New Zealand No 303970
International No PCT/EP96/01009
TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION
Priority dates 17 03 1995,
Complete Specification Filed 11 03 1996
Classification (6) C12S3/02, A61K38/47, A23L1/29
Publication date 29 September 1999
Journal No 1444
new zealand patents act 1953
COMPLETE SPECIFICATION
Title of Invention Aspergillus arabinofuranosidase
Name, address and nationality of applicant(s) as in international application form
DANISCO A/S, Langebrogade 1, P O Box 17, DK-1001 Copenhagen K, Denmark
New Zealand No 303970
International NoPCT/EP96/01009
NEW ZEALAND PATENTS ACT 1953
complete specification
Title of Invention Aspergillus arabinofuranosidase
Name, address and nationality of applicant(s) as in international application form
DANISCO A/S, Langebrogade 1, P 0 Box 17, DK-1001 Copenhagen K, Denmark
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 arabmoxylan 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 filamentous fungus (such as Aspergillus Niger) 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 ammo 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 alternative, it may be desirable to isolate the resultant 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 organism, such as a filamentous fungus or a crop plant, to express non-plant genes, such as for the same purposes
Also, it may be desirable to use an organism, such as a filamentous fungus or a crop plant, to express mammalian genes Examples of the latter products include interferons, insulin, blood factors and plasminogen activators It is also desirable to use microorganisms, such as filamentous fungi, to prepare products from GOIs by use of promoters 30 that are active in the micro-organisms
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Fruit and vegetable cell walls largely consist of polysaccharide, 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, 5 1975, P Albersheim, Plant Biochem 3rd Edition (Bonner and Vamer), 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 10 the plant cell wall), hemicellulose (comprising various B-xylan chains) and pectic substances (consistmg of galacturonans and rh.imnogalacturonans, arabtnans, and galactans and arabmogalactans) From the standpoint of the food industry, the pectic substances, arabmans m 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 arabman A review of arabtnans may be found m EP-A-0506190 According to this document, arabmans consist of a mam cham of a-(l-»5) groups linked to one another Side chains are linked ce-(l-+3) or sometimes a-(l-*2) to the main a-(l-*5)-L-arabinan backbone In apple, for example, one third of the 20 total arabinose is present in the side chains The molecular weight of arabman is normally about 15 kDa
Arabmans are degraded by enzymes collectively called arabmases In this regard, arabinan-degradmg activity is the ability of an enzyme to release arabinose residues, 25 either monomers or oligomers, from arabman backbones or from arabman-contauung side chains of other hemicellulose backbone structures such as arabmogalactans, or even the release of arabinose monomers via the cleavage of the l-*6 linkage between the terminal arabmofuranosyl unit and the intermediate glucosyl unit of monoterpenyl a-L-arabmofuranosyl glucosides
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The activity of the arabman degrading enzymes of EP-A-0506190 include a) the ability to cleave (l-»2)-a-L-arabinosidic linkages, b) the ability to cleave (l-»3)-o;-L-arabinosidic linkages, c) the ability to cleave (l-»5)-o:-L-arabinosidic linkages, d) the ability to cleave the l-»6 linkage between the terminal arabmofuranosyl unit and the intermediate glucosyl 5 unit of monoterpenyl a-L-arabinofuranosyl glucosides
Arabinan-degradmg enzymes are known to be produced by a variety of plants and microorganisms, among these, fungi such as those of the genera Aspergillus, Corticium, Rhodotorula (Kaji,A (1984) Adv Carbohydr Chem Biochem . 42, 383). Dichotomitus 10 (Brillouet et al (1985) Carbohydrate Research, 144, 113), Ascomycetes and Basidomycetes (Sydow, G (1977) DDR Patent Application No 124,812)
Ajiother 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 arabmoxylan, in which arabinose side chains are attached to a backbone of xylose residues
Arabinoxylans are carbohydrates found m 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 Arabmoxylans, Xylans and Xylanases pages 51-67, edited by J Visser published by Elsevier Science Publishers)
Typically, arabinoxylans comprise a xylose backbone linked together via /3-1,4- bonds The xylose backbone is substituted with L-arabinose residues which are linked via a-l 25 bonds to the 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 coumanc acid The degree and kmd of substitution depends on the source of the 30 particular arabmoxylan
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Arabmoxylans are found in cereal cell wall where they are part of the secondary cell wall Arabmoxylans form about 3 % of wheat flour - part of it is water soluble (WSP), pan of it is water insoluble (WIP)
Despite the fact that the arabinoxylans amount to only about 3 % of wheat the importance of the arabmoxylan fraction is much higher This is because the arabmoxylans 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 m a dough despite the fact that they amount to only 3 % of the dry matter When arabmoxylans 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 mampulated using enzymes that degrade arabmoxylans 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 arabmoxylans
Enzymes that only cleave the arabinose side chains from the xylan backbone of 20 arabmoxylan are, for the purposes of this application, collectively called arabmoxylan degrading enzymes
In feeds based on cereals, arabmoxylans m the cereals can mcrease the viscosity of the fluids in the intestines of the ammals 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 arabmoxylan (such as xylanases) to the feed to avoid indigestion and to mcrease the nutritive value of the feed However, some enzymes that degrade the arabinoxylans (especially some of the xylanases) require the presence of unsubstituted 30 backbones and so their activity can be limited
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Further discussions on arabmoxylans can be found m Xylans and Xylanases (1992, edited by J Visser published by Elsevier Science Publishers)
An arbinoxylan degrading enzyme is (l,4)-fl-D-arabinoxylan arabinofuranohydrolase 5 (AXH), as described by Kormelink et al 1991 (Kormehnk, FJM , Searle-Van Leeuwen M J F , Wood T M , Voragen, A G J (1991) Purification and characterization of a (1,4)-/3-D-arabmoxylan arabinofuranohydrolase from Aspergillus awamori Appl Microbiol 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 arabmoxylans, preferably by use of recombinant DNA techniques
The present invention seeks to provide an enzyme having arabmoxylan degrading activity, 15 preferably wherem the enzyme can be prepared in certain or specific cells or tissues, such as in just a specific cell or tissue, of an organism, 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 m specific cells or tissues, such as in certain or specific cells or tissues, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant
In ar* tion, the present mvention seeks to provide a promoter that is capable of directing 25 expression of a GOI, such as a nucleotide sequence codmg for the enzyme according to the present invention, preferably in certain specific cells or tissues, such as m just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant Preferably, the promoter is used in Aspergillus wherem the product encoded by the GOI is excreted from the host 30 organism into the surrounding medium
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Furtheraiore, the present invention seeks to provide constructs, vectors, plasnuds, cells, tissues, organs and organisms 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 organism, typically a filamentous fungus, preferably of the 5 genus Aspergillus, or even a plant
According to a first aspect of the present invention there is provided an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics a MW of 33,270 D ± 50 D, a pi value of about 3 7, arabmoxylan degrading activity, a pH 10 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 wherem the enzyme is capable of cleaving arabinose from the xylose backbone of an arabmoxylan
According to a second aspect of the present invention there is provided an enzyme having the sequence shown as SEQ ID No 1 or a variant, homologue or fragment thereof
According to a third aspect of the present invention there is provided an enzyme coded by the nucleotide sequence shown as SEQ ID 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
According to a fifth aspect of the present invention there is provided a nucleotide sequence having the sequence shown as SEQ ID No 2 or a variant, homologue or fragment thereof or a sequence complementary thereto
According to a sixth aspect of the present invention there is provided a promoter having 30 the sequence shown as SEQ ID No 3 or a variant, homologue or fragment thereof or a sequence complementary thereto
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7
According to a seventh aspect of the present invention there is provided a terminator having the nucleotide sequence shown as SEQ ID No 13 or a variant, homologue or fragment thereof or a sequence complementary thereto
According to an eighth aspect of the present invention there is provided a signal sequence having the nucleotide sequence shown as SEQ ID 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, wherem 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 arabmoxylan
According to an eleventh aspect of the present invention there is provided a combination of enzymes to degrade an arabmoxylan, the combination comprising an enzyme accordmg to the present invention and a xylanase
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 arabmoxylan or for controlling the expression thereof or for controlling the expression of another GOI
Accordmg to a thirteenth aspect of the present invention there is provided a signal sequence having the sequence shown as SEQ ID 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 mvention m the manufacture of a medicament or foodstuff to reduce or prevent indigestion and/or increase digestibility and/or mcrease nutrient absorption
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According to a fifteenth aspect of the present invention there is provided an arabinofuranosidase enzyme having arabmoxylan degrading activity, which is immunologically 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 intermediate m xylose metabolism
Accordmg to a seventeenth aspect of the present invention there is provided a process of reducing the viscosity of a branched substrate wherem 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 mvention as a viscosity modifier
According to a further aspect of the present mvention there is provided the use of the enzyme of the present mvention to reduce the viscosity of pectin
Other aspects of the present mvention include constructs, vectors, plasmids, cells, tissues, organs and transgenic organisms compnsmg the aforementioned aspects of the present invention
Other aspects of the present mvention include methods of expressing or allowing 25 expression or transforming any one of the nucleotide sequence, the construct, the plasmid, the vector, the"cell, the tissue, the organ or the organism, as well as the products thereof
Additional aspects of the present invention include uses of the promoter for expressing GOIs in culture media such as a broth or m a transgenic organism
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Furtlier 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 ID No 2 or a variant, homologue or fragment thereof or a sequence complementary thereto
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 vanant, homologue or fragment thereof or a sequence complementary thereto
Preferably, the promoter comprises the 100 bps sequence from the Xma 111 to the BamHl sites
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 transgenic organism is a plant
Preferably, in the use, the enzyme is used m combination with a xylanase, preferably an endoxylanase
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Highly preferred embodiments of each of the aspects 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 orgamsm or the transgenic orgamsm, the promoter is present in combination with at least one GOI
Preferably the promoter and the GOI are stably incorporated within the transgenic 10 orgamsm's genome
Preferably the transgenic 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, wherem the enzyme has the following characteristics a MW of 33,270 D ± 50 D, a pi value of about 3 7, arabmoxylan 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 20 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 wherem the enzyme is capable of cleavmg arabinose from the xylose backbone of an arabmoxylan, wherein the enzyme has the sequence shown as SEQ ID No 1 or a variant, homologue or fragment thereof
Another highly preferred embodiment is an enzyme obtainable from Aspergillus, wherem the enzyme has the following characteristics a MW of 33,270 D ± 50 D, a pi value of about 3 7, arabmoxylan 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 temperamre optima of from about 40°C to about 60°C (more especially from about 45°C to about 30 55°C, more especially about 50°C), and wherem the enzyme is capable of cleaving arabinose from the xylose backbone of an arabmoxylan, wherein the enzyme is coded by the nucleotide sequence shown as SEQ ID No 2 or a variant, homologue or fragment
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thereof or a sequence complementary thereto
The advantages of the present invention are that it provides a means for preparing an arabinofuranosidase enzyme having arabmoxylan degrading activity and the nucleotide 5 sequence coding for the same 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 mvention can affect the viscosity of ground cereals, such as dough, to ease the handling 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 arabmoxylan and thus increases the nutritive value of the feed In addition, it reduces the viscosity of the arabmoxylan in the intestine of the animals 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 mvention and the xylanase have a surprising and unexpected synergistic effect with each other
In this regard, the enzyme of the present mvention 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 mcreased because the enzyme of the present mvention provides a polysaccharide substrate having fewer substituted 25 groups
The present invention therefore provides an enzyme having arabmoxylan degrading activity wherein the enzyme can be prepared in certain or specific cells or tissues, such as in just a specific cell or tissue, of an orgamsm, typically a filamentous fungus, 30 preferably of the genus Aspergillus, such as Aspergillus niger The enzyme may even be prepared m a plant
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More m pamcuiar, the enzyme of the present invention is capable of specifically cleaving arabinose from the xylose backbone of arabmoxylan
The arabinofuranosidase of the present invention is different from the 5 arabinofuranosidases previously known In this regard, the previous described arabmofuranosidases - such as those of EP-A-0506190 - are characterised by their ability to degrade unbranched arabinan, and are assayed using p-rutrophenyl-arabinoside
The arabinofuranosidase of the present invention does not degrade unbranched arabinan, 10 and only a minor activity is seen on mtrophenyl-arabmoside In contrast, the arabinofuranosidase of the present invention is useful for degrading arabmoxylan Therefore, the arabinofuranosidase of the present invention is quite different from the previous isolated arabinofuranosidases
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 organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger The GOI may even be expressed in a plant
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 m certain specific ~ells or tissues, such as m just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant Preferably, the promoter 25 is used in Aspergillus wherein the product encoded by the GOI is excreted from the host organism mto the surrounding medium The promoter may even be tailored (if necessary) to express a GOI in a plant
The present invention also provides constructs, vectors, plasmids, cells, tissues, organs 30 and organisms comprising the GOI and/or the promoter, and methods of expressing the same, preferably m specific cells or tissues, such as expression in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus
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Aspergillus, 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) ammo acid from or to the sequence providing the resultant ammo acid sequence has arabmoxylan degrading activity, preferably having at least the same activity of the enzyme shown in the sequence listings (SEQ ID No 1 or 12) In particular, the term "homologue" covers homology with respect to structure and/or function providing the resultant enzyme has arabmoxylan 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 m the attached sequence listings More preferably there is at least 95 %, more preferably at least 98 %, homology to SEQ ID NO 1 shown m the attached sequence listings
The terms "variant", "homologue" or "fragment" m 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 arabmoxylan degrading activity, preferably having at least the same activity of the 20 enzyme shown in the sequence listings (SEQ ID 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 havmg arabmoxylan 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 m the 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 ftom or to the sequence providing the resultant nucleotide sequence has the ability to act as a promoter m an expression system - such as the transformed cell or the transgenic orgamsm accordmg 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 5 attached sequence listings More preferably there is at least 95%, more preferably at least 98%, homology to SEQ ID NO 3 shown in the attached sequence listings
The terms "variant", "homologue" or "fragment" m relation to the terminator or signal nucleotide sequences include any substitution of, variation of, modification of, 10 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 terminator or codes for an ammo acid sequence that has the ability to act as a signal sequence respectively m an expression system - such as the transformed cell or the transgenic organism according to the present invention In particular, the term "homologue" covers 15 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 m the attached sequence listings More preferably there is at least 95%, more 20 preferably at least 98%, homology to SEQ ID NO s 13 and 14 (respectively) shown m the attached sequence listings
The terms "variant", "homologue" or "fragment" in relation to the signal ammo acid sequence include any substitution of, variation of, modification of, replacement of, 25 deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant sequence haslhe ability to act as a signal sequence m 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 providmg the resultant nucleotide sequence has the ability to act as or 30 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 attached sequence listings More preferably there is at least
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95%, more preferably at least 98%, homology to SEQ ID NO 15 shown in the attached sequence listings
The above terms are synonymous with allelic variations of the sequences
The term "complementary" means that the present invention also covers nucleotide sequences that can hybridise to the nucleotide sequences of the coding sequence or the promoter sequence, respectively
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 mvention
The term "construct" - which is synonymous with terms such as "conjugate", "cassette" 15 and "hybrid" - includes a GOI directly or indirectly attached to a promoter An example of an indirect attachment is the provision of a suitable spacer group such as an mtron sequence, such as the S/zi-mtron or the ADH mtron, intermediate the promoter and the GOI The same is true for the term "fused" m relation to the present invention which includes direct or indirect attachment 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 m their natural environment A highly preferred embodiment is the or a GOI being operatively linked to a or the promoter
The construct may even contam or express a marker which allows for the selection of the genetic construct m, for example, a filamentous fungus, preferably of the genus Aspergillus, 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 resistance -eg resistance to G418, hygromycin, bleomycin, kanamycm and gentamycin
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The term "vector" includes expression vectors and transformation vectors
The terra "expression vector" means a construct capable of in vivo or in vitro expression
The term "transformation vector" means a construct capable of being transferred from one species to another - such as from an E coh plasmid to a filamentous fungus, preferably of the genus Aspergillus It may even be a construct capable of being transferred from an E coh plasmid to an Agrobactenum to a plant
The term "tissue" includes tissue per se and organ
The term "orgamsm" m relation to the present invention includes any orgamsm 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, wherem the promoter can allow expression of a GOI and/or wherein the nucleotide sequence according to the present mvention can be expressed when present m the orgamsm
Preferably the orgamsm 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 orgamsm that compnses the promoter accordmg to the present invention and/or the nucleotide sequence codmg for the enzyme according to the present invention and/or products 25 obtained therefrom, wherem the promoter can allow expression of a GOI and/or wherem the nucleotide sequence according to the present invention can be expressed within the orgamsm Preferably the promoter and/or the nucleotide sequence is (are) incorporated in the genome of the orgamsm
Preferably the transgenic orgamsm 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 organism comprising any one of, or combinations of, the promoter according to the present invention, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present invention, vectors according to the present 5 invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present mvention or the products thereof For example the transgenic orgamsm 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 compnse 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 preferred embodiment, the transgenic orgamsm does not comprise the combination of the promoter according to the present invention and the nucleotide 15 sequence coding for the enzyme accordmg to the present invention, wherein both the promoter and the nucleotide sequence are native to that organism and are in their natural environment Thus, in these highly preferred embodiments, 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 us 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 m its natural environment
The term "promoter" is used in the normal sense of the an, 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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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 necessarily 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 a-Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 10 promoter or the a-Amy 637 promoter) as described m our co-pending UK patent application 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 15 the a-Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the a-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 m Figure 2
Preferably, the promoter is the promoter of the present mvention
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 Pnbnow Box or a TATA box The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the GOI For example, suitable other sequences include the Shi-mtron or an ADH mtron Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements
Also, suitable elements to enhance transcnption or translation may be present An example of the latter element is the TMV 5' signal sequence (see Sleat Gene 217 [1987]
9
19
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 5 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
The present mvention also encompasses the use of promoters to express a nucleotide sequence coding for the enzyme according to the present invention, wherein a pan of the promoter is inactivated but wherein the promoter can still function as a promoter Partial mactivation of a promoter in some instances is advantageous
In particular, with the Amy 351 promoter mentioned earlier it is possible to inactivate a pan of it so that the partially inactivated promoter expresses GOIs in a more specific manner such as m just one specific tissue type or organ
The term "inactivated" means paraal mactivation in the sense that the expression pattern
of the promoter is modified but wherem the partially inactivated promoter still functions as a promoter However, as mentioned above, the modified promoter is capable of expressing a GOI in at least one (but not all) specific tissue of the original promoter One such promoter is the Amy 351 promoter descnbed above
Examples of partial mactivation include altering the folding pattern of the promoter sequence, or bindmg 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
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 catabolite repression, and thus abolish the catabolite repression of the native promoter
The term "GOI" with reference to the present mvention 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 catabohc processes The GOI may code for an agent for introducing or 0 increasing pathogen resistance The GOI may even be an antisense construct for modifying the expression of natural transcripts present m 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 humans
For example, the GOI could code for a pharmaceutically active protein or enzyme such as any one of the therapeutic compounds insulin, interferon, 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-nutntive factors and plant proteins that have a more desirable amino acid composition (eg 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, thaumatm and a-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 glucanase
The GOI can be the nucleotide sequence coding for the a-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 a-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 m Figure 4 The
WO 96/29416 PCT/EP96/01009
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 a-5 glucan lyase 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 orgamsm 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 Leach,J 1987 (Recombinant 15 DNA in filamentous fungi Progress and Prospects CRC Cnt Rev Biotechnol 6 357-393), Davis R W 1994 (Heterologous gene expression and protein secretion in Aspergillus In Martinelh S D , Kinghorn J R ( Editors) Aspergillus 50 years on Progress m industrial microbiology vol 29 Elsevier Amsterdam 1994 pp 525-560), Ballance,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 Martinelh S D , Kinghorn J R ( Editors) Aspergillus 50 years on Progress m industrial microbiology vol 29 Elsevier Amsterdam 1994 pp 641-666) However, the following commentary provides 25 a summary of those teachings for producing transgenic Aspergillus accordmg to the present mvention
Filamentous fungi have during almost a century been widely used m industry for production 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 citnc 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 m 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 a-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 m 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'mtroduced 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 m at least 30 some Aspergilh Such a fusion leads to cleavage in vivo resulting m 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 intracellular^ if the GOI is not fused to a signal sequence Such proteins will accumulate 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 accumulate extracellulary
With regard to product stability and host strain modifications, some heterologous proteins are not veiy 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 filamentous fungi, several transformation protocols have been 15 developed for many filamentous fungi (Ballance 1991, ibid) Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca2+ ions The transformed 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, irpC, maD and pyrG, antibiotic 20 resistance markers such as benomyl resistance, hygromycm resistance and phleomycm resistance A very common used transformation marker is the amdS gene of A mdulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source
Even though the enzyme, the nucleotide sequence coding for same and the promoter of the present invention are not disclosed m EP-B-0470145 and CA-A-2006454, those two documents 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 teachings are now included in the following commentary
The basic principle m the construction of genetically modified plants is to insert genetic information m the plant genome so as to obtain a stable maintenance 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 Potrylcus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42 205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27)
Thus, in one aspect, the present mvention 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 organism, 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 m further detail m 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 tvmefaciens or a Ri plasmid from Agrobactenum rhizogenes An et al (1986), Plant Physiol 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
Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above A non-limiting example of such a Ti plasmid is pGV3850
The promoter or nucleotide sequence or construct of the present invention should 30 preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential
WO 96/29416 PCT/EP96/01009
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 which contains the sequences 5 necessary to infect the plant (e g the vir region) and at least one border pan 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 m 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 m a microorganism m 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 microorganisms having the above properties may be used When a vector of a vector system as defined above has been constructed in E coh, it is transferred, if necessary, into a suitable Agrobacterium strain, e g Agrobacterium tumefaciens The Ti-plasmid harbouring the promoter or nucleotide 20 sequence or construct of the mvention is thus preferably transferred into a suitable Agrobacterium strain, eg 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 m E coll and a marker which allows a selection of the transformed cells The vectors contain for example pBR 322, pUC senes, Ml3 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 m the vector The contained plasmid is used for the transformation in E coh The E coh 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 connected 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 accordmg to the present invention in the plants the presence and/or insertion of further DNA sequences may be necessary If, for example, for the transformation the 10 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 flanking areas of the introduced genes, can be connected The use of T-DNA for the transformation of plant cells has been intensively studied and is descnbed m EP-A-120516, Hoekema, m The Binary Plant "Vector System Offset-drukkenj Kanters B B , Alblasserdam, 1985, 15 Chapter V, Fraley, et al , Cnt Rev Plant Sci , 4 1-46, and An et al , EMBO J (1985) 4 277-284
Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is descnbed m Butcher D N et al (1980), Tissue Culture 20 Methods for Plant Pathologists, eds D S Ingrams and J P Helgeson, 203-208 For further teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42 205-225) and Chnstou (Agro-Food-Industry Hi-Tech March/April 199417-27) With this technique, infection of a plant may be done on a certain pan or tissue of the plant, l e on a part of a leaf, a root, a stem or another pan of the 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 punctunng the plant with a needle or rubbing the plant with an abrasive The wound is then inoculated with the Agrobacterium The inoculated plant or plant pan is then 30 grown on a suitable culture medium and allowed to develop into mature plants
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When plant cells are constructed, these cells may be grown and maintained m accordance with well-known tissue cultunng methods such as by cultunng the cells m a suitable culture medium supplied with the necessary growth factors such as ammo 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 by subculturmg the shoots on a medium containing the appropriate nutrients, plant 10 hormones, etc
Further teachings on plant transformation may be found m EP-A-0449375
In summation, the present invention provides an arabinofuranosidase enzyme having 15 arabmoxylan 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 terminator and signal sequences for the same
The following sample was deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1RY on 16 January 1995
E coli containing plasmid pB53 1 {i e E coh DH5a-
pB53 1} The deposit number is NCIMB 40703
The present invention will now be descnbed by way of example
In the following Examples reference is made to the accompanying figures m which
WO 96/29416 PCT/EP96/01009
28
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 5 NCIMB 40703,
Figure 12 is a schematic diagram of deletions made to the promoter of the present mvention,
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,
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.
The following text discusses the use of inter aha recombinant DNA techniques, General teachings of recombinant DNA techniques may be found in Sambrook, J , Fntsch, E F , Maniatis T (Editors) Molecular Cloning A laboratory manual Second edition Cold
#
29
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 arabinofuranosidase
Aspergillus niger 3M43 was grown in medium containing 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 25X100mm Q-SEPHAROSE (Pharmacia) high Performance column, equilibrated with 20 mM Tns, HC1 pH 7 5, and a 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 containing the arabinofuranosidase were combined and desalted using a 50x200 mm G-25 SEPHAROSE Superfine (Pharmacia) The column was eluted with distilled water
After desalting 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
The fractions containing arabinofuranosidase were combined and desalted and concentrated as descnbed above The combined fractions were loaded on a 16X100 mm Phenylsepharose High Performance column (Pharmacia), equilibrated with 50 mM Phosphate buffer pH 6 0, containing 1 5 M (NH4)2S04 A gradient where the (NH4):S04 concentration was vaned from 1 5 - 0 M was applied and the eluate collected m fractions 30 The fractions containing Arabinofuranosidase were combined The purity of the arabinofuranosidase was evaluated by SDS-PAGE usmg the Phast system gel (Pharmacia)
Characterization
The molecular weight of the purified arabinofuranosidase was determined by mass spectrometry using laser desorption technology The MW of the arabinofuranosidase was 5 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 Davnl (1992) Electrophoresis 13 pp 97-99 Activity Studies
Activity of AbfC as a function of water soluble pentosan (WSP) concentrations (mg/ml) was determined The results are shown in Figure 21 The results show that AbfC activity reached maximum at substrate concentration of 8 mg/ml WSP
r pH Activity Studies
The effect of pH on the activity of the arabinofuranosidase of the present invention was mvestigated usmg water soluble pentosan (10 mg/ml) from, wheat as a substrate in 50 mM citric acid sodium phosphate buffer The incubation time was 15 minutes The arabinofuranosidase of the present invention was observed to have a wide pH optima 25 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
Temperature Activity Studies
The effect of temperature on the activity of the arabinofuranosidase of the present mvention was investigated usmg water soluble pentosan (10 mg/ml) from wheat as a substrate m 50 mM sodium acetate at a pH of 5 0 The incubation time was 15 minutes
WO 96/29416 PCT/EP96/01009
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 endoprotemase 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 Matsudaira P (Editor) A practical Guide to Protein and Peptide Purification for Microsequencmg Second Edition Academic Press, San Diego 1993 pp 45-73)
Freeze dried /3-arabinofuranosidase (0 4 nig) was dissolved in 50 n\ of 8M urea, 0 4 M NH4HCO3, pH 8 4 After overlay with N2 and addition of 5 p\ of 45 Mm DTT, the protein was denatured and reduced for 15 min at 50°C under N2 After cooling to RT, 5 (il of 100 Mm lodoacetamide was added for the cysteines to be denvatised for 15 mm at RT in the dark under N2 Subsequently, 90 /il of water and 5 ng of endoprotemase 20 Lys-C in 50 y\ of 50 Mm Tncine and 10 mM EDTA, pH 8 0, was added and the digestion was carried out for 24h at 37°C under N; The resulting peptides were separated by reversed phase HPLC on a YYDAC C18 column (0 46 x 15 cm, 10 nm, The Separations Group, California) usmg solvent A 0 1 % TFA in water and solvent B, 0 1 % TFA in acetomtrile Selected peptides were rechromatographed on a Develosil CI 8 25 column (0 46 x 10 cm, 3/xm) using the same solvent system prior to sequencmg 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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SEQ ID No 7 SEQ I.D. No, 8
Isolation of a PCR clone of a fragment of the gene
PCR primers were synthesised using an Applied Biosystems DNA synthesiser model 392 In this regard, PCR primers were synthesized from one of the found peptide sequences, namely SEQ ID No 5 The primers were
One pnmer from EMTAQA (reversed)
SEQ ID NO 9 GCY TGN GCN GTC ATY TC
17 mer 64 mix
One pnmer from MIVEAIG
SEQ ID NO 10 ATG ATH GTN GAR GCN ATH GG
mer 288 mix
PCR amplification was performed with 100 pmol of each of these primers in 100 /ul reactions using Amphtaq polymerase (PERKIN ELMER) The following program was
STEP TEMP TIME
1
94°C
2 min
2
94°C
1 mm
3
55°C
2 nun
4
72°C
2 mm
72°C
nun
6
°C
SOAK
Steps 2-4 were repeated for 40 cycles
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PCR reactions were performed on a PERKIN ELMER DNA Thermal Cycler
A 100 bp amplified fragment was isolated and cloned into a pT7-Blue T-vector, according to the manufacturers instructions (Novagen)
Isolation of A niger genomic DNA
lg of frozen A niger mycelium was ground in a mortar under liquid nitrogen Following evaporation of the nitrogen cover, the ground mycelium was extracted with 15ml of 10 an extraction buffer (lOOmM Tns Hcl, pH 8 0, O 50mM EDTA, 500mM NaCl, lOmM B-mercaptoethanol) containing 1ml 20% sodium dodecyl sulphate After mcubation at 65°C for 10 min 5ml 5M KAc pH 5 0, was added and the mixture further incubated, after mixmg, on ice for 20 mins After extraction, the mixture was centrifuged for 20 mms and the supernatant mixed with 0 6 vol isopropanol to precipitate the extracted 15 DNA After further centnfiigation for 15 mms the DNA pellet was dissolved in 0 7 ml TE (lOmM Tns, HC1 pH 8 0, ImM EDTA) and precipitated with 75 ixl 3M NaAc, pH 4 8, and 500 pi isopropanol
After centnfugation the pellet was washed with 70% ETOH and dried under vacuum 20 The DNA was dissolved m 200 pi TE and stored at -20°C
Construction of a library
pg genomic DNA was partly digested with Tsp509I, which gives ends which are 25 compatible with EcoKL ends The digested DNA was separated on a 1 % agarose gel and fragments of 4-10 kb was purified A XZAPII EcoRl/ClAP kit from Stratagene was used for library construction according to the manufacturers instructions 2 pi of the ligation (totally 5 pi) was packed with Gigapack Gold II packing extract from Stratagene The library contained 650,000 independent clones
Screening of the library
34
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 detected on both sheets The gene was sequenced, and the found sequence showed that all of the peptides sequenced were coded by the found sequence
Sequence information
SEQ ID No 12 presents the promoter sequence, the enzyme coding sequence, the terminator sequence and the signal sequence and the amino acid sequence of the enzyme of the present invention
Arabinofuranosidase assay
Two different arabmoxylan preparations from wheat flour, Wheat Insoluble Pentosan (WIP) and Wheat soluble Pentosan (WSP), were degraded with the arabinofuranosidase 20 enzyme of the present mvention alone and m combination with an endoxylanase purified from A niger The assays were done on 1 % substrate m 50 Mm 50 Mm Na-acetate buffer at pH 5 0 The reactions were performed 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 supernatants were collected, dried under 25 vacuum and resuspended in 0 5 ml distilled water The samples were diluted 1 1 in water and analysed on a"Chromopack Carbohydrate Pb column (300X7 8 mm, cat 29010) using Shimadzu C-R4A Chromatopac HPLC system usmg a Shimadzu 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 xylotnose, 0 48 mg/ml xylobiose, 0 60 mg/ml xylose and 0 58 mg/ml L-arabraose The peaks were identified and quantified usmg 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 5 0 Values are expressed in mg/ml
xylotnose 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 m 50 Mm Na-acetate buffer pH 5 0 Values are expressed in mg/ml
xylotriose xylobiose xylose arabinose no enzyme
0 0
00
0 0
0 0
abfC
00
00
0 0
0 30
xyl
■0*08
0 14
0 0
0 0
abfC + xyl
0 42
0 47
0 0
0 42
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 synergistically 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, X2- xylobiose, X3- xylotnose, 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 these experiments provide two important findings
First the enzyme of the present invention liberates arabinose, in particular L-arabmose, from arabmoxylan
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 m a synergistic fashion
Induction of the AbfC gene: Identification of inducers
The regulation of transcription of the AbfC encoding gene of Aspergillus niger was studied using a strain containing a fusion of the AbfC promoter to the /^-glucuronidase encoding gene (uid A) of E coh
GUS producing transformants were grown on different carbon sources and assayed both qualitatively and quantitatively 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
xyhtol
1 49
arabinose
6 66
arabitol
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
arabmogalactan
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 carbon catabolite repression However, unlike all the published promoters for 25 arabinofuranosidases, which are induced by arabinose and arabitol, the AbfC promoter of the present mvention is regulated strongly by the intermediates in xylose metabolism Accordingly, the present invention also covers an arabinofuranosidase promoter wherein the promoter is inducible by an intermediate m 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 m the 5' upstream region of the gene was constructed (see Figure 12) The E coh uid A gene was used as the reporter gene and a qualitative GUS assay was performed
The results indicated that the truncated AbfC promoter of 590 bp contains sufficient information for the mducibihty of the AbfC gene and its regulation Deletion of 100 bps sequence from the Xma 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 be 15 important but not sufficient to abolish the activity of this promoter All the transformants analysed containing this promoter construct showed very pale blue when tested (+-GUS) This region is as follows
-170 TCATCCAATAT
As seen, this region contains the CCAAT element and is a putative target for a general transcriptional 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
WO 96/29416 PCT/EP96/01009
^ 39
HETEROLOGOUS PROTEIN PRODUCTION USING ASPERGILLUS NIGER TRANSFORMED WITH THE AbfC PROMOTER AND THE AbfC SIGNAL SEQUENCE
Transformation of Aspergillus 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), DaboussuM J , Djebalh,A. , Gerlmger, C , 10 Blaiseau, P L , Cassan, M , Lebrun, M H , Pansot, D , Brygoo.Y 1989 (Transformation of seven species of filamentous fungi using the mtrate reductase gene of Aspergillus nidulans Curr Genet 15 453-456) and Punt, P J , van den Hondel, 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 Schtmmelcultures, 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 20 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 stenle petn dish with 10 ml STC (1 2 M sorbitol, 10 mM Tns Hcl pH 7,5, 50 Mm CaCl2) with 75 mg lysmg enzymes (Sigma L-2265) and 4500 units lyticase (Sigma L-25 8012)
The mycelium is mcubated with the enzyme until the mycelium is degraded and the protoplasts are released The degraded mycelium is then filtered through a stenle 60 /xm mesh filter The protoplasts are harvested by centnfugation 10 mm at 2000 rpm m a 30 swing out rotor The supernatant is discarded and the pellet is dissolved m 8 mil 5 M MgS04, and then centnfuged at 3000 rpm for 10 mm
WO 96/29416 PCT/EP96/01009
The upper band, containing the protoplasts is transferred to another tube, using a transfer pipette and 2 ml 0 6 M KC1 is added Carefully 5 ml 30% sucrose is added on the top and the tube is centnfuged 15 mm at 3000 rpm
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 pi protoplast solution (106-107 protoplasts) are mixed with 10 pi DNA solution containing 5- 10 pg DNA and incubated 25 min at room temperature Then 60 % PEG-4000 is carefully added m portions of 200 pi, 200 pi and 800 pi The mixture is incubated 20 mm at room temperature 3 ml STC is added to the mixture and carefully mixed The mixture is centnfuged 3000 rpm for 10 min
The supernatant is removed and the protoplasts are solubilized in the remaining of the supernatant 3-5 ml topagarose is added and the protoplasts are quickly spread on selective plates
AbfC promoter and heterologous gene expression
The expression vector pXP-Amy (Figure 13) contains the 2 1 kb a-amylase encoding gene from Thermomyces lanuginosus cloned downstream of the AbfC promoter (2 1 kb) and upstream of the Xylanase A terminator This vector together with the hygromycm
gene as a selectable marker was used for co-transformation experiments to test the functionality of the AbfG-promoter
The best transformant v/as accumulated in shake flask experiments at least 1 gram per litre of a-amylase in the culture media Starch degrading activity was then detected
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 m protein secretion
An expression construct containing the signal peptide of the AbfC gene translationally fused to the mature a-amylase from T lanugmosus was prepared and expression of this 5 construct in the production strains was observed In this regard, the translauonal 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 hygromycm resistance marker The endogenous signal 10 peptide directed the secretion of amylase out of the cell
Substrate Specificity of AbfC Protein
The substrate specificity of the punfied AbfC was determined using arabinose contaimng 15 hemicelluloses arabinoxylans from wheat, oat and larch, branched and debranched arabmans, arabmogalactan, sugar beet pectin, and xyloglucan
The HPLC and HP-TLC results are shown m Figure 16, in which the following abbreviations are used WSP - water-soluble pentosan, WIP - water-msoluble pentosan, 20 AG - arabmogalactan, deB-A - debranched arabinan The standards used were A-arabmose, X- xylose
The results indicate that arabmose is the hydrolysis product from arabmoxylans No hydrolysis products were released from arabmogalactan, debranched arabinan or 25 xyloglucan Arabmose was released as a hydrolysis product from branched arabinan AbfC is therefore a 1,2/1,2 debranchmg enzyme and it has no activity towards linear 1,5 a-linked L-arabinofuranose residues found in debranched arabmans and arabmogalactan This enzyme also releases a product when pectm is used as the substrate It is believed that this product is an arabmose contaimng ferulic acid or an arabmobiose
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42
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 earned 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 /il of AbfC for 20 hours, 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
Accordingly, the present invention covers the use of the enzyme of the present invention to reduce the viscosity of-pectin
ANTIBODY PRODUCTION
Antibodies were raised against the enzyme of the present invention by injecting rabbits with the punfied enzyme and isolating the immunoglobulins from antiserum according to procedures descnbed according to N Harboe and A Ingild ("Immunization, Isolation
WO 96/29416 PCT/EP96/01009
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of Immunoglobulins, Estimation of Antibody Titre" In A Manual of Quantitative Immunoelectrophoresis, 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 York, 1977)
SUMMARY
Even though it is known that Aspergillus niger produces arabinofuranosidases, the present mvention 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 mvention 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 terminator) can be used to express or can be used in the expression of GOIs m 15 organisms, such as in A. niger
The arabinofuranosidase of the present mvention 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-arabmoside
The arabinofuranosidase of the present mvention does not degrade arabinan, and only a minor activity is seen on p-nitrophenyl-arabinoside
In contrast, the arabinofuranosidase of the present invention is useful for degrading arabmoxylan Therefore, the arabinofuranosidase of the present invention is quite different from the previous isolated arabinofuranosidases
More m particular, the enzyme of the present invention is capable of specifically cleaving 30 arabmose from the xylose backbone of arabmoxylan
WO 96/29416 PCI 'EP96/01009
O 44 \
The enzyme of the present mvention 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 arabmoxylans When added to feeds (including silage) for monogastic animals (e g 5 poultry or svvine) which contain cereals such as barlev, wheat, maize, rye or oats or cereal by-products such as wheat bran or maize bran, the enzyme significantly 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-degradmg enzymes may be used to reduce the viscosity of feeds 10 containing arabmans The arabinoxylan-degradmg enzyme may be added beforehand to the feed or silage if pre-soaking or wet diets are preferred
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 mvention is m 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-degradmg enzymes, produced according to the present invention, to the xylanase treatment step should assist m the degradation of an arabman-contaimng hemicellulose backbone and thus facilitate an improved, more efficient removal of both ligmns and terpenoids The application of arabinoxylan-degradmg enzymes should be particularly 25 advantageous in the processing of soft woods m which the hemicellulose backbone contains glucuronic acid—
The enzyme accordmg 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 mvention will be apparent to those skilled in the an without departing from the scope of the mvention
45
SEQUENCE LISTINGS SEQ ID NO 1 ENZYME SEQUENCE
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 296 amino acids
(B) TYPE amino acid (D) TOPOLOGY linear
(n) MOLECULE TYPE protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO 1
Lys Cys Ser Leu Pro Ser 1 5
Ser Tyr Ser Trp Ser Ser Thr Asp Ala Leu Ala Thr Pro Lys Ser Gly
15 20
Trp Thr Ala Leu Lys Asp Phe Thr Asp Val Val Ser Asp Gly Lys His
30 35
lie Val Tyr Ala Ser Thr Thr Asp Glu Ala Gly Asn Tyr Gly Ser Met
40 45 50
Thr Phe Gly Ala Phe Ser Glu Trp Ser Asn Met Ala Ser Ala Ser Lys 55 60 65 70
Thr Ala Thr Pro Tyr Asn Ala Val Ala Pro Thr Leu Phe Tyr Phe Lys
75 80 85
Pro Lys Ser lie Trp Val Leu Ala Tyr Gin Trp Gly Ser Ser Thr Phe
90 95 100
Thr Tyr Arg Thr Ser Gin 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
He Asp Gin Thr Val He Gly Asp Asp Thr Asn Met Tyr Leu Phe Phe 135 140 145 150
Ala Gly Asp Asn Gly Lys lie Tyr Arg Ser Ser Met Ser He Asp Glu 155 160 165
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Phe Pro Gly Ser Phe Gly Ser Gin Tyr Glu Glu lie Leu Ser Gly Ala
170 175 180
Thr Asn Asp Leu Phe Glu Ala Val Gin Val Tyr Thr Val Asp Gly Gly
185 190 195
Glu Gly Asn Ser Lys Tyr Leu Met lie Val Glu Ala lie 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
Trp Thr Ala Gin Ala Ala Ser Glu Asp Lys Pro Phe Ala Ala Lys Pro
235 240 245
Thr Val Ala Pro Pro Gly Pro Lys Thr Leu Ala Met Val Thr Trp Phe
250 255 260
Ala Thr Tnr Leu He Lys Pro *
255 270
47
SEQ ID NO 2
NUCLEOTIDE CODING SEQUENCE
AM
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
TTT
ACT
GAT
GTT
GTC
TCT
GAC
GGC
AM
CAT
ATC
GTC
TAT
GCG
TCC
ACT
ACT
GAT
GM
GCG
GGA
AAC
TAT
GGC
TCG
ATG
ACC
TTT
GGC
GCT
TTC
TCA
GAG
TGG
TCG
MC
ATG
GCA
TCT
GCT
AGC
MG
ACA
GCC
ACC
CCC
TAC
MT
GCC
GTG
GCT
CCT
ACC
CTG
TTC
TAC
ITC
MG
CCG
AM
AGC
ATC
TGG
GTT
CTG
GCC
TAC
CM
TGG
GGC
TCC
AGC
ACA
TTC
ACC
TAC
CGC
ACC
TCC
CM
GAT
CCC
ACC
MT
GTC
AAC
GGC
TGG
TCG
TCG
GAG
MG
GCG
CTT
TTC
ACC
GGA
AM
CTC
AGC
GAC
TCA
AGC
ACC
GGT
GCC
ATT
GAC
CAG
ACG
GTG
ATT
GGC
GAC
GAT
ACG
AAT
ATG
TAT
CTC
TTC
TTT
GCT
GGC
GAC
MC
GGC
MG
ATC
TAC
CGA
TCC
AGC
ATG
TCC
ATC
GAT
GM
TTT
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
MC
AGC
MG
TAC
CTC
ATG
ATC
GTT
GAG
GCG
ATC
GGG
TCC
ACT
GGA
CAT
CGT
TAT
TTC
CGC
TCC
nc
ACG
GCC
AGC
AGT
CTC
GGT
GGA
GAG
TGG
ACA
GCC
CAG
GCG
GCA
AGT
GAG
GAT
MA
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
AM
CCA
TGA
WO 96/29416 PCT/EP96/01009
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SEQ ID NO 3 PROMOTER SEQUENCE
CTGCAGAAGA TGGCAGTCGC CACAGCCGAT CACCCGATCC ATACTGGATG TTGTAACTTG 60
GAGACAGCCT GCAGATGCTC TGATGAAGGT CTGCAAATAG TTCCTGGACC TCGATAGTGA 120
AGTATACCGA TTCGTCAATG TTGTATATCC AGCCACTTTG AAAGTACCAA CTTTTAGTTC 180
GATTGATCAG AATACTTTTG GTGTGTAACA TTGACAAGCC AAATTATCAA TCTCTTCTAC 240
CGGTAAGGTG TCAACTACCC GGCCGAAAGT ACCGGAAGGT CGTGGTGTTT TAAGGTGAM 300
CAACTATCAG GGCGGCAATG TGTCAAAGTA GAACCAGTTT GCTTAGCGCC ATTAGGATCC 360
ACGCCTAGAC CCTTGATGCC CGGGAGTTAT CCGTCCTGTC ACAGCAATTA TTTCCCCGAG 420
TCTACTGCCG AAGAACAGCC ATTGTGGCGT ACTCACGGAA TTACCCACTG TGTAGGGTAG 480
TCTTGAACGC CGTTCTAGAC ACGGCAACGC TCCGGTGGAC GATCGTTTCT GGCTAATGTA 540
CTCCGTAGTT TAGGCAGCAT GCTGATCATC TTCCCCCTAG GGAAAGGCCC CTGAATAGTG 600
CGCCAAAATG AGCTTGAGCA AAGGAATGTT CTTTCTAAGC CAAAGTGAGG GAAATAACCA 660
AGCAGCCCAC TTTTATCCGA AACGTTTCTG GTGTCATCCA ATATGGATAA ATCCCGATTG 720
TTCTTCTGCA CATATCTCTA TTGTCATAAG TGCAACTACA TATATTTGAA CATGGTTTGG 780
TCCTCTTTCC AAGTTATTCG-TTCTCCGTGA CCAGCGATTT CAGCCATTGA TTCTTTTGTT 840 TCTTTCCCCG CGGATAAACT CATACGAAG
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 (n) 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 15 10 15
Ala Thr Pro Lys 20
INFORMATION FOR SEQ ID NO 5
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 41 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear (il) MOLECULE TYPE peptide
(v) FRAGMENT TYPE internal (xi) SEQUENCE DESCRIPTION SEQ ID NO 5
Tyr Leu Met lie Val Glu Ala lie Gly Ser Thr Gly His Arg Tyr Phe 15 10 15
Arg Ser Phe Thr Ala Ser Ser Leu Gly Gly Glu Met Thr Ala Gin Ala
25 30
Ala Ser Glu Asp'tys Pro Phe Xaa Gly 35 40
50
INFORMATION FOR SEQ ID NO 6
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 25 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear (n) MOLECULE TYPE peptide
(v) FRAGMENT TYPE internal (xi) SEQUENCE DESCRIPTION SEQ ID NO 6
Ser lie Trp Val Leu Ala Tyr Gin Trp Gly Ser Ser Thr Phe Thr Tyr 15 10 15
Arg Thr Ser Gin Asp Pro Thr Asn Val 20 25
INFORMATION FOR SEQ ID NO 7
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 30 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear (n) MOLECULE TYPE peptide
(v) FRAGMENT TYPE internal (xi) SEQUENCE DESCRIPTION SEQ ID NO 7
Asp lie Val Tyr Ala Ser Thr Thr Asp Glu Ala Gly Asn Tyr Gly Ser 15 10 15
Met Thr Phe Gly Ala Phe Ser Glu Xaa Ser Asn Met Ala Ser 20 25 30
51
INFORMATION FOR SEQ ID NO B
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 41 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear (n) MOLECULE TYPE peptide
(v) FRAGMENT TYPE internal (xi) SEQUENCE DESCRIPTION SEQ ID NO 8
lie Tyr Arg Ser Ser Met Ser lie Asp Glu Phe Pro Gly Ser Phe Gly 15 10 15
Ser Gin Tyr Glu Glu lie Leu Ser Gly Ala Thr Asn Asp Leu Phe Glu
25 30
Ala Val Gin Val Tyr Thr Val Asp Gly 35 40
INFORMATION FOR SEQ ID NO 9
(D SEQUENCE CHARACTERISTICS
(A) LENGTH 17 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(li) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = "OLIGONUCLEOTIDE"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 6 GCYTGNGCNG TCATYTC
INFORMATION FOR SEQ ID NO 10 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH~"20 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(n) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc « "OLIGONUCLEOTIDE"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 10 ATG ATH GTN GAR GCN ATH GG 20
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
(n) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /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
(n) MOLECULE TYPE DNA (genomic)
(vi) ORIGINAL SOURCE
(A) ORGANISM Aspergillus niger
(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/KE1!4 mat_peptide
(B) LOCATION 948 1754
(xi) SEQUENCE DESCRIPTION SEQ ID NO 12
CTGCAGAAGA TGGCAGTCGC CACAGCCGAT CACCCGATCC ATACTGGATG TTGTAACTTG 60
GAGACAGCCT GCAGATGCTC TGATGAAGGT CTGCAAATAG TTCCTGGACC TCGATAGTGA 120
AGTATACCGA TTCGTCAATG TTGTATATCC AGCCACTTTG AAAGTACCAA CTTTTAGTTC 180
GATTGATCAG AATACTTTTG GTGTGTAACA TTGACAAGCC AAATTATCAA TCTCTTCTAC 240
53
CGGTAAGGTG TCAACTACCC GGCCGAAAGT ACCGGAAGGT CGTGGTGTIT TAAGGTGAAA CAACTATCAG GGCGGCAATG TGTCAAAGTA GAACCAGTTT GCTTAGCGCC ATTAGGATCC ACGCCTAGAC CCTTGATGCC CGGGAGTTAT CCGTCCTGTC ACAGCAATTA TTTCCCCGAG TCTACTGCCG AAGAACAGCC ATTGTGGCGT ACTCACGGAA TTACCCACTG TGTAGGGTAG TCTTGAACGC CGTTCTAGAC ACGGCAACGC TCCGGTGGAC GATCGTTTCT GGCTAATGTA CTCCGTAGTT TAGGCAGCAT GCTGATCATC TTCCCCCTAG GGAAAGGCCC CTGAATAGTG CGCCAAAATG AGCTTGAGCA AAGGAATGTT CTTTCTAAGC CAAAGTGAGG GAAATAACCA AGCAGCCCAC TTTTATCCGA AACGTTTCTG GTGTCATCCA ATATGGATAA ATCCCGATTG TTCTTCTGCA CATATCTCTA TTGTCATAAG TGCAACTACA TATATTTGAA CATGGTTTGG TCCTCTTTCC AAGTTATTCG TTCTCCGTGA CCAGCGATTT CAGCCATTGA TTCTTTTGTT TCTTTCCCCG CGGATAAACT CATACGAAG ATG AAG TTC TTC AAT GCC AAA GGC
Met Lys Phe Phe Asn Ala Lys Gly -26 -25 -20
AGC TTG CTG TCA TCA GGA ATC TAC CTC AH GCA TTA ACC CCC TTT GTT Ser Leu Leu Ser Ser Gly lie Tyr Leu lie Ala Leu Thr Pro Phe Val
-15 -10 -5
AAC GCC AAA TGC TCT CTT CCA TCG TCC TAT AGT TGG AGT TCA ACC GAT Asn Ala Lys Cys Ser Leu Pro Ser Ser Tyr Ser Trp Ser Ser Thr Asp
1 5 10
GCT CTC GCA ACT CCT AAG TCA GGA TGG ACC GCA CTG AAG GAC TTT ACT 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 AAA CAT ATC GTC TAT GCG TCC ACT ACT GAT Asp Val Val Ser Asp Gly Lys His lie Val Tyr Ala Ser Thr Thr Asp
40 45
GAA GCG GGA AAC TAT GGC TCG ATG ACC TTT GGC GCT TTC TCA GAG TGG Glu Ala Gly Asn Tyr Gly Ser Met Thr Phe Gly Ala Phe Ser Glu Trp
50 55 60
TCG AAC ATG GCA TCT GCT AGC AAG ACA GCC ACC CCC TAC AAT GCC GTG 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 AAG CCG AAA AGC ATC TGG GTT CTG GCC Ala Pro Thr Leu Phe Tyr Phe Lys Pro Lys Ser lie Trp Val Leu Ala
80 85 90
TAC CAA TGG GGC TCC AGC ACA TTC ACC TAC CGC ACC TCC CAA GAT CCC Tyr Gin Trp Gly Ser Ser Thr Phe Thr Tyr Arg Thr Ser Gin Asp Pro 95 100 105 110
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ACC AAT GTC AAC GGC TGG TCG TCG GAG AAG GCG CTT TTC ACC GGA AAA 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 AH GGC GAC 1373 Leu Ser Asp Ser Ser Thr Gly Ala lie Asp Gin Thr Val lie Gly Asp
130 135 140
GAT ACG AAT ATG TAT CTC TTC TTT GCT GGC GAC AAC GGC AAG ATC TAC 1421 Asp Thr Asn Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly Lys lie Tyr
145 150 155
CGA TCC AGC ATG TCC ATC GAT GAA TTT CCC GGA AGC TTC GGC AGC CAG 1469 Arg Ser Ser Met Ser lie Asp Glu Phe Pro Gly Ser Pne Gly Ser Gin
160 165 170
TAC GAG GAA ATT CTG AGT GGT GCC ACC AAC GAC CTA TTC GAG GCG GTC 1517 Tyr Glu Glu lie Leu Ser Gly Ala Thr Asn Asp Leu Phe Glu Ala Val 175 180 185 190
CAA GTG TAC ACG GTT GAC GGC GGC GAG GGC AAC AGC AAG TAC CTC ATG 1565 Gin 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 lie Val Glu Ala He 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 Gin Ala Ala Ser Glu
225 230 235
GAT AAA CCC TTC GCA GCA AAG CCA ACA GTG GCG CCA CCT GGA CCG AAG 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 AAA CCA TGA 1757 Thr Leu Ala Met Val Thr Trp Phe Ala Thr Thr Leu He Lys Pro * 255 2-60 265 270
CTGTCGATCC TTGCAACCTC CAGTTGCTCT ATCAGGGCCA TGACCCCCAA CAGCAGTGGC 1817 GACTACAACC TCTTGCCATG GAAGCCGGGC GTCCTTACCT TGAAGCAGTG ACGAGCTTAT 1877 CTTTAGTTGC AGATCGTGTT TCTCCTTTCT TCTTCAAGTA GTTTTAGTGG TGGAAGACAG 1937 CAGAAGGTGG TCATCATCTT AGGCTCAGTT GGGGTGGGCT CCTGCCACGT TTTGTCCATA 1997 GGCTAGTAAT TTGCACGGAA TTCAGTTCAT TGGCAAGGAG TGCGGTACGA ATACCTGTTT 2057 TCACAATAGC AATTAGGCCC AGTAGTTATA CTACGTACTG GAATTGAGTA CTCGTAGTAG 2117 CAAGATTGTT TGCCTCAGAG GGAATGGCCG ACACGTGAGC AAGTCACCTT CATCAGCTAG 2177
55
TCGCGTTCCA CATAGACAAT GGTCCAGCTC CAGAGTGGAA TTTGGGCTAC TTTGAACGAT 2237
GGCCGATTGA ATCGCGCGTC TCCTCAATTG TATTTAACCA CAATAGGCCA GGTATTGGCA 2297
nCACTCTCC GCCTTTGCGG GTGCCGGCAC GAGATGTCTC CTGAAGAAAC TAGGCAACGA 2357
GCAGACTGTG GATATGGGAG ATGGTTGACG ATGTGCTTCT TGGTAAATTT GAAGCCTCCA 2417
GGGCCTCTAG AAAGGCGGGA ATTTAAATCT CAAGTGCCCT AACGTGTCCG ACCACGGTGT 2477
TGATCATCAT TCATTGAATC GGATAACAGT CTTGGTTCGG AAACTGAACA GGCGGCTCTT 2537
GAATGACACT CTGGATCC 2555
(2) INFORMATION FOR SEQ ID NO 13 TERMINATOR SEQUENCE
CTGTCGATCC TTGCAACCTC CAGTTGCTCT ATCAGGGCCA TGACCCCCAA CAGCAGTGGC 60
GACTACAACC TCTTGCCATG GAAGCCGGGC GTCCTTACCT TGAAGCAGTG ACGAGCTTAT 120
CTTTAGTTGC AGATCGTGTT TCTCCTTTCT TCTTCAAGTA GTTTTAGTGG TGGAAGACAG 180
CAGAAGGTGG TCATCATCTT AGGCTCAGTT GGGGTGGGCT CCTGCCACGT TTTGTCCATA 240
GGCTAGTAAT TTGCACGGAA TTCAGTTCAT TGGCAAGGAG TGCGGTACGA ATACCTGTTT 300
TCACAATAGC AATTAGGCCC AGTAGTTATA CTACGTACTG GAATTGAGTA CTCGTAGTAG 360
CAAGATTGTT TGCCTCAGAG GGAATGGCCG ACACGTGAGC AAGTCACCTT CATCAGCTAG 420
TCGCGTTCCA CATAGACAAT GGTCCAGCTC CAGAGTGGAA TTTGGGCTAC TTTGAACGAT 480
GGCCGATTGA ATCGCGCGTC TCCTCAATTG TATTTAACCA CAATAGGCCA GGTATTGGCA 540
TTCACTCTCC GCCTTTGCGG GTGCCGGCAC GAGATGTCTC CTGAAGAAAC TAGGCAACGA 600
GCAGACTGTG GATATGGGAG ATGGTTGACG ATGTGCTTCT TGGTAAATTT GAAGCCTCCA 660
GGGCCTCTAG AAAGGCGGGA ATTTAAATCT CAAGTGCCCT AACGTGTCCG ACCACGGTGT 720
TGATCATCAT TCATTGAATC GGATAACAGT CTTGGTTCGG AAACTGAACA GGCGGCTCTT 780
GAATGACACT CTGGATCC 798
(2) INFORMATION FOR SEQ ID NO 14 Signal SEQUENCE
ATG AAG TTC TTC AAT GCC AAA GGC AGC TTG CTG TCA TCA GGA ATC TAC 48 CTC ATT GCA TTA ACC CCC TTT GTT AAC GCC 78
56
SEQ ID NO 15
SIGNAL SEQUENCE
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 26 amino acids
(B) TYPE amino acid (D) TOPOLOGY linear
(n) MOLECULE TYPE protein
(xt) SEQUENCE DESCRIPTION SEQ ID NO 15
Met Lys Phe Phe Asn Ala Lys Gly Ser Leu Leu Ser Ser Gly lie Tyr Leu lie Ala Leu Thr Pro Phe Val Asn Ala
20 26
WO 96/29416 57 PCI7EP96/01009
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13btr)
A The indications rnaae oelow relate lo iht microorganism referred »o in Ibe description on P*Se _
_ , line S a^d
B IDENTIFICATION OF DEPOSIT further aeoesiis arc iQentified on an additional sots I |
Name of aepositary institution
The National Collections of Industrial and Marine Bacteria Limned
AdQrcis of oeposiiary institution (tnctuatng postal coacani country)
23 St Macnar En\e AberQeen Scotland A.B2 1RY
United Kingdom
Date of deposit ib jAt-joAty ms
Accession Nurnoer
MCIM& 14010b
C ADDTTIONAL INDICATIONS (lesveolant if not applicable) This information is continued on an additional so;:' | j
In respect of those designations in which a EuroDean patent is sought, and other designated state having equivalent legislation, a samDie of tne ceocsited microorganism will be maae available untxl the ouDlication of the mention zz tne grant of the European patent or until the date on whicn tne apolicaticr nas oeen refused or vxthdrawn or is deemed to be v,itndra\r, ori, b\ tne issue cf sr_- a sample to an expert. nominated by tne person reauestmg tne sassDle. (P.ule 25 EPC).
D DESIGNATED STATES FOR WHICH INDICATIONS ARE Mj\DE fifthe inmceuons arc not (or all acsinatcz Steal
I
E SEPARATE FURNISHING OF INDICATIONS ileave oiar-Kil not aconzaoi:
tneinoications tutec Di ow uin oesLDnittsc'to tne inicnanona Bureau ta'e-upc: n-irrt-jromi mrc ot ttt tnzizz crs ; ; i.-:-Sumocr of ucDosil')
For receiving Office use oniy ro* Irternauonsi Bureau use oni\
I M" 1"Q1S s^eei wai*~csivec witn tae international aooucauon 1 | Tms sneet wis received ov tre Inte-nauor.al Eu-ss- "
Autnortzsc office"
rc—*CT/RO']3fi rJU i co~