MXPA97007112A - Endo-beta-1,4-glucanase from aspergil - Google Patents

Abstract

A glucanase enzyme is described, and a nucleotide sequence coding for it and a promoter to control the expression of the same are described.

Description

END0-BET -l, 4-GLUCANASfl fl PARTIR OF ASPERGILLUS DESCRIPTIVE MEMORY The present invention relates to an enzyme.

In addition, the present invention relates to a nucleotide sequence coding for the enzyme. Also, the present invention relates to a promoter, wherein the promoter can be used to control the expression of the nucleotide sequence encoding the enzyme. In particular, the enzyme of the present invention is a glucanase enzyme, that is, an enzyme that can degrade β-1, -glucosidic bonds. It is known that it is desirable to direct the expression of a gene of interest ("GOT") in certain tissues of an organism - such as a filamentous fungus (such as Aspergillus niger) or even a crop plant. The resulting protein or enzyme may be useful for the organism itself. For example, it may be desirable to produce culture protein products with an optimized amino acid composition and thus increase the nutritional value of a culture. For example, the crop can be made more useful as a food. Alternatively, it may be desirable to isolate the resulting protein or enzyme and then use the protein or enzyme to prepare, for example, food compositions. To this aspect, the resulting protein or enzyme may be a component of the food composition or may be used to prepare food compositions, including altering the characteristics or appearance of the 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, 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 in vivo gene activators. It is also desirable to use microorganisms, such as filamentous fungi, to prepare products from genes of interest through the use of promoters that are active in the microorganisms. The cell walls of vegetables and fruits consist largely of polysaccharide, the main components being pectin, cellulose and xyloglucan (R.R. Selvendran and 3. Robertson, IFR Report 1989). Numerous cell wall models have been proposed that attempt to incorporate the essential properties of strength and flexibility (P. Albersheirn, Sci. Am. 232, 81-95, 1975, P. Albersheim, Plant Biochem, 3rd edition (Bonner and Varner), Ac. Press, 1976; T. Hayashi, Ann. Rev. Plant Physiol. S Plant Mol. Biol., 40, 139-158, 1989). The composition of the cell wall of the plant is complex and variable. The polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the cell wall of the plant), hernicellulose (comprising several β-xylan chains, such as xyloglucans) and pectic substances (consisting of galacturonans and rhamnogalacturonans; arabinanos, and galactanos and arabinogalactanos). In particular, glucans are polysaccharides made exclusively from glucose subunits. Typical examples of glucans are starch and cellulose. Enzymes that degrade glucans are collectively known as glucanases. A typical glucanase is β-1,4-endoglucanase. Β-1, 4-endoglucanases have uses in many industries. For example, in the brewing industry, barley is used for the production of malt and, in recent years, as an aid in the brewing process. When the quality of the malt is not good, or the barley has been used as auxiliary, problems may arise with the high viscosity in the must due to ß-glucanoe from the barley. In this regard, barley contains large amounts of β-1,3 / 1, 4-glucans of very high molecular weight. When dissolved, these glucans produce high viscosity solutions, which can cause problems in some applications. For example, the high viscosity reduces the filtration capacity of the wort and can lead to unacceptably long filtration times. To avoid these problems, ß-glucanase has been traditionally added to the myoste to avoid such problems, that is, the problem with glucans can be avoided by the addition of enzymes, in particular, glucanases, which degrade the polymers. Additional information about these problems can be found in the Grinsted booklet called "Glucanase GV", the revisions made by Dr. CU. Bamforth (Brewers Digest, June 1982, pages 22-28, and Brewer's Guardian, September 1985, pages 21-26), and T. Godfrey's paper (Industrial Enzyology, The Application of Enzy in Industry, chapter 4.5). , pages 221-259). In the food industry, barley can be used for chicken feed as it is cheap, but again, ß-glucan can cause problems for the digestion of chickens. By adding ß-glucanase to the food, the degree of digestion efficiency can be increased. In addition, the feces of the hens on the food containing barley is sticky, which makes it difficult to remove and makes the eggs dirty. UO 93/2019 describes endo-β-l, 4-glucanase (EC No. 3.2.1.4). In accordance with UO 93/2019, these glucanases are a group of hydrolases that catalyze endo hydrolysis of 1,4-β-D-glycosidic bonds in cellulose, lichenine, β-D-glucans of cereals and other plant material containing parts cellulose Endo-l, 4-ß-D-glucan hydrolase is sometimes called endo-β-1,4-glucanase. UO 93/2019 endo-ß-1, 4-glucanase shows an optimum pH of 2.0 to 4.0, an isoelectric point of 2.0 to 3.5, a molecular weight between 30,000 and 50,000 and an optimum temperature of 30 to 70 ° C. Additional teachings about gl? Canoe can be found in UO 93/17101, in particular xyloglucans. In accordance with IO 93/17101, xyloglucans are 1,4-ß-gl? Rans which have been extensively substituted by α-1, 6-xylyl side chains, some of which are 1,2-ß-galactosylated. They are found in large quantities in the primary cell walls of dicotyledonous plants but also in certain seeds, where they have different functions. The xyloglucan of the primary cell wall is fucosylated. Xyloglucan is tightly bound with hydrogen to cellulose microfibrils and requires concentrated alkali or strong swelling agents to release it. CÁ- >; He believes that the xyloglucan forms transversal bridges between cellulose microfibrils, the cellulose network / xyloglucan for anso the larger load / elastic network of the wall. The suspension culture cells ruled with DCB (cell walls lacking cellulose) release xyloglucan into their medium, suggesting that xyloglucan is normally bound to cellulose. Hydrolysis of the xyloglucan from the primary cell wall has been demonstrated in segments of yellow squash hypocotyls growing in the dark, during growth induced by mitochondrial acid (K. Wabaybayashi et al., Plant Physiol., 95, 1070-1076 , 1991). It is believed that the endohydrolysis of xyloglucan from the wall contributes to the loss of the wall that accompanies the expansion of the cell (T. Hyashi, Ann, Rev. Plant Physiol., To Plant Mol. Biol., 40, 139- 168, 1989). It has also been shown that the average olecular weight of xyloglucan decreases during the ripening of the tomato fruit and this may contribute to the softening of the tissue that accompanies the ripening process (D ... J. Huber, 3. Amer. Soc. Hort Sci., 108 (3), 405-409, 1983). Certain seeds, v. gr. , Nast? Rt, contains up to 30% by weight of xyloglucan, stored in cotyledon cell walls, which serves as a reserve polysaccharide and is rapidly depolymerized during germination. It would be useful to increase the activity of glucanase, for example having a plant with a high concentration of glucanase for use in food, preferably using recombinant DNA techniques. The present invention seeks to provide an enzyme which has glucanase activity; preferably wherein the enzyme can be prepared in certain cells or tissues or in specific cells or tissues, such as in only one specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Asperglus, such as Aspergil us niger, or even a plant.

Also, the present invention seeks to provide an enzyme gene encoding the enzyme and that can be expressed preferably in specific cells or tissues, such as in certain or specific cells or tissues, of an organism, typically a filamentous fungus, preferably of the Aspergillus genus, such as Aspergillus niger or even a plant. In addition, the present invention seeks to provide a promoter that is capable of directing the expression of a gene of interest, such as a nucleotide sequence coding for the enzyme according to the present invention, preferably in certain or specific cells or tissues, such as in only a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergill's niger, or even a plant. Preferably the promoter is used in Aspergi 1 lus, where the product encoded by the gene of interest is excreted from the host organism to the surrounding medium. Moreover, the present invention attempts to provide constructs, vectors, plasmids, cells, tissues, organs and organisms comprising the gene of interest and / or the promoter, and methods for expressing thereto, preferably in specific cells or tissues, such a co the expression in only one specific cell or tissue of an organism, typically a filamentous fungus, preferably of the Aspergill's genus, or even a plant. In accordance with a first aspect of the present invention, an enzyme obtainable from Aspergillus is provided, wherein the enzyme has the following characteristics: a MW of 24,235 D ± 50 D; ? pl value of approximately 4; glucanase activity; and wherein the glucanase activity is endo-β-1,4-glucanase activity. In accordance with a second aspect of the present invention, an enzyme having the sequence shown as I.D. DE SEC No. 1 or a variant, homologue or fragment thereof. According to a third aspect of the invention, an enzyme encoded by the nucleotide sequence shown as I.D. DE SEC No. 2 or a variant, homologue or fragment thereof, or a sequence complementary thereto. According to a fourth aspect of the present invention, a nucleotide sequence encoding the enzyme according to the present invention is provided. According to a fifth aspect of the present invention, a nucleotide sequence having the sequence shown as co or I.D. is provided. DE SEC No. 2, or a variant, homologue or fragment thereof or a complementary sequence for it. In accordance with the sixth aspect of the present invention, a promoter having the sequence shown as T.D. DE SEC No. 3, or a variant, homologue or fragment thereof or a complementary sequence for the same. According to a seventh aspect of the present invention, a terminator having the nucleotide sequence shown as I.D. DE SEC No. 13, or a variant, homologous or fragment thereof or a complementary sequence for it. In accordance with an eighth aspect of the present invention, a signal sequence having the nucleotide sequence shown as I.D. is provided. DE SEC No. 14, or a variant, homologue or fragment thereof or a sequence complementary thereto. According to a ninth aspect of the present invention, there is provided a method for expressing a gene of interest through the use of a promoter, wherein the promoter is the promoter according to the present invention. According to a tenth aspect of the present invention, the use of a co-enzyme with the present invention to degrade a glucan is provided. According to an eleventh aspect of the present invention, NCIMB 40704 plasmid is provided, or a nucleotide sequence obtainable therefrom to express an enzyme capable of degrading arabinoxylan or to control the expression thereof or to control the expression of another gene of interest. In accordance with a twelfth aspect of the present invention,, a signal sequence having the sequence shown as I.D. DE SEC No. 15, or a variant, homologue or fragment thereof. According to thirteenth aspect of the present invention, a glucanase enzyme is provided which has the ability to degrade ß-l, 4-gl? Cosidic bonds, which is immunologically reactive with an antibody created against a glucanase enzyme Purified q? e has the sequence shown as ID DE SEQ No. 1. In accordance with a fourteenth aspect of the present invention, a promoter is provided which is inductible by glucose. In accordance with the fifteenth aspect of the present invention, the use of glucose is provided to induce a promoter. Other aspects of the present invention include constructs, vectors, plasmids, cells, tissues, organs and transgenic organisms that comprise the aforementioned aspects of the present invention. Other aspects of the present invention include methods for expressing or allowing expression or transforming any of the nucleotide sequence, the construct, the plasmid, the vector, the cell, the tissue, the organ or the organism, as well as the products. thereof. Additional aspects of the present invention include uses of the promoter to express genes of interest in culture media such as broth or in a transgenic organism.

Additional aspects of the present invention include uses of the enzyme to prepare or treat food products, including animal feeds. In the following text, the enzyme of the present invention is sometimes called Egla enzyme and the coding sequence for it is sometimes called the Egla gene.

In addition, the promoter of the present invention is sometimes referred to as the Egla promoter. Preferably, the enzyme is encoded by the nucleotide sequence shown as I.D. DE SEC No. 2, or a variant, homologue or fragment thereof or a sequence complementary thereto. Preferably, the nucleotide sequence has the sequence shown as I.D. DE SEC No. 2, or a variant, homologue or fragment thereof or a sequence complementary thereto. Preferably, the nucleotide sequence is operably linked to a promoter. Preferably, the promoter is the promoter having the sequence shown as I.D. DE SEC No. 3, or a variant, homologue or fragment thereof or a complementary sequence for the same. Preferably, the promoter of the present invention is operably linked to a gene of interest. Preferably, the gene of interest comprises a nucleotide sequence according to the present invention. In a preferred embodiment, the transgenic organism is homgo. For example, the organism can be a yeast, which would then be useful for example in the brewing industry. Preferably, the transgenic organism is a filamentous fungus, most preferably of the genus Aspergillus. In another preferred embodiment, the transgenic organism is a plant. In another preferred embodiment, the transgenic organism is a yeast. In this regard, yeast has been widely used as a vehicle for expression of heterologous genes. The species Saccharo yces cerevisiae has a long history of industrial use, including the use for expression of heterologous genes. The expression of heterologous genes in Saccharomycee cerevisiae has been reviewed by Goodey et al. (1987, Yeaet Biotechnology, DR Berry et al., Eds. Pp. 401-429, Alien and Un in, London) and by King et al. (1989, Molecular and Cell Biology of Yeasts, EF, Uton GT Yarronton, eds, pp. 107-133, Blackie, Glasgow). For several reasons, Saccharomyces cerevisiae is well studied for the expression of heterologous genes. First, it is not pathogenic for humans and is incapable of producing certain endotoxins. Second, it has a long history of safe use after centuries of commercial exploitation for various purposes. This has led to widespread acceptance by the public. Third, the extensive commercial use and research n dedicated to the organism have resulted in a wealth of knowledge about genetics and physiology, as well as large-scale fermentation characteristics of Saccharomyces cerevisiae. An additional advantage is that the yeasts are capable of post-translational modifications of proteins and therefore have the potential for glycosylation and / or secretion of heterologous gene products in the growth medium. A review of the principles of heterologous gene expression on Saccharomyces cerevisiae and the secretion of gene products is given by E. Hmchcliffe F. Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol. 5, Anthony H. Rose and 3. St? Art Harpson, ede, 2nd edition, Acadenic Press Ltd.) It is known that the glycosylation of enzymes expressed on yeast increases the heat stability of the enzyme. The increase in heat stability of the glucanase according to the present invention will make this enzyme suitable for use in the brewing industry and for use in the preparation of animal feed, ie, feed for chickens. It is known that yeast secrete a few proteins into the culture medium. This makes yeast a very attractive host for expression of heterologous genes, since the products of secretable genes can be easily recovered and purified.

Several types of yeast vectors are available, including vector integrators, which require recharge with the host genome for maintenance, and plasmids vectors that replicate autonomously. In order to prepare transgenic Saccharomyces, expression constructs are prepared by inserting a gene of interest (such as an alanase or SEQ ID No. 2) into a construct designated for expression in yeast. The constructs contain an active promoter in yeast fused to the gene of interest, usually using a promoter of yeast origin, such as the GAL1 promoter. The gene of interest can be fused to the signal sequence that directs the protein encoded by the gene of interest to be secreted. Typically, a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. An active ternate in the yeast gives f n to the expression system. Heterologous expression in yeast has been reported for several genes. The gene products can be glycosylated, which is advantageous for some enzymes intended for a specific application where heat tolerance is desirable. The proteins can be deposited intracellularly if the gene of interest is not fused to a signal sequence, or they can be secreted extracellularly if the gene of interest is equipped with an e-signal sequence. For the transformation of yeasts, several transformation protocols have been developed. For example, the transgenic Saccharornyces according to the present invention can be prepared following the teachings of Hinnen et al. (1978, Proceedings of the National Academy of Sciences of the U.S.: A: 75, 1929) Beggs, 3.O. (1978, Nature, London, 275, 104); and Ito, H. and others (1983, 3. Bacteriology 153, 163-168). The transformed yeast cells are selected using several selective markers. Among the markers used for transformation are a number of auxotrophic narcators such as LEU2, HIS4 and TRP1, and markers of resistance to dominant antibiotics such as markers of aminoglycoside antibiotics, e.g., G418. The highly preferred embodiments of each of the aspects of the present invention do not include any of the native enzyme, the native promoter or the native nucleotide sequence in its natural environment. Preferably, in any of the plasmid, the vector, such as an expression vector or a transformation vector, the cell, tissue, organ, organism or transgenic organism, the promoter is present in combination with at least one gene of interest. Preferably, the promoter and the gene of interest are stably incorporated into the genome of the transgenic organism. Preferably, the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, most preferably Aspergillus niger. The transgenic organism can even be a plant, such as a broad-leaved or dicotyledonous onoco plant. A highly preferred embodiment is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 24,235 D ± 50 D; a value of p [of about 4; glase activity; and wherein the glucanase activity is endo-β-1,4-glucanase activity; wherein the enzyme has the sequence shown as T.D. DE SEC No. 1, or a variant, homologue or fragment thereof. Another highly preferred embodiment is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 24,235 D ± 50 D; a pT-value of about 4; glase activity; and wherein the activity of gl? canase is endo? -l, -gl? canase activity; wherein the enzyme is encoded by the nucleotide sequence shown as I.D. DE SEC No.?, Or a variant, homologue or fragment thereof, or a sequence complementary thereto. The advantages of the present invention are that it provides a means for preparing a gl? Canase enzyme and the n-cleotide sequence encoding it. In addition, it provides a promoter that can control the expression of that, or of another nucleotide sequence.

Other advantages of the present invention are that the enzyme can be used to prepare useful foods containing cereals, such as barley, corn, rice, etc. Therefore, the present invention provides an enzyme having glucanase activity, wherein the enzyme can be prepared in certain or specific cells or tissues, such as in only a specific cell or tissue, of an organism, typically a filamentous fungus. , preferably of the genus Aspergillus, such as Aspergillus niger. The enzyme can even be prepared in a plant. Also, the present invention provides a gene of interest that encodes the enzyme and that can be expressed preferably in specific cells or tissues, such as certain or specific cells or tissues, of the organism, typically a filamentous fungus, preferably of the genus. Aspergill? S, such as Aspergillus niger. The gene of interest can even be expressed in a plant. In addition, the present invention provides a promoter that is capable of directing the expression of a gene of interest, such as a nucleotide sequence encoded for the enzyme according to the present invention, preferably in certain specific cells or tissues, such as or in only one specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus. us niger, or even a plant. Preferably, the promoter is used in Aspergill? S wherein the product encoded by the gene of interest is excreted by the host organism to the surrounding medium. The promoter may even be designed (if necessary) to express a gene of interest in a plant. The present invention also provides consorrers, vectors, plasmids, cells, tissues, organs? organisms comprising the gene of interest and / or the promoter, as well as methods for expressing thereto, preferably in specific cells or tissues, such as expression in only one cell or tissue specific to an organism, typically a filamentous fungus, preferably of the genus Aspergill? s, or even a plant. The terms "variant", "homologous" or "fragment" in relation to the enzyme include any substitution of, variation of, modification of, replacement of, deletion from, or addition of, one (or more) amino acids of or for the sequence as long as the resulting amino acid sequence has glycanase activity, preferably having at least the same enzyme activity as shown in the sequence listings (SEQ ID No. 1 or 12). In particular, the term "homologous" covers homology with respect to structure and / or function, as long as the resulting enzyme has glucanase activity. With respect to sequence homology, preferably there is at least 75%, most preferably at least 85%, more preferably at least 90% homology with SEQ ID NO. 1 shown in the attached sequence listings. Very preferably there is at least 95%, more preferably at least 98% homology with SEQ ID NO. 1 shown in the attached sequence lists. The terms "variant", "homologous" or "fragment" in relation to the nucleotide sequence coding for the enzyme include any substitution, variation, modification, replacement, deletion, or addition of, one (or more) nucleic acids of or for the sequence, as long as the resulting nucleotide sequence codes for an enzyme which has glycanase activity, preferably having at least the same enzyme activity as shown in the sequence listings ( SEC ID No. 2 or 12). In particular, the term "homologous" covers homology with respect to structure and / or function, so long as the resulting nucleotide sequence codes for an enzyme having gl? Canase activity. With respect to sequence homology, preferably there is at least 75%, most preferably at least 85%, more preferably at least 90% homology with SEQ ID NO. 2 shown in the attached sequence listings. Most preferably there is at least 95%, more preferably at least 98% homology with SEQ ID NO. 2 shown in the attached sequence lists. The terms "variant", "homologue" or "fragment" in relation to the promoter include any substitution, variation, modification, replacement, deletion, or addition of, one (or more) nucleic acids of or for the sequence as long as the resulting nucleotide sequence has the ability to act as a promoter in an expression system-such as the transformed cell or the transgenic organism in accordance with the present invention. In particular, the term "homologous" covers homology with respect to structure and / or function, as long as the resulting nucleotide sequence has the ability to act as a promoter. With respect to sequence homology, preferably there is at least 75%, and preferably at least 85%, more preferably at least 90% homology with SEQ ID NO. 3 shown in the attached sequence listings. Most preferably there is at least 95%, more preferably at least 98% homology with SEQ ID NO. 3 shown in the attached sequence lists. The terms "variant", "homologous" or "fragment" in relation to the terminator or the signal nucleotide sequences include any substitution of, variation of, modification of, replacement of, deletion of, or addition of, one (or more) nucleic acids of or for the sequence as long as the resulting nucleotide sequence has the ability to act as a terminator or code for an amino acid sequence having the ability to act co or a signal sequence respectively in an expression system - such as the transformed cell or the transgenic organism according to the present invention. In particular, the term "homologous" covers homology with respect to structure and / or function, as long as the resulting nucleotide sequence has the ability to act as or to code for a terminator or signal respectively. With respect to sequence homology, preferably there is at least 75%, most preferably at least 85%, more preferably at least 90% homology with SEQ ID NOS. 13 and 14 (respectively) shown in the attached sequence listings. Most preferably there is at least 95%, more preferably at least 98% homology with SEQ ID NOS. 13 and 14 (respectively) shown in the attached sequence lists. The terms "variant", "homologue" or "fragment" in relation to the signal amino acid sequence include any substitution of, variation of, modification of, replacement of, deletion from, or addition of, one (or more) amino acids of or for the sequence, as long as the resulting sequence has the ability to act as a signal sequence in an expression system such as the transformed cell or the transgenic organism in accordance with the present invention. In particular, the term "homologous" covers homology with respect to structure and / or function, so long as the resulting nucleotide sequence has the ability to act as, or code for, a signal. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, most preferably at least 90% homology with TD of SEQ. 15 shown in the attached sequence listings. More preferably, there is at least 95%, most preferably at least 98% homology with SEQ ID NO. 15 shown in the attached sequence listings. The above terms are synonymous with allelic variations of the sequences. The term "complementary" means that the present invention also covers nucleotide sequences that can hybridize to the nucleotide sequences of the coding sequence, the promoter sequence, the terminator sequence or the signal sequence, respectively. The term "nucleotide" in connection with the present invention includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence of the present invention, by virtue of which the genomic coding sequence has two introñes and its removal would allow the expression in bacteria. The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes a 601 linked directly or indirectly to a promoter. An example of an indirect linkage is the provision of a suitable spacer group, such as a sequence of .intron, such as the intron Shl or the .intron ADH, intermediate between the promoter and the GOI. The same is true for the term "merged" in relation to the present invention, which includes direct or indirect linkage. In each case, it is highly preferred that the terms not cover the natural combination of the gene encoding the enzyme ordinarily associated with the wild-type gene promoter and when both are in their natural environment. A highly preferred embodiment is that the, or a GOI, are operatively linked to a, or the promoter. The shell may even contain or express a marker that allows selection of the genetic construct in, for example, a filamentous fungus, preferably of the genus Aspergillus, such as Aspergill's niger, or plants, preferably cereals, such as corn, rice , barley, etc., in which it has been transferred. There are several markers that can be used, such as for example those that code for mannose-6-phosphate isomerase (especially for plants), or those markers that provide resistance to an ibotic-for example, resistance to G418, hi grornicin , bleomycin, kanarnicin and gentamicin The term "vector" includes expression vectors and transformation vectors. The term "expression vector" means a construct capable of expression in vivo or in vitro. 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 a plasmid of E. coli to an Agrobacterium to a plant. The term "tissue" includes tissue per se and organ. The term "organism" in relation to the present invention, includes any organism that may comprise the promoter according to the present invention and / or the nucleotide sequence encoding the enzyme according to the present invention and / or products obtained therefrom, wherein the promoter may allow expression of a 601 and / or wherein the nucleotide sequence according to the present invention can be expressed when it is present in the organism. Preferably, the organism is a filamentous fungus, preferably of the genus Aspergillus, most preferably Aspergill's niger. The term "transgenic organism" in relation to the present invention includes any organism comprising the promoter according to the present invention and / or the nucleotide sequence encoding the enzyme according to the present invention and / or products obtained from the same, wherein the promoter can allow the 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 nucleotide sequence is incorporated into the genome of the organism. Preferably, the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, most preferably flspergilus niger. Therefore, the tranegenic 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 encoding the enzyme in accordance with the present invention, constructs in accordance with 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 products thereof. For example, the transgenic organism may comprise a GOI, preferably an exogenous nucleotide sequence, under the control of the promoter according to the present invention. The transgenic organism may also comprise the nucleic acid 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. In a highly preferred embodiment, the transgenic organism does not comprise the combination of the promoter according to the present invention and the nucleotide sequence encoding the enzyme according to the present invention, wherein both the promoter and the nucleotide sequence are native to that organism and they are in their natural environment. Thus, in these highly preferred embodiments, the present invention does not cover the sequence encoding the native nucleotides according to the present invention in their natural environment when it is under the control of its native promoter which is also in its natural environment. . Furthermore, in this highly preferred embodiment, the present invention does not cover the native enzyme according to the present invention when it is in its natural environment and when it has been expressed by its native nucleotide coding sequence which is also in its natural environment and when that nucleotide sequence is under the control of eu promoter-native which is also in its natural environment. The term "promoter" is used in the normal sense of the technique, for example, 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 GOI, which may be the sequence of nucleotides encoding 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 the expression of the nucleotide sequence. In this regard, the promoter does not necessarily need to be the same promoter as the one of the present invention. In this aspect, the promoter can be a cell or tissue-specific promoter. For example, if the organism is a plant, then the promoter may be one that affects the expression of the nucleotide sequence in one or more tissues of stem, shoot, root and leaf. By way of example, the promoter for the nucleotide sequence of the present invention may be the -Arny 1 promoter (also known as Arny 1 promoter, Amy 673 promoter or α-Amy 637 promoter), as described in the application copending patent of U.S. U.S. No. 9421292.5 filed on October 21, 1994. That promoter comprises the sequence shown in Figure 1. Alternatively, the promoter for the nucleotide sequence of the present invention may be the α-Arny 3 promoter ( also known as Arny 3 promoter, Amy promoter 351 or a-Amy promoter 351), as described in copending U.S. patent application No. 9421286.7 filed October 21, 1994. That promoter comprises the sequence shown in the figure 2. 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, may additionally include features to ensure or enhance expression in a suitable host. For example, the features may be conserved regions such as a Pribnow region, or a TATA region. The promoters may even contain other sequences to affect (eg to maintain, increase, decrease) the expression levels of the GOI. For example, other suitable sequences include the Shl intron or an ADH intron. Other sequences include inducible elements - such as inducible temperature elements, chemical agents, light or voltage. Also suitable elements may be present to increase transcription or translation. An example of the last element is the TMV 5 'signal sequence (see Sleat Gene 217 C1987) 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97). In addition, the present invention also encompasses combinations of promoters and / or nucleotide sequences that encode proteins or enzymes and / or elements. For example, the present invention encompasses the combination of a promoter according to the present invention linked in operation to a GOI, which may be a nucleotide sequence according to the present invention, and another promoter such as a specific tissue promoter. united in operation at the same or different GOT. The present invention also encompasses the use of promoters to express a nucleotide sequence encoding the enzyme according to the present invention, wherein a portion of the promoter is inactivated, but wherein the promoter can still function as a promoter. In some cases, partial inactivation of a promoter is advantageous. In particular, with the Arny promoter 351 mentioned above, it is possible to inactivate a part of it, when the partially inactivated promoter expresses GOIs in a more specific manner, such as in a specific tissue type or organ. The term "inactivated" means partial inactivation in the sense that the expression pattern of the promoter is modified, but where the partially inactivated promoter still functions as a promoter. However, as mentioned above, the modified promoter is capable of expressing a GOT in at least one of (but not all) of the specific tissues of the original promoter. One such promoter is the Amy 351 promoter described above. Examples of partial inactivation include altering the fold pattern of the promoter sequence, or joining species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognized by the RNA poly erase, for example. Another, and preferred, form of partially inactivating the promoter, will truncate it AGA to form fragrant thereof. Another way may be to mutate at least a part of the sequence, since polyrase RNA can not bind to that part? other part. Another modification is to mutate the binding sites for the regulatory proteins, for example the known CreA protein of filamentous fungi, to exert catabolic repression of carbon, and thus suppress the catabolic repression of the native promoter. The term "GOI" with reference to the present invention means any gene of interest. A GOI can be any nucleotide whether foreign or natural to the organism in question (for example filamentous fungi, preferably of the genus Aspergill? S, or a plant). Typical examples of a GOI include genes that code for proteins and enzymes that modify etabólicoe and catabolic processes. The GOI can code for an agent to introduce or increase pathogenic resistance. The GOI may even be an antisense construct to modify the expression of natural transcripts present in the relevant tissues. The GOI may even encode an unnatural protein of a filamentous fungus, preferably of the genus Aspergillus, or a compound that is of benefit to animals or humans. For example, GOT may code for a pharmaceutically active protein or enzyme such as any of the therapeutic compounds insulin, methylferon, human serum albumin, human growth factor and blood coagulation factors. In this regard, the transformed cell or organism could prepare acceptable amounts of the desired composition that can be easily recovered from the cell? organism. The GOI may even be a protein that gives nutritional value to a food or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors, and vegetable proteins having a more convenient amino acid composition (e.g., a higher lysine content than a non-transgenic plant). The GOI can even encode an enzyme that can be used in food processing such as quirnosine, taurnantrna and cf-galactosidase. The GOI may be a gene encoding any of a pest toxin, an antisense transcript such as patatma or α-amylase, ADP-glucose pyrophosphorylase (eg, see EP-A-0455316), an antisense protease or a glucanase. The GOI may be the nucleotide sequence encoding the enzyme or-a -lase q? E is the subject of co-pending UK patent application 9413439.2, filed on July 4, 1994, the sequence of which is shown in FIG. Figure 3. The 601 may be the nucleotide sequence encoding the enzyme or-amylase, which is the subject of the p &g application., copending U.S. Patent No. 9421290.9, filed October 21, 1994, the sequence of which is shown in Figure 4. The GOI can be any of the n? cleoti sequences or that encode the ADP- enzymes. gl? thing pyrophospholases which are the subject of the copending application PCT / EP94 / 01082, filed on April 7, 1994, the sequences of which are shown in figures 5 and 6. The GOI can be any of the sequences of nucleotide q? e encode for the enzyme a-glucan lyase, which are disclosed in co-pending Patent Application PCT No. EP94 / 03397, filed on October 15, 1994, the sequences of which are shown in the figures 7-10.

In a preferred embodiment, the GOI is a nucleotide sequence encoding the enzyme according to the present invention. As mentioned above, a preferred host organism is of the genus Aspergillus, such as Aspergillus niger. The transgenic Aspergillus according to the present invention can be prepared following the teachings of Rambosek, 3. and Leach, 3. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects, CRC Cpt. Rev. Biotechnol., 6: 357-393), David R: U > : 1994 (Heterologoue gene expression and protein secretion in Aspergillus, Martinelli S.D., Kinghorn J.R. (Editors) Aspergillus: 50 years on, Progress in industrial microbiology, vol.2 29 Elsevier Arnsterdam 1994. pp 525-560), Ballance, D.3. 1991 (Transformation syetems for Fila entous Fungi and an Overview of "Fungal Gene etrust" in Leong, SA, Berk-a RM (Editors) Molecular Industrial Mycology, Syetems and Applications for Filarnentous Fungi, Marcel Dekker Inc. New York 1991. pp 1-29 and Turner G. 1994 (Vectors for genetic rnampulation) In: Matinelli SD, Kmghorn 3.R. (Editors) Aepergill? e: 50 yeare on Progress in industrial icrobiology vol 29 Elsevier Amsterdam 1994. pp. 641- 666. However, the following commentary provides a summary of those teachings for producing transgenic Aspergillus in accordance with the present invention.For almost a century, filamentous fungi have been widely used in the industry for the production of organic compounds and enzymes. Traditional fermentations of Japanese kojí and soybean have used Aspergillus sp for hundreds of years.In this century, Aspergillus niger has been used for the production of organic acids, particularly citric acid and for to the production of different enzymes to be used in the industry. There are two main reasons why filamentous fungi have been used so widely in the industry. First, filarnentoeoe fungi can produce high amounts of extracellular products, for example, enzymes and organic compounds such as antibiotics or organic acids. Second, filamentous fungi can grow on low cost substrates, such as grains, bran, beet pulp, etc. The same reasons have made filamentous fungi organisms attractive as huéepedee for heterologous expression in accordance with the present invention. n. To prepare the transgenic Asp rgillus, expretion conet rs are prepared by inserting a GOI (such as an arnilasa or identification of sequence No. 2) into a construct designed for expression in filamentous fungi. Several types of constructions 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 those of the present invention include a fungal promoter for a highly expressed extracellular enzyme, such as the glucoarnilase promoter or the a-arnilasse promoter. The GOI can be fused to a signal sequence (such as that of the present invention or other suitable sequence) that directs the protein to be secreted encoded by the GOI. Finally, a signal sequence of fungal origin, such as that of the present invention, is used. An active agent in fungi ends the expression line, such as that of the present invention. Another type of fungal expression system has been developed where 601 is fused to a smaller or larger part of a fungal gene encoding a stable protein. This can stabilize the protein encoded by 601. In said seventh, a cut-off site, recognized by a specific protease, can be introduced between the fungal protein and the protein encoded by the GOI, since the protein < The fusion produced can be cut in this position by the specific protease, thus releasing the protein encoded by the GOI ("POI"). By way of example, a site can be introduced that is recognized by a KEX-2-like peptidase found in at least some Aspergill i. This merger leads to breakdown in vivo, causing the protection of the POI and the production of POI and not a larger fusion protein. Heterologous expression in Aspergill? S has been reported for vapoe genee q? E encode for bacteria, fungi, vertebrates and plant proteins. Proteins can be deposited intracellarly if the GOI is not fused to a signal sequence. These proteins will accumulate in the cytoplasm and, ultimately, they will not be glycosylated, which may be an advantage for some bacterial proteins. If the GOI is designed with a signal sequence, the protein will accumulate extracellularly. With respect to the stability of the product and modifications of the host strain, some heterologous proteins are not very stable when secreted in the fungal culture fluid. Most fungi produce several extracellular proteases that degrade heterologous proteins. To avoid this problem, special strains of fungi have been used with reduced production of protease, co or hosts for heterologous production. For the transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi (Ballance 1991, ibidem). Many of them are prepared based on protoplasts and DNA is introduced into the protoplasts using PEG and Ca2 + ions. Traneformadoe protoplasts are then regenerated, and transformed fungi are selected using different selective markers. Among the markers used for transformation are several auxotrophic markers such as argB, trpC, niaD and pyrG, markers of antibiotic resistance, such as resistance to benoyl, resistance to hygromycin and resistance to fleornicin. A commonly used transformation marker is the amdS gene of A. nid? Lans, which in high numbers of copies allows the fungus to grow with acrylamide as the sole source of nitrogen. Although the enzyme, the n-cleotide sequence coding for it, and the promoter of the present invention are not described in EP-B-0470145 and CA-A-200645, these two documents provide some useful background information on the types of techniques that can be employed to practice the present invention. Some of this background teachings are now included in the following comment. The basic principle in the construction of genetically modified plants is to insert genetic information into the genome of the plant, in order to obtain a stable maintenance of the inserted genetic material. There are several techniques for inserting genetic information, the two main being the direct introduction of genetic information and the introduction of genetic information through the use of a vector system. A review of the general techniques can be found in the articles of Potrykus (Annu Rev Plant Physiol Plant Mol Biol 111991] 42: 205-225) and Chpetou (Agro-Food-Ind? Stry Hi-Tech March / April 1994 17-27 ). Thus, in an aspect, the present invention relates to a vector system carrying a nucleotide promoter or entity or construct according to the present invention and which is capable of introducing the promoter or nucleotide sequence. or a construct in the genome of an organism, such a co or a plant. The vector system may comprise a vector *, but may comprise two vectors. In the case of the two vectors, the vector system normally refers to a binary vector system. Binary vector systems are described in greater detail in Gynhe? Ng An et al. (1980), Bmay Vectors, Plant Molecular Biology Manual A3, 1-19. A system used extensively for the transformation of plant cells with a promoter or sequence of given nucleotides or connects is based on the use of a Ti plasmid of Agrobacterium tumefaciens, or a plasmid Ri of A robacteriurn rhizogenes, An et al. (1986 ), Plant Physiol. 81, 301-305 and Butcher D.N. and others (1980), Tisseu Culture Methods for Plant Pathology sts, eds .: D.S. Ingrams and 3. P. Helgeeon, 203-208. Different Ti and Ri plasmids have been assembled which are suitable for producing the vegetale or plant cell constructs described above. A non-limiting example of such Ti plasmid is pGV3850. The promoter or nucleotide sequence or construct of the present invention should preferably be inserted into the Ti plasmid between the terminal sequences of the T-DNA or adjacent to a T-DNA sequence, in order to avoid interruption of the sequences immediately surrounding the T-DNA. T-DNA limits, since at least one of these regions appears to be essential for the 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 that contains the sequences necessary to infect the plant (e.g. the vir region), and at least a borderline part of a T-DNA sequence, the borderline part being located on the same vector as the genetic construct. In addition, the vector system is preferably a Ti plasmid of Agrobacterium tumefaciens or a Ri plasmid of Agrobacterium rhizogenes or a derivative thereof; In view of the fact that these plasmids are well known and widely used in the context of transgenic plants, there are many vectors made from plastic seeds or derivatives thereof. In the connection of a tranegenic plant, the promoter or nucleotide or linker sequence of the present invention can first be produced in a microorganism in which the vector ee 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 can be used. When a vector system has been constructed as defined above in E. coli, it is transferred, if necessary, to a suitable strain of Agrobacterium, for example, Agrobacterium turnef ciens. The Ti plasmid hosting the promoter or nucleotide sequence or construct of the invention is thus preferably transferred into a suitable strain of Agrobacterium, eg, A. turnefaciens, in order to obtain an Agrobacterium cell that hosts the promoter. or nucleotide sequence or construct of the invention, the DNA of which is subsequently transferred into the plant cell to be modified. As reported in CA-A-2006454, a large number of cloning vectors containing a replication system in E. coli are available, and a marker allowing a selection of the transformed cells. The vectors contain, for example, the series pBR 322, pUC, M13 rnp, pACYC 184, etc. In this way, the nucleotide or construct or promoter of the present invention can be introduced into a suitable restriction position in the vector. The contained plasmid is used for transformation in E. coli. The E. coli cells are cultured in an appropriate nutrient medium and then harvested and lysed. Then, the plasmid is recovered. As methods of analysis, the methods of sequence analysis, restriction analysis, electrophoresis and other biological methods of molecular biochemistry are generally used. After each manipulation, the used DNA sequence can be restricted and connected to the next DNA sequence. Each sequence can be cloned into the same plasmid or a different plasmid. After each method of introduction into the plants of the desired promoter or nucleotide construct or sequence according to the present invention, the presence and / or insertion of additional DNA sequences may be necessary. For example, if the plasmid Ti or Ri of the cells of the plant is used for the transformation, they can be connected as flanking areas of the genes introduced into at least the right boundary and frequently also the right and left boundaries of the T-DNA of the plant. the Ti and Ri plasmids. The use of T-DNA for the transformation of plant cells has been studied intensively and is described in EP-A-120516; Hoekema, in: The Bmary Plant Vector System Offset-dr? Kkerij Kanters B.B, Alblasserdarn, 1985, Chapter V; Fraley et al., Cpt. Rev. Plant Sci., 4: 1-46 and An et al., EMEO 3. (1985) 4: 277-284. Direct infection of plant tissues by Agrobacter uni is a simple technique that has been widely used and described in Bu che D.N. and others (1980), Tissue Culture Methods for Plant Pathologists, eds .: D.S. Ingra s and 3. P. Helgeson, 203-208. For further teaching on this topic see Potrykus (Ann? Rev Plant Physiol Plant Mol Biol [1991] 42: 205-225) and Cristou (Agro-Food-Ind? Stry HiTech March / April 1994 17-27). With this technique, the infection of a plant on a certain part or tissue of the plant, that is, on a part of a leaf, a root, a stem or another part of the plant can be performed.

Typically, the plant to be infected is injured, with direct infection of plant tissues by means of Agrobactep? M carrying the promoter and / or the GOI, for example, by cutting the plant with a knife or by pricking the plant with a needle. or rubbing the plant with an abrasive. The lesion is then inoculated with the Agrobacterium. The plant or part of the inoculated plant is then grown in a suitable culture medium and allowed to grow on mature plants. When plant cells are constructed, these cells can be grown and maintained in accordance with well-known tissue culture methods, such as by cultivating the cells in a suitable culture medium supplied with the necessary growth factors, such as amino acids, plant hormones, vitamins. , etc. The regeneration of transformed cells in genetically modified plants can be achieved using known methods for the regeneration of plants from tissue or cell culture, for example, by selecting transformed offspring using an antibiotic and subclaring lobes. in a medium containing the appropriate nutrients, plant hormones, etc. Additional teachings on vegetable tranaformation can be found in EP-A-0449375. In summary, the present invention provides a glucanase enzyme, and the nucleotide sequence encoding it. In addition, it provides a promoter that can control the expression of that, or another, nucleotide sequence. In addition, it includes termination and signal sequences for them. The following sample was deposited in accordance with the Budapest Treaty in the recognized deposit The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1RY, January 16, 1995: E. coli containing plasmid pEGLA-3. { that is, DH5a-? EGLA-3 from E. coli} . The deposit number is NCIMB 40704. The present invention will now be described by way of example. In the following examples reference is made to the accompanying figures in which Figures 1-10 are sequences of promoters and genes of interest from previous patent applications which are useful for use with the aspects of the present invention; Figure 11 is a plasmid screen of pEGLA-3; Figure 12 is a schematic diagram of some deletions of promoters; Figure 13 is a plasmid map of pGPAMY; Figure 14 is a graph; Figure 15 is a plasmid map of pGP-GssAMY-Hyg; Figure 16 is a graph; and Figure 17 is a Western Blot analysis.

The following examples discuss techniques of recog ned DNA. General teachings of recombinant DNA techniques can be found in Sambrook, 3., Fptsch, E.F., Mamatis T. (Editors) Molecular Cloning. Laboratory manual. Second edition. Cold Spring Harbor Laboratory Press. New York 1989.

PURIFICATION OF LR ß-GLUCANfíSfl Aspergillus niger 3M43 was grown in a medium containing wheat bran and beet pulp. The fermentation broth was separated from the solid part of the broth by filtration. The concentrated fermentation broth was then poured onto a high performance Q-SEPHAROSE column (Pharmacia) of 25X100 nm, equilibrated with 20 mM Tris. HCl pH 7.5 0-500 mM NaCl and fractions of the elution were collected. The ß-gl? Canase was eluted at approximately 100 M NaCl. The fractions containing gl? Canase were changed and desalted using a G-25 SEPHAROSE Superfine (Pharmacia) of 50x200 nm. The column was eluted afterwards with distilled water. After desalting, the enzyme was concentrated using high entrapment rotation columns. Then, the concentrated and desalted fractions were subjected to gel filtration on a SUPERDEX 50 column of 50x600 nm. The sample was loaded and the column was eluted with 0.2 M phosphate buffer with a pH of 7.0 plus 0.2 N NaCl and the elution fractions were pooled. The fractions containing glucanaea were combined, desalted and concentrated as described above. The combined fractions were loaded onto a high performance column PhenylSEPHAROSE of 16x100 rnrn (Pharmacia), equilibrated with phosphate buffer to 20 mm with pH of 6.0, containing (NHi ^ O * at 1.5 M. A gradient was applied in which the concentration of (NH / I) 2S0A varied from 1.5 to 0 M and the elution was collected in fractions The fractions containing glucanaea were combined The purity of the β-1, 4-glucanase (SDS-PAGE) was evaluated using the gel of the Phast system (Pharmaci).

CHARACTERIZATION The molecular weight of the purified glucanase was determined by mass spectrometry used in the laser desorption technology. It was found that the molecular weight of the glucanase was 24,235 D ± 50 D. The pl value was determined using a Broad pl Kit (Phar acia). Glucanase has a pl value of approximately 4. After the SDS-PAGE analysis, the PAS treatment reagent showed that the glucanase is not glycosylated. The staining of PAS was done according to the T procedure.

Van Seuningen and M. Davril (1992) Electrophoresis 13 pp 97-99.

SEQUENCING AMINO ACIDS OF Lfl ß-GLUCANASE The enzyme was digested with the sort of sequence in the Lys-C protein from Boehpnger Mannheim using a modification of the method described by Stone to Williams 1993 (Stone, KL and Williams, KR (1993).) Enzymatic digestion of Proteins and HPLC Peptide Isolation In: Matsudaira P. (Editor) A Practical Guide to Protein and Peptide P rification for Microsequencmg Second Edition Academic Press, San Diego 1993. pp 45-73 Frozen dry ß-glucan (0.4 mg) was dissolved in 50 ul of urea at 8M, NH4HCO3, at 0.4M, with a pH of 8.4 After the annealing with N and the addition of 5 μl of DTT at 45 nm, the protein was denatured and reduced for 15 minutes to 50 ° C under N2.After cooling to room temperature, 5 μl of iodine-acetylide was added to 100 M so that the cisterns were depurated for 15 minutes at room temperature in the low dark N.sub.2 Subsequently, 90 μl of water was added and 5 μl endoprotemase Lye-C in 50 μl of tricine at 50 μM and EDTA at 10 rnM, with a pH of 8.0, and digestion was carried out for 24 hours at 37 ° C under N2. The resulting peptides were separated by reverse phase HPLC a column of VYDAC C18 (0.46 x 15 cm; 10 μ; The Separations Gruop; California) was opened using a solvent A: 0.1% TFA in water and a solvent B: TFA 0.1% in acetonite p 1 o. The selected peptides were re-chromathographed on a Develoeil C18 column (0.46 x 10 crn; 3 μm) using the same solvent system before sequencing on a Biosysterns 476A sequencer using rapid cycles of pulsed liquid. The following sequence sequences were found: SEQUENCE ID No. 4 SEQUENCE TD No. 5 SEQUENCE ID No. 6 SEQUENCE ID No. 7 SEQUENCE ID No. 8 ISOLATION OF A PCR CLON OF A GENE FRAGMENT The PVR primers were synthesized using an Applied Biosystems model 392 DNA synthesizer. In this reep > ect, the PCR indicators were detected from two of the peptide sequences found, WEVUYGT from Sequence ID No. 4 and UTUSGG from Sequence ID No. .. The initiator derived from WEVWYGT (inverted) is shown in the ID of Sequence No. 9 and the UTWSGG derivative initiator is shown in Sequence ID No. 10 - see below: SEQUENCE ID No. 10 TGG ACN TGG WSN GGN GG Mixture 256 of 17 rner SEQUENCE ID No. 9 CTN CCR TAC CAN ACY TOC CA Mixture 64 of 20 mer PCR amplification was performed with 100 pmol of each of these primers in 100 μl reactions using Arnplitaq II kit (Perkin Elmer). The program was: Step Temperature Time 1 94 ° C 2 min. 2 94 ° C 1 rnin. 3 55 ° C 2 min 4 72 ° C 2 min. 5 72 ° C 5 min. 6 5 ° C REMOVAL Steps 2-4 were repeated for 40 cycles. The program was conducted in a PERKIN ELMER DNA thermal pencil. An amplified fragment was isolated at 350 bp and cloned into a Tazul-pT7 vector according to the manufacturer's instructions (Novagen). A fragment was isolated and sequenced. The sequence found showed that it was indeed part of a glucanase gene.

ISOLATION OF THE GENOMIC FLDN OF A. NIGER One gram of frozen mycelium of A. niger was crushed in a mortar under liquid nitrogen. After evaporation of the nitrogen cover, the crushed mycelium was extracted with 15 nl of an extraction buffer (Tris-HCl at 100 mM, pH 8.0, EDTA at 0.50 mM, NaCl at 500 mM, β-mercaptoethanol at 100 rnM) containing 1 Ml of 20% sodium dodecylsulfate. After incubation at 65 ° C for 10 minutes, 5 ml of 5 M KAc, pH 5.0, was added, and the mixture was again covered, after mixing, on ice for 20 minutes. The mixture was then centrifuged for 20 minutes and the supernatant was mixed with 0.6 volume of isopropanol to precipitate the extracted DNA. Deepuée of the largest centrifugation for 15 minutes, the DNA pellet was dissolved in 0.7 nm of TE (TPS-HCl at 10 M, pH of 8.0, EDTA at 1 mM) and precipitated with 75 μl of NaAc at 3 M, pH of 4.8 and 500 μl of isopropanol. After centrifugation, the pellet was washed with 70% ETOH and dried under vacuum. The DNA was dissolved in 200 μl of TE and stored at -20 ° C.

CONSTRUCTION OF Lfl GENOTECA Partially 20 μg of genomic DNA was digested with Tsp509I, which gives ends that are compatible with the EcoRI ends. The ingested DNA was separated on a 1% agarose gel and 4-10 kb fragments were purified. A \ ZAPII EcoRI / CIAP Stratagene kit will be used for the library construct according to the manufacturer's instructions. 2 μl of the ligation (in total 5 μl) was packed with Gigapack Gold II packaging extract according to the manufacturer's instructions (Stratagene). The library contained 650,000 independent clones.

SELECTION OF Lfl GENOTECfl 2 x 50,000 pfu were cultured on NZY sheets (5 g of NaCl, 2 rng of MgSO ".7H2 ?, 5 g of yeast extract, 10 g of casein hydrolyzate, 15 g of agar per liter) and lifts were made. plates on leaves Hybond N (Anersharn). The leaves were hybridized with PCR clone labeled with 32pdctP (Arnersham) using Pharmacia Ready-to-go labeling equipment. Plate lifts and hybridization were done in duplicate and positive clones were considered only when hybridization could be detected on both leaves. The nucleotide sequence of the present invention was sequenced using an ALF laser fluorescence sequencer (Pharmacia). The sequence contained the entire amino acid sequence found, confirming that the isolated gene encoded β-1,4-in gl ueon sa.

INFORMATION OF THE SEQUENCE The. Sequence identification No. 12. presents the promoter sequence, the enzyme coding sequence, the terninar sequence and the sequence of the sequence and the amino acid sequence of the enzyme of the present invention.

PROOF OF THE ACTIVITY OF ENZYMES The purified protein was analyzed for the activity of endo-β-1,4-glucanase using a barley ß-glucan tablet between azurine loop (factory name: Glucazyme tablets requested by Megazyme, Australia) following the instructional given by the manufacturer. The purified enzyme gave a high activity on this sulfate. Typically, the enzyme has a specific activity of 2,250 mg / minute glucose per minute per milligram of protein.

INDUCTION OF THE GENE Eqlfl: IDENTIFICATION OF THE SOURCE OF CARBON IN UCTORfl The following table shows the identification of a high and low molecular weight inductoree number of the gl? Conase promoter. This analysis was carried out using the full-length glucanase promoter of the present invention fused to the E-coli β-glucuronidase gene. The inducing resistance of the different carbon sources was determined quantitatively by measuring the specific activity of the intracellular GUS to hydrolyze p-nitrophenol glucuronide.

CARBON FUETE GUS ACTIVITY (1%) (units / rn) -24 - hoxyl xylose 12.91 xylitol 10.62 arabinose 8.50 arabitol 14.40 glucose 20.25 celubiosa 19.44 x? Lo-ol? Gòtner 70 11. RO gl? Copí ranoside 19.70 rneti l-xylopi ranoside 12.60 xyloglcan 13.90 pectin 9.70 arabinogalactan 30.20 arabitol + glucose 29.50 Surprisingly, glucose, which is normally a potent repressor of catabolitoe, induces the gluconean promoter. Accordingly, the present invention also relates to a promoter that is inducible by glucose. In addition, the present invention relates to the use of glucose to induce a promoter. These aspects of the present invention are different from the teachings of WO 94/04673 which discloses a fungino promoter that is active in the presence of glucose. In this R2 respect, the promoter of the present invention is not only active in the presence of glucose, but is also inducible by glucose. One of the advantages of having a glucose promoter that is inducible by glucose is that the promoter can be used to express a gene of interest and thus used to prepare a POI (such as a heterologous POI), in a medium environment that contains glucose. This is important because glucose is one of the preferred sources of carbon for the accumulation of biomass. In addition, glucose-containing media are expected to produce lower amounts of proteases, thus providing better POI yields. In addition, the use of a derepressed promoter in a derepressed host strain will increase the speed and efficiency of the reaction media, such as a fermentation reaction medium. In addition, the use of mixed sources of carbon during fermentation will allow efficient induction of this promoter, for example at low levels of glucose and an economical source of carbon (eg beet pulp).

EFFECTS OF SUPPRESSION OF THE PROMOTERS ON THE REGULATION OF THE EXPRESSION OF THE GLUCflNflSfl GENE A series of suppression studies were performed, which are shown in Figure 12. In those studies, the different promoter suppression constructs shown in Figure 12 were fused to the GUS gene. The activity of the reporter gene was analyzed qualitatively. The results showed that none of the deletions eliminated the inductance of the glucase promoter. These results indicate the presence of multiple sites for the activation of transcription and the initiation of transcription.

PROTEIN PRODUCTION HETEROLOGA USING ASPERGILLUS NIGER TRANSFORMANTS THAT COMPRISE THE GLUCANASE PROMOTER (PG) AND GLUCANASE SIGNAL SEQUENCE (SSG) Transformation of flspergillus niger The protocol for the transformation of A. niger was based on the teachings of Buxton, F.P. Gwynne D.I., Davis, R.U. 1985 (Transformation of ñspergillus niger using the argB gene of ñspergillus nid? Lans. Gene 37: 207-214), Daboussi, M.J., Djeballi, A. Gerlinger, C, Blaiseau. P.L., Cassan. M., Lebrun, MH, Parisot, D.-, Brygoo, Y. 1989 (Transformation of seven species of filarnentous f? Ngi ueing the nitrate reductase gene of Aspergillus nidulans, Curr. Genet. 15: 453-456) and Punt, PJ, van der Hondel, CAMJJ 1992 (Transformation of filament? S fungi based on hygromycin B and Phlenycin resistance arkers, Meth. Ensym 216: 447-457). For the purification of protoplaetoe, spores from? N PDA (Potato dextroea agar - from Difco, Lab. Detroit) fresh sporulated plate N400 (CBS 120.49, Centraalburea? Voor Schimrnelc? Ltures, Baarn) (from 7 days) they were washed in 5-10 rnl of water. A shake flask was inoculated with 200 rnl of PDC (Potato dextrose broth, Di feo 0549--17-9, Di feo Lab. Detroit), with eeta e suspension of spore and agitation (250 rpm) for 16- 20 hours at 30 ° C. The mycelium is grown using a Miracloth paper and 3-4 g of wet mycelium are transferred to a petri dish with 10 rnl of STC (1.2 M sorbitol, 10 M Tris Hcl pH 7.5, 50 Mm CaCl2) with 75 ng of enzymes of lysate (Sigina L-2265) and 4500 units of liticase (Sigrna L-R012). The mycelium is incubated with the enzyme until the mycelium degrades and the protoplasts are released. The degraded mycelium is then filtered through a sterile 60 μrn sieve filter. The protoplasts were cultured for 10 minutes of centifugation at 2000 rprn in an external rolling rotor. The supernatant is discarded and the pellet is dissolved in 8 ml of 1.5 M Mg or *, and then I fired 3000 rprn for 10 minutes. The upper band containing the protoplasts is transferred to another tube, using a transfer pipette and 2 ml 0.6 M KCl are added. 5 rnl of 30% sucrose is carefully added on top and the tube is centrifuged 15 minutes at 3000 rpm. The protoplasts, which are located in the interface band, are transferred to a new tube and diluted with 1 vol. of STC. The solution is centrifuged 10 minutes at 3000 rpm. The pellet is washed twice with STC, and finally solubilized in 1 ml of STC. The protoplasts are counted and finally concentrated before the transformation. For the transformation, 100 μl of protoplast solution (106-107 protoplasts) is mixed with 10 μl of DNA solution containing 5-10 μg of DNA and incubated 25 minutes at room temperature. Then, 60% of PEG-4000 is carefully added in 200 μl, 200 μl and 800 μl positions. The mixture is incubated 20 minutes at room temperature. Add 3 ml of STC to the mixture and stir carefully. The mixture is centrifuged at 3000 rprn for 10 minutes. The supernatant is removed and the protoplasts are solubilized in the remaining supernatant. Thirteen ml of topagarose are added and the protoplasts are quickly spread on selective plates.

Glucanase promoter and heterologous gene expression Figure 13 shows the pGP Amy expression vector that was used in the studies. This expression vector comprises the glucanase promoter fused to the Thrmomyces lanuginosus precursor from the α-amylase gene. Transcription of the promoter is terminated using the terminator Xylanase A. The construct was used in a transformation experiments with the hygro icine resistance gene as a selectable marker. The production of ct-amylase using independent transformants containing the expression vector pGPAmy when grown on beet pulp and wheat savior is shown in Figure 14. The a-amylase activity was detected for the first time in the culture medium after 48 hours of culture. A peak in enzyme activity was observed after 3 and 4 days.

Glucanase signal sequence and heterologous protein production For these studies, the expression vector pGPGssArnyHyg was used. The vector pGPGssArnyHyg is shown in Figure 15. This vector comprises the mature cf-amylase gene translationally fused to the glucanase signal peptide (labeled ss). In addition, this vector comprises the promoter of the present invention (labeled EGl.A) and the xylanase A terminator. The transcription of this vector is therefore under the control of the glucanase promoter and termination by the xylanase A terminator. This link was used to test, inter alia, the efficiency of the signal peptide in the secretion of heterologous protein. Figure 16 shows the results of the induction of of-arnilasa by using the construct in strain 6M179 when it is grown in beet pulp and wheat bran. The results show that the activity of the enzyme was located in the culture medium and was detected for the first time after 48 hours of culture. The accu- mulation of enzyme activity on day k was observed.

Western Blot Analysis Figure 17 shows a Weetern Blot analysis of proteine from the supernatant of three independent formant rans separated by SDS-PAGE and stained to a membrane. A synthetic peptide with 15 amino acid residues of cf-ami laea of T. lanuginosue recognized an individual band in the Weetern Blot analysis of supernatants in culture from part of the transformants.

Antibody production The antibodies were cultured against the enzyme of the present invention by injecting rabbits with the purified enzyme and isolating the immunoglobulins from the antiserum in accordance with the procedures described in accordance with N Harboe and A Ingild ("Immunization, Isolation of Imunoglobulin, Estimation of Antibody Titre "in A Manual of Quantitative Imm? Noelectrophoresis, Methods and Applications, NH Axelsen., And others (eds ..) Univereitetsforlaget, Oslo, 1973) and by TG Cooper (" The Tools of Biochemistry ", John Wiley S Sons, New York, 1977). conclusion Although it is known that Aepergill's niger produces several enzymes that degrade ß-glucan, the present invention provides a novel and ingenious ß-1, -endogl ucanase as well as the coding sequence for it, the terminator sequence of the same, the signal sequence for the ism and the promoter for eeae eecuenciae. An important advantage of the present invention is that the enzyme can be produced in large quantities. In addition, the promoter and the regulatory sequences (such as the signal sequence and the terminator) can be used to express or can be used in the expression of nerve genes in organisms, such as in A. niger. The enzyme of the present invention is advantageous for food supplements. In addition, it can be used in the brewing industry, since it has a high fiber conversion potential. In addition, there are fewer treatment problems when the enzyme is used, particularly with polysaccharides. In addition, the enzyme effectively degrades the β-glucans, so it can be used advantageously in the brewing industry to reduce the viscosity and also improve the filterability of the beer. This is important, since the high molecular weight glucans are beer and the like can cause filtration difficulties and give rise to sediments, gels and toughness. The signal sequence of the present invention is useful for the secretion of a POI, such as a heterologous POI, thereby improving the quality and quantity of the POT. Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

INFORMATION ABOUT THE SEQUENCES SEQUENCE OF THE ENZYME ID. OF SEQUENCE NO. 1: Glp Thr Met Cys Ser Gln Tyr A.SP Ser Ala Ser Ser Pro Pro Tyr Ser I 5 10 15 Val Asp Gln Asn Leu Trp Gly Glu Tyr Gln Gly Thr Gly Ser Gln Cys 20 25 30 Val Tyr Val Asp Lys Leu Ser Ser Ser Gly Wing Ser Trp His Thr Lys 35 40 45 Trp Thr Trp Ser Gly Gly Glu Gly Thr Val Lys Ser Tyr Ser Asn Ser 50 55 60 Gly Leu Thr Phe Asp Lys Lys Leu Val Ser Asp Val Ser Ser He Pro 65 70 75 80 Thr Ser Val Thr Trp Ser Gln Asp Asp Thr Asn Val Gln Wing Asp Val 85 90 95 Ser Tyr Asp Leu Phe Thr Wing Wing Asn Wing Asp His Wing Thr Ser Ser 100 105 110 Gly Asp Tyr Glu Leu Met He Trp Leu Wing Arg Tyr Gly Ser Val Gln 115 120 125 Pro He Gly Lys Gln He Wing Thr Wing Thr Val Gly Gly Lys Ser Trp 130 135 140 GTu Val Trp Tyr Gly Thr Ser Thr Gln Wing Gly Wing Glu Gln Lys Thr 145 150 155 160 Tyr Ser Phe Val Wing Gly Ser Pro He Asn Ser Trp Ser Gly Asp He 165 170 175 Lys Asp Phe Phe Asn Tyr Leu Thr Gln Asn Gln Gly Phe Pro Wing Ser 180 185 190 Ser Gln His Leu He Thr Leu Gln Phe Gly Thr Glu Pro Phe Thr Gly 195 200 205 Gly Pro Wing Thr Phe Thr Va "Asp Asn T> -p Thr Wing Ser Val Asn * 210 215 220 ENZYME CODING SEQUENCE SEQUENCE ID NO. 2: CAG ACG ATG TGC TGT CAG TAT GAC AGT GCC TCG AGC CCC CCA TAC TCG GTG AAC CAG AAC CTC TGG GGC GAA TAC CAG GGC ACT GGC AGC CAG TGT GTC TAC GTC GAC AAG cp AGC AGC GGT GCC TCA TGG CAT ACC AAA TGG ACC TGG AGT GGT GGC GAG GGA ACÁ GTG AAA AGC TAC TCT AAC TCC GGC cp ACG pr GAC AAG AAG CTA GTC AGC GAT GTG TCA AGC Ap CCC ACC TCG GTG ACA TGG AGC CAG GAC GAC ACC AAT GTC CA GCC GAT GTC TCA TAT GAT CTG PC ACC AAT GCG GAT CAT GCC ACT TCC AGC GGT GAC TAT GAG cp ATG Ap TGG CGC TAC GGC TCA GTC CAG CCT Ap GGC AAG CAG Ap GCC ACG GCC ACT GTG GGA GGC AAG TCC TGG GAG GTG TGG TAT GGT ACC AGC ACC CAG GCC GGT GCG GAG CAA AAG ACAT TAT AGC GTG GCA GGA TCT CCT ATC AAC TCG TGG AGT GGG GAC Ap AAG GAC pc pc AAC TAT CTC ACC CAG AAC CAG GGC pc CCG GCT AGC TCT CAG CAT pG ATC ACT CTG CAG pr GGA ACT GAG CCG pc ACC GGT GGC CCG GCA ACC PC GG AC GAC AAC TGG GCT AGCT GTC AAC sz OR 0201 3W33391LV 1113913133 LL9339131V 9VU13319V V93U1331I V1313D 31 096 1V3VW3311 V1L3191931191933133333913913131931939139W V1LLV1S1V9 006 119V9V31V911V33939W 19V3W11919V1W1W3V 99319WV993V3V9911V1 ST Ofr? 93391399133193113V11399119V139 V1V139133V V393991V91 V9119D9913 08¿ 99V391191V V3191V3V1L 1V99V993W V91LLV3131999W91V93313919WV 02Z 9U3 9939 W13931V33 V9V3V33913 Y9339119911391 3V33131193331V 099 9V9913111V JJJ.W1H333W9V399V3 V39V31931V 1V991W1V391V1V13199 009 11W331V9V 131933313V 133W33W33W1V933V311 1V9331311V1V9L 9 OFRS 13139 33313V11V3W331VW199313LLV3V119V 91V1V3WV1 V91V199193 02V 133 V1L3V 9V9913333991V33J1991 V1911W13331V91V13V 1VWV9W99 OL 02fr 1V3V11V1V9 V3913W1993WV131 91VW9V9V31 V3399991W 9 V99V1 09C 913V319W3 V9W3VW3931V19 99V V99V99913V 99WV9W399V191V1VSV OOC 1V19V9LLL91W19V9V9V V1V99193 V99V99V399 V9913399W 9V39993313 0 * 3 1WV933W339V3V13 1 V991 081 1V39191V9V19319W93139313V13V 3SV vUSVlWil 9V913V3111939199139V 3V991U131113111 3V 9V131999 02T 2993W993999V391W399V199V1V91 U9999931V W919991993V99V9119V 09 V9Y39U9V9319V91V9911V1V99V31V 11199V1V9UJ V1339. V39W911W and 29 F) 3 INFORMATION FOR ID. OF SEQUENCE NO. 4: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TTPO: amino acid (C) TYPE OF CHAIN: individual (D) TOPOLOGY: linear (II) TYPE OF MOLECULE: pep + ao (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Aeperegillus niger (xi) SEQUENCE DESCRIPTION: ID. OF SEQUENCE NO. 4: Ser- Trp Glu Val Trp Tyr Gly Thr Ser Thr Gln Wing Gly Wing Glu Gln 1 5 10 15 Lys INFORMATION FOR TD .. OF SEQUENCE NO. 5: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: individual (D) TOPOLOGY: linear (II) TYPE OF MOLECULE: peptide (v) FRAGMFNTO TTPO: intorno (xi) SEQUENCE DESCRIPTION: ID. OF SECUFNCTA NO. 5: Thr Tyr Ser- Phe Val Wing Gly Ser 'Pro Tie 1 5 10 INFORMATION FOR ID. OF SEQUENCE NO. 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: TD. OF SEQUENCE NO. 6: Lys Leu Val Ser Asp Val Ser Ser lie Pro Thr Ser Val Thr Xaa Ser 1 5 10 15 Gln Asp Asp Thr Asn Xaa Xaa Wing Wing Val Ser Tyr Xaa Leu Phe Thr 20 25 30 Wing Wing Asn 35 INFORMATION FOR ID. OF SECUENCTA NO. 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) I ONTTTUD: 11 amino acids (B) TYPE: amino acid (C) TYPE OF CADFNA: individual (D) TOPOLOGY: linear (ll) TYPE OF MOLECULE: peptide (v) TYPE DF FRAGMENT : i terno (l) DESCRIPTION OF SECUENCTA: ID. OF SECUENCTA NO. 7: Trp Thr Trp Ser- Gly Gly Glu Gly Thr Val Lye 1 5 10 INFORMATION FOR TD. OF SECUENCTA NO. 8: (l) CHARACTERISTICS OF THE SEQUENCE: (A) LONGITUÜ: 11 arni noac i two (B) TYPE: amino acid (C) TYPE OF CHAIN: individual (D) TOPOLOGY: linear () TYPE OF MOLECULE: pept i do (v) ) FRAGMENT TTPO: internal (xi) SEQUENCE DESCRIPTION: TD. OF SEQUENCE NO. 8: Leu Ser Ser Ser Gly Ala Ser Trp His Thr Lys 1 5 10 INFORMATION FOR ID. OF SEQUENCE NO. 9: (l) CHARACTERISTICS DF IA SFCUFNCTA • (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN TTPO: INDIVIDUAL (D) TOPOLOGY: linear (li) MOLECULE TTPO: another nucleic acid ( A) DESCRIPTION: / desc = "oligon? Cl eotido" (Xl) SEQUENCE DESCRIPTION: ID. OF SEQUENCE NO. 9: GTN CCR TAC CAN ACY TCC CA 17 INFORMATION FOR TD. OF SEQUENCE NO. 10: (l) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TTPO: nucleic acid (C) CHAIN TTPO: individual (D) TOPOLOGY: linear iii) MOLECULE TTPO: nucleic acid (A) ) DESCRIPTION: / desc = "gone oligonucleot" (xi) DESCRIPTION OF SECUENCTA: TD. OF SEQUENCE NO. 10: TGG ACN TGG USN GGN GG 17 INFORMATION FOR ID. OF SEQUENCE NO. eleven: (l) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1 ^ base pairs (B) TTPO: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGT: linear (ll) TYPE OF MOLECULE: peptide (A) DESCRIPTION: / desc = "PCR fragment" (v) FRAGMENT TTPO: i tern (vi) ORIGINAL SOURCE: (A) ORGANISM: Aspe regí ll? S ni e (xi) SEQUENCE DESCRIPTION: ID. OF SEQUENCE NO. eleven GTGGAGTGGT GGCGAGGGAA CAGTGAAAAG CTACTC'AAC TCGGSCCp- S TG,: 5 GAAGCTAGTC AGCGATGTGT CAAGCApCC CACCTCGGTG ACATGGAGCC AGGACGACAC 12C CAATGTCCAA GCCGATGTCT CATATGATCT GpCACCGCG GCGAATGC2G ATCATGCCAI 180 i CLAGUGGT GACTATGAGC I I A TGA I I G G I ATGTuACG TCG I GAACAA GATAGA ~ GG- 2 > »C JUM b iMM IAALLAUUL OL OLLU "* UÜU» I ~ "U.U LrtO ^ .ml? O J. GUL-ÜG", • J A JU C ACTGTGGGAG GCAAG i CC i or GGAGG. _ • uG • CGG 3-? INFORMATION FOR ID. OF SECUENCTA NO. 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2360 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANIZATION: Aeperegillue niger (B) CEPA: 3M43 (IX) RASGO: (A) NAME / KEY: CDS (B) LOCATION: union (1021..1427, 1476..1708, 1778 ..1857) (D) OTHER INFORMATION: / product = "Endoglucanaea" gen / = "eglA" (ix) RASK: (A) NAME / KEY: exon (B) LOCATION: 1021..1427 (ix) TRAIT: ( A) NAME / KEY: i nt r (B) LOCATION: 1428..1475 (ix) RASK: (A) NAME / KEY: exon (B) LOCATION: 1476..1708 dx) TRAIT: (A) NO BRE / CLAVF: in rum (B) LOCATION: 1709..1777 (lx) RASK: (A) NAME / KEY: exon (B) LOCATION: 1778..1854 (ix) RASK: (A) NAME / KEY: sig_pept? D ? (B) LOCATION: 1021..1068 (? X) RASK: (A) NAME / KEY: mat_pept? Do (B) LOCATION: union (1,069..1427, 1476..1708 1777. .1854) (xi.) SEQUENCE DESCRIPTION: ID. OF SEQUENCE NO. 12: AApGAAGCA píTGATAGG pTAAGCCTA ATCAGGATAT TGGATGAGTC GAGpGCAGA 60 AGpGAGGAC GGTGGGTGAA ATCGGGGGp TGATAGGTAG GCAATGCAGG GCGGAACGGG 120 AAGGGTCTAG ACAATpcp TCTpTGGAC AGCTGGTGCG pTCACTGAG ATTAATAGTA 130 AGCAAACTAC TCGCTCGAAG TCGTAGATGT GCATAATGGA TAACTACAGC CAACCGAAAT 2¿0 CTCCGGGCAG AAGGCCTGGA GGCAGGAGGA AACGTGGATA AGAGAGTAAT GpTGAGTAT 3C0 AGATATGTAG GCAAGAAAGG ACTGGGAGG AGGAAGTATC GCAAACAAGA CAA.GTCACTG 360 AATAGGAAAG AATGGGGCCA TCAGAGAAAT GAATCTAAAC GGTAACTGCA GATAHACAT 420 GGAAGAAAAT ACTATGATCC CTAApGATA TGGpCCATG GCCCCTGGAG ACpAAACCT 480 CGTGGTATGA TAAACATATG A.GpAC.ApC TCGGTAAATC CAACApACT CCCAAGCTCT 540 GpGATA.pC TCCGATAAp CACCGATAAC CAACCAACCT ACTCCCGTCT AGATCCAAp 600 GGTCTATATG CATAATGGAT ATCGTCAGCA CAGGCAGAAC CCpTAApT ATpCTGGAG 660 ATCCCGpCT CCACAATGCT TGGTT3CCGA CTGCCACAGA CCATCGCTAA CpGAAGCGG 720 AAAGTGCTCC GATGAAGGGT CTCATpTGA AACGGAGGAT pACATGTCA ATGpGCAGG 780 CTGGCGpGA TGATGGCGCA ACCTGCTATA GCTAGpGGC pACpCGTC CTGGCTGCCG 840 TApGGACAC GGAAAGTCGG ACAATAATAG TGpAACAGT AAGCGCCAp GATCAGAGp 900 GATGTATpA AAGCTGCGTC GTCTGCTGCC CCCTCCGTGT TCGTGTCpA pCCAAACAT 960 TCAACCTCTA pCCpTCGA AGTCCTGTAG ATCTGCCGp CCTCTGCTp ApGCCCAAC 1020 ATG AAG CTC TCC ATG ACÁ Cp TCC CTG Tp GCG GCC ACT GCC ATG GGC 1068 Met Lys Leu Ser Met Thr Leu Ser Leu Phe Wing Wing Thr Wing Met Gly -16 -15 -10 -5 CAG ACG ATG TGC TCT CAG TAT GAC AGT GCC TCG AGC CCC CCA TAC TCG 1116 Gln Thr Met Cys Ser Gln Tyr Asp Ser Wing Ser Pro Pro Tyr Ser 1 5 10 15 GTG AAC CAG AAC CTC TGG GGC GAA TAC CAG GGC ACT GGC AGC CAG TGT 1164 Val Asn Gln Asn Leu Trp Gly Glu Tyr Gln Gly Thr Gly Ser Gln Cys 20 25 30 GTC TAC GTC GAC AAG Cp AGC AGC GGT GCC TCA TGG CAT ACC AAA 1212 Val Tyr Val Aso Lys Leu Ser Ser Ser Gly Wing Ser TrD His Thr Lys 35 40 45 TGG ACC TGG AGT GGT GGC GAG GGA ACÁ GTG AAA AGC TAC TC ^ AAC "CC 125: Trp Thr 7GD Ser Gly Gly Glu G'y thr Val Lys Ser Tyr Ser Asn Ser? ° 55 60 _ _ "^ Z C. ACG ~ GAC AAG AAG CTA GTC AGC GAT GTG "CA AGC i. CCC -uc Gly Leu Thr Phe Aso Lys Lys Leu Val Ser Aso val Ser Ser He P- * c 65 70 75 30 • C TCG GTG ACÁ TGG AGC CAG GAC GAC ACC AAT GTC CAÁ GCC GAT GTC 1356 Thr Ser Val Thr Trp Ser Gln Asp Asp Thr Asn Val Gln Ala ASD Val 85 90 95 TCA TAT GAT CTG pc ACC GCG GCG AAT GCG GAT CAT GCC ACT TCC AGC 1404 Zer Tyr Asp Leu Phe Tr Ala Ala Asn Ala Asp His Ala Tnr Be Ser 100 105 110 GGT GAC TAT GAG Cp ATG p TG GTATGTGACG TCGTGAACAA 1447 Gly Asp Tyr Glu Leu Met He Trp 115 120 10 GATAGATGGA GGAGGCTAAC GTAACCAG G Cp GCC CGC TAC GGC TCA GTC CAG 1500 Leu Ala Arg Tyr Gly Ser Val Gln 125 CCT Ap GGC AAG CAG Ap GCC ACG GCC ACT GTG GGA GGC AAG TCC TGG 1548 Pro He Gly Lys Gln I Ala Thr Ala Thr Val Gly Gly Lys Ser Trp 130135140 GAG GTG TGG TAT GGT ACC AGC ACC CAG GCC GGT GCG GAG CAA AAG ACÁ 1596 15 Glu Val Trp Tyr Gly Thr Ser Thr Gln Ala Gly Ala Glu Gln Lys Thr 145 150 155 160 TAT AGC GTG GCA GGA TCT CCT ATC AAC TCG TGG AGT GGG GAC Ap 1644 Tyr Ser Phe Val Wing Gly Ser Pro He Asn Ser Trp Ser Gly Asp He 165 170 175 AAG GAC pc pc AAC TAT CTC ACC AAC CAG CAA GGC CCG GCT AGC pc Lys Asp Phe 2Q 1692 Phe Tyr Asn Gln Thr Leu Gln Gly Asn Phe Pro Ala Ser 180 185 190 CAG CAT TCT ATC pG A GTGAGTpTC CTAApCTAC TAGCGAGCGC 1738 Ser Gln His Leu He 195 CGGCAGTTGA AApGGTCAC TAACAGAAGT GATGApAG CT CTG CAÁ Tp GGA 1791 Thr Leu Gln Phe Gly? 5 200 * f ^ -, • »« / "» ("r" TT f. Rt r-1"" * rt * r. rrr "" -? f "" T "» * r - - n> * * r -r »-« - > »•) (' My l rtÜ o I l L Ü UU. ÜUL Lu ÜUn A L i i '«. . UJ U L I ML 'V? II JU ior r'nr Glu Phe Thr Gly Gly Dro Ala Pro Thr Phe Asn Tr * Aso Tro ACC GCT 205 210 215 AGT G "C AAC TAA AAGGCTpAG GCGCGGCTGG GGTAAATAAC 1857' nr Val Ala Ser Sn w 220"'Ut. J i i. . .j. ,. AU AUXJ i v-UUÜ U I 'j i nAünU-. UWr i n, ..- ^. ? . Ur. : • * • pGGAAACAC TCApCAAGA TCGGTACTCC TCpCAGCCG AGAAAGGCAC AGATAGTGTA 2007 TCGAATCCAA TCAAATCTAT pGGTGpGC pAAApCCG AGCCAGTCCT pccpGAAA 2067 GGTAATCCAC CCGTAGCGAT TGATCApAA CAGATCCGAG TGGTGCTAGG pAAA.pGCT 2127 AACCCGATCC CGCTCCAAp .AGCTAGCGCA TCCGGCAGAT TCAAACpGA CAGTGGGCCG 2137 GGCApACCT GAACCTGTAG AAGGAACAGA CCCTTGTCTA GAAATCTCTA AATAGTATAA 22: GCCGAAA.Cp GCCCCGGACG TACCCT.AAGC TAAGApGCT ZT? ÍXATTZ CCAGGGGGGT 2307 GAACTCTCTA AAGAGGGAGC ATCGCpGCC GATGTCTGGT TCGGGGATCA TGA 2360TION FOR ID. OF SEQUENCE NO. 13 TERMINATOR SEQUENCE AAGGCTpAG GCGCGGCTGG GGTAAATAAC AGCpGpTC TTCGTTCTAG_50_AACGTCGGGC GTGTAAGAGC TAGAAATCCA CCCACTCTGA pGGAAACAC 100 TCApCAAGA TCGGTACTCC TCpCAGCCG AGAAAGGCAC AGATAGTGTA 150 TCGAATCCAA TCAAATCTAT pGGTGpGC pAAApCCG AGCCAGTCCT 200 pcCpGAAA GGTAATCCAC CCGTAGCGAT TGATCApAA CAGATCCGAG 250 TGGTGCTAGG pAAApGCT AACCCGATCC CGCTCCAAp AGCTAGCGCA 300 • - TCCGGCAGAT TCAAACpGA CAGTGGGCCG GGCApACCT GAACCTGTAG_350_AAGGAACAGA CCCpGTCTA GAAATCTCTA AATAGTATAA GCCGAAACp 400 GCCCCGGACG TACCCTAAGC TAAGApGCT CpCGCApC CCAGGGGGGT 450 GAACTCTCTA AAGAGGGAGC ATCGCpGCC GATGTCTGGT TCGGGGATCA 500 • TGA 5037 (2) INFORMATION FOR ID. OF SEQUENCE NO. 14: SEQUENCE OF SEÑOLS ATG AAG CTC TCC ATG ACÁ Cp TCC CTG Tp GCG GCC ACT GCC ATG GGC 48 (2) INFORMATION FOR ID. OF SEQUENCE NO. 15: SEQUENCE OF SIGNALS Met _ s Leu Se z Thr Leu Ser Leu Phe Ala Ala Thr Ala Mo *

Claims (29)

NOVELTY OF THE INVENTION CLAIMS

1. - An extract obtainable from Aspergillus, also characterized because the enzyme has the following characteristics: a. a MW of 24,235 D ± D; b. a pT value of approximately 4; c. activity of the glase; wherein the activity of gl? canase is endo-β-1, 4-gl? canase activity.

2. An enzyme that has the sequence shown as T.D. SEC. No. l or a variant, homologo or fragment of the same.

3. An enzyme encoded by means of the nucleotide sequence shown as T.D. SEC. No. 2 or a variant, homologue or fragment thereof or a sequence or structure thereof.

4. A sequence of nucleotides encoding the enzyme pair according to claim 1.

5. A nucleotide sequence that encodes the enzyme according to the rei indication 2.

6. A nucleotide sequence that has the sequence shown as ID SEC. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.

7.- A nucleoi sequence. two in accordance with any of claims 4 to 6 operatively linked to a promoter.

8. A nucleotide sequence according to claim 7, further characterized in that the promoter is the promoter that has the sequence shown as I.D. SEC. No. or a variant, homologous or fragment thereof or a sequence complementary to it.

9.- A promoter that has the sequence shown as I.D. SEC. No. or a variant, homologue or fragment of it or a sequence complementary to it.

10.- A promoter in accordance with the claim 9 operatively linked to a gene of interest.

11. A promoter according to claim 10, further characterized in that the gene of interest comprises a nucleotide sequence according to any of claims 4-6.

12.- A terrnmador that has the sequence of nucleotides shown in I.D. SEC. No. 13 or a variant, homologous or fragment thereof or a sequence complementary to it.

13.- A signal sequence that has the nucleotide sequence shown in I.D. SEC. No. 14 or a variant, homologue or fragment thereof or a sequence complementary thereto.

14. A construct that comprises or expresses the invention of co-form with any of claims 1 to 13.

15. - A vector comprising or expressing the invention according to any of claims 1 to 14.

16. A plasmid comprising or expressing the invention according to any of claims 1 to 15.

17.- A transgenic organism. which comprises or expresses the invention in accordance with any of claims 1 to 16.

18. A transgenic organism according to claim 17, further characterized in that the organism is a fungus.

19. A transgenic organism according to claim 17, further characterized in that the organism is a filamentous fungus, preferably of the genus Aspergillus.

20. A method according to claim 17, further characterized in that the organism is a plant.

21. A transgenic organism according to claim 17, further characterized in that the organism is a yeast.

22. A process for preparing an enzyme according to any of claims 1 to 3, which comprises expressing a nucleotide sequence according to any of claims 4-8.

23. A process according to claim 22, characterized in that the enzyme has the sequence shown as I.D. SEC. No. 1 or a variant, homolog or fragment thereof, and the nucleotide sequence has the sequence shown as T.D. SEC. No. 2 or a variant, homolog or fragment or a sequence complementary to it.

24. A method according to claim 22 or 23, further characterized in that the expression is controlled (partially or completely) by means of the use of a promoter according to claim 9.

25.- A method for expressing a gene for interest through the use of a promoter, further characterized in that the promoter is the promoter according to claim 9.,

26. The use of an enzyme according to any of claims 1 to 3 or prepared by means of a method according to any of claims 22. to 25 to degrade a glucan. 27.- The plasmid NCIMB 407004, or a nucleotide sequence obtainable therefrom to express a glucanase enzyme or to control the expression thereof or to control the expression of another gene of interest. 28.- An enzyme of gl? Canase that has the ability to degrade the β-l, 4-gl? Cosidic inlays, which is immunologically reactive with? N antibody cultured against a purified glycanase enzyme having the sequence shown in the TD SEC. No. 1 .. 29.- A signal sequence that has the sequence -? W. shown as I.D. SEC. No. 15 or a variant, homologue or fragment thereof. iX