BETA- 1.4-ENDOGLUCANASE FROM ASPERGILLUS NICER

The present invention relates to an enzyme. In addition, the present invention relates to a nucleotide sequence coding for the enzyme. The present invention also relates to one or more uses of the enzyme. The present invention also relates to a promoter suitable for expressing that nucleotide sequence, as well as other nucleotide sequences.

In particular, the enzyme of the present invention is a glucanase enzyme, more especially an enzyme that can degrade ]S-l ,4-glucosidic bonds.

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, 1975;, P. Albersheim, Plant Biochem. 3rd Edition (Bonner and Varner), Ac. Press, 1976; T. Hayashi, Ann. Rev. Plant Physiol. & Plant Mol. Biol., 40, 139-168, 1989).

The composition of the plant cell wall is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose which is the main structural component of the plant cell wall; hemicellulose which comprises various β-xylan chains, such as xyloglucans; and pectic substances consisting of galacturonans, rhamnogalacturonans, arabinans, galactans and arabinogalactans.

In particular, glucans are polysaccharides made up exclusively of glucose subunits. Typical examples of glucans are starch and cellulose.

The enzymes that degrade glucans are collectively referred to as glucanases. A typical glucanase is /3-1,4-endoglucanase.

j3-l ,4-endoglucanases have uses in many industries.

For example, β-1 ,4-endoglucanases are used in the pulp industry, the textile industry, and in the formulation of detergents, such as laundry detergents.

By way of further example, 0-1 ,4-endoglucanases are used in the food industry, such as the brewing industry. In this regard, barley is used for production of malt, and, in recent years, as adjunct in the brewing process. When the quality of the malt is poor, or barley has been used as an adjunct, problems with high viscosity in the wort can arise because of β -glucans from the barley . This is because barley contains large quantities of mixed 0-1,3/1 ,4- glucans of very high molecular weight. When dissolved, these glucans produce high viscosity solutions, which can cause troubles in some applications. For example, the high viscosity reduces the filterability of the wort and can lead to unacceptable long filtration times. To avoid these problems β- glucanase has been traditionally added to wort to avoid such problems - i.e. the problem with glucans can be avoided by addition of enzymes, in particular, glucanases, which degrade the polymers.

Further information on these problems may be found in a brochure called "Glucanase GV" (supplied by Danisco Ingredients), the reviews by Dr. C.W. Bamforth (Brewers Digest June 1982 pages 22-28; and Brewers' Guardian September 1985 pages 21-26), and the paper by T.Godfrey (Industrial Enzymology The Application of Enzymes in

Industry Chapter 4.5 pages 221-259).

In the feed industry barley can be used for chicken feed because it is cheap, but again the 0-glucan can give problems for the digestion of the chicken. In addition, the faeces of chickens feeding on feed containing barley can be very sticky making it difficult to remove and results in dirty eggs. By addition of β-glucanase to the feed the digestibility of the feed can be increased. This, in turn, makes the faeces less sticky.

Still with regard to the feed industry, hydrolysis of primary cell wall xyloglucan has also been demonstrated in segments of dark grown squash hypocotyl, during IAA induced growth (K. Wakabayashi el al, Plant Physiol. , 95, 1070-1076, 1991).

Furthermore, endohydrolysis of wall xyloglucan is thought to contribute to wall loosening which accompanies cell expansion (T. Hyashi. Ann. Rev. Plant Physiol. & Plant Mol. Biol., 40, 139-168, 1989). In addition, the average molecular weight of xyloglucan has also been shown to decrease during tomato fruit ripening and this may contribute to the tissue softening which accompanies the ripening process (D.J.

Huber, J. Amer. Soc. Hort. Sci. , 108(3), 405-409, 1983). Also, certain seeds - e.g. Nasturtium - contain up to 30% by weight of xyloglucan, stored in thickened cotyledonary cell walls, which serves as a reserve polysaccharide and is rapidly depolymerised during germination. Thus, it would be useful to increase glucanase activity, for example to have a plant with high concentration of glucanase for use in feed.

WO 93/20193 discusses endo-β-l,4-glucanases (EC No. 3.2.1.4). According to WO 93/20193, these glucanases are a group of hydrolases which catalyse endo hydrolysis of 1 ,4-β-D-glycosidic linkages in cellulose, lichenin, cereal β-D-glucans and other plant material containing cellulosic parts. Endo-l,4-β-D-glucan 4-glucano hydrolase is sometimes called endo-β-l,4-glucanase.

The endo-β-l,4-glucanase of WO 93/20193 exhibits a pH-optimum of 2.0 to 4.0, an isoelectric point of 2.0 to 3.5, a molecular weight of between 30,000 and 50,000, and a temperature optimum between 30 and 70°C.

Further teachings on glucans may be found in WO 93/17101, in particular xyloglucans. According to WO 93/17101, xyloglucans are 1 ,4-β-glucans that have been extensively substituted with α-l,6-xylosyl side chains, some of which are 1,2-β- galactosylated. They are found in large amounts in the primary cell walls of dicots but also in certain seeds, where they serve different roles.

Primary cell wall xyloglucan is fucosylated. Xyloglucan is tightly hydrogen bonded to cellulose microfibrils and requires concentrated alkali or strong swelling agents to release it.

Xyloglucan is thought to form cross-bridges between cellulose microfibrils, the cellulose/xyloglucan network forming the major load-bearing/elastic network of the wall. DCB mutated suspension culture cells (cell walls lacking cellulose) release xyloglucan into their media, suggesting that xyloglucan is normally rightly bound to cellulose.

EP-A-0339550 reports on an alkaline cellulase produced by Bacillus sp. No sequence data for either the enzyme or the coding sequence are provided. The enzyme is said to be effective for laundry detergent compositions.

EP-A-0458162 reports on an proteinase-resistant cellulase produced by Aspergillus niger. No sequence data for either the enzyme or the coding sequence are provided. The enzyme is said to be effective for laundry detergent compositions.

Akiba et al in J Ferment and Bioengin 79 No.2 125-130 (1995) report on an endo-β-

1 ,4-glucanase obtained from Aspergillus Niger. This enzyme has a molecular weight of 40kDa when measured by both gel filtration and SDS-polyacrylamide gel electrophoresis. Very limited sequence data are provided for the enzyme. The enzyme is said to be effective for laundry detergent compositions.

Akiba et al in Biosci. Biotech. Biochem. 59 No.6 1048-1051 (1995) report on the preparation of three different carbohydrate-depleted enzymes from an endo-β-1,4- glucanase obtained from Apergillus Niger. No sequence data for either the enzymes or their coding sequences are provided.

WO 90/09436 discloses a thermostable Bacillus (l,3-l,4)-β-glucanase.

WO 97/13862 discloses two cDNA clones coding for cellulase enzymes that are said to possess β-glucanase activity. The nucleotide sequences of these clones are presented as SEQ ID NO: l: and as SEQ ID NO:3: in WO 97/13862. The amino acid sequences of these cellulase enzymes are presented as SEQ ID NO:2: and as SEQ ID NO:4: in WO 97/13862.

Each of the specific sequences of WO 97/13862 (especially SEQ ID NO:3: and SEQ ID NO:4: of WO 97/13862) lly SEQ ID NO:3: of WO 97/13862) will hereinafter be referred to as the "cellulase sequence of WO 97/13862" .

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 (e.g. Aspergillus niger) or even a plant crop. The resultant protein or enzyme may then be used in industry. Alternatively, the resultant protein or enzyme may be useful for the organism itself. For example, it may be desirable to produce crop protein products with an optimised amino 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, 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 micro-organisms, such as filamentous fungi, to prepare products from GOIs by use of promoters that are active in the micro-organisms.

The present invention seeks to provide an enzyme that is useful for industry and also a nucleotide sequence coding for same.

The present invention also seeks to provide a promoter that is useful for expressing GOIs.

According to a first aspect of the present invention there is provided an enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 or SEQ ID No. 4.

According to a second aspect of the present invention there is provided an enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 and the sequence shown as SEQ ID No. 4.

According to a third aspect of the present invention there is provided an enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 and the sequence shown as SEQ ID No. 4, wherein SEQ ID No. 3 is nearer the N terminal end than SEQ ID No. 4.

Preferably the enzyme according to any one of the above-mentioned aspects is capable of exhibiting 3-1,4-endoglucanase activity.

According to a fourth aspect of the present invention there is provided an enzyme capable of exhibiting β-l,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

Preferably, the enzyme comprises at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably all of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,

SEQ ID No. 7, and SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

Any two or more of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 may be separated by with another sequence or other sequences.

Preferably, if two or more of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 are present then the location of SEQ ID No. 3 is nearer the N terminal end than SEQ ID No. 4 which is nearer the N terminal end than SEQ ID No. 5 which is nearer the N terminal end than SEQ ID No. 6 which is nearer the N terminal end than SEQ ID No. 7 which is nearer the N terminal end than SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

According to a fifth aspect of the present invention there is provided an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics:

a. a MW of 37,000 D ± 1000 D (as determined by using an SDS gel) b. glucanase activity

wherein the glucanase activity is endo 3-1,4-glucanase activity.

According to a sixth aspect of the present invention there is provided an enzyme having a sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

According to a seventh aspect of the present invention there is provided an enzyme capable of exhibiting j3-l,4-endoglucanase activity and being encoded by at least any one or more of the nucleotide sequences shown as: SEQ. I.D. No. 2, a variant, homologue or fragment thereof, SEQ. I.D. No. 9, SEQ. I.D. No. 10, SEQ. I.D. No. 11 , SEQ. I.D. No. 12, SEQ. I.D. No. 13, SEQ. I.D. No. 14, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

According to an eighth aspect of the present invention there is provided a nucleotide sequence coding for the enzyme according to the present invention.

According to a ninth aspect of the present invention there is provided a nucleotide sequence comprising at least the sequence shown as SEQ ID No. 9 or SEQ ID No. 10.

According to a tenth aspect of the present invention there is provided a nucleotide sequence comprising at least the sequences shown as SEQ ID No. 9 and SEQ ID No. 10.

Preferably SEQ ID No. 9 is nearer the 5' end than SEQ ID No. 10.

Preferably the nucleotide sequence codes for an enzyme capable of exhibiting 0-1 ,4- endoglucanase activity.

According to a eleventh aspect of the present invention there is provided a nucleotide sequence coding for an enzyme capable of exhibiting 0-1 ,4-endoglucanase activity, wherein the nucleotide sequence comprises at least the sequence shown as SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

Preferably the nucleotide nucleotide sequence comprises at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably all of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

Any two or more of SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14 may be separated by another sequence or other sequences.

Preferably if two or more of SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ

ID No. 12, SEQ ID No. 13, and SEQ ID No. 14 are present then the location of SEQ ID No. 9 is nearer the 5' end than SEQ ID No. 10 which is nearer the 5' end than

SEQ ID No. 11 which is nearer the 5' end than SEQ ID No. 12 which is nearer the 5' end than SEQ ID No. 13 which is nearer the 5' end than SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

According to a twelfth aspect of the present invention there is provided a nucleotide sequence having the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

According to a thirteenth aspect of the present invention there is provided a nucleotide sequence according to the present invention operatively linked to a promoter.

Additional nucleotide(s) may be present to ensure expression of the nucleotide sequence - such as a start codon and/or a signal sequence.

According to a fourteenth aspect of the present invention there is provided a process of preparing an enzyme according to the present invention comprising expressing a nucleotide sequence according to the present invention.

According to a fifteenth aspect of the present invention there is provided the use of an enzyme according to the present invention or an enzyme prepared by a process according to the present invention to degrade a glucan.

According to a sixteenth aspect of the present invention there is provided the use of an enzyme according to the present invention or an enzyme prepared by a process according to the present invention to prepare a foodstuff (such as a feed).

According to a seventeenth aspect of the present invention there is provided a foodstuff comprising or prepared from the enzyme according to the present invention or an enzyme prepared by a process according to the present invention.

According to an eighteenth aspect of the present invention there is provided a promoter having the sequence shown as or contained within SEQ. I.D. No. 15 or a variant, homologue or fragment thereof.

According to a ninteenth aspect of the present invention there is provided a glucanase enzyme having the ability to degrade 0-1 ,4-glucosidic bonds, which is immunologically reactive with an antibody raised against a purified glucanase enzyme having the sequence shown as SEQ. I.D. No. 1.

According to a twentieth aspect of the present invention there is provided a nucleotide sequence comprising at least the sequence shown as SEQ ID No. 19, or the sequence shown as SEQ ID No. 20, or the sequence shown as SEQ ID No. 21 , or the sequence shown as SEQ ID No. 22.

Other aspects of the present invention include constructs, vectors, plasmids, cells, tissues, organs and transgenic organisms comprising or expressing the aforementioned aspects of the present invention.

Other aspects of the present invention include methods of expressing or allowing expression or transforming any one of the nucleotide sequence, the promoter, 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 in a transgenic organism.

Further aspects of the present invention include uses of the enzyme for preparing or treating foodstuffs, including animal feed.

Some of the key advantages of the present invention are that it provides an enzyme having glucanase activity. 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.

The present invention also provides a GOI coding for the enzyme that can be expressed preferably in specific cells or tissues, such as those of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even 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 in certain 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. Preferably, the promoter is used in Aspergillus wherein the product encoded by the GOI is excreted from the host organism into the surrounding medium.

Also, the present invention provides constructs, vectors, plasmids, 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 genus Aspergillus, or even a plant.

In the following text, the enzyme of the present invention is sometimes referred to as the endo-0-l,4-glucanase II enzyme or the glucanase II enzyme or the egl-B enzyme or the egl-2 enzyme; whereas the coding sequence therefor is sometimes referred to as the eglB gene or the glucanase II gene or the gene encoding the egl-B enzyme or gene encoding the egl-2 enzyme.

Preferably the enzyme does not have a cellulose binding domain.

Preferably the transgenic organism is a fungus.

Preferably the transgenic organism is a filamentous fungus, preferably Aspergillus.

Preferably the transgenic organism is a plant.

Preferably the transgenic organism is a yeast.

Preferably in the process, the enzyme has the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof, and the nucleotide sequence has the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof.

Preferably the promoter of the present invention is operatively linked to a GOI.

Preferably the promoter is operatively linked to a GOI, wherein the GOI comprises a nucleotide sequence according to the present invention.

Preferably, the enzyme of the present invention is used in the preparation of a foodstuff - particularly a foodstuff comprising a 0-glucan. Typical foodstuffs include dairy products, meat products, poultry products, fish products and bakery products. Preferably, the foodstuff is a feed. Preferably, the foodstuff is a beverage.

The terms "variant" , "homologue" or "fragment" in relation to the enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant amino acid sequence has 0-1,4-endoglucanase activity, preferably having at least the same activity as the enzyme shown as SEQ

I.D. No. 1 - but wherein the enzyme is not the cellulase sequence of WO 97/13862. In particular, the term "homologue" covers homology with respect to structure and/or sequence and/or function providing the resultant enzyme has 0-1 ,4-endoglucanase activity. With respect to sequence homology (i.e. similarity), preferably there is at least 75 % , more preferably at least 85 % , more preferably at least 90% homology to the sequence shown as SEQ ID No. l in the attached sequence listings. More preferably there is at least 95 %, such as at least 98% , homology to the sequence

shown as SEQ ID No. 1 in the attached sequence listings.

The terms "variant", "homologue" or "fragment" in relation to the nucleotide sequence coding for the enzyme of the present invention 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 0-1 ,4-endoglucanase activity, preferably having at least the same activity as the enzyme shown as SEQ I.D. No. 1 - but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862. In particular, the term "homologue" covers homology with respect to sequence and/or structure and/or function providing the resultant nucleotide sequence codes for an enzyme having 0- 1,4-endoglucanase activity. With respect to sequence homology (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequence shown as SEQ ID. No. 2 in the attached sequence listings. More preferably there is at least 95%, such as at least 98%, homology to the sequence shown as SEQ ID. No. 2.

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 one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence has the ability to act as a promoter in an expression system - such as the transformed cell or the transgenic organism according to the present invention, preferably having at least the same ability to act as a promoter as the promoter contained within or as shown as SEQ ID No. 15 in the attached sequence listings. In particular, the term "homologue" covers homology with respect to sequence and/or 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 the promoter contained within or as shown as SEQ ID NO. 15 shown in the attached sequence listings. More preferably there is at least 95 %, such as at least 98% , homology to SEQ ID NO. 15 shown in the attached sequence listings.

Preferably, the promoter comprises at least the nucleotides 567 to 1 136 as shown in Figure 1. More preferably, the promoter comprises at least the nucleotides 369 to 1136 as shown in Figure 1.

The above terms are synonymous with allelic variations of the sequences.

The present invention also covers sequences that are complementary to the above- mentioned sequences.

The term "complementary" means that the present invention also covers nucleotide sequences that can hybridise - preferably under stringent conditions - 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 it means cDNA.

The term "construct" - which is synonymous with terms such as "conjugate" , "cassette" and "hybrid" - includes the nucleotide sequence according to the present invention directly or indirectly attached to a promoter. It also includes a GOI directly or indirectly attached to the promoter of the present invention. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the SΛ7-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention or the GOI. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In each case, the terms do not cover the natural combination of the gene coding for the enzyme ordinarily associated with the wild type gene promoter and when they are both in their natural environment. A highly preferred embodiment is the or a GOI being operably linked to a or the promoter.

The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a filamentous fungus, preferably of the genus Aspergillus. such as Aspergillus niger, or plants, such as potatoes, sugar beet etc. , into which it has been transferred. Alternatively, or in addition, the construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant seed, such as corn, wheat or barley, 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 plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.

The term "vector" includes expression vectors and transformation vectors.

The term "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.coli plasmid to a filamentous fungus, preferably of the genus Aspergillus. It may even be a construct capable of being transferred from an E.coli plasmid to an Agrobacterium to a plant.

The term "tissue" includes isolated tissue and tissue within an organ.

The term "organism" in relation to the present invention includes any organism that could comprise the promoter according to the present invention and/or the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, wherein the promoter can allow expression of a GOI and/or wherein the nucleotide sequence according to the present invention can be expressed when present in the organism.

Preferably the organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.

Other preferred organisms include any one of Bacillus, Aspergillus oryzae, A. tubigensis, A. awamori, Trichoderma reesei, T. viride and T. longibrachiatum.

The term "transgenic organism" in relation to the present invention includes any organism that comprises the promoter according to the present invention and/or the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, wherein the promoter can allow expression of a GOI and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism. Preferably the promoter and/or the nucleotide sequence is (are) incorporated in the genome of the organism.

Preferably the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.

Other preferred transgenic organisms include any one of Bacillus, Aspergillus oryzae,

A. tubigensis, A. awamori, Trichoderma reesei, T. viride and T. longibrachiatum.

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 invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention or the products thereof. For example the transgenic organism can comprise a GOI, preferably an exogenous nucleotide sequence, under the control of the promoter according to the present invention. The transgenic organism can also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a promoter, which may be the promoter according to the present invention.

The term "transgenic organism" does not cover the native nucleotide coding sequence according to the present invention in its natural environment when it is under the control of its native promoter which is also in its natural environment. In addition,

the present invention does not cover the native enzyme according to the present invention when it is in its natural environment and when it has been expressed by its native nucleotide coding sequence which is also in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment.

The transformed cell or organism could prepare acceptable quantities of the desired compound which would be easily retrievable from, the cell or organism.

Preferably the construct of the present invention comprises the nucleotide sequence of the present invention and a promoter.

The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression.

In one aspect, the promoter of the present invention is capable of expressing a GOI, which can be the nucleotide sequence coding for the enzyme of the present invention.

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, the organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of seed, stem, sprout, root and leaf tissues.

By way of example, the promoter for the nucleotide sequence of the present invention can be the α-Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 promoter or the α-Amy 637 promoter) as described in PCT patent application PCT/EP95/02195 (incorporated herein by reference). Alternatively, the promoter for the nucleotide sequence of the present invention can be the α-Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the α-Amy 351

promoter) as described in PCT patent application PCT/EP95/02196 (incorporated herein by reference). Alternatively, the promoter could be the glucanase promoter - sometimes referred to as the egla promoter - as described in PCT patent application PCT/EP96/01008 (incorporated herein by reference). Alternatively, the promoter could be the arabinofuranosidase promoter as described in PCT patent application

PCT/EP96/01009 (incorporated herein by reference).

Preferably, the promoter is the promoter of the present invention.

In addition to the nucleotide sequences described above, the promoters, particularly that of the present invention, could additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a Pribnow 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 5Λ/-intron or an ADH intron. Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' signal sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).

The present invention also encompasses combinations of promoters and/or nucleotide sequences coding for proteins or recombinant enzymes and/or elements.

The present invention also encompasses the use of promoters to express a nucleotide sequence coding for the enzyme according to the present invention or the GOI, wherein a part of the promoter is inactivated but wherein the promoter can still function as a promoter. Partial inactivation of a promoter in some instances is advantageous. In particular, with the Amy 351 promoter mentioned earlier it is possible to inactivate a part of it so that the partially inactivated promoter expresses

the nucleotide of the present invention or a GOI in a more specific manner such as in just one specific tissue type or organ.

The term "partial inactivation" means that the expression pattern of the promoter is modified but wherein the partially inactivated promoter still functions as a promoter.

However, as mentioned above, the modified promoter is capable of expressing the nucleotide of the present invention or a GOI in at least one (but not all) specific tissue of the original promoter. One such promoter is the Amy 351 promoter described above. Examples of partial inactivation include altering the folding pattern of the promoter sequence, or binding species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognised by, for example, RNA polymerase. Another, and preferable, way of partially inactivating the promoter is to truncate it to form fragments thereof. Another way would be to mutate at least a part of the sequence so that the RNA polymerase can not bind to that part or another part. 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 combination of constructs according to the present invention means any gene of interest. A GOI can be any nucleotide that is either foreign or natural to the organism (e.g. filamentous fungus, preferably of the genus Aspergillus, or a plant) in question. Typical examples of a GOI include genes encoding for proteins and enzymes that modify metabolic and catabolic processes.

The GOI may code for an agent for introducing or increasing pathogen resistance. The GOI may even be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues. The GOI may even code for a non-native protein of a filamentous fungus, preferably of the genus Aspergillus, or a compound that is of benefit to animals or humans. Examples of GOIs include pectinases, pectin depolymerases, polygalacturonases, pectate lyases, pectin lyases, rhamno- galacturonases, hemicellulases, endo-0-glucanases. arabinases, or acetyl esterases, or combinations thereof, as well as antisense sequences thereof. The GOI may be a protein giving nutritional value to a food or crop. Typical examples include plant

proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non-transgenic plant).

The GOI may even code for an enzyme that can be used in food processing such as chymosin, thaumatin and α-galactosidase. The GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for patatin or α-amylase, ADP-glucose pyrophosphorylase (e.g. see EP-A-0455316), a protease antisense, a glucanase or genomic 0-1,4-endoglucanase.

The GOI may even code for an intron of a particular enzyme but wherein the intron can be in sense or antisense orientation. In the latter instance, the particular enzyme could be genomic 0-1 ,4-endoglucanase. Antisense expression of genomic exon or intron sequences as the GOI would mean that the natural 0-1 ,4-endoglucanase expression would be reduced or eliminated but wherein the expression of the 0-1,4- endoglucanase gene according to the present invention would not be affected.

The GOI can be the nucleotide sequence coding for the arabinofuranosidase enzyme which is the subject of PCT patent application PCT/EP96/01009 (incorporated herein by reference). The GOI can be any of the nucleotide sequences coding for the ADP- glucose pyrophosphorylase enzymes which are the subject of PCT patent application PCT/EP94/01082 (incorporated herein by reference). The GOI can be any of the nucleotide sequences coding for the α-glucan lyase enzyme which are described in PCT patent application PCT/EP94/03397 (incorporated herein by reference). The GOI can be any of the nucleotide sequences coding for the glucanse enzyme which are described in PCT patent application PCT/EP96/01008 (incorporated herein by reference) .

The host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see

Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold

Spring Harbor Laboratory Press). If a prokaryotic host is used then the gene may need to be suitably modified before transformation - such as by removal of introns.

Preferably, in any one of the plasmid, the vector such as an expression vector or a transformation vector, the cell, the tissue, the organ, the organism or the transgenic organism, the promoter is present in combination with at least one GOI.

Preferably the promoter and the GOI are stably maintained within the transgenic organism. By way of example, the promoter and/or the GOI (such as the nucleotide sequence according to the present invention) may be maintained within the transgenic organism in a stable extrachromosomal construct. This is preferred for transgenic bacteria and yeast, or even some filamentous fungi. Alternatively, the promoter and/or the GOI (such as the nucleotide sequence according to the present invention) may be stably incorporated within the transgenic organism's genome. This is preferred for some transgenic bacteria and yeast, and most filamentous fungi.

Preferably the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger. Alternatively, the transgenic organism can be a yeast. The transgenic organism can even be a plant, such as a monocot or dicot plant.

Thus, a preferred embodiment according to the present invention is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ± 1000 D (as determined by using an SDS gel); and glucanase activity; wherein the glucanase activity is endo 0-1,4-glucanase activity; and wherein the enzyme comprises at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably all of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 - but wherein the enzyme is not the cellulase sequence of WO 97/13862.

Another preferred embodiment according to the present invention is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ± 1000 D (as determined by using an SDS gel); and glucanase activity; wherein the glucanase activity is endo 0-1 ,4-glucanase activity; and wherein the enzyme is encoded by a nucleotide nucleotide sequence which comprises at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably all of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14 - but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

A highly preferred embodiment according to the present invention is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ± 1000 D (as determined by using an SDS gel); and glucanase activity; wherein the glucanase activity is endo 0-1 ,4-glucanase activity; and wherein the enzyme has the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof - but wherein the enzyme is not the cellulase sequence of WO 97/13862.

Another highly preferred embodiment is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ± 1000 D

(as determined by using an SDS gel); and glucanase activity; and wherein the glucanase activity is endo 0-1 ,4-glucanase activity; wherein the enzyme is coded by the nucleotide sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof - but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

Another highly preferred embodiment is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ± 1000 D (as determined by using an SDS gel); and glucanase activity; and wherein the glucanase activity is endo 0-1 ,4-glucanase activity; wherein the enzyme has the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof; and wherein the enzyme is coded by the nucleotide sequence shown as SEQ. I.D. No.

2 or a variant, homologue or fragment thereof - but wherein the enzyme is not the cellulase sequence of WO 97/13862 and wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

Preferably the enzyme sequence is that shown as SEQ. I.D. No. 1.

Preferably the nucleotide sequence is that shown as SEQ. I.D. No. 2.

A preferred host organism for the expression of the nucleotide sequence of the present invention and/or for the preparation of the enzyme according to the present invention is an organism of the genus Aspergillus, such as Aspergillus niger. In this regard, a transgenic Aspergillus according to the present invention can be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In:

Martinelli S.D. , Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in 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 structure. In: Leong, S.A., Berka R.M. (Editors) Molecular Industrial Mycology. Systems and Applications for Filamentous Fungi. Marcel Dekker Inc.

New York 1991. pp 1-29) and Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S.D. , Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666). The following commentary provides a summary of those teachings for producing transgenic Aspergillus according to the present invention.

For almost a century, filamentous fungi have been widely used in many types of industry for the production of organic compounds and enzymes. For example, traditional Japanese koji and soy fermentations have used Aspergillus sp. Also, in this century Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry.

There are two major reasons why filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracelluar 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. 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 a nucleotide sequence coding for an amylase enzyme or even SEQ. I.D. No. 2) into a construct designed for 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 active in fungi. Examples of promoters other than that of the present invention include a fungal promoter for a highly expressed extracellulary enzyme, such as the glucoamylase promoter or the α-amylase promoter. The GOI can be fused to a signal sequence which directs the protein encoded by the GOI to be secreted. Usually a signal sequence of fungal origin is used, such as that of the present invention. A terminator active in fungi ends the expression system, such as that of the present invention.

Another type of expression system has been developed in fungi where the nucleotide sequence according to the present invention (or even the GOI) is fused to a smaller or a larger part of a fungal gene encoding a stable protein. This aspect can stabilize the protein encoded by the nucleotide sequence according to the present invention (or even another GOI which encodes a protein of interest (POI)). In such a system a cleavage site, recognized by a specific protease, can be introduced between the fungal protein and the protein encoded by the nucleotide sequence according to the present invention (or even the GOI), so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein encoded by the nucleotide sequence according to the present invention (or even another GOI). By way of

example, one can introduce a site which is recognized by a KEX-2 like peptidase found in at least some Aspergilli (Broekhuijsen et al 1993 J Biotechnol 31 135-145). Such a fusion leads to cleavage in vivo resulting in protection of the expressed product and not a larger fusion protein.

Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins. The proteins can be deposited intracellularly if the nucleotide sequence according to the present invention (or another 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 bacterial proteins. If the nucleotide sequence according to the present invention (or another GOI) is equipped with a signal sequence the protein will accumulate extracelluarly.

With regard to product stability and host strain modifications, some heterologous proteins are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracelluar 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 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 Ca ²⁺ 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, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A commonly used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.

In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast

Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).

For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.

A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5,

Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).

Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.

In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. The constructs contain a promoter active in yeast fused to the nucleotide sequence of the present invention, usually a promoter of yeast origin, such as the GAL1 promoter, is used. Usually a signal sequence of yeast origin, such as

the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system.

For the transformation of yeast several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).

The transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1 , and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.

Another host organism is a plant.

Even though the enzyme, the nucleotide sequence coding therefor and the promoter of the present invention are not disclosed in 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 in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted 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 information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205- 225) and Christou (Agro-Food-Industry Hi-Tech March April 1994 17-27).

Thus, in one aspect, the present invention relates to a vector system which carries a nucleotide sequence or promoter or construct according to the present invention and which is capable of introducing the nucleotide sequence or promoter 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 of two vectors, the vector system is normally referred to as a binary vector system. Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3 , 1-19.

One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant 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 nucleotide sequence or promoter or construct of the present invention should 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 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 necessary to infect the plant (e.g. the vir region) and at least one border part of a T- DNA sequence, the border part being located on the same vector as the genetic construct. Preferably, the vector system is an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids

are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.

In the construction of a transgenic plant the promoter or nucleotide sequence or construct of the. present invention may be first constructed in a microorganism in which the vector 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. coli. it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens. The

Ti-plasmid harbouring the promoter or nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the promoter or nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.

As reported in CA-A-2006454, a large amount of cloning vectors are available which contain a replication system in E. coli and a marker which allows a selection of the transformed cells. The vectors contain for example pBR 322, the pUC series, the M13 mp series, pACYC 184 etc.

In this way, the promoter or nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector. The contained plasmid is used for the transformation in E. coli. The E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered and then analysed - such as by any one or more of the following techniques: 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 according 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 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 described in EP-A- 120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B. , Alblasserdam, 1985, Chapter V; Fraley, et al , Crit. Rev. Plant Sci. , 4: 1-46; and An et al. , EMBO J. (1985) 4:277-284.

Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds. : D.S. Ingrams and J.P. Helgeson, 203- 208. For further teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant

Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/ April 1994 17-27). With this technique, infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of 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 puncturing 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 part is then grown on a suitable culture medium and allowed to develop into mature plants.

When plant cells are constructed, these cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc. Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the

regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.

Further teachings on plant transformation may be found in EP-A-0449375.

In summation, the present invention provides a glucanase enzyme and a nucleotide sequence coding for the same. In addition, it provides a promoter that can control the expression of that, or another, nucleotide sequence.

The following samples were 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 13 August 1996:

1. E.coli containing plasmid pEglB-1. The deposit number is NCIMB 40825.

2. E.coli containing plasmid pEglB-2. The deposit number is NCIMB 40826.

Highly preferred aspects of the present invention therefore relate to nucleotide coding sequences and promoter sequences obtainable from those deposits, including expression vectors, constructs, organisms and transgenic organisms comprising those same sequences or plasmids.

The present invention will now be described only by way of example.

In the following Examples reference is made to the accompanying Figures in which

Figure 1 presents nucleotide and enzyme sequences;

Figure 2 presents nucleotide and enzyme sequences;

Figure 3 presents nucleotide and enzyme sequences;

Figure 4 presents a plasmid map of pEgl-Bl;

Figure 5 presents a plasmid map of pEgl-B2;

Figure 6 presents a genomic map of Egl-B;

Figure 7 presents a plasmid map of pPR59;

Figure 8 presents a graph; and

Figure 9 presents a graph.

The following Examples discuss recombinant DNA techniques. General teachings of recombinant DNA techniques may be found in Sambrook, J. , Fritsch, E.F. , Maniatis T. (Editors) Molecular Cloning. A laboratory manual. Second edition. Cold Spring Harbour Laboratory Press. New York 1989.

Enzyme purification

The enzyme was purified as described as follows.

The egl-B enzyme was prurified from the crude fermentation broth by using successively Sephadex G25 M gel filtration media for desalting, Q-Sepharose BB,

SOURCE 15Q and MONO-P media for ion exchange and SOURCE PHE medium for hydrophobic interaction chromatography.

Purity was checked by runing egl-B on MONO-Q, HR 5/5, MONO-P, HR 5/5, Superdex 75, 10/30 columns and by isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis. (All media for chromatography and electrophoresis from Pharmacia-LKB Biotechnology.)

N-Terminal sequence analysis

The N-terminal sequence was obtained by sequencing the purified enzyme which gave following sequence:

ASVFEWFGSNESGAEFGT

The enzyme was digested using endopeptidase lys-C and three peptides were purified and sequenced. The following sequences were found:

1 AVTDGGAHALVDPHNYGRY

2 ERVTEATQWLK

3 EATQWLKDNKKVGFIGE

where peptides 2 and 3 are overlapping.

The degree of sequence similarity (homology) for the enzyme according to the present invention with those enzymes and proteins on the SWISS-PROT database (release no.

31) was not very high. For example, there was no similarity to the N-terminal sequence.

Further analysis revealed that the enzyme of the present invention shows some similarities with microbial glucanases which belong to the glycosyl hydrolase Family

5. Family 5 classification is discussed by Henrissat et al (1993 Biochem J. 293 781- 786). In contrast to most of the glucanases in Family 5, the glucanase of the present invention does not have a cellulose binding domain.

PCR cloning

The following four different PCR primers were constructed:

1. B249 of peptide 2/3 sequence KDNKKV, (Reversed)

ACY TTY TTR TTR TCY TT 17 mer, 32 mixture

2. B250 of peptide 2/3 sequence TEATQW, (Reversed)

CCA YTG NGT NGC YTC NGT 18 mer, 256 mixture

3. B263 of peptide 1 sequence DPHNYG

GAY CCN CAY AAY TAY GG 17 mer, 64 mixture

4. B264 of N-terminal sequence VFEWFG

GTN TTY GAR TGG TTY GG 17 mer, 32 mixture

The localisation of the primers can be described schematically as follows:

PCR amplification was performed over two rounds of amplifications

In first round of amplification 100 pmol of each primer was used with 0.5 μg genomic Aspergillus niger DNA as a template in 100 μl reactions.

#1 B264+B249

#2 B264+B250

The reactions were mixed and amplification was done by the following program:

94°C 5 min

These three steps repeated 35 times

10 μl of each reaction was run on an analytical agarose gel. If no bands were clearly visible a second round of amplifications were performed. For this round 100 pmol of each primer and 2 μl of a first round reaction was used in 100 μl reactions. The following combinations of primers were used with the 1st round PCR reaction as template:

Template Primers

#2.1 #1 B263+B250

#2.2 #1 B263 +B249 #2.3 #2 B263 +B250

The PCR amplification was done using the same program as in first round.

10 μl of each reaction was run on an agarose gel and in reaction #2.2 and #2.3 distinct bands was seen. These fragments were cloned using pT7-Blue T-vector kit, and sequenced on an ALF sequencer. The fragment in #2.3 contained, in addition to the primers, DNA encoding the remaining parts of the peptides from which the primers had been deduced. This fragment comprises part of the eglB gene encoding glucanase II.

The complete sequence of the PCR fragment is shown in Figure 3.

In Figure 3, the sequence shown in lower case letters is the pT7blue sequence. The underlined nucleic acid sequences are the primer sequences. The double underlined amino acid sequences are peptides 1 and 2/3. The sequence is a combination of two independent pT7-blue clones each containing the PCR fragment in opposite direction.

The cloned PCR fragment was radiolabelled and used as a probe to screen an A. niger 4M147 library (see below), and two independent cloned were found. Both clones contained the entire coding sequence.

In addition, these clones contained the glucanse II promoter. SEQ ID No. 15 comprises the glucanase II promoter.

Construction of a library

20 μg genomic DNA was partly digested with Tsp509I, which gives ends which are compatible with EcoPΛ ends. The digested DNA was separated on a 1 % agarose gel and fragments of 4-10 kb was purified. A λZAPII EcoRI/ClAP kit from Stratagene was used for library construction according to the manufacturers instructions. 2 μl of the ligation (totally 5 μl) was packed with Gigapack Gold II packing extract according to the manufacturer's instructions (Stratagene). The library contained

650,000 independent clones.

Screening of the A. niger library

2X 50.000 pfu of a lamdba ZAPII library of Aspergillus niger 4M147 was plated on large (22x22 cm) NZY plates.

The inoculated NZY plates were incubated over night at 37°C and plaque lifts of the plates were made. Two lifts were made on each plate on Hybond N (Amersham) sheets. The DNA was fixed using UV radiation for 3 min and the sheets were hybridised, using radiolabelled PCR clone as probe. The filters were prehybridised in 25 ml prehybridisation buffer (6.25 ml 20XSSC, 1.25ml 100X Denhard (2%

Bovine serum albumin, 2% Ficol™, 2% polyvinylpyrrolidone), 1.25 ml 10 % SDS and 16.25 ml water. 150 μl 10 mg/ml denatured Salmon sperm DNA was added immediately before use for one hour at 65°C. The prehybridisation buffer was discarded and the filters hybridised over night at 65°C in 25 ml prehybridisation buffed with the PCR fragment labelled with ³² P-dCTP using Ready-to-Go labelling kit (Pharmacia).

The following day the filters were washed as follows:

2X 15 min with 2xSSC + 0.1 % SDS

15 min with lxSSC + 0.1 % SDS 10 min with O. lxSSC + 0.1 % SDS

(SDS = sodium dodecyl sulphate; SSC = saline sodium citrate made from a stock solution of 20 x SSC (3M NaCl, 0.3M sodium citrate))

All washes were done at 65°C. The sheets were autoradiographed for 16 hours and positive clones were isolated. A clone was reckoned to be positive only if there was a hybridisation signal on both of the plaque lifts of the plate in question.

The positive clones were isolated using a Pasteur pipette and the phages were then eluted. The clones were purified by plating them on to small petri dishes with NZY and hybridising plaquelifts by essentially the same procedure.

Two positive clones with EglB were found. These two clones were subcloned using the Rapid excision system from Stratagene where the ZAP II phages were converted to pBluescript based plasmids containing the same insert. The two clones were named pEglB-1 and pEglB-2 - which are shown in Figures 4 and 5 respectively.

Characterisation of the clones

The plasmids of Figure 4 and 5 were characterised by restriction mapping. EglB was then sequenced. In total 3447 bases were determined to ensure sufficient sequence of the entire promoter, the coding sequence and the terminator. The complete sequence is shown in Figure 1 and Figure 2.

A reading frame with all the sequenced peptides was found. This reading frame was interrupted by 5 small introns and encoded a 332 amino acid long enzyme. The structure of the glucanse II gene is shown diagramatically in Figure 6. The structure of the coding gene is discussed in more detail below.

Sequence analysis

The genomic DNA sequence for the enzyme of the present invention is shown in

Figure 1 and Figure 2. Figure 1 and Figure 2 also show the coding sequence and the enzyme sequence. The full enzyme sequence according to the present invention is also shown as SEQ ID No. 1 and the nucleotide coding sequence according to the present invention is shown as SEQ ID No. 2. SEQ ID No. 16 presents the full genomic sequence. SEQ ID No. 17 presents the N-terminal sequence. SEQ ID No.

18 presents the the endo-0-1 ,4-glucanase II precursor polypeptide. The first 28 amino acids are believed to act as a signal and/or secretory sequence. As shown in Figures 1 and 2 the mature enzyme comprises 304 amino acids.

In Figure 2 the intron sequences of the eglB gene are written in lower case letters.

The ATG start codon is underlined. The N-terminal of the mature enzyme is double underlined. The sequences of the peptides are written in bold. A possible signal sequence cleavage site is marked by an arrow.

Further details on the nucleotide sequence are as follows:

(A) LENGTH: 3447 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(E) CODING SEQUENCE: join 1137..1236, 1280..1335, 1379..1531, 1575..1639, 1683..1780, 1824..2350

(F) SIGNAL PEPTIDE: LOCATION: 1137..1220

(G) MATURE ENZYME: join 1221..1236, 1280..1335, 1379..1531, 1575..1639, 1683..1780, 1824..2347 (H) EXON:

LOCATION: 1137..1236 (I) INTRON:

LOCATION: 1237..1279 (J) EXON:

LOCATION: 1280..1335 (K) INTRON: LOCATION: 1336..1378

(L) EXON:

LOCATION: 1379..1531 (M) INTRON:

LOCATION: 1532..1574 (N) EXON:

LOCATION: 1575..1639 (O) INTRON:

LOCATION: 1640..1682 (P) EXON: LOCATION: 1683..1780

(Q) INTRON:

LOCATION: 1781..1823 (R) EXON:

LOCATION: 1824..2350

Characterization of the 0-glucanase

The molecular weight of the glucanase II enzyme was determined to be 37 kDa using SDS-PAGE. This measured molecular weight is higher than the calculated molecular weight of 33.8 kDa. We believe that the difference is due glycosylation (possibly to up to 10% glycosylation) of the glucanase as evidenced by rapid periodic acid-Schiff staining - i.e. PAS reaction (Van Seuningen and Davril 1992 Electrophoresis L3 97- 99) - of the purified enzyme.

Testing enzyme activity

The purified protein was assayed for endo 0-1,4 glucanase activity using AZO-CM- Cellulose (supplied by Megazyme, Australia) by the instructions given by the manufacturer. The purified enzyme gave a high activity on this substrate.

Thermostability of egl-B enzyme and egl-A enzyme

As mentioned above, the egl-B (or egl-2) enzyme is that of the present invention. The egl-A (or egl-1) enzyme is that of PCT/EP96/01008.

1. Assay for measuring endo-0-glucanase activity

The method comprised measuring the number of reducing end-groups produced by the action of 0-glucanase enzymes on barley-0-glucan. Potassium ferricyanide was used as a colouring agent.

2. Assay for measuring residual activity of 0-glucanase after heat treatment

Egl-1 and egl-2 were diluted in acetate buffer, pH 5.5, and pre-treated at 50, 60, 70 and 80°C for 30 minutes. After cooling to ambient temperature the residual activity is determined.

3. Results

The results are shown the Table below and in Figure 8. The results show that the enzyme of the present invention displays a favourable thermostability profile.

TEMPERATURE 50°C 60°C 70°C 80°C

% RESIDUAL ACTIVITY

egl-A 100 90 3 0

egl-B 100 96 96 68

Thus, the enzyme of the present invention is heat stable, even though it is produced by a mesophile organism.

Construction of pPR59

The trpC terminator was purified from the pBARGTEl (Pall, M.L and Brunelli, J.P. (1993) A series of six fungal transformtion vectors containing polylinkers with multiple unique restriction sites. Fungal genetic Newsletter 40, 59-63). pBARGPEl was digested with Notl and Kpnl and a fragment on ca. 700 bp was purified. The fragment was bluntended using Mung Bean nuclease.

pBluescriptIISK+ was digested with Notl and bluntended using Klenow Polymerase and the TrpC terminator fragment was inserted into this vector.

The plasmid pEglA-2 was digested with Bglil, and the ends completely filled in using Klenow polymerase. Next the plasmid was digested with Pstl and a fragment on 583 nucleotides was purified.

The eglB gene was isolated by amplification of the coding sequence from pEglB-1. A PCR reaction was performed using primer:

CTCTTACTCGAGCAGTCATCATG

binding to nucleotides 1124-1147 in Figure 1 and primer:

CCGTATAGTTCTCCGATGCAC

binding to the complementary strand in position 2375-2395.

The PCR amplification was done using the Pfu polymeraseXNew England) according to the instructions of the manufacturer.

Thus, this reaction amplified a fragment on 1279 nucleotides containing the entire eglB gene (nucleotides 1124-2395 in Figure 1).

The PCR fragment was inserted into pBluescript-TrpC construct digested with Smal and dephosphorylates with alkaline phosphatase. The resultant construct was digested with EcoRl and the ends completely filled in using Klenow polymerase, and subsequently digested with Pstl, and the vector purified. The fragment containing the egl-A promoter was inserted into this vector which gave the expression plasmid pPR59.

Digestions and other manipulations were performed according to the procedures described by J.Sambrook, Fristch, E.F. and Maniatis, T: Molecular cloning. A Laboratory manual. Second edition, Cold Spring Harbour Press, New York 1989.

Thus, expression plasmid pPR59 - as shown in Figure 7 - was constructed where egl- B was expressed under control of the egl-a promoter.

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), Daboussi, M.J. , Djeballi, A. ,

Gerlinger, C , Blaiseau, P.L. , Cassan, M. , Lebrun, M.H., Parisot, D.. Brygoo, Y. 1989 (Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans. Curr. Genet. 15:453-456) and Punt, P.J., van den 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 Schimmelcultures, Baarn) (7 days old) were washed off in 5-10 ml water. A shake flask with 200 ml Potato Dextrose Broth (difco 0549-17-9, Difco Lab. Detroit) was inoculated with this spore suspension and shaken (250 φm) for 16-20 hours at 30°C.

The mycelium was harvested using Miracloth paper and 3-4 g wet mycelium were transferred to a sterile petri dish with 10 ml STC (1.2 M sorbitol, 10 mM Tris HC1 pH 7,5, 50 mM CaCL) with 75 mg lysing enzymes (Sigma L-2265) and 4500 units lyticase (Sigma L-8012).

The mycelium was incubated with the enzyme until the mycelium was degraded and the protoplasts were released. The degraded mycelium was then filtered through a sterile 60 μm mesh filter. The protoplasts were harvested by centrifugation 10 min at 2000 φm in a swing out rotor. The supernatant was discarded and the pellet was dissolved in 8 ml 1.5 M MgSO ₄ and then centrifuged at 3000 m for 10 min.

The upper band, containing the protoplasts was transferred to another tube, using a transfer pipette and 2 ml 0.6 M KC1 was added. Carefully 5 ml 30% sucrose was added on the top and the tube was centrifuged 15 min at 3000 φm.

The protoplasts, lying in the interface band, were transferred to a new tube and diluted with 1 vol. STC. The solution was centrifuged 10 min at 3000 φm. The pellet was washed twice with STC, and finally solubilised in 1 ml STC. The protoplasts were counted and eventually concentrated before transformation.

For the transformation, 100 μl protoplast solution (10 ⁶ -10 ⁷ protoplasts) were mixed with 10 μl DNA solution containing 5 - 10 μg DNA and incubated 25 min at room temperature. Then 60 % PEG-4000 was carefully added in portions of 200 μl, 200 μl and 800 μl. The mixture was incubated 20 min at room temperature. 3 ml STC was added to the mixture and carefully mixed. The mixture was centrifuged 3000 φm for 10 min.

The supernatant was removed and the protoplasts were solubilized in the remaining of the supernatant. 3-5 ml topagarose was added and the protoplasts were quickly spread on selective plates.

Properties of the transformed A. niger

Transformants of A. niger 3M43 were prepared (as described above) and analysed for glucanase II expression. Those transformants expressed the heat stable glucanase enzyme of the present invention.

Glucanase promoter and heterologous gene expression

An expression vector was made by fusing fragments of the glucanase promoter to the gene encoding 0-glucuronidase from E. Coli. These constructs were used in a co- transformation experiment with the hygromycin resistance gene as the selectable marker. 0-glucuronidase activity was detected when the promoter fragments comprised at least the nucleotides 567 to 1136, such as fragments comprising at least the nucleotides 369 to 1136 as shown in Figure 1.

Enzyme Production

Experiments showed that the enzyme was produced efficiently in solid state fermentation. However, by use of a transformed organism, such as transformed A. niger (e.g. transformed A. niger as mentioned above), the enzyme can be produced in a more convenient way - such as by use of submerge fermentation techniques.

Antibody Production

Antibodies were raised against the enzyme of the present invention by injecting rabbits with the purified enzyme and isolating the immunoglobulins from antiserum according to procedures described according to N Harboe and A Ingild ("Immunization, Isolation 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).

Preparation of a foodstuff

A foodstuff (such as a feed) was prepared and tested as described in the following text.

1. Stability of egl-A and egl-B by pelleting at different temperatures

As mentioned above, the egl-B (or egl-2) enzyme is that of the present invention.

The egl-A (or egl-1) enzyme is that of PCT/EP96/01008.

To a standard feed preparation was added egl-A to contain 5 BGU/gram and pelleted at 65, 75, 85 and 90°C. Likewise, to a similar standard feed preparation was added egl-B to contain 5 BGU/gram and pelleted at 65, 75, 85 and 90°C. Residual activity was measured by the viscosity reduction of a barley-0-glucan solution in an Ostwald viscometer at 50°C, pH 5.0.

2. Results

The results are shown the Table below and in Figure 9. The results show that the enzyme of the present invention displays favourable properties for food production at elevated temperatures.

TEMPERATURE 50°C 65°C 75°C 85°C 90C

% RESIDUAL ACTIVITY

egl-A 100 45 11 3 2

egl-B 100 100 90 __ 61 24

The heat stability of the enzyme of the present invention makes it attractive for feed use since feeds often have to be heat treated and pelleted before use to avoid

Salmonella infections.

SUMMARY

Even though it is known that Aspergillus niger produces several enzymes which can degrade 0-glucan, the present invention provides a novel and inventive 0-1,4- endoglucanase, as well as the coding sequence therefor. An important advantage of the present invention is that the enzyme can be produced in high amounts.

The present invention therefore provides an enzyme having glucanase 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 organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger. The enzyme may even be prepared in a plant.

Furthermore, we have isolated a gene encoding glucanase II from Aspergillus niger. The gene has been transformed into A. niger under the control of another promoter and glucanase II activity was found in several transformants.

In addition, the present invention provides a promoter that is capable of directing expression of a GOI, such as a nucleotide sequence coding for the enzyme according to the present invention, preferably in certain specific cells or tissues, such as in just a specific cell or tissue, of an 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 wherein the product encoded by the GOI is excreted from the host organism into the surrounding medium. The promoter may even be tailored (if necessary) to express a GOI in a plant.

Some of the advantages of the present invention are that it provides a means for preparing a glucanase enzyme 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.

The enzyme of the present invention is advantageous as it has a high fibre-conversion potential. In addition, there are fewer processing problems when the enzyme is used, particularly with non-starchy polysaccharides. In addition, the enzyme efficiently degrades 0-glucans. Thus, the enzyme of the present invention is advantageous for preparing or for use as feed supplements. The enzyme of the present invention is advantageous for use in the brewing industry as it can be used to lower the viscosity of beer, and also to improve the filterability of beer. This is important as large molecular weight glucans in beer and the like can cause filtration difficulties and give rise to sediments, gels and hazes.

Other advantages of the present invention are that the enzyme can be used to prepare useful feeds containing cereals, such as barley, maize, rice etc.

For ease of reference, the present invention will be described by way of numbered paragraphs.

1. An enzyme having a sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof, but wherein the enzyme is not the cellulase sequence of WO

97/13862.

2. An enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3.

3. An enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 4.

4. An enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 and the sequence shown as SEQ ID No. 4.

5. An enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 and the sequence shown as SEQ ID No. 4, wherein SEQ ID No. 3 is nearer the N terminal end than SEQ ID No. 4.

6. An enzyme according to any one of paragraphs 1 to 4 wherein the enzyme is capable of exhibiting 0-1,4-endoglucanase activity.

7. An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 5.

8. An enzyme capable of exhibiting 0-1 ,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 6, but wherein the enzyme is not the cellulase sequence of WO

97/13862.

9. An enzyme capable of exhibiting 0-1 ,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 7.

10. An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

11. An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least two of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

12. An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least three of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.

13. An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least four of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.

14. An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least five of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.

15. An enzyme capable of exhibiting 0-1 ,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least all of the

sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.

16. An enzyme according to any one of paragraphs 11 to 15 wherein if two or more of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 are present then the location of SEQ ID No. 3 is nearer the N terminal end than SEQ ID No. 4 which is nearer the N terminal end than SEQ ID No. 5 which is nearer the N terminal end than SEQ ID No. 6 which is nearer the N terminal end than SEQ ID No. 7 which is nearer the N terminal end than SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

17. An enzyme obtainable from Aspergillus, wherein the zyme has the following characteristics:

a. a MW of 37,000 D ± 1000 D (as determined by using an SDS gel) b. glucanase activity

wherein the glucanase activity is endo 0-1,4-glucanase activity.

18. An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being encoded by at least any one or more of the nucleotide sequences shown as: SEQ. I.D. No. 2, a variant, homologue or fragment thereof, SEQ. I.D. No. 9, SEQ. I.D. No. 10, SEQ. I.D. No. 11, SEQ. I.D. No. 12, SEQ. I.D. No. 13, SEQ. I.D. No. 14, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

19. An enzyme according to any one of paragraphs 1 to 18 wherein the enzyme does not have a cellulose binding domain.

20. A nucleotide sequence coding for the enzyme according to any one of paragraphs 1 to 19.

21. A nucleotide sequence comprising at least the sequence shown as SEQ ID No. 9.

22. A nucleotide sequence comprising at least the sequence shown as SEQ ID No. 10.

23. A nucleotide sequence comprising at least the sequences shown as SEQ ID No. 9 and SEQ ID No. 10.

24. A nucleotide sequence comprising at least the sequences shown as SEQ ID No.

9 and SEQ ID No. 10, wherein SEQ ID No. 9 is nearer the 5' end than SEQ ID No. 10.

25. A nucleotide sequence according to any one of paragraphs 21 to 24 wherein the nucleotide sequence codes for an enzyme capable of exhibiting 0-1,4-endoglucanase activity.

26. A nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity, wherein the nucleotide sequence comprises at least the sequence shown as SEQ ID No. 11.

27. A nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity, wherein the nucleotide sequence comprises at least the sequence shown as SEQ ID No. 12, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

28. A nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity, wherein the nucleotide sequence comprises at least the sequence shown as SEQ ID No. 13.

29. A nucleotide sequence coding for an enzyme capable of exhibiting 0-1 ,4- endoglucanase activity, wherein the nucleotide sequence comprises at least the

sequence shown as SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

30. A nucleotide sequence coding for an enzyme capable of exhibiting 0-1 ,4- endoglucanase activity, wherein the nucleotide sequence comprises at least two of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

31. A nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity, wherein the nucleotide sequence comprises at least three of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.

32. A nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity, wherein the nucleotide sequence comprises at least four of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.

33. A nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity, wherein the nucleotide sequence comprises at least five of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.

34. A nucleotide sequence coding for an enzyme capable of exhibiting 0-1 ,4- endoglucanase activity, wherein the nucleotide sequence comprises at least all of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.

35. A nucleotide sequence according to any one of paragraphs 30 to 34 wherein if two or more of SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14 are present then the location of SEQ ID No.

9 is nearer the 5' end than SEQ ID No. 10 which is nearer the 5' end than SEQ ID No. 11 which is nearer the 5' end than SEQ ID No. 12 which is nearer the 5' end than SEQ ID No. 13 which is nearer the 5' end than SEQ ID No. 14, but wherein the enzyme is not the cellulase sequence of WO 97/13862.

36. A nucleotide sequence having the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.

37. A nucleotide sequence comprising at least the sequence shown as SEQ ID No.

19.

38. A nucleotide sequence comprising at least the sequence shown as SEQ ID No. 20.

39. A nucleotide sequence comprising at least the sequence shown as SEQ ID No. 21.

40. A nucleotide sequence comprising at least the sequence shown as SEQ ID No. 22.

41. A nucleotide sequence according to any one of paragraphs 20 to 40 operatively linked to a promoter.

42. A construct comprising or capable of expressing the invention according to any one of paragraphs 1 to 41.

43. A vector comprising or capable of expressing the invention of any one of paragraphs 1 to 42.

44. A plasmid comprising or capable of expressing the invention of any one of paragraphs 1 to 43.

45. A transgenic organism comprising or capable of expressing the invention according to any one of paragraphs 1 to 44.

46. A transgenic organism according to paragraph 45 wherein the organism is a fungus.

47. A transgenic organism according to paragraph 46 wherein the organism is a filamentous fungus, preferably Aspergillus.

48. A transgenic organism according to paragraph 45 wherein the organism is a plant.

49. A transgenic organism according to paragraph 45 wherein the organism is a yeast or a bacterium.

50. A process of preparing an enzyme according to any one of paragraphs 1 to 19 comprising expressing a nucleotide sequence according to any one of paragraphs 20 to 41.

51. A process according to paragraph 50 wherein the enzyme has the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof, and the nucleotide sequence has the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof.

52. Use of an enzyme according to any one of paragraphs 1 to 19 or an enzyme prepared by a process according to paragraph 50 or 51 to degrade a glucan.

53. A glucanase enzyme having the ability to degrade 0-1,4-glucosidic bonds, which is immunologically reactive with an antibody raised against a purified glucanase enzyme having the sequence shown as SEQ. I.D. No. 1.

54. Use of an enzyme according to any one of paragraphs 1 to 19 or an enzyme prepared by a process according to paragraph 50 or 51 to prepare a foodstuff (such as a feed).

55. A foodstuff comprising or prepared from the enzyme according to any one of paragraphs 1 to 19 or an enzyme prepared by a process according to paragraph 50 or 51.

56. A promoter having the sequence shown as or contained within SEQ. I.D. No. 15 or a variant, homologue or fragment thereof.

57. A promoter according to paragraph 56 operatively linked to a GOI.

58. A promoter according to paragraph 57 wherein the promoter is operatively linked to a GOI, wherein the GOI comprises a nucleotide sequence according to any one of paragraphs 20-40.

59. NCIMB 40825.

60. NCIMB 40826.

61. A terminator sequence obtainable from the sequence presented in Figure 1.

Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

In the following pages, a series of sequences are presented.

These sequences are:

SEQ. ID. No. 1. Enzyme amino acid sequence

SEQ. ID. No. 2. Nucleotide encoding sequence for the enzyme SEQ. ID. No. 3. Peptide sequence

SEQ. ID. No. 4. Peptide sequence

SEQ. ID. No. 5. Peptide sequence

SEQ. ID. No. 6. Peptide sequence

SEQ. ID. No. 7. Peptide sequence SEQ. ID. No. 8. Peptide sequence

SEQ. ID. No. 9. Nucleotide encoding sequence for a peptide sequence

SEQ. ID. No. 10. Nucleotide encoding sequence for a peptide sequence

SEQ. ID. No. 11. Nucleotide encoding sequence for a peptide sequence

SEQ. ID. No. 12. Nucleotide encoding sequence for a peptide sequence SEQ. ID. No. 13. Nucleotide encoding sequence for a peptide sequence

SEQ. ID. No. 14. Nucleotide encoding sequence for a peptide sequence

SEQ. ID. No. 15. Nucleotide sequence

SEQ. ID. No. 16. Nucleotide and enzyme sequences

SEQ. ID. No. 17. N terminal sequence SEQ. ID. No. 18. Precursor protein sequence

SEQ. ID. No. 19. Nucleotide encoding sequence for a peptide sequence

SEQ. ID. No. 20. Nucleotide encoding sequence for a peptide sequence

SEQ. ID. No. 21. Nucleotide encoding sequence for a peptide sequence

SEQ. ID. No. 22. Nucleotide encoding sequence for a peptide sequence

SEQUENCES

SEQUENCE ID NO . 1

Ala Ser Val Phe 4 Glu Trp Phe Gly Ser Asn Glu Ser Gly Ala Glu Phe Gly Thr Asn He 5 10 15 20

Pro Gly Val Trp Gly Thr Asp Tyr He Phe Pro Asp Pro Ser Ala He 25 30 35

Ser Thr Leu He Asp Lys Gly Met Asn Phe Phe Arg Val Gin Phe Met

40 45 50

Met Glu Arg Leu Leu Pro Asp Ser Met Thr Gly Ser Tyr Asp Glu Glu 55 60 65 Tyr Leu Ala Asn Leu Thr Thr Val He Lys Ala Val Thr Asp Gly Gly 70 75 80

Ala H.s Ala Leu Val Asp Pro His Asn Tyr Gly Arg Tyr Asn Gly Glu 85 90 95 100

He lie Ser Ser Thr Ser Asp Phe Gin Thr Phe Trp Glu Asn Leu Ala 105 110 115

Gly Gin Tyr Lys Asp Asn Asp Leu Val Met Phe Asp Thr Asn Asn Glu

120 125 130

Tyr His Asp Met Asp Gin Asp Leu Val Leu Asn Leu Asn Gin Ala Ala 135 140 145 He Asn Gly He Arg Ala Ala Gly Ala Thr Ser Gin Tyr He Phe Val 150 155 160

Glu Gly Asn Ser Trp Thr Gly Ala Trp Thr Trp Val Asp Val Asn Asp 165 170 175 180

Asn Met Lys Asn Leu Thr Asp Pro Glu Asp Lys He Val Tyr Glu Met 185 190 195

His Gin Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Glu Thr Cys Val

200 205 210

Ser Glu Thr He Gly Lys Glu Arg Val Thr Glu Ala Thr Gin Trp Leu 215 220 225 Lys Asp Asn Lys Lys Val Gly Phe He Gly Glu Tyr Ala Gly Gly Ser 230 235 240

Asn Asp Val Cys Arg Ser Ala Val Ser Gly Met Leu Glu Tyr Met Ala 245 250 255 260

Asn Asn Thr Asp Val Trp Lys Gly Ala Ser Trp Trp Ala Ala Gly Pro 265 270 275

Trp Trp Gly Asp Tyr He Phe Ser Met Glu Pro Pro Asp Gly Thr Ala

280 285 290

Tyr Thr Gly Met Leu Asp He Leu Glu Ala Tyr Leu * 295 300 305

SEQUENCE ID NO 2

GCG TCT GTG TTT GAA GGG TTC GGA TCG AAT GAG TCG GGT GCT GAG TTT GGA ACC AAT ATC CCT GGG GTT TGG GGA ACC GAC TAC ATC TTC CCC GAC CCC TCT GCC ATC TCT ACG TTG ATT GAC AAG GGG ATG AAC TTC TTC CGC GTC CAG TTC ATG ATG GAG AGG TTG CTG CCC GAC TCG ATG ACT GGT TCA TAT GAT GAG GAG TAT CTG GCC AAC TTG ACG ACA GTG ATA AAA GCG GTA ACG GAC GGA GGC GCC CAT GCG CTT GTC GAC CCT CAT AAC TAT GGC AGA TAG AAC GGC GAG ATC ATC TCC AGC ACG TCA GAC πc CAG ACC TTC TGG GAG AAC CTG GCG GGC CAG TAC AAA GAT AAC GAC CTG GTC ATG TTT GAC ACT GAC AAC GAA TAT CAC GAC ATG GAC CAG GAT CTC GTG CTG AAC CTC AAC CAA GCA GCC ATT AAC GGC ATC CGC GCC GCA GGT GCG ACC AGC CAG TAC ATC TTC GTC GAA GGC AAC TCC TGG ACC GGC GCC TGG ACG TGG GTC GAC GTC AAC GAC AAC ATG AAG AAT TTG ACC GAC CCC GAA GAC AAG ATC GTC TAT GAA ATG CAC CAG TAC CTA GAC TCC GAC GGT TCC GGC ACT TCG GAG ACC TGC GTG TCC GAG ACC ATC GGA AAA GAG CGG GTC ACT GAA GCT ACA CAG TGG CTG AAG GAC AAT AAG AAG GTC GGC TTC ATA GGC GAA TAT GCC GGG GGT TCC AAT GAT GTA TGT CGG AGT GCC GTG TCG GGG ATG CTG GAG TAC ATG GCG AAT AAC ACC GAC GTA TGG AAG GGT GCG TCG TGG TGG GCA GCC GGG CCA TGG TGG GGA GAC TAC ATT TTC AGC ATG GAG CCC CCA GAT GGA ACT GCG TAC ACG GGT ATG CTG GAT ATC CTG GAG GCA TAT cπ

SEQUENCE ID

Asp Ser Met Thr Gly Ser Tyr

SEQUENCE ID NO 4

Thr Val He Lys Ala Val

SEQUENCE ID NO 5 Ser Ser Thr Ser Asp

SEQUENCE ID NO 6 Gly Gin Tyr Lys Asp

SEQUENCE ID NO 7

Glu Tyr Met Ala Asn

SEQUENCE ID NO 8 Gly Met Leu Asp He

SEQUENCE ID NO 9

GAC TCG ATG ACT GGT TCA TAT

SEQUENCE ID NO 10 ACA GTG ATA AAA GCG GTA

ACGGGGCAGG AAGTTCCTGT TCTGAATGTT CCGCCGACGA GTCGCAGTCC GTGTTTTGAT 60 CAGGTAGAGT CGTCAGCAGC ATGCGGAGTT ATπGGCCGG TTTTTAATGA GAATTTGGAG 120

GGGATGGTGG ATGGATTTAA TAAGGAGAAG GGGGGATGTA AGTCTTTCTT TTTCTTTCTT 180

TCTTTCTTTC TTTCTTTCTT TTTTTTTTTT TTTTTTTTTT GGTTTCTTTG TGTGTGATGG 240

GTGGAAAGGA GAAAAGCTAA CTAGGATGTG TACGTACTAA GGTAACTACC GTCCTCTATG 300

ACTCGTGGTC ATTCATGACG AAAATTTTGG ATGATCCGAC AGCGTACGGA TTCCCCAATG 360 CCACGTGTAT CGATGATGAT GGGACGTCGT GTATTTGGTG GAATAATTAT CATCCCGGAA 420

TGAAGTATCA TCTTTTGCAG GCAGAGGATA TGAAGTGGGG TAGATGGGGG GGTTTGGGGC 480

TTGGTAGTAG TATTATTATT ATAGTATATT ATTTAAAGGT TGGATAGTAT TGCTGCTATA 540

CTACTATATT TGAGATGATA TATATACTAG TAGTGGTTTG GTATGGAACT TCTAGTTACT 600

GTTGTCAATG CTCAGCTCAG TTTGTTCATT GGTTCATTGG TTCATTGGTT CATTGTTGAT 660 ATGTAGTATA GACTGCACCG GTTATATATT TTGAGGAGGG ACTGCCTACG GTATTCTGGC 720

TGAATGCATC GCTACTATTA CCATGTCACT GACAGGAAAG CTCCACCTAT GCACTACTCT 780

ATCCTCTCAG ATGTTGATCC TTCTTCTCCC GTCACGGACC TCATTTCCGT ATCAGTTTCA 840

TGACCCTGAC CCTGCCAAGG CGGATCATTG ACCGAGCGTA GTCCCTGCTT CGACTTTATC 900

CGCCCTATAG GCATTGATGA TATCTGCAGG TGTGAAGAAC TGCAACATTT CCTGTTTGAA 960 TATCCGCTAT TCTCTACATT CGGATGTTTC AACTTGAATA AACGGCGGAG GAGATGTCTG 1020

GCTAAGTTGC TACCTCCGTG CTGGATTGGG ATAGTTGGAG TAGGAACGAA AATATAAAGG 1080

ATGGATACTC TCATCTCCTT GGACACTAAA TGTAGACTCT TAATCGAGCA GTCATC 1136

SEQUENCE ID NO 16

0) SEQUENCE CHARACTERISTICS

(A) LENGTH 3447 base pairs

(B) TYPE nucleic acid (O STRANDEDNESS double

(D) TOPOLOGY linear (n) MOLECULE TYPE. DNA (genomic) (xi) SEQUENCE DESCRIPTION ACGGGGCAGG AAGTTCCTGT TCTGAATGTT CCGCCGACGA GTCGCAGTCC GTGTTTTGAT 60 CAGGTAGAGT CGTCAGCAGC ATGCGGAGTT ATTTGGCCGG TTTTTAATGA GAATTTGGAG 120 GGGATGGTGG ATGGATTTAA TAAGGAGAAG GGGGGATGTA AGTCTTTCTT TTTCTTTCTT 180 TCTTTCTTTC TTTCTTTCTT TTTTTTTTTT TTTTTTTTπ GGTTTCTTTG TGTGTGATGG 240 GTGGAAAGGA GAAAAGCTAA CTAGGATGTG TACGTACTAA GGTAACTACC GTCCTCTATG 300 ACTCGTGGTC ATTCATGACG AAAATTTTGG ATGATCCGAC AGCGTACGGA TTCCCCAATG 360 CCACGTGTAT CGATGATGAT GGGACGTCGT GTATTTGGTG GAATAATTAT CATCCCGGAA 420 TGAAGTATCA TCTTTTGCAG GCAGAGGATA TGAAGTGGGG TAGATGGGGG GGπTGGGGC 480 TTGGTAGTAG TATTATTATT ATAGTATATT ATTTAAAGGT TGGATAGTAT TGCTGCTATA 540 CTACTATAπ TGAGATGATA TATATACTAG TAGTGGTπG GTATGGAACT TCTAGTTACT 600 GTTGTCAATG CTCAGCTCAG TπGTTCAπ GGπCATTGG πCAπGGπ CATTGTTGAT 660 ATGTAGTATA GACTGCACCG GTTATATATT πGAGGAGGG ACTGCCTACG GTAπCTGGC 720 TGAATGCATC GCTACTAπA CCATGTCACT GACAGGAAAG CTCCACCTAT GCACTACTCT 780 ATCCTCTCAG ATGπGATCC TTCπCTCCC GTCACGGACC TCAπTCCGT ATCAGTTTCA 840 TGACCCTGAC CCTGCCAAGG CGGATCAπG ACCGAGCGTA GTCCCTGCπ CGACπTATC 900 CGCCCTATAG GCATTGATGA TATCTGCAGG TGTGAAGAAC TGCAACATπ CCTGπTGAA 960 TATCCGCTAT TCTCTACATT CGGATGTTTC AACTTGAATA AACGGCGGAG GAGATGTCTG 1020 GCTAAGTTGC TACCTCCGTG CTGGATTGGG ATAGπGGAG TAGGAACGAA AATATAAAGG 1080 ATGGATACTC TCATCTCCTT GGACACTAAA TGTAGACTCT TAATCGAGCA GTCATC 1136 ATG AAG Tπ CAG AGC ACT CTG CTT CTT GCC GCC GCG GCT GGT TCC GCG 1184 Met Lys Phe Gin Ser Thr Leu Leu Leu Ala Ala Ala Ala Gly Ser Ala -28 -25 -20 -15 πG GCT GTG CCT CAT GGT CCT GGA CAT AAG AAG AGG GCG TCT GTG TTT 1232 Leu Ala Val Pro His Gly Pro Gly His Lys Lys Arg Ala Ser Val Phe

-10 -5 1

GAA T GTAAGGGTCC ATTACCTCGT CGTTGCGAAG CTGAACAGAA TAG GG TTC 1284 Glu Trp Phe

5 GGA TCG AAT GAG TCG GGT GCT GAG Tπ GGA ACC AAT ATC CCT GGG GTT 1332 Gly Ser Asn Glu Ser Gly Ala Glu Phe Gly Thr Asn He Pro Gly Val 10 15 20 TGG GTATGTATCA TTGCCCAAGT ATAGACGTAT ACTGATGTCG CAG GGA ACC GAC 1387 Trp Gly Thr Asp

25 TAC ATC TTC CCC GAC CCC TCT GCC ATC TCT ACG TTG ATT GAC AAG GGG 1435 Tyr He Phe Pro Asp Pro Ser Ala He Ser Thr Leu He Asp Lys Gly 30 35 40

ATG AAC TTC TTC CGC GTC CAG TTC ATG ATG GAG AGG TTG CTG CCC GAC 1483 Met Asn Phe Phe Arg Val Gin Phe Met Met Glu Arg Leu Leu Pro Asp

45 50 55

TCG ATG ACT GGT TCA TAT GAT GAG GAG TAT CTG GCC AAC TTG ACG ACA 1531 Ser Met Thr Gly Ser Tyr Asp Glu Glu Tyr Leu Ala Asn Leu Thr Thr 60 65 70 75

GTAAGATGAC TCCAGTCTAT GTTGAGTAGT ACTGACGAGA TAG GTG ATA AAA GCG 1586

Val He Lys Ala GTA ACG GAC GGA GGC GCC CAT GCG CTT GTC GAC CCT CAT AAC TAT GGC 1634 Val Thr Asp Gly Gly Ala His Ala Leu Val Asp Pro His Asn Tyr Gly 80 85 90 95

AGA TA GTAAGTATGC AGTCCCCGTA GGTGATGCCT GCTAAAAAAA CAG C AAC 1686 Arg Tyr Asn

GGC GAG ATC ATC TCC AGC ACG TCA GAC TTC CAG ACC TTC TGG GAG AAC 1734 Gly Glu lie He Ser Ser Thr Ser Asp Phe Gin Thr Phe Trp Glu Asn 100 105 110

CTG GCG GGC CAG TAC AAA GAT AAC GAC CTG GTC ATG TTT ^{^} GAC ACT A 1780 Leu Ala Gly Gin Tyr Lys Asp Asn Asp Leu Val Met Phe Asp Thr 115 120 125 GTAAGTACCC ACAATCCTGT CAAGAATCAT GCTGACAAGG CAG AC AAC GAA TAT 1834

Asn Asn Glu Tyr 130 CAC GAC ATG GAC CAG GAT CTC GTG CTG AAC CTC AAC CAA GCA GCC ATT 1882 His Asp Met Asp Gin Asp Leu Val Leu Asn Leu Asn Gin Ala Ala He 135 140 145

AAC GGC ATC CGC GCC GCA GGT GCG ACC AGC CAG TAC ATC πc GTC GAA 1930 Asn Gly He Arg Ala Ala Gly Ala Thr Ser Gin Tyr He Phe Val Glu 150 155 160 165

GGC AAC TCC TGG ACC GGC GCC TGG ACG TGG GTC GAC GTC AAC GAC AAC 1978 Gly Asn Ser Trp Thr Gly Ala Trp Thr Trp Val Asp Val Asn Asp Asn

170 175 180

ATG AAG AAT πG ACC GAC CCC GAA GAC AAG ATC GTC TAT GAA ATG CAC 2026 Met Lys Asn Leu Thr Asp Pro Glu Asp Lys He Val Tyr Glu Met His 185 190 195 CAG TAC CTA GAC TCC GAC GGT TCC GGC ACT TCG GAG ACC TGC GTG TCC 2074 Gin Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Glu Thr Cys Val Ser

200 205 210

GAG ACC ATC GGA AAA GAG CGG GTC ACT GAA GCT ACA CAG TGG CTG AAG 2122 Glu Thr He Gly Lys Glu Arg Val Thr Glu Ala Thr Gin Trp Leu Lys 215 220 225

GAC AAT AAG AAG GTC GGC TTC ATA GGC GAA TAT GCC GGG GGT TCC AAT 2170 Asp Asn Lys Lys Val Gly Phe He Gly Glu Tyr Ala Gly Gly Ser Asn 230 235 240 245

GAT GTA TGT CGG AGT GCC GTG TCG GGG ATG CTG GAG TAC ATG GCG AAT 2218 Asp Val Cys Arg Ser Ala Val Ser Gly Met Leu Glu Tyr Met Ala Asn

250 255 260

AAC ACC GAC GTA TGG AAG GGT GCG TCG TGG TGG GCA GCC GGG CCA TGG 2266 Asn Thr Asp Val Trp Lys Gly Ala Ser Trp Trp Ala Ala Gly Pro Trp

265 270 275

TGG GGA GAC TAC ATT TTC AGC ATG GAG CCC CCA GAT GGA ACT GCG TAC 2314 Trp Gly Asp Tyr He Phe Ser Met Glu Pro Pro Asp Gly Thr Ala Tyr 280 285 290

ACG GGT ATG CTG GAT ATC CTG GAG GCA TAT CTT TGA GGTCTGGGTG 2360 Thr Gly Met Leu Asp He Leu Glu Ala Tyr Leu * 295 300 305

GGTCGCAAGA TGCGGTGCAT CGGAGAACTA TACGGAGTAT CTTGTCCGGG TGGGCGGTGG 2420

TGGTACAGAG AGGAGTACTA GTATACATTA GTGGCAGCGC ACTGACTGAC GTCACAAGGC 2480

ATTCCGTTTT GGACGCTCCA TCCπGTGCA TTCTGTTGAG TGTTACCTGT AGGCACTAGG 2540

CAGTGAGGCA CGCCTGTCGC TGAAATAGTG CACGTTGTAC ACCGTAATAA GTAGTCAGTG 2600

GATTCTTATC AACGATCATC GAGTATAGCT CAACGAATTG CGTATGAAGT AGGGATCTGA 2660 CGACCCACTG GTCATAGGAT ATACATAGπ CTCCTCATCA GCCπATCGG ATAGATGATG 2720 πCGTGGATG AACACCGCGA TGTGAATGCA AGAGAAAACA ACGAGTGCAG GTATGGCAGT 2780

GATGGGATGG TCGAGGCCGA AGAGGAACCA TGGATGAGAG ACGGGCCCGC CAAGACGCTC 2840

AACGTAAGCC CGGGπAATA GTCTGGTCGA TCGGATGAGG GTCAATCCCG GCACTTTCCC 2900

GTCTGCCGCG ACGGTTGAGA ATTGAAACAG CAGAπTGCT TCTπTCATA GGTGGTCATG 2960 AAGTCATGAC TCGACTAGTC GTGGTTGATT ATGCCAGTTG CCπGCTAAG AATACTCCGA 3020

GGTGATGATC CATCAAGCAC TGπGAGATA ACTCACGGGG CACTTCATCG GACGTCCCTG 3080

AGGCAGTACC ATGGGACGGA CAGCAGGGAA TATGTCGATG CAGAAGAAAC TTCCGAGAAT 3140

GTCATGCATA CCAATAAAAG CATCCAACAT GGACCGCGTA TTGCCAAGCT CAGAATGATG 3200

AAGCCTAGTC AGTGGAGGTT CCGTGGTTGG TGGGCTGAGG GTGAGACACG CTCATCCAGA 3260 GATCCAGGCT CACAGGAAAT CTTGGGAATG AGATGGTTAA GCAGATTCCC GATGATGACG 3320

ATCGCTACTT CACTGTATGG TGCCTCTTTG GTCCACCGTG TATCAGATAG GTTGGTTGTG 3380

GTGATACGAT CCAACTCGAC CCGATCGACA TAAAπGCGC AGGACTACTC AGTTAATAGC 3440

CAGAATT 3447

SEQUENCE ID NO 17

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 18 amino acids

(B) TYPE amino acid (C) STRANDEDNESS. single

(D) TOPOLOGY: linear (n) MOLECULE TYPE: peptide (v) FRAGMENT TYPE. N-terminal (xi) SEQUENCE DESCRIPTION' Ala Ser Val Phe Glu Trp Phe Gly Ser Asn Glu Ser Gly Ala Glu Phe 1 5 10 15

Gly Thr

SEQUENCE ID NO. 18 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH 333 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:

Met Lys Phe Gin Ser Thr Leu Leu Leu Ala Ala Ala Ala Gly Ser Ala

-28 -25 -20 -15

Leu Ala Val Pro His Gly Pro Gly His Lys Lys Arg Ala Ser Val Phe

-10 -5 1 Glu Trp Phe Gly Ser Asn Glu Ser Gly Ala Glu Phe Gly Thr Asn He

5 10 15 20

Pro Gly Val Trp Gly Thr Asp Tyr He Phe Pro Asp Pro Ser Ala He

25 30 35

Ser Thr Leu He Asp Lys Gly Met Asn Phe Phe Arg Val Gin Phe Met 40 45 50

Met Glu Arg Leu Leu Pro Asp Ser Met Thr Gly Ser Tyr Asp Glu Glu

55 60 65

Tyr Leu Ala Asn Leu Thr Thr Val He Lys Ala Val Thr Asp Gly Gly

70 75 80 Ala His Ala Leu Val Asp Pro His Asn Tyr Gly Arg Tyr Asn Gly Glu

85 90 95 100

He He Ser Ser Thr Ser Asp Phe Gin Thr Phe Trp Glu Asn Leu Ala

105 110 115

Gly Gin Tyr Lys Asp Asn Asp Leu Val Met Phe Asp Thr Asn Asn Glu 120 125 130

Tyr His Asp Met Asp Gin Asp Leu Val Leu Asn Leu Asn Gin Ala Ala

135 140 145

He Asn Gly He Arg Ala Ala Gly Ala Thr Ser Gin Tyr He Phe Val 150 155 160

Glu Gly Asn Ser Trp Thr Gly Ala Trp Thr Trp Val Asp Val Asn Asp

165 170 175 180

Asn Met Lys Asn Leu Thr Asp Pro Glu Asp Lys He Val Tyr Glu Met

185 190 195 His Gin Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Glu Thr Cys Val

200 205 210

Ser Glu Thr He Gly Lys Glu Arg Val Thr Glu Ala Thr Gin Trp Leu

215 220 225

Lys Asp Asn Lys Lys Val Gly Phe He Gly Glu Tyr Ala Gly Gly Ser 230 235 240

Asn Asp Val Cys Arg Ser Ala Val Ser Gly Met Leu Glu Tyr Met Ala

245 250 255 260

Asn Asn Thr Asp Val Trp Lys Gly Ala Ser Trp Trp Ala Ala Gly Pro

265 270 275 Trp Trp Gly Asp Tyr He Phe Ser Met Glu Pro Pro Asp Gly Thr Ala

280 285 290

Tyr Thr Gly Met Leu Asp He Leu Glu Ala Tyr Leu * 295 300 305

SEQUENCE ID NO. 19

AAC CTG GCG GGC CAG TAC AAA GAT

SEQUENCE ID NO. 20

GGC CAG TAC AAA GAT AAC GAC CTG

SEQUENCE ID NO. 21

GCG TAC ACG GGT ATG CTG GAT ATC

SEQUENCE ID NO. 22 GGT ATG CTG GAT ATC CTG GAG GCA

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

sheet [ I

an additional sheet [~~]

for all designated States)

nature ofth indicanora e.g. "Accession

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3ύu)

sheet [x ^*

an additional sheet | |

for all designated States)

nature of the indications e g . "Accession

RE'."-_tl :' :u« _'r .- ^• .-_ . ;._ ^■ -; FOR THE PURPOSES Or .TINT PROCEDURE

Danisco Biotechnoloj Langebrogade 1 , PO Box 17, INTERNA IONAL FORM DK-1001 Copenhagen, Denmark. RECEIPT IN THE CAΞϊ J!" AN ORIGINAL DEPOSI". issued pursuanc i: =..._.« 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified a. cue :.cc:om of tills page

IDENTIFICATION OF THE HICROORCANISH

Iden lϊlca ion reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:

Escherichia coli

NCIMB 40825 K12 XLOLR pEglB-1

II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by: I I a scientific description

I λ I a proposed taxonomic designation

(Hark with a cross where applicable)

III. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by It on j_3 Au LB --i" ¹ *" °' ^the original deposit) ¹

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International

Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion)

INTERNATIONAL DEPOSITARY AUTHORITT

Na tei Signature(s) of person(s) having the power

*- :_*_> Ltd to represent the International Depositary Authority or of author iied official(s)ι

23 St £&ά*33 Cffcrθ

Address: Abβ ^j -d-sβfl ^β cceiαπci Date: * ⁶ August 1996

UK ΛB» 1«Y JΛ t iSi j/q -fl <&

¹ Where Rule 6.4(d) applies, such date is the date on which the status of international depo isltaϊy authority was acquired.

Form BP/4 (sole page)

3- =AJ Ξ3

Danisco Biotechnology, "c?-. ^v .

Langebrogade 1,

P0 Box 17,

DK-1001 Copenhagen,

Denmark. VIABILITY STATE_:E::T issued pursuant to Rule 10 .2 by the INTERNATIONAL DEPOSITARY AUTHORITY identif ied on the following page

SAMS A.ND ADDRESS OF THE PARTY TO WHOM THE VI ABILITY STATEMENT is isscεo J

I . DEPOSITOR II. IDEN IFICATION OF THE MICRCORCAKISM

Naise: Accession nu_r.ber given by the

INTERNATIONAL DEPOSITARY AOTHORITY:

NCIMB 40825

Address: AS ABOVE Date of the deposit ar of the trans

13 August 1996

III. VIA3XLΣTY STATEMENT

The viability of the microorganism identified under II above was tested on 15 August 1996 ■ ⁰ⁿ -*t date, the said aicrcorganism was

CD viable

no longer viable

¹ Indicate the date of the original deposit or, where a new deposit or a transfer has been nude, the most recent relevant date (date of the new deposit or date of the transfer).

² In the cases referred to tn Rule 10.2(a) (ti) and (til), refer to the most reeent viabtlity test.

Mark with a cross the applicable box.

For= 3.V3 (first page)

rtll in if the information has been requested and if the results of the test were negative .

?ora 3?/9 ( second and last patje)

Danisco Biotechnology, angebrogade 1,

P0 Box 17,

DK-1001 Copenhagen, INTERNATIONA FORM

Denmark.

RECEIPT IN THE CASE Of AN GR-C-NΛ-. DEPOSI'. issued pursuant tj RuLe 7.1 by the INTERNATIONAL CE?0S:7AJ.Y AUTHORITY identic led _t tπe bottom of this page

Form BP/4 (sole page)

3U A.? — ώ • TΛ_-__ATV W -1 - .--.i

R£CCC:;:T:C or ΓHΪ

P' PJCS ΞS

Danisco Biotechnology, 1

Langebrogade 1,

PO Box 17,

DK-1001 Copenhagen ,

Denmark. VIABILITY STATΪ:Ξ::T issued pursuant to Rule 10 . 2 by t e ΣΪJTΣRNATICNAL DEPOSITARY AUTHORITY identified on the following page

_IA. ^U _E AND ADDRESS OF HE PARTY

TO WHOM TKE VIABILITY STATEMENT IS ISSUED

I . DEPOSITOR . II . IDENTIFICATION OF TKΞ IC CORCλϊ.IS.V

Naee: Accession aiaber given by the INTERNATIONAL DEPOSITARY ACTKOZUTY:

Address: AS ABOVE NCIMB 40826 Date of the deposit or of the transfer:

13 August 1996

XII. VIABILITY STATESEST

The viability of the 3U.eroorgan-_.sm identified under IX above was tested on 15 August 1996 On that date , the said aicroorganism was

I ³

I ^X I viable

no longer viable

¹ Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the aost recent relevant date (date of the new deposit or date of the transfer).

² In the cases referred to in Rule 10.2(a) (ii) and (iii) , refer to the most recent viability test.

Mark with a cross the applicable box.

For= 3?/9 (first pace)

ISTE-CIATIONAL OEPOSITA.T. AUTHORITY

Nacre: Signature (s) of person. s) havlr.5 the powe: to represent -the International Depositary Authority or of authorised official(s):

Addres : NCfcB Ld

23 St fc ββε* D«w owman θαctftaπe

'IK AOO t-HY Date: 16 August 1996 <—

Fill in if the information has been requested and if the results of the test were negative.

"ora ^' 3?/9 (second and last pace)