Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ASPERGILLUS ARABINOFURANOSIDASE
The present invention relates to an enzyme. In addition, the present invention relates to
a nucleotide sequence coding for the enzyme. Also, the present invention relates to a
5 promoter, wherein the promoter can be used to control the expression of the nucleotide
sequence coding for the enzyme.
In particular, the enzyme of the present invention is an arabinofuranosidase enzyme
having arabinoxylan degrading activity.
It is known that it is desirable to direct expression of a gene of interest ("GOI") in certain
tissues of an organism - such as a fil~m~ontous fungus (such as Aspergillus N~ger) or~even
a plant crop. The resultant protein or enzyme may be useful for the organism itself. For
example, it may be desirable to produce crop protein products with an optimised amino
15 acid composition and so increase the nutritive value of a crop. For example, the crop
may be made more useful as a feed.
In the ~ ve, it may be desirable to isolate the rçs~llt~nt protein or enzyme and then
use the protein or enzyme to prepare, for example, food compositions. In this regard,
20 the resultant protein or enzyme can be a component of the food composition or it can be
used to prepare food compositions, including altering the characteristics or appearance
of food compositions. It may even be desirable to use the organicm, such as a
fil~m~ntous fungus or a crop plant, to express non-plant genes, such as for the same
purposes.
Also, it may be desirable to use an org~nicm, such as a filamentous fungus or a crop
plant, to express m~mm~ n genes. Examples of the latter products include h~ relulls,
insulin, blood factors and placmin~gen activators. It is also desirable to use micro-
org~nicmc, such as fil~ment~us fungi, to prepare products from GOIs by use of promoters
30 that are active in the micro-org~nicm.c.
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Fruit and vegetable cell walls largely consist of polys~rch~ri-l~, the major components
being pectin, cellulose and xyloglucan (R.R. Selvendran and J.A. Robertson, IFR Report
1989). Numerous cell wall models have been proposed which attempt to incorporate the
essential properties of strength and flexibility (P. Albersheim, Sci. Am. 232, 81-95,
1975; P. Albersheim, Plant Biochem. 3rd Edition (Bonner and Varner), Ac. Press, 1976;
T. Hayashi, Ann. Rev. Plant Physiol. & Plant Mol. Biol., 40, 139-168, 1989).
The composition of the plant cell wall is complex and variable. Polysaccharides are
mainly found in the form of long chains of cellulose (the main structural component of
the plant cell wall), hemicellulose (comprising various ~3-xylan chains) and pectic
~llbst~n- ~c (consisting of gal~ch-ronans and rhamnogalacturonans; arabinans; and
g~l~r.t~n.c and arabinogal~t~n.c). From the standpoint of the food industry, the pectic
substances, arabinans in particular, have become one of the most important constituents
of plant cell walls (Whitaker, J.R. (1984) Enzyme Microb. Technol., 6,341).
One form of plant polysaccharide is arabinan. A review of arabinans may be found in
EP-A-0506190. According to this document, arabinans consist of a main chain of ~-
(1 ~5) groups linked to one another. Side chains are linked ~-(1~3) or sometimes c~-
(1~2) to the main cY-(1 5)-L-arabinan backbone. In apple, for example, one third of the
total arabinose is present in the side chains. The molecular weight of arabinan is
normally about 15 kDa.
Arabinans are degraded by enzymes collectively called arabinases. In this regard,
arabinan-degrading activity is the ability of an enzyme to release arabinose residues,
either monomers or oligomers, from arabinan backbones or from arabinan-cont~ining side
chains of other hemicellulose backbone structures such as arabinog~l~rt~nc, or even the
release of arabinose monomers via the cleavage of the 1~6 linkage between the terminal
arabinofuranosyl unit and the int~rrn~ t~ glucosyl unit of monoterpenyl c~-L-
arabinofuranosyl glucosides.
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The activity of the arabinan degrading enzymes of EP-A-0506190 include: a) the ability
to cleave (1 ~2)-cY-L-arabinosidic linkages; b) the ability to cleave (1 3)-c~-L-arabinosidic
linkages; c) the ability to cleave (1~5)-cY-L-arabinosidic linkages; d) the ability to cleave
the 1 6 linkage between the terminal arabinofuranosyl unit and the intermediate glucosyl
unit of monoterpenyl ~-L-arabinofuranosyl glucosides.
Arabinan-degrading enzymes are known to be produced by a variety of plants and
microolgalli~,llls, among these, fungi such as those of the genera Aspergillus, Corticium,
Rhodotorula (Kaji, A. (1984) Adv. Carbohydr. Chem. Biochem., 4~, 383), Dic~.otomitus
10 (Brillouet et al. (1985) Carbohydrate Research, 144, 113), Ascomycetes and
Basidomycetes (Sydow, G. (1977) DDR Patent Application No. 124,812).
Another plant polysaccharide is xylan, whose major monosaccharide unit is xylose.
Xylans are abundant components of the hemicelluloses. In monocotyledonous plants the
15 dominant hemicellulose is an arabinoxylan, in which arabinose side chains are attached
to a backbone of xylose residues.
Arabinoxylans are carbohydrates found in the cell wall of cereals. A review of
arabinoxylans and the enzymatic degradation thereof may be found in Voragen et al
20 (1992 Characterisation of Cereal Arabinoxylans, Xylans and Xylanases pages 51-67,
edited by J. Visser published by Elsevier Science Publishers).
Typically, arabinoxylans comprise a xylose backbone linked together via ,B-1,4- bonds.
The xylose backbone is substituted with L-arabinose residues which are linked via ~-1
25 bonds to tne 2 or 3 position of the xylose residues. The xylose residues can be single or
double substituted. In addition to substitution with arabinose the xylose residues can be
substituted with acetyl groups, glucuronic acid and various other carbohydrates. The
arabinose residues can be further substituted with phenolic acids such as ferulic acid and
coumaric acid. The degree and kind of substitution depends on the source of the
30 particular arabinoxylan.
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Arabinoxylans are found in cereal cell wall where they are part of the secondary cell
wall. Arabinoxylans form about 3 % of wheat flour - part of it is water soluble (WSP),
part of it is water insoluble (WIP).
5 Despite the fact that the arabinoxylans amount to only about 3 % of wheat the importance
of the arabinoxylan fraction is much higher. This is because the arabinoxylans of cereals
act as hydrocolloids, as they form a gel like structure with water. For example, the
arabinoxylans of wheat flour bind up to 30 % of the water in a dough despite the fact that
they amount to only 3 % of the dry matter. When arabinoxylans bind water they
10 increase the viscosity of the ground cereals and to such an extent that the cereals can
become difficult to manage.
The rheological properties of several systems where ground cereals are used can be
manipulated using enzymes that degrade arabinoxylans. In modern bakery it is
15 advantageous to reduce the viscosity of the dough in order to reduce the energy needed
to process the doughs and also to get a higher volume of the bread. This is usually
achieved by using enzymes that can degrade the xylose backbone of arabinoxylans.
Enzymes that omy cleave the arabinose side chains from the xylan backbone of
20 arabinoxylan are, for the purposes of this application, collectively called arabinoxylan
degrading enzymes.
In feeds based on cereals, arabinoxylans in the cereals can increase the viscosity of the
fluids in the intestines of the animals after the feeds have been ingested. This is a
25 problem as it causes discomfort, such as indigestion, to the animals. Also, the nutritive
value of the feeds is reduced. These problems can be avoided by addition of enzymes
that degrade the arabinoxylan (such as xylanases) to the feed to avoid indigestion and to
increase the nutritive value of the feed. However, some enzymes that degrade thearabinoxylans (especially some of the xylanases) require the presence of unsubstituted
30 backbones and so their activity can be limit~l
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Further discussions on arabinoxylans can be found in Xylans and Xylanases (1992, edited
by J. Visser published by Elsevier Science Publishers).
An arbinoxylan degrading enzyme is (1,4)-B-D-arabinoxylan arabinofuranohydrolase~, 5 (AXH), as described by Kormelink et al 1991 (Kormelink, F.J.M., Searle-Van Leeuwen
M.J.F., Wood. T.M., Voragen, A.G.J.(1991) Purification and characterization of a(1 ,4)-~B-D-arabinoxylan arabinofuranohydrolase from Aspergillus awamori. Appl. Micro-
biol. Biotechnol. 25:753-758). However, this document provides no sequence data for
the enzyme or the nucleotide sequence coding for same or for the promoter for the same.
Clearly, it would be useful to be able to degrade arabinoxylans, preferably by use of
recombinant DNA techniques.
The present invention seeks to provide an enzyme having arabinoxylan degrading activity;
15 preferably wherein the enzyme can be prepared in certain or specific cells or tissues, such
as in just a specific cell or tissue, of an org~ni.~m, typically a filamentous fungus,
preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant.
Also, the present invention seeks to provide a GOI coding for the enzyme that can be
20 expressed preferably in specific cells or tissues, such as in certain or specific cells or
tissues, of an org~ni.~m, typically a filamentous fungus, preferably of the genus
Aspergillus, such as Aspergillus niger, or even a plant.
In addition, the present invention seeks to provide a promoter that is capable of directing
25 expression of a GOI, such as a nucleotide sequence coding for the enzyme according to
the present invention, preferably in certain specific cells or tissues, such as in just a
specific cell or tissue, of an org~ni.~m, typically a filamentous fungus, preferably of the
genus Aspergillus, such as Aspergillus niger, or even a plant. Preferably, the promoter
is used in Aspergillus wherein the product encoded by the GOI is excreted from the host
30 organism into the surrounding m~ m
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Furthermore, the present invention seeks to provide constructs, vectors, plasmids, cells,
tissues, organs and org~ni~m~ comprising the GOI and/or the promoter, and methods of
expressing the same, preferably in specific cells or tissues, such as expression in just a
specific cell or tissue, of an org~ni~m, typically a filamentous fungus, preferably of the
S genus Aspergillus, or even a plant.
According to a first aspect of the present invention there is provided an enzymeobtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW
of 33,270 D + 50 D; a pI value of about 3 7; arabinoxylan degrading activity; a pH
optima of from about 2.5 to about 7.0 (more especially from about 3.3 to about 4.6,
more especially about 4); a temperature optima of from about 40~C to about 60~C (more
especially from about 45~C to about 55~C, more especially about 50~C); and wherei-n tne
enzyme is capable of cleaving arabinose from the xylose backbone of an arabinoxylan.
15 According to a second aspect of the present invention there is provided an enzyme having
the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof.
According to a t'nird aspect of the present invention there is provided an enzyme coded
by the nucleotide sequence shown as SEQ. I.D. No. 2 or a variant, homologue or
20 fragment thereof or a sequence complementary thereto.
According to a fourth aspect of the present invention there is provided a nucleotide
sequence coding for the enzyme according to the present invention.
25 According to a fifth aspect of the present invention there is provided a nucleotide
sequence having the sequence shown as SEQ. I.D. No. 2 or a variant, homologue orfragment thereof or a sequence complement~ry thereto.
According to a sixth aspect of the present invention there is provided a promoter having
30 the sequence shown as SEQ. I.D. No. 3 or a variant, homologue or fragment thereof or
a sequence complem~nt"ry thereto.
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According to a seventh aspect of the present invention there is provided a lerminator
having the nucleotide sequence shown as SEQ. I.D. No. 13 or a variant, homologue or
fragment thereof or a sequence complementary thereto.
5 According to an eighth aspect of the present invention there is provided a signal sequence
having the nucleotide sequence shown as SEQ. I.D. No. 14 or a variant, homologue or
fragment thereof or a sequence complementary thereto.
According to a ninth aspect of the present invention there is provided a process for
10 expressing a GOI by use of a promoter, wherein the promoter is the promoter according
to the present invention.
According to a tenth aspect of the present invention there is provided the use of an
enzyme according to the present invention to degrade an arabinoxylan.
According to an eleventh aspect of the present invention there is provided a combination
of enzymes to degrade an arabinoxylan, the combination comprising an enzyme according
to the present invention and a xylanase.
20 According to a twelfth aspect of the present invention there is provided plasmid NCIMB
40703, or a nucleotide sequence obtainable therefrom for expressing an enzyme capable
of degrading arabinoxylan or for controlling the expression thereof or for controlling the
expression of another GOI.
25 According to a thirteenth aspect of the present invention there is provided a signal
sequence having the sequence shown as SEQ. I.D. No. 15 or a variant, homologue or
fragment thereof.
According to a fourteenth aspect of the present invention there is provided the use of the
30 enzyme according to the present invention in the m~mlf~ctllre of a mP~lic~ment or
foodstuff to reduce or prevent indigestion and/or increase digestibility and/or increase
mltrient absorption.
,
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According to a fifteenth aspect of the present invention there is provided an
arabinofuranosidase enzyme having arabinoxylan degrading activity, which is
immllnologically reactive with an antibody raised against a purified arabinofuranosidase
enzyme having the sequence shown as SEQ. I.D. No. 1.
According to a sixteenth aspect of the present invention there is provided an
arabinofuranosidase promoter wherein the promoter is inducible by an interm.o~ tf~ in
xylose metabolism.
10 According to a seventeenth aspect of the present invention there is provided a process of
reducing the viscosity of a branched substrate wherein the enzyme degrades the branches
of the substrate but not the backbone of the substrate.
According to a further aspect of the present invention there is provided the use of the
15 enzyme of the present invention as a viscosity modifier.
According to a further aspect of the present invention there is provided the use of the
er~7yme of the present invention to reduce the viscosity of pectin.
20 Other aspects of the present invention include constructs, vectors, plasmids, cells, tissues,
organs and transgenic org~nisms comprising the aforementioned aspects of the present
invention.
Other aspects of the present invention include methods of expressing or allowing25 expression or transforming any one of the nucleotide sequence, the construct, the
plasmid, the vector, the cell, the tissue, the organ or the org~ni~m, as well as the
products thereof.
Additional aspects of the present invention include uses of the promoter for expressing
30 GOIs in culture media such as a broth or in a transgenic org~nism
-
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Further aspects of the present invention include uses of the enzyme for preparing or
treating foodstuffs, including animal feed.
Preferably the enzyme is coded by the nucleotide sequence shown as SEQ. I.D. No. 2
5 or a variant, homologue or fragment thereof or a sequence complementary thereto.
Preferably the nucleotide sequence has the sequence shown as SEQ. I.D. No. 2 or a
variant, homologue or fragment thereof or a sequence complementary thereto.
10 Preferably the nucleotide sequence is operatively linked to a promoter.
Preferably the promoter comprises the sequence CCAAT.
Preferably the promoter is the promoter having the sequence shown as SEQ. I.D. No.
15 3 or a variant, homologue or fragment thereof or a sequence complementary thereto.
Preferably, the promoter comprises the 100 bps sequence from the Xm~ 111 to the
BamHl sites.
20 Preferably the promoter of the present invention is operatively linked to a GOI.
Preferably the GOI comprises a nucleotide sequence according to the present invention.
Preferably the transgenic organism is a fungus.
Preferably the transgenic organism is a filamentous fungus, more preferably of the genus
Aspergillus.
Preferably the t.dnsgel.ic organism is a plant.
Preferably, in the use, the enzyme is used in combination with a xylanase, preferably an
endoxylanase.
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Highly pl~r~ d embo-liments of each of the aspecls of the present invention do not
include any one of the native enzyme, the native promoter or the native nucleotide
sequence in its natural environment.
Preferably, in any one of the plasmid, the vector such as an expression vector or a
transformation vector, the cell, the tissue, the organ, the organism or the transgenic
org~ni~m, the promoter is present in combination with at least one GOI.
Preferably the promoter and the GOI are stably incorporated within the transgenic
organism's genome.
Preferably the L~ iC organism is a filamentous fungus, preferably of the genus
Aspergillus, more preferably Aspergillus niger. The transgenic organism can even be a
plant, such as a monocot or dicot plant.
A highly preferred embodiment is an enzyme obtainable from Aspergillus, wherein the
enzyme has the following characteristics: a MW of 33,270 D + 50 D; a pI value ofabout 3.7; arabinoxylan degrading activity; a pH optima of from about 2.5 to about 7.0
(more especially from about 3.3 to about 4.6, more especially about 4); a temperature
optima of from about 40~C to about 60~C (more especially from about 45~C to about
55~C, more especially about 50~C); and wherein the enzyme is capable of cleavingarabinose from the xylose backbone of an arabinoxylan; wherein the enzyme has the
sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof.
Another highly L.l~rel~d embodiment is an enzyme obtainable from Aspergil~us, wherein
the enzyme has the following characteristics: a MW of 33,270 D + 50 D; a pI value of
about 3.7; arabinoxylan degrading activity; a pH optima of from about 2.5 to about 7.0
(more especially from about 3.3 to about 4.6, more especially about 4); a temperature
optima of from about 40~C to about 60~C (more especially from about 45~C to about
55~C, more especially about 50~C); and wherein the enzyme is capable of cleavingarabinose from the xylose backbone of an arabinoxylan; wherein the enzyme is coded by
the nucleotide sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment
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11
thereof or a sequence complementary thereto.
The advantages of the present invention are that it provides a means for preparing an
arabinofuranosidase enzyme having arabinoxylan degrading activity and the nucleotide
5 sequence coding for the sarne. In addition, it provides a promoter that can control the
expression of that, or another, nucleotide sequence.
Other advantages are that the enzyme of the present invention can affect the viscosity of
ground cereals, such as dough, to ease the h~n~lling thereof and for example to get a
10 higher volume of the bread.
The enzyme of the present invention is also advantageous for feed because it degrades
arabinoxylan and thus increases the nutritive value of the feed. In addition, it reduces
the viscosity of the arabinoxylan in the intestine of the ~nim~l.c and so reduces or prevents
15 indigestion.
The combination of the use of the enzyme of the present invention with a xylanase is
particularly advantageous because the enzyme of the present invention and the xylanase
have a surprising and unexpected synergistic effect with each other.
In this regard, the enzyme of the present invention increases the degradative effect of the
xylanase, and the xylanase increases the degradative effect of the enzyme of the present
invention. It is believed that the activity of the xylanase is increased because the enzyme
of the present invention provides a polysaccharide substrate having fewer substituted
25 groups.
The present invention therefore provides an enzyme having arabinoxylan degradingactivity wherein the enzyme can be ~le~aled in certain or specific cells or tissues, such
as in just a specific cell or tissue, of an org~nicm, typically a filamentous fungus,
30 preferably of the genus Aspergillus, such as Aspergillus niger. The enzyme may even be
L~lc~dled in a plant.
,
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12
More in particular, the enzyme of the present invention is capable of specifically cleaving
arabinose from the xylose backbone of arabinoxylan.
The arabinofuranosidase of the present invention is different from the
S arabinofuranosidases previously known. In this regard, the previous described
arabinofuranosidases - such as those of EP-A-0506190 - are characterised by their ability
to degrade unbranched arabinan, and are assayed using p-nitrophenyl-arabinoside.
The arabinofuranosidase of the present invention does not degrade unbranched arabinan,
10 and only a minor activity is seen on nitrophenyl-arabinoside. In contrast, the
arabinofuranosidase of the present invention is useful for degrading arabinoxylan.
Therefore, the arabinofuranosidase of the present invention is quite different from the
previous isolated arabinofuranosidases.
15 Also, the present invention provides a GOI coding for the enzyme that can be expressed
preferably in specific cells or tissues, such as in certain or specific cells or tissues, of an
org~nicm, typically a fil~mentous fungus, preferably of the genus Aspergillus, such as
Aspergillus niger. The GOI may even be expressed in a plant.
20 In addition, the present invention provides a promoter that is capable of directing
expression of a GOI, such as a nucleotide sequence coding for the enzyme according to
the present invention, preferably in certain specific cells or tissues, such as in just a
specific cell or tissue, of an org~nicm, typically a filamentous fungus, preferably of the
genus Aspergillus, such as Aspergillus niger, or even a plant. Preferably, the promoter
25 is used in AspergiUus wherein the product encoded by the GOI is excreted from the host
organism into the ~u~ u~lding m~ rn The promoter may even be tailored (if
n~C~cc~ry) to express a GOI in a plant.
The present invention also provides constructs, vectors, plasmids, cells, tissues, organs
30 and org~nicmc comprising the GOI and/or the promoter, and methods of expressing the
same, preferably in specific cells or tissues, such as expression in just a specific cell or
tissue, of an org~nicm, typically a filamentous fungus, preferably of the genus
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13
As~ergillus, or even a plant.
The terms "variant", "homologue" or "fragment" in relation to the enzyme include any
substitution of, variation of, modification of, replacement of, deletion of or addition of
5 one (or more) amino acid from or to the sequence providing the resultant amino acid
sequence has arabinoxylan degrading activity, preferably having at least the same activity
of the enzyme shown in the sequence listings (SEQ I.D. No. 1 or 12). In particular, the
term "homologue" covers homology with respect to structure and/or function providing
the resultant enzyme has arabinoxylan degrading activity. With respect to sequence
10 homology, preferably there is at least 75 %, more preferably at least 85 %, more
preferably at least 90% homology to SEQ ID NO. 1 shown in the attached sequence
listings. More preferably there is at least 95 %, more preferably at least 98 %, homology
to SEQ ID NO. 1 shown in the attached sequence listings.
15 The terms "variant", "homologue" or "fragment" in relation to the nucleotide sequence
coding for the enzyme include any substitution of, variation of, modification of,
replacement of, deletion of or addition of one (or more) nucleic acid from or to the
sequence providing the resultant nucleotide sequence codes for an enzyme having
arabinoxylan degrading activity, preferably having at least tne same activity of the
20 enzyme shown in the sequence listings (SEQ I.D. No. 2 or 12). In particular, the term
"homologue" covers homology with respect to structure and/or function providing the
resultant nucleotide sequence codes for an enzyme having arabinoxylan degrading
activity. With respect to sequence homology, preferably there is at least 75%, more
preferably at least 85 %, more preferably at least 90 % homology to SEQ ID NO . 2 shown
25 in the attached sequence listings. More preferably there is at least 95 %, more preferably
at least 98%, homology to SEQ ID NO. 2 shown in tne attached sequence listings.
The terms "variant", "homologue" or "fragment" in relation to the promoter include any
substitution of, variation of, modification of, replacement of, deletion of or addition of
30 one (or more) nucleic acid from or to the sequence providing the resultant nucleotide
sequence has the ability to act as a promoter in an expression system - such as the
transformed cell or the transgenic organism according to the present invention. In
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14
particular, the term "homologue" covers homology with respect to structure and/or
function providing the resultant nucleotide sequence has the ability to act as a promoter.
With respect to sequence homology, preferably there is at least 75 %, more preferably at
least 85%, more preferably at least 90% homology to SEQ ID NO. 3 shown in the
attached sequence listings. More preferably there is at least 95%, more preferably at
least 98%, homology to SEQ ID NO. 3 shown in the ~tt~ch~l sequence listings.
The terms "variant", "homologue" or "fragment" in relation to the terrnin~tor or signal
nucleotide sequences include any substitution of, variation of, modification of,replacement of, deletion of or addition of one (or more) nucleic acid from or to the
sequence providing the resultant nucleotide sequence has the ability to act as a terrnin~tor
or codes for an amino acid sequence that has the ability to act as a signal sequence
respectively in an expression system - such as the transformed cell or the transgenic
organism according to the present invention. In particular, the term "homologue" covers
homology with respect to structure and/or function providing the resultant nucleotide
sequence has the ability to act as or code for a terminator or signal respectively. With
respect to sequence homology, preferably there is at least 75 %, more preferably at least
85%, more preferably at least 90% homology to SEQ ID NO.s 13 and 14 (respectively)
shown in the ~ h~ sequence listings. More preferably there is at least 95%, morepreferably at least 98%, homology to SEQ ID NO.s 13 and 14 (respectively) shown in
the ~tt~rh-od sequence listings
The terms "variant", "homologue" or "fragment" in relation to the signal amino acid
sequence include any substitution of, variation of, modification of, replacement of,
deletion of or addition of one (or more) amino acid from or to the sequence providing
the resultant sequence has the ability to act as a signal sequence in an expression system -
such as the transformed cell or the transgenic organism according to the presentinvention. In particular, the term "homologue" covers homology with respect to structure
and/or function providing the resultant nucleotide sequence has the ability to act as or
code for a signal respectively. With respect to sequence homology, preferably there is
at least 75%, more preferably at least 85%, more preferably at least 90% homology to
SEQ ID NO 15 shown in the ~tt~ch~-l sequence listings. More preferably there is at least
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95%, more preferably at least 98%, homology to SEQ IDN015 shown in the attached
sequence listings.
The above terms are synonymous with allelic variations of the sequences.
S
The term "complementary" means that the present invention also covers nucleotidesequences that can hybridise to the nucleotide sequences of the coding sequence or the
promoter sequence, respectively.
10 The term "nucleotide" in relation to the present invention includes genomic DNA,cDNA,
synthetic DNA, and RNA Preferably it means DNA, more preferably cDNA for the
coding sequence of the present invention.
The term "construct" - which is synonymous with terms such as "conjugate", "cassette"
15 and "hybrid" - includes a GOI directly or indirectly att~rh~l to a promoter. An example
of an indirect "tr~chment is the provision of a suitable spacer group such as an intron
sequence, such as the Shl-intron or the ADH intron, intermediate the promoter and the
GOI. The same is true for the term "fused" in relation to the present invention which
includes direct or indirect ~tt~c.hment In each case, it is highly preferred that the terms
20 do not cover the natural combination of the gene coding for the enzyme ordinarily
associated with the wild type gene promoter and when they are both in their natural
environment. A highly ~le~ll~d embodiment is the or a GOI being operatively linked
to a or the promoter.
25 The construct may even contain or express a marker which allows for the selection of the
genetic construct in, for example, a filamentous fungus, preferably of the genusAspergillus, such as Aspergillus niger, or plants, preferably cereals, such as maize, rice,
barley etc., into which it has been transferred. Various markers exist which may be
used, such as for example those encoding mannose-6-phosphate isomerase (especially for
30 plants) or those markers that provide for antibiotic re.sist~nre - e.g. resistance to G418,
hygromycin, bleomycin, k~ldlllycin and gentamycin.
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16
The term "vector" includes expression vectors and tran~r~nlllation vectors.
The term "expression vector" means a construct capable of in vivo or in vitro expression.
5 The term "transformation vector" means a construct capable of being transferred from
one species to another - such as from an ~. coli plasmid to a filamentous fungust
preferably of the genus Aspergillus. It may even be a construct capable of beingtransferred from an E. coli plasmid to an Agrobacterium to a plant.
10 The term "tissue" includes tissue per se and organ.
The term "organism" in relation to the present invention includes any organism that could
comprise the promoter according to the present invention and/or the nucleotide sequence
coding for the enzyme according to the present invention and/or products obtained
15 therefrom, wherein the promoter can allow expression of a GOI and/or wherein the
nucleotide sequence according to the present invention can be expressed when present in
the or~;ani.cm
Preferably the organism is a filamentous fungus, preferably of the genus Aspergillus~
20 more preferably Aspergillus niger.
The term "transgenic organism" in relation to the present invention includes any organism
that comprises the promoter according to the present invention and/or the nucleotide
sequence coding for the enzyme according to the present invention and/or products
25 obtained thel~rl.Jlll, wherein the promoter can allow expression of a GOI and/or wherein
the nucleotide sequence according to the present invention can be expressed within the
org~ni~m Preferably the promoter and/or the nucleotide sequence is (are) incorporated
in the genome of the org~ni~m
30 Preferably the transgenic organism is a filamentous fungus, preferably of the genus
Aspergillus, more preferably Aspergillus niger.
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Therefore, the transgenic organism of the present invention includes an organismcomprising any one of, or combinations of, the promoter according to the presentinvention, the nucleotide sequence coding for the enzyme according to the present
invention, constructs according to the present invention, vectors according to the present
S invention, plasmids according to the present invention, cells according to the present
invention, tissues according to the present invention or the products thereof. For example
the transgenic organism can comprise a GOI, preferably an exogenous nucleotide
sequence, under the control of the promoter according to the present invention. The
transgenic organism can also comprise the nucleotide sequence coding for the enzyme of
10 the present invention under the control of a promoter, which may be the promoter
according to the present invention.
In a highly ~,efell~d embodiment, the transgenic organism does not comprise the
combination of the promoter according to the present invention and the nucleotide
15 sequence coding for the enzyme according to the present invention, wherein both the
promoter and the nucleotide sequence are ~lative to that organism and are in their natural
environment. Thus, in these highly L)l~fell~d embo~1im~ntc, the present invention does
not cover the native nucleotide coding sequence according to the present invention in its
natural environment when it is under the control of its native promoter which is also in
20 its natural environment. In addition, in this highly preferred embodiment, the present
invention does not cover the native enzyme according to the present invention when it is
in its natural environment and when it has been expressed by its native nucleotide coding
sequence which is also in its natural environment and when that nucleotide sequence is
under the control of its native promoter which is also in its natural environment.
The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase
binding site in the Jacob-Mond theory of gene expression.
In one aspect, the promoter of the present invention is capable of expressing a GOI,
30 which can be the nucleotide sequence coding for the enzyme of the present invention.
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18
In another aspect, the nucleotide sequence according to the present invention is under the
control of a promoter that allows expression of the nucleotide sequence. In this regard,
the promoter need not nPces~,.rily be the same promoter as that of the present invention.
In this aspect, the promoter may be a cell or tissue specific promoter. If, for example,
5 the organism is a plant then the promoter can be one that affects expression of the
nucleotide sequence in any one or more of stem, sprout, root and leaf tissues.
By way of example, the promoter for the nucleotide sequence of the present invention can
be the ~-Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637
10 promoter or the a!-Amy 637 promoter) as described in our co-pending UK patentapplication No. 9421292.5 filed 21 October 1994. That promoter comprises the sequence
shown in Figure 1.
Alternatively, the promoter for the nucleotide sequence of the present invention can be
the cY-Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter
or the c~-Amy 351 promoter) as described in our co-pending UK patent application No.
9421286.7 filed 21 October 1994. That promoter comprises the sequence shown in
Figure 2.
20 Preferablyt the promoter is the promoter of the present invention.
In addition to the nucleotide sequences described above, the promoters, particularly that
of the present invention, could additionally include features to ensure or to increase
expression in a suitable host. For example, the features can be conserved regions such
25 as a Pribnow Box or a TATA box. The promoters may even contain other sequences to
affect (such as to m~int,.in, enhance, decrease) the levels of expression of the GOI. For
example, suitable other sequences include the Shl-intron or an ADH intron. Othersequences include inducible elements - such as temperature, chemical, light or stress
inducible elements.
Also, suitable elements to enhance transcription or translation may be present. An
example of the latter element is the TMV 5' signal sequence (see Sleat Gene 217 [1987]
-
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217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
In addition the present invention also encompasses combinations of promoters and/or
nucleotide sequences coding for proteins or enzymes and/or elements. For example, the
S present invention encompasses the combination of a promoter according to the present
invention operatively linked to a GOI, which could be a nucleotide sequence according
to the present invention, and another promoter such as a tissue specific promoter
operatively linked to the same or a different GOI.
10 The present invention also encompasses the use of promoters to express a nucleotide
sequence coding for the enzyme according to the present invention, wherein a part of the
promoter is inactivated but wherein the promoter can still function as a promoter. Partial
inactivation of a promoter in some instances is advantageous.
15 In particular, with the Amy 351 promoter mentioned earlier it is possible to inactivate a
part of it so that the partially inactivated promoter e~Lesses GOIs in a more specific
manner such as in just one specific tissue type or organ.
The term "inactivated" means partial inactivation in the sense that the expression pattern
20 of the promoter is modified but wherein the partially inactivated promoter still functions
as a promoter. However, as mentioned above, the modified promoter is capable of
e~ e~sillg a GOI in at least one (but not all) specific tissue of the original promoter.
One such promoter is the Amy 351 promoter described above.
25 Examples of partial inactivation include altering the folding pattern of the promoter
sequence, or binding species to parts of the nucleotide sequence, so that a part of the
nucleotide sequence is not recognised by, for example, RNA polymerase. Another, and
preferable, way of partially inactivating the promoter is to truncate it to form fragments
thereof. Another way would be to mutate at least a part of the sequence so that the RNA
30 polymerase can not bind to that part or another part.
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Another modification is to mutate the binding sites for regulatory proteins for example
the CreA protein known from filamentous fungi to exert carbon catabolile repression, and
thus abolish the catabolite lc~lc;ssion of the native promoter.
5 The term "GOI" with reference to the present invention means any gene of interest. A
GOI can be any nucleotide that is either foreign or natural to the organism (e . g .
filamentous fungus, preferably of the genus Aspergillus, or a plant) in question. Typical
examples of a GOI include genes encoding for proteins and enzymes that modify
metabolic and catabolic processes. The GOI may code for an agent for introducing or
10 increasing pathogen recict~nre. The GOI may even be an antisense construct for
modifying the expression of natural tralls~ L~ present in the relevant tissues. The GOI
may even code for a non-natural protein of a filamentous fungus, preferably of the genus
Aspergillus, or a compound that is of benefit to animals or hllm~nc.
15 For example, the GOI could code for a ph~rrn~rel-rir~lly active protein or enzyme such
as any one of the therapeutic compounds insulin, inlelr~ , human serum albumin,
human growth factor and blood clotting factors. In this regard, the transformed cell or
organism could prepare acceptable quantities of the desired compound which would be
easily retrievable from, the cell or organism. The GOI may even be a protein giving
20 nutritional value to a food or crop. Typical examples include plant proteins that can
inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable
amino acid composition (e.g. a higher lysine content than a non-transgenic plant). The
GOI may even code for an enzyme that can be used in food processing such as chymosin,
th~nm~tin and ~-galactosidase. The GOI can be a gene encoding for any one of a pest
25 toxin, an antisense transcript such as that for patatin or a!-amylase, ADP-glucose
pyrophosphorylase (e.g. see EP-A-0455316), a protease antisense or a glllr~n~ce
The GOI can be the nucleotide sequence coding for the ~x-amylase enzyme which is the
subject of our co-pending UK patent application 9413439.2 filed on 4 July 1994, the
30 sequence of which is shown in Figure 3. The GOI can be the nucleotide sequence coding
for the cY-amylase enzyme which is the subject of our co-pending UK patent application
9421290.9 filed on 21 October 1994, the sequence of which is shown in Figure 4. The
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~1
GOI can be any of the nucleotide sequences coding for the ADP-glucose
pyrophosphorylase enzymes which are the subject of our co-pending PCT patent
application PCT/EP94/01082 filed 7 April 1994, the sequences of which are shown in
Figures 5 and 6. The GOI can be any of the nucleotide sequences coding for the ~-
5 glucan Iyase enzyme which are described in our co-pending PCT patent application
PCT/EP94/03397 filed 15 October 1994, the sequences of which are shown in Figures
7-10.
In one preferred embodiment, the GOI is a nucleotide sequence coding for the enzyme
10 according to the present invention.
As mentioned above, a preferred host organism is of the genus Aspergillus, such as
Aspergillus niger. The transgenic Aspergillus according to the present invention can be
prepared by following the teachings of Rambosek,J. and T~ch,T. 1987 (Recombinant15 DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-
393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Asperg-
illus. In: Martinelli S.D., Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in
industrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525-560), R~ n~e,D.J.
1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene
20 structure. In :Leong,S .A., Berka R.M. (Editors) Molecular Industrial Mycology. Systems
and Applications for Filamentous Fungi. Marcel Dekker Inc. New York 1991. pp 1-29)
and Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S.D., Kinghorn
J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29.
Elsevier Arnsterdam 1994. pp. 641-666). However, the following commentary provides
25 a summary of those t~hin~ for producing transgenic Aspergillus according to the
present invention.
Filamentous fungi have during almost a century been widely used in industry for produc-
tion of organic compounds and enzymes. Traditional japanese koji and soy fermentations
30 have used Aspergillus sp for hundreds of years. In this century Aspergillus niger has
been used for production of organic acids particular citric acid and for production of
various enzymes for use in industry.
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There are two major reasons for that filamentous fungi have been so widely used in
industry. First filamentous fungi can produce high amounts of extracellular products, for
example enzymes and organic compounds such as antibiotics or organic acids. Second
filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc.
5 The same reasons have made filamentous fungi attractive organisms as hosts for heterologous expression according to the present invention.
In order to prepare the transgenic Aspergillus, expression constructs are prepared by
inserting a GOI (such as an amylase or SEQ. I.D. No. 2) into a construct designed for
10 expression in filamentous fungi.
Several types of constructs used for heterologous expression have been developed. The
constructs contain the promoter according to the present invention (or if desired another
promoter if the GOI codes for the enzyme according to the present invention) which is
15 active in fungi. Examples of promoters other than that of the present invention include
a fungal promoter for a highly expressed extracellular enzyme, such as the glucoamylase
promoter or the ~-amylase promoter. The GOI can be fused to a signal sequence (such
as that of the present invention or another suitable sequence) which directs the protein
encoded by the GOI to be secreted. Usually a signal sequence of fungal origin is used,
20 such as that of the present invention. A terminator active in fungi ends the expression
system, such as that of the present invention.
Another type of expression system has been developed in fungi where the GOI is fused
to a smaller or a larger part of a fungal gene encoding a stable protein. This can stabilize
25 the protein encoded by the GOI. In such a system a cleavage site, recognized by a
specific protease, can be introduced between the fungal protein and the protein encoded
by the GOI, so the produced fusion protein can be cleaved at this position by the specific
protease thus liberating the protein encoded by the GOI ("POI"). By way of example,
one can introduce a site which is recognized by a KEX-2 like peptidase found in at least
30 some Aspergilli. Such a fusion leads to cleavage in vivo resulting in protection of the
POI and production of POI and not a larger fusion protein
-
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Heterologous expression in Aspergillus has been reported for several genes coding for
bacterial, fungal, vertebrate and plant proteins. The proteins can be deposited
intracellularly if the GOI is not fused to a signal sequence. Such proteins will accllmnl~t~
in the cytoplasm and will usually not be glycosylated which can be an advantage for some
5 bacterial proteins. If the GOI is equipped with a signal sequence the protein will
accl-m~ t~ extracellulary.
With regard to product stability and host strain modifications, some heterologous proteins
are not very stable when they are secreted into the culture fluid of fungi. Most fungi
10 produce several extracellular proteases which degrade heterologous proteins. To avoid
this problem special fungal strains with reduced protease production have been used as
host for heterologous production.
For the transformation of fil~mentQus fungi, several transformation protocols have been
15 developed for many filamentous fungi (R~ n~e 1991, ibi~). Many of them are based
on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and
Ca2+ ions. The Lld,~rol.lled protoplasts then regenerate and the transformed fungi are
selected using various selective markers. Among the markers used for transformation are
a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic
20 resistance markers such as benomyl resistance, hygromycin resistance and phleomycin
resistance. A very common used transformation marker is the amdS gene of A. nidulans
which in high copy number allows the fungus to grow with acrylamide as the sole
nitrogen source.
25 Even though the enzyme, the nucleotide sequence coding for same and the promoter of
the present invention are not disclosed in EP-B-0470145 and CA-A-2006454, those two
docnm~nt~ do provide some useful background commentary on the types of techniques
that may be employed to prepare transgenic plants according to the present invention.
Some of these background te~hing~ are now included in the following commentary.
The basic principle in the construction of genetically modified plants is to insert genetic
information in the plant genome so as to obtain a stable m~ e of the inserted
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genetic material.
Several techniques exist for inserting the genetic information, the two main principles
being direct introduction of the genetic information and introduction of the genetic
5 information by use of a vector system. A review of the general techniques may be found
in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991l 42:205-225) and
Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27)
Thus, in one aspect, the present invention relates to a vector system which carries a
10 promoter or nucleotide sequence or construct according to the present invention and
which is capable of introducing the promoter or nucleotide sequence or construct into the
genome of an org~ni~m, such as a plant.
The vector system may comprise one vector, but it can comprise two vectors. In the case
15 of two vectors, the vector system is normally referred to as a binary vector system.
Binary vector systems are described in further detail in Gynheung An et al. (1980),
Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
One extensively employed system for transformation of plant cells with a given promoter
20 or nucleotide sequence or construct is based on the use of a Ti plasmid from
Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al.
(1986), Plant Plrysiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture
Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P Helgeson, 203-208.
25 Several dirr~l~.lt Ti and Ri plasmids have been constructed which are suitable for the
construction of the plant or plant cell constructs described above. A non-limitin~
example of such a Ti plasmid is pGV3850.
The promoter or nucleotide sequence or construct of the present invention should30 preferably be inserted into the Ti-plasmid between the t~-rmin~l sequences of the T-DNA
or adjacent a T-DNA sequence so as to avoid disruption of the sequences imm~ t~ly
surrounding the T-DNA borders, as at least one of these regions appear to be essential
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for insertion of modified T-DNA into the plant genome.
As will be understood from the above explanation, if the organism is a plant, then the
vector system of the present invention is preferably one wnich contains the sequences
5 necessary to infect the plant (e.g. the vir region) and at least one border part of a T-DNA
sequence, the border part being located on the same vector as the genetic construct.
Furthermore, the vector system is preferably an Agrobacterium tumefaciens Ti-plasmid
or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids are
10 well-known and widely employed in the construction of transgenic plants, many vector
systems exist which are based on these plasmids or derivatives thereof.
In the construction of a transgenic plant the promoter or nucleotide sequence or construct
of the present invention may be first constructed in a microorganism in which the vector
15 can replicate and which is easy to manipulate before insertion into the plant. An example
of a useful microorganism is E. coli, but other microorg~ni.~m~ having the aboveproperties may be used. When a vector of a vector system as defined above has been
constructed in E. coCi, it is lld~,relled, if n~cess"ry, into a suitable Agrobacterium strain,
e.g. Agrobacterium tumefaciens. The Ti-plasmid harbouring the promoter or nucleotide
20 sequence or construct of the invention is thus preferably transferred into a suitable
Agrobacterium strain, e . g . A. tumefaciens, so as to obtain an Agrobacterium cell
harbouring the promoter or nucleotide sequence or construct of the invention, which
DNA is subsequently transferred into the plant cell to be modified.
.
As reported in CA-A-2006454, a large amount of cloning vectors are available which
contain a replication system in E. coli and a marker which allows a selection of the
transformed cells. The vectors contain for example pBR 322, pUC series, M13 mp
series, pACYC 184 etc.
In this way, the nucleotide or construct or promoter of the present invention can be
introduced into a suitable restriction position in the vector. The contained plasmid is
used for the transformation in E. coli. The E. coli cells are cultivated in a suitable nutrient
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26
medium and then harvested and lysed. The plasmid is then recovered. As a method of
analysis there is generally used sequence analysis, restriction analysis, electrophoresis and
further biochemical-molecular biological methods. After each manipulation, the used
DNA sequence can be restricted and conn.-cterl with the next DNA sequence. Each
sequence can be cloned in the same or different plasmid.
After each introduction method of the desired promoter or construct or nucleotide
sequence according to the present invention in the plants the presence and/or insertion of
further DNA sequences may be n~3cess~ry. If, for example, for the transformation the
Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however
the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as fl~nking areas of
the introduced genes, can be conn~cte~1. The use of T-DNA for the transformati~n of
plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in:
The Binary Plant Vector System Offset-drukkerij Kanters B.B., Alblasserdam, 1985,
Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985)
4:277-284.
Direct infection of plant tissues by Agrobacterir~m is a simple technique which has been
widely employed and wnich is described in Butcher D.N. et al. (1980), Tissue Culture
Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208. For
further teachings on t'nis topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol
[1991] 42:205-225~ and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27) .
With this technique, infection of a plant may be done on a certain part or tissue of the
plant, i.e. on a part of a leaf, a root, a stem or another part of tne plant.
Typically, with direct infection of plant tissues by Agrobacterium carrying the promoter
and/or the GOI, a plant to be infected is wounded, e.g. by cutting the plant with a razor
or puncturing the plant with a needle or rubbing the plant with an abrasive The wound
is then inoculated with the Agrobacrerium. The inoc ll~t.-d plant or plant part is then
30 grown on a suitable culture m~ m and allowed to develop into mature plants.
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When plant cells are constructed, these cells may be grown and m~int~in~-d in accordance
with well-known tissue culturing methods such as by culturing the cells in a suitable
culture medium supplied with the necessary growth factors such as amino acids, plant
hormones, vitamins, etc.
Regeneration of the transformed cells into genetically modified plants may be
accomplished using known methods for the regeneration of plants from cell or tissue
cultures, for example by selecting transformed shoots using an antibiotic and bysubculturing the shoots on a medium cont~ining the apL)l~,pliate nutrients, plant
10 hormones, etc.
Further te~chings on plant transformation may be found in EP-A-0449375.
In s-lmm~tion, the present invention provides an arabinofuranosidase enzyme having
15 arabinoxylan degrading activity and the nucleotide sequence coding for the same. In
addition, it provides a promoter that can control the expression of that, or another,
nucleotide sequence. In addition it includes te.rmin~tor and signal sequences for the
same.
20 The following sample was deposited in accordance with the Budapest Treaty at the
recognised depositary The National Collections of Industrial and Marine Bacteria T.imite~l
(NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 lRY on
16 January 1995:
E. coli cont~ining plasmid pB53.1 {i.e. E.coli DH5~-
pB53.1}. The deposit number is NCIMB 40703.
The present invention will now be described by way of example.
.
In the following Examples reference is made to the accompanying figures in which:
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Figures 1-10 are sequences of promoters and GOIs of earlier patent applications that are
useful for use with the aspects of the present invention;
Figure 11 is a plasmid map of the plasmid pB53.1, which is the subject of deposit
NCIMB 40703;
Figure 12 is a schematic diagram of deletions made to the promoter of the present
invention;
10 Figure 13 is a plasmid map of pXP-AMY;
Figure 14 is a plasmid map of pXP-XssAMY;
Figure 15 is a graph;
Figure 16 is an HP-TLC profile;
Figure 17 is an HP-TLC profile;
20 Figure 18 iS an HPLC profile;
Figure 19 is a viscosity plot;
Figure 20 is an activity plot;
Figure 21 is an activity plot; and
Figure 22 is an activity plot.
30 The following text ~ ccll~ses the use of inter alia recombinant DNA techniques. General
te~l~hin~.~ of recombinant DNA techniques may be found in Sambrook, J., Fritsch, E.F.,
Maniatis T. (Editors) Molecular Cloning. A laboratory manual. Second edition. Cold
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Spring Harbour Laboratory Press. New York 1989.
In these Examples, the enzyme of the present invention is sometimes referred to as AbfC.
In addition, the promoter of the present invention is sometimes referred to as the AbfC
5 promoter.
Purification of the arabinofuranosida_e
Aspergillus niger 3M43 was grown in medium cont~ining wheat bran and beet pulp. The
10 fermentation broth was separated from the solid part of the broth by filtration.
Concentrated fermentation broth was loaded on a 25XlOOmm Q-SEPHAROSE
(Ph~ ci~) high Performance column, equilibrated with 20 mM Tris, HCl pH 7.S, anda linear gradient from 0-500 Mm NaCl was performed and fractions of the eluate was
collected. The Arabinofuranosidase was eluted at 130-150 Mm NaCl.
The fractions cont~ining the arabinofuranosidase were combined and desalted using a
50x200 inm G-25 SEPHAROSE Superfine (Pharmacia). The column was eluted with
distilled water.
20 After clec~lting the enzyme was concentrated using High-Trap spin columns. Next the
concentrated and desalted fractions were subjected to gel filtration on a 50x600 mm
SUPERDEX 50 column. The sample was loaded and the column was eluted with 0.2 M
Phosphate buffer pH 7.0 plus 0.2 M NaCl, and fractions of the eluate were collected.
25 The fractions cont~ining arabinofuranosidase were combined and desalted and
concentrated as described above. The combined fractions were loaded on a 16X100 mm
Phenylsepharose High Pelrollnallce column (Pharmacia), equilibrated with 50 mM
Phosphate buffer pH 6.0, cont~ining 1.5 M (NH4)2SO4. A gradient where the (NH4).,SO4
concentration was varied from 1.5 - 0 M was applied and the eluate collected in fractions.
30 The fractions cont~ining Arabinof~lranosidase were combined. The purity of the
arabinofuranosidase was evaluated by SDS-PAGE using the Phast system gel
(Pharmacia) .
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Characterization
The molecular weight of the purified arabinofuranosidase was determined by mass
spectrometry using laser desorption technology. The MW of the arabinofuranosidase was
found to be 33,270 D + 50 D.
The pI value was determined by use of a Broad pI Kit (Pharmacia). The
arabinofuranosidase has a pI value of about 3.7.
After SDS-PAGE analysis, treatment PAS reagent showed that the arabinofuranosidase
was glycosylated. The PAS staining was done according to the procedure of I. Van-
Seuningen and M. Davril (1992) Electrophoresis 13 pp 97-99.
Activity Studies
Activity of AbfC as a function of water soluble pentosan (WSP) concentrations (mg/ml)
was ~let~rrnin~rl The results are shown in Figure 21. The results show that AbfCactivity reached maximum at substrate concentration of 8 mg/ml WSP.
pH Activity Studies
The effect of pH on the activity of the arabinofuranosidase of the present invention was
invt-stig~tt-rl using water soluble pentosan (10 mg/ml) from wheat as a substrate in 50 mM
citric acid sodium phosphate buffer. The inrllb~tion time was 15 mimltt~s. The
arabinofuranosidase of the present invention was observed to have a wide pH optima
range of from about 2.5 to about 7.0 (see Figure 20)~ more especially from about 3.3 to
about 4.6, more especially about 4.
Tempel;alule Activity Studies
The effect of temperature on the activity of the arabinofuranosidase of the present
invention was investig~tt~d using water soluble pentosan (10 mg/ml) from wheat as a
substrate in 50 mM sodium acetate at a pH of 5Ø The incubation time was 15 mimltes.
~ ~ = ~
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The arabinofuranosidase of the present invention was observed to have an optimal activity
at a temperature of from about 40~C to about 60~C, more especially from about 45~C to
about 55~C, more especially about 50~C (Figure 22). The enzyme is still active at about
10~C and showed residual activity at 70~C and 80~C.
Amino acid sequencing of the arabinofuranosidase
The enzyme was digested with endoproteinase Lys-C sequencing grade from Boehringer
Mannheim using a modification of the method described by Stone & Williams 1993
10 (Stone, K.L. and Williams, K.R. (1993). Enzymatic digestion of Proteins and HPLC
Peptide Isolation. In: M~tc~ ira P. (Editor). A practical Guide to Protein and Peptide
Purification for Microsequencing. Second Edition. Academic Press, San Diego 1993. pp
45-73)-
Freeze dried ,B-arabinofuranosidase (0.4 mg) was dissolved in 50 ,~l of 8M urea, 0.4 M
NH4HCO3, pH 8.4. After overlay with N2 and addition of 5 ,ul of 45 Mm DTT, the
protein was denatured and reduced for 15 min at 50~C under N2. After cooling to RT,
5 ,ul of 100 Mm iodo~et~mide was added for the cysteines to be derivatised for 15 min
at RT in the dark under N2. Subsequently, 90 ,Ll of water and 5 ~g of endoproteinase
Lys-C in 50 ,Ll of 50 Mm Tricine and 10 mM EDTA, pH 8.0, was added and the
digestion was carried out for 24h at 37~C under N2. The resulting peptides were
separated by reversed phase HPLC on a VYDAC C18 column (0.46 x 15 cm; 10 ~m;
The Separations Group; California) using solvent A: 0.1% TFA in water and solvent B:
0.1 % TFA in acetonitrile. Selected peptides were rechromatographed on a Develosil C18
column (0.46 x 10 cm; 3,um) using the same solvent system prior to sequencing on an
Applied Biosystems 476A sequencer using pulsed-liquid fast cycles.
The following peptide sequences were found:
SEQ I.D. No. 4
SEQ I.D. No. 5
SEQ I.D. No. 6
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32
SEQ I.D. No. 7
SEQ I.D. No. 8
Isolation of a PCR clone of a fr~ nt of the gene
PCR primers were synthesised using an Applied Biosystems DNA synth~si~er model 392.
In this regard, PCR primers were synthesized from one of the found peptide sequences,
namely SEQ ID No. 5. The primers were:
10 One primer from EMTAQA (reversed)
SEQ ID NO. 9 GCY TGN GCN GTC ATY TC
17 mer 64 mix
15 One primer from MIVEAIG
SEQ ID NO. 10 ATG ATH GTN GAR GCN ATH GG
20 mer 288 mix
PCR amplification was performed with 100 pmol of each of these primers in 100 ,ul
reactions using Amplitaq polymerase (PERKIN ELMER). The following program was:
STEP TEMP TIME
1 94~C 2 min
2 94~C 1 min
3 55~C 2 min
4 72~C 2 min
72~C 5 min
6 5~C SOAK
Steps 2~ were repeated for 40 cycles.
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PCR reactions were performed on a PE~KIN ELMER DNA Thermal Cycler.
A 100 bp amplified fragment was isolated and cloned into a pT7-Blue T-vector, according
to the m~mlf~t~lrers instructions (Novagen).
S
Isolation of A. niger genomic DNA
lg. of frozen A. niger mycelium was ground in a mortar under liquid nitrogen. Follow-
ing evaporation of the nitrogen cover, the ground mycelium was extracted with 15ml of
an extraction buffer (lOOmM Tris Hcl, pH 8.0, O.SOrnM EDTA, SOOmM NaCl, lOmM
B-mercaptoethanol) cont~ining lml 20% sodium dodecyl sulphate. After incubation at
65~C for 10 min. Sml SM KAc. pH S.O, was added and the mixture further incub-ated,
after mixing, on ice for 20 mins. After extraction, the mixture was centrifuged for 20
mins. and the supernatant mixed with 0.6 vol. isopropanol to precipitate the extracted
DNA. After further centrifugation for lS mins. the DNA pellet was dissolved in 0.7 ml
TE (lOmM Tris, HCl pH 8.0, lmM EDTA) and precipitated with 75 ~l 3M NaAc, pH
4.8, and 500 ~l isopropanol.
After centrifugation the pellet was washed with 70% ETOH and dried under vacuum.The DNA was dissolved in 200 ~l TE and stored at -20~C.
Construction of a library
20 ~g genomic DNA was partly digested with Tsp509I, which gives ends which are
25 compatible with EcoRI ends. The digested DNA was separated on a 1 % agarose gel and
fragments of 4-10 kb was purified. A ~ZAPII EcoRI/CIAP kit from Stratagene was used
for library construction according to the m~mlf~ lrers instructions. 2 ,~Ll of the ligation
(totally 5 ,Ll) was packed with Gigapack Gold II packing extract from Stratagene. The
library contained 650,000 independent clones.
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Screening of the library
2 X 50,000 pfu was plated on NZY plates and plaquelifts were done on Hybond N sheets
(Amersham). Plaquelifts were done in duplicates. The sheets were hybridized with the
5 PCR clone labelled with 32p dCTP (Amersham) using Ready-to-go labelling kit from
Pharmacia. Positive clones were reckoned only when hybridization was cletect~d on both
sheets. The gene was sequenced, and the found sequence showed that all of the peptides
sequenced were coded by the found sequence.
10 Sequence information
SEQ. ID. No. 12 presents the promoter sequence, the enzyme coding sequence-, thetermin~tor sequence and the signal sequence and the amino acid sequence of the enzyme
of the present invention.
Arabinofuranosidase assay
Two different arabinoxylan preparations from wheat flour, Wheat Insoluble Pentosan
(WIP) and Wheat soluble Pentosan (WSP), were degraded with the arabinofuranosidase
20 enzyme of the present invention alone and in combination with an endoxylanase purified
from A. r~iger. The assays were done on 1% substrate in 50 Mm 50 Mm Na-acetate
buffer at pH 5Ø The reactions were ~elrolllled at 30 ~C for 2.5 hours. The reactions
were stopped by addition of 3 vol. ethanol which precipitates the high molecular weight
material. The samples were centrifuged and the supern~t~ntC were collected, dried under
25 vacuum and resuspended in 0.5 ml ~ictille~l water. The samples were diluted 1:1 in
water and analysed on a Chromopack Carbohydrate Pb column (300X7.8 mm, cat.
29010) using Shim~ C-R4A Chromatopac HPLC system using a Shim~ RI D-6A
refractive index detector in accordance with the suppliers instructions.
The column was calibrated using a standard composed of 0.48 mg/ml xylotriose, 0.48
mg/ml xylobiose, 0.60 mg/ml xylose and 0.58 mg/ml L-arabinose. The peaks were
identifled and qll~ntifiefl using the software supplied with the equipment.
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Results - Liberated saccharides from Wheat Insoluble Pentosan
Substrate 1% WIP in 50 Mm Na-acetate buffer pH 5Ø Values are expressed in mg/ml.
xylotriose xylobiose xylose arabinose
no enzyme 0.0 0.0 0.0 0.0
abfC 0.0 0.0 0.0 0.11
xyl 0.09 0. 14 0.0 0.0
abfC + xyl 0.37 0.41 0.0 0.30
abfC denotes the enzyme according to the present invention; and xyl denotes the xylanase
described before.
Results - Saccharides liberated from Wheat Soluble Pentosan
Substrate 1 % WSP in 50 Mm Na-acetate buffer pH 5Ø Values are expressed in
mg/ml.
xylotriose xylobiose xylose arabinose
no enzyme 0.0 0.0 0.0 0.0
abfC 0.0 0.0 0.0 0.30
xyl 0.08 0. 14 0.0 0.0
abfC + xyl 0.42 0.47 0.0 0.42
- 25 abfC denotes the enzyme according to the present invention; and xyl denotes the xylanase
described before.
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Figure 17 shows HP-TLC profiles of the AbfC enzyme acting syner~icti~ y with
Xylanase A. In this Figure, the following abbreviations are used: water-soluble pentosan
(WSP); water-insoluble pentosan (WIP); and oat xylan as substrate. The standards were:
X- xylose; X,- xylobiose; X3- xylotriose; A- arabinose.
Figure 18 shows the HPLC analysis of hydrolysis products using 1% oat spelt xylan as
the substrate. Figure 18(a) and Figure 18(b) show the products when the AbfC enzyme
and the xylanase enzyme respectively were used alone. Figure 18(c) show the products
when the AbfC enzyme and the xylanase enzyme when combined.
The results of t'nese experiments provide two important findings.
First the enzyme of the present invention liberates arabinose, in particular L-arabinose,
from arabinoxylan.
Second the combined actions of the enzyme according to the present invention with the
endoxylanase is significantly higher than the sum of their individual action. Accordingly,
the two enzymes affect each others enzymatic activities in a synergistic fashion.
20 Induction of the AbfC gene~ ntific~ti~n of inducers
The regulation of Lldns~ Lion of the AbfC encoding gene of Aspergi~lus niger wasstudied using a strain Cont,.inin~ a fusion of the AbfC promoter to the ,B-glucuronidase
encoding gene (uid A) of E coli.
GUS producing transformants were grown on different carbon sources and assayed both
qualitatively and qll,.ntit,.tively for the ability to hydrolyse p-nitrophenol glucuronide.
The results are shown below:
-
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CARBON SOURCE GUS ACTIVITY AFTER 24 HOURS INDUCTION
(1 %) (units/mg)
xylose 12 37
xylitol 1.49
arabinose 6 . 66
arabitol 5.30
glucose 0. 70
cellubiose 0 . 95
xylo-oligomer 70 17 .26
glucopyranoside 0.40
methyl-xylopyranoside 24.20
xyloglucan 1.00
pectin 0.27
arabinogalal~t~n 2.60
arabitol + glucose 2.20
The results show that the AbfC promoter is switched on after 24 hours when grown in
the presence of xylose, xylo-oligomer 70, methyl-xylopyranoside, arabinose and arabitol.
20 These studies also suggest that methyl-xylopyranoside is the natural and strongest inducer
of this promoter.
The AbfC promoter is strongly repressed by glucose and is therefore under carboncatabolite l~pl~s~ion. However, unlike all the published promoters for
25 arabinofuranosidases, which are in~ cerl by arabinose and arabitol, the AbfC promoter
of the present invention is regulated strongly by the inte~n~ t~-s in xylose metabolism.
Accordingly, the present invention also covers an arabinofuranosidase promoter wherein
the promoter is inducible by an i,lL~llllediate in xylose metabolism.
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Effects of different promoter deletions on the regulation of the expression of the
AbfC gene
To study the regulation at the molecular level, experiments were set up to detect possible
5 upstream regulating sequences required for expression of the AbfC gene. A series of
plasmids with deletions in the 5' L~ LI~a~l~ region of the gene was constructed (see Figure
12). The E coli uid A gene was used as the reporter gene and a qualitative GUS assay
was performed.
10 The results indicated that the truncated AbfC promoter of 590 bp contains sufficient
information for the inducibility of the AbfC gene and its regulation. Deletion of 100 bps
sequence from the X~na 111 to the BamHl sites of the promoter led to a reduction in
activity of this promoter. Therefore, this 100 bps area is important for good levels of
gene expression. Deletion of 290 bps before the ATG identified this region to be15 important but not sufficient to abolish the activity of this promoter. All the kansformants
analysed cont~ining this promoter construct showed very pale blue when tested (+-
GUS). This region is as follows:
-170 T C A T C C A A T A T
As seen, this region contains the CCAAT element and is a putative target for a general
L,d"~c.i~ional activator. This sequence is similar to the nuclear protein binding sites
found in two starch inducible promoters: the Aspergillus niger glucoamylase gene and the
Aspergillus oryzae amylase gene as well as the amdS gene of Aspergillus nidulans.
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HETEROLOGOUS PROTEIlY PRODUCTION USING ASPERGILLUS N7GER
TRANSFORMED WITH THE AbfC PROMOTER AND THE AbfC SIGNAL
SEQUENCE
Transformation of Asperg~llus Niger
The protocol for transformation of A. niger was based on the teachings of Buxton,F.P.,
Gwynne D . I ., Davis ,R. W . 1985 (Transformation of Aspergillus niger using the argB gene
of Aspergillus nidulans. Gene 37:207-214), Daboussi,M.J., Djeballi,A., Gerlinger, C.,
Blaiseau, P.L., Cassan, M., Lebrun, M.H., Parisot, D., Brygoo,Y. 1989
(Transformation of seven species of filamentous fungi using the nitrate reductase gene of
Aspergillus nidulans. Curr. Genet. 15:453-456) and Punt, P.J., van den Handel,
C.A.M.J.J. 1992 (Transformation of filamentous fungi based on hygromycin B and
Phleomycin resistance markers. Meth. Enzym. 216:447-457).
For the purification of protoplasts, spores from one PDA (Potato Dextrose Agar - from
Difco Lab. Detroit) plate of fresh sporulated N400 (CBS 120.49, Centraalbureau voor
Schimmelcultures, Baarn) (7 days old) are washed off in 5-10 ml water. A shake flask
with 200 ml PDC (Potato Dextrose Broth, Difco 0549-17-9, Difco Lab. Detroit) is
inoculated with this spore suspension and shaken (250 rpm) for 16-20 hours at 30~C.
The mycelium is harvested using Miracloth paper and 3-4 g wet mycelium are transferred
to a sterile petri dish with 10 ml STC (1.2 M sorbitol, 10 mM Tris Hcl pH 7,5, 50 Mm
CaCl2) with 75 mg lysing enzymes (Sigma L-2265) and 4500 units lyticase (Sigma L-
8012).
The mycelium is incubated with the enzyme until the mycelium is degraded and theprotoplasts are released. The degraded mycelium is then filtered through a sterile 60 ,um
mesh filter. The protoplasts are harvested by centrifugation 10 min at 2000 rpm in a
30 swing out rotor. The supelllaL~llL is discarded and the pellet is dissolved in 8 ml 1.5 M
MgSO4, and then centrifuged at 3000 rpm for 10 min.
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The upper band, cont~ining the protoplasts is transferred to another tube, using a transfer
pipette and 2 ml 0.6 M KCl is added. Carefully 5 ml 30% sucrose is added on the top
and the tube is centrifuged 15 min at 3000 rpm.
5 The protoplasts, lying in the interface band, are transferred to a new tube and diluted
with 1 vol. STC. The solution is centrifuged 10 min at 3000 rpm. The pellet is washed
twice with STC, and finally solubilized in 1 ml STC. The protoplasts are counted and
eventually concentrated before transformation.
For the transformation, 100 ~l protoplast solution (106-107 protoplasts) are mixed with
10 ~1 DNA solution cont~ining 5- 10 ~g DNA and incubated 25 min at room
temperature. Then 60 % PEG4000 is carefully added in portions of 200 ~l, 200 ~1 and
800 ,~l. The mixture is incubated 20 min at room temperature. 3 ml STC is added to the
mixture and carefully mixed. The mixture is centrifuged 3000 rpm for 10 min.
The supernatant is removed and the protoplasts are solubilized in the rem~ining of the
supernatant. 3-5 ml topagarose is added and the protoplasts are quickly spread on
selective plates.
20 AbfC promoter and heterologous gene e~le~ion
The expression vector pXP-Amy (Figure 13) contains the 2.1 kb ~-amylase encodinggene from Thermon~ces lanuginosus cloned downstream of the AbfC promoter (2.1 kb)
and upstream of the Xylanase A terminator. This vector together with the hygromycin
25 gene as a selectable marker was used for co-transformation experiments to test the
functionality of the AbfC promoter.
The best transformant was ~rcllm~ trrl in shake flask experiments at least 1 gram per
litre of ~x-amylase in the culture media. Starch degrading activity was then detected
30 within 48 hours and a peak of enzyme activity is observed at 4 days of growth on sugar
beet pulp and wheat bran (Figure 15).
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AbfC signal sequence functions in protein secretion
An expression construct cont~ining the signal peptide of the AbfC gene translationally
fused to the mature ~-amylase from T. Ianuginosus was prepared and expression of this
5 construct in the production strains was observed. In this regard, the translational fusion
construct pXPXss-Amy (Figure 14) was placed under the transcriptional control of the
AbfC promoter and the xylanase A termination signal. The incorporation of an
endogenous signal peptide resulted in increased detectability of co-transformants
expressing both amylase and the hygromycin resistance marker. The endogenous signal
10 peptide directed the secretion of amylase out of the cell.
Substrate Speci~lcity of AbfC Protein
The substrate specificity of the purified AbfC was determined using arabinose cont~ining
15 hemicelluloses: arabinoxylans from wheat, oat and larch, branched and debranched
arabinans; arabinog~lar.t~n, sugar beet pectin, and xyloglucan.
The HPLC and HP-TLC results are shown in Figure 16, in which the following
abbreviations are used: WSP - water-soluble pentosan, WIP - water-insoluble pentosan,
20 AG - arabinogalactan, deB-A - debranched arabinan. The standards used were: A-
arabinose, X- xylose.
The results indicate that arabinose is the hydrolysis product from arabinoxylans. No
hydrolysis products were released from arabinogalactan, debranched arabinan or
25 xyloglucan. Arabinose was released as a hydrolysis product from branched arabinan.
AbfC is therefore a 1,2tl,2 debr~nr~ing enzyme and it has no activity towards linear 1,5
cY-linked L-arabinofuranose residues found in debranched arabinans and arabinog~lart~n
This enzyme also releases a product when pectin is used as the substrate. It is believed
that this product is an arabinose cont~ining ferulic acid or an arabinobiose.
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Reduction of Viscosity By AbfC
The results for the substrate specificity studies also suggest that the enzyme of the present
invention could be used to reduce the viscosity of feeds. In this regard, the enzyme
5 would reduce the viscosity of branched substrates by removing the branches but not the
backbone of that substrate. This is in contrast to the known viscosity modifiers which
degrade the substrate backbone.
Accordingly, the present invention covers a process of reducing the viscosity of a
10 branched substrate wherein the enzyme degrades the branches of the substrate but not the
backbone of the substrate.
In particular, the present invention covers the use of the enzyme of the present invention
as a viscosity modifier.
In this regard, an experiment was carried out to investigate the reduction of viscosity of
the water-soluble pentosan fraction from wheat flour by arabinofuranosidase. In this
experiment, 6 ml water-soluble pentosan was incubated with 100 ~1 of AbfC for 20hours, 20~C at pH 5 .5 .
The results (see Figure 19) show that the enzyme of the present invention can be used
to reduce the viscosity of pectins, especially pectins that are used in beverages - such as
fruit juices.
25 Accordingly, the present invention covers the use of the enzyme of the present invention
to reduce the viscosity of pectin.
ANTIBODY PRODUCTION
30 Antibodies were raised against the enzyme of the present invention by injecting rabbits
with the purified enzyme and isolating the immnn~globulins from antiserum according
to procedures described according to N Harboe and A Ingild ("T"",.l"~ tion, Isolation
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of Tmmllnnglobulins, Estimation of Antibody Titre" In A Manual of Qll~ntir~tive
Tmmllnoelectrophoresis, Methods and Applications, N H Axelsen, et al (eds.),
Universitetsforlaget, Oslo, 1973) and by T G Cooper ("The Tools of Biochemistry", John
Wiley & Sons, New ~ork, 1977).
S~lMMA~Y
Even though it is Known that Aspergillus niger produces arabinofuranosidases, the present
invention provides a novel and inventive arabinofuranosidase, as well as the coding
10 sequence therefor and the promoter for that sequence. An important advantage of the
present invention is that the enzyme can be produced in high amounts.
In addition, the promoter and the regulatory sequences (such as the signal sequence and
the te~;min~tor) can be used to express or can be used in the expression of GOIs in
15 org~ni~m~, such as in A. niger.
The arabinofuranosidase of the present invention is different from the
arabinofuranosidases previously known. In this regard, the previous described
arabinofuranosidases - such as those of EP-A-0506190 - are characterised by their ability
20 to degrade arabinan, and are assayed using p-nitrophenyl-arabinoside.
The arabinofuranosidase of the present invention does not degrade arabinan, and only a
minor activity is seen on p-nitrophenyl-arabinoside.
25 In contrast, the arabinofuranosidase of the present invention is useful for degrading
arabinoxylan. Therefore, the arabinofuranosidase of the present invention is quite
different from the previous isolated arabinofuranosidases.
More in particular, the erl7yme of the present invention is capable of specifically cleaving
30 arabinose from the xylose backbone of arabinoxylan.
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The enzyme of the present invention is useful as it can improve processes for preparing
foodstuffs and feeds as well as the foodstuffs and feeds themselves. For example, the
enzyme of the present invention may be added to animal feeds which are rich in
arabinoxylans. When added to feeds (including silage) for monogastic animals (e.g.
5 poultry or swine) which contain cereals such as barley, wheat, maize. rye or oats or
cereal by-products such as wheat bran or maize bran, the enzyme signific ~ntly improves
the break-down of plant cell walls which leads to better utilization of the plant nutrients
by the animal. As a consequence, growth rate and/or feed conversion are improved.
Moreover, arabinoxylan-degrading enzymes may be used to reduce the viscosity of feeds
10 cont~ining arabinans. The arabinoxylan-degrading enzyme may be added beforehand to
the feed or silage if pre-soaking or wet diets are ~L~r~ d.
Of particular benefit is the use of the enzyme according to the present invention in
combination with a xylanase, especially an endoxylanase.
A possible further application for the enzyme according to the present invention is in the
pulp and paper industry. The application of xylanases is often reported to be beneficial
in the removal of lignins and terpenoids from the cellulose and hemicellulose residues of
a hemicellulose backbone, an essential step in the processing of wood, wood pulp or
20 wood derivative product for the production of paper. The addition of arabinoxylan-
degrading enzymes, produced according to the present invention, to the xylanase
tre~tn~nt step should assist in the degradation of an arabinan-cont~ining hemicellulose
backbone and thus facilitate an improved, more efficient removal of both lignins and
terpenoids. The application of arabinoxylan-degrading enzymes should be particularly
25 advantageous in the processing of soft woods in which the hemicellulose backbone
contains glucuronic acid.
The enzyme according to the present invention is also useful as it acts in a synergistic
manner with endoxylanase (see results presented above).
Other modifications of the present invention will be apparent to those skilled in the art
without departing *om the scope of the invention.
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SEQUENCE LISTINGS
SEQ ID NO: 1
ENZYME SEQUENCE
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 296 amino aclds
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Lys Cys Ser Leu Pro Ser
1 5~er Tyr Ser Trp Ser Ser Thr Asp Ala Leu Ala Thr Pro Lys Ser Gly
Trp Thr Ala Leu Lys Asp Phe Thr Asp Val Val Ser Asp Gly Lys His
Z5 30 35
Ile Val Tyr Ala Ser Thr Thr Asp Glu Ala Gly Asn Tyr Gly Ser Met
Thr Phe Gly Ala Phe Ser Glu Trp Ser Asn Met Ala Ser Ala Ser Lys
Thr Ala Thr Pro Tyr Asn Ala Val Ala Pro Thr Leu Phe Tyr Phe Lys
85~ro Lys Ser Ile Trp Val Leu Ala Tyr Gln Trp Gly Ser Ser Thr Phe
100
Thr Tyr Arg Thr Ser Gln Asp Pro Thr Asn Val Asn Gly Trp Ser Ser
105 110 115
Glu Lys Ala Leu Phe Thr Gly Lys Leu Ser Asp Ser Ser Thr Gly Ala
120 125 130
Ile Asp Gln Thr Val Ile Gly Asp Asp Thr Asn Met Tyr Leu Phe Phe
135 140 145 150
Ala Gly Asp Asn Gly Lys.Ile Tyr Arg Ser Ser Met Ser Ile Asp Glu
155 160 165
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Phe Pro Gly Ser Phe Gly Ser Gln Tyr Glu Glu Ile Leu Ser Gly Ala
170 175 180~hr Asn Asp Leu Phe Glu Ala Val Gln Val Tyr Thr Val Asp Gly Gly
185 190 195
Glu Gly Asn Ser Lys Tyr Leu Met Ile Val Glu Ala Ile Gly Ser Thr
200 205 210
Gly His Arg Tyr Phe Arg Ser Phe Thr Ala Ser Ser Leu Gly Gly Glu
215 220 225 230~rp Thr Ala Gln Ala Ala Ser Glu Asp Lys Pro Phe Ala Ala Lys Pro
235 240 245~hr Val Ala Pro Pro Gly Pro Lys Thr Leu Ala Met Val Thr Trp Phe
250 255 260~la Thr Thr Leu Ile Lys Pro *
265 270
_
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SEQ ID NO: 2
NUCLEOTIDE CODING SEQUENCE
AAA TGC TCT CTT CCA TCG TCC TAT AGT TGG AGT TCA ACC GAT GCT CTC
GCA ACT CCT AAG TCA GGA TGG ACC GCA CTG MG GAC m ACT GAT GTT
GTC TCT GAC GGC AAA CAT ATC GTC TAT GCG TCC ACT ACT GAT GAA GCG
GGA MC TAT GGC TCG ATG ACC m GGC GCT TTC TCA GAG TGG TCG AAC
ATG GCA TCT GCT AGC MG ACA GCC ACC CCC TAC MT GCC GTG GCT CCT
ACC CTG TTC TAC TTC AAG CCG AAA AGC ATC TGG GTT CTG GCC TAC CAA
TGG GGC TCC AGC ACA TTC ACC TAC CGC ACC TCC CM GAT CCC ACC MT
GTC MC GGC TGG TCG TCG GAG MG GCG CTT TTC ACC GGA MM CTC AGC
GAC TCA AGC ACC GGT GCC ATT GAC CAG ACG GTG ATT GGC GAC GAT ACG
AAT ATG TAT CTC TTC 111 GCT GGC GAC MC GGC MG ATC TAC CGA TCC
AGC ATG TCC ATC GAT GM 111 CCC GGA AGC TTC GGC AGC CAG TAC GAG
GAA ATT CTG AGT GGT GCC ACC MC GAC CTA TTC GAG GCG GTC CM GTG
TAC ACG GTT GAC GGC GGC GAG GGC AAC AGC MG TAC CTC ATG ATC GTT
GAG GCG ATC GGG TCC ACT GGA CAT CGT TAT TTC CGC TCC TTC ACG GCC
AGC AGT CTC GGT GGA GAG TGG ACA GCC CAG GCG GCA AGT GAG GAT MM
CCC TTC GCA GCA MG CCA ACA GTG GCG CCA CCT GGA CCG MG ACA TTA
GCC ATG GTG ACT TGG TTC GCA ACA ACC CTG ATC AAA CCA TGA
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48
SEQ ID NO: 3
PROMOTER SEQUENCE
CTGCAGMGA TGGCAGTCGC CACAGCCGAT CACCCGATCC ATACTGGATG TTGTMCTTG 60
GAGACAGCCT GCAGATGCTC TGATGMGGT CTGCMMTAG TTCCTGGACC TCGATAGTGA 120
AGTATACCGA TTCGTCMTG TTGTATATCC AGCCACIIIG MMGTACCM C~lilAGTTC 180
GATTGATCAG MTACl IIIG GTGTGTMCA TTGACMGCC MMTTATCM TCTCTTCTAC 240
CGGTMGGTG TCMCTACCC GGCCGMMGT ACCGGMGGT CGTGGTGIII TMGGTGMM 300
CMCTATCAG GGCGGCMTG TGTCMMGTA GMCCAGill GCTTAGCGCC ATTAGGATCC 360
ACGCCTAGAC CCTTGATGCC CGGGAGTTAT CCGTCCTGTC ACAGCMTTA IIICCCCGAG 420
TCTACTGCCG MGMCAGCC ATTGTGGCGT ACTCACGGM TTACCCACTG TGTAGGGTAG 480
TCTTGMCGC CGTTCTAGAC ACGGCMCGC TCCGGTGGAC GATCGmCT GGCTMTGTA 540
CTCCGTAGTT TAGGCAGCAT GCTGATCATC TTCCCCCTAG GGMMGGCCC CTGMTAGTG 600
CGCCMMTG AGCTTGAGCA MGGMTGTT CmCTMGC CMMGTGAGG GMMTMCCA 660
AGCAGCCCAC TTTTATCCGA MCGliICTG GTGTCATCCA ATATGGATM ATCCCGATTG 720
TTCTTCTGCA CATATCTCTA TTGTCATMG TGCMCTACA TATAIIIGM CATGGIIIGG 780
TCCTCIIICC MGTTATTCG TTCTCCGTGA CCAGCGAIII CAGCCATTGA TT(:llll(ill 840
TCIIICCCCG CGGATMMCT CATACGMG
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49
INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Lys Cys Ser Leu Pro Ser Ser Tyr Ser Trp Ser Ser Thr Asp Ala Leu
1 5 10 15
Ala Thr Pro Lys
INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Tyr Leu Met Ile Val Glu Ala Ile Gly Ser Thr Gly His Arg Tyr Phe
1 5 10 15
Arg Ser Phe Thr Ala Ser Ser Leu Gly Gly Glu Met Thr Ala Gln Ala
Ala Ser Glu Asp Lys Pro Phe Xaa Gly
CA 02214~91 1997-09-03
WO 96/29416 PCT/li~P96/OlOO9
INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Ser Ile Trp Val Leu Ala Tyr Gln Trp Gly Ser Ser Thr Phe Thr Tyr
1 5 10 15
Arg Thr Ser Gln Asp Pro Thr Asn Val
INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Asp Ile Val Tyr Ala Ser Thr Thr Asp Glu Ala Gly Asn Tyr Gly Ser
1 5 10 15
Met Thr Phe Gly Ala Phe Ser Glu Xaa Ser Asn Met Ala Ser
CA 02214~91 1997-09-03
WO 96/29416 PCT/EP96/01009
51
INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Ile Tyr Arg Ser Ser Met Ser Ile Asp Glu Phe Pro Gly Ser Phe Gly
1 5 10 15
Ser Gln Tyr Glu Glu Ile Leu Ser Gly Ala Thr Asn Asp Leu Phe Glu
20 25 30
Ala Val Gln Val Tyr Thr Val Asp Gly
35 40
INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "OLIGONUCLEOTIDE"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GCYTGNGCNG TCATYTC 17
INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "OLIGONUCLEOTIDE"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
ATG ATH GTN GAR GCN ATH GG 20
CA 02214~91 1997-09-03
WO 96/29416 PCT/EP96tOlOO9
52
INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 89 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS~ double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIFTION: /desc = "PCR fragment"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ATGATTGTGG AGGCGATCGG GTCCACTGGA CATCGTTATT TCCGCTCCTT CACGGCCAGC 60
AGTCTCGGTG GAGAGATGAC CGCACAGGC 89
INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2555 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
~vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus nïger
(B) STRAIN: 3M43
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:870..1757
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION:870..947
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION:948..1754
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CTGCAG M GA TGGCAGTCGC CACAGCCGAT CACCCGATCC ATACTGGATG TTGT M CTTG 60
GAGACAGCCT GCAGATGCTC TGATG MGGT CTGCA M TAG TTCCTGGACC TCGATAGTGA 120
AGTATACCGA TTCGTC M TG TTGTATATCC AGCCACIIIG MM GTACC M CllilAGTTC 180
GATTGATCAG M TACIIIIG GTGTGTAACA TTGAC MGCC MM TTATC M TCTCTTCTAC 240
CA 02214~91 1997-09-03
WO 96/29416 PCT/EP96101009
53
CGGTMGGTG TCMCTACCC GGCCGAMGT ACCGGMGGT CGTGGTGI I I TMGGTGMM 300
CMCTATCAG GGCGGCMTG TGTCMMGTA GMCCAGI I I GCTTAGCGCC ATTAGGATCC 360
ACGCCTAGAC CCTTGATGCC CGGGAGTTAT CCGTCCTGTC ACAGCMTTA l l ICCCCGAG 420
TCTACTGCCG MGMCAGCC ATTGTGGCGT ACTCACGGM TTACCCACTG TGTAGGGTAG 480
TCTTGMCGC CGTTCTAGAC ACGGCMCGC TCCGGTGGAC GATCGmCT GGCTMTGTA 540
CTCCGTAGTT TAGGCAGCAT GCTGATCATC TTCCCCCTAG GGMMGGCCC CTGMTAGTG 600
CGCCMMTG AGCTTGAGCA MGGMTGTT CTI ICTMGC CAMGTGAGG GMMTMCCA 660
AGCAGCCCAC I I I IATCCGA MCG~ I I CTG GTGTCATCCA ATATGGATM ATCCCGATTG 720
TTCTTCTGCA CATATCTCTA TTGTCATMG TGCMCTACA TATAI rlGM CATGGI I IGG 780
TCCTCl I ICC MGTTATTCG TTCTCCGTGA CCAGCGAI I I CAGCCATTGA TTCI I I IGTT 840
TCl l ICCCCG CGGATMMCT CATACGAAG ATG MG TTC TTC MT GCC MM GGC 893
Met Lys Phe Phe Asn Ala Lys Gly
-26 -25 -20
AGC TTG CTG TCA TCA GGA ATC TAC CTC ATT GCA TTA ACC CCC l l l GTT 941
Ser Leu Leu Ser Ser Gly Ile Tyr Leu Ile Ala Leu Thr Pro Phe Val
-15 -10 -5
MC GCC AM TGC TCT CTT CCA TCG TCC TAT AGT TGG AGT TCA ACC GAT 989
Asn Ala Lys Cys Ser Leu Pro Ser Ser Tyr Ser Trp Ser Ser Thr Asp
5 10
GCT CTC GCA ACT CCT MG TCA GGA TGG ACC GCA CTG MG GAC l l l ACT 1037
Ala Leu Ala Thr Pro Lys Ser Gly Trp Thr Ala Leu Lys Asp Phe Thr
15 20 25 30
GAT GTT GTC TCT GAC GGC MM CAT ATC GTC TAT GCG TCC ACT ACT GAT 1085
Asp Val Val Ser Asp Gly Lys His Ile Val Tyr Ala Ser Thr Thr Asp
35 40 45
GM GCG GGA MC TAT GGC TCG ATG ACC m GGC GCT TTC TCA GAG TGG 1133
Glu Ala Gly Asn Tyr Gly Ser Met Thr Phe Gly Ala Phe Ser Glu Trp
50 55 60
TCG MC ATG GCA TCT GCT AGC MG ACA GCC ACC CCC TAC AAT GCC GTG 1181
Ser Asn Met Ala Ser Ala Ser Lys Thr Ala Thr Pro Tyr Asn Ala Val
65 70 75
GCT CCT ACC CTG TTC TAC TTC MG CCG AAA AGC ATC TGG GTT CTG GCC 1229
Ala Pro Thr Leu Phe Tyr Phe Lys Pro Lys Ser Ile Trp Val Leu Ala
80 85 90
TAC CM TGG GGC TCC AGC ACA TTC ACC TAC CGC ACC TCC CM GAT CCC 1277
Tyr Gln Trp Gly Ser Ser Thr Phe Thr Tyr Arg Thr Ser Gln Asp Pro
100 105 110
CA 02214~91 1997-09-03
WO 96/29416 PCT/EP96/01009
54
ACC M T GTC AAC GGC TGG TCG TCG GAG M G GCG CTT TTC ACC GGA M A 1325
Thr Asn Val Asn Gly Trp Ser Ser Glu Lys Ala Leu Phe Thr Gly Lys
115 120 125
CTC AGC GAC TCA AGC ACC GGT GCC ATT GAC CAG ACG GTG ATT GGC GAC 1373
Leu Ser Asp Ser Ser Thr Gly Ala Ile Asp Gln Thr Val Ile Gly Asp
130 135 140
GAT ACG M T ATG TAT CTC TTC m GCT GGC GAC M C GGC MG ATC TAC 1421
Asp Thr Asn Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly Lys Ile Tyr
145 150 155
CGA TCC AGC ATG TCC ATC GAT G M lll CCC GGA AGC TTC GGC AGC CAG 1469
Arg Ser Ser Met Ser Ile Asp Glu Phe Pro Gly Ser Phe Gly Ser Gln
160 165 170
TAC GAG G M ATT CTG AGT GGT GCC ACC M C GAC CTA TTC GAG GCG GTC 1517
Tyr Glu Glu Ile Leu Ser Gly Ala Thr Asn Asp Leu Phe Glu Ala Val
175 180 185 ~ 190
C M GTG TAC ACG GTT GAC GGC GGC GAG GGC MC AGC MG TAC CTC ATG 1565
Gln Val Tyr Thr Val Asp Gly Gly Glu Gly Asn Ser Lys Tyr Leu Met
195 200 205
ATC GTT GAG GCG ATC GGG TCC ACT GGA CAT CGT TAT TTC CGC TCC TTC 1613
Ile Val Glu Ala Ile Gly Ser Thr Gly His Arg Tyr Phe Arg Ser Phe
210 215 220
ACG GCC AGC AGT CTC GGT GGA GAG TGG ACA GCC CAG GCG GCA AGT GAG 1661
Thr Ala Ser Ser Leu Gly Gly Glu Trp Thr Ala Gln Ala Ala Ser Glu
225 230 235
GAT MM CCC TTC GCA GCA M G CCA ACA GTG GCG CCA CCT GGA CCG M G 1709
Asp Lys Pro Phe Ala Ala Lys Pro Thr Val Ala Pro Pro Gly Pro Lys
240 245 250
ACA TTA GCC ATG GTG ACT TGG TTC GCA ACA ACC CTG ATC M A CCA TGA 1757
Thr Leu Ala Met Val Thr Trp Phe Ala Thr Thr Leu Ile Lys Pro *
255 260 265 270
CTGTCGATCC TTGC M CCTC CAGTTGCTCT ATCAGGGCCA TGACCCCCAA CAGCAGTGGC 1817
GACTAC M CC TCTTGCCATG G M GCCGGGC GTCCTTACCT TG M GCAGTG ACGAGCTTAT 1877
CIIIAGTTGC AGATCGTGTT TCTCCIIICT TCTTC MGTA GIIIIAGTGG TGG M GACAG 1937
CAG M GGTGG TCATCATCTT AGGCTCAGTT GGGGTGGGCT CCTGCCACGT m GTCCATA 1997
GGCTAGT M T TTGCACGG M TTCAGTTCAT TGGC M GGAG TGCGGTACGA ATACCTGIII 2057
TCAC MTAGC M TTAGGCCC AGTAGTTATA CTACGTACTG G M TTGAGTA CTCGTAGTAG 2117
C M GATTGTT TGCCTCAGAG GG MTGGCCG ACACGTGAGC MGTCACCTT CATCAGCTAG 2177
CA 02214~91 1997-09-03
WO 96/29416 PCT/EP96/01009
TCGCGTTCCA CATAGACMT GGTCCAGCTC CAGAGTGGM l l IGGGCTAC l l IGMCGAT 2237
GGCCGATTGA ATCGCGCGTC TCCTCMTTG TAmMCCA CAATAGGCCA GGTATTGGCA 2297
TTCACTCTCC GCCI I iGCGG GTGCCGGCAC GAGATGTCTC CTGMGMMC TAGGCAACGA 2357
GCAGACTGTG GATATGGGAG ATGGTTGACG ATGTGCTTCT TGGTMAI I I GMGCCTCCA 2417
GGGCCTCTAG MMGGCGGGA Al l IMMTCT CMGTGCCCT MCGTGTCCG ACCACGGTGT 2477
TGATCATCAT TCATTGMTC GGATMCAGT CTTGGTTCGG MACTGMCA GGCGGCTCTT 2537
GAATGACACT CTGGATCC 2555
(2) INFORMATION FOR SEQ ID NO: 13:
TERMINATOR SEQUENCE
CTGTCGATCC TTGCMCCTC CAGTTGCTCT ATCAGGGCCA TGACCCCCAA CAGCAGTGGC 60
GACTACMCC TCTTGCCATG GMGCCGGGC GTCCTTACCT TGMGCAGTG ACGAGCTTAT 120
CI I IAGTTGC AGATCGTGTT TCTCCI I ICT TCTTCMGTA GTTTTAGTGG TGGMGACAG 180
CAGMGGTGG TCATCATCTT AGGCTCAGTT GGGGTGGGCT CCTGCCACGT mGTCCATA 240
GGCTAGTMT TTGCACGGM TTCAGTTCAT TGGCMGGAG TGCGGTACGA ATACCTG l I l 300
TCACMTAGC MTTAGGCCC AGTAGTTATA CTACGTACTG GMTTGAGTA CTCGTAGTAG 360
CMGATTGTT TGCCTCAGAG GGMTGGCCG ACACGTGAGC MGTCACCTT CATCAGCTAG 420
TCGCGTTCCA CATAGACMT GGTCCAGCTC CAGAGTGGM l l IGGGCTAC l l IGMCGAT 480
GGCCGATTGA ATCGCGCGTC TCCTCMTTG TAmMCCA CMTAGGCCA GGTATTGGCA 540
TTCACTCTCC GCCmGCGG GTGCCGGCAC GAGATGTCTC CTGMGMMC TAGGCMCGA 600
GCAGACTGTG GATATGGGAG ATGGTTGACG ATGTGCTTCT TGGTMAI I l GMGCCTCCA 660
GGGCCTCTAG MAGGCGGGA AmMATCT CMGTGCCCT MCGTGTCCG ACCACGGTGT 720
TGATCATCAT TCATTGMTC GGATMCAGT CTTGGTTCGG MMCTGMCA GGCGGCTCTT 780
GMTGACACT CTGGATCC 798
(2) INFORMATION FOR SEQ ID NO: 14
Signal SEQUENCE
ATG MG TTC TTC MT GCC MM GGC AGC TTG CTG TCA TCA GGA ATC TAC 48
CTC ATT GCA TTA ACC CCC m GTT AAC GCC 78
~ =
CA 022l459l l997-09-03
WO 96/29416 PCT/EP96/OlO09
56
SEQ ID NO: 15
SIGNAL SEQUENCE
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Met Lys Phe Phe Asn Ala Lys Gly Ser Leu 10
Leu Ser Ser Gly Ile Tyr Leu Ile Ala Leu 20
Thr Pro Phe Val Asn Ala 26
CA 02214591 1997-09-03
WO 96/29416 57 PCT/EP96/01009
rNDIcATIoNs RErA~:~G TO ~ DEPOSITI~D MICROORGANIS~
(F'CT Rulc 13~
A. Thc inr(i~ior~ made bciow reiale to the microorE~.snism referred ro in Ibc descs-sption
on page ~ ~ line S 2~ a~d 2~
~. IDENTrFICATlON OF DEI'OSrr . unher deDos~ts arc iden~it~ed on an additional SDe::
Name o~ depositary Institutlon
The National Collections of Industrial and Ma~ine B2ct~-ri2 Llm-ted (,'~-T~3)
Addrtss of depositary Insututlon (irciualn~ poslal coa~ ana crJ Sntry)
23 St. Mach2- D~ive
Aberdeen
Scotland
AB2 lRY
United Kingdom
D~tc of dcposi~ Acc ssion s~iumDe.
16, ~ JA2 Y 1'7~ 1 s~~l ~B 40~
C. ADDITIONAL INDICATIONS (I, ovr l~lanS~ ir'nr~s nrq r' ~ ~lis intosrnation is ~on~ ed on an sd~ition~l sn::t O
In respect of those design2tions in which a European paten~ is soug~.~, 2n~ 2ns
other designated st2te haYing equivalent legisl2,ion, a sam~;e or the de?os-ned
microorganism ~ be made available until the publication o~ the men.~on c- thegrant of the Europe2n paten~ or until the date on wh~_h the apDlic2t~c,. h2s be~refused or ~ithdrawn or is deemed to S~e with~lra~.., or.; ~v the issue c. su~:- 2
sample to an exDe-~ nomin2ted by the person resSues. ng the s~mple. (?~u~ ~(4)
EPC).
D. DEsIGNATED STATES FOR WHIC~ INDICATIONS ARE MADE fifllu inr;iCaLiOnSarcnof forall acsl-rn~ ;la~
E. SEPARATE F~ 5~G OF Ih'DICAllONS ;Icovco~ar~iinolaDD~I.aolc:
rncinn~ on~ilslecoc:oww~ suom~ o~n inSerna~lOnair~ureaulaS-~s~ ul_~c~c~na~:rco;lrc~nsJC-:~Or~ r
~r umoer orLi CDoSI ~ )
For r~lvlng Os~ " e use oniy ro. Ir.sernaslon.i 3ureau use oni-,
SS~ rni5 shees waS f~ceivec Yvith tr;e inres~ationzl appiiCaliOn O rnis snce~ Yr.Ss re~ived bv t;~se Internationai Bu-~u c..:
_ \
Autnonzc~i osrilc~ r c~,~ Autnorszec os-tlc:.
. vzSn~
i c~.. p~,~Orl 3C (; u ! ~ I C~
