction in viscosity is obtained or the
solubilized polymers (obtained from dissolving AXinsol) cannot
contribute to increasing viscosity. This latter effect is essential
in some of the claimed applications.
[0048] Without wishing to be bound by theory, the enzyme of the
present invention mainly releases polymers, which do not contribute
to viscosity, because the released polymers are short.
[0049] For the first time, the present inventors have isolated and
sequenced this novel xylanase.
[0050] Typically, conventional xylanases may breakdown AXinsol, but
will often lead to an increase in viscosity of the mixture. This
increased viscosity is disadvantageous in many applications.
[0051] Without wishing to be bound by theory, although some
conventional xylanases breakdown AXinsol, they lead to an increase
in soluble degradation products of high molecular weight, which
leads to an increase in viscosity in the mixture.
[0052] Furthermore or alternatively and again without wishing to be
bound by theory, conventional xylanase enzymes may breakdown
AXinsol, but because they do not degrade the solubilised products
of high molecular weight fast enough the viscosity in the mixture
is not ideal. In contrast, with the methods and uses of the present
invention, the xylanases breakdown AXinsol without increasing
viscosity and/or whilst reducing viscosity quickly compared with
conventional enzymes. Without wishing to be being bound by theory,
it is believed that high molecular weight products are not formed
by the enzymes of the present invention.
[0053] The enzymes of the present invention and as described herein
have been found to not only breakdown (solubilise) insoluble
arabinoxylans (AXinsol) from a wide range of substrates, including
corn, wheat, DDGS, etc, in particular corn and corn-based
substrates, in particular both wheat (including wheat-based)
products and corn (including corn-based products), but also
efficiently ensuring that viscosity is not raised and/or reducing
viscosity. Without wishing to be being bound by theory, it is
believed that high molecular weight products are not formed by the
enzymes of the present invention.
[0054] Thus the present invention relates to enzymes capable of
solubilising pentosans, in particular xylan-containing materials,
such as arabinoxylans, in particular insoluble arabinoxylans. In
particular the enzyme is particularly good at solubilising
pentosans in particular xylan-containing materials, such as
arabinoxylans, in particular insoluble arabinoxylans, in a broad
spectrum of substrates, including corn based substrates.
[0055] The present invention further relates to enzymes capable of
degrading AXsol or the breakdown products of AXinsol to ensure
viscosity is not increased and/or is reduced in the reaction
mixture.
[0056] Many of the xylanases commercialized for use in feedstuffs
for solubilizing pentosans are GH11 enzymes. It had been considered
by those skilled in the art that GH10 xylanases were not as strong
at solublizing pentosans, particularly AXinsol, compared with GH11
xylanases.
[0057] Surprisingly it has been found that the novel xylanase
disclosed herein which is a GH10 xylanase is particularly good at
degrading AXinsol in a broad spectrum of substrates, including corn
based substrates. Surprisingly, the present inventors have found
that the GH10 xylanases of the present invention outperform
commercial GH11 xylanases in their ability to solubilize
pentosans.
[0058] The fact that the present enzymes efficiently degrade
AXinsol from corn and corn-based substrates is significantly
advantageous as corn holds much more AX in the insoluble form
compared with other cereals, such as wheat and rye for example.
Therefore only xylanases that can breakdown AXinsol can show
significant benefit to animals fed on corn-based diet, such as
corn-soy diet for example.
[0059] It was completely unexpected for a GH10 xylanase to be so
good on degrading AXinsol in cereals, particularly in corn or
corn-based substrates.
[0060] The enzymes of the present invention are able to efficiently
(and quickly) degrade the polymers and oligomers that are produced
from degradation of AXinsol or that are present in grain-based
material. This leads to an unexpected advantage for the GH10
xylanases taught herein in that they are particularly good in a
number of applications to keep viscosity low or to reduce
viscosity, e.g. in feedstuffs; in brewing and/or malting; in
grain-based production of glucose, e.g. for further processing to
biofuels and/or biochemicals (e.g. bio-based isoprene); or in the
wheat gluten-starch separation industry for the production of
starch for example.
[0061] Notably it has been found that the degradation product on
average is shorter for the GH10 enzymes tested herein compared with
GH11 enzymes. This means that the degradation products do not
contribute to or cause an increase in viscosity.
[0062] Based on these findings, the xylanases according to the
present invention can be used to degrade a xylan-containing
material, particularly arabinoxylans, particularly AXinsol. In
addition or alternatively, the xylanases according to the present
invention can be used to degrade soluble polymers (e.g. oligomers)
that are produced from degradation of AXinsol or that are
(naturally) present in grain-based materials. Surprisingly it has
been found that the xylanases according the present invention can
be used to both degrade a xylan-containing material, particularly
arabinoxylans, particularly AXinsol, and to degrade soluble
polymers (e.g. oligomers) that are produced from degradation of
AXinsol.
[0063] Such enzymes finds useful application in many industries,
including feedstuffs, malting and brewing, in the treatment of
arabinoxylan containing raw materials like grain-based materials,
in the wheat gluten-starch separation industry, in the production
of starch derived syrups, in biofuel production, and the like.
[0064] Only through the isolation and testing of FveXyn 4 (SEQ ID
No.3) were these surprising and unexpected technical effects found.
Thereafter it was possible to identify other similar acting enzymes
(e.g. SEQ ID No. 11 and/or SEQ ID No. 15).
STATEMENTS OF THE INVENTION
[0065] According to a first aspect the present invention provides a
xylanase comprising (or consisting of) a polypeptide sequence shown
herein as SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, or a variant,
homologue, fragment or derivative thereof having at least 98.5%
(e.g. at least 98.8 or 99 or 99.1 or 99.5%) identity with SEQ ID
No. 1 or SEQ ID No. 2 or SEQ ID No. 3; or a xylanase which is
encoded by a nucleotide sequence shown herein as SEQ ID No. 4, SEQ
ID No. 5 or SEQ ID No. 6, or a nucleotide sequence which has at
least 97.7% (e.g. at least 98%, 98.5% or 99%) identity with SEQ ID
No. 4, SEQ ID No. 5 or SEQ ID No. 6.
[0066] In a further aspect the present invention provides an
isolated or recombinant nucleic acid molecule comprising (or
consisting of) a polynucleotide sequence selected from the group
consisting of: [0067] a. a polynucleotide sequence which encodes a
polypeptide sequence selected from the group consisting of SEQ ID
No. 1, SEQ ID No. 2 or SEQ ID No. 3, or a variant, homologue,
fragment or derivative thereof having at least 98.5% (e.g. at least
98.8 or 99 or 99.1 or 99.5%) identity with SEQ ID No. 1 or SEQ ID
No. 2 or SEQ ID No. 3; or [0068] b. a polynucleotide sequence shown
herein as SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6; or a
nucleotide sequence which has at least 97.7% (e.g. at least 98%,
98.5% or 99%) identity with SEQ ID No. 4, SEQ ID No. 5 or SEQ ID
No. 6.
[0069] In one aspect of the present invention there is provided a
vector (e.g. a plasmid) comprising a polynucleotide sequence
selected from the group consisting of: [0070] a. a polynucleotide
sequence which encodes a polypeptide sequence selected from the
group consisting of SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, or
a variant, homologue, fragment or derivative thereof having at
least 98.4% (e.g. at least 98.5% or 98.8 or 99 or 99.1 or 99.5%)
identity with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3; or
[0071] b. a polynucleotide sequence shown herein as SEQ ID No. 4,
SEQ ID No. 5 or SEQ ID No. 6; or a nucleotide sequence which has at
least 97.7% (e.g. at least 98%, 98.5% or 99%) identity with SEQ ID
No. 4, SEQ ID No. 5 or SEQ ID No. 6.
[0072] A further aspect of the present invention provides a host
cell comprising a vector of the present invention or a nucleic acid
comprising (or consisting of) a nucleotide sequence shown herein as
SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6; or a nucleotide
sequence which has at least 97.7% (e.g. at least 98%, 98.5% or 99%)
identity with SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.
[0073] The present invention further relates to a method for
degrading arabinoxylan-containing material comprising admixing an
arabinoxylan-containing material with a xylanase, which xylanase is
a GH10, fungal xylanase and degrades insoluble arabinoxylan
(AXinsol) as well as degrading the polymers, oligomers or
combinations thereof produced from the degradation of the AXinsol,
and wherein the xylanase degrades the polymers, oligomers or
combinations thereof produced from the degradation of the AXinsol
immediately or substantially immediately upon their production.
[0074] The present invention further relates to a method of
degrading insoluble arabinoxylan-containing material comprising
admixing the material with a xylanase comprising (or consisting of)
a polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15, or a variant, homologue, fragment or derivative thereof
having at least 75% identity (such as at least 80%, 85%, 90%, 95%,
98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2, SEQ ID No.
3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or
a polypeptide sequence which comprises SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18; or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic
code.
[0075] The present invention further relates to a method for
producing glucose or starch (e.g. starch from wheat or glucose from
starch), comprising admixing a xylan-containing material
(preferably an arabinoxylan-containing material, such as an
insoluble arabinoxylan-containing material) with a xylanase
comprising (or consisting of) a polypeptide sequence shown herein
as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID
No. 10, SEQ ID No. 11, or SEQ ID No. 15, or a variant, homologue,
fragment or derivative thereof having at least 75% identity (such
as at least 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID
No. 1 or SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10,
SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide sequence which
comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,
SEQ ID No. 10, SEQ ID No. 11, or SEQ ID No. 15 with a conservative
substitution of at least one of the amino acids; or a xylanase
which is encoded by a nucleotide sequence shown herein as SEQ ID
No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13,
SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a
nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No.
5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ
ID No. 16, SEQ ID No.17 or SEQ ID No. 18 under high stringency
conditions, or a nucleotide sequence which has at least 75%
identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No.
5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.
14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the
degeneracy of the genetic code.
[0076] In one embodiment the xylan-containing material further
comprises starch.
[0077] Use of a xylanase comprising (or consisting of) a
polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15, or a variant, homologue, fragment or derivative thereof
having at least 75% identity (such as at least 80%, 85%, 90%, 95%,
98% or 99% identity) with SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3,
SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a
polypeptide sequence which comprises SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or, SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic code
for degrading a xylan-containing material (preferably an
arabinoxylan-containing material, preferably an insoluble
arabinoxylan-containing material), optionally without increasing
the viscosity or also decreasing the viscosity of the reaction
mixture.
[0078] According to another aspect of the present invention, there
is provided a method for improving the performance of a subject or
for improving digestibility of a raw material in a feed (e.g.
nutrient digestibility) or for improving feed efficiency in a
subject or for reducing the viscosity of the intestinal content,
the method comprising administering a subject with a xylanase
comprising (or consisting of) a polypeptide sequence shown herein
as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID
No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,
fragment or derivative thereof having at least 75% identity (such
as at least 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID
No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ
ID No. 11 or SEQ ID No. 15; or a polypeptide sequence which
comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,
SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a conservative
substitution of at least one of the amino acids; or a xylanase
which is encoded by a nucleotide sequence shown herein as SEQ ID
No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13,
SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a
nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No.
5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ
ID No. 16, SEQ ID No.17 or SEQ ID No. 18 under high stringency
conditions, or a nucleotide sequence which has at least 75%
identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No.
5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.
14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the
degeneracy of the genetic code or administering a subject with a
xylanase obtainable (or obtained) from Fusarium verticilloides.
[0079] A yet further aspect of the present invention is use of a
xylanase comprising (or consisting of) a polypeptide sequence shown
herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,
SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant,
homologue, fragment or derivative thereof having at least 75%
identity (such as at least 80%, 85%, 90%, 95%, 98% or 99% identity)
with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9
or SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide
sequence which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3,
SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a
conservative substitution of at least one of the amino acids; or a
xylanase which is encoded by a nucleotide sequence shown herein as
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID
No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18 under high stringency
conditions, or a nucleotide sequence which has at least 75%
identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No.
5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.
14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the
degeneracy of the genetic code or use of a xylanase obtainable (or
obtained) from Fusarium verticilioides for improving the
performance of a subject or for improving digestibility of a raw
material in a feed (e.g. nutrient digestibility) or for improving
feed efficiency in a subject or for reducing the viscosity of the
intestinal content.
[0080] In another aspect the present invention provides a feed
additive composition comprising (or consisting essentially of or
consisting of) a xylanase comprising (or consisting of) a
polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, or SEQ ID
No. 15 or a variant, homologue, fragment or derivative thereof
having at least 75% identity (such as at least 80%, 85%, 90%, 95%,
98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID
No. 3 or SEQ ID No. 9 or SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No.
15; or a polypeptide sequence which comprises SEQ ID No. 1, SEQ ID
No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or
SEQ ID No. 15 with a conservative substitution of at least one of
the amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic code;
or a xylanase obtainable (or obtained) from Fusarium
verticilloides.
[0081] In a further aspect the present invention provides a kit
comprising xylanase comprising (or consisting of) a polypeptide
sequence shown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3,
SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a
variant, homologue, fragment or derivative thereof having at least
75% identity (such as at least 80%, 85%, 90%, 95%, 98% or 99%
identity) with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ
ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or a
polypeptide sequence which comprises SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic code
and instructions for administration.
[0082] In a yet further aspect the present invention provides a
feedstuff comprising a feed additive composition comprising (or
consisting essentially of or consisting of) a xylanase comprising
(or consisting of) a polypeptide sequence shown herein as SEQ ID
No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ
ID No. 11 or SEQ ID No. 15, or a variant, homologue, fragment or
derivative thereof having at least 75% identity (such as at least
80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQ
ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID
No. 11 or SEQ ID No. 15; or a polypeptide sequence which comprises
SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No.
10, SEQ ID No. 11 or SEQ ID No. 15 with a conservative substitution
of at least one of the amino acids; or a xylanase which is encoded
by a nucleotide sequence shown herein as SEQ ID No. 4, SEQ ID No.
5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ
ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence
which can hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18 under high stringency conditions, or a
nucleotide sequence which has at least 75% identity (such as at
least 80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID
No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID
No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code, or a xylanase obtainable (or obtained) from
Fusarium verticilloides.
[0083] A premix comprising a feed additive composition comprising
(or consisting essentially of or consisting of) a xylanase
comprising (or consisting of) a polypeptide sequence shown herein
as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID
No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,
fragment or derivative thereof having at least 75% identity (such
as at least 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID
No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No.
10 or SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide sequence
which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID
No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a
conservative substitution of at least one of the amino acids; or a
xylanase which is encoded by a nucleotide sequence shown herein as
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID
No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18 under high stringency
conditions, or a nucleotide sequence which has at least 75%
identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No.
5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.
14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the
degeneracy of the genetic code; or a xylanase obtainable (or
obtained) from Fusarium verticilloides, and at least one mineral
and/or at least one vitamin.
[0084] In another aspect, the present invention provides a method
of preparing a feedstuff comprising admixing a feed component with
a feed additive composition comprising (or consisting essentially
of or consisting of) a xylanase comprising (or consisting of) a
polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15, or a variant, homologue, fragment or derivative thereof
having at least 75% identity (such as at least 80%, 85%, 90%, 95%,
98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID
No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID
No. 15; or a polypeptide sequence which comprises SEQ ID No. 1, SEQ
ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11
or SEQ ID No. 15 with a conservative substitution of at least one
of the amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic code,
or a xylanase obtainable (or obtained) from Fusarium
verticilloides.
[0085] According to another aspect of the present invention, there
is provided a method for degrading grain-based material (including
whole grains or partial grains or malted grains, e.g. malted
barley) and reducing viscosity in the reaction medium (e.g. grain
mash), the method comprising admixing said grain-based material
with a xylanase comprising (or consisting of) a polypeptide
sequence shown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3,
SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a
variant, homologue, fragment or derivative thereof having at least
75% identity (such as at least 80%, 85%, 90%, 95%, 98% or 99%
identity) with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ
ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or a
polypeptide sequence which comprises SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic code,
or a xylanase obtainable (or obtained) from Fusarium
verticilloides.
[0086] A yet further aspect of the present invention is use of a
xylanase comprising (or consisting of) a polypeptide sequence shown
herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,
SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant,
homologue, fragment or derivative thereof having at least 75%
identity (such as at least 80%, 85%, 90%, 95%, 98% or 99% identity)
with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9
or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or a xylanase
which is encoded by a nucleotide sequence shown herein as SEQ ID
No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13,
SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a
nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No.
5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ
ID No. 16, SEQ ID No.17 or SEQ ID No. 18 under high stringency
conditions, or a nucleotide sequence which has at least 75%
identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No.
5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.
14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the
degeneracy of the genetic code, or use of a xylanase obtainable (or
obtained) from Fusarium verticilloides for degrading grain-based
material (including whole grains or partial grains or malted
grains, e.g. malted barley) whilst reducing viscosity in the
reaction medium (e.g. grain mash).
[0087] According to another aspect of the present invention, there
is provided a method for producing biofuel (e.g. bioethanol) or a
biochemical (e.g. bio-based isoprene) comprising admixing a
grain-based material with a xylanase comprising (or consisting of)
a polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15, or a variant, homologue, fragment or derivative thereof
having at least 75% identity (such as at least 80%, 85%, 90%, 95%,
98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID
No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID
No. 15; or a polypeptide sequence which comprises SEQ ID No. 1, SEQ
ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11
or SEQ ID No. 15 with a conservative substitution of at least one
of the amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic code,
or a xylanase obtainable (or obtained) from Fusarium
verticilloides.
[0088] A yet further aspect of the present invention is use of a
xylanase comprising (or consisting of) a polypeptide sequence shown
herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,
SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant,
homologue, fragment or derivative thereof having at least 75%
identity (such as at least 80%, 85%, 90%, 95%, 98% or 99% identity)
with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9
or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or a xylanase
which is encoded by a nucleotide sequence shown herein as SEQ ID
No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13,
SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a
nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No.
5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ
ID No. 16, SEQ ID No.17 or SEQ ID No. 18 under high stringency
conditions, or a nucleotide sequence which has at least 75%
identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with
SEQ ID No. 4, SEQ ID No. 5, SEQ ID-No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No.
5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.
14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the
degeneracy of the genetic code, or use of a xylanase obtainable (or
obtained) from Fusarium verticilloides for producing biofuel (e.g.
bioethanol) or a biochemical (e.g. bio-based isoprene).
[0089] According to another aspect of the present invention, there
is provided a method for separating a cereal flour (e.g. wheat
flour) into starch and gluten fractions the method comprising
admixing a cereal flour (e.g. wheat flour), water and a xylanase
comprising (or consisting of) a polypeptide sequence shown herein
as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID
No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,
fragment or derivative thereof having at least 75% identity (such
as at least 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID
No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No.
10 or SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide sequence
which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID
No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a
conservative substitution of at least one of the amino acids; or a
xylanase which is encoded by a nucleotide sequence shown herein as
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID
No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18 under high stringency
conditions, or a nucleotide sequence which has at least 75%
identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No.
5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.
14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the
degeneracy of the genetic code, or a xylanase obtainable (or
obtained) from Fusarium verticilloides.
[0090] A yet further aspect of the present invention provides the
use of a xylanase comprising (or consisting of) a polypeptide
sequence shown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3,
SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a
variant, homologue, fragment or derivative thereof having at least
75% identity (such as at least 80%, 85%, 90%, 95%, 98% or 99%
identity) with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ
ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or a
polypeptide sequence which comprises SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No.6 r SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic code,
or use of a xylanase obtainable (or obtained) from Fusarium
verticilloides for separating a cereal flour (e.g. wheat flour)
into starch and gluten fractions whilst reducing viscosity in the
reaction medium (e.g. grain mash).
[0091] In a further aspect there is provided the use of the
xylanase comprising (or consisting of) a polypeptide sequence shown
herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,
SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant,
homologue, fragment or derivative thereof having at least 75%
identity (such as at least 80%, 85%, 90%, 95%, 98% or 99% identity)
with SEQ ID No. 1 or SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ
ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide
sequence which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3,
SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a
conservative substitution of at least one of the amino acids; or a
xylanase which is encoded by a nucleotide sequence shown herein as
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or
a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID
No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18 under high stringency
conditions, or a nucleotide sequence which has at least 75%
identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18; or
a nucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No.
5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.
14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the
degeneracy of the genetic code, in the production of a fermented
beverage, such as a beer.
[0092] In a yet further aspect, the present invention provides a
method of producing a fermented beverage (e.g. beer) comprising the
step of contacting a mash and/or a wort with a xylanase comprising
(or consisting of) a polypeptide sequence shown herein as SEQ ID
No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ
ID No. 11 or SEQ ID No. 15, or a variant, homologue, fragment or
derivative thereof having at least 75% identity (such as at least
80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQ
ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11
or SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID
No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ
ID No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
can hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID
No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17
or SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18; or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic
code.
[0093] A further aspect of the present invention provides a method
of producing a fermented beverage (e.g. beer) comprising the steps
of: (a) preparing a mash, (b) filtering the mash to obtain a wort,
and (c) fermenting the wort to obtain a fermented beverage, such as
a beer, wherein xylanase comprising (or consisting of) a
polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15, or a variant, homologue, fragment or derivative thereof
having at least 75% identity (such as at least 80%, 85%, 90%, 95%,
98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2, SEQ ID No.
3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or
a polypeptide sequence which comprises SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity (such as at least 80%,
85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18; or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic code,
is added to: (i) the mash of step (a) and/or (ii) the wort of step
(b) and/or (iii) the wort of step (c).
[0094] The present invention yet further provides a fermented
beverage, such as a beer, produced by a method of present
invention.
[0095] For the avoidance of doubt, SEQ ID No. 3 is the mature form
of SEQ ID No. 1 or SEQ ID No. 2.
[0096] Likewise, SEQ ID No. 11 is the mature form of SEQ ID No. 9
or SEQ ID No.10.
DETAILED DISCLOSURE OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0097] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale
& Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, NY (1991) provide one of skill with a general dictionary
of many of the terms used in this disclosure.
[0098] This disclosure is not limited by the exemplary methods and
materials disclosed herein, and any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of embodiments of this disclosure. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, any nucleic acid sequences are written left to right in
5' to 3' orientation; amino acid sequences are written left to
right in amino to carboxy orientation, respectively.
[0099] The headings provided herein are not limitations of the
various aspects or embodiments of this disclosure which can be had
by reference to the specification as a whole. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification as a whole.
[0100] Amino acids are referred to herein using the name of the
amino acid, the three letter abbreviation or the single letter
abbreviation.
[0101] The term "protein", as used herein, includes proteins,
polypeptides, and peptides.
[0102] As used herein, the term "amino acid sequence" is synonymous
with the term "polypeptide" and/or the term "protein". In some
instances, the term "amino acid sequence" is synonymous with the
term "peptide". In some instances, the term "amino acid sequence"
is synonymous with the term "enzyme".
[0103] The terms "protein" and "polypeptide" are used
interchangeably herein. In the present disclosure and claims, the
conventional one-letter and three-letter codes for amino acid
residues may be used. The 3-letter code for amino acids as defined
in conformity with the IUPACIUB Joint Commission on Biochemical
Nomenclature (JCBN). It is also understood that a polypeptide may
be coded for by more than one nucleotide sequence due to the
degeneracy of the genetic code.
[0104] Other definitions of terms may appear throughout the
specification. Before the exemplary embodiments are described in
more detail, it is to understand that this disclosure is not
limited to particular embodiments described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting, since the scope of the
present disclosure will be limited only by the appended claims.
[0105] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within this disclosure. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within this disclosure, subject to any specifically excluded limit
in the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in this disclosure.
[0106] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an enzyme" includes a plurality of such
candidate agents and reference to "the feed" includes reference to
one or more feeds and equivalents thereof known to those skilled in
the art, and so forth.
[0107] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
such publications constitute prior art to the claims appended
hereto.
[0108] Increasing prices of raw material traditionally used as
energy source in animal feed, as a feedstock in biofuel production,
as an ingredient in brewing or malting, or as a feedstock in wheat
gluten-starch separation processes for instance have resulted in
inclusion of low-cost fibrous materials in the starting substrates
for these industries, particularly the use of low-cost fibrous
by-products in animal feed.
[0109] Fibre addition may cause several disadvantageous effects.
For example in animal feed fibre addition may cause
anti-nutritional effects. The presence of un-degraded polymers
present in the animal's intestine causes a highly viscous content
and impeded diffusion with reduced nutrient absorption as a result.
Also, the polymers possess a high water holding capacity hindering
an effective re-absorption of water, and the water retention
increases the volume of the gut content, which leads to a decrease
intestinal transit time (Englyst & Kingman (1993) in Human
Nutrition and Dietetics, 9th edition (Garrow J. S., James W. P. T.,
eds.) p. 53).
[0110] In feedstuffs, hemicellulose and cellulose (including
insoluble arabinoxylan) also form physical barriers encapsulating
(or entrapping) nutrients like starch and protein and thereby
retaining access to these nutrients for the animal.
[0111] Hemicellulose and cellulose (including insoluble
arabinoxylans (AXinsol)) by themselves are also potential energy
sources, as they consist of C5- and C6-saccharides. Mono
C6-saccharides can be used as energy source by the animal, while
oligo C5-saccharides can be transformed into short chain fatty
acids by the micro flora present in the animal gut (van den Broek
et al., 2008 Molecular Nutrition & Food Research, 52, 146-63),
which short chain fatty acids can be taken up and digested by the
animal's gut.
[0112] Release of nutrients and water from feedstuffs as a
consequence of physical barrier degradation is dependent on the
ability of the xylanase to degrade insoluble fibre components (e.g.
insoluble arabanoxylans (AXinsol)).
[0113] In one aspect, the present invention relates to a novel
xylanase.
[0114] In one embodiment the present invention provides a xylanase
enzyme comprising (or consisting of) a polypeptide sequence shown
herein as SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or a variant,
homologue, fragment or derivative thereof having at least 98.4%
(e.g. at least 98.5 or 98.8 or 99 or 99.1 or 99.5%) identity with
SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3.
[0115] The present invention also relates to a xylanase which is
encoded by a nucleotide sequence shown herein as SEQ ID No. 4, SEQ
ID No. 5 or SEQ ID No. 6, or a nucleotide sequence which can
hybridize to SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 under high
stringency conditions, or a nucleotide sequence which has at least
97.7% (e.g. at least 98%, 98.5% or 99%) identity with SEQ ID No. 4,
SEQ ID No. 5 or SEQ ID No. 6.
[0116] In one aspect, the xylanase enzyme of the present invention
may be obtainable from (or obtained from) a fungus, namely Fusarium
verticilloides.
[0117] In another aspect, the present invention provides a xylanase
obtainable from (or obtained from) Fusarium verticilioides for use
in feed or a feed additive composition.
[0118] In one embodiment the xylanase enzyme of the present
invention may be referred to herein as FveXyn4 or Hifi168.
[0119] The present invention yet further provides a nucleic acid
comprising (or consisting of) a nucleotide sequence shown herein as
SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6; or a nucleotide
sequence which has at least 97.7% (e.g. at least 98%, 98.5% or 99%)
identity with SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.
[0120] Both the polypeptide sequences and the nucleic acid
sequences taught herein are preferably isolated.
[0121] The xylanase of the present invention is preferably a GH10
xylanase. In other words the xylanase may have a molecular weight
in the range of 32-39 kDa and/or the catalytic domain of the
xylanase consists of an eightfold p/a barrel structure (as taught
in Harris et al 1996--Acta. Crystallog. Sec. D 52, 393-401).
[0122] In one aspect, the present invention provides a composition
comprising a xylanase enzyme comprising (or consisting of) a
polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2 or
SEQ ID No. 3 or a variant, homologue, fragment or derivative
thereof having at least 98.5% (e.g. at least 98.8 or 99 or 99.1 or
99.5%) identity with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3
or a xylanase which is encoded by a nucleotide sequence shown
herein as SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6, or a
nucleotide sequence which has at least 97.7% (e.g. at least 98%,
98.5% or 99%) identity with SEQ ID No. 4, SEQ ID No. 5 or SEQ ID
No. 6.
[0123] In one aspect, the xylanase enzyme for use in the methods
and uses of the present invention comprises (or consists of) a
polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, or SEQ ID
No. 15 or a variant, homologue, fragment or derivative thereof
having at least 75% (suitably at least 85% or at least 90% or at
least 99%) identity with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No.
3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No.
15, or a polypeptide sequence which comprises SEQ ID No. 1, SEQ ID
No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or
SEQ ID No. 15 with a conservative substitution of at least one of
the amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which has at least
75% (suitably at least 85%, or at least 90% or at least 98%)
identity with SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18, or a nucleotide sequence which differs from SEQ ID
No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID
No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID
No. 18 due to the degeneracy of the genetic code.
[0124] In one aspect, the xylanase enzyme for use in the methods
and uses of the present invention comprises (or consists of) a
polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 or a variant, homologue, fragment or derivative thereof
having at least 85% identity with SEQ ID No. 1 or SEQ ID No. 2 or
SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or
SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No.
1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18 or a nucleotide sequence which
has at least 85% identity with SEQ ID No. 4, SEQ ID No. 5, SEQ ID
No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,
SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic
code.
[0125] In one aspect, the xylanase enzyme for use in the methods
and uses of the present invention comprises (or consists of) a
polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 or a variant, homologue, fragment or derivative thereof
having at least 90% identity with SEQ ID No. 1 or SEQ ID No. 2 or
SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or
SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No.
1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18 or a nucleotide sequence which
has at least 90% identity with SEQ ID No. 4, SEQ ID No. 5, SEQ ID
No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,
SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic
code.
[0126] In one aspect, the xylanase enzyme for use in the methods
and uses of the present invention comprises (or consists of) a
polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 or a variant, homologue, fragment or derivative thereof
having at least 99% identity with SEQ ID No. 1 or SEQ ID No. 2 or
SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or
SEQ ID No. 15, or a polypeptide sequence which comprises SEQ ID No.
1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
has at least 98% identity with SEQ ID No. 4, SEQ ID No. 5, SEQ ID
No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,
SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID
No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID
No.17 or SEQ ID No. 18 due to the degeneracy of the genetic
code.
[0127] In one embodiment the xylanase enzyme of the present
invention may be referred to herein as FveXyn4 (this term refers to
the active protein, e.g. the mature protein).
[0128] In one embodiment the xylanase enzyme of the present
invention may be referred to herein as FoxXyn2 (this term refers to
the active protein, e.g. the mature protein).
[0129] In one embodiment preferably the xylanase is a fungal
xylanase.
[0130] In one embodiment preferably the xylanase is a GH10
xylanase.
[0131] In one embodiment preferably the xylanase is a fungal GH10
xylanase.
[0132] In one embodiment preferably the xylanase in an
endoxylanase, e.g. an endo-1,4-.beta.-d-xylanase. The
classification for an endo-1,4-.beta.-d-xylanase is E.C.
3.2.1.8.
[0133] Preferably the xylanase of the present invention has an
optimum pH at about 6.
[0134] Preferably the xylanase of the present invention retains
greater than 70% of maximum activity between pH 4.6 and 7,
preferably between 5.1 and 7.
[0135] Without wishing to be bound by theory, pH may also have an
important effect on enzyme efficacy and efficiency. For feed
applications in particular the pH profile of the xylanases of the
present invention favour activity in the small intestine, under
neutral conditions.
[0136] Preferably the xylanase of the present invention has an
optimum temperature of about 60.degree. C.
[0137] Preferably the xylanase of the present invention retains
greater than 70% of maximum activity between 45.degree. C. and
64.degree. C.
[0138] In one embodiment, the xylanases according to the present
invention is capable of degrading (or degrades) a xylan-containing
material, particularly arabinoxylans, particularly isoluble
arabinoxylans (AXinsol).
[0139] In another embodiment the xylanases according to the present
invention is capable of degrading (or degrades) soluble polymers
(e.g. oligomers) that are produced from degradation of AXinsol or
that are (naturally) present in grain-based material.
[0140] In a further embodiment the xylanases according the present
invention is capable of degrading (or degrades) both a
xylan-containing material, particularly arabinoxylans, particularly
AXinsol, and soluble polymers (e.g. oligomers) that are produced
from degradation of AXinsol.
[0141] In one embodiment the xylanases of the present invention are
unaffected by wheat xylanases inhibitors, e.g. proteinaceous
inhibitors, e.g TAXI-like proteinaceous inhibitors in wheat. Prior
art fungal xylanases can be inhibited by as much as 70-95% by wheat
proteinaceous inhibitors. Preferably the xylanases of the present
invention are only inhibited by 20-30% at most in wheat
applications.
[0142] TAXI are Triticum aestivum xylanases inhibitors, present in
cereals.
[0143] The term "consisting essentially of" as used herein means
that unspecified components may be present if the characteristics
of the claimed composition are thereby not materially affected.
[0144] The term "consisting of" means that the proportions of the
specific ingredients must total 100%.
[0145] The term "comprising" used herein may be amended in some
embodiments to refer to consisting essentially of or consisting of
(both having a more limited meaning that "comprising").
[0146] In one embodiment the insoluble arabinoxylan containing
material is not wheat straw.
[0147] Uses
[0148] The xylanase of the present invention can be suitably used
in any one of the following applications:
[0149] a) An additive in animal feedstuffs; and/or
[0150] b) A feed supplement for an animal; and/or
[0151] c) Breakdown of grain-based material (e.g. this can be whole
grain or part of grain). The breakdown products (e.g. glucose) can
be used as a feedstock for any fermentation process, such as in
biofuel (e.g. bioethanol) production or in the production of other
products such as biochemicals (e.g., bio-based isoprene). Therefore
in one embodiment the present invention relates to the production
of biofuel (e.g. bioethanol) and to the enhanced utilisation of
grain-based material in the biofuel industry; and/or
[0152] c) Cereal (e.g. wheat) gluten-starch separation industry.
The resultant product(s) may be starch (e.g. purified starch)
and/or gluten and/or fibres and/or water solubles (such as soluble
pentosans). In one embodiment the present invention relates to the
production of starch and/or gluten; and/or
[0153] d) Improving malting and brewing, e.g. by breaking down
grain-based material (e.g. malted barley).
[0154] In one embodiment the xylanase of the present invention is
used in a feedstuff. Preferably a feedstuff comprising corn or is a
corn-based feedstuff.
[0155] In one embodiment the xylanase of the present invention is
used in malting or brewing.
[0156] In a further embodiment the xylanase of the present
invention is used in wheat gluten-starch separation.
[0157] In a yet further embodiment the xylanase of the present
invention is used in the breakdown of grain-based material and may
be part of the biofuel (e.g. bioethanol) production process.
[0158] For the avoidance of doubt, SEQ ID No. 3 is the mature form
of SEQ ID No. 1 or SEQ ID No. 2.
[0159] Likewise, SEQ ID No. 11 is the mature form of SEQ ID No. 9
or SEQ ID No.10.
[0160] Advantages
[0161] The novel xylanase taught herein has many advantages
compared with known xylanases.
[0162] The xylanases as taught herein and of the present invention
are unexpectedly good at solubilising pentosans.
[0163] The xylanases as taught herein and of the present invention
are unexpectedly good at solubilising AXinsol.
[0164] Surprisingly it has been found that the xylanase of the
present invention is particularly good at degrading
xylan-containing materials, such as arabinoxylans, e.g. AXinsol, in
a broad spectrum of substrates, corn, wheat, DDGS, etc, in
particular corn and corn based substrates, in particular both wheat
(including wheat-based) products and corn (including corn-based
products). Compared with the benchmark xylanases which are all
commercially produced and marketed xylanases, the novel xylanase
taught herein was capable of much more efficient degradation and
pentosan release from more plant based materials (in particular
corn-based substrates) compared with the marketed xylanases. This
was completely unexpected. This contrasts with prior-known enzymes,
which are often inferior at solubilising AXinsol in corn or
corn-based substrates or which are not as efficient in both wheat-
and corn-based substrates.
[0165] In addition, the enzyme of the present invention is
particularly good at not only breaking down (solubilising) AXinsol,
but also breaking down (or degrading) the solubilized polymers
efficiently. By being able to efficiently (quickly) breakdown
(degrade) the solubilized polymers (obtained from dissolving
AXinsol) a reduction in viscosity is obtained. This latter effect
is essential in some of the claimed applications.
[0166] Typically, conventional xylanases may breakdown AXinsol, but
will lead to an increase is the polymer production products which
will lead to an increase in viscosity of the mixture. This
increased viscosity is disadvantageous in many applications.
[0167] The enzymes of the present invention and as described herein
have been found to not only breakdown (solubilise) insoluble
arabinoxylans (AXinsol) from a wide range of substrates, including
corn, wheat, DDGS, etc, in particular corn and corn-based
substrates, in particular both wheat (including wheat-based)
products and corn (including corn-based products), but also
efficiently breakdown the thus solubilised polymers to ensure
viscosity is not raised and/or to reduce viscosity.
[0168] The xylanases of the present invention and as described
herein are capable of degrading AXsol or the breakdown products of
AXinsol to ensure viscosity is not increased and/or viscosity is
reduced in the reaction mixture.
[0169] Many of the xylanases commercialized for use in feedstuffs
for solubilizing pentosans are GH11 enzymes. It had been considered
by those skilled in the art that GH10 xylanases were not as strong
at solubilizing pentosans, particularly AXinsol, compared with GH11
xylanases. Surprisingly it has been found that the novel xylanase
disclosed herein which is a GH10 xylanase is particularly good at
solubilizing AXinsol in a broad spectrum of substrates, including
corn based substrates. Surprisingly, the present inventors have
found that the GH10 xylanases of the present invention (and taught
herein) outperform commercial GH11 xylanases in their ability to
solubilize pentosans.
[0170] The fact that the present enzymes efficiently solubilize
AXinsol from corn and corn-based substrates is significantly
advantageous as corn holds much more AX in the insoluble form
compared with other cereals, such as wheat and rye for example.
Therefore only xylanases that can breakdown AXinsol can show
significant benefit to animals fed on corn-soy diet for
example.
[0171] It was completely unexpected for a GH10 xylanase to be so
good on solubilizing AXinsol in cereals, particularly in corn or
corn-based substrates.
[0172] The enzymes of the present invention are able to efficiently
(and quickly) degrade the polymers and/or oligomers that are
produced from solubilisation of AXinsol or that are present in
grain-based materials. This leads to an unexpected advantage for
the GH10 xylanases taught herein in that they are particularly good
in a number of applications to keep viscosity low or to reduce
viscosity, e.g. in feedstuffs; in brewing and/or malting; in
grain-based production of glucose, e.g. for further processing to
biofuels and/or biochemicals (e.g., bio-based isoprene); or in the
wheat gluten-starch separation industry for the production of
starch for example.
[0173] Notably it has been found that the degradation product on
average is shorter for the GH10 enzymes tested herein compared with
GH11 enzymes. This enhances the lowering of viscosity effect.
[0174] In addition, a further advantage of the the GH10 xylanases
of the present invention (unlike many GH11 xylanases) are
unaffected by wheat xylanase inhibitors, e.g., TAXI like
proteinaceous inhibitors, which occur in wheat.
[0175] One advantage of the present invention is that it improves
wheat gluten-starch separation.
[0176] The enzyme of the present invention is particularly
effective at enhancing the performance of a subject or improving
the digestibility of a raw material in a feed and/or for improving
feed efficiency in a subject.
[0177] Xylan-Containing Material
[0178] The xylanase of the present invention (or composition
comprising the xylanase of the present invention) maybe used to
degrade any xylan-containing material.
[0179] In one embodiment the xylan-containing material is any plant
material comprising arabinoxylan.
[0180] In one embodiment the xylan-containing material is any plant
material comprising insoluble arabinoxylan (AXinsol).
[0181] In one embodiment the xylan-containing material is a
feedstuff or feed component.
[0182] In one embodiment the xylan-containing material is a
grain-based material (including whole grains or partial grains or
malted grains, e.g. malted barley). When the method relates to
biofuel production (e.g. bioethanol production) then preferably the
xylan-containing material is a grain-based material.
[0183] In another embodiment the xylan-containing material may be a
barley malt or mash, or malted barley or combinations thereof.
[0184] In a yet further embodiment the xylan-containing material
may be a cereal flour (e.g. wheat, oat, rye or barley flour). When
the method relates to a gluten-starch separation process preferably
the xylan-containing material is a cereal flour (e.g. wheat oat,
rye or barley flour).
[0185] Breakdown or Degradation
[0186] The enzyme (or composition comprising the enzyme) of the
present invention or as disclosed herein may be used to breakdown
(degrade) AXinsol or AXsol or degradation products of AXinsol.
[0187] The term "breakdown" or "degrade" in synonymous with
hydrolyses.
[0188] Solubilisation/Degradation
[0189] The present invention relates to a method of degrading a
xylan-containing material (preferably an arabinoxylan-containing
material, preferably an insoluble arabinoxylan (AXinsol)-containing
material) to produce soluble pentosans (which can be polymeric,
oligomeric or monomeric).
[0190] This method may be described herein as pentosan
solubilisation or arabinoxylan solubilisation or AXinsol
solubilisation or degradation of AXinsol.
[0191] In one embodiment, the present invention relates to a method
of degrading (or breaking down) insoluble arabinoxylan (AXinsol).
This can also be referred to as solubilisation of insoluble
arabinoxylan and/or solubilisation of pentosans.
[0192] In a further embodiment of the present invention the method
relates to degrading (e.g. breaking down) polymers derived from the
degradation of insoluble arabinoxylans.
[0193] Arabinoxylan (AX)
[0194] The term "arabinoxylans" (AX) as used herein means a
polysaccharide consisting of a xylan backbone (1,4-linked xylose
units) with L-arabinofuranose (L-arabinose in its 5-atom ring form)
attached randomly by 1a.fwdarw.2 and/or 1a.fwdarw.3 linkages to the
xylose units throughout the chain. Arabinoxylan is a hemicellulose
found in both the primary and secondary cell walls of plants.
Arabinoxylan can be found in the bran of grains such as wheat,
maize (corn), rye, and barley.
[0195] Arabinoxylan (AX) is found in close association with the
plant cell wall, where it acts as a glue linking various building
blocks of the plant cell wall and tissue, give it both structural
strength and rigidity.
[0196] The term "pentosan" as used herein is any of a group of
carbohydrates which yield pentoses on complete hydrolysis.
[0197] Since xylose and arabinose (the constituents of
arabinoxylans) are both pentoses, arabinoxylans are usually
classified as pentosans.
[0198] AX is the principal Non Starch Polysaccharide (NSP)-fraction
in several of the most important feed raw material, including wheat
and corn.
[0199] Its abundance, location within vegetable material and
molecular structure cause AX to have a severe, negative impact on
feed digestibility, effectively reducing the nutritional value of
the raw materials in which it is present. This makes AX an
important anti-nutritional factor, reducing animal production
efficiency.
[0200] In addition AX can have a severe, negative impact when
trying to breakdown plant material for example in processes such as
brewing, malting, biofuel manufacture, effectively reducing the
amount of substrate accessible in the raw plant material.
[0201] AXs can also hold substantial amounts of water (which can be
referred to as their water holding capacity)--this can cause
soluble arabinoxylans to result in (high) viscosity--which is a
disadvantage in many applications.
[0202] The term "Hemicellulose"--as used herein means the
polysaccharide components of plant cell walls other than cellulose.
The term "hemicellulose" as used herein may mean polysaccharides in
plant cell walls which are extractable by dilute alkaline
solutions. Hemicelluloses comprise almost one-third of the
carbohydrates in woody plant tissue. The chemical structure of
hemicelluloses consists of long chains of a variety of pentoses,
hexoses, and their corresponding uronic acids. Hemicelluloses may
be found in fruit, plant stems, and grain hulls. Xylan is an
example of a pentosan consisting of D-xylose units with
1.beta..fwdarw.4 linkages.
[0203] Water Insoluble Arabinoxylan (AXinsol)
[0204] Water-insoluble arabinoxylan (AXinsol) also known as
water-unextractable arabinoxylan (WU-AX) constitutes a significant
proportion of the dry matter of plant material.
[0205] In wheat AXinsol can account for 6.3% of the dry matter. In
wheat bran and wheat DDGS AXinsol can account for about 20.8% or
13.4% of the dry matter (w/w).
[0206] In rye AXinsol can account for 5.5% of the dry matter.
[0207] In corn AXinsol can account for 3.5-6% (e.g. 5.1%) of the
dry matter. In corn DDGS AXinsol can account for 10-20% (e.g.
12.6%) of the dry matter.
[0208] AXinsol causes nutrient entrapment in feed. Large quantities
of well digestible nutrients such as starch and proteins remain
either enclosed in clusters of cell wall material or bound to side
chains of the AX. These entrapped nutrients will not be available
for digestion and subsequent absorption in the small intestine.
[0209] Water-Soluble Arabinoxylan (AXsol)
[0210] Water-soluble arabinoxylan (AXso) also known as water
extractable arabinoxylan (WE-AX) can cause problems in biofuel
production and/or malting and/or brewing and/or in feed as they can
cause increased viscosity due to the water-binding capacity of
AXsol.
[0211] In feed AXsol can have an anti-nutritional effect
particularly in monogastrics as they cause a considerable increase
of the viscosity of the intestinal content, caused by the
extraordinary water-binding capacity of AXsol. The increase
viscosity can affect feed digestion and nutrient use as it can
prevent proper mixing of feed with digestive enzymes and bile salts
and/or it slows down nutrient availability and absorption and/or it
stimulates fermentation in the hindgut.
[0212] In wheat AXsol can account for 1.8% of the dry matter. In
wheat bran and wheat DDGS AXsol can account for about 1.1% or 4.9%
of the dr matter (w/w).
[0213] In rye AXsol can account for 3.4% of the dry matter.
[0214] In barley AXsol can account for 0.4-0.8% of the dry
matter.
[0215] In corn AXsol can account for 0.1-0.4% (e.g. 0.1%) of the
dry matter. In corn DDGS AXinsol can account for 0.3-2.5% (e.g.
0.4%) of the dry matter.
[0216] In addition, however, to the amount of AXsol present in
plant material, when a xylanase solubilises AXinsol in the plant
material this can release pentosans and/or oligomers which
contribute to AXsol content of the plant material.
[0217] One significant advantage of the xylanases disclosed herein
is that they have the ability to solubilise AXinsol without
increasing viscosity. It is presently believed that high molecular
weight products are not formed A breakdown of AXsol can decrease
viscosity.
[0218] A breakdown of AXsol can release nutrients.
[0219] Viscosity
[0220] The present invention can be used to ensure that the
viscosity is not increased and/or to reduce viscosity in any
process where the water-binding capacity of AXsol causes an
undesirable increase in viscosity.
[0221] The present invention relates to ensuring that viscosity is
not increased and/or to reducing viscosity by breaking down
(degrading) AXsol or by breaking down (degrading) the polymers
and/or oligomers produced by solubilising AXinsol.
[0222] Without wishing to be bound by theory, by being able to
efficiently (quickly) breakdown (degrade) the solubilized polymers
(e.g. oligomers) obtained from dissolving AXinsol an undesirable
increase in viscosity can be avoided and/or a reduction in
viscosity can be obtained. The term "efficiently" as used herein
means that the enzyme is capable of degrading the polymers (e.g.
oligomers) being formed by solubilisation of the AXinsol faster
than the speed with which the AXinsol is degraded (or
solubilized).
[0223] Reducing viscosity has advantages in many applications as
taught herein.
[0224] An in vitro assay which attempts to mimic the environment in
the small intestine of a chicken was originally described by
Bedford & Classen (1993 Poultry Sci., 72, 137-143). The assay
consists of a two steps incubation of the feed first at low pH with
pepsin followed by incubation with pancreatin at neutral pH. It is
generally accepted that the viscosity of the supernatant after end
incubation correlates with the viscosity created in vivo in
broilers.
[0225] Without increasing viscosity and/or a reduction in viscosity
as taught herein for feed applications means that addition of the
xylanase will result in an unchanged or lower viscosity measured by
the method described in Example 7. By unchanged it is meant that
the measured value, being the average of three repetitions, falls
within two standard deviation of the measured value for a wheat
sample without xylanase addition.
[0226] Viscosity can be measured using the following devices: Rapid
ViscoAnalyzer (RVA) (e.g in bioethanol processing) and Haake VT550
viscometer (Thermofisher) (e.g. is wheat-gluten starch processing).
Both devices can monitor viscosity profiles of fuel ethanol
processes and wheat starch separation processes, of which the
experimental conditions are taught in Example 8 and 9,
respectively.
[0227] In the present invention a reduction in viscosity can be
calculated by comparing one sample comprising the xylanase of the
present invention (or taught herein) compared with another
comparable sample without the xylanase of the present invention (or
taught herein).
[0228] Comparing the viscosity reduction profiles of the xylanase
of the present invention with those of the market benchmark
xylanase(s) demonstrates the enzyme performance. The aim is to
improve enzyme performance compared to the market benchmark. The
benchmark enzyme(s) for the individual applications are provided in
the examples below.
[0229] The benchmark enzyme for feed applications is
Econase.RTM.XT.
[0230] The benchmark enzyme for bioethanol processing is
Xylathin.TM..
[0231] The benchmark enzyme for wheat-gluten starch separation is
Shearzyme Plus.TM. In one embodiment of the present invention the
xylanases taught herein are viscosity reducers.
[0232] Generally, wheat (or other cereal) is first dry-milled to
separate the bran and germ from the endosperm, which is ground into
flour. This endosperm flour is then further fractionated through a
wheat starch separation process into several product streams of
varying commercial value. The major aim is to produce a refined
grade of A-starch, consisting of large, lenticular granules of
15-40 .mu.m. The second stream B-starch consists of less purified
starch granules, which are spherical and small (1-10 .mu.m). (C. C.
Maningat, P. A. Seib, S. D. Bassi, K. S. Woo, G. D. Lasater,
Chapter 10 from the book "Starch" (2009) 441-451, Wheat starch:
production, properties, modification and uses). Isolated wheat
starch forms the starting material for modified starch production
with applications in both food- and nonfood-applications. Vital
gluten is the third product of added-value in wheat separation
processes.
[0233] The vitality of the isolated wheat gluten is determined by
the ability to form viscoelastic networks, required for
breadmaking. Vital gluten encapsulate the carbon dioxide formed in
dough preparation during baking, and consequently increase the
bread volume. (Anne van der Borght, Hans Goesaert, Wim S.
Veraverbeke, Jan A. Delcour, Journal of Cereal Science 41 (2005)
221-237, Fractionation of wheat and wheat flour into starch and
gluten: overview of the main processes and the factors involved.)
It is therefore often used to enrich flours for bread making, to
achieve improved bread products. Other markets for gluten include
as an additive in vegetarian, meat, fish or poultry products,
including those in pet-food industry; in cereal breakfast; or in
soy sauce. Due to its thermoplasticity and good film-forming
properties, gluten is also used in non-food markets as adhesives.
(L. Day, M. A. Augustin, I. L. Batey, C. W. Wrigley, Trends in Food
Science &Technology 17 (2006) 82-90, Wheat-gluten uses and
industry needs.).
[0234] The xylanases taught herein can be used to reduce the
viscosity (or not increase viscosity) in processes for separating
cereal flour (e.g. wheat, oat, rye or barley flour) into starch and
gluten fractions and to improve the separation by degrading
oligosaccharides that hinder gluten agglomeration.
[0235] Wort viscosity, and the viscosity of barley mash and barley
malt in brewing and malting can cause significant disadvantages
during brewing and/or malting. The present invention relates to
reducing the viscosity (or not increase the viscosity) of wort,
barley mash, barley malt or a combination thereof.
[0236] Feed or Feedstuff
[0237] The enzyme or feed additive composition of the present
invention may be used as--or in the preparation of--a feed.
[0238] The term "feed" is used synonymously herein with
"feedstuff".
[0239] Preferably the arabinoxylan-containing material of the
present invention is a feedstuff, or a constituent of a feedstuff,
or a feed component.
[0240] The feed may be in the form of a solution or as a solid or
as a semi-solid--depending on the use and/or the mode of
application and/or the mode of administration.
[0241] When used as--or in the preparation of--a feed--such as
functional feed--the enzyme or composition of the present invention
may be used in conjunction with one or more of: a nutritionally
acceptable carrier, a nutritionally acceptable diluent, a
nutritionally acceptable excipient, a nutritionally acceptable
adjuvant, a nutritionally active ingredient.
[0242] In a preferred embodiment the enzyme or feed additive
composition of the present invention is admixed with a feed
component to form a feedstuff.
[0243] The term "feed component" as used herein means all or part
of the feedstuff. Part of the feedstuff may mean one constituent of
the feedstuff or more than one constituent of the feedstuff, e.g. 2
or 3 or 4. In one embodiment the term "feed component" encompasses
a premix or premix constituents.
[0244] Preferably the feed may be a fodder, or a premix thereof, a
compound feed, or a premix thereof. In one embodiment the feed
additive composition according to the present invention may be
admixed with a compound feed, a compound feed component or to a
premix of a compound feed or to a fodder, a fodder component, or a
premix of a fodder.
[0245] The term "fodder" as used herein means any food which is
provided to an animal (rather than the animal having to forage for
it themselves). Fodder encompasses plants that have been cut.
[0246] The term fodder includes silage, compressed and pelleted
feeds, oils and mixed rations, and also sprouted grains and
legumes.
[0247] Fodder may be obtained from one or more of the plants
selected from: corn (maize), alfalfa (Lucerne), barley, birdsfoot
trefoil, brassicas, Chau moellier, kale, rapeseed (canola),
rutabaga (swede), turnip, clover, alsike clover, red clover,
subterranean clover, white clover, fescue, brome, millet, oats,
sorghum, soybeans, trees (pollard tree shoots for tree-hay), wheat,
and legumes.
[0248] The term "compound feed" means a commercial feed in the form
of a meal, a pellet, nuts, cake or a crumble. Compound feeds may be
blended from various raw materials and additives. These blends are
formulated according to the specific requirements of the target
animal.
[0249] Compound feeds can be complete feeds that provide all the
daily required nutrients, concentrates that provide a part of the
ration (protein, energy) or supplements that only provide
additional micronutrients, such as minerals and vitamins.
[0250] The main ingredients used in compound feed are the feed
grains, which include corn, wheat, canola meal, rapeseed meal,
lupin, soybeans, sorghum, oats, and barley.
[0251] Suitably a premix as referred to herein may be a composition
composed of microingredients such as vitamins, minerals, chemical
preservatives, antibiotics, fermentation products, and other
essential ingredients. Premixes are usually compositions suitable
for blending into commercial rations.
[0252] Any feedstuff of the present invention may comprise one or
more feed materials selected from the group comprising a) cereals,
such as small grains (e.g., wheat, barley, rye, oats, triticale and
combinations thereof) and/or large grains such as maize or sorghum;
b) by products from cereals, such as corn gluten meal, wet-cake
(particularly corn based wet-cake), Distillers Dried Grain (DDG)
(particularly corn based Distillers Dried Grain (cDDG)), Distillers
Dried Grain Solubles (DDGS) (particularly corn based Distillers
Dried Grain Solubles (cDDGS)), wheat bran, wheat middlings, wheat
shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus
pulp; c) protein obtained from sources such as soya, sunflower,
peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried
plasma protein, meat and bone meal, potato protein, whey, copra,
sesame; d) oils and fats obtained from vegetable and animal
sources; e) minerals and vitamins.
[0253] In one embodiment the feedstuff comprises or consists of
corn, DDGS (such as cDDGS), wheat, wheat bran or a combination
thereof.
[0254] In one embodiment the feed component may be corn, DDGS (e.g.
cDDGS), wheat, wheat bran or a combination thereof.
[0255] In one embodiment the feedstuff comprises or consists of
corn, DDGS (such as cDDGS) or a combination thereof.
[0256] In one embodiment a feed component may be corn, DDGS (such
as cDDGS) or a combination thereof.
[0257] A feedstuff of the present invention may contain at least
30%, at least 40%, at least 50% or at least 60% by weight corn and
soybean meal or corn and full fat soy, or wheat meal or sunflower
meal.
[0258] A feedstuff of the present invention may contain between
about 5 to about 40% corn DDGS.
[0259] For poultry--the feedstuff on average may contain between
about 7 to 15% corn DDGS. For swine (pigs)--the feedstuff may
contain on average 5 to 40% corn DDGS.
[0260] A feedstuff of the present invention may contain corn as a
single grain, in which case the feedstuff may comprise between
about 35% to about 80% corn.
[0261] In feedstuffs comprising mixed grains, e.g. comprising corn
and wheat for example, the feedstuff may comprise at least 10%
corn.
[0262] In addition or in the alternative, a feedstuff of the
present invention may comprise at least one high fibre feed
material and/or at least one by-product of the at least one high
fibre feed material to provide a high fibre feedstuff. Examples of
high fibre feed materials include: wheat, barley, rye, oats, by
products from cereals, such as corn gluten meal, corn gluten feed,
wet-cake, Distillers Dried Grain (DDG), Distillers Dried Grain
Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice
bran, rice hulls, oat hulls, palm kernel, and citrus pulp. Some
protein sources may also be regarded as high fibre: protein
obtained from sources such as sunflower, lupin, fava beans and
cotton.
[0263] In one embodiment the feedstuff of the present invention
comprises at least one high fibre material and/or at least one
by-product of the at least one high fibre feed material selected
from the group consisting of Distillers Dried Grain Solubles
(DDGS)--particularly cDDGS, wet-cake, Distillers Dried Grain
(DDG)--particularly cDDG, wheat bran, and wheat for example.
[0264] In one embodiment the feedstuff of the present invention
comprises at least one high fibre material and/or at least one
by-product of the at least one high fibre feed material selected
from the group consisting of Distillers Dried Grain Solubles
(DDGS)--particularly cDDGS, wheat bran, and wheat for example.
[0265] In the present invention the feed may be one or more of the
following: a compound feed and premix, including pellets, nuts or
(cattle) cake; a crop or crop residue: corn, soybeans, sorghum,
oats, barley copra, straw, chaff, sugar beet waste; fish meal; meat
and bone mea; molasses; oil cake and press cake; oligosaccharides;
conserved forage plants: silage; seaweed; seeds and grains, either
whole or prepared by crushing, milling etc.; sprouted grains and
legumes; yeast extract.
[0266] The term "feed" in the present invention encompasses in some
embodiments pet food. A pet food is plant or animal material
intended for consumption by pets, such as dog food or cat food. Pet
food, such as dog and cat food, may be either in a dry form, such
as kibble for dogs, or wet canned form. Cat food may contain the
amino acid taurine.
[0267] The term "feed" in the present invention encompasses in some
embodiments fish food. A fish food normally contains macro
nutrients, trace elements and vitamins necessary to keep captive
fish in good health. Fish food may be in the form of a flake,
pellet or tablet. Pelleted forms, some of which sink rapidly, are
often used for larger fish or bottom feeding species.
[0268] Some fish foods also contain additives, such as beta
carotene or sex hormones, to artificially enhance the color of
ornamental fish.
[0269] The term "feed" in the present invention encompasses in some
embodiment bird food. Bird food includes food that is used both in
birdfeeders and to feed pet birds. Typically bird food comprises of
a variety of seeds, but may also encompass suet (beef or mutton
fat).
[0270] As used herein the term "contacted" refers to the indirect
or direct application of the enzyme (or composition comprising the
enzyme) of the present invention to the product (e.g. the feed).
Examples of the application methods which may be used, include, but
are not limited to, treating the product in a material comprising
the feed additive composition, direct application by mixing the
feed additive composition with the product, spraying the feed
additive composition onto the product surface or dipping the
product into a preparation of the feed additive composition.
[0271] In one embodiment the feed additive composition of the
present invention is preferably admixed with the product (e.g.
feedstuff). Alternatively, the feed additive composition may be
included in the emulsion or raw ingredients of a feedstuff.
[0272] For some applications, it is important that the composition
is made available on or to the surface of a product to be
affected/treated. This allows the composition to impart one or more
of the following favourable characteristics: performance
benefits.
[0273] The enzyme (or composition comprising the enzyme) of the
present invention may be applied to intersperse, coat and/or
impregnate a product (e.g. feedstuff or raw ingredients of a
feedstuff) with a controlled amount of said enzyme.
[0274] Preferably, the enzyme (or composition comprising the
enzyme) of the present invention will be thermally stable to heat
treatment up to about 70.degree. C.; up to about 85.degree. C.; or
up to about 95.degree. C. The heat treatment may be performed for
up to about 1 minute; up to about 5 minutes; up to about 10
minutes; up to about 30 minutes; up to about 60 minutes. The term
thermally stable means that at least about 75% of the enzyme that
was present/active in the additive before heating to the specified
temperature is still present/active after it cools to room
temperature. Preferably, at least about 80% of the enzyme that is
present and active in the additive before heating to the specified
temperature is still present and active after it cools to room
temperature.
[0275] In a particularly preferred embodiment the enzyme (or
composition comprising the enzyme) of the present invention is
homogenized to produce a powder.
[0276] In an alternative preferred embodiment, the enzyme (or
composition comprising the enzyme) of the present invention is
formulated to granules as described in WO2007/044968 (referred to
as TPT granules) or WO1997/016076 or WO1992/012645 incorporated
herein by reference.
[0277] In another preferred embodiment when the feed additive
composition is formulated into granules the granules comprise a
hydrated barrier salt coated over the protein core. The advantage
of such salt coating is improved thermo-tolerance, improved storage
stability and protection against other feed additives otherwise
having adverse effect on the enzyme.
[0278] Preferably, the salt used for the salt coating has a water
activity greater than 0.25 or constant humidity greater than 60% at
20.degree. C.
[0279] Preferably, the salt coating comprises a
Na.sub.2SO.sub.4.
[0280] The method of preparing an enzyme (or composition comprising
the enzyme) of the present invention may also comprise the further
step of pelleting the powder. The powder may be mixed with other
components known in the art. The powder, or mixture comprising the
powder, may be forced through a die and the resulting strands are
cut into suitable pellets of variable length.
[0281] Optionally, the pelleting step may include a steam
treatment, or conditioning stage, prior to formation of the
pellets. The mixture comprising the powder may be placed in a
conditioner, e.g. a mixer with steam injection. The mixture is
heated in the conditioner up to a specified temperature, such as
from 60-100.degree. C., typical temperatures would be 70.degree.
C., 80.degree. C., 85.degree. C., 90.degree. C. or 95.degree. C.
The residence time can be variable from seconds to minutes and even
hours.
[0282] Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1
minutes 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes
and 1 hour.
[0283] It will be understood that the enzyme (or composition
comprising the enzyme) of the present invention is suitable for
addition to any appropriate feed material.
[0284] It will be understood by the skilled person that different
animals require different feedstuffs, and even the same animal may
require different feedstuffs, depending upon the purpose for which
the animal is reared.
[0285] Optionally, the feedstuff may also contain additional
minerals such as, for example, calcium and/or additional
vitamins.
[0286] Preferably, the feedstuff is a corn soybean meal mix.
[0287] In one embodiment, preferably the feed is not pet food.
[0288] In another aspect there is provided a method for producing a
feedstuff. Feedstuff is typically produced in feed mills in which
raw materials are first ground to a suitable particle size and then
mixed with appropriate additives. The feedstuff may then be
produced as a mash or pellets; the later typically involves a
method by which the temperature is raised to a target level and
then the feed is passed through a die to produce pellets of a
particular size. The pellets are allowed to cool. Subsequently
liquid additives such as fat and enzyme may be added. Production of
feedstuff may also involve an additional step that includes
extrusion or expansion prior to pelleting--in particular by
suitable techniques that may include at least the use of steam.
[0289] The feedstuff may be a feedstuff for a monogastric animal,
such as poultry (for example, broiler, layer, broiler breeders,
turkey, duck, geese, water fowl), and swine (all age categories), a
ruminant such as cattle (e.g. cows or bulls (including calves)),
horses, sheep, a pet (for example dogs, cats) or fish (for example
agastric fish, gastric fish, freshwater fish such as salmon, cod,
trout and carp, e.g. koi carp, marine fish such as sea bass, and
crustaceans such as shrimps, mussels and scallops). Preferably the
feedstuff is for poultry.
[0290] Corn Based Feedstuff
[0291] In a preferred embodiment the feedstuff may be a corn based
feedstuff. The term "corn based feedstuff" as used herein means a
feedstuff which comprises or consists of corn (maize) or a
by-product of corn.
[0292] Preferably the corn based feedstuff comprises corn or a
by-product of corn as the major constituent. For example the corn
based feedstuff may comprise at least 35% corn or a by-product of
corn, such as at least 40% corn or a by-product of corn, such as at
least 50% corn or a by-product of corn, such as at least 60% corn
or a by-product of corn, such as at least 70% corn or a by-product
of corn, such as at least 80% or a by-product of corn, such as at
least 90% corn or a by-product of corn, for example 100% corn or a
by-product of corn.
[0293] In some embodiments the corn based feedstuff may comprise
corn or a by-product of corn as a minor constituent; in which case
the feedstuff may be supplemented with corn or a by-product of
corn. By way of example only the feedstuff may comprise for example
wheat supplemented with corn or a by-product of corn.
[0294] When corn or the by-product of corn is a minor constituent
of the feedstuff, the corn or by-product of corn is at least 5%,
preferably at least 10%, preferably at least 20%, preferably at
least 30% of the feedstuff.
[0295] For the avoidance of doubt the term "corn" as used herein is
synonymous with maize, e.g. Zea mays.
[0296] In one embodiment the by-product of corn may be corn
Distillers Dried Grain Solubles (cDDGS) or corn wet-cake or corn
Distillers Dried Grain (DDG) or corn gluten meal or corn gluten
feed or combinations thereof.
[0297] In one embodiment preferably the arabinoxylan-containing
material of the present invention comprises a by-product of corn,
such as corn Distillers Dried Grain Solubles (cDDGS) or corn
wet-cake or corn Distillers Dried Grain (DDG) or corn gluten meal
or corn gluten feed or combinations thereof.
[0298] Wheat Based Feedstuff
[0299] In a preferred embodiment the feedstuff may be a wheat based
feedstuff. The term "wheat based feedstuff" as used herein means a
feedstuff which comprises or consists of wheat or a by-product of
wheat.
[0300] Preferably the wheat based feedstuff comprises wheat or a
by-product of wheat as the major constituent. For example the wheat
based feedstuff may comprise at least 40% wheat or a by-product of
wheat, such as at least 60% wheat or a by-product of wheat, such as
at least 80% or a by-product of wheat, such as at least 90% wheat
or a by-product of wheat, for example 100% wheat or a by-product of
wheat.
[0301] In some embodiments the wheat based feedstuff may comprise
wheat or a by-product of wheat as a minor constituent; in which
case the feedstuff may be supplemented with wheat or a by-product
of wheat. By way of example only the feedstuff may comprise for
example wheat supplemented with wheat or a by-product of wheat.
[0302] When wheat or the by-product of wheat is a minor constituent
of the feedstuff, the wheat or by-product of wheat is at least 5%,
preferably at least 10%, preferably at least 20%, preferably at
least 30% of the feedstuff.
[0303] In one embodiment the by-product of wheat may be wheat bran,
wheat middlings, wheat fibres for example.
[0304] Bran is the hard outer layer of grain and consists of
combined aleurone and pericarp. Along with germ, it is an integral
part of whole grains, and is often produced as a by-product of
milling in the production of refined grains. When bran is removed
from grains, the grains lose a portion of their nutritional value.
Bran is present in and may be milled from any cereal grain,
including rice, corn (maize), wheat, oats, barley and millet. Bran
is particularly rich in dietary fiber and essential fatty acids and
contains significant quantities of starch, protein, vitamins and
dietary minerals.
[0305] Wheat middlings is coarse and fine particles of wheat bran
and fine particles of wheat shorts, wheat germ, wheat flour and
offal from the "tail of the mill".
[0306] Wheat middlings is an inexpensive by-product intermediate of
human food and animal feed.
[0307] In one embodiment preferably the arabinoxylan-containing
material of the present invention comprises wheat bran and/or wheat
middlings.
[0308] Wet-Cake, Distillers Dried Grains (DDG) and Distillers Dried
Grain Solubles (DDGS)
[0309] Wet-cake, Distillers Dried Grains and Distillers Dried
Grains with Solubles are products obtained after the removal of
ethyl alcohol by distillation from yeast fermentation of a grain or
a grain mixture by methods employed in the grain distilling
industry.
[0310] Stillage coming from the distillation (e.g. comprising
water, remainings of the grain, yeast cells etc.) is separated into
a "solid" part and a liquid part.
[0311] The solid part is called "wet-cake" and can be used as
animal feed as such.
[0312] The liquid part is (partially) evaporated into a syrup
(solubles).
[0313] When the wet-cake is dried it is Distillers Dried Grains
(DDG).
[0314] When the wet-cake is dried together with the syrup
(solubles) it is Distillers Dried Grans with Solubles (DDGS).
[0315] Wet-cake may be used in dairy operations and beef cattle
feedlots.
[0316] The dried DDGS may be used in livestock, e.g. dairy, beef
and swine) feeds and poultry feeds.
[0317] Corn DDGS is a very good protein source for dairy cows.
[0318] Corn Gluten Meal
[0319] In one aspect, the by-product of corn may be corn gluten
meal (CGM).
[0320] CGM is a powdery by-product of the corn milling inductry.
CGM has utility in, for example, animal feed. It can be used as an
inexpensive protein source for feed such as pet food, livestock
feed and poultry feed. It is an especially good source of the amino
acid cysteine, but must be balanced with other proteins for
lysine.
[0321] Feed Additive Composition
[0322] The feed additive composition of the present invention
and/or the feedstuff comprising same may be used in any suitable
form.
[0323] The feed additive composition of the present invention may
be used in the form of solid or liquid preparations or alternatives
thereof. Examples of solid preparations include powders, pastes,
boluses, capsules, pellets, tablets, dusts, and granules which may
be wettable, spray-dried or freeze-dried. Examples of liquid
preparations include, but are not limited to, aqueous, organic or
aqueous-organic solutions, suspensions and emulsions.
[0324] In some applications, the feed additive compositions of the
present invention may be mixed with feed or administered in the
drinking water.
[0325] In one aspect the present invention relates to a method of
preparing a feed additive composition, comprising admixing a
xylanase as taught herein with a feed acceptable carrier, diluent
or excipient, and (optionally) packaging.
[0326] Premix
[0327] The feedstuff and/or feed additive composition may be
combined with at least one mineral and/or at least one vitamin. The
compositions thus derived may be referred to herein as a
premix.
[0328] Malting and Brewing
[0329] The enzyme (or composition comprising the enzyme) of the
present invention may be used in malting and brewing.
[0330] Barley grains contain 1.7 to 4.1% (w/w) water-extractable
and 3.6 to 6.4% (w/w) total beta-glucan (Anderson, M. A., Cook, J.
A., & Stone, B. A., Journal of the Institute of Brewing, 1978,
84, 233-239; Henry, J., Journal of the Science of Food and
Agriculture, 1985, 36, 1243).
[0331] Wheat grains contain 0.1 to 0.8% (w/w) water-extractable and
0.6 to 1.4% (w/w) total beta-glucan (Anderson, M. A. et al (1978)
supra).
[0332] Efficient hydrolysis of arabinoxylans (AXsol) and
beta-glucan is important because such compounds can be involved in
production problems such as wort viscosity (Ducroo, P. &
Frelon, P. G., Proceedings of the European Brewery Convention
Congress, Zurich, 1989, 445; Vietor, R. J. & Voragen, A. G. J.,
Journal of the Institute of Brewing, 1993, 99, 243) and
filterability and haze formation (Coote, N. & Kirsop, B. H.
1976., Journal of the Institute of Brewing, 1976, 82, 34; Izawa,
M., Kano, Y. & Kanimura, M. 1991. Proceedings Aviemore
Conference on Malting, brewing and Distillling, 1990, 427).
[0333] The present invention provides a method of hydrolysing
arabinoxylans (e.g. AXinsol and AXsol) during malting and brewing
wherein wheat grains, barley grains or a combination thereof, or
portions of the wheat and/or barley grains, are admixed with the
enzyme of the present invention.
[0334] In one aspect of the present invention may relate to a food
composition that is a beverage, including, but not limited to, a
fermented beverage such as beer and wine, comprising a xylanase
comprising (or consisting of) a polypeptide sequence shown herein
as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID
No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,
fragment or derivative thereof having at least 75% identity (such
as at least 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID
No. 1 or SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10,
SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide sequence which
comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,
SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a conservative
substitution of at least one of the amino acids; or a xylanase
which is encoded by a nucleotide sequence shown herein as SEQ ID
No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13,
SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a
nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No.
5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ
ID No. 16, SEQ ID No.17 or SEQ ID No. 18 under high stringency
conditions, or a nucleotide sequence which has at least 75%
identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with
SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No.
13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18; or
a nucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No.
5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.
14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the
degeneracy of the genetic code.
[0335] In another aspect of the present invention may relate to a
food composition that is a beverage, including, but not limited to,
a fermented beverage such as beer and wine, comprising a xylanase
enzyme comprising (or consisting of) a polypeptide sequence shown
herein as SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or a variant,
homologue, fragment or derivative thereof having at least 98.5%
(e.g. at least 98.8 or 99 or 99.1 or 99.5%) identity with SEQ ID
No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or a xylanase which is
encoded by a nucleotide sequence shown herein as SEQ ID No. 4, SEQ
ID No. 5 or SEQ ID No. 6, or a nucleotide sequence which has at
least 97.7% (e.g. at least 98%, 98.5% or 99%) identity with SEQ ID
No. 4, SEQ ID No. 5 or SEQ ID No. 6.
[0336] In the context of the present invention, the term "fermented
beverage" is meant to comprise any beverage produced by a method
comprising a fermentation process, such as a microbial
fermentation, such as a bacterial and/or yeast fermentation.
[0337] In an aspect of the invention the fermented beverage is
beer. The term "beer" is meant to comprise any fermented wort
produced by fermentation/brewing of a starch-containing plant
material. Often, beer is produced from malt or adjunct, or any
combination of malt and adjunct as the starch-containing plant
material. As used herein the term "malt" is understood as any
malted cereal grain, such as malted barley or wheat.
[0338] As used herein the term "adjunct" refers to any starch
and/or sugar containing plant material which is not malt, such as
barley or wheat malt. As examples of adjuncts, mention can be made
of materials such as common corn grits, refined corn grits,
brewer's milled yeast, rice, sorghum, refined corn starch, barley,
barley starch, dehusked barley, wheat, wheat starch, torrified
cereal, cereal flakes, rye, oats, corn (maize), potato, tapioca,
cassava and syrups, such as corn syrup, sugar cane syrup, inverted
sugar syrup, barley and/or wheat syrups, and the like may be used
as a source of starch.
[0339] As used herein, the term "mash" refers to an aqueous slurry
of any starch and/or sugar containing plant material such as grist,
e. g. comprising crushed barley malt, crushed barley, and/or other
adjunct or a combination hereof, mixed with water later to be
separated into wort and spent grains.
[0340] As used herein, the term "wort" refers to the unfermented
liquor run-off following extracting the grist during mashing.
[0341] In another aspect the invention relates to a method of
preparing a fermented beverage such as beer comprising mixing the
xylanase of the present invention with malt or adjunct.
[0342] Examples of beers comprise: full malted beer, beer brewed
under the "Reinheitsgebot", ale, IPA, lager, bitter, Happoshu
(second beer), third beer, dry beer, near beer, light beer, low
alcohol beer, low calorie beer, porter, bock beer, stout, malt
liquor, non-alcoholic beer, non-alcoholic malt liquor and the like,
but also alternative cereal and malt beverages such as fruit
flavoured malt beverages, e. g. citrus flavoured, such as lemon-,
orange-, lime-, or berry-flavoured malt beverages, liquor flavoured
malt beverages, e. g., vodka-, rum-, or tequila-flavoured malt
liquor, or coffee flavoured malt beverages, such as
caffeine-flavoured malt liquor, and the like.
[0343] Breakdown of Grain-Based Material e.g. for Biofuel
Production
[0344] The enzyme (or composition comprising the enzyme) of the
present invention or as disclosed herein may be used to breakdown
(degrade) AXinsol and AXsol during grain processing from e.g.
grain-based material. The grain-based material may be whole grains
(e.g. whole wheat, barley, rye, triticale or corn grains or
mixtures thereof) or portions of the whole grains, or mixtures
thereof.
[0345] In one embodiment the enzyme (or composition comprising the
enzyme) of the present invention or as disclosed herein may be used
to breakdown (degrade) AXinsol and AXsol in grain-based materials
or whole grains.
[0346] For the avoidance of doubt the whole grains can be
mechanically broken.
[0347] The grain-based material may be broken down or degraded to
glucose. The glucose may subsequently be used as a feedstock for
any fermentation process, e.g. for biofuel (e.g. bioethanol)
production and/or biochemicals (e.g., bio-based isoprene)
production.
[0348] The grain-based material may be feedstock for a biofuel
(e.g. bioethanol) production process.
[0349] Today most fuel ethanol is produced from corn (maize) grain,
which is milled, treated with amylase enzymes to hydrolyse starch
to sugars, fermented, and distilled. While substantial progress has
been made in reducing costs of ethanol production, substantial
challenges remain. Improved techniques are still needed to reduce
the cost of biofuel feedstocks for ethanol production. For example,
in grain-based ethanol production degradation of arabinoxylans may
increase accessibility of starch.
[0350] The present invention provides a xylanase for use in the
breakdown of hemicelluloses, e.g. arabinoxylan--particularly
AXinsol and AXsol.
[0351] By way of example only, in the European fuel alcohol
industry, small grains like wheat, barley and rye are common raw
materials, in the US corn is mainly used. Wheat, barley and rye
contain, next to starch, high levels of non-starch polysaccharide
polymers (NSP), like cellulose, beta-glucan and hemicellulose.
[0352] The ratio in which the different NSPs are represented differ
for each feedstock. The table below shows the different amounts of
NSPs in wheat, barley and rye compared to some other
feedstocks.
TABLE-US-00001 TABLE 1 Non-starch Polysaccharides present in
different feedstocks (g kg.sup.-1 dry matter) Barley Oats Hul- Hul-
Corn Wheat Rye Hulled less Hulled less Beta-Glucan 1 8 16 42 42 28
41 Cellulose 22 17-20 15-16 43 10 82 14 Soluble and 75 89-99
116-136 144 114 150 113 Non-soluble NCP.sup.1 Total NSP 97 107-119
132-152 186 124 232 116 .sup.1Non Cellulosic Polysaccharides:
pentosans, (arabino)xylans and other hemicelluloses
[0353] NSPs can give high viscosity to grain mashes due to their
large water-binding capacity. High viscosity has a negative impact
on ethanol production since it will limit the solid concentration
that can be used in mashing and it will reduce the energy
efficiency of the process. In addition, residual hemicelluloses
present throughout the process may contribute to fouling in heat
exchangers and distillation equipment. The largest impact of a high
viscosity is seen when a mash is cooled to fermentation temperature
(32.degree. C.). This explains that the viscosity needs to be
reduced in the process anywhere before the cooling step.
[0354] In one embodiment of the present invention the method for
degrading grain-based material comprises admixing the xylanase as
disclosed herein as early as possible in the biofuel (e.g.
bioethanol) production process, e.g. preferably during mixing of
the grain-based material at the start of the process. One advantage
of adding the xylanases as disclosed herein at an early stage in
the process is that the enzymes breakdown initial viscosity.
[0355] In one embodiment of the present invention the method for
degrading grain-based material comprises admixing the xylanase as
disclosed herein prior to or during saccharification, fermentation,
or a combination thereof.
[0356] In one embodiment of the present invention the method for
degrading grain-based material comprises admixing the xylanase as
disclosed herein during liquefaction (e.g. a high temperature step
that follows mixing).
[0357] Therefore in one embodiment the present invention relates to
reducing viscosity when degrading grain-based materials, e.g. in
biofuel (e.g. bioethanol) production processes.
[0358] The benefits of using the xylanases taught herein to reduce
viscosity when degrading grain-based materials, e.g. in biofuel
(e.g. bioethanol) production processes are multiple: [0359] Higher
dry substance mash can be used in the process [0360] Higher solids
content of final syrup can be obtained [0361] Better heat transfer,
lower energy requirement [0362] Reduced evaporator fouling leading
to reduced cleaning costs [0363] Increased final ethanol yields
[0364] Improved quality of DDGS (by-product) [0365] Better
separation between the solid and liquid part during stillage
separation (after distillation). The lower viscosity increases
separation efficiency.
[0366] A further significant advantage of the present invention is
that use of the xylanase described herein in biofuel production can
also result in improved (by)products from that process such as
wet-cake, Distillers Dried Grains (DDG) or Distillers Dried Grains
with Solubles (DDGS). Therefore one advantage of the present
invention is since the wet-cake, DDG and DDGS are (by)products of
biofuel (e.g. bioethanol) production the use of the present
invention can result is improved quality of these (by)products. For
example the arabinoxylans in the (by)products can be already
dissolved during the biofuel production process.
[0367] Cereal (e.g. Wheat) Gluten-Starch Separation
[0368] The enzyme (or composition comprising the enzyme) of the
present invention or as disclosed herein may be used to breakdown
(degrade) AXinsol and AXsol during wheat starch and gluten
separation.
[0369] After initial separation of the wheat bran and germ from the
endosperm, fractionation of wheat endosperm flour into starch and
gluten fractions is industrially applied on large scale to obtain
high quality A-starch and byproducts B-starch and vital gluten.
[0370] The product of the degradation of the cereal flour (e.g.
wheat flour) in the present invention is starch (high quality
A-starch).
[0371] In addition, by-products B-starch and vital gluten are also
produced. Each individual product is then further processed to
supplement or modify food product characteristics to the market
needs.
[0372] There are several wheat separation processes used by
industry described in literature. These industrial processes differ
mainly in the forms of the flour-water mixtures presented to the
fractionation equipment (centrifuge, hydrocyclone, or screen) or in
the initial reaction conditions as temperature and applying of
shear (Abdulvahit Sayaslan, Lebensm.-Wiss. U.-Technol 37 (2004)
499-515, Wetmilling of wheat flour: industrial processes and
small-sacale test methods).
[0373] In the method for separating a cereal flour (e.g. wheat
flour) into starch and gluten fractions the method comprises
admixing a cereal flour (e.g. wheat flour), water and a xylanase.
The cereal flour, water and xylanase may be mixed simultaneously or
sequentially. In some embodiments the cereal flour (e.g. wheat
flour) and water may be admixed before admixing with the
xylanase.
[0374] In general, cereal flour (e.g. wheat flour) is either mixed
to a dough or batter, varying between 35 to 63% Dry solids, at
temperatures of .about.20-45.degree. C. The mixture is then further
processed either by: [0375] 1) letting the mixture rest for some
time (.about.30 minutes) and sequentially washing out the starch
from the mixture using a screen, centrifuge or hydrocyclone to
separate the starch milk from the gluten, or [0376] 2) applying
shear to the mixture, optionally diluting the mixture further and
then separating the wheat flour by a hydrocyclone, or a 2- or
3-phase decanter centrifuge.
[0377] The term "dry solids" as used herein means total solids
(dissolved and undissolved) of a slurry (in %) on a dry weight
basis.
[0378] In one embodiment of the present invention the method or use
as claimed may include the steps of mixing wheat flour to form a
dough or batter between 35-63% dry solids, at a temperature of
about 20 to about 45.degree. C. and separating the starch from the
gluten.
[0379] The method of the present invention may further
comprise:
[0380] a) resting the mixture for about 30 minutes and sequentially
washing out the starch from the mixture using either a screen, a
centrifuge or a hydrocyclone to separate the starch milk from the
gluten; or
[0381] b) applying shear to the mixture and optionally diluting the
mixture further, separating the starch from the gluten using a
hydrocyclone or a 2- or 3-phase decanter centrifuge.
[0382] The present invention provides for improving the separation
of the starch and the gluten by adding xylanases as taught herein
suitably during the initial mixing step of flour and water in the
various processes described above used for wheat starch separation.
Separation is improved by adding xylanases during the initial
mixing step due to viscosity reduction and the hydrolysis of AXsol
and/or AXinsol interfering with the gluten particles. By degrading
these poly- and oligosaccharides, gluten agglomeration is enhanced,
improving the gluten yield. (S. A. Frederix, C. M. Courtin, J. A.
Delcour, J. Cereal Sci. 40 (2004) 41-49, Substrate selectivity and
inhibitor sensitivity affect xylanase functionality in wheat flour
gluten-starch separation).
[0383] One advantage of the present invention is that it results in
higher A-starch yields and/or better quality gluten (e.g. better
quality vital gluten).
[0384] One advantage of the present invention is that it improves
wheat gluten-starch separation.
[0385] One of the ways to evaluate gluten quality is by monitoring
gluten agglomeration. When a certain amount of friction through
kneading of the dough or mixing of the batter is applied, gluten
particles tend to agglomerate into larger particles that form a
polymeric network, called "vital gluten". "Vital gluten" can be
added to food products to improve properties of baked goods such as
dough strength, shelf-life and bread volume (L. Day, M. A.
Augustin, I. L. Batey and C. W. Wrigley; Wheat-gluten uses and
industry needs; Trends in Food Science & Technology 17 (2006)
82-90).
[0386] In the bakery industry, the quality and quantity of the
gluten in a wheat flour is determined by the ICC standard assay No.
155 (AACC 38-12) using a Glutomatic. In this device, a dough is
formed from wheat flour (10.0 gr) mixed with a small amount of 2%
NaCl solution (4.2-4.8 ml). After 20 seconds of mixing step, the
dough is continuously kneaded while being washed for 5 minutes with
a 2% NaCl solution at room temperature (.about.22.degree. C.)
pumped through the mixing cup at a flow rate of .about.70
ml/minute. During this washing step, the wash water containing
starch is collected and the gluten particles form a gluten ball
within the Glutomatic sieve holder.
[0387] The quality of the gluten is measured by evaluating the
gluten agglomeration. This is done by centrifuging the gluten ball
in a special centrifuge containing a small sieve. The gluten
particles that pass this sieve are weighed (small gluten) and the
total amount of gluten is weighed. The gluten index is calculated
by (total wet gluten--small wet gluten)/total wet gluten. The more
gluten agglomeration is improved, the smaller the small gluten
fraction will be and the higher the gluten index value is. A high
gluten index, with a theoretical maximum of 100%, indicates a high
quality gluten ball.
[0388] Another value to quantify the amount of gluten is the dried
gluten yield (%). This value is calculated by dividing the grams of
total dried gluten by the total amount of dry flour which was used
in the experiment. The more dried gluten is recovered, the better
the separation is.
[0389] This industrial assay is currently under adaptation to
simulate a dough separation process used in industry.
[0390] Dosages
[0391] Preferably, the xylanase is present in the xylan-containing
material (e.g. feedstuff) in the range of about 500.times.U/kg to
about 16,000.times.U/kg xylan-containing material (e.g. feed), more
preferably about 750.times.U/kg feed to about 8000.times.U/kg
xylan-containing material (e.g. feed), preferably about
1500.times.U/kg feed to about 3000.times.U/kg xylan-containing
material (e.g. feed), preferably about 2000.times.U/kg feed to
about 2500.times.U/kg xylan-containing material (e.g. feed), and
even more preferably about 1000.times.U/kg xylan-containing
material (e.g. feed) to about 4000.times.U/kg xylan-containing
material (e.g. feed).
[0392] In one embodiment the xylanase is present in the
xylan-containing material (e.g. feedstuff) at more than about
500.times.U/kg xylan-containing material (e.g. feed), suitably more
than about 600.times.U/kg xylan-containing material (e.g. feed),
suitably more than about 700.times.U/kg xylan-containing material
(e.g. feed), suitably more than about 800.times.U/kg
xylan-containing material (e.g. feed), suitably more than about
900.times.U/kg xylan-containing material (e.g. feed), suitably more
than about 1000.times.U/kg xylan-containing material (e.g. feed),
suitably more than about 2000.times.U/kg, suitably more than about
2500.times.U/kg, suitably more than about 3000.times.U/kg
xylan-containing material (e.g. feed),
[0393] In one embodiment the xylanase is present in the
xylan-containing material (e.g. feedstuff) at a concentration of
between about 2000.times.U/kg to about 2500.times.U/kg.
[0394] In one embodiment the xylanase is present in the
xylan-containing material (e.g. feedstuff) at less than about
16,000.times.U/kg xylan-containing material (e.g. feed), suitably
less than about 8000.times.U/kg xylan-containing material (e.g.
feed), suitably less than about 7000.times.U/kg xylan-containing
material (e.g. feed), suitably less than about 6000.times.U/kg
xylan-containing material (e.g. feed), suitably less than about
5000.times.U/kg xylan-containing material (e.g. feed), suitably
less than about 4000.times.U/kg xylan-containing material (e.g.
feed).
[0395] Preferably, the xylanase may be present in a feed additive
composition in range of about 100.times.U/g to about
320,000.times.U/g composition, more preferably about 300.times.U/g
composition to about 160,000.times.U/g composition, and even more
preferably about 500.times.U/g composition to about 50,000 XU/g
composition, and even more preferably about 500.times.U/g
composition to about 40,000 XU/g composition.
[0396] In one embodiment the xylanase is present in the feed
additive composition at more than about 100.times.U/g composition,
suitably more than about 200.times.U/g composition, suitably more
than about 300.times.U/g composition, suitably more than about
400.times.U/g composition, suitably more than about 500.times.U/g
composition.
[0397] In one embodiment the xylanase is present in the feed
additive composition at less than about 320,000.times.U/g
composition, suitably less than about 160,000 XU/g composition,
suitably less than about 50,000.times.U/g composition, suitably
less than about 40,000.times.U/g composition, suitably less than
about 30000.times.U/g composition.
[0398] The xylanase activity can be expressed in xylanase units
(XU) measured at pH 5.0 with AZCL-arabinoxylan (azurine-crosslinked
wheat arabinoxylan, Xylazyme tablets, Megazyme) as substrate.
Hydrolysis by endo-(1-4)-.beta.-D-xylanase (xylanase) produces
water soluble dyed fragments, and the rate of release of these
(increase in absorbance at 590 nm) can be related directly to
enzyme activity. The xylanase units (XU) are determined relatively
to an enzyme standard (Danisco Xylanase, available from Danisco
Animal Nutrition) at standard reaction conditions, which are
40.degree. C., 5 min reaction time in McIlvaine buffer, pH 5.0.
[0399] The xylanase activity of the standard enzyme is determined
as amount of released reducing sugar end groups from an
oat-spelt-xylan substrate per min at pH 5.3 and 50.degree. C. The
reducing sugar end groups react with 3, 5-Dinitrosalicylic acid and
formation of the reaction product can be measured as increase in
absorbance at 540 nm. The enzyme activity is quantified relative to
a xylose standard curve (reducing sugar equivalents). One xylanase
unit (XU) is the amount of standard enzyme that releases 0.5
.mu.mol of reducing sugar equivalents per min at pH 5.3 and
50.degree. C.
[0400] In one embodiment suitably the enzyme is classified using
the E.C. classification above, and the E.C. classification
designates an enzyme having that activity when tested in the assay
taught herein for determining 1 XU.
[0401] Preferably, the xylanase is present in the mixing step of a
wheat starch separation process in the dough or batter in the range
of about 0.01 kg/MT DS dough or batter to about 0.60 kg/MT DS, more
preferably about 0.05 kg/MT DS to about 0.45 kg/MT DS dough or
batter, and even more preferably about 0.10 kg/MT DS to about 0.25
kg/MT DS dough or batter.
[0402] In some embodiments (particularly in the wheat starch
separation embodiment) the xylanase may be dosed in the range of
about 0.019 g protein/MT DS wheat flour (which is equivalent to
0.019 mg/kg DS) to about 119 g protein/MT DS wheat flour (which is
equivalent to 119 mg/kg DS--where DS means dry solids content and
MT means metric ton.
[0403] In some embodiments (particularly in the wheat starch
separation embodiment) the xylanase may be dosed at about 1.19 g
protein/MT DS wheat flour (which is equivalent to about 1.19 mg/kg
DS)--where DS means dry solids content and MT means metric ton.
[0404] In some embodiments (particularly in the wheat starch
separation embodiment) the xylanase may be dosed in the range of
about 9 to about 120000 units/kg wheat flour, suitably between
about 500-2400 units/kg wheat flour, suitably between about
900-1200 units/kg wheat flour (wherein 1 unit is defined as the
amount of enzyme required to generate 1 micromole of xoylose
reducing sugar equivalents per minute under the conditions of the
birch wood assay of Example 3).
[0405] In some embodiments (particularly in degrading grain-based
material) the xylanase may be dosed in the range of about 0.29
g/protein/MT DS wheat (which is equivalent to 0.29 mg/kg DS) to
about 0290 g/protein/MT DS wheat (which is equivalent to 290 mg/kg
DS).
[0406] In some embodiments (particularly in degrading grain-based
material) the xylanase may be dosed at 2.9 g/protein/MT DS wheat
(which is equivalent to 2.9 mg/kg DS).
[0407] In some embodiments (particularly in degrading grain-based
material) the xylanase may be dosed in the range of about 22 to
about 285000 units/kg, suitably about 1100 to about 5700 units/kg,
suitably about 2200 to about 2850 units/kg (wherein 1 unit is
defined as the amount of enzyme required to generate 1 micromole of
xoylose reducing sugar equivalents per minute under the conditions
of the birch wood assay of Example 3).
[0408] The enzyme and/or composition comprising the enzyme
according to the present invention may be designed for one-time
dosing or may be designed for use (e.g. feeding) on a daily
basis.
[0409] The optimum amount of the enzyme and/or composition
comprising the enzyme to be used in the present invention will
depend on the product to be treated and/or the method of contacting
the product with the composition and/or the intended use for the
same.
[0410] The amount of enzyme used in the compositions should be a
sufficient amount to be effective.
[0411] The amount of enzyme used in the compositions should be a
sufficient amount to be effective and to remain sufficiently
effective in for example improving the performance of an animal fed
feed products containing said composition. This length of time for
effectiveness should extend up to at least the time of utilisation
of the product (e.g. feed additive composition or feed containing
same).
[0412] Formulation
[0413] In one embodiment the enzyme may be formulated as a liquid,
a dry powder or a granule.
[0414] The dry powder or granules may be prepared by means known to
those skilled in the art, such as, in top-spray fluid bed coater,
in a bottom spray Wurster or by drum granulation (e.g. High sheer
granulation), extrusion, pan coating or in a microingredients
mixer.
[0415] For some embodiments the enzyme may be coated, for example
encapsulated.
[0416] In one embodiment the coating protects the enzyme from heat
and may be considered a thermoprotectant.
[0417] In one embodiment the feed additive composition is
formulated to a dry powder or granules as described in
WO2007/044968 (referred to as TPT granules) or WO1997/016076 or
WO1992/012645 (each of which is incorporated herein by
reference).
[0418] In one embodiment the feed additive composition may be
formulated to a granule for feed compositions comprising: a core;
an active agent; and at least one coating, the active agent of the
granule retaining at least 50% activity, at least 60% activity, at
least 70% activity, at least 80% activity after conditions selected
from one or more of a) a feed pelleting process, b) a steam-heated
feed pretreatment process, c) storage, d) storage as an ingredient
in an unpelleted mixture, and e) storage as an ingredient in a feed
base mix or a feed premix comprising at least one compound selected
from trace minerals, organic acids, reducing sugars, vitamins,
choline chloride, and compounds which result in an acidic or a
basic feed base mix or feed premix.
[0419] With regard to the granule at least one coating may comprise
a moisture hydrating material that constitutes at least 55% w/w of
the granule; and/or at least one coating may comprise two coatings.
The two coatings may be a moisture hydrating coating and a moisture
barrier coating. In some embodiments, the moisture hydrating
coating may be between 25% and 60% w/w of the granule and the
moisture barrier coating may be between 2% and 15% w/w of the
granule. The moisture hydrating coating may be selected from
inorganic salts, sucrose, starch, and maltodextrin and the moisture
barrier coating may be selected from polymers, gums, whey and
starch.
[0420] The granule may be produced using a feed pelleting process
and the feed pretreatment process may be conducted between
70.degree. C. and 95.degree. C. for up to several minutes, such as
between 85.degree. C. and 95.degree. C.
[0421] In one embodiment the feed additive composition may be
formulated to a granule for animal feed comprising: a core; an
active agent, the active agent of the granule retaining at least
80% activity after storage and after a steam-heated pelleting
process where the granule is an ingredient; a moisture barrier
coating; and a moisture hydrating coating that is at least 25% w/w
of the granule, the granule having a water activity of less than
0.5 prior to the steam-heated pelleting process.
[0422] The granule may have a moisture barrier coating selected
from polymers and gums and the moisture hydrating material may be
an inorganic salt. The moisture hydrating coating may be between
25% and 45% w/w of the granule and the moisture barrier coating may
be between 2% and 10% w/w of the granule.
[0423] The granule may be produced using a steam-heated pelleting
process which may be conducted between 85.degree. C. and 95.degree.
C. for up to several minutes.
[0424] In some embodiments the enzyme may be diluted using a
diluent, such as starch powder, lime stone or the like.
[0425] In one embodiment, the enzyme or composition comprising the
enzyme is in a liquid formulation suitable for consumption
preferably such liquid consumption contains one or more of the
following: a buffer, salt, sorbitol and/or glycerol.
[0426] In another embodiment the enzyme or composition comprising
the enzyme may be formulated by applying, e.g. spraying, the
enzyme(s) onto a carrier substrate, such as ground wheat for
example.
[0427] In one embodiment the enzyme or composition comprising the
enzyme according to the present invention may be formulated as a
premix. By way of example only the premix may comprise one or more
feed components, such as one or more minerals and/or one or more
vitamins.
[0428] In one embodiment the enzyme for use in the present
invention are formulated with at least one physiologically
acceptable carrier selected from at least one of maltodextrin,
limestone (calcium carbonate), cyclodextrin, wheat or a wheat
component, sucrose, starch, Na.sub.2SO.sub.4, Talc, PVA, sorbitol,
benzoate, sorbiate, glycerol, sucrose, propylene glycol,
1,3-propane diol, glucose, parabens, sodium chloride, citrate,
acetate, phosphate, calcium, metabisulfite, formate and mixtures
thereof.
[0429] Packaging
[0430] In one embodiment the enzyme and/or composition comprising
same (e.g. feed additive composition) and/or premix and/or feed or
feedstuff according to the present invention is packaged.
[0431] In one preferred embodiment the feed additive composition
and/or premix and/or feed or feedstuff is packaged in a bag, such
as a paper bag.
[0432] In an alternative embodiment the feed additive composition
and/or premix and/or feed or feedstuff may be sealed in a
container. Any suitable container may be used.
[0433] Forms
[0434] The enzyme or composition comprising the enzyme (e.g. the
feed additive composition) of the present invention and other
components and/or the feedstuff comprising same may be used in any
suitable form.
[0435] The enzyme or composition comprising same (e.g. feed
additive composition) of the present invention may be used in the
form of solid or liquid preparations or alternatives thereof.
[0436] Examples of solid preparations include powders, pastes,
boluses, capsules, pellets, tablets, pills, capsules, ovules,
solutions or suspensions, dusts, and granules which may be
wettable, spray-dried or freeze-dried. Examples of liquid
preparations include, but are not limited to, aqueous, organic or
aqueous-organic solutions, suspensions and emulsions.
[0437] The composition comprising the enzyme may contain flavouring
or colouring agents, for immediate-, delayed-, modified-,
sustained-, pulsed- or controlled-release applications.
[0438] By way of example, if the composition of the present
invention is used in a solid, e.g. pelleted form, it may also
contain one or more of: excipients such as microcrystalline
cellulose, lactose, sodium citrate, calcium carbonate, dibasic
calcium phosphate and glycine; disintegrants such as starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex silicates;
granulation binders such as polyvinylpyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),
sucrose, gelatin and acacia; lubricating agents such as magnesium
stearate, stearic acid, glyceryl behenate and talc may be
included.
[0439] Examples of nutritionally acceptable carriers for use in
preparing the forms include, for example, water, salt solutions,
alcohol, silicone, waxes, petroleum jelly, vegetable oils,
polyethylene glycols, propylene glycol, liposomes, sugars, gelatin,
lactose, amylose, magnesium stearate, talc, surfactants, silicic
acid, viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, petroethral fatty acid esters,
hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
[0440] Preferred excipients for the forms include lactose, starch,
a cellulose, milk sugar or high molecular weight polyethylene
glycols.
[0441] For aqueous suspensions and/or elixirs, the composition of
the present invention may be combined with various sweetening or
flavouring agents, colouring matter or dyes, with emulsifying
and/or suspending agents and with diluents such as water, propylene
glycol and glycerin, and combinations thereof.
[0442] Subject
[0443] The term "subject", as used herein, means an animal that is
to be or has been administered with a feed additive composition
according to the present invention or a feedstuff comprising said
feed additive composition according to the present invention.
[0444] The term "subject", as used herein, means an animal.
[0445] In one embodiment, the subject is a mammal, bird, fish or
crustacean including for example livestock or a domesticated animal
(e.g. a pet).
[0446] In one embodiment the "subject" is livestock.
[0447] The term "livestock", as used herein refers to any farmed
animal. Preferably, livestock is one or more of ruminants such as
cattle (e.g. cows or bulls (including calves)), mono-gastric
animals such as poultry (including broilers, chickens and turkeys),
pigs (including piglets), birds, aquatic animals such as fish,
agastric fish, gastric fish, freshwater fish such as salmon, cod,
trout and carp, e.g. koi carp, marine fish such as sea bass, and
crustaceans such as shrimps, mussels and scallops), horses
(including race horses), sheep (including lambs).
[0448] In another embodiment the "subject" is a domesticated animal
or pet or an animal maintained in a zoological environment.
[0449] The term "domesticated animal or pet or animal maintained in
a zoological environment" as used herein refers to any relevant
animal including canines (e.g. dogs), felines (e.g. cats), rodents
(e.g. guinea pigs, rats, mice), birds, fish (including freshwater
fish and marine fish), and horses.
[0450] Performance
[0451] As used herein, "animal performance" may be determined by
the feed efficiency and/or weight gain of the animal and/or by the
feed conversion ratio and/or by the digestibility of a nutrient in
a feed (e.g. amino acid digestibility) and/or digestible energy or
metabolizable energy in a feed and/or by nitrogen retention and/or
by animals ability to avoid the negative effects of necrotic
enteritis and/or by the immune response of the subject.
[0452] Preferably "animal performance" is determined by feed
efficiency and/or weight gain of the animal and/or by the feed
conversion ratio.
[0453] By "improved animal performance" it is meant that there is
increased feed efficiency, and/or increased weight gain and/or
reduced feed conversion ratio and/or improved digestibility of
nutrients or energy in a feed and/or by improved nitrogen retention
and/or by an improved immune response in the subject resulting from
the use of feed additive composition of the present invention in
feed in comparison to feed which does not comprise said feed
additive composition.
[0454] Preferably, by "improved animal performance" it is meant
that there is increased feed efficiency and/or increased weight
gain and/or reduced feed conversion ratio.
[0455] As used herein, the term "feed efficiency" refers to the
amount of weight gain per unit of feed when the animal is fed
ad-libitum or a specified amount of feed during a period of
time.
[0456] By "increased feed efficiency" it is meant that the use of a
feed additive composition according the present invention in feed
results in an increased weight gain per unit of feed intake
compared with an animal fed without said feed additive composition
being present.
[0457] Feed Conversion Ratio (FCR) As used herein, the term "feed
conversion ratio" refers to the amount of feed fed to an animal to
increase the weight of the animal by a specified amount.
[0458] An improved feed conversion ratio means a lower feed
conversion ratio.
[0459] By "lower feed conversion ratio" or "improved feed
conversion ratio" it is meant that the use of a feed additive
composition in feed results in a lower amount of feed being
required to be fed to an animal to increase the weight of the
animal by a specified amount compared to the amount of feed
required to increase the weight of the animal by the same amount
when the feed does not comprise said feed additive composition.
[0460] Nutrient Digestibility
[0461] Nutrient digestibility as used herein means the fraction of
a nutrient that disappears from the gastro-intestinal tract or a
specified segment of the gastro-intestinal tract, e.g. the small
intestine. Nutrient digestibility may be measured as the difference
between what is administered to the subject and what comes out in
the faeces of the subject, or between what is administered to the
subject and what remains in the digesta on a specified segment of
the gastro intestinal tract, e.g. the ileum.
[0462] Nutrient digestibility as used herein may be measured by the
difference between the intake of a nutrient and the excreted
nutrient by means of the total collection of excreta during a
period of time; or with the use of an inert marker that is not
absorbed by the animal, and allows the researcher calculating the
amount of nutrient that disappeared in the entire gastro-intestinal
tract or a segment of the gastro-intestinal tract. Such an inert
marker may be titanium dioxide, chromic oxide or acid insoluble
ash. Digestibility may be expressed as a percentage of the nutrient
in the feed, or as mass units of digestible nutrient per mass units
of nutrient in the feed.
[0463] Nutrient digestibility as used herein encompasses starch
digestibility, fat digestibility, protein digestibility, and amino
acid digestibility.
[0464] Energy digestibility as used herein means the gross energy
of the feed consumed minus the gross energy of the faeces or the
gross energy of the feed consumed minus the gross energy of the
remaining digesta on a specified segment of the gastro-intestinal
tract of the animal, e.g. the ileum. Metabolizable energy as used
herein refers to apparent metabolizable energy and means the gross
energy of the feed consumed minus the gross energy contained in the
faeces, urine, and gaseous products of digestion. Energy
digestibility and metabolizable energy may be measured as the
difference between the intake of gross energy and the gross energy
excreted in the faeces or the digesta present in specified segment
of the gastro-intestinal tract using the same methods to measure
the digestibility of nutrients, with appropriate corrections for
nitrogen excretion to calculate metabolizable energy of feed.
[0465] Combination with Other Components
[0466] The enzyme of the present invention (or the xylanase as
taught herein) may be used in combination with other
components.
[0467] In one embodiment the enzyme of the present invention (or
the xylanase as taught herein) may be used in combination with a
probiotic or a direct fed microbial (DFM), e.g. a direct fed
bacteria.
[0468] The combination of the present invention comprises the
enzyme of the present invention (or the xylanase as taught herein
or a composition comprising the enzyme, e.g. a feed additive
composition) and another component which is suitable for human or
animal consumption and is capable of providing a medical or
physiological benefit to the consumer.
[0469] In one embodiment the "another component" may be one or more
further enzymes (e.g. further feed enzymes or brewing or malting
enzymes, or grain processing enzymes or wheat gluten-starch
separation enzymes).
[0470] Suitable additional enzymes for use in the present invention
may be one or more of the enzymes selected from the group
consisting of: endoglucanases (E.C. 3.2.1.4); celliobiohydrolases
(E.C. 3.2.1.91), .beta.-glucosidases (E.C. 3.2.1.21), cellulases
(E.C. 3.2.1.74), lichenases (E.C. 3.1.1.73), lipases (E.C.
3.1.1.3), lipid acyltransferases (generally classified as E.C.
2.3.1.x), phospholipases (E.C. 3.1.1.4, E.C. 3.1.1.32 or E.C.
3.1.1.5), phytases (e.g. 6-phytase (E.C. 3.1.3.26) or a 3-phytase
(E.C. 3.1.3.8), alpha-amylases (E.C. 3.2.1.1), ther xylanases (E.C
3.2.1.8, E.C. 3.2.1.32, E.C. 3.2.1.37, E.C. 3.1.1.72, E.C.
3.1.1.73), glucoamylases (E.C. 3.2.1.3), proteases (e.g. subtilisin
(E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline
serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x))
and/or mannanases (e.g. a .beta.-mannanase (E.C. 3.2.1.78)).
[0471] In one embodiment (particularly for feed applications) the
other component may be one or more of the enzymes selected from the
group consisting of an amylase (including .alpha.-amylases (E.G.
3.2.1.1), G4-forming amylases (E.C. 3.2.1.60), .beta.-amylases
(E.C. 3.2.1.2) and .gamma.-amylases (E.C. 3.2.1.3); and/or a
protease (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C.
3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a
keratinase (E.C. 3.4.x.x)).
[0472] In one embodiment (particularly for feed applications) the
other component may be a combination of an amylase (e.g.
.alpha.-amylases (E.C. 3.2.1.1)) and a protease (e.g. subtilisin
(E.C. 3.4.21.62)).
[0473] In one embodiment (particularly for feed applications) the
other component may be a .beta.-glucanase, e.g. an
endo-1,3(4)-.beta.-glucanases (E.C. 3.2.1.6).
[0474] In one embodiment (particularly for feed applications) the
other component may be a mannanases (e.g. a .beta.-mannanase (E.C.
3.2.1.78)).
[0475] In one embodiment (particularly for feed applications) the
other component may be a lipase lipase (E.C. 3.1.1.3), a lipid
acyltransferase (generally classified as E.C. 2.3.1.x), or a
phospholipase (E.C. 3.1.1.4, E.C. 3.1.1.32 or E.C. 3.1.1.5),
suitably a lipase (E.C. 3.1.1.3).
[0476] In one embodiment (particularly for feed applications) the
other component may be a protease (e.g. subtilisin (E.C. 3.4.21.62)
or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease
(E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)).
[0477] In one embodiment the additional component may be a
stabiliser or an emulsifier or a binder or carrier or an excipient
or a diluent or a disintegrant.
[0478] The term "stabiliser" as used here is defined as an
ingredient or combination of ingredients that keeps a product (e.g.
a feed product) from changing overtime.
[0479] The term "emulsifier" as used herein refers to an ingredient
(e.g. a feed ingredient) that prevents the separation of emulsions.
Emulsions are two immiscible substances, one present in droplet
form, contained within the other. Emulsions can consist of
oil-in-water, where the droplet or dispersed phase is oil and the
continuous phase is water; or water-in-oil, where the water becomes
the dispersed phase and the continuous phase is oil. Foams, which
are gas-in-liquid, and suspensions, which are solid-in-liquid, can
also be stabilised through the use of emulsifiers.
[0480] As used herein the term "binder" refers to an ingredient
(e.g. a feed ingredient) that binds the product together through a
physical or chemical reaction. During "gelation" for instance,
water is absorbed, providing a binding effect. However, binders can
absorb other liquids, such as oils, holding them within the
product. In the context of the present invention binders would
typically be used in solid or low-moisture products for instance
baking products: pastries, doughnuts, bread and others. Examples of
granulation binders include one or more of: polyvinylpyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),
sucrose, maltose, gelatin and acacia.
[0481] "Carriers" mean materials suitable for administration of the
enzyme and include any such material known in the art such as, for
example, any liquid, gel, solvent, liquid diluent, solubilizer, or
the like, which is non-toxic and which does not interact with any
components of the composition in a deleterious manner.
[0482] The present invention provides a method for preparing a
composition (e.g. a feed additive composition) comprising admixing
an enzyme of the present invention with at least one
physiologically acceptable carrier selected from at least one of
maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or
a wheat component, sucrose, starch, Na.sub.2SO.sub.4, Talc, PVA,
sorbitol, benzoate, sorbiate, glycerol, sucrose, propylene glycol,
1,3-propane diol, glucose, parabens, sodium chloride, citrate,
acetate, phosphate, calcium, metabisulfite, formate and mixtures
thereof.
[0483] Examples of "excipients" include one or more of:
microcrystalline cellulose and other celluloses, lactose, sodium
citrate, calcium carbonate, dibasic calcium phosphate, glycine,
starch, milk sugar and high molecular weight polyethylene
glycols.
[0484] Examples of "disintegrants" include one or more of: starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex
silicates.
[0485] Examples of "diluents" include one or more of: water,
ethanol, propylene glycol and glycerin, and combinations
thereof.
[0486] The other components may be used simultaneously (e.g. when
they are in admixture together or even when they are delivered by
different routes) or sequentially (e.g. they may be delivered by
different routes) to the xylanase of the present invention.
[0487] Preferably, when the feed additive composition of the
present invention is admixed with another component(s), the DFM
remains viable.
[0488] In one embodiment preferably the feed additive composition
according to the present invention does not comprise chromium or
organic chromium
[0489] In one embodiment preferably the feed additive according to
the present invention does not contain glucanase.
[0490] In one embodiment preferably the feed additive according to
the present invention does not contain sorbic acid.
[0491] Isolated
[0492] In one aspect, preferably the amino acid sequence, or
nucleic acid, or enzyme according to the present invention is in an
isolated form. The term "isolated" means that the sequence or
enzyme or nucleic acid is at least substantially free from at least
one other component with which the sequence, enzyme or nucleic acid
is naturally associated in nature and as found in nature. The
sequence, enzyme or nucleic acid of the present invention may be
provided in a form that is substantially free of one or more
contaminants with which the substance might otherwise be
associated. Thus, for example it may be substantially free of one
or more potentially contaminating polypeptides and/or nucleic acid
molecules.
[0493] Purified
[0494] In one aspect, preferably the sequence, enzyme or nucleic
acid according to the present invention is in a purified form. The
term "purified" means that the given component is present at a high
level. The component is desirably the predominant component present
in a composition. Preferably, it is present at a level of at least
about 90%, or at least about 95% or at least about 98%, said level
being determined on a dry weight/dry weight basis with respect to
the total composition under consideration.
[0495] Nucleotide Sequence
[0496] The scope of the present invention encompasses nucleotide
sequences encoding proteins having the specific properties as
defined herein.
[0497] The term "nucleotide sequence" as used herein refers to an
oligonucleotide sequence or polynucleotide sequence, and variant,
homologues, fragments and derivatives thereof (such as portions
thereof). The nucleotide sequence may be of genomic or synthetic or
recombinant origin, which may be double-stranded or single-stranded
whether representing the sense or anti-sense strand.
[0498] The term "nucleotide sequence" in relation to the present
invention includes genomic DNA, cDNA, synthetic DNA, and RNA.
Preferably it means DNA, more preferably cDNA sequence coding for
the present invention.
[0499] In a preferred embodiment, the nucleotide sequence when
relating to and when encompassed by the per se scope of the present
invention does not include the native nucleotide sequence according
to the present invention when in its natural environment and when
it is linked to its naturally associated sequence(s) that is/are
also in its/their natural environment. For ease of reference, we
shall call this preferred embodiment the "non-native nucleotide
sequence". In this regard, the term "native nucleotide sequence"
means an entire nucleotide sequence that is in its native
environment and when operatively linked to an entire promoter with
which it is naturally associated, which promoter is also in its
native environment. However, the amino acid sequence encompassed by
scope the present invention can be isolated and/or purified post
expression of a nucleotide sequence in its native organism.
Preferably, however, the amino acid sequence encompassed by scope
of the present invention may be expressed by a nucleotide sequence
in its native organism but wherein the nucleotide sequence is not
under the control of the promoter with which it is naturally
associated within that organism.
[0500] Typically, the nucleotide sequence encompassed by the scope
of the present invention is prepared using recombinant DNA
techniques (i.e. recombinant DNA). However, in an alternative
embodiment of the invention, the nucleotide sequence could be
synthesised, in whole or in part, using chemical methods well known
in the art (see Caruthers M H et al., (1980) Nuc Acids Res Symp Ser
215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser
225-232).
[0501] Preparation of the Nucleotide Sequence
[0502] A nucleotide sequence encoding either a protein which has
the specific properties as defined herein or a protein which is
suitable for modification may be identified and/or isolated and/or
purified from any cell or organism producing said protein. Various
methods are well known within the art for the identification and/or
isolation and/or purification of nucleotide sequences. By way of
example, PCR amplification techniques to prepare more of a sequence
may be used once a suitable sequence has been identified and/or
isolated and/or purified.
[0503] By way of further example, a genomic DNA and/or cDNA library
may be constructed using chromosomal DNA or messenger RNA from the
organism producing the enzyme. If the amino acid sequence of the
enzyme is known, labelled oligonucleotide probes may be synthesised
and used to identify enzyme-encoding clones from the genomic
library prepared from the organism. Alternatively, a labelled
oligonucleotide probe containing sequences homologous to another
known enzyme gene could be used to identify enzyme-encoding clones.
In the latter case, hybridisation and washing conditions of lower
stringency are used.
[0504] Alternatively, enzyme-encoding clones could be identified by
inserting fragments of genomic DNA into an expression vector, such
as a plasmid, transforming enzyme-negative bacteria with the
resulting genomic DNA library, and then plating the transformed
bacteria onto agar plates containing a substrate for enzyme (i.e.
maltose), thereby allowing clones expressing the enzyme to be
identified.
[0505] In a yet further alternative, the nucleotide sequence
encoding the enzyme may be prepared synthetically by established
standard methods, e.g. the phosphoroamidite method described by
Beucage S. L. et al., (1981) Tetrahedron Letters 22, p 1859-1869,
or the method described by Matthes et al., (1984) EMBO J. 3, p
801-805. In the phosphoroamidite method, oligonucleotides are
synthesised, e.g. in an automatic DNA synthesiser, purified,
annealed, ligated and cloned in appropriate vectors.
[0506] The nucleotide sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin, or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate) in accordance with standard
techniques. Each ligated fragment corresponds to various parts of
the entire nucleotide sequence. The DNA sequence may also be
prepared by polymerase chain reaction (PCR) using specific primers,
for instance as described in U.S. Pat. No. 4,683,202 or in Saiki R
K et al., (Science (1988) 239, pp 487-491).
[0507] Amino Acid Sequences
[0508] The scope of the present invention also encompasses amino
acid sequences of enzymes having the specific properties as defined
herein.
[0509] As used herein, the term "amino acid sequence" is synonymous
with the term "polypeptide" and/or the term "protein". In some
instances, the term "amino acid sequence" is synonymous with the
term "peptide". In some instances, the term "amino acid sequence"
is synonymous with the term "enzyme".
[0510] The amino acid sequence may be prepared/isolated from a
suitable source, or it may be made synthetically or it may be
prepared by use of recombinant DNA techniques.
[0511] Preferably the amino acid sequence when relating to and when
encompassed by the per se scope of the present invention is not a
native enzyme. In this regard, the term "native enzyme" means an
entire enzyme that is in its native environment and when it has
been expressed by its native nucleotide sequence.
[0512] Sequence Identity or Sequence Homology
[0513] The present invention also encompasses the use of sequences
having a degree of sequence identity or sequence homology with
amino acid sequence(s) of a polypeptide having the specific
properties defined herein or of any nucleotide sequence encoding
such a polypeptide (hereinafter referred to as a "homologous
sequence(s)"). Here, the term "homologue" means an entity having a
certain homology with the subject amino acid sequences and the
subject nucleotide sequences. Here, the term "homology" can be
equated with "identity".
[0514] The homologous amino acid sequence and/or nucleotide
sequence should provide and/or encode a polypeptide which retains
the functional activity and/or enhances the activity of the
enzyme.
[0515] In the present context, in some embodiments a homologous
sequence is taken to include an amino acid or a nucleotide sequence
which may be at least 97.7% identical, preferably at least 98 or
99% identical to the subject sequence.
[0516] In some embodiments a homologous sequence is taken to
include an amino acid or a nucleotide sequence which may be at
least 85% identical, preferably at least 90 or 95% identical to the
subject sequence.
[0517] Typically, the homologues will comprise the same active
sites etc. as the subject amino acid sequence for instance.
Although 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.
[0518] In one embodiment, a homologous sequence is taken to include
an amino acid sequence or nucleotide sequence which has one or
several additions, deletions and/or substitutions compared with the
subject sequence.
[0519] In the present context, "the subject sequence" relates to
the nucleotide sequence or polypeptide/amino acid sequence
according to the invention.
[0520] Preferably, the % sequence identity with regard to a
polypeptide sequence is determined using SEQ ID No. 3 as the
subject sequence in a sequence alignment. In one embodiment, the
polypeptide subject sequence is selected from the group consisting
of SEQ ID No. 3, SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 9, SEQ ID
No. 10, SEQ ID No. 11 or SEQ ID No. 15. In a preferred embodiment
the polypeptide subject sequence is selected from the mature
sequences SEQ ID No. 3, SEQ ID No. 11 or SEQ ID No. 15.
[0521] Preferably, the % sequence identity with regard to a
nucleotide sequence is determined using SEQ ID No. 6 as the subject
sequence in the sequence alignment. In one embodiment, the subject
sequence for nucleotide sequences may be selected from the group
consisting of SEQ ID No. 4, SEQ ID No. 5. SEQ ID No. 6, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 and
SEQ ID No. 18. In a preferred embodiment the subject sequence is
sequence SEQ ID No. 6.
[0522] A "parent nucleic acid" or "parent amino acid" means a
nucleic acid sequence or amino acid sequence, encoding or coding
for the parent polypeptide, respectively.
[0523] In one embodiment the present invention relates to a protein
whose amino acid sequence is represented herein or a protein
derived from this (parent) protein by substitution, deletion or
addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7,
8, 9 amino acids, or more amino acids, such as 10 or more than 10
amino acids in the amino acid sequence of the parent protein and
having the activity of the parent protein.
[0524] Suitably, the degree of identity with regard to an amino
acid sequence is determined over at least 20 contiguous amino
acids, preferably over at least 30 contiguous amino acids,
preferably over at least 40 contiguous amino acids, preferably over
at least 50 contiguous amino acids, preferably over at least 60
contiguous amino acids, preferably over at least 100 contiguous
amino acids, preferably over at least 200 contiguous amino
acids.
[0525] In one embodiment the present invention relates to a nucleic
acid sequence (or gene) encoding a protein whose amino acid
sequence is represented herein or encoding a protein derived from
this (parent) protein by substitution, deletion or addition of one
or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids,
or more amino acids, such as 10 or more than 10 amino acids in the
amino acid sequence of the parent protein and having the activity
of the parent protein.
[0526] In the present context, in one embodiment a homologous
sequence or foreign sequence is taken to include a nucleotide
sequence which may be at least 97.7% identical, preferably at least
98 or 99% identical to a nucleotide sequence encoding a polypeptide
of the present invention (the subject sequence).
[0527] In another embodiment, a homologous sequence is taken to
include a nucleotide sequence which may be at least 85% identical,
preferably at least 90 or 95% identical to a nucleotide sequence
encoding a polypeptide of the present invention (the subject
sequence).
[0528] Typically, the homologues will comprise the same sequences
that code for the active sites etc. as the subject sequence.
Although 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.
[0529] 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 or % identity between two or more sequences. %
homology or % identity 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.
[0530] This is called an "ungapped" alignment. Typically, such
ungapped alignments are performed only over a relatively short
number of residues.
[0531] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology or %
identity when a global alignment is performed.
[0532] Consequently, most sequence comparison methods are designed
to produce optimal alignments that take into consideration possible
insertions and deletions without penalising unduly the overall
homology score. This is achieved by inserting "gaps" in the
sequence alignment to try to maximise local homology.
[0533] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons.
[0534] Calculation of maximum % homology or % identity therefore
firstly requires the production of an optimal alignment, taking
into consideration gap penalties. A suitable computer program for
carrying out such an alignment is the Vector NTI (Invitrogen
Corp.). Examples of software that can perform sequence comparisons
include, but are not limited to, the BLAST package (see Ausubel et
al 1999 Short Protocols in Molecular Biology, 4th Ed--Chapter 18),
BLAST 2 (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS
Microbiol Lett 1999 177(1): 187-8 and tatiana(a)ncbi.nlm.nih.gov),
FASTA (Altschul et al 1990 J. Mol. Biol. 403-410) and AlignX for
example. At least BLAST, BLAST 2 and FASTA are available for
offline and online searching (see Ausubel et al 1999, pages 7-58 to
7-60), such as for example in the GenomeQuest search tool
(www.genomequest.com).
[0535] Although the final % homology or % identity can be measured
in terms of identity, the alignment process itself is typically not
based on an all-or-nothing pair comparison. Instead, 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. Vector NTI programs generally use either the public
default values or a custom symbol comparison table if supplied (see
user manual for further details). For some applications, it is
preferred to use the default values for the Vector NTI package.
[0536] Alternatively, percentage homologies may be calculated using
the multiple alignment feature in Vector NTI (Invitrogen Corp.),
based on an algorithm, analogous to CLUSTAL (Higgins D G &
Sharp P M (1988), Gene 73(1), 237-244).
[0537] Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
[0538] Should Gap Penalties be used when determining sequence
identity, then preferably the following parameters are used for
pairwise alignment:
TABLE-US-00002 FOR BLAST GAP OPEN 9 GAP EXTENSION 2
TABLE-US-00003 FOR CLUSTAL DNA PROTEIN Weight Matrix IUB Gonnet 250
GAP OPENING 15 10 GAP EXTEND 6.66 0.1
[0539] In one embodiment, CLUSTAL may be used with the gap penalty
and gap extension set as defined above.
[0540] Suitably, the degree of identity with regard to a nucleotide
sequence or protein sequence is determined over at least 20
contiguous nucleotides/amino acids, preferably over at least 30
contiguous nucleotides/amino acids, preferably over at least 40
contiguous nucleotides/aminio acids, preferably over at least 50
contiguous nucleotides/amino acids, preferably over at least 60
contiguous nucleotides/amino acids, preferably over at least 100
contiguous nucleotides/amino acids.
[0541] Suitably, the degree of identity with regard to a nucleotide
sequence sequence is determined over at least 100 contiguous
nucleotides, preferably over at least 200 contiguous nucleotides,
preferably over at least 300 contiguous nucleotides, preferably
over at least 400 contiguous nucleotides, preferably over at least
500 contiguous nucleotides, preferably over at least 600 contiguous
nucleotides, preferably over at least 700 contiguous nucleotides,
preferably over at least 800 contiguous nucleotides.
[0542] Suitably, the degree of identity with regard to a nucleotide
sequence may be determined over the whole sequence taught
herein.
[0543] Suitably, the degree of identity with regard to a nucleotide
sequence may be determined over the whole sequence taught herein as
the mature sequence, e.g. SEQ ID No. 6 or SEQ ID No. 14 or SEQ ID
No. 18. Suitably, the degree of identity with regard to a
nucleotide sequence may be determined over the whole sequence as
taught herein as SEQ ID No. 6.
[0544] Suitably, the degree of identity with regard to a protein
(amino acid) sequence is determined over at least 100 contiguous
amino acids, preferably over at least 200 contiguous amino acids,
preferably over at least 300 contiguous amino acids.
[0545] Suitably, the degree of identity with regard to an amino
acid or protein sequence may be determined over the whole sequence
taught herein.
[0546] Suitably, the degree of identity with regard to an amino
acid or protein sequence may be determined over the whole sequence
taught herein as the mature sequence, e.g. SEQ ID No. 3, SEQ ID No.
11 or SEQ ID No. 15. Suitably, the degree of identity with regard
to an amino acid or protein sequence may be determined over the
whole sequence taught herein as SEQ ID No. 3.
[0547] In the present context, the term "query sequence" means a
homologous sequence or a foreign sequence, which is aligned with a
subject sequence in order to see if it falls within the scope of
the present invention. Accordingly, such query sequence can for
example be a prior art sequence or a third party sequence.
[0548] In one preferred embodiment, the sequences are aligned by a
global alignment program and the sequence identity is calculated by
identifying the number of exact matches identified by the program
divided by the length of the subject sequence.
[0549] In one embodiment, the degree of sequence identity between a
query sequence and a subject sequence is determined by 1) aligning
the two sequences by any suitable alignment program using the
default scoring matrix and default gap penalty, 2) identifying the
number of exact matches, where an exact match is where the
alignment program has identified an identical amino acid or
nucleotide in the two aligned sequences on a given position in the
alignment and 3) dividing the number of exact matches with the
length of the subject sequence.
[0550] In yet a further preferred embodiment, the global alignment
program is selected from the group consisting of CLUSTAL and BLAST
(preferably BLAST) and the sequence identity is calculated by
identifying the number of exact matches identified by the program
divided by the length of the subject sequence.
[0551] The sequences may also have deletions, insertions or
substitutions of amino acid residues result in a functionally
equivalent substance. 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. For example, 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.
[0552] 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:
TABLE-US-00004 ALIPHATIC Non-polar G A P I L V Polar - uncharged C
S T M N Q Polar - charged D E K R AROMATIC H F W Y
[0553] The present invention also encompasses homologous
substitution (substitution and replacement are both used herein to
mean the interchange of an existing amino acid residue, with an
alternative residue) that may occur i.e. like-for-like substitution
such as basic for basic, acidic for acidic, polar for polar etc.
Non-homologous substitution may also occur i.e. from one class of
residue to another or alternatively involving the inclusion of
unnatural amino acids such as ornithine (hereinafter referred to as
Z), diaminobutyric acid ornithine (hereinafter referred to as B),
norleucine ornithine (hereinafter referred to as O), pyriylalanine,
thienylalanine, naphthylalanine and phenylglycine.
[0554] Replacements may also be made by unnatural amino acids
include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino
acids*, lactic acid*, halide derivatives of natural amino acids
such as trifluorotyrosine*, p-Cl-phenylalanine*,
p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*,
.beta.-alanine*, L-.alpha.-amino butyric acid*, L-.gamma.-amino
butyric acid*, L-.alpha.-amino isobutyric acid*, L-.epsilon.-amino
caproic acid.sup.#, 7-amino heptanoic acid*, L-methionine
sulfone.sup.#*, L-norleucine*, L-norvaline*,
p-nitro-L-phenylalanine*, L-hydroxyproline.sup.#, L-thioproline*,
methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*,
pentamethyl-Phe*, L-Phe (4-amino).sup.#, L-Tyr (methyl)*, L-Phe
(4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl
acid)*, L-diaminopropionic acid # and L-Phe (4-benzyl)*. The
notation * has been utilised for the purpose of the discussion
above (relating to homologous or non-homologous substitution), to
indicate the hydrophobic nature of the derivative whereas # has
been utilised to indicate the hydrophilic nature of the derivative,
#* indicates amphipathic characteristics.
[0555] Variant amino acid sequences may include suitable spacer
groups that may be inserted between any two amino acid residues of
the sequence including alkyl groups such as methyl, ethyl or propyl
groups in addition to amino acid spacers such as glycine or
3-alanine residues. A further form of variation, involves the
presence of one or more amino acid residues in peptoid form, will
be well understood by those skilled in the art. For the avoidance
of doubt, "the peptoid form" is used to refer to variant amino acid
residues wherein the .alpha.-carbon substituent group is on the
residue's nitrogen atom rather than the .alpha.-carbon. Processes
for preparing peptides in the peptoid form are known in the art,
for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and
Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.
[0556] In one embodiment the xylanase for use in the present
invention may comprise a polypeptide sequence shown as SEQ ID No.
1, SEQ ID No. 2 or SEQ ID No. 3. SEQ ID No. 9, SEQ ID No. 10 SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids.
[0557] Suitably there may be at least 2 conservative substitutions,
such as at least 3 or at least 4 or at least 5.
[0558] Suitably there may be less than 15 conservative
substitutions, such as less than 12, less than 10, or less than 8
or less than 5.
[0559] The nucleotide sequences for use in the present invention
may include within them synthetic or modified nucleotides. A number
of different types of modification to oligonucleotides are known in
the art. These include methylphosphonate and phosphorothioate
backbones and/or the addition of acridine or polylysine chains at
the 3' and/or 5' ends of the molecule.
[0560] For the purposes of the present invention, it is to be
understood that the nucleotide sequences described herein may be
modified by any method available in the art. Such modifications may
be carried out in order to enhance the in vivo activity or life
span of nucleotide sequences of the present invention.
[0561] The present invention also encompasses the use of nucleotide
sequences that are complementary to the sequences presented herein,
or any derivative, fragment or derivative thereof. If the sequence
is complementary to a fragment thereof then that sequence can be
used as a probe to identify similar coding sequences in other
organisms etc.
[0562] Polynucleotides 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 individuals, for example
individuals from different populations. In addition, other
homologues 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. 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 any one of the sequences in
the attached sequence listings under conditions of medium to high
stringency. Similar considerations apply to obtaining species
homologues and allelic variants of the polypeptide or nucleotide
sequences of the invention.
[0563] 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.
[0564] 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.
[0565] Alternatively, such polynucleotides may be obtained by site
directed mutagenesis of characterised sequences. This may be useful
where for example silent codon sequence changes are required to
optimise codon preferences for a particular host cell in which the
polynucleotide sequences are being expressed. Other sequence
changes may be desired in order to introduce restriction enzyme
recognition sites, or to alter the property or function of the
polypeptides encoded by the polynucleotides.
[0566] Polynucleotides (nucleotide sequences) of the 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 polynucleotides 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
polynucleotides of the invention as used herein.
[0567] Polynucleotides such as 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.
[0568] In general, primers will be produced by synthetic means,
involving a stepwise 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.
[0569] Longer polynucleotides will generally be produced using
recombinant means, for example using a PCR (polymerase chain
reaction) cloning techniques. 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.
[0570] Amino Acid Numbering
[0571] In the present invention, a specific numbering of amino acid
residue positions in the xylanases used in the present invention
may be employed. By alignment of the amino acid sequence of a
sample xylanases with the xylanase of the present invention
(particularly SEQ ID No. 3) it is possible to allot a number to an
amino acid residue position in said sample xylanase which
corresponds with the amino acid residue position or numbering of
the amino acid sequence shown in SEQ ID NO:3 of the present
invention.
[0572] Hybridisation
[0573] The present invention also encompasses sequences that are
complementary to the nucleic acid sequences of the present
invention or sequences that are capable of hybridising either to
the sequences of the present invention or to sequences that are
complementary thereto.
[0574] The term "hybridisation" as used herein 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.
[0575] The present invention also encompasses the use of nucleotide
sequences that are capable of hybridising to the sequences that are
complementary to the sequences presented herein, or any fragment or
derivative thereof.
[0576] The term "variant" also encompasses sequences that are
complementary to sequences that are capable of hybridising to the
nucleotide sequences presented herein.
[0577] Preferably, the term "variant" encompasses sequences that
are complementary to sequences that are capable of hybridising
under stringent conditions (e.g. 50.degree. C. and 0.2.times.SSC
{1.times.SSC=0.15 M NaCl, 0.015 M Na.sub.3citrate pH 7.0}) to the
nucleotide sequences presented herein.
[0578] More preferably, the term "variant" encompasses sequences
that are complementary to sequences that are capable of hybridising
under high stringency conditions (e.g. 65.degree. C. and
0.1.times.SSC {1.times.SSC=0.15 M NaCl, 0.015 M Na.sub.3citrate pH
7.0}) to the nucleotide sequences presented herein.
[0579] The present invention also relates to nucleotide sequences
that can hybridise to the nucleotide sequences of the present
invention (including complementary sequences of those presented
herein).
[0580] The present invention also relates to nucleotide sequences
that are complementary to sequences that can hybridise to the
nucleotide sequences of the present invention (including
complementary sequences of those presented herein).
[0581] Preferably hybridisation is analysed over the whole of the
sequences taught herein.
[0582] Expression of Enzymes
[0583] The nucleotide sequence for use in the present invention may
be incorporated into a recombinant replicable vector. The vector
may be used to replicate and express the nucleotide sequence, in
protein/enzyme form, in and/or from a compatible host cell.
[0584] Expression may be controlled using control sequences e.g.
regulatory sequences.
[0585] The protein produced by a host recombinant cell by
expression of the nucleotide sequence may be secreted or may be
contained intracellularly depending on the sequence and/or the
vector used. The coding sequences may be designed with signal
sequences which direct secretion of the substance coding sequences
through a particular prokaryotic or eukaryotic cell membrane.
[0586] Expression Vector
[0587] The term "expression vector" means a construct capable of in
vivo or in vitro expression.
[0588] Preferably, the expression vector is incorporated into the
genome of a suitable host organism. The term "incorporated"
preferably covers stable incorporation into the genome.
[0589] The nucleotide sequence of the present invention may be
present in a vector in which the nucleotide sequence is operably
linked to regulatory sequences capable of providing for the
expression of the nucleotide sequence by a suitable host
organism.
[0590] The vectors for use in the present invention may be
transformed into a suitable host cell as described below to provide
for expression of a polypeptide of the present invention.
[0591] The choice of vector e.g. a plasmid, cosmid, or phage vector
will often depend on the host cell into which it is to be
introduced.
[0592] The vectors for use in the present invention may contain one
or more selectable marker genes--such as a gene, which confers
antibiotic resistance e.g. ampicillin, kanamycin, chloramphenicol
or tetracyclin resistance. Alternatively, the selection may be
accomplished by co-transformation (as described in WO91/17243).
[0593] Vectors may be used in vitro, for example for the production
of RNA or used to transfect, transform, transduce or infect a host
cell.
[0594] Thus, in a further embodiment, the invention provides a
method of making nucleotide sequences 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.
[0595] The vector may further comprise a nucleotide sequence
enabling the vector to replicate in the host cell in question.
Examples of such sequences are the origins of replication of
plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and plJ702.
[0596] Regulatory Sequences
[0597] In some applications, the nucleotide sequence for use in the
present invention is operably linked to a regulatory sequence which
is capable of providing for the expression of the nucleotide
sequence, such as by the chosen host cell. By way of example, the
present invention covers a vector comprising the nucleotide
sequence of the present invention operably linked to such a
regulatory sequence, i.e. the vector is an expression vector.
[0598] The term "operably linked" refers to a juxtaposition wherein
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.
[0599] The term "regulatory sequences" includes promoters and
enhancers and other expression regulation signals.
[0600] The term "promoter" is used in the normal sense of the art,
e.g. an RNA polymerase binding site.
[0601] Enhanced expression of the nucleotide sequence encoding the
enzyme of the present invention may also be achieved by the
selection of heterologous regulatory regions, e.g. promoter,
secretion leader and terminator regions.
[0602] Preferably, the nucleotide sequence according to the present
invention is operably linked to at least a promoter.
[0603] Other promoters may even be used to direct expression of the
polypeptide of the present invention.
[0604] Examples of suitable promoters for directing the
transcription of the nucleotide sequence in a bacterial, fungal or
yeast host are well known in the art.
[0605] The promoter can additionally include features to ensure or
to increase expression in a suitable host. For example, the
features can be conserved regions such as a Pribnow Box or a TATA
box.
[0606] Constructs
[0607] The term "construct"--which is synonymous with terms such as
"conjugate", "cassette" and "hybrid"--includes a nucleotide
sequence for use according to the present invention directly or
indirectly attached to a promoter.
[0608] An example of an indirect attachment is the provision of a
suitable spacer group such as an intron sequence, such as the
Sh1-intron or the ADH intron, intermediate the promoter and the
nucleotide sequence of the present invention. The same is true for
the term "fused" in relation to the present invention which
includes direct or indirect attachment. In some cases, the terms do
not cover the natural combination of the nucleotide sequence coding
for the protein ordinarily associated with the wild type gene
promoter and when they are both in their natural environment.
[0609] The construct may even contain or express a marker, which
allows for the selection of the genetic construct.
[0610] For some applications, preferably the construct of the
present invention comprises at least the nucleotide sequence of the
present invention operably linked to a promoter.
[0611] Host Cells
[0612] The term "host cell"--in relation to the present invention
includes any cell that comprises either the nucleotide sequence or
an expression vector as described above and which is used in the
recombinant production of a protein having the specific properties
as defined herein.
[0613] In one embodiment the organism is an expression host.
[0614] Thus, a further embodiment of the present invention provides
host cells transformed or transfected with a nucleotide sequence
that expresses the protein of the present invention.
[0615] The cells will be chosen to be compatible with the said
vector and may for example be prokaryotic (for example bacterial),
fungal or yeast cells.
[0616] Examples of suitable bacterial host organisms are gram
positive or gram negative bacterial species.
[0617] In one embodiment the xylanases taught herein are expressed
in the expression host Trichoderma reesei.
[0618] In some embodiments the expression host for the xylanases
taught herein may be one or more of the following fungal expression
hosts: Fusarium spp. (such as Fusarium oxysporum); Aspergillus spp.
(such as Aspergillus niger, A. oryzae, A. nidulans, or A. awamon)
or Trichoderma spp. (such as T. reesei).
[0619] In some embodiments the expression host may be one or more
of the following bacterial expression hosts: Streptomyces spp. or
Bacillus spp. (e.g. Bacillus subtilis or B. licheniformis).
[0620] The use of suitable host cells--such as yeast and fungal
host cells--may provide for post-translational modifications (e.g.
myristoylation, glycosylation, truncation, lipidation and tyrosine,
serine or threonine phosphorylation) as may be needed to confer
optimal biological activity on recombinant expression products of
the present invention.
[0621] Organism
[0622] The term "organism" in relation to the present invention
includes any organism that could comprise the nucleotide sequence
coding for the polypeptide according to the present invention
and/or products obtained therefrom, and/or wherein a promoter can
allow expression of the nucleotide sequence according to the
present invention when present in the organism.
[0623] In one embodiment the organism is an expression host.
[0624] Suitable organisms may include a prokaryote, fungus, yeast
or a plant.
[0625] The term "transgenic organism" in relation to the present
invention includes any organism that comprises the nucleotide
sequence coding for the polypeptide according to the present
invention and/or the products obtained therefrom, and/or wherein a
promoter can allow expression of the nucleotide sequence according
to the present invention within the organism. Preferably the
nucleotide sequence is incorporated in the genome of the
organism.
[0626] The term "transgenic organism" does not cover native
nucleotide coding sequences in their natural environment when they
are under the control of their native promoter which is also in its
natural environment.
[0627] Therefore, the transgenic organism of the present invention
includes an organism comprising any one of, or combinations of, the
nucleotide sequence coding for the polypeptide according to the
present invention, constructs according to the present invention,
vectors according to the present invention, plasmids according to
the present invention, cells according to the present invention,
tissues according to the present invention, or the products
thereof.
[0628] For example the transgenic organism may also comprise the
nucleotide sequence coding for the polypeptide of the present
invention under the control of a heterologous promoter.
[0629] Transformation of Host Cells/Organism
[0630] As indicated earlier, the host organism can be a prokaryotic
or a eukaryotic organism. Examples of suitable prokaryotic hosts
include E. coli, Streptomyces spp. and Bacillus spp., e.g. Bacillus
subtilis.
[0631] Teachings on the transformation of prokaryotic hosts is well
documented in the art, for example see Sambrook et al (Molecular
Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor
Laboratory Press). If a prokaryotic host is used then the
nucleotide sequence may need to be suitably modified before
transformation--such as by removal of introns.
[0632] Filamentous fungi cells may be transformed using various
methods known in the art--such as a process involving protoplast
formation and transformation of the protoplasts followed by
regeneration of the cell wall in a manner known. The use of
Aspergillus as a host microorganism is described in EP 0 238
023.
[0633] Transformation of prokaryotes, fungi and yeasts are
generally well known to one skilled in the art.
[0634] A host organism may be a fungus--such as a mould. Examples
of suitable such hosts include any member belonging to the genera
Trichoderma (e.g. T. reesei), Thermomyces, Acremonium, Fusarium,
Aspergillus, Penicillium, Mucor, Neurospora and the like.
[0635] In one embodiment, the host organism may be a fungus. In one
preferred embodiment the host organism belongs to the genus
Trichoderma, e.g. T. reesei).
[0636] Culturing and Production
[0637] Host cells transformed with the nucleotide sequence of the
present invention may be cultured under conditions conducive to the
production of the encoded polypeptide and which facilitate recovery
of the polypeptide from the cells and/or culture medium.
[0638] The medium used to cultivate the cells may be any
conventional medium suitable for growing the host cell in questions
and obtaining expression of the polypeptide.
[0639] The protein produced by a recombinant cell may be displayed
on the surface of the cell.
[0640] The protein may be secreted from the host cells and may
conveniently be recovered from the culture medium using well-known
procedures.
[0641] Secretion
[0642] Often, it is desirable for the protein to be secreted from
the expression host into the culture medium from where the protein
may be more easily recovered. According to the present invention,
the secretion leader sequence may be selected on the basis of the
desired expression host. Hybrid signal sequences may also be used
with the context of the present invention.
[0643] Large Scale Application
[0644] In one preferred embodiment of the present invention, the
amino acid sequence is used for large scale applications.
[0645] Preferably the amino acid sequence is produced in a quantity
of from 1 g per litre to about 2 g per litre of the total cell
culture volume after cultivation of the host organism.
[0646] Preferably the amino acid sequence is produced in a quantity
of from 100 mg per litre to about 900 mg per litre of the total
cell culture volume after cultivation of the host organism.
[0647] Preferably the amino acid sequence is produced in a quantity
of from 250 mg per litre to about 500 mg per litre of the total
cell culture volume after cultivation of the host organism.
[0648] General Recombinant DNA Methodology Techniques
[0649] The present invention employs, unless otherwise indicated,
conventional techniques of chemistry, molecular biology,
microbiology, recombinant DNA and immunology, which are within the
capabilities of a person of ordinary skill in the art. Such
techniques are explained in the literature. See, for example, J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning:
A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor
Laboratory Press; Ausubel, F. M. et al. (1995 and periodic
supplements; Current Protocols in Molecular Biology, ch. 9, 13, and
16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree,
and A. Kahn, 1996, DNA Isolation and Sequencing: Essential
Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984,
Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D.
M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA
Structure Part A: Synthesis and Physical Analysis of DNA Methods in
Enzymology, Academic Press. Each of these general texts is herein
incorporated by reference.
[0650] The invention also relates to the following aspects as
defined in the following numbered paragraphs: [0651] 1. A method
for degrading arabinoxylan-containing material comprising admixing
an arabinoxylan-containing material with a xylanase, which xylanase
is a GH10, fungal xylanase and degrades insoluble arabinoxylan
(AXinsol) as well as degrading the polymers, oligomers or
combinations thereof produced from the degradation of the AXinsol,
and wherein the xylanase degrades the polymers, oligomers or
combinations thereof produced from the degradation of the AXinsol
immediately or substantially immediately upon their production.
[0652] 2. A method for degrading arabinoxylan-containing material
in a xylan-containing material, comprising admixing said
xylan-containing material with a xylanase comprising a polypeptide
sequence shown herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1,
SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No.15; or a
variant, homologue, fragment or derivative thereof having at least
75% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1 or
SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or
a polypeptide sequence which comprises SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 6, SEQ ID No. 5. SEQ ID No. 4,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity with SEQ ID No. 6, SEQ ID
No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID
No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code, or a xylanase obtainable (or obtained) from
Fusarium verticilloides. [0653] 3. Use of a xylanase comprising a
polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, or or SEQ
ID No. 15; or a variant, homologue, fragment or derivative thereof
having at least 75% identity with SEQ ID No. 3 or SEQ ID No. 2 or
SEQ ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or
SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No.
3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ
ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
can hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID
No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17
or SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity with SEQ ID No. 6, SEQ ID
No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID
No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code, or a xylanase obtainable (or obtained) from
Fusarium verticilloides for solubilizing arabinoxylan in a
xylan-containing material. [0654] 4. The method or use according to
any one of paragraphs 2 or 3 wherein the arabinoxylan is insoluble
arabinoxylan (AXinsol). [0655] 5. The method or use according to
any one of paragraphs 1-4 wherein the xyan-containing material is
selected from one or more of the group consisting of: a feed or
feedstuff; a feed component; a grain-based material; a mash; a
wort; a malt; malted barely; an adjunct, a barley mash; and a
cereal flour. [0656] 6. The method or use according to any one of
the preceding paragraphs wherein the arabinoxylans are solubilized
without increasing viscosity in the reaction medium. [0657] 7. The
method or use according to any one of the preceding paragraphs
wherein the method or use is (or is part of) a wheat gluten-starch
separation process. [0658] 8. The method or use according to any
one of paragraphs 1-6 wherein the method or use is (or is part of)
a biofuel (e.g. bioethanol) or biochemical (e.g. bio-based
isoprene) production process. [0659] 9. The method or use according
to any one of paragraphs 1-6 wherein the method or use is (or is
part of) a malting or brewing process. [0660] 10. The method or use
according to any one of paragraphs 1-6 wherein the method or use is
for improving the performance of a subject or for improving
digestibility of a raw material in a feed (e.g. nutrient
digestibility) or for improving feed efficiency in a subject or for
reducing the viscosity of the intestinal content of a subject,
[0661] 11. The method or use according to any one of the preceding
paragraphs wherein the xylanase comprises a polypeptide sequence
shown herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID
No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a variant,
homologue, fragment or derivative thereof having at least 85%
identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1 or SEQ
ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or a
polypeptide sequence which comprises SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No.
12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 85% identity with SEQ ID No. 6, SEQ ID
No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID
No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code. [0662] 12. The method or use according to any one
of the preceding paragraphs wherein the xylanase comprises a
polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15; or a variant, homologue, fragment or derivative thereof
having at least 95% identity with SEQ ID No. 3 or SEQ ID No. 2 or
SEQ ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or
SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No.
3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ
ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
can hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID
No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17
or SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 95% identity with SEQ ID No. 6, SEQ ID
No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID
No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code. [0663] 13. The method or use according to any one
of the preceding paragraphs wherein the xylanase comprises a
polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15; or a variant, homologue, fragment or derivative thereof
having at least 99% identity with SEQ ID No. 3 or SEQ ID No. 2 or
SEQ ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or
SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No.
3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ
ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
can hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID
No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17
or SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 97% (e.g. at least 97.7% or at least
98%) identity with SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID
No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17
or SEQ ID No. 18, or a nucleotide sequence which differs from SEQ
ID No. 6 or SEQ ID No. 5 or SEQ ID No. 4 or SEQ ID No. 12 or SEQ ID
No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No.17 or SEQ ID
No. 18 due to the degeneracy of the genetic code. [0664] 14. The
method or use according to paragraph 5 wherein the feed or
feedstuff comprises or consists of corn, DDGS (such as cDDGS),
wheat, wheat bran or a combination thereof. [0665] 15. The method
or use according to paragraph 14 wherein the feed or feedstuff is a
corn-based feedstuff. [0666] 16. The method or use according to any
one of the preceding paragraphs wherein the xylanase is used in
combination with one or more of the enzymes selected from the group
consisting of a protease (e.g. subtilisin (E.C. 3.4.21.62) or a
bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C.
3.4.21.x) or a keratinase (E.C. 3.4.x.x)) and/or an amylase
(including .alpha.-amylases (E.C. 3.2.1.1), G4-forming amylases
(E.C. 3.2.1.60), .beta.-amylases (E.C. 3.2.1.2) and
.gamma.-amylases (E.C. 3.2.1.3); and/or a protease (e.g. subtilisin
(E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline
serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)).
[0667] 17. The method or use according to any one of the preceding
paragraphs wherein the xylanase is used in combination with an
amylase (e.g .alpha.-amylases (E.C. 3.2.1.1)) and a protease (e.g.
subtilisin (E.C. 3.4.21.62)). [0668] 18. The method or use
according to any one of paragraphs 1-15 wherein the enzyme is used
in combination with a .beta.-glucanase, e.g. an
endo-1,3(4)-.beta.-glucanases (E.C. 3.2.1.6). [0669] 19. The method
or use according to any one of the preceding paragraphs wherein the
xylanase is contacted with a mash and/or a wort. [0670] 20. The
method or use according to any one of the preceding paragraphs
wherein the method comprises the steps of: (a) preparing a mash,
(b) filtering the mash to obtain a wort and (c) fermenting the wort
to obtain a fermented beverage, wherein said xylanase is added to:
(i) the mash of step (a) and/or (ii) the wort of step (b) and/or
(iii) the wort of step (c). [0671] 21. The method or use according
to any one of the preceding paragraphs wherein the xylanase
comprises (or consists of) a polypeptide sequence shown herein as
SEQ ID No. 3, SEQ ID No. 2 or SEQ ID No. 1, or a variant,
homologue, fragment or derivative thereof having at least 99%
identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1; or a
polypeptide which is encoded by a nucleotide sequence shown herein
as SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4, or a nucleotide
sequence which has at least 97% (e.g. at least 97.7% or at least
98%) identity with SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4.
[0672] 22. A fermented beverage, e.g. beer, produced by a method
according to any one of the preceding paragraphs. [0673] 23. A
polypeptide having xylanase activity comprising (or consisting of)
a polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2
or SEQ ID No. 1, or a variant, homologue, fragment or derivative
thereof having at least 99% identity with SEQ ID No. 3 or SEQ ID
No. 2 or SEQ ID No. 1; or a polypeptide which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5 or
SEQ ID No. 4, or a nucleotide sequence which has at least 97% (e.g.
at least 97.7% or at least 98%) identity with SEQ ID No. 6, SEQ ID
No. 5 or SEQ ID No. 4. [0674] 24. The polypeptide according to
paragraph 23 wherein the xylanase in an endo-1,4-.beta.-d-xylanase.
[0675] 25. The polypeptide according to paragraph 23 or paragraph
24 wherein the enzyme has an optimum temperature in the range of
50-70.degree. C., preferably about 60.degree. C. [0676] 26. A
polypeptide according to any one of paragraphs 23 to 25 wherein the
enzyme has a pH optimum in the range of 4.6 to 7, preferably about
6. [0677] 27. A polypeptide according to any one of paragraphs
23-26 wherein the enzyme is formulated with a coating or is
encapsulated. [0678] 28. An isolated or recombinant nucleic acid
molecule comprising (or consisting of) a polynucleotide sequence
selected from the group consisting of: [0679] a. a polynucleotide
sequence which encodes a polypeptide sequence selected from the
group consisting of SEQ ID No. 3, SEQ ID No. 2 or SEQ ID No. 1, or
a variant, homologue, fragment or derivative thereof having at
least 99% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No.
1; or [0680] b. a polynucleotide sequence shown herein as SEQ ID
No. 6, SEQ ID No. 5 or SEQ ID No. 4; or a nucleotide sequence which
has at least 97% (e.g. at least 97.7% or at least 98%) identity
with SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4. [0681] 29. A
vector (e.g. a plasmid) comprising a polynucleotide sequence
selected from the group consisting of: [0682] a. a polynucleotide
sequence which encodes a polypeptide sequence selected from the
group consisting of SEQ ID No. 3, SEQ ID No. 2 or SEQ ID No. 1, or
a variant, homologue, fragment or derivative thereof having at
least 99% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No.
1; or [0683] b. a polynucleotide sequence shown herein as SEQ ID
No. 6, SEQ ID No. 5 or SEQ ID No. 4; or a nucleotide sequence which
has at least 97% (e.g. at least 97.7% or at least 98%) identity
with SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4.
[0684] 30. A host cell comprising the nucleic acid of paragraph 28
or a vector of paragraph 29. [0685] 31. A method of degrading a
xylan-containing material (preferably an insoluble
arabinoxylan-containing material) comprising admixing the material
with a polypeptide according to any one of paragraphs 23-27. [0686]
32. A feed additive composition comprising (or consisting
essentially of or consisting of) a xylanase comprising a
polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15; r a variant, homologue, fragment or derivative thereof
having at least 75% identity with SEQ ID No. 3 or SEQ ID No. 2 or
SEQ ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or
SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No.
3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ
ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
can hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID
No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17
or SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity with SEQ ID No. 6, SEQ ID
No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID
No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code, or a xylanase obtainable (or obtained) from
Fusarium verticilloides or a polypeptide according to any one of
paragraphs 23 to 27. [0687] 33. The feed additive composition
according to paragraph 32 which further comprises one or more of
the enzymes selected from the group. consisting of an amylase
(including .alpha.-amylases (E.C. 3.2.1.1), G4-forming amylases
(E.C. 3.2.1.60), .beta.-amylases (E.C. 3.2.1.2) and
.gamma.-amylases (E.C. 3.2.1.3); and/or a protease (e.g. [0688]
subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or
an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C.
3.4.x.x)). [0689] 34. The feed additive composition according to
paragraph 32 or paragraph 33 which further comprises an amylase
(e.g .alpha.-amylases (E.C. 3.2.1.1)) and a protease (e.g.
subtilisin (E.C. 3.4.21.62)). [0690] 35. The feed additive
composition according to paragraph 32 which further comprises a
.beta.-glucanase, e.g. an endo-1,3(4)-.beta.-glucanases (E.C.
3.2.1.6). [0691] 36. A premix comprising a xylanase comprising a
polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15; or a variant, homologue, fragment or derivative thereof
having at least 75% identity with SEQ ID No. 3 or SEQ ID No. 2 or
SEQ ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or
SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No.
3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ
ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
can hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID
No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17
or SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity with SEQ ID No. 6, SEQ ID
No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID
No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code; a polypeptide according to any one of paragraphs
23 to 27; or a feed additive composition comprising according to
any one of paragraphs 32 to 35; or a xylanase obtainable (or
obtained) from Fusarium verticilloides, and at least one mineral
and/or at least one vitamin. [0692] 37. The method or use according
to any one of paragraphs 1-21 comprising administering a subject
with a polypeptide according to any one of paragraphs 23-27 or a
feed additive composition according to any one of paragraphs 32 to
35 or a premix according to paragraph 36 or a xylanase obtainable
(or obtained) from Fusarium verticilloides. [0693] 38. A kit
comprising a polypeptide according to any one of paragraphs 23-27
or a feed additive composition according to any one of paragraphs
32-35 or a premix according to paragraph 36 and instructions for
administration. [0694] 39. A feedstuff comprising a feed additive
composition according to any one of paragraphs 32-35 or a feed
additive composition comprising (or consisting essentially of or
consisting of) a xylanase comprising a polypeptide sequence shown
herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9,
SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a variant,
homologue, fragment or derivative thereof having at least 75%
identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1 or SEQ
ID No. 9 or SEQ ID No. 10 or SEQ ID No.11 or SEQ ID No. 15; or a
polypeptide sequence which comprises SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15 with a conservative substitution of at least one of the
amino acids; or a xylanase which is encoded by a nucleotide
sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4,
SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No.17 or SEQ ID No. 18, or a nucleotide sequence which can
hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No.
12, SEQ ID No. 13 or SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17 or
SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity with SEQ ID No. 6, SEQ ID
No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13. SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID
No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code; a polypeptide according to any one of paragraphs
23-27; or a xylanase obtainable (or obtained) from Fusarium
verticilioides. [0695] 40. A method of preparing a feedstuff
comprising admixing a feed component with a xylanase comprising a
polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2,
SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID
No. 15; or a variant, homologue, fragment or derivative thereof
having at least 75% identity with SEQ ID No. 3 or SEQ ID No. 2 or
SEQ ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or
SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No.
3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11 or SEQ ID No. 15 with a conservative substitution of at
least one of the amino acids; or a xylanase which is encoded by a
nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ
ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.
16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide sequence which
can hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID
No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No.17
or SEQ ID No. 18 under high stringency conditions, or a nucleotide
sequence which has at least 75% identity with SEQ ID No. 6, SEQ ID
No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No.17 or SEQ ID No. 18, or a nucleotide
sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID
No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID
No. 16 or SEQ ID No.17 or SEQ ID No. 18 due to the degeneracy of
the genetic code; a polypeptide according to paragraphs 23-27, or a
xylanase obtainable (or obtained) from Fusarium verticilloides or a
feed additive composition according to any one of paragraphs 32-35.
[0696] 41. A polypeptide, nucleic acid, vector, host cells,
methods, uses and kits as generally described herein with reference
to the Figures and Examples.
[0697] The invention will now be described, by way of example only,
with reference to the following Figures and Examples.
EXAMPLES
Example 1
[0698] Cloning of Fusarium Verticillioides Xylanase (FveXyn4)
[0699] Genomic DNA isolated from a strain of Fusarium
verticilioides was used for amplifying a xylanase gene. The
sequence of the cloned gene, called the FveXyn4 gene, is depicted
in SEQ ID No. 4. The protein encoded by the FveXyn4 gene is
depicted in SEQ ID No. 1. The protein product of gene FveXyn4
belongs to glycosyl hydrolase family 10 (GH10) based on the PFAM
search (http://pfam.sanger.ac.uk/). At the N-terminus, FveXyn4
protein has a 15 amino acid signal peptide predicted by SignalP-NN
(Emanuelsson et al., Nature Protocols, 2:953-971, 2007). This
indicates that FveXyn4 is a secreted glycosyl hydrolase.
Example 2
[0700] Expression of FveXyn4 Protein
[0701] The FveXyn4 gene was amplified from genomic DNA of Fusarium
verticillioides using the following primers: Primer 1
5'-caccATGAAGCTGTCTTCTTTCCTCTA-3' (SEQ ID No. 7), and Primer 2
5'-TTTTTAGCGGAGAGCGTTGACAACAGC-3' (SEQ ID No. 8). The PCR product
was cloned into pENTR/D-TOPO vector (Invitrogen K2400) to generate
the FveXyn4 pEntry plasmid. The expression plasmid pZZH254 was
obtained by Gateway cloning reaction between the FveXyn4 pEntry
plasmid and pTrex3gM expression vector (described in US
2011/0136197 A1) using Gateway.RTM. LR Clonase.RTM. II enzyme kit
(Invitrogen 11791). A map of plasmid pZZH254 is provided as FIG.
10. The sequence of the FveXyn4 gene was confirmed by DNA
sequencing (SEQ ID No. 4). The plasmid pZZH254 was transformed into
a quad deleted Trichoderma reesei strain (described in WO
05/001036) using biolistic method (Te'o V S et al., J Microbiol
Methods, 51:393-9, 2002).
[0702] Following sequence confirmation, protoplasts of a quad
deleted T. reesei strain (described in WO 05/001036) were
transformed with the expression plasmid pTTT-Ate CA1 using the PEG
protoplast method (Penttila et al, Gene, 61:155-164, 1987). For
protoplast preparation, spores were grown for about 10 hours at
24.degree. C. in Trichoderma Minimal Medium MM (20 g/L glucose, 15
g/L KH.sub.2PO.sub.4, pH 4.5, 5 g/L (NH.sub.4).sub.2SO.sub.4, 0.6
g/L MgSO.sub.4.times.7H.sub.2O, 0.6 g/L CaCl.sub.2.times.2H.sub.2,
1 ml of 1000.times.T. reesei Trace elements solution (175 g/L
Citric Acid anhydrous, 200 g/L FeSO.sub.4.times.7H.sub.2, 16 g/L
ZnSO.sub.4.times.7H.sub.2, 3.2 g/L CuSO.sub.4, 1.4 g/L
MnSO.sub.4.times.H.sub.2, and 0.8 g/L Boric Acid). Germinating
spores were harvested by centrifugation and treated with 30 mg/mL
Vinoflow FCE (Novozymes, AG Switzerland) solution for from 7 hours
to overnight at 30.degree. C. at 100 rpm to lyse the fungal cell
walls. Protoplasts were washed in 0.1 M Tris HC buffer (pH 7)
containing 0.6 M sorbitol and resuspended in 10 mM Tris HCl buffer
(pH 7.5) containing 1.2 M sorbitol and 10 mM calcium chloride. For
PEG transformation, approximately 1 .mu.g of DNA and
1-5.times.10.sup.7 protoplasts in a total volume of 200 .mu.l were
treated with 2 ml of 25% PEG solution, diluted with 2 volumes of
1.2 M sorbitol/10 mM Tris, pH 7.5/10 mM CaCl.sub.2) solution.
Transformants were selected on a medium containing acetamide as a
sole source of nitrogen (acetamide 0.6 g/L; cesium chloride 1.68
g/L; glucose 20 g/L; potassium dihydrogen phosphate 15 g/L;
magnesium sulfate heptahydrate 0.6 g/L; calcium chloride dihydrate
0.6 g/L; iron (II) sulfate 5 mg/L; zinc sulfate 1.4 mg/L; cobalt
(II) chloride 1 mg/L; manganese (II) sulfate 1.6 mg/L; agar 20 g/L;
pH 4.25). Transformed colonies (about 50-100) appeared in about 1
week. After growth on acetamide plates, the spores were collected
and reselected on acetamide plates. After 5 days, the spores were
collected using 10% glycerol, and 1.times.10.sup.8 spores were
inoculated in a 250 ml shake flask with 30 ml Glucose/Sophorose
defined medium for protein expression. Protein expression was
confirmed by SDS-PAGE. The spore suspension was subsequently grown
in a 7 L fermentor in a defined medium containing 60%
glucose-sophorose feed. Glucose/Sophorose defined medium (per
liter) consists of (NH.sub.4).sub.2SO.sub.4 5 g, PIPPS buffer 33 g,
Casamino Acids 9 g, KH.sub.2PO.sub.4 4.5 g, CaCl.sub.2 (anhydrous)
1 g, MgSO.sub.4.7H.sub.2O 1 g, pH to 5.5 adjusted with 50% NaOH
with Milli-Q H.sub.2O to bring to 966.5 mL. After sterilization,
the following were added: 26 mL 60% Glucose/Sophrose, and
400.times.T. reesei Trace Metals 2.5 mL.
[0703] FveXyn4 was purified from concentrated fermentation broth of
a 7 L fermentor culture using two chromatography columns.
Concentrated fermentation broth buffered in 20 mM sodium phosphate
buffer pH 6.0 containing 1 M ammonium sulfate was loaded on a
hydrophobic interaction chromatography column (Sepharose Phenyl FF,
26/10). The protein was eluted from the column using a linear
gradient of equilibration/wash buffer to 20 mM sodium phosphate
buffer pH 6.0. The fraction containing FveXyn4 protein was loaded
onto a gel filtration column (HiLoad Superdex 75 pg 26/60), and the
mobile phase used was 20 mM sodium phosphate, pH 7.0, containing
0.15 M NaCl. The purified protein was concentrated using a 3K
Amicon Ultra-15 device and the concentrated protein fraction was
used in further studies.
[0704] The nucleotide sequence of FveXyn4 gene from expression
plasmid pZZH254 is set forth as SEQ ID No. 4. The signal sequence
is shown in bold (upper case), and the predicted intron is shown in
bold and lowercase.
[0705] The amino acid sequence of FveXyn4 protein expressed from
plasmid pZZH254 is set forth as SEQ ID No. 1. The signal sequence
predicted by SignalP-NN software is shown underlined. This is the
pre-pro-protein.
[0706] The amino acid sequence of the predicted mature form of
FveXyn4 protein is set forth as SEQ ID No. 3. This is the active
form of the enzyme. SEQ ID No. 2 shows the pro-protein, i.e. before
post-translational modification. Depending on the host the
post-translation modification may vary and therefore the present
invention also encompasses mature, active forms of SEQ ID No.
2.
Example 3
[0707] Xylanase Activity of FveXyn4
[0708] FveXyn4 belongs to the glycosyl hydrolase 10 family (GH10,
CAZy number). The beta 1-4 xylanase activity of FveXyn4 was
measured using 1% xylan from birch wood (Sigma 95588) or 1%
arabinoxylan from wheat flour (Megazyme P-WAXYM) as substrates. The
assay was performed in 50 mM sodium citrate pH 5.3, 0.005% Tween-80
buffer at 50.degree. C. for 10 minutes.
[0709] The released reducing sugar was quantified by reaction with
3, 5-Dinitrosalicylic acid and measurement of absorbance at 540 nm.
The enzyme activity is quantified relative to a xylose standard
curve. In this assay, one xylanase unit (U) is defined as the
amount of enzyme required to generate 1 micromole of xylose
reducing sugar equivalents per minute under the conditions of the
assay.
Example 4
[0710] pH Profile of FveXyn4
[0711] The pH profile of FveXyn4 was determined using xylan from
birch wood (Sigma 95588) as substrate. The assay was performed in
Sodium Citrate/Sodium Phosphate buffer solution adjusted to pH
values between 2 and 9. Birchwood xylan (2% solution) dissolved in
water was mixed with same volume of 50 mM Citrate/Phosphate buffer
solution in a 96-well plate, and the substrate was equilibrated at
50.degree. C. before adding enzyme. After 10 minutes, the enzyme
reaction was stopped by transferring 60 microliters of reaction
mixture to a 96-well PCR plate containing 100 microliters of DNS
solution. The PCR plate was heated at 95.degree. C. for 5 minutes
in a Bio-Rad DNA Engine. Then plate was cooled to room temperature
and 100 microliters were transferred from each well to a new
96-well plate. Release of reducing sugars from the substrate was
quantified by measuring the optical density at 540 nm in a
spectrophotometer. Enzyme activity at each pH was reported as
relative activity where the activity at the pH optimum was set to
100%. The pH profile of FveXyn4 is shown in FIG. 11. FveXyn4 was
found to have an optimum pH at about 6, and was found to retain
greater than 70% of maximum activity between pH 4.6 and 7.
Example 5
[0712] Temperature Profile of FveXyn4
[0713] The temperature optimum of purified FveXyn4 was determined
by assaying for xylanase activity at temperatures varying between
40.degree. C. and 75.degree. C. for 10 minutes in 50 mM sodium
citrate buffer at pH 5.3. The activity was reported as relative
activity where the activity at the temperature optimum was set to
100%. The temperature profile of FveXyn4 is shown in FIG. 12.
FveXyn4 was found to have an optimum temperature of 60.degree. C.,
and was found to retain greater than 70% of maximum activity
between 45.degree. C. and 64.degree. C.
Example 6
[0714] Pentosan Solubilisation (Breakdown or Solubilisation of
Insoluble Arabinoxylan (AXinsol))
[0715] The new xylanase (FveXyn4) was cloned, expressed, purified
and characterized and tested against a benchmark xylanase product
(Econase.RTM.XT).
[0716] The ability to solubilize insoluble arabinoxylan from 4
substrates, namely wheat, wheat bran, corn and corn DDGS was used
as the key selection criteria.
[0717] The new xylanase showed strong performance on pentosan
solubilisation from relevant feed raw materials (wheat, wheat bran,
corn, corn DDGS). Surprisingly the new xylanase was far better than
the benchmarks (e.g. the commercially available xylanase products)
on a greater number of substrates.
[0718] In particular the enzyme was surprisingly far superior to
the benchmark on the corn based materials.
[0719] The new xylanase also shows numerous advantageous properties
for its industrial use including unexpectedly high pentosan
solubilisation, particularly in feedstuffs comprising corn.
[0720] 6.1 Materials and Methods
[0721] Enzyme Samples
[0722] The xylanases used in this study are:
[0723] A new GH10 xylanase from Fusarium verticilloides (designated
FveXyn4) expressed in Trichoderma reesei, wherein the xylanase was
used in purified form--this enzyme may be referred to herein as
FveXyn4, and the following benchmark, commercially available
xylanase: Econase.RTM. XT. This benchmark enzyme was extracted from
commercial dry formulated samples. The xylanase component from
Econase.RTM. XT commercial dry formulated samples was extracted in
a 33% (w/w) slurry using McIlvain buffer, pH 5.0. The extract was
cleared using centrifugation (3000 RCF for 10 min) and filtered
using a PALL Acrodisc PF syringe filter (0.8/0.2 .mu.m Supor
membrane) and subsequently heated 20 min at 70.degree. C. After
removable of precipitation by centrifugation (38 724 RCF for 15
min) the buffer was replaced by passage through a Sephadex G25
column (PD10 from Pharmacia) equilibrated with 20 mM Na Citrate, 20
mM NaCl, pH 3.4. Purification of the xylanase component was
performed using Source 15S resin, followed by elution with a linear
increasing salt gradient (NaCl in 20 mM Na Citrate buffer pH
3.4).
[0724] Econase XT.RTM. is an endo-1,4-.beta.-xylanase (EC 3.2.1.8)
produced by the strain Trichoderma reesei RF5427 (CBS 114044),
available from ABVista.
[0725] Protein concentration was determined by measuring absorption
at 280 nm. The extinction coefficients were estimates from the
amino acid sequences. For Econase XT the absorption at 280 nm of 1
mg/ml was calculated to be 2.84 AU.
[0726] Feed Raw Materials
[0727] The feed used in these experiments is raw material. The
feeds are either corn, corn DDGS, wheat or wheat bran.
[0728] Pentosan Solubilisation (AXinsol Solubilisation)
[0729] The method used for pentosan solubilisation was: 100 mg of
feed raw material was transferred to a 2 ml Eppendorf centrifuge
tube and the precise weight recorded. 750 .mu.L incubation buffer
(200 mM HEPES, 100 mM NaCl, 2 mM CaCl, pH 6.0) and 900 .mu.l
chloramphenicol solution (40 .mu.g/ml in incubation buffer) was
added. Enzyme of choice was added to make a total volume of 1.8
mL.
[0730] Each sample was assayed in doublets and in parallel with a
blank (incubation without exogenously added enzyme). The samples
were incubated on an Eppendorf thermomixer at 40.degree. C. with
shaking. After 2 or 18 hours of incubation the supernatant was
filtered using 96 wells filterplates (Pall Corporation, AcroPrep 96
Filter Plate, 1.0 .mu.m Glass, NTRL, 1 mL well). After filtration
the samples were stored at 4.degree. C. until analysis for total
amount of C5 sugars, arabinose and xylose.
[0731] Quantification of C5 Sugars (Pentosans)
[0732] The total amount of pentoses brought into solution was
measured using the method of Rouau and Surget (1994, A rapid
semi-automated method of the determination of total and
water-extractable pentosan in wheat flours. Carbohydrate Polymers,
24, 123-32) with a continuous flow injection apparatus (FIG. 7).
The supernatants were treated with acid to hydrolyse
polysaccharides to monosugars. Phloroglucinol (1, 3,
5-trihydroxybenzen) was added for reaction with monopentoses and
monohexoses, which forms a coloured complex. By measuring the
difference in absorbance at 550 nm compared to 510 nm, the amount
of pentoses in the solution was calculated using a standard curve.
Unlike the pentose-phloroglucinol complex, the absorbance of the
hexose-phloroglucinol complex is constant at these wavelengths.
Glucose was added to the phloroglucinol solution to create a
constant glucose signal and further ensure no interference from
hexose sugars.
[0733] 6.2 Results and Discussion
[0734] Pentosan solubilisation was monitored in a dose response
setup using fibrous by-products of wheat (namely wheat bran) and a
fibrous by-product of corn (namely cDDGS).
[0735] The results from benchmark Econase.RTM. XT and of the novel
xylanase (FveXyn4) are shown in FIG. 8 (in wheat bran) and FIG. 9
(in corn DDGS).
[0736] FIG. 8 shows pentosan (C-5 sugar) release (solubilisation of
pentosans) from wheat bran as a function of xylanase dosage. The
xylanases used were the xylanase of the present invention (FveXyn4)
compared with the benchmark xylanase namely Econase.RTM. XT.
[0737] FIG. 9 shows solubilisation of pentosans from cDDGS as a
function of xylanase dosage. The xylanases used were the xylanase
of the present invention (FveXyn4) compared with the benchmark
xylanase namely Econase.RTM. XT.
[0738] Econase.RTM. XT performs well on wheat but shows no or
limited effect on corn.
[0739] This indicates a clear difference in substrate specificity
compared to for instance FveXyn4, surprisingly FveXyn4 is good at
breaking down AXinsol (e.g. solubilising pentosans) in both wheat
and corn based substrates. Typically xylanase enzymes are poor
performers on corn based products. Surprisingly the present enzyme
is capable of dissolving insoluble arabinoxylans (AXinsol) in both
wheat and corn based substrates.
[0740] It is worth noting that the tested commercially available
xylanase (Econase.RTM. XT) did not show significant solubilisation
of corn. In fact, there are very few commercially available
xylanases that show significant ability to dissolve AXinsol in corn
(or solubilisation of corn).
[0741] This is where the present enzyme differs significantly.
Example 7
[0742] Viscosity Reduction in In Vitro Animal Model Assay
[0743] Viscosity reduction on wheat was determined using a modified
version of the procedure described by Bedford & Classen (1993
Poultry Sci., 72, 137-143). 3.6 mL of pepsin solution (2000 U/mL in
0.1 N HCl) was mixed with 2.4 g wheat prior to addition of the
indicated amount of xylanase (FveXyn4) followed by 45 min
incubation at 40.degree. C. 1.2 ml pancreatin solution (8 mg/mL in
1 M MES, pH 6.8) was then mixed into the slurry resulting in a
final pH at 6.0. The sample was allowed to incubate for 60 min at
40.degree. C. with mixing after 30 and 60 min. The sample was then
placed on ice for 5 min to stop the reaction and centrifuged 10 min
at 3320 RCF followed by filtration through a 0.45 .mu.m filter to
obtain a clear supernatant. Sample viscosity was then measured at
20.degree. C. using a Brookfield digital viscometer (model DV-I+,
Brookfield Engineering Laboratories, Stoughton, Mass. 02172, USA)
fitted with a CPE-40 cone and plate. Each data point is the average
of three repetitions.
[0744] The results are shown in FIG. 13. As can be seen even under
these conditions (which conditions attempt to mimic the environment
in the small intestine of an animal) FveXyn4 reduces the
viscosity.
Example 8
[0745] Viscosity Reduction in Grain-Based Material (e.g. for
Biofuel Production)
[0746] In the European fuel alcohol industry, small grains like
wheat, barley and rye are common raw materials, in contrast to the
US where mainly corn is used. These small grains contain, next to
starch, high levels of non-starch polysaccharide polymers (NSP),
like cellulose, beta-glucan and hemicellulose.
[0747] The ratio in which the different NSPs are represented differ
for each feedstock.
[0748] NSPs give high viscosity to grain mashes due to their large
water-binding capacity. High viscosity has a negative impact on
ethanol production since it will limit the solid concentration that
can be used in mashing and it will reduce the energy efficiency of
the process. In addition, residual hemicelluloses present
throughout the process may contribute to fouling in heat exchangers
and distillation equipment. The largest impact of a high viscosity
is seen when a mash is cooled to fermentation temperature
(32.degree. C.). This explains that the viscosity needs to be
reduced in the process anywhere before the cooling step.
[0749] 8.1 Materials and Methods
[0750] A Rapid Visco Analyzer (RVA 4500) from Perten Instruments
was used to measure viscosity profiles of a wheat mash. The RVA
4500 is a cooking stirring viscometer with ramped temperature and
variable shear that can be used to determine the quality and
processing characteristics of starch in grains, tubers, flours and
extruded and cooked foods and feeds.
[0751] There are also applications for protein foods, ingredients
such as modified starches and hydrocolloids and malting and
brewing.
[0752] The wheat mash (50 grams of a 30% DS, 34.86% `as is` slurry)
was prepared according to the following protocol: [0753] Weigh
17.42 grams of wheat [0754] In a 50 ml beaker glass, weigh 32.58
grams of tap water and add 114 .mu.l 4N H.sub.2SO.sub.4 [0755] Add
the wheat to the water and stir for 5 minutes at maximum speed
(approx. 500 rpm) with an overhead stirrer [0756] Transfer 25.0
grams of this slurry (pH 5.2) to an RVA cup, add 50-fold diluted
enzymes and start RVA run [0757] Divide all slurry over two 15-ml
Greiner centrifuge tubes and centrifuge 10 minutes at 3500 rpm
(2547.times.g) [0758] Determine layer separation (read volume scale
on centrifuge tube)
[0759] The term "dry solids content (DS)" refers to the total
solids (dissolved and undissolved) of a slurry (in %) on a dry
weight basis. At the onset, "initial DS" refers to the dry solids
in the slurry at time zero. As the hydrolysis reaction proceeds,
the portion of DS that are dissolved can be referred to as "Syrup
DS" as well as "Supernatant DS".
[0760] In each experiment (25 grams of slurry), xylanases FveXyn4
and FoxXyn2 were dosed at 25 .mu.g protein (per 8.71 g wheat `as
is`), corresponding to 2.9 g protein per g wheat `as is`.
XYLATHIN.TM. was dosed at 4 g protein per g wheat `as is`.
SPEZYME.RTM. CL was dosed at 2.0 AAU/g DS (2.3 AAU/g `as is`).
[0761] A standard wheat liquefaction was mimicked in the RVA.
Pretreatment was performed for 20 minutes at 60.degree. C.,
followed by a liquefaction step for 30 minutes at 85.degree. C.
After pretreatment and liquefaction, slurry was cooled down to
32.degree. C., to determine viscosity at fermentation conditions.
Tables below contain the viscosities after each step, complete RVA
profiles are shown in FIGS. 14 and 18.
[0762] In one experiment, the performance of FveXyn4, Fusarium
verticilloides was compared to Verenium's Xylathin.TM. (benchmark).
Enzyme in Xylathin.TM. is a glycosyl hydrolase family 11 from
uncultured (metagenomic) bacterium (U.S. Pat. No. 7,504,120).
TABLE-US-00005 TABLE 8.1 Viscosity (mPa*s) Xylathin .TM., Blank
benchmark FveXyn4 After pretreatment 626 361 183 (1200 sec. process
time) After liquefaction 495 177 106 (3120 sec. process time) At
fermentation 1005 379 222 temperature (3660 sec. process time) Brix
after RVA run 27.50 .+-. 0.27 27.89 28.18
[0763] The Brix value is a measure of the concentration of
dissolved substance, for instance sugars, in a watery liquid, or
syrup in case of grain processing. It is used to quantify the
solubilization/degradation process of the sugars. The higher the
values, the more sugars are dissolved. Reference values are needed
for comparison. Brix values are expressed in degrees and are
measured using a refractometer.
[0764] These data together with FIG. 14 show that FveXyn4
outperforms Xylathin.TM. on viscosity.
[0765] In another experiment, the performance of FoxXyn2, Fusarium
oxysporum) was compared to Verenium's Xylathin.TM. (benchmark). The
enzyme in Xylathin.TM. is a glycosyl hydrolase family 11 from
uncultured (metagenomic) bacterium (U.S. Pat. No. 7,504,120).
TABLE-US-00006 TABLE 8.2 Viscosity (mPa*s) Xylathin .TM., Blank
benchmark FoxXyn2 After pretreatment 626 361 204 (1200 sec. process
time) After liquefaction 495 177 115 (3120 sec. process time) At
fermentation 1005 379 237 temperature (3660 sec. process time) Brix
after RVA run 27.50 .+-. 0.27 27.89 30.20
[0766] These data together with FIG. 18 show that FoxXyn2
outperforms Xylathin.TM. on viscosity.
Example 9
[0767] Wheat Gluten-Starch Separation
[0768] Separation of wheat flour into starch and gluten fractions
is industrially applied on large scale to obtain high quality
A-starch and byproducts B-starch and vital gluten.
[0769] Separation is improved by addition of xylanases.
[0770] 9.1 Materials and Methods
[0771] The following assay simulates the wheat starch separation of
a batter process at 40.degree. C. In this assay, industrial wheat
flour (Cargill) was added to preheated tap water (50.degree. C.) to
create a 35% DS slurry by mixing 1 minute in a kitchen blender
(Braun). pH of the slurry remained `as is` at .about.6.1. 100 gram
of this slurry was transferred to the Haake VT550 viscometer, which
was calibrated at 40.degree. C. After 1 minute of incubation, the
enzyme solution was added to the slurry. Meanwhile, the viscosity
profile was monitored before and after enzyme addition for 15
minutes in total. After incubation, triplicate samples of the
incubated slurry and one sample f to slurry were taken for a spin
test. Each spin test sample has a total weight of 22.5 g, which
contains 15.8-15.9 g slurry sample added to 6.6-6.7 g of disposable
centrifuge tube (15 ml). All samples were centrifuged in a Hermle
Z400 centrifuge for 15 minutes at 3500 rpm. Brix values were
determined from the syrup of the centrifuged samples.
Example 9A
[0772] In one experiment, the performance of xylanase FveXyn4 in
accordance with the present invention in a wheat gluten-starch
separation process was compared to the commercial enzymes,
Shearzyme Plus.TM. (Novozymes). The commercial enzyme Shearzyme
Plus.TM. was dosed at 0.20 kg/MT DS. Xylanase FveXyn4 was tested at
a dose of 1.19 g protein/MT DS.
[0773] A 35% dry solids batter (pH `as is` at .about.6.1) was
prepared by adding Cargill wheat flour to demineralized water
preheated to 50.degree. C., while continuously mixing it in a Braun
blender for 1 minute at 18900 r.p.m. From this batter, 100 g was
transferred to the Haake VT550 viscometer, preheated to 40.degree.
C. and stirring at .gamma.=50 l/s. The viscosity was measured for 1
minute, after which enzymes were added and the viscosity was
monitored for in total 15 minutes, see results in Table 1. The
underlined values are viscosity values lower, or in other words
better in performance, than those of the benchmark, Shearzyme
Plus.TM.. It is clear that xylanase FveXyn4 is significantly
performing better than the benchmark Shearzyme Plus.TM..
[0774] In succession, three samples of the batter were immediately
after incubation put in 15 ml disposable centrifuge tubes, each
with a total weight of 22.5 g. The tubes were centrifuged for 15
minutes at 3500 r.p.m. in a Hermle Z400 centrifuge, and the
individual layers were measured and averaged for the triplicates.
Table 2 shows the percentage of each layer for the blanc, a batter
without enzyme addition incubated in the Haake VT550 viscometer
under the same conditions, and for all the xylanases tested. The
underlined values of supernatant layer (%) are considerably larger
than those of the benchmark, Shearzyme Plus.TM..
TABLE-US-00007 TABLE 9.1 The effect of xylanases on wheat flour
viscosity in time, during a 15 minute incubation at 40.degree. C.
Viscosity in time (mPA*s) Shearzyme Time Plus .TM. (min) Blanc
(Benchmark) FveXyn4 0 481.13 513.00 494.1 5 393.50 295.90 204.1 10
375.83 238.70 155.5 15 354.97 210.60 130.7 Brix at 5.88 6.07 6.32
t.sub.15
TABLE-US-00008 TABLE 9.2 The effect of xylanases on wheat starch
separation after 15 minute incubation at 40.degree. C. Average of
Shearzyme Separated Plus .TM. layers (%) Blanc (Benchmark) FveXyn4
Starch 33.74 34.6 34.3 Fiber 16.84 14.0 13.2 Sludge 24.22 23.3 20.2
Supernatant 25.20 28.1 32.3
Example 9B
[0775] In a second experiment, the performance of xylanase FoxXyn2
in a wheat starch separation process was compared to the commercial
enzyme, Shearzyme Plus.TM.. The commercial enzyme Shearzyme
Plus.TM. was dosed at 0.20 kg/MT DS. Xylanase FoxXyn2 was tested at
a dose of 1.19 gr protein/MT DS.
[0776] A 35% dry solids batter (pH `as is` at .about.6.1) was
prepared by adding Cargill wheat flour to demineralized water
preheated to 50.degree. C., while continuously mixing it in a Braun
blender for 1 minute at 18900 r.p.m. From this batter, 100 gr. was
transferred to the Haake VT550 viscometer, preheated to 40.degree.
C. and stirring at .gamma.=50 l/s. The viscosity was measured for 1
minute, after which enzymes were added and the viscosity was
monitored for in total 15 minutes, see results in Table 1. The
underlined values are viscosity values lower, or in other words
better in performance, than those of the benchmark, Shearzyme
Plus.TM.. It is clear that xylanase FoxXyn2 is significantly
performing better than the benchmark Shearzyme Plus.TM..
[0777] In succession, three samples of the batter were immediately
after incubation put in 15 ml disposable centrifuge tubes, each
with a total weight of 22.5 gr. The tubes were centrifuged for 15
minutes at 3500 r.p.m. in a Hermle Z400 centrifuge, and the
individual layers were measured and averaged for the triplicates.
Table 2 shows the percentage of each layer for the blanc, a batter
without enzyme addition incubated in the Haake VT550 viscometer
under the same conditions, and for all the xylanases tested. The
underlined values of supernatant layer (%) are considerably larger
than those of the benchmark, Shearzyme Plus.
TABLE-US-00009 TABLE 9.3 The effect of xylanases on wheat flour
viscosity in time, during a 15 minute incubation at 40.degree. C.
Viscosity in time (mPA*s) Shearzyme Time Plus (min) Blanc
(Benchmark) FoxXyn2 0 481.13 513.00 488.4 5 393.50 295.90 203.05 10
375.83 238.70 152.25 15 354.97 210.60 126.35 Brix 5.88 6.07 6.35 at
t.sub.15
TABLE-US-00010 TABLE 9.4 The effect of xylanases on wheat starch
separation after 15 minute incubation at 40.degree. C. Average of
Shearzyme Separated Plus layers (%) Blanc (Benchmark) FoxXyn2
Starch 33.74 34.6 34.1 Fiber 16.84 14.0 13.5 Sludge 24.22 23.3 19.9
Supernatant 25.20 28.1 32.5
Example 10
[0778] Cloning of Fusarium oxysporum Xylanase FoxXyn2
[0779] The nucleotide sequence of the FoxXyn2 gene isolated from
Fusarium oxysporum is set forth as SEQ ID Nos 12, 13 and 14. The
predicted intron is shown in SEQ ID No. 12 (FIG. 19) in italics and
lowercase.
[0780] The amino acid sequence of the FoxXyn2 precursor protein is
set forth as SEQ ID No. 9 (FIG. 15). The predicted signal sequence
is shown in italics and lowercase.
[0781] The amino acid sequence of the predicted mature forms of
FoxXyn2 is set forth as SEQ ID No.s 10 and 11 (shown in FIGS. 15
and 17). SEQ ID No. 10 shows a section of the polypeptide that may
be cleaved before full maturation of the protein. The active form
of the protein may be with or without this section, and thus the
active protein may have SEQ ID No. 10 or SEQ ID No. 11.
[0782] The protein product of gene FoxXyn2 belongs to glycosyl
hydrolase family 10. This suggests that FoxXyn2 is a secreted
glycosyl hydrolase.
Example 11
[0783] Expression of FoxXyn2 Protein
[0784] The FoxXyn2 gene was amplified from genomic DNA of Fusarium
oxysporum using the following primers: Primer 1
5'-ccgcggccgcaccATGAAGCTGTCTTCCTTCCTCTACACC-3' (SEQ ID NO:19), and
Primer 2 5'-ccggcgcgcccttaTTAGCGGAGAGCGTTGACAACAG-3' (SEQ ID
NO:20). After digested with Not I and Asc I, the PCR product was
cloned into pTrex3gM expression vector (described in US
2011/0136197 A1) digested with the same restriction enzymes, and
the resulting plasmid was labeled pZZH135. A plasmid map of pZZH135
is provided in FIG. 22. The sequence of the FoxXyn2 gene was
confirmed by DNA sequencing.
[0785] The plasmid pZZH135 was transformed into a quad deleted
Trichoderma reesei strain (described in WO 05/001036, incorporated
herein by reference) using biolistic method (taught in Te'o V S et
al., J Microbiol Methods, 51:393-9, 2002). The protein isolated
from the culture supernatant after filtration was used to perform
SDS-PAGE analysis and xylanase activity assay to confirm enzyme
expression.
[0786] The nucleotide sequence of FoxXyn2 gene from expression
plasmid pZZH135 is set forth as SEQ ID No. 12 (FIG. 19). The signal
sequence is shown in bold, and the predicted intron is shown in
italics and lowercase.
[0787] The amino acid sequence of FoxXyn2 protein expressed from
plasmid pZZH135 is set forth as SEQ ID No. 9 (FIG. 15). The signal
sequence is shown in italics.
[0788] The amino acid sequence of the mature form of FoxXyn2
protein is set forth as SEQ ID No. 10 (FIG. 16).
[0789] FoxXyn2 protein was purified from culture supernatant using
affinity chromatography resin Blue Sepharose, 6FF, and samples were
used for biochemical characterization as described in subsequent
examples.
Example 12
[0790] Xylanase Activity of FoxXyn2
[0791] FoxXyn2 belongs to the glycosyl hydrolase 10 family (GH10,
CAZy number). The beta 1-4 xylanase activity of FoxXyn2 was
measured using 1% xylan from birch wood (Sigma 95588) or 1%
arabinoxylan from wheat flour (Megazyme P-WAXYM) as substrates. The
assay was performed in 50 mM sodium citrate pH 5.3, 0.005% Tween-80
buffer at 50.degree. C. for 10 minutes.
[0792] The released reducing sugar was quantified by reaction with
3, 5-Dinitrosalicylic acid and measurement of absorbance at 540 nm.
The enzyme activity is quantified relative to a xylose standard
curve. In this assay, one xylanase unit (U) is defined as the
amount of enzyme required to generate 1 micromole of xylose
reducing sugar equivalents per minute under the conditions of the
assay.
Example 13
[0793] pH Profile of FoxXyn2
[0794] The pH profile of FoxXyn2 was determined using xylan from
birch wood (Sigma 95588) as substrate. The assay was performed in
Sodium Citrate/Sodium Phosphate buffer solution adjusted to pH
values between 2 and 9. Birchwood xylan (2% solution) dissolved in
water was mixed with equal volume of 50 mM Citrate/Phosphate buffer
solution in a 96-well plate, and the substrate was equilibrated at
50.degree. C. before adding enzyme. After 10 minutes, the enzyme
reaction was stopped by transferring 60 microliters of reaction
mixture to a 96-well PCR plate containing 100 microliters of DNS
solution. The PCR plate was heated at 95.degree. C. for 5 minutes
in a Bio-Rad DNA Engine. Then plate was cooled to room temperature
and 100 microliters were transferred from each well to a new
96-well plate. Release of reducing sugars from the substrate was
quantified by measuring the optical density at 540 nm in a
spectrophotometer. Enzyme activity at each pH was reported as
relative activity where the activity at the pH optimum was set to
100%. The pH profile of FoxXyn2 is shown in FIG. 23. FoxXyn2 was
found to have an optimum pH at about 6, and was found to retain
greater than 50% of maximum activity between pH 4.5 and 6.5.
Example 14
[0795] Temperature Profile of FoxXyn2
[0796] The temperature optimum of purified FoxXyn2 was determined
by assaying for xylanase activity at temperatures varying between
45.degree. C. and 94.degree. C. for 10 minutes in 50 mM sodium
citrate buffer at pH 5.3. The activity was reported as relative
activity where the activity at the temperature optimum was set to
100%. The temperature profile of FoxXyn2 is shown in FIG. 24.
FoxXyn2 was found to have an optimum temperature of 60.degree. C.,
and was found to retain greater than 50% of maximum activity
between 40.degree. C. and 65.degree. C.
Example 15
[0797] FveXyn4 in Animal Feed--Pigs
[0798] 15.1 Materials and Methods Two experiments are conducted to
evaluate the efficacy of FveXyn4 in corn/corn DDGS based diets
(Experiment 1) and wheat/wheat bran based diets (Experiment 2). An
Animal Care and Use Committee approves the use of the pigs and
relevant welfare guidelines for the Country are used. Each
experiment used a total of 48 pigs
([Yorkshire.times.Landrace].times. Duroc) housed in groups of two
and each pen contained a gilt and a barrow. Each pen has a smooth
transparent plastic sides and plastic-covered expanded metal sheet
flooring in a temperature-controlled room (22.+-.2.degree. C.).
TABLE-US-00011 TABLE 15.1 Basal diets Item Experiment 1 Experiment
2 Corn 41.44 US corn DDGS 40.00 Hard Wheat 56.57 Wheat Bran 5.06
Wheat middlings 19.94 Soybean Meal 15.00 13.50 Tallow 0.99 L-Lysine
HCl 0.75 0.73 DL-Methionine 0.10 0.17 L-Threonine 0.25 0.30
L-Tryptophan 0.06 0.01 Digestibility marker (celite) 0.30 0.30 Salt
0.30 0.47 Limestone 1.30 1.38 Monocalcium Phosphate 0.00 0.08
Vitamins/Trace minerals premix.sup.1 0.50 0.50 Calculated
provisions Crude protein, % 21.44 19.11 Net energy, MJ/kg 8.88 8.88
SID Lysine g/NE MJ 1.31 1.31 SID Lysine, % 1.16 1.16 SID
Methionine, % 0.35 0.35 Neutral detergent fibre, % 20.63 17.93
Calcium, % 0.67 0.68 Available phosphorous, % 0.22 0.22 .sup.1The
vitamin and trace mineral premix provided the following (per kg of
diet): vitamin A, 11,000 IU; vitamin D3, 2,756 IU; vitamin E, 55
IU; vitamin B12, 55 .mu.g; riboflavin, 16,000 mg; pantothenic acid,
44.1 mg; niacin, 82.7 mg; Zn, 150 mg; Fe, 175 mg; Mn, 60 mg; Cu,
17.5 mg; I, 2 mg; and Se, 0.3 mg
[0799] Respective basal diets are formulated to meet the NRC
nutrients recommendations for swine (NRC, 1998 Table 15.1). In each
experiment, one batch of the basal diet is manufactured and split
into four portions and each portion subsequently mixed with
additives identified in Table 15.2. In experiment 1, pigs are
offered the experimental diets for 42 days, whilst in experiment 2
pigs are offered the experimental diets of for 21 days. Feed and
water is freely available at all times during experimentation.
There are 4 replicate pens per treatment in each experiment; pen
allocation to the treatments is randomized based on pig body weight
at the start of the experiment. Body weight and Feed intake are
recorded on aweekly basis and used to calculate feed conversion
ratio. The growth performance data (BW, ADFI, ADG and FCR) are
subjected to mixed-liner model using the GLM procedures of SAS.
TABLE-US-00012 TABLE 15.2 Treatments identification Treatment
Phytase.sup.1 Diet ID (FTU/kg of feed) Xylanase Control 1 500 FTU 0
Control + FveXyn4 2 500 FTU 2000 U/kg Control + Commercial 3 500
FTU 75 ppm xylanase.sup.2 .sup.1Phytase from Danisco Animal
Nutrition .sup.2Econase XT .RTM. from AB Vista (sometimes referred
to herein as AB Vista)
[0800] 15.2 Results and Discussion
[0801] Compared with the control, FveXyn4 improves animal weight
gain in both corn (Table 15.3) and wheat (Table 15.4) based diets.
Furthermore, FveXyn4 has numerically superior effects on growth
performance relative to commercially available feed xylanase,
particularly in corn based diets. This demonstrates the
efficaciousness of FveXyn4 in mitigating the negative effects of
the insoluble and soluble fibrous fractions in cereal ingredients
on swine performance and shows better utilization of energy
contained in the fibre component of cereal grains.
TABLE-US-00013 TABLE 15.3 Experiment 1: Effect of new xylanase on
growth performance of growing pigs fed corn-corn DDGS based diets
Initial Final Average Average Feed body body daily daily conversion
weight, weight, feed intake, gain, ratio, kg kg grams/day grams/day
g/g Control 32.5 68.18b 2285 849b 2.69 FveXyn4 32.9 74.09a 2391
972a 2.46 Commercial 32.8 71.0ab 2220 909ab 2.43 xylanase (Econase
.RTM. XT) SEM 0.69 2.18 154 43.5 0.11
TABLE-US-00014 TABLE 15.4 Experiment 2: Effect of new xylanase on
growth performance of growing pigs fed wheat-wheat bran based diets
Initial Final Average Average Feed body body daily daily conversion
weight, weight, feed intake, gain, ratio, kg kg grams/day grams/day
g/g Control 50.5 66.3 2221 753b 2.98a FveXyn4 49.0 69.1 2332 955a
2.46ab Commercial 49.7 69.4 2464 935a 2.66ab xylanase (Econase
.RTM. XT) SEM 1.53 1.99 172 56.4 0.24
Example 16
[0802] FveXyn4 in Animal Feed--Pigs
[0803] 16.1 Materials and Methods
[0804] An experiment is conducted to evaluate the efficacy of
FveXyn4 in wheat/wheat bran based diets. Animal use and
experimental procedures are approved by an Animal Care Committee
and relevant welfare guidelines for the Country were used. Growing
gilts ([Yorkshire.times.Landracey ].times.Duroc ) are obtained from
a Research Unit and upon arrival, pigs are weighed and based on
body weight randomly assigned to pens to give 8 pens per treatment.
The pens are equipped with a feeder, a nipple type drinker and
plastic-covered expanded metal floors and a wall partitioning
between pens that allowed visual contact with pigs in adjacent
pens. The room temperature was maintained at 22.+-.2.degree. C.
throughout the experiment.
TABLE-US-00015 TABLE 16.1 Composition of the basal diet Item Level
Hard Wheat 56.57 Wheat Bran 5.06 Wheat middlings 19.94 Soybean Meal
13.50 Soybean oil 0.99 L-Lysine HCl 0.73 DL-Methionine 0.17
L-Threonine 0.30 L-Tryptophan 0.01 Digestibility marker (celite)
0.30 Salt 0.47 Limestone 1.38 Monocalcium Phosphate 0.08
Vitamins/Trace minerals premix.sup.1 0.50 Calculated provisions
Crude protein, % 19.11 Net energy, MJ/kg 8.88 SID Lysine g/NE MJ
1.31 SID Lysine, % 1.16 SID Methionine, % 0.35 Neutral detergent
fibre, % 17.93 Calcium, % 0.68 Available phosphorous, % 0.22
.sup.1Provided per kg of complete diet: vitamin A, 8255 IU; vitamin
D3, 1000 IU; vitamin E, 20 IU; vitamin K, 1.5 mg; riboflavin, 7.5
mg; niacin, 30 mg; vitamin B12, 25 .mu.g; pyridoxine, 4.5 mg;
biotin, 200 .mu.g; folic acid, 1 mg; thiamin, 4 mg; choline, 781
mg; copper, 10 mg; iodine, 0.6 mg; iron, 130 mg; manganese, 40 mg;
selenium, 0.3 mg; zinc, 130 mg.
[0805] A basal diet is formulated to meet the NRC nutrients
recommendations for swine (NRC, 1998; Table 1.1). One batch of the
basal diet is manufactured and split into four portions and each
portion subsequently mixed with additives identified in Table 16.2.
The 4 treatments are allotted in a completely randomized design to
give 8 pens per treatment. Pigs are offered the experimental diets
for 42 days during which feed and water are freely available at all
times. Body weight and Feed intake are recorded on aweekly basis
and used to calculate feed conversion ratio. The growth performance
data (BW, ADFI, ADG and FOR) are subjected to mixed-liner model
using the GLM procedures of SAS
TABLE-US-00016 TABLE 16.2 Treatments identification Treatment
Phytase.sup.1 Diet ID (FTU/kg of feed) Xylanase Control 1 500 FTU 0
Control + FveXyn4 2 500 FTU 2000 U/kg Control + Commercial 3 500
FTU 75 ppm xylanase.sup.2 .sup.1Phytase from Danisco Animal
Nutrition .sup.2Econase XT .RTM. from AB Vista
[0806] 16.2 Results and Discussion
[0807] FveXyn4 results in better body weight gain, specifically
9.9% higher average daily gain (over 42 days) than a commercial
xylanase from AB Vista. Pigs exhibited a better FCR and heavier
final body weight when receiving FveXyn4.
TABLE-US-00017 TABLE 16.3 Effect of new xylanase on growth
performance of growing pigs fed wheat based diets Initial Final
Average Average Feed body body daily daily conversion weight,
weight, feed intake, gain, ratio, kg kg grams/day grams/day g/g
Control 23.9 61.8 2104 904ab 2.33 FveXyn4 23.0 63.1 2075 954a 2.18
Commercial 23.2 59.6 2021 868b 2.33 xylanase (Econase .RTM. XT) SEM
0.48 1.16 61.2 23.7 0.07
Example 17
[0808] FveXyn4 in Animal Feed--Pigs
[0809] 17.1 Materials and Methods
[0810] The experiment is conducted to evaluate the efficacy of
FveXyn4 on ileal nutrients and energy digestibility in growing pigs
fed wheat/wheat bran based diets. The protocol for the experiment
is reviewed and approved by an Institutional Animal Care and Use
Committee and relevant welfare guidelines for the Country are used.
Six growing barrows (initial body weight of 30 kg) are equipped
with a T-cannula in the distal ileum for the purpose of the
experiment. Pigs are of the off springs of G-Performer boars that
were mated to Fertilium 25 females (Genetiporc, Alexandria, Minn.,
USA) and are housed in individual pens (1.2.times.1.5 m) in an
environmentally controlled room. Each pen is equipped with a feeder
and a nipple drinker and has fully slatted concrete floors.
TABLE-US-00018 TABLE 17.1 Composition of the basal diet Item Level
Hard Wheat 56.57 Wheat Bran 5.06 Wheat middlings 19.94 Soybean Meal
13.50 Soybean oil 0.99 L-Lysine HCl 0.73 DL-Methionine 0.17
L-Threonine 0.30 L-Tryptophan 0.01 Digestibility marker (celite)
0.30 Salt 0.47 Limestone 1.38 Monocalcium Phosphate 0.08
Vitamins/Trace minerals premix.sup.1 0.50 Calculated provisions
Crude protein, % 19.11 Net energy, MJ/kg 8.88 SID Lysine g/NE MJ
1.31 SID Lysine, % 1.16 SID Methionine, % 0.35 Neutral detergent
fibre, % 17.93 Calcium, % 0.68 Available phosphorous, % 0.22
.sup.1Provided the following quantities of vitamins and trace
minerals per kilogram of complete diet: Vitamin A as retinyl
acetate, 11,128 IU; vitamin D.sub.3 as cholecalciferol, 2,204 IU;
vitamin E as DL-alpha tocopheryl acetate, 66 IU; vitamin K as
menadione nicotinamide bisulfite, 1.42 mg; thiamin as thiamine
mononitrate, 0.24 mg; riboflavin, 6.58 mg; pyridoxine as pyridoxine
hydrochloride, 0.24 mg; vitamin B.sub.12, 0.03 mg; D-pantothenic
acid as D-calcium pantothenate, 23.5 mg; niacin as nicotinamide and
nicotinic acid, 44 mg; folic acid, 1.58 mg; biotin, 0.44 mg; Cu, 10
mg as copper sulfate; Fe, 125 mg as iron sulfate; I, 1.26 mg as
potassium iodate; Mn, 60 mg as manganese sulfate; Se, 0.3 mg as
sodium selenite; and Zn, 100 mg as zinc oxide.
TABLE-US-00019 TABLE 17.2 Treatments identification Treatment
Phytase.sup.1 Diet ID (FTU/kg of feed) Xylanase Control 1 500 FTU 0
Control + FveXyn4 2 500 FTU 2000 U/kg Control + Commercial 3 500
FTU 75 ppm xylanase.sup.2 .sup.1Phytase from Danisco Animal
Nutrition .sup.2Econase XT .RTM. from AB Vista
[0811] A basal diet is formulated to meet the NRC nutrients
recommendations for swine (Table 17.1). One batch of the basal diet
is manufactured and split into four portions and each portion
subsequently mixed with additives identified in Table 17.2. The
experiment was designed and conducted according to a 4.times.4
Latin square design with 2 added columns to give 6 replicates per
diet. All pigs are fed at a level of 3 times their maintenance
energy requirement (106 kcal ME per kg.sup.0.75; NRC, 1998), and
provided at 0800 and 1700 h. Animals have free access to water
through a bowl-type drinker. Pig weights are recorded at the
beginning and at the end of each period and the amount of feed
supplied each day is recorded. Each experimental period lasted for
7 d. The initial 5 days of each period is considered an adaptation
period to the diet. Ileal digesta are collected for 8 h on d 6 and
7 using standard operating procedures. In brief, a plastic bag is
attached to the cannula barrel and digesta flowing into the bag is
collected. Bags are removed whenever they are filled with
digesta--or at least once every 30 min and are immediately frozen
at -20.degree. C. On the completion of one experimental period,
animals are deprived of feed overnight and the following morning, a
new experimental diet is offered.
[0812] At the end of the experiment, ileal samples are thawed,
mixed within animal and diet, and a sub-sample is collected for
chemical analysis. A sample of basal diet is also collected and
analyzed. Digesta samples are lyophilized and finely ground prior
to chemical analysis. All samples are analyzed for dry matter,
Titanium, gross energy, crude protein, fat and neutral detergent
fibre according to standard procedures (AOAC, 2005). The values for
apparent ileal digestibility of energy and nutrients are calculated
as described previously (Stein et al., 2007 Livestock Science, 2007
vol. 109, issue 1 part 3 p 282-285 and J ANIM SCI January 2007 vol.
85 no. 1 172-180). Data are analyzed using the MIXED procedures of
SAS.
[0813] 17.2 Results and Discussion
[0814] Results indicate that pigs fed FveXyn4 have significantly
higher (P<0.05) apparent ileal digestibility of crude protein
and fat than control and the commercial xylanase fed pigs (Table
17.3). Results suggest that FveXyn4 is effective in unlocking
energy in fibrous feed fed to growing pigs by breaking down fibres.
Indeed, pigs fed FveXyn4 show increased fibre digestibility by a
range of 6 to 11 percentage units relative to the control and
commercial xylanase (Table 17.3). Subsequently pigs fed FveXyn4
extract 106 and 219 kcal extra (Table 17.3) energy compared to pigs
fed the control and commercial xylanase, respectively.
TABLE-US-00020 TABLE 17.3 Effect of new xylanase on apparent ileal
nutrients and fibre energy digestibility (%) and energy utilization
(kcal/kg) in growing pigs fed wheat based diets Neutral Dry Crude
detergent matter protein Fat fibre Energy Control 68.0ab 74.7b
62.2b 66.3ab 3,168ab FveXyn4 71.2a 77.0a 67.7a 72.1a 3,274a
Commercial 65.8b 74.6b 56.3b 61.3b 3,055b xylanase (Econase .RTM.
XT) SEM 1.44 0.84 2.03 2.65 47.6
Example 18
[0815] FveXyn4 in Animal Feed--Pigs
[0816] 18.1 Materials and Methods
[0817] The efficacy of FveXyn4 on ileal and total tract nutrients
and energy digestibility in growing pigs fed corn-corn DDGS and
wheat/wheat bran based diets is studied. An Animal Care and Use
Committee approves the use of the pigs and pigs are cared for
according to the relevant welfare guidelines for the Country. A
total of 24 barrows ([ Yorkshire.times.Landrace ].times. Duroc;
initial body weight of 30 kg) are equipped with a T-cannula in the
distal ileum for the purpose of the experiment. The pigs are
individually housed in metabolism crates that a smooth transparent
plastic sides and plastic-covered expanded metal sheet flooring in
a temperature-controlled room (22.+-.2.degree. C.).
TABLE-US-00021 TABLE 18.1 Basal diets used Corn Wheat Item based
based Corn 41.44 US corn DDGS 40.00 Hard Wheat 56.57 Wheat Bran
5.06 Wheat middlings 19.94 Soybean Meal 15.00 13.50 Tallow 0.99
L-Lysine HCl 0.75 0.73 DL-Methionine 0.10 0.17 L-Threonine 0.25
0.30 L-Tryptophan 0.06 0.01 Digestibility marker (celite) 0.30 0.30
Salt 0.30 0.47 Limestone 1.30 1.38 Monocalcium Phosphate 0.00 0.08
Vitamins/Trace minerals premix.sup.1 0.50 0.50 Calculated
provisions Crude protein, % 21.44 19.11 Net energy, MJ/kg 8.88 8.88
SID Lysine g/NE MJ 1.31 1.31 SID Lysine, % 1.16 1.16 SID
Methionine, % 0.35 0.35 Neutral detergent fibre, % 20.63 17.93
Calcium, % 0.67 0.68 Available phosphorous, % 0.22 0.22 .sup.1The
vitamin and trace mineral premix provided the following (per kg of
diet): vitamin A, 11,000 IU; vitamin D3, 2,756 IU; vitamin E, 55
IU; vitamin B12, 55 .mu.g; riboflavin, 16,000 mg; pantothenic acid,
44.1 mg; niacin, 82.7 mg; Zn, 150 mg; Fe, 175 mg; Mn, 60 mg; Cu,
17.5 mg; I, 2 mg; and Se, 0.3 mg
[0818] Respective basal diets are formulated to meet the NRC
nutrient recommendations for swine (NRC, 1998 Table 18.1). In each
experiment, one batch of the basal diet is manufactured and split
into four portions and each portion subsequently mixed with
additives identified in Table 18.2.
TABLE-US-00022 TABLE 18.2 Treatments identification Treatment
Phytase.sup.1 Diet ID (FTU/kg of feed) Xylanase Control 1 500 FTU 0
Control + FveXyn4 2 500 FTU 4000 U/kg Control + Commercial 3 500
FTU 100 ppm xylanase.sup.2 .sup.1Phytase from Danisco Animal
Nutrition .sup.2Econase XT .RTM. from AB Vista
[0819] The experiment is designed and conducted as two period
cross-over design in which all corn diets are run in period one and
all wheat diets run in period two. Within a period, the 4
treatments will be allocated to pigs in a completely randomized
design to give 6 replicates per treatment. Pigs are fed a common
commercial diet for a week before commencement of the second
period. The pigs are fed their respective diets in two equal
portions at 0830 and 1630. Daily feed allowance is based on the
pig's BW at the beginning of the period and is calculated to supply
2.6 times the estimated maintenance requirements. Each experimental
period lasts for 14 d: d 7 for adaptation, d 8 and 9 for grab fecal
collection and d 10 and 11 for ileal digesta collection to examine
coefficient of apparent ileal and total tract digestibility of N,
DM, energy and crude fat. Pigs are allowed free accessible to water
from nipple drinkers located in each pen at all times. Digesta flow
measurements and blood samples collection are conducted from d 12
to 14. Data id analysed using GLM procedures of SAS. Statistical
significance is accepted at P<0.05.
[0820] 18.2 Results and Discussion
[0821] Preliminary results indicate that pigs fed FveXyn4 have
significantly improved energy digestibility.
Example 19
[0822] FveXyn4 in Animal Feed (Poultry)
[0823] 19.1 Materials and Methods
[0824] An experiment is conducted to evaluate the efficacy of
FveXyn4 on growth performance of broiler chickens fed corn/corn
DDGS based diets and wheat/wheat bran based diets. The experimental
procedures are approved by an Animal Ethics Committee and, complied
with relevant welfare guidelines for the Country. A two-phase
feeding programme (starter and finisher) is used (Table 1). The
starter and finisher diets are offered from d 0 to 21 and 22 to 42,
respectively.
TABLE-US-00023 TABLE 19.1 Composition of the basal diets.sup.1
Starter, d 0-21 Finisher, d 22-42 Corn Wheat Corn Wheat Corn 57.39
-- 58.50 -- Corn DDGS 11.00 -- 15.00 -- Wheat -- 60.17 -- 63.30
Wheat bran -- 9.00 -- 13.00 Soybean meal, 45% 26.50 22.34 19.00
14.00 Tallow 1.75 4.85 3.20 5.95 Vitamin-mineral premix.sup.2 0.33
0.33 0.33 0.33 Sodium bicarbonate 0.20 0.22 0.20 0.29 Salt 0.38
0.38 0.34 0.35 Monocalcium phosphate 0.35 0.37 0.13 0.20 Limestone
1.700 1.69 1.70 1.65 L-Lysine-HCl 0.135 0.25 0.20 0.34
DL-methionine 0.185 0.23 0.13 0.19 L-threonine 0.100 0.19 0.09 0.22
Calculated provisions Crude protein 21.0 21.1 18.6 18.2 ME (MJ/kg)
12.5 12.2 12.9 12.5 Calcium 0.81 0.80 0.75 0.73 Available
Phosphorous 0.25 0.25 0.21 0.21 Sodium 0.23 0.22 0.23 0.22
Digestible Lysine 1.01 0.99 0.90 0.87 Digestible Methionine 0.47
0.47 0.39 0.39 Digestible Threonine 0.74 0.74 0.64 0.66 Digestible
Tryptophan 0.19 0.21 0.16 0.17 .sup.1A commercial phytase from
Danisco Animal Nutrition top dressed to supply 500 FTU/kg of final
feed .sup.2Supplied per kilogram of diet: antioxidant, 100 mg;
biotin, 0.2 mg; calcium pantothenate, 12.8 mg; cholecalciferol, 60
.mu.g; cyanocobalamin, 0.017 mg; folic acid, 5.2 mg; menadione, 4
mg; niacin, 35 mg; pyridoxine, 10 mg; trans-retinol, 3.33 mg;
riboflavin, 12 mg; thiamine, 3.0 mg; dl-.alpha.-tocopheryl acetate,
60 mg; choline chloride, 638 mg; Co, 0.3 mg; Cu, 3.0 mg; Fe, 25 mg;
I, 1 mg; Mn, 125 mg; Mo, 0.5 mg; Se, 200 .mu.g; Zn, 60 mg.
[0825] Two basal diets, one based on wheat/wheat bran and soybean
meal, and the other based on corn/corn DDGS and soybean meal, are
formulated to meet or exceed the recommended requirements for
nutrients, except AME, for broilers (Table 19.1). From each basal
diet, four experimental diets are developed to constitute control,
FveXyn4, commercial xylanases 1 and commercial xylanases 2 as
identified in Table 19.2.
TABLE-US-00024 TABLE 19.2 Treatments identification Treatment
Phytase.sup.1 Diet ID (FTU/kg of feed) Xylanase Control 1 500 FTU 0
Control + FveXyn4 2 500 FTU 1250 U/kg Control + Commercial 3 500
FTU 50 ppm xylanase .sup.2 .sup.1Phytase from Danisco Animal
Nutrition .sup.2 Econase XT .RTM. from AB Vista
[0826] Male broiler (Ross 308) chicks are obtained as day-olds from
a commercial hatchery. The chicks are individually weighed and
allocated to 72 brooder cages (8 chicks per cage) and the 8 dietary
treatments randomly assigned to eight cages each. On day 12, the
birds are transferred to grower cages. The space allocation per
bird in brooder and grower cages is 530 and 640 cm.sup.2,
respectively. The brooder and grower cages are housed in
environmentally controlled rooms. The temperature is maintained at
31.degree. C. in the first week and then gradually reduced to
22.degree. C. by the end of third week. The birds receive 20 hours
fluorescent illumination and, allowed free access to the diets and
water. Body weights and feed intake are recorded at weekly
intervals throughout the 42-day experimental period. Mortality is
recorded daily. Any bird that dies is weighed and the weight is
used to adjust FCR. Feed conversion ratios are calculated by
dividing total feed intake by weight gain of live plus dead birds.
Data are analysed as a two-way factorial arrangement of treatments
using the General Linear Models procedure of SAS (2004).
[0827] 19.2 Results and Discussion
[0828] Compared with the control and commercial xylanase 2, FveXyn4
improves gain and FCR in both corn and wheat based diets (Table
19.3). This demonstrates the efficaciousness of FveXyn4 in
mitigating the negative effects of the insoluble and soluble
fibrous fractions in cereal ingredients that may limit poultry
performance.
TABLE-US-00025 TABLE 19.3 Effect of new xylanase on growth
performance of broiler chickens fed corn-corn DDGS and wheat/wheat
bran based diets Initial Final Body Feed Treatments body body Feed
weight conversion Grain Xylanase weight, g weight, g intake, g
gain, g ratio, g/g Corn Control 38.3 2151 3738 2113 1.81 Corn
FveXyn4 38.3 2306 3946 2268 1.76 Corn Commercial 38.5 2304 3969
2265 1.78 xylanase Wheat Control 38.4 2477 4056 2438 1.72 Wheat
FveXyn4 38.3 2678 4294 2639 1.69 Wheat Commercial 38.2 2456 4077
2418 1.71 xylanase SEM 0.17 56.5 105 56.5 0.02 Main effects, grains
Corn 38.3 2252b 3851b 2214b 1.77a Wheat 38.3 2588a 4165a 2550a
1.68b SEM 0.09 28.2 52.5 28.2 0.01 Main effects, xylanases Control
38.3 2314b 3847 2275b 1.76a FveXyn4 38.3 2492a 4120 2453a 1.72b
Commercial xylanase 38.3 2380b 4023 2342b 1.74ab SEM 0.12 39.9 74.3
39.9 0.01 Probabilities Grain -- <0.01 <0.01 <0.01
<0.01 Xylanase -- <0.01 0.22 <0.01 <0.01 Grain and
xylanase interaction -- 0.04 0.37 0.04 0.05 Within a column, means
with different letters are significantly different, (P <
0.05).
Example 20
[0829] FveXyn4 in Animal Feed (Poultry)
[0830] 20.1 Material and Methods
[0831] The experiment is conducted to evaluate the efficacy of
FveXyn4 on energy and nutrient utilization/retention in broiler
chickens fed corn/corn DOGS based diets. An Animal Care and Use
Committee approved all bird handling and collection procedures.
Two-hundred fifty-six male broiler chicks (Ross 708, Aviagen,
Huntsville, Ala.) are housed in electrically heated battery cages
(model SIB 4T, Alternative Design Manufacturing, Siloam Springs,
Ark.) in an environmentally controlled room for a 21 day trial.
Battery temperatures on d 1 to 8, d 8 to 15, and d 15 to 21 are
kept at 35, 32, and 27.degree. C., respectively.
[0832] A corn-based basal diet (Table 20.1) is formulated to meet
the nutrient requirements of the broiler chicken. From the basal
diet, four experimental diets are developed to constitute control,
FveXyn4, and commercial xylanase as identified in Table 20.2.
TABLE-US-00026 TABLE 20.1 Composition of the basal diet
Ingredients, g/kg Levels Corn 554.4 Corn-DDGS 110.0 Soybean Meal
244.4 Soybean Oil 10.0 L-Lysine HCl 4.3 DL-Methionine 2.7
L-Threonine 1.1 Sodium Bicarbonate 2.0 Salt 2.2 Limestone (A) 15.3
MCP (B) 5.6 Vitamin-Min premix (C) 3.0 TiO2 Premix (D) 25.0 Phytase
premix (E) 10.0 Calculated provisions Crude protein, g/kg 211.4
MEP, MJ/kg 115.1 Calcium, g/kg 8.9 Available phosphorous, g/kg 2.8
Digestible Lysine, g/kg 11.5 Digestible Methionine, g/kg 5.5 (A)
38% Ca (B) 16% Ca, 21% P. (C) Supplies the following per kg DIET:
Vit. A, 5484 IU; Vit. D3, 2643 ICU; Vit E, 11 IU; Menadione sodium
bisulfite, 4.38 mg; Riboflavin, 5.49 mg; d-pantothenic acid, 11 mg;
Niacin, 44.1 mg; Choline chloride, 771 mg; Vit B12, 13.2 ug;
Biotin, 55.2 ug; Thiamine mononitrate, 2.2 mg; Folic acid, 990 ug;
Pyridoxine hydrochloride, 3.3 mg; I, 1.11 mg; Mn, 66.06 mg; Cu,
4.44 mg; Fe, 44.1 mg; Zn, 44.1 mg; Se, 300 ug. Also contains per g
of premix: Vit. A, 1828 IU; Vit. D3, 881 ICU; Vit E, 3.67 IU;
Menadione sodium bisulfite, 1.46 mg; Riboflavin, 1.83 mg;
d-pantothenic acid, 3.67 mg; Niacin, 14.69 mg; Choline chloride,
257 mg; Vit B12, 4.4 ug; Biotin, 18.4 ug; Thiamine mononitrate, 735
ug; Folic acid, 330 ug; Pyridoxine hydrochloride, 1.1 mg; I, 370
ug; Mn, 22.02 mg; Cu, 1.48 mg; Fe, 14.69 mg; Zn, 14.69 mg; Se, 100
ug. (D) Prepared as 5 g of TiO2 added to 20 g of fine ground SBM.
(E) Prepared as 0.2 g of phytase added to 9.8 g of fine ground
SBM
TABLE-US-00027 TABLE 20.2 Treatments identification Treatment
Phytase.sup.1 Diet ID (FTU/kg of feed) Xylanase Control 1 500 FTU 0
Control + FveXyn4 2 500 FTU 1250 U/kg Control + Commercial 3 500
FTU 50 ppm xylanase .sup.2 .sup.1Phytase from Danisco Animal
Nutrition .sup.2 Econase XT .RTM. from AB Vista
[0833] Upon arrival at the research facility, chicks are weighed,
grouped into 4 blocks by blocks, and randomly allocated to 4
dietary groups in each block with 8 birds per cage in a randomized
complete block design. The chicks are fed experimental diets (mash)
ad libitum from d 1 and are also allowed ad libitum access to clean
drinking water. Excreta are collected twice daily on d 19, 20, and
21 post-hatch. During collection, waxed paper is placed in trays
under the cages and excreta on the waxed paper are collected. The
collected excreta samples are pooled per cage over the 3 d, stored
in a freezer, dried, and ground to pass through a 0.5-mm screen
using a mill grinder (Retsch ZM 100, GmbH & Co. K.C., Haan,
Germany). Excreta and diet samples are analyzed for gross energy,
dry matter, fat and neutral detergent fibre using standard
procedures (AOAC, 2005) for calculation of apparent retentions and
AME. Data were subsequently subjected to GLM procedures of SAS.
[0834] 20.2 Results and Discussion
[0835] Birds fed FveXyn4 retain more fat and fibre and subsequently
extracted more energy (.about.57 kcal/kg) compared with the control
fed birds (Table 6). Furthermore, FveXyn4 fed birds have
significantly higher fibre retention compared to the commercial
xylanase.
TABLE-US-00028 TABLE 20.3 Effect of new xylanase on apparent
nutrients and fibre retention (%) and energy utilization (kcal/kg)
in broiler chickens fed corn-corn DDGS based diets Neutral Dry
Crude detergent matter protein Fat fibre Energy Control 67.5ab 60.4
51.6b 27.4b 3107b FveXyn4 68.2ab 60.1 57.8a 30.1a 3164a Commercial
68.8a 60.5 52.2ab 26.8b 3156ab xylanase (Econase .RTM. XT) SEM 0.48
0.91 2.05 1.15 21.8 Within a column, means with different letters
are significantly different, (P < 0.05).
Example 21
[0836] FveXyn4 in Animal Feed (Poultry)
[0837] 21.1 Material and Methods
[0838] An experiment is conducted to evaluate the efficacy of
FveXyn4 on energy and nutrient utilization/retention in broiler
chickens fed corn/corn DDGS based diets.
[0839] The experiment is approved by an Animal Experimentation
Committee and conforms to the protocol required by relevant
regulations. The birds are allocated to dietary treatments when
they are 14 days old. At day old, the birds are brooded together
and receive a commercial corn-soybean meal pre-experimental diet.
The experiment is carried out in an environmentally controlled
house.
TABLE-US-00029 TABLE 21.1 Composition of the basal diet Ingredient,
% Level Corn 54.4 Corn DDGS 11.0 Soybean Meal 28.9 Soybean Oil 1.00
L-Lysine HCl 0.43 DL-Methionine 0.27 L-Threonine 0.11 Sodium
Bicarbonate 0.20 Salt 0.22 Limestone 1.53 Monocalcium phosphate
0.56 Vitamin mineral premix 1.00 Titanium dioxide 0.30 Calculated
provisions 100 Crude protein, % 21.1 MEP, MJ/kg 11.5 Calcium, %
0.89 Available phosphorous 0.28 Digestible lysine 1.15 Digestible
methionine 0.55
[0840] The premix provided (units kg.sup.-1 diets): Vit A 16,000
iu; Vit D.sub.3 3,000 iu; Vit E 75 iu; Vit B.sub.1 mg; Vit B.sub.2
10 mg; Vit B.sub.6 3 mg; Vit B.sub.12 15 .mu.g; Vit K.sub.3 5 mg;
Nicotinic acid 60 mg; Pantothenic acid 14.5 mg; Folic acid 1.5 mg;
Biotin 275 .mu.g; Choline chloride 250 mg; Iron 20 mg; Copper 10
mg; Manganese 100 mg; Cobalt 1 mg; Zinc 82 mg; Iodine 1 mg;
Selenium 0.2 mg; Molybdenum 0.5 mg.
TABLE-US-00030 TABLE 21.2 Treatments identification Treatment
Phytase.sup.1 Diet ID (FTU/kg of feed) Xylanase Control 1 500 FTU 0
Control + FveXyn4 2 500 FTU 1250 U/kg Control + Commercial 4 500
FTU 50 ppm xylanase.sup.2 .sup.1Phytase from Danisco Animal
Nutrition .sup.2Econase XT .RTM. from AB Vista
[0841] A corn-based basal diet (Table 21.1) is formulated to meet
the nutrient requirements of the broiler chicken. From the basal
diet, four experimental diets are developed to constitute control,
FveXyn4 and commercial xylanase as identified in Table 21.2.
One-hundred and ninety-two 14-d old birds are wing tagged and
allocated to 4 treatments, with 8 replicate cages per treatment and
6 birds per replicate cage. The experimental design is a randomized
complete block with blocks randomly allocated to spaces within a
room. Birds are fed experimental diets from day 14 to day 21. Water
from the mains is provided to the birds ad libitum. The total
excreta voided are collected on days 19 to 21. This enables
calculation of apparent total tract retention using total
collection. The excreta are dried in forced air oven prior to
chemical analyses. The diets and excreta are chemically analyzed.
Dry matter (DM) is determined by drying in a force draft oven at
100.degree. C. for 24 hours. The crude fat is determined using
Soxhlet extractor system (AOAC 920.39). Nitrogen content is
determined by combustion method using a Leco system (Sweeney,
1998). Titanium is determined using the method of Short et al.
(1996). Gross energy is determined in an adiabatic calorimeter.
Data are subsequently subjected to GLM procedures of SAS.
[0842] 21.2 Results and Discussion
[0843] Compared with the control, FveXyn4 increases (P<0.05)
retention of dry matter, crude protein and fat (Table 21.3) in a
corn-corn DDGS based diet. Furthermore, FveXyn4 has higher
retention of dry matter, protein and fat relative to commercially
available feed xylanase. This demonstrates the efficaciousness of
FveXyn4 in mitigating the negative effects of the insoluble and
soluble fibrous fractions in corn grain and corn-co-products.
Indeed, addition of FveXyn4 in the control diet results in
significant energy uplift of 116 kcal/kg
TABLE-US-00031 TABLE 21.3 Effect of new xylanase on apparent
nutrients and fibre retention (%) and energy utilization (kcal/kg)
in broiler chickens fed corn-corn DDGS based diets Dry Crude matter
protein Fat Energy Control 73.5bc 69.2ab 76.1b 3431b FveXyn4 76.1a
72.2a 79.6a 3547a Commercial 72.7c 66.2b 78.9ab 3428b xylanase
(Econase .RTM. XT) SEM 0.70 1.32 1.12 30.6 Within a column, means
with different letters are significantly different, (P <
0.05).
Example 22
[0844] FveXyn4 in Animal Feed (Poultry)
[0845] 22.1 Materials and Methods
[0846] An experiment is conducted to evaluate dose response
efficacy of FveXyn4 on growth performance of broiler chickens fed
corn/corn DDGS based diets and wheat/wheat bran based diets. The
experimental procedures are approved by the Institutional Animal
Ethics Committee and, comply with the relevant welfare guidelines
of the Country. A two-phase feeding programme (starter and
finisher) is used (Table 22.1). The starter and finisher diets are
offered from d 0 to 21 and 22 to 42, respectively.
TABLE-US-00032 TABLE 22.1 Composition of the basal diets.sup.1
Starter, d 0-21 Finisher, d 22-42 Corn Wheat Corn Wheat Corn 57.39
-- 58.50 -- Corn DDGS 11.00 -- 15.00 -- Wheat -- 60.17 -- 63.30
Wheat bran -- 9.00 -- 13.00 Soybean meal, 45% 26.50 22.34 19.00
14.00 Tallow 1.75 4.85 3.20 5.95 Vitamin-mineral premix.sup.2 0.33
0.33 0.33 0.33 Sodium bicarbonate 0.20 0.22 0.20 0.29 Salt 0.38
0.38 0.34 0.35 Monocalcium phosphate 0.35 0.37 0.13 0.20 Limestone
1.700 1.69 1.70 1.65 L-Lysine-HCl 0.135 0.25 0.20 0.34
DL-methionine 0.185 0.23 0.13 0.19 L-threonine 0.100 0.19 0.09 0.22
Calculated provisions Crude protein 21.0 21.1 18.6 18.2 ME (MJ/kg)
12.5 12.2 12.9 12.5 Calcium 0.81 0.80 0.75 0.73 Available
Phosphorous 0.25 0.25 0.21 0.21 Sodium 0.23 0.22 0.23 0.22
Digestible Lysine 1.01 0.99 0.90 0.87 Digestible Methionine 0.47
0.47 0.39 0.39 Digestible Threonine 0.74 0.74 0.64 0.66 Digestible
Tryptophan 0.19 0.21 0.16 0.17 .sup.1Phytase B top dressed to
supply 500 FTU/kg of final feed .sup.2Supplied per kilogram of
diet: antioxidant, 100 mg; biotin, 0.2 mg; calcium pantothenate,
12.8 mg; cholecalciferol, 60 .mu.g; cyanocobalamin, 0.017 mg; folic
acid, 5.2 mg; menadione, 4 mg; niacin, 35 mg; pyridoxine, 10 mg;
trans-retinol, 3.33 mg; riboflavin, 12 mg; thiamine, 3.0 mg;
dl-.alpha.-tocopheryl acetate, 60 mg; choline chloride, 638 mg; Co,
0.3 mg; Cu, 3.0 mg; Fe, 25 mg; I, 1 mg; Mn, 125 mg; Mo, 0.5 mg; Se,
200 .mu.g; Zn, 60 mg.
[0847] Two basal diets, one based on wheat/wheat bran and soybean
meal, and the other based on corn/corn DDGS and soybean meal, are
formulated to meet or exceed the recommended requirements for
nutrients, except AME, for broilers (Table 22.1). From each basal
diet, three experimental diets are developed to constitute control,
3 doses of FveXyn4 and commercial xylanase as identified in Table
22.2.
TABLE-US-00033 TABLE 22.2 Treatments identification Treatment
Phytase Diet ID (FTU/kg of feed) Xylanase Control 1 500 FTU 0
Control + FveXyn4-dose 1 2 500 FTU 1250 U/kg Control + FveXyn4-dose
2 3 500 FTU 2500 U/kg Control + FveXyn4-dose 3 4 500 FTU 5000 U/kg
Control + Commercial 5 500 FTU 100 ppm xylanase.sup.1 .sup.1AB
Vista
[0848] Male broiler (Ross 308) chicks are obtained as day-olds from
a commercial hatchery. The chicks are individually weighed and
allocated to 60 brooder cages (8 chicks per cage) and the 10
dietary treatments randomly assigned to six cages each. On day 12,
the birds are transferred to grower cages. The space allocation per
bird in brooder and grower cages is 530 and 640 cm.sup.2,
respectively. The brooder and grower cages are housed in
environmentally controlled rooms. The temperature is maintained at
31.degree. C. in the first week and then gradually reduced to
22.degree. C. by the end of third week. The birds receive 20 hours
fluorescent illumination and, allowed free access to the diets and
water. Body weights and feed intake are recorded at weekly
intervals throughout the 42-day experimental period. Mortality is
recorded daily. Any bird that die is weighed and the weight is used
to adjust FCR. Feed conversion ratios are calculated by dividing
total feed intake by weight gain of live plus dead birds. Data are
analysed as a two-way factorial arrangement of treatments using the
General Linear Models procedure of SAS (2004).
[0849] 22.2 Results and Discussion
[0850] Compared with the control and commercial xylanase, FveXyn4
improves feed conversion efficiency in a dose dependent manner in
both corn and wheat based diets (Table 22.3). These observations
suggested more value of the xylanase is derived at higher inclusion
rate. This demonstrates the efficaciousness of FveXyn4 in
mitigating the negative effects of the insoluble and soluble
fibrous fractions in cereal ingredients that may limit poultry
ability to utilize dietary nutrients to support growth
performance.
TABLE-US-00034 TABLE 22.3 Dose response effect of new xylanase on
growth performance of broiler chickens fed corn-corn DDGS and
wheat/wheat bran based diets Initial Final Body Feed Treatments
body body Feed weight conversion Grain Xylanase weight, g weight, g
intake, g gain, g ratio, g/g Corn Control 33.50 2569 4162 2536
1.726 Corn FveXyn4- Dose1 33.54 2584 4335 2551 1.708 Corn FveXyn4-
dose2 33.65 2585 4224 2552 1.671 Corn FveXyn4- Dose3 33.54 2580
4177 2546 1.683 Corn Commercial 33.98 2646 4295 2612 1.677 xylanase
2 Wheat Control 33.56 2508 4456 2474 1.824 Wheat FveXyn4- Dose1
33.98 2485 4285 2451 1.814 Wheat FveXyn4- dose2 33.63 2674 4413
2641 1.656 Wheat FveXyn4- Dose3 33.81 2707 4330 2673 1.629 Wheat
Commercial 34.13 2590 4297 2556 1.694 xylanase 2 SEM 0.22 55.2 56.6
91.3 56.5 Main effects, grains Corn 33.64 2593 4238b 2559 1.693
Wheat 33.82 2593 4356a 2559 1.723 SEM 0.10 25.30 40.82 25.28 0.018
Main effects, xylanases Control 33.53 2539 4309 2505 1.775a
FveXyn4-Dose1 33.76 2535 4310 2501 1.761ab FveXyn4-Dose2 33.64 2630
4318 2596 1.664c FveXyn4-dose3 33.68 2643 4254 2610 1.656c
Commercial Xylanase 2 34.05 2618 4296 2584 1.686bc SEM 0.152 40.00
64.55 39.98 0.028 Probabilities Grain -- 0.993 0.046 0.989 0.237
Xylanase -- 0.165 0.958 0.166 0.008 Grain and Xylanase interaction
-- 0.190 0.329 0.189 0.212 Within a column, means with different
letters are significantly different, (P < 0.05)
Example 23
[0851] FveXyn4 in Combination with Protease
[0852] The effect of FveXyn4 in combination with protease was
investigated on the solubilization of pentosan and protein from
prepared insoluble DDGS
[0853] 23.1 Materials and Methods
[0854] Enzyme Samples
[0855] The xylanase used in this study is a new GH10 xylanase from
Fusarium verticilloides (designated FveXyn4) expressed in
Trichoderma reesei, wherein the xylanase was used in purified
form--this enzyme may be referred to herein as FveXyn4.
[0856] The protease used in this study is the Multifect P-3000
product (available from Danisco Animal Nutrition). The preparation
of protease was performed just prior to loadings. Proper amount of
stock solution was diluted in cooled MQ-water and mixed while kept
on ice. One protease unit (U) was defined as the release of 1.0
.mu.g of phenolic compound (expressed as tyrosine equivalents) from
a casein substrate per minute.
[0857] Substrate Preparation
[0858] For the preparation of insoluble DDGS substrate, removal of
soluble non-starch polysaccharides (S-NSP) was performed according
to Bach Knudsen (Bach Knudsen, K. E., Carbohydrate and lignin
contents of plant materials used in animal feeding. Animal Feed
Science and Technology 1997, 67, 319-338.); Milled DDGS (<212
.mu.m) and acetate/CaCl.sub.2-buffer (0.1 M/20 mM, pH 5.0) was
added together with thermostable .alpha.-amylase (E-BLAAM 53.7
U/mg, Megazyme International) and incubated for 1 h at 100.degree.
C. with frequent mixing. Complete degradation of starch was done by
incubation with amyloglucosidase (E-AMGDF 36 U/mg, Megazyme
International) for 2 h at 60.degree. C. After removal of the
starch, the S-NSP was extracted by a phosphate buffer (0.2 M, pH
7.0) and placed at 100.degree. C. for 1 h, followed by
centrifugation. The pellet was then thoroughly washed with
phosphate buffer, ethanol (85% v/v), and finally acetone, with
centrifugation and discard of supernatant in between washes. The
sample was placed at room temperature until completely dry.
[0859] Procedure
[0860] FveXyn4 alone or in combination with protease was
investigated on the solubilization of pentosan and protein of
prepared insoluble DDGS. 87.5 mg of the prepared insoluble DDGS
substrate was weighed into 1.5 ml eppendorf tubes and mixed with
citrate buffer (25 mM, pH 6), xylanase (217 mg/kg substrate and 206
mg/kg substrate for corn- and wheat DDGS, respectively), and
protease (8.6.times.10.sup.5 U/kg substrate) to a final reaction
volume of 1.0 ml. The incubations were carried out at 4 h,
39.degree. C. and 1300 rpm by use of Eppendorf ThermoMixer
incubator (Eppendorf). After incubation, samples were filtered and
analyzed for soluble pentosan and--protein content, as described
below. Reactions were performed in duplicates.
[0861] Protein Quantification
[0862] Soluble protein was quantified using the BCA (bicinchoninic
acid) Protein Assay Kit from Pierce. The samples were prepared in
microtiter plates (25 .mu.l/well) and incubated with 200 .mu.l
premixed assay reagent for 30 minutes at 37.degree. C., 1100 rpm.
The absorbance was measured spectrophotometrically at 562 nm
against a 0-2000 .mu.g/ml Bovine Serum Albumin (BSA) standard, as
described in the manual. Values were corrected for the amount of
added enzymes.
[0863] Quantification of C5 Sugars (Pentosans)
[0864] The total amount of pentoses brought into solution was
measured using the method of Rouau and Surget (1994, A rapid
semi-automated method of the determination of total and
water-extractable pentosan in wheat flours. Carbohydrate Polymers,
24, 123-32) with a continuous flow injection apparatus (FIG. 7).
The supernatants were treated with acid to hydrolyse
polysaccharides to monosugars. Phloroglucinol (1, 3,
5-trihydroxybenzen) was added for reaction with monopentoses and
monohexoses, which forms a coloured complex.
[0865] By measuring the difference in absorbance at 550 nm compared
to 510 nm, the amount of pentoses in the solution was calculated
using a standard curve. Unlike the pentose-phloroglucinol complex,
the absorbance of the hexose-phloroglucinol complex is constant at
these wavelengths. Glucose was added to the phloroglucinol solution
to create a constant glucose signal and further ensure no
interference from hexose sugars.
[0866] Statistical Analysis
[0867] A one-way ANOVA was applied on the experimental data for
comparison of treatments on both the solubilization of pentosan and
protein, with pairwise comparisons performed by Holm-Sidak method,
using SigmaPlot 12.0 (SyStat Software Inc.). Overall significance
level at P=0.05.
[0868] 23.2 Results and Discussion
[0869] Pentosan and protein solubilization was measured by
incubation of insoluble corn and wheat DDGS with xylanase and
protease alone and in combination.
[0870] The results are shown in FIG. 27 (insoluble corn DDGS) and
in FIG. 28 (insoluble wheat DDGS).
[0871] FIG. 27 shows the effect of the xylanase and protease
treatments alone and in combination on the solubilization of
pentosan and protein from insoluble corn DDGS. Letters a-d are
significant different according to on-way ANOVA and Holm-Sidak
comparisons with overall significance level at P=0.05. Error bars
indicate S.D.
[0872] FIG. 28 shows the effect of the xylanase and protease
treatments alone and in combination on the solubilization of
pentosan and protein from insoluble wheat DDGS. Letters a-d are
significant different according to on-way ANOVA and Holm-Sidak
comparisons with overall significance level at P=0.05. Error bars
indicate S.D.
[0873] When compared to the effects of xylanase treatment by
itself, the combination of xylanase and protease further increased
the solubillization of protein from both corn and wheat DDGS. More
interestingly, addition of protease also significantly increased
the solubilization of pentosan from both corn and wheat DDGS,
indicating a synergistic effect where addition of protease increase
the accessibility of the xylanase towards the substrate by opening
up the feed matrix structure through protein degradation.
Furthermore, xylanase by itself and in combination with protease
also increase the solubilization of protein as compared to control
and protease alone, respectively. This further supports the theory
of a synergistic effect between xylanase and protease.
Example 24
[0874] Comparison of FveXyn4 and FoxXyn2 in Pentosan Solubilisation
(Breakdown or Solubilisation of Insoluble Arabinoxylan
(AXinsol))
[0875] The ability to solubilize insoluble arabinoxylan from corn
DDGS was used as one of the key selection criteria and in this
experiment the ability to solubilise arabinoxylan from corn DDGS is
compared for the two xylanases of the present invention (FveXyn4
and FoxXyn2).
[0876] The two xylanases showed strong and equal performance on
pentosan solubilisation from corn DDGS, indicating equal
performance of these homologous xylanases on solubilisation of
arabinoxylan from fibrous by-products.
[0877] 24.1 Materials and Methods
[0878] Enzyme Samples
[0879] The xylanases used in this study are:
[0880] A GH10 xylanase from Fusarium verticilloides (designated
FveXyn4) expressed in Trichoderma reesei, wherein the xylanase was
used in purified form--this enzyme may be referred to herein as
FveXyn4, and a GH10 xylanase from Fusarium oxysporum (designated
FoxXyn2) expressed in Trichoderma reesei, wherein the xylanase was
used in purified form--this enzyme may be referred to herein as
FoxXyn2
[0881] Feed Raw Materials
[0882] The feed used in this experiments is corn DDGS.
[0883] Pentosan Solubilisation (AXinsol Solubilisation)
[0884] The method used for pentosan solubilisation was: 100 mg of
feed raw material was transferred to a 2 ml Eppendorf centrifuge
tube and the precise weight recorded. 750 .mu.L incubation buffer
(200 mM HEPES, 100 mM NaCl, 2 mM CaCl, pH 6.0) and 900 .mu.l
chloramphenicol solution (40 .mu.g/ml in incubation buffer) was
added. Enzyme of choice was added to make a total volume of 1.8
mL.
[0885] Each sample was assayed in doublets and in parallel with a
blank (incubation without exogenously added enzyme). The samples
were incubated on an Eppendorf thermomixer at 40.degree. C. with
shaking. After 2 nr 18 hours of incubation the supernatant was
filtered using 96 wells filterplates (Pall Corporation, AcroPrep 96
Filter Plate, 1.0 .mu.m Glass, NTRL, 1 mL well). After filtration
the samples were stored at 4.degree. C. until analysis for total
amount of C5 sugars, arabinose and xylose.
[0886] Quantification of C5 Sugars (Pentosans)
[0887] The total amount of pentoses brought into solution was
measured using the method of Rouau and Surget (1994, A rapid
semi-automated method of the determination of total and
water-extractable pentosan in wheat flours. Carbohydrate Polymers,
24, 123-32) with a continuous flow injection apparatus (FIG. 7).
The supernatants were treated with acid to hydrolyse
polysaccharides to monosugars. Phloroglucinol (1, 3,
5-trihydroxybenzen) was added for reaction with monopentoses and
monohexoses, which forms a coloured complex. By measuring the
difference in absorbance at 550 nm compared to 510 nm, the amount
of pentoses in the solution was calculated using a standard curve.
Unlike the pentose-phloroglucinol complex, the absorbance of the
hexose-phloroglucinol complex is constant at these wavelengths.
Glucose was added to the phloroglucinol solution to create a
constant glucose signal and further ensure no interference from
hexose sugars.
[0888] 24.2 Result and Discussion
[0889] Pentosan solubilisation was monitored in a dose response
setup using fibrous by-product of corn (namely cDDGS).
[0890] The results comparing the two xylanases of the present
invention (FveXyn4 and FoxXyn2) are shown in FIG. 29 (in corn
DDGS).
[0891] FIG. 29 shows solubilisation of pentosans from cDDGS as a
function of xylanase dosage. The xylanases used were the xylanases
of the present invention (FveXyn4 and FoxXyn2).
[0892] Both xylanases performs well on corn and are equally good a
breaking down AXinsol (e.g. solubilising pentosans) in corn based
substrates which clearly indicates that these two homologous
xylanases perform equal on solubilisation of arabinoxylan from
fibrous by-products.
[0893] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the present invention.
Although the present invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in biochemistry and biotechnology or related fields
are intended to be within the scope of the following claims.
Sequence CWU 1
1
201328PRTFusarium verticillioides 1Met Lys Leu Ser Ser Phe Leu Tyr
Thr Ala Ser Leu Val Ala Ala Ile1 5 10 15Pro Thr Ala Ile Glu Pro Arg
Gln Ala Ala Asp Ser Ile Asn Lys Leu 20 25 30Ile Lys Asn Lys Gly Lys
Leu Tyr Tyr Gly Thr Ile Thr Asp Pro Asn 35 40 45Leu Leu Gly Val Ala
Lys Asp Thr Ala Ile Ile Lys Ala Asp Phe Gly 50 55 60Ala Val Thr Pro
Glu Asn Ser Gly Lys Trp Asp Ala Thr Glu Pro Ser65 70 75 80Gln Gly
Lys Phe Asn Phe Gly Ser Phe Asp Gln Val Val Asn Phe Ala 85 90 95Gln
Gln Asn Gly Leu Lys Val Arg Gly His Thr Leu Val Trp His Ser 100 105
110Gln Leu Pro Gln Trp Val Lys Asn Ile Asn Asp Lys Ala Thr Leu Thr
115 120 125Lys Val Ile Glu Asn His Val Thr Gln Val Val Gly Arg Tyr
Lys Gly 130 135 140Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Ile Phe
Glu Trp Asp Gly145 150 155 160Thr Leu Arg Lys Asp Ser His Phe Asn
Asn Val Phe Gly Asn Asp Asp 165 170 175Tyr Val Gly Ile Ala Phe Arg
Ala Ala Arg Lys Ala Asp Pro Asn Ala 180 185 190Lys Leu Tyr Ile Asn
Asp Tyr Ser Leu Asp Ser Gly Ser Ala Ser Lys 195 200 205Val Thr Lys
Gly Met Val Pro Ser Val Lys Lys Trp Leu Ser Gln Gly 210 215 220Val
Pro Val Asp Gly Ile Gly Ser Gln Thr His Leu Asp Pro Gly Ala225 230
235 240Ala Gly Gln Ile Gln Gly Ala Leu Thr Ala Leu Ala Asn Ser Gly
Val 245 250 255Lys Glu Val Ala Ile Thr Glu Leu Asp Ile Arg Thr Ala
Pro Ala Asn 260 265 270Asp Tyr Ala Thr Val Thr Lys Ala Cys Leu Asn
Val Pro Lys Cys Ile 275 280 285Gly Ile Thr Val Trp Gly Val Ser Asp
Lys Asn Ser Trp Arg Lys Glu 290 295 300His Asp Ser Leu Leu Phe Asp
Ala Asn Tyr Asn Pro Lys Pro Ala Tyr305 310 315 320Thr Ala Val Val
Asn Ala Leu Arg 3252313PRTFusarium verticillioides 2Ile Pro Thr Ala
Ile Glu Pro Arg Gln Ala Ala Asp Ser Ile Asn Lys1 5 10 15Leu Ile Lys
Asn Lys Gly Lys Leu Tyr Tyr Gly Thr Ile Thr Asp Pro 20 25 30Asn Leu
Leu Gly Val Ala Lys Asp Thr Ala Ile Ile Lys Ala Asp Phe 35 40 45Gly
Ala Val Thr Pro Glu Asn Ser Gly Lys Trp Asp Ala Thr Glu Pro 50 55
60Ser Gln Gly Lys Phe Asn Phe Gly Ser Phe Asp Gln Val Val Asn Phe65
70 75 80Ala Gln Gln Asn Gly Leu Lys Val Arg Gly His Thr Leu Val Trp
His 85 90 95Ser Gln Leu Pro Gln Trp Val Lys Asn Ile Asn Asp Lys Ala
Thr Leu 100 105 110Thr Lys Val Ile Glu Asn His Val Thr Gln Val Val
Gly Arg Tyr Lys 115 120 125Gly Lys Ile Tyr Ala Trp Asp Val Val Asn
Glu Ile Phe Glu Trp Asp 130 135 140Gly Thr Leu Arg Lys Asp Ser His
Phe Asn Asn Val Phe Gly Asn Asp145 150 155 160Asp Tyr Val Gly Ile
Ala Phe Arg Ala Ala Arg Lys Ala Asp Pro Asn 165 170 175Ala Lys Leu
Tyr Ile Asn Asp Tyr Ser Leu Asp Ser Gly Ser Ala Ser 180 185 190Lys
Val Thr Lys Gly Met Val Pro Ser Val Lys Lys Trp Leu Ser Gln 195 200
205Gly Val Pro Val Asp Gly Ile Gly Ser Gln Thr His Leu Asp Pro Gly
210 215 220Ala Ala Gly Gln Ile Gln Gly Ala Leu Thr Ala Leu Ala Asn
Ser Gly225 230 235 240Val Lys Glu Val Ala Ile Thr Glu Leu Asp Ile
Arg Thr Ala Pro Ala 245 250 255Asn Asp Tyr Ala Thr Val Thr Lys Ala
Cys Leu Asn Val Pro Lys Cys 260 265 270Ile Gly Ile Thr Val Trp Gly
Val Ser Asp Lys Asn Ser Trp Arg Lys 275 280 285Glu His Asp Ser Leu
Leu Phe Asp Ala Asn Tyr Asn Pro Lys Pro Ala 290 295 300Tyr Thr Ala
Val Val Asn Ala Leu Arg305 3103305PRTFusarium verticillioides 3Gln
Ala Ala Asp Ser Ile Asn Lys Leu Ile Lys Asn Lys Gly Lys Leu1 5 10
15Tyr Tyr Gly Thr Ile Thr Asp Pro Asn Leu Leu Gly Val Ala Lys Asp
20 25 30Thr Ala Ile Ile Lys Ala Asp Phe Gly Ala Val Thr Pro Glu Asn
Ser 35 40 45Gly Lys Trp Asp Ala Thr Glu Pro Ser Gln Gly Lys Phe Asn
Phe Gly 50 55 60Ser Phe Asp Gln Val Val Asn Phe Ala Gln Gln Asn Gly
Leu Lys Val65 70 75 80Arg Gly His Thr Leu Val Trp His Ser Gln Leu
Pro Gln Trp Val Lys 85 90 95Asn Ile Asn Asp Lys Ala Thr Leu Thr Lys
Val Ile Glu Asn His Val 100 105 110Thr Gln Val Val Gly Arg Tyr Lys
Gly Lys Ile Tyr Ala Trp Asp Val 115 120 125Val Asn Glu Ile Phe Glu
Trp Asp Gly Thr Leu Arg Lys Asp Ser His 130 135 140Phe Asn Asn Val
Phe Gly Asn Asp Asp Tyr Val Gly Ile Ala Phe Arg145 150 155 160Ala
Ala Arg Lys Ala Asp Pro Asn Ala Lys Leu Tyr Ile Asn Asp Tyr 165 170
175Ser Leu Asp Ser Gly Ser Ala Ser Lys Val Thr Lys Gly Met Val Pro
180 185 190Ser Val Lys Lys Trp Leu Ser Gln Gly Val Pro Val Asp Gly
Ile Gly 195 200 205Ser Gln Thr His Leu Asp Pro Gly Ala Ala Gly Gln
Ile Gln Gly Ala 210 215 220Leu Thr Ala Leu Ala Asn Ser Gly Val Lys
Glu Val Ala Ile Thr Glu225 230 235 240Leu Asp Ile Arg Thr Ala Pro
Ala Asn Asp Tyr Ala Thr Val Thr Lys 245 250 255Ala Cys Leu Asn Val
Pro Lys Cys Ile Gly Ile Thr Val Trp Gly Val 260 265 270Ser Asp Lys
Asn Ser Trp Arg Lys Glu His Asp Ser Leu Leu Phe Asp 275 280 285Ala
Asn Tyr Asn Pro Lys Pro Ala Tyr Thr Ala Val Val Asn Ala Leu 290 295
300Arg30541039DNAFusarium verticillioides 4atgaagctgt cttctttcct
ctacaccgcc tcgctggtcg cggccattcc caccgccatc 60gagccccgcc aggctgccga
cagcatcaac aagctgatca agaacaaggg caagctctac 120tacggaacca
tcaccgaccc caacctgctc ggcgtcgcaa aggacaccgc catcatcaag
180gccgactttg gcgccgttac ccccgagaac tcgggcaagt gggacgccac
cgagcccagc 240cagggcaagt tcaacttcgg tagcttcgac caggttgtca
actttgccca gcagaatggc 300ctcaaggtcc gaggtcacac tctggtctgg
cactctcagc tccctcagtg ggttaagaac 360atcaacgaca aggctactct
gaccaaggtc attgagaacc acgtcaccca agtcgttgga 420cgctacaagg
gcaagatcta cgcctgggta tgttttattc ccccagactt cttcgaaatg
480actttgctaa catgttcagg acgtcgtcaa cgagatcttc gagtgggacg
gtaccctccg 540aaaggactct cacttcaaca acgtcttcgg caacgacgac
tacgttggca ttgccttccg 600cgccgcccgc aaggctgacc ccaacgccaa
gctgtacatc aacgactaca gcctcgactc 660cggcagcgcc tccaaggtca
ccaagggtat ggttccctcc gtcaagaagt ggctcagcca 720gggcgttccc
gtcgacggca ttggctctca gactcacctt gaccccggtg ccgctggcca
780aatccagggt gctctcactg ccctcgccaa ttctggtgtc aaggaggttg
ccatcaccga 840gctcgacatc cgcactgccc ccgccaacga ctacgctacc
gtcaccaagg cctgcctcaa 900cgtccccaag tgcattggta tcaccgtctg
gggtgtctct gacaagaact cttggcgcaa 960ggagcacgac agtcttctgt
tcgatgctaa ctacaacccc aagcctgctt acactgctgt 1020tgtcaacgct
ctccgctaa 10395987DNAFusarium verticillioides 5atgaagctgt
cttctttcct ctacaccgcc tcgctggtcg cggccattcc caccgccatc 60gagccccgcc
aggctgccga cagcatcaac aagctgatca agaacaaggg caagctctac
120tacggaacca tcaccgaccc caacctgctc ggcgtcgcaa aggacaccgc
catcatcaag 180gccgactttg gcgccgttac ccccgagaac tcgggcaagt
gggacgccac cgagcccagc 240cagggcaagt tcaacttcgg tagcttcgac
caggttgtca actttgccca gcagaatggc 300ctcaaggtcc gaggtcacac
tctggtctgg cactctcagc tccctcagtg ggttaagaac 360atcaacgaca
aggctactct gaccaaggtc attgagaacc acgtcaccca agtcgttgga
420cgctacaagg gcaagatcta cgcctgggac gtcgtcaacg agatcttcga
gtgggacggt 480accctccgaa aggactctca cttcaacaac gtcttcggca
acgacgacta cgttggcatt 540gccttccgcg ccgcccgcaa ggctgacccc
aacgccaagc tgtacatcaa cgactacagc 600ctcgactccg gcagcgcctc
caaggtcacc aagggtatgg ttccctccgt caagaagtgg 660ctcagccagg
gcgttcccgt cgacggcatt ggctctcaga ctcaccttga ccccggtgcc
720gctggccaaa tccagggtgc tctcactgcc ctcgccaatt ctggtgtcaa
ggaggttgcc 780atcaccgagc tcgacatccg cactgccccc gccaacgact
acgctaccgt caccaaggcc 840tgcctcaacg tccccaagtg cattggtatc
accgtctggg gtgtctctga caagaactct 900tggcgcaagg agcacgacag
tcttctgttc gatgctaact acaaccccaa gcctgcttac 960actgctgttg
tcaacgctct ccgctaa 9876942DNAFusarium verticillioides 6attcccaccg
ccatcgagcc ccgccaggct gccgacagca tcaacaagct gatcaagaac 60aagggcaagc
tctactacgg aaccatcacc gaccccaacc tgctcggcgt cgcaaaggac
120accgccatca tcaaggccga ctttggcgcc gttacccccg agaactcggg
caagtgggac 180gccaccgagc ccagccaggg caagttcaac ttcggtagct
tcgaccaggt tgtcaacttt 240gcccagcaga atggcctcaa ggtccgaggt
cacactctgg tctggcactc tcagctccct 300cagtgggtta agaacatcaa
cgacaaggct actctgacca aggtcattga gaaccacgtc 360acccaagtcg
ttggacgcta caagggcaag atctacgcct gggacgtcgt caacgagatc
420ttcgagtggg acggtaccct ccgaaaggac tctcacttca acaacgtctt
cggcaacgac 480gactacgttg gcattgcctt ccgcgccgcc cgcaaggctg
accccaacgc caagctgtac 540atcaacgact acagcctcga ctccggcagc
gcctccaagg tcaccaaggg tatggttccc 600tccgtcaaga agtggctcag
ccagggcgtt cccgtcgacg gcattggctc tcagactcac 660cttgaccccg
gtgccgctgg ccaaatccag ggtgctctca ctgccctcgc caattctggt
720gtcaaggagg ttgccatcac cgagctcgac atccgcactg cccccgccaa
cgactacgct 780accgtcacca aggcctgcct caacgtcccc aagtgcattg
gtatcaccgt ctggggtgtc 840tctgacaaga actcttggcg caaggagcac
gacagtcttc tgttcgatgc taactacaac 900cccaagcctg cttacactgc
tgttgtcaac gctctccgct aa 942727DNAArtificial SequencePrimer 1
7caccatgaag ctgtcttctt tcctcta 27827DNAArtificial SequencePrimer 2
8tttttagcgg agagcgttga caacagc 279328PRTFusarium oxysporum 9Met Lys
Leu Ser Ser Phe Leu Tyr Thr Ala Ser Leu Val Ala Ala Ile1 5 10 15Pro
Thr Ala Ile Glu Pro Arg Gln Ala Ser Asp Ser Ile Asn Lys Leu 20 25
30Ile Lys Asn Lys Gly Lys Leu Tyr Tyr Gly Thr Ile Thr Asp Pro Asn
35 40 45Leu Leu Gly Val Ala Lys Asp Thr Ala Ile Ile Lys Ala Asp Phe
Gly 50 55 60Ala Val Thr Pro Glu Asn Ser Gly Lys Trp Asp Ala Thr Glu
Pro Ser65 70 75 80Gln Gly Lys Phe Asn Phe Gly Ser Phe Asp Gln Val
Val Asn Phe Ala 85 90 95Gln Gln Asn Gly Leu Lys Val Arg Gly His Thr
Leu Val Trp His Ser 100 105 110Gln Leu Pro Gln Trp Val Lys Asn Ile
Asn Asp Lys Ala Thr Leu Thr 115 120 125Lys Val Ile Glu Asn His Val
Thr Asn Val Val Gly Arg Tyr Lys Gly 130 135 140Lys Ile Tyr Ala Trp
Asp Val Val Asn Glu Ile Phe Asp Trp Asp Gly145 150 155 160Thr Leu
Arg Lys Asp Ser His Phe Asn Asn Val Phe Gly Asn Asp Asp 165 170
175Tyr Val Gly Ile Ala Phe Arg Ala Ala Arg Lys Ala Asp Pro Asn Ala
180 185 190Lys Leu Tyr Ile Asn Asp Tyr Ser Leu Asp Ser Gly Ser Ala
Ser Lys 195 200 205Val Thr Lys Gly Met Val Pro Ser Val Lys Lys Trp
Leu Ser Gln Gly 210 215 220Val Pro Val Asp Gly Ile Gly Ser Gln Thr
His Leu Asp Pro Gly Ala225 230 235 240Ala Gly Gln Ile Gln Gly Ala
Leu Thr Ala Leu Ala Asn Ser Gly Val 245 250 255Lys Glu Val Ala Ile
Thr Glu Leu Asp Ile Arg Thr Ala Pro Ala Asn 260 265 270Asp Tyr Ala
Thr Val Thr Lys Ala Cys Leu Asn Val Pro Lys Cys Ile 275 280 285Gly
Ile Thr Val Trp Gly Val Ser Asp Lys Asn Ser Trp Arg Lys Glu 290 295
300His Asp Ser Leu Leu Phe Asp Ala Asn Tyr Asn Pro Lys Ala Ala
Tyr305 310 315 320Thr Ala Val Val Asn Ala Leu Arg
32510313PRTFusarium oxysporum 10Ile Pro Thr Ala Ile Glu Pro Arg Gln
Ala Ser Asp Ser Ile Asn Lys1 5 10 15Leu Ile Lys Asn Lys Gly Lys Leu
Tyr Tyr Gly Thr Ile Thr Asp Pro 20 25 30Asn Leu Leu Gly Val Ala Lys
Asp Thr Ala Ile Ile Lys Ala Asp Phe 35 40 45Gly Ala Val Thr Pro Glu
Asn Ser Gly Lys Trp Asp Ala Thr Glu Pro 50 55 60Ser Gln Gly Lys Phe
Asn Phe Gly Ser Phe Asp Gln Val Val Asn Phe65 70 75 80Ala Gln Gln
Asn Gly Leu Lys Val Arg Gly His Thr Leu Val Trp His 85 90 95Ser Gln
Leu Pro Gln Trp Val Lys Asn Ile Asn Asp Lys Ala Thr Leu 100 105
110Thr Lys Val Ile Glu Asn His Val Thr Asn Val Val Gly Arg Tyr Lys
115 120 125Gly Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Ile Phe Asp
Trp Asp 130 135 140Gly Thr Leu Arg Lys Asp Ser His Phe Asn Asn Val
Phe Gly Asn Asp145 150 155 160Asp Tyr Val Gly Ile Ala Phe Arg Ala
Ala Arg Lys Ala Asp Pro Asn 165 170 175Ala Lys Leu Tyr Ile Asn Asp
Tyr Ser Leu Asp Ser Gly Ser Ala Ser 180 185 190Lys Val Thr Lys Gly
Met Val Pro Ser Val Lys Lys Trp Leu Ser Gln 195 200 205Gly Val Pro
Val Asp Gly Ile Gly Ser Gln Thr His Leu Asp Pro Gly 210 215 220Ala
Ala Gly Gln Ile Gln Gly Ala Leu Thr Ala Leu Ala Asn Ser Gly225 230
235 240Val Lys Glu Val Ala Ile Thr Glu Leu Asp Ile Arg Thr Ala Pro
Ala 245 250 255Asn Asp Tyr Ala Thr Val Thr Lys Ala Cys Leu Asn Val
Pro Lys Cys 260 265 270Ile Gly Ile Thr Val Trp Gly Val Ser Asp Lys
Asn Ser Trp Arg Lys 275 280 285Glu His Asp Ser Leu Leu Phe Asp Ala
Asn Tyr Asn Pro Lys Ala Ala 290 295 300Tyr Thr Ala Val Val Asn Ala
Leu Arg305 31011305PRTFusarium oxysporum 11Gln Ala Ser Asp Ser Ile
Asn Lys Leu Ile Lys Asn Lys Gly Lys Leu1 5 10 15Tyr Tyr Gly Thr Ile
Thr Asp Pro Asn Leu Leu Gly Val Ala Lys Asp 20 25 30Thr Ala Ile Ile
Lys Ala Asp Phe Gly Ala Val Thr Pro Glu Asn Ser 35 40 45Gly Lys Trp
Asp Ala Thr Glu Pro Ser Gln Gly Lys Phe Asn Phe Gly 50 55 60Ser Phe
Asp Gln Val Val Asn Phe Ala Gln Gln Asn Gly Leu Lys Val65 70 75
80Arg Gly His Thr Leu Val Trp His Ser Gln Leu Pro Gln Trp Val Lys
85 90 95Asn Ile Asn Asp Lys Ala Thr Leu Thr Lys Val Ile Glu Asn His
Val 100 105 110Thr Asn Val Val Gly Arg Tyr Lys Gly Lys Ile Tyr Ala
Trp Asp Val 115 120 125Val Asn Glu Ile Phe Asp Trp Asp Gly Thr Leu
Arg Lys Asp Ser His 130 135 140Phe Asn Asn Val Phe Gly Asn Asp Asp
Tyr Val Gly Ile Ala Phe Arg145 150 155 160Ala Ala Arg Lys Ala Asp
Pro Asn Ala Lys Leu Tyr Ile Asn Asp Tyr 165 170 175Ser Leu Asp Ser
Gly Ser Ala Ser Lys Val Thr Lys Gly Met Val Pro 180 185 190Ser Val
Lys Lys Trp Leu Ser Gln Gly Val Pro Val Asp Gly Ile Gly 195 200
205Ser Gln Thr His Leu Asp Pro Gly Ala Ala Gly Gln Ile Gln Gly Ala
210 215 220Leu Thr Ala Leu Ala Asn Ser Gly Val Lys Glu Val Ala Ile
Thr Glu225 230 235 240Leu Asp Ile Arg Thr Ala Pro Ala Asn Asp Tyr
Ala Thr Val Thr Lys 245 250 255Ala Cys Leu Asn Val Pro Lys Cys Ile
Gly Ile Thr Val Trp Gly Val 260 265 270Ser Asp Lys Asn Ser Trp Arg
Lys Glu His Asp Ser Leu Leu Phe Asp 275 280 285Ala Asn Tyr Asn Pro
Lys Ala Ala Tyr Thr Ala Val Val Asn Ala Leu 290 295
300Arg305121039DNAFusarium oxysporum 12atgaagctgt cttccttcct
ctacaccgcc tcgctggtcg cggccattcc caccgccatc 60gagccccgcc aggcctccga
cagcatcaac aagctgatca agaacaaggg caagctctac 120tacggaacca
tcaccgaccc caacctgctc ggcgtcgcaa aggacactgc catcatcaag
180gctgactttg gcgccgtcac acccgagaac tcgggtaagt gggatgccac
cgagcccagc 240cagggcaagt tcaacttcgg cagcttcgac caggtcgtca
actttgctca gcagaatggc 300ctcaaggtcc gaggtcacac tctagtctgg
cactcccagc tccctcagtg ggttaagaac 360atcaacgaca aggctacttt
gaccaaggtc atcgagaacc acgtcaccaa cgtcgttgga 420cgctacaagg
gcaagatcta cgcctgggta tgttttcttc actcgaactt cttataaatg
480gctttactaa catgttcagg acgtcgttaa cgagatcttc gactgggatg
gtaccctccg 540aaaggactct cacttcaaca acgtcttcgg caacgacgac
tacgttggca ttgccttccg 600cgctgcccgc aaggctgacc ccaacgccaa
gctgtacatc aacgactaca gcctcgactc 660cggcagcgcc tccaaggtca
ccaagggcat ggttccctct gtcaagaagt ggctcagcca 720gggcgtcccc
gtcgacggta ttggttctca gactcacctt gaccccggtg ccgctggcca
780aatccagggt gctctcactg ccctcgccaa ctctggtgtg aaggaggttg
ccatcaccga 840gctcgacatc cgcactgccc ccgccaacga ctacgctacc
gttaccaagg cctgcctcaa 900cgtccccaag tgcattggta tcaccgtctg
gggcgtatct gacaagaact cttggcgcaa 960ggagcacgac agccttctgt
tcgatgctaa ctacaacccc aaggctgctt acactgctgt 1020tgtcaacgct
ctccgctaa 103913987DNAFusarium oxysporum 13atgaagctgt cttccttcct
ctacaccgcc tcgctggtcg cggccattcc caccgccatc 60gagccccgcc aggcctccga
cagcatcaac aagctgatca agaacaaggg caagctctac 120tacggaacca
tcaccgaccc caacctgctc ggcgtcgcaa aggacactgc catcatcaag
180gctgactttg gcgccgtcac acccgagaac tcgggtaagt gggatgccac
cgagcccagc 240cagggcaagt tcaacttcgg cagcttcgac caggtcgtca
actttgctca gcagaatggc 300ctcaaggtcc gaggtcacac tctagtctgg
cactcccagc tccctcagtg ggttaagaac 360atcaacgaca aggctacttt
gaccaaggtc atcgagaacc acgtcaccaa cgtcgttgga 420cgctacaagg
gcaagatcta cgcctgggac gtcgttaacg agatcttcga ctgggatggt
480accctccgaa aggactctca cttcaacaac gtcttcggca acgacgacta
cgttggcatt 540gccttccgcg ctgcccgcaa ggctgacccc aacgccaagc
tgtacatcaa cgactacagc 600ctcgactccg gcagcgcctc caaggtcacc
aagggcatgg ttccctctgt caagaagtgg 660ctcagccagg gcgtccccgt
cgacggtatt ggttctcaga ctcaccttga ccccggtgcc 720gctggccaaa
tccagggtgc tctcactgcc ctcgccaact ctggtgtgaa ggaggttgcc
780atcaccgagc tcgacatccg cactgccccc gccaacgact acgctaccgt
taccaaggcc 840tgcctcaacg tccccaagtg cattggtatc accgtctggg
gcgtatctga caagaactct 900tggcgcaagg agcacgacag ccttctgttc
gatgctaact acaaccccaa ggctgcttac 960actgctgttg tcaacgctct ccgctaa
98714942DNAFusarium oxysporum 14attcccaccg ccatcgagcc ccgccaggcc
tccgacagca tcaacaagct gatcaagaac 60aagggcaagc tctactacgg aaccatcacc
gaccccaacc tgctcggcgt cgcaaaggac 120actgccatca tcaaggctga
ctttggcgcc gtcacacccg agaactcggg taagtgggat 180gccaccgagc
ccagccaggg caagttcaac ttcggcagct tcgaccaggt cgtcaacttt
240gctcagcaga atggcctcaa ggtccgaggt cacactctag tctggcactc
ccagctccct 300cagtgggtta agaacatcaa cgacaaggct actttgacca
aggtcatcga gaaccacgtc 360accaacgtcg ttggacgcta caagggcaag
atctacgcct gggacgtcgt taacgagatc 420ttcgactggg atggtaccct
ccgaaaggac tctcacttca acaacgtctt cggcaacgac 480gactacgttg
gcattgcctt ccgcgctgcc cgcaaggctg accccaacgc caagctgtac
540atcaacgact acagcctcga ctccggcagc gcctccaagg tcaccaaggg
catggttccc 600tctgtcaaga agtggctcag ccagggcgtc cccgtcgacg
gtattggttc tcagactcac 660cttgaccccg gtgccgctgg ccaaatccag
ggtgctctca ctgccctcgc caactctggt 720gtgaaggagg ttgccatcac
cgagctcgac atccgcactg cccccgccaa cgactacgct 780accgttacca
aggcctgcct caacgtcccc aagtgcattg gtatcaccgt ctggggcgta
840tctgacaaga actcttggcg caaggagcac gacagccttc tgttcgatgc
taactacaac 900cccaaggctg cttacactgc tgttgtcaac gctctccgct aa
94215305PRTFusarium sp. 15Gln Ala Ala Asp Ser Ile Asn Lys Leu Ile
Lys Asn Lys Gly Lys Leu1 5 10 15Tyr Tyr Gly Thr Ile Thr Asp Pro Asn
Leu Leu Gly Val Ala Lys Asp 20 25 30Thr Ala Val Ile Lys Ala Asp Phe
Gly Ala Val Thr Pro Glu Asn Ser 35 40 45Gly Lys Trp Asp Ala Thr Glu
Pro Ser Gln Gly Asn Phe Asn Phe Gly 50 55 60Ser Phe Asp Gln Val Val
Asn Phe Ala Gln Gln Asn Gly Leu Lys Val65 70 75 80Arg Gly His Thr
Leu Val Trp His Ser Gln Leu Pro Gln Trp Val Lys 85 90 95Asn Ile Asn
Asp Lys Ala Thr Leu Thr Lys Val Ile Glu Asn His Val 100 105 110Thr
Gln Val Val Gly Arg Tyr Lys Gly Lys Ile Tyr Ala Trp Asp Val 115 120
125Val Asn Glu Ile Phe Asp Trp Asp Gly Thr Leu Arg Lys Asp Ser His
130 135 140Phe Asn Asn Val Phe Gly Asn Asp Asp Tyr Val Gly Ile Ala
Phe Arg145 150 155 160Ala Ala Arg Lys Ala Asp Pro Asn Ala Lys Leu
Tyr Ile Asn Asp Tyr 165 170 175Ser Leu Asp Ser Ala Ser Ala Ser Lys
Val Thr Lys Gly Met Val Pro 180 185 190Ser Val Lys Lys Trp Leu Ser
Gln Gly Val Pro Val Asp Gly Ile Gly 195 200 205Ser Gln Ser His Leu
Asp Pro Gly Ala Ala Gly Gln Val Gln Gly Ala 210 215 220Leu Thr Ala
Leu Ala Asn Ser Gly Val Lys Glu Val Ala Ile Thr Glu225 230 235
240Leu Asp Ile Arg Thr Ala Pro Ala Asn Asp Tyr Ala Thr Val Thr Lys
245 250 255Ala Cys Leu Asn Val Pro Lys Cys Ile Gly Ile Thr Val Trp
Gly Val 260 265 270Ser Asp Lys Asn Ser Trp Arg Lys Glu His Asp Ser
Leu Leu Phe Asp 275 280 285Ser Asn Tyr Asn Pro Lys Pro Ala Tyr Thr
Ala Val Val Asn Ala Leu 290 295 300Arg305161039DNAFusarium sp.
16atgaagctgt cttctttcct ctacaccgcc tcgctggtcg cggccattcc caccgccatc
60gagccccgcc aggccgccga cagcatcaac aagctgatca agaacaaggg caagctctac
120tacggaacca tcaccgaccc caacctgctc ggcgtcgcaa aggacaccgc
cgtcatcaag 180gccgactttg gcgccgtcac ccccgagaac tcgggcaagt
gggacgccac cgagcccagc 240cagggcaact tcaacttcgg tagcttcgac
caggtcgtca actttgctca gcagaatggc 300ctcaaggtcc gaggtcacac
tctggtctgg cactctcagc tccctcagtg ggttaagaac 360atcaacgaca
aggctactct gaccaaggtc attgagaacc acgtcaccca agtcgttgga
420cgctacaagg gcaagatcta cgcctgggta tgttttcttg cctcgacctt
ctcaaagatg 480aatttgctaa catgttcagg acgttgtcaa cgagatcttc
gactgggacg gtaccctccg 540aaaggattct cacttcaaca acgtcttcgg
caacgatgac tacgttggca ttgccttccg 600cgccgcccgc aaggctgacc
ccaacgccaa gctgtacatc aacgactaca gcctcgactc 660cgccagcgcc
tccaaggtca ccaagggcat ggtcccctcc gtcaagaagt ggctcagcca
720gggcgttccc gtcgacggca ttggctccca gtctcacctt gaccccggtg
ccgctggcca 780agtccagggt gctctcactg ccctcgccaa ctctggtgtc
aaggaggttg ccatcaccga 840gctcgacatc cgcactgccc ccgccaacga
ctacgccacc gtcaccaagg cctgcctaaa 900cgtccccaag tgcattggta
tcaccgtctg gggtgtctct gacaagaact cttggcgcaa 960ggagcacgac
agccttctgt tcgactccaa ctacaacccc aagcctgctt acactgctgt
1020tgtcaacgct ctccgctaa 103917987DNAFusarium sp. 17atgaagctgt
cttctttcct ctacaccgcc tcgctggtcg cggccattcc caccgccatc 60gagccccgcc
aggccgccga cagcatcaac aagctgatca agaacaaggg caagctctac
120tacggaacca tcaccgaccc caacctgctc ggcgtcgcaa aggacaccgc
cgtcatcaag 180gccgactttg gcgccgtcac ccccgagaac tcgggcaagt
gggacgccac cgagcccagc 240cagggcaact tcaacttcgg tagcttcgac
caggtcgtca actttgctca gcagaatggc 300ctcaaggtcc gaggtcacac
tctggtctgg cactctcagc tccctcagtg ggttaagaac 360atcaacgaca
aggctactct gaccaaggtc attgagaacc acgtcaccca agtcgttgga
420cgctacaagg gcaagatcta cgcctgggac gttgtcaacg agatcttcga
ctgggacggt 480accctccgaa aggattctca cttcaacaac gtcttcggca
acgatgacta cgttggcatt 540gccttccgcg ccgcccgcaa ggctgacccc
aacgccaagc tgtacatcaa cgactacagc 600ctcgactccg ccagcgcctc
caaggtcacc aagggcatgg tcccctccgt caagaagtgg 660ctcagccagg
gcgttcccgt cgacggcatt ggctcccagt ctcaccttga ccccggtgcc
720gctggccaag tccagggtgc tctcactgcc ctcgccaact ctggtgtcaa
ggaggttgcc 780atcaccgagc tcgacatccg cactgccccc gccaacgact
acgccaccgt caccaaggcc 840tgcctaaacg tccccaagtg cattggtatc
accgtctggg gtgtctctga caagaactct 900tggcgcaagg agcacgacag
ccttctgttc gactccaact acaaccccaa gcctgcttac 960actgctgttg
tcaacgctct ccgctaa 98718942DNAFusarium sp. 18attcccaccg ccatcgagcc
ccgccaggcc gccgacagca tcaacaagct gatcaagaac 60aagggcaagc tctactacgg
aaccatcacc gaccccaacc tgctcggcgt cgcaaaggac 120accgccgtca
tcaaggccga ctttggcgcc gtcacccccg agaactcggg caagtgggac
180gccaccgagc ccagccaggg caacttcaac ttcggtagct tcgaccaggt
cgtcaacttt 240gctcagcaga atggcctcaa ggtccgaggt cacactctgg
tctggcactc tcagctccct 300cagtgggtta agaacatcaa cgacaaggct
actctgacca aggtcattga gaaccacgtc 360acccaagtcg ttggacgcta
caagggcaag atctacgcct gggacgttgt caacgagatc 420ttcgactggg
acggtaccct ccgaaaggat tctcacttca acaacgtctt cggcaacgat
480gactacgttg gcattgcctt ccgcgccgcc cgcaaggctg accccaacgc
caagctgtac 540atcaacgact acagcctcga ctccgccagc gcctccaagg
tcaccaaggg catggtcccc 600tccgtcaaga agtggctcag ccagggcgtt
cccgtcgacg gcattggctc ccagtctcac 660cttgaccccg gtgccgctgg
ccaagtccag ggtgctctca ctgccctcgc caactctggt 720gtcaaggagg
ttgccatcac cgagctcgac atccgcactg cccccgccaa cgactacgcc
780accgtcacca aggcctgcct aaacgtcccc aagtgcattg gtatcaccgt
ctggggtgtc 840tctgacaaga actcttggcg caaggagcac gacagccttc
tgttcgactc caactacaac 900cccaagcctg cttacactgc tgttgtcaac
gctctccgct aa 9421940DNAArtificial SequencePrimer 1 19ccgcggccgc
accatgaagc tgtcttcctt cctctacacc 402037DNAArtificial SequencePrimer
2 20ccggcgcgcc cttattagcg gagagcgttg acaacag 37
* * * * *
References