MXPA96001430A - Enzymes that degradate arabinoxil - Google Patents

Abstract

The present invention provides methods and constructs or expression constructs for the cloning and overexpression of an arabinoxylan degrading enzyme of fungal origin, in a selected microbial host cell. The enzyme shows to be active in the degradation of insoluble solids in water obtained from corn. The enzyme can be used in the preparation of compositions for animal feed, human food or industrial processes

Description

REF: 22323 ENZYMES THAT DEGRADATE ARABINOXILANO TECHNICAL FIELD The present invention relates to the field of molecular biology. In particular, the present invention relates to the cloning and expression of the genes encoding the polypeptides that show degradation activity of the arabinoxylan. These enzymes are used appropriately in industrial processes such as baking bread, paper and pulp processing and in the preparation of forage and food (additives).

BACKGROUND OF THE INVENTION The composition of the cell wall of a plant is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the cell wall of plants), hemicellulose (comprising several chains of ß-xylan) and pectin- The existence, distribution and structural characteristics of polysaccharides The cell wall of plants are determined by (1) the species of the plant; (2) the variety; (3) the type of tissue, (4) the growth conditions; (5) age and (6) processing of plant material before feeding. There are basic differences between monocots (eg cereals and grasses) and dicotyledons (eg cloves, rapeseed and soybeans) and between seed and vegetative parts of the plant (Chesson, 1987, Carré and Brillouet, 1986). The monocotyledoniae are characterized by the presence of a complex of arabinoxylan as the main hemicellulose skeleton. The main structure of helicellulose in the dicotyledons is a xyloglucan complex. In addition, higher concentrations of pectin are found in the dicotyledons than in the monocotyledons. The seeds are generally very high in pectic substances but relatively low in cellulosic material. Three or more polysaccharide structures that interact in the cell wall can be distinguished: (1) the central lamella that forms the outer cell wall. This also serves as the anchoring point of the individual cells to each other within the tissue matrix of the plant. The central lamella consists mainly of calcium salts of highly esterified pectins; (2) The primary wall that is located just to the side of the central lamella. This is a well-organized structure of cellulose microfibrils embedded in an amorphous matrix of hemicellulose pectin, phenolic esters and proteins; (3) The secondary wall that forms as the plant matures. During the growth and aging phase of the plant, microfibrils of cellulose, hemicellulose and lignin are deposited. The primary cell wall of the cells of metabolically active plants (eg mesophyll and epidermis) is more susceptible to enzymatic hydrolysis than the secondary cell wall, which by its state, has become highly lignified. There is a high degree of interaction between cellulose, hemicellulose and pectin in a cell wall.

Enzymatic degradation of these highly crosslinked polysaccharide structures is not a simple process. At least five different enzymes are needed to completely decompose an arabinoxylan, for example. Endo cleavage is effected by the use of an endo-β (1α4) -D-xylanase. The exo- (1-> 4) -D-xylanase liberates xylose units at the non-reducing end of the polysaccharide. Three other enzymes (α-glucuronidase, α-L-arabinofuranosidase and acetyl esterase) are used to attack substituents on the xylan backbone. The choice of specific enzymes depends of course on the specific hemicellulose to be degraded (McCleary and Matheson, 1986). Enzymes that attack the lateral chains of the xylan skeleton may be of interest because they change the characteristics of the polymer, making it more suitable for certain applications. Furthermore, these enzymes can act synergistically with the endoxylanases that separate the main chain (for an extensive review see Kormelin, 1992, Doctoral Thesis, Wageningen University). A DNA fragment encoding a degradation activity of the arabinoxylan is known. In the European patent application 463 706, the isolation, characterization and cloning of an endoxylanase gene from Aspergillus tubigensi s is described. This enzyme is not capable of attacking the side chains of the arabinoxylan skeleton.

Enzymatic activities capable of attacking the side chains from Aspergillus niger are also known (Kormelink, 1992, supra, Chapters 6 and 7). An enzyme called arabinofuranosidase A (ArafurA) is characterized by the ability to release arabinose residues from oligosaccharide structures obtained from arabinoxylans. However, Arafur A is not active on high molecular weight substrates. In addition, Aspergill us niger produces an enzyme called arafur B which is active on the oligosaccharide, as well as on the high molecular weight arabirazoxy structures. The enzymatic action of ArafurB is confined to the release of arabinose residues from terminal monosubstituted xylose residues. DNA fragments are not known until now. An activity that is capable of releasing arabinose residues from non-terminal monosubstituted xylose residues in both oligosaccharides as well as in the high molecular weight arabinoxylan structures of Aspergill us awamori has been isolated. This enzyme is called arabinoxylan arabinofuranose hydrolase (AXH). So far the data of the fragments and / or the DNA sequence is not available. It is clear that not all the enzymes involved in the degradation of arabinoxylan have already been detected (Kormelink 1990).

For example, enzymes that attack the xylose molecules disubstituted with arabinose have not yet been found. The reason for this is that these enzymes are often secreted in low amounts. Molecular cloning and overproduction in an adequate host of these enzymes, although not easy, is a way to obtain sufficient quantities of pure enzyme, which in turn allows to evaluate its importance in several applications.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides the recombinant DNA comprising a DNA fragment encoding a polypeptide having arabinoxylan degrading activity, or a polypeptide precursor thereof, characterized in that the DNA fragment is selected from: (a) a DNA fragment encoding a polypeptide having the amino acid sequence represented by amino acids 1 to 306, or a polypeptide precursor of such polypeptide represented by amino acids -27 to 306 in IDSECNO: 5; (b) a DNA fragment encoding a polypeptide having the amino acid sequence represented by amino acids 1 to 306, or a precursor of such polypeptide represented by amino acids -27 to 306 in IDSECNO: 7; (c) A DNA fragment encoding a variant or a portion of the polypeptides represented by the residual amino acids 1 to 306 described in IDSECNO: 5 or 7, which still has arabinoxylan degrading activity, or a polypeptide precursor thereof; (d) A DNA fragment encoding a polypeptide having arabinoxylan degrading activity and having the nucleotide sequence represented by nucleotides 784 to 1779 in IDSECNO: 5 or nucleotides 823 to 1818 in IDSECNO: 7; (e) A DNA fragment encoding a polypeptide having arabinoxylan degrading activity, or a portion of such a polypeptide, a DNA fragment which is capable of hybridizing to a DNA fragment as represented by nucleotides 784 through 1779 in IDSECNO: 5 or nucleotides 823 to 1818 in the IDSECNO: 7. The recombinant DNA according to the invention is preferably obtained from a filamentous fungus, more in particular from a species of Aspergillus. Especially preferred recombinant DNA sequences comprise DNA fragments encoding the AXDA of Aspergillus niger or tubigensis. According to another embodiment, the recombinant DNA according to the invention comprises the 51 and 3 'regulatory DNA sequences required for the expression of the DNA fragment in a prokaryotic or eukaryotic host cell when present therein. The regulatory DNA sequences are preferably heterologous with respect to the sequence encoding the polypeptide of such DNA fragment, more preferably such regulatory DNA sequences are selected to increase the expression of the DNA fragment in a host as compared to the expression of the DNA fragment in such a host when they bind to their homologous regulatory DNA sequences. According to another embodiment such recombinant DNA is in the form of a vector. The invention further provides a transformed eukaryotic or prokaryotic host cell comprising the recombinant DNA according to the invention, preferably of the genus Aspergillus, as well as a method for obtaining a host cell capable of increasing the expression of an enzyme that degrades arabinoxylan by the treatment of a host cell under conditions of transformation with a recombinant DNA according to the invention and selecting the increase of expression of the enzyme that degrades the arabinoxylan. The invention also provides a method for obtaining an enzyme that degrades arabinoxylan comprising the steps of growing host cells capable of producing such an enzyme under conditions that lead to this and recovering the enzyme, characterized in that the host cells, or their ancestors, have been transformed with a recombinant DNA according to the invention. According to yet another embodiment, the invention provides a substantially pure polypeptide having arabinoxylan degrading activity and characterized by the amino acid sequence described in the IDSECNO: 6 or the IDSECNO: 8, as well as the variants and genetic portions thereof. that still have such activity. The invention also focuses on a composition comprising a substantially pure polypeptide according to claim 14 formulated for use in forages, foods, or paper and pulp processing, optionally, wherein the enzyme is immobilized. Methods of using a polypeptide according to the invention are also provided to aid in the degradation of arabinoxylan, as an additive for forages or foods, in the processing of paper and pulp and in baking of bread. Fodders and foods containing a polypeptide according to the invention are also claimed. According to a further embodiment, the recombinant DNA is provided, comprising a DNA fragment represented by nucleotides 1 to 783 of the IDSECNO: 5, or a subfragment thereof, capable of regulating the expression of a DNA sequence linked to this , as well as the recombinant DNA 'comprising a DNA fragment represented by nucleotides 1 to 822 of IDSECNO: 7, or a subfragment thereof, capable of regulating the expression of a DNA sequence linked thereto. The invention is better illustrated by the description of the following figures.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 elution profile (OD280) of the degradation activity of arabinoxylan on CLAP. Figure 2 diagram showing the activity of the AXDA on the fraction of insoluble solids in water (SIA) of corn as a function of pH.

Figure 3 Percentage of in vitro digestion of maize SIA as a function of the amount of crude AXDA enzyme Figure 4 Map of restriction sites in pIM3001 Figure 5 Map of restriction sites in pIM3002 DETAILED DESCRIPTION OF THE INVENTION The present invention provides molecules of Isolated and purified DNAs comprising the sequence of the genes of the enzyme that degrades arabinoxylan and genetic variants thereof. The DNA molecules can include the region encoding the enzyme that degrades the arabyloxylan as well as its adjacent 5 'and 3' regulatory regions. Genetic variants include DNA molecules that encode mutant arabinoxylan proteins and degenerate DNA molecules, in which the desired activity of the enzyme expressed in them is retained. The present invention also provides DNA constructs for the expression of enzymes that degrade arabinoxylan in a desired expression host. Such expression constructs include hybrid DNA molecules that contain the regions encoding enzymes that degrade arabinoxylan operably linked to regulatory regions, such as a promoter, secretion and termination signals that originate in homologous or heterologous organisms. , those regulatory regions are capable of directing the increased expression of the enzyme encoded by the DNA molecule encoding the enzyme which degrades arabyloxylan in an appropriate host. Preferably, the expression construct should be integrated into the genome of the selected expression host. The present invention further provides vectors, preferably plasmids, for the cloning and / or transformation of the microbial hosts via the introduction into the microbial host of the DNA constructs for the expression of the enzymes that degrade the arabyloxylan. In addition, the present invention provides homologous or heterologous hosts transformed with vectors containing the DNA constructs described above. Heterologous hosts can be selected from bacteria, yeast or fungi. Within the context of the present invention, it is understood that the term "homologous" is intended to include everything that is native to the DNA molecule that codes for the enzyme that degrades the arabinoxylan of interest, including its regulatory regions. A homologous host is defined as the species from which such DNA molecules can be isolated. The term "heterologous" thus defines everything that is not native to the DNA molecule that codes for the enzyme that degrades the arabinoxylan of interest itself, including the regulatory regions. A "heterologous" host is defined as any microbial species different from that of which the genes encoding the enzyme that degrades aravyoxylan have been isolated. Within the context of the present invention, the phrase "increased expression of the arachloxylane-degrading enzyme of interest" is defined as the expression of the enzyme that degrades the arabinoxylan of interest at concentrations higher than those ordinarily found in the homologous natural organisms. In the same context, the augmented expression is also intended to include the expression of the enzymes that degrade the arabinoxylan of interest in a heterologous organism that does not normally produce such enzymes that degrade arabinoxylan except by the introduction of the DNA molecule or expression construct that encodes enzymes that degrade the arabinoxylan of interest in the heterologous expression host. It should also be understood, of course, that the descendants of those expression hosts are encompassed by the present invention. The present invention also includes DNA sequences that hybridize to the DNA sequences that can be obtained from the fungi described above., but that may differ in the sequence of the codon due to the degeneration of the genetic code or variation of crossed species. Thus, the invention includes the DNA fragments that code for the enzymes that degrade the arabyloxylan that can be obtained from other species than Aspergillus. Typically, the processes for obtaining similar DNA fragments involve the selection of bacteria or plaques of bacteriophages transformed with recombinant plasmids containing DNA fragments from an organism that is known or expected to produce an enzyme that degrades arabinoxylan according to the invention. After duplication of the DNA in situ, the DNA is released from the cells or plates, and immobilized on filters (usually nitrocellulose). The filters can then be selected to detect the complementary DNA fragments using a labeled nucleic acid probe based on any of the sequences determined for the axperA genes of Aspergillus. Depending on whether or not the organism to be selected for being distant or closely related, the conditions of hybridization and washing should be adapted to extract the true positives and reduce the amount of false positives. A typical procedure for the hybridization of DNA immobilized on the filter is described in Chapter 5, Table 3, pp. 120, and 121 in: Hybridization of nucleic acid - a practical method, B.D. Hames &; S.J. Higgins Eds., 1985, IRL Press, Oxford). Although optimal conditions are usually determined empirically, few empirical rules may be useful for narrow and less closely related sequences. To identify DNA fragments very closely related to the probe, hybridization was carried out as described in Table 3 of Hames & Higgins, supra, (the essential of which is reproduced below) with a final wash step under highly stringent conditions in 0.1 * SET buffer (20 times of SET = 3M NaCl, 20mM EDTA, 0.2M Tris-HCl, pH 7.8, 0.1% SDS) at 68 ° Célsius. To identify the sequences with limited homology with the probe, the process to be followed is that of Table 3 of Hames & Higgins, supra, but with a lower temperature of hybridization and washing. A final wash with buffer 2 * SET, 50 ° C for example could allow the identification of the sequences having approximately 75% homology. As is well known to those skilled in the art, the exact relationship between homology and hybridization conditions depends on the length of the probe, the composition of bases (% of G + C) and the distribution of inconsistencies; a random distribution has a strong merging effect on the Tm resulting in a non-random or agglomerated pattern of inconsistencies. The above conditions apply to probes having a length of at least 300 bp, preferably at least 500 bp, more preferably approximately 1 kbp, and a GC content of the DNA to be included in the probe of approximately 50 +. 10% If the GC content of a given organism is known, then the conditions can be empirically used taking into account the following equation (at 1M NaCl, with the 35% interval <(% of G + C) <75%) : Tm = 81.5 ° C + 0.41 * (% of G + C). Approximately, this means that if the GC content (% of G + C) is greater than 10% on average, the (strict) hybridization conditions can be adjusted by increasing the hybridization and washing temperature by about 4 ° C. For the purposes of this disclosure, a DNA fragment that is said to hybridize to the DNA fragment according to the invention is defined as that which gives a positive DNA signal immobilized after hybridization with a probe of at least 300 p. / preferably 500 bp, more preferably 1 kbp of any of the sequences described in IDSECNO: 5 or 7, and washing following the procedure of Table 3 of Chapter 5 of Hames & Higgins (as reproduced in essence below) at a temperature of 50 ° C, buffer 2 * SET (ie NaCl 0.3 M). The essential aspects of the procedure described in Table 3, Chapter 5 of Hames & Higgins are the following: (1) prehybridization of the filters in the absence of a probe, (2) Hybridization at a temperature between 50 and 68 ° C in SET buffer between 0.1 and 4 * SET (depending on requirements), 10 * Denhardt solution (100 * Denhardt solution contains 2% bovine serum albumin, 2 Ficoll%, 2% polyvinyl pyrrolidone), 0.1% SDS, 0.1% sodium pyrophosphate, salmon sperm DNA 50 g / ml (from a standard obtained by dissolving 1 mg / ml of salmon sperm DNA, sonicated to a length of 200 to 500 bp, allowed to stand in a water bath for 20 min., and diluted with water to a final concentration of 1 mg / ml); the hybridization time is not very critical and can be any between 1 and 24 hours, preferably about 16 hours (o / n); the probe is typically labeled by slot translation using 32p as the radioactive label at a specific activity of between 5 * 107 and 5 * 108 c.p.m./μg; (3) (repeated) washing the filter with 3 * SET, 0.1% SDS, 0.1% sodium pyrophosphate at 68 ° C at a temperature between 50 ° C and 68 ° C (depending on the desired requirement), Repeat washing while decreasing the SET concentration to 0.1%., Wash once for 20 min. in 4 * SET at room temperature, dry the filters on 3MM paper, expose the filters to an X-ray film in a cassette at -70 ° C for between 1 hour and 96 hours (depending on the strength of the signal), and reveal the movie.

In general, the volume of prehybridization and hybridization mixtures should be kept to a minimum. All wet steps should be carried out in small sealed bags in a preheated water bath.

The above procedure serves to define the DNA fragments that hybridize according to the invention. Obviously, numerous modifications to the method can be made to identify and isolate the DNA fragments according to the invention. It should be understood that the DNA fragments thus obtained fall under the terms of the claims since they can be detected following the above procedure, regardless of whether they have already been identified and / or isolated using this method. Numerous protocols, which can be used suitably to identify and isolate the DNA fragments according to the invention, have been described in the literature and in manuals, including the Hames & amp; Higgins, supra). The above procedure is for polynucleotide probes. When oligonucleotide probes are used the relationship between T < ¿The temperature at which a perfectly matched hybrid is semi-dissociated is estimated by the relationship: T¿ = 4 ° C per base pair of GC + 2 ° C per base pair AT. Based on existing protocols and using this empirical rule, oligonucleotide probes can also be designed to effectively isolate the DNA fragments encoding enzymes that degrade arabinoxylan according to the invention from related and more distant organisms. The procedure using oligonucleotides is particularly useful if an enzyme has been purified from a different source and part of the amino acid sequence has been determined. Using this sequence a group of degenerate probes can be produced for the selection of a DNA library or Southern blot, essentially as described above. Good candidates for selection are other filamentous fungi, such as Trichoderma, Penicillium, Dichotomi tud squalus, Disporotrichum dimorphosporum, and bacteria, such as Bacillus species, and the like. If the initial selection results in clones that appear to contain hybridizing fragments, such fragments can be isolated and, if desired, sequenced to determine similarities in the sequence. Once an enzyme has been identified that degrades the arabinoxylan of interest, the DNA sequence encoding such an enzyme can be obtained from the filamentous fungi that naturally produce it by growing the fungi in a medium containing arabinoxylan., isolating the enzyme that degrades the desired arabinoxylan using known methods such as those outlined in Example 1 and determining at least a portion of the amino acid sequence of the purified protein. The DNA probes can then be obtained by designing oligonucleotide sequences based on the deduced partial amino acid sequence. The amino acid sequences can be determined from the N-terminus of the entire protein and / or from the N-terminals of the internal peptide fragments obtained via the proteolytic or chemical digestion of the entire protein. Once obtained, the DNA probes are used to select a genomic or cDNA library. A genomic library can be prepared by partially digesting the chromosomal DNA of the fungi with a restriction enzyme that recognizes a DNA sequence of four successive nucleotides, for example Sau3A, and cloning the resultant fragments of a suitable neoplasm or phage lambda vector, for example GEM-11 lambda. Alternatively, a cDNA library can be prepared by cloning the cDNA, synthesized from the mRNA isolated from the fungal cells induced for the synthesis of an enzyme that degrades the arabinoxylan, into an appropriate phage vector, for example gt 10 lambda. Subsequently, after plating a sufficient number of colonies or plaques, the genomic or cDNA library can be selected with a suitable DNA probe. If this method is not successful, the. The genomic or cDNA library can be differentially selected with the cDNA probes obtained from the mRNA of uninduced and induced cells. The induced mRNA is prepared from the cells growing in the medium containing arabinoxylan as the carbon source, while the non-induced mRNA must be isolated from the cells growing on a carbon source other than the arabinoxylan, for example glucose. Among the clones that hybridize only with the induced cDNA probe, a clone containing the gene encoding the enzyme degrading the desired arabinoxylan can be recovered. Alternatively, a gene for the enzyme that degrades arabinoxylan can be identified by cross-hybridization with a related sequence. Preferably, the oligonucleotide probes are obtained from the N-terminal amino acid sequence (see Example 1.5.1) of an enzyme that degrades arabinoxylan having an apparent molecular weight of 32 kDa purified from a filtrate of Aspergillus niger and / or of the amino acid sequence of an internal peptide fragment (see Example 1.5.2) obtained by the digestion of the enzyme with CNBr. The developed oligonucleotide mixtures can be used to hybridize with both genomic and cDNA libraries. Alternatively, as illustrated here. The purified AXDA enzyme was used to produce antibodies. The antibodies are used in the immunoselection of expression libraries. In this way, expression clones of AXDA are identified. It should be noted that the protein was isolated from A. niger variant tubigensis and the cDNA was isolated from A. niger N400 illustrating that the different Aspergilli contain AXDA activity. With the advent of new DNA amplification techniques, such as direct or inverted PCR, it is also possible to clone DNA fragments in vi tro once the terminal sequences of the coding region are known. The availability of a DNA sequence encoding an enzyme that degrades arabinoxylan allows the construction of mutant enzyme molecules by site-directed mutagenesis. If the tertiary structure of the enzyme that degrades arabinoxylan is known, and if its catalytic and substrate-binding domains are localized, amino acids can be selected by mutagenesis (for example with the help of computer modeling) that are likely to affect the catalytic and / or binding functions of the substrate. If the tertiary structure of the protein is not available, either random mutants along with the entire coding sequence can be generated, or the tertiary structure of the protein can be predicted by comparison with similar known enzymes isolated from other microorganisms. To facilitate the insertion of the DNA fragment containing the sequence encoding AXDA into the expression constructs comprising one or more of the heterologous regulatory regions, the polymerase chain reaction (PCR) can be used (PCR Technology: Principies and Applications for DNA Amplification, (1989) HA Ehrlich, ed., Stockton Press, New York) to introduce the appropriate restriction enzyme sites at the 5 'and 3' ends of the sequence encoding the AXDA. The choice of restriction sites depends on the DNA sequence of the expression vector, ie the presence of other restriction sites within the DNA molecule. To obtain the increased or amplified expression of the AXDA proteins in the original production species (homologs), or alternatively in the heterologous fungal strain, the DNA regions encoding the AXDA, including its control region, are introduced into the selected expression host to increase the number of copies of the gene and, consequently, the expression of the protein. If a heterologous expression host is preferred, and a yeast or bacterial strain is selected, an uninterrupted DNA molecule (without intron) is used for the construction of a heterologous expression vector to avoid the possibility of binding signals or splicing residues in the genomic fragment are not recognized by the heterologous host. This uninterrupted DNA molecule can be obtained from a cDNA library constructed from the mRNA isolated from the cells, induced for the synthesis of the AXDA. This library can be selected with an oligonucleotide or a cDNA probe obtained as described above. Alternatively, an uninterrupted DNA molecule can be obtained by applying a polymerase chain reaction using the appropriate 5 'and 3' oligonucleotides on the first strand of cDNA synthesized from the RNA of the cells induced with arabinoxylan. Enhanced or amplified expression of the AXDA of interest can also be achieved by selecting the heterologous regulatory regions, for example the promoter, secretory and terminator-conducting regions, which serve to increase expression and, if desired, concentrations of secretion of the protein of interest of the chosen expression host and / or to provide the inducible control of the expression of the AXDA of interest. In addition to the AXDA of the native promoter of interest, other promoters may be used to direct its expression. The promoter can be selected for its efficiency in the direction of expression of the AXDA of interest in the desired expression host. In another embodiment, a constitutive promoter can be selected to direct the expression of the desired AXDA, relatively free of other enzymes. Such an expression construct is additionally advantageous since it avoids the need to culture the expression hosts in a medium containing arabinoxylans as the inducing substrate.

Examples of strongly constitutive and / or inducible promoters that are preferred for use in fungal expression hosts are the promoters of ATP synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi), alcohol dehydrogenase (adhA), a-amylase (amy), glucoamylase (gam), acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd). Examples of strong yeast promoters are the promoters of the alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase and triosephosphate isomerase. Examples of strong bacterial promoters are the a-amylase and Spo2 promoters as well as the promoters of the protease genes. Hybrid promoters can also be used advantageously to improve the inducible regulation of the expression construct. Frequently, it is desirable that the AXDA of interest be secreted from the expression host into the culture medium. In accordance with the present invention, the AXDA of the native secretory sequence of interest can be used to affect the secretion of the expressed AXDA. However, sometimes an increase in the expression of the enzyme results in levels of protein production beyond what the expression host is capable of processing and segregating, creating a bottleneck such that protein products accumulate inside the cell. Accordingly, the present invention also provides heterologous leader sequences to provide more efficient secretions of the chosen expression host enzyme. In accordance with the present invention, the secretion director can be selected based on the desired expression host. A heterologous secretion director which is homologous to the other regulatory regions of the expression construct may be chosen. For example, the leader of the highly secreted glucoamylase protein can be used in combination with the glucoamylase promoter itself, as well as the combination with other promoters. Hybrid signal sequences may also be advantageously used within the context of the present invention. Examples of heterologous secretory leader sequences are those that originate from the glucoamylase gene (fungi), the a-factor gene (yeast) or the α-amylase gene (Bacillus). In general, terminators are not considered to be critical elements for the augmented or amplified expression of genes. If desired, a terminator of the same genes may be selected as a promoter, or alternatively, the homologous terminator may be employed. In addition to the genomic fragment mentioned above, the transforming DNA may contain a selection marker to discriminate cells that have incorporated the desired gene from all untransformed cells. This selection marker, provided with the appropriate 5 'and 3' regulatory sequences may reside on the same DNA molecule that contains the desired gene or may be present on a molecule other than the molecule. In the latter case, a cotransformation must be carried out. The ratio of the expression vector / selection vector must be adjusted in such a way that a high percentage of the selected transformants have also incorporated the vector containing the expression construct of the AXDA of interest. The most suitable selection systems for industrial microorganisms are those formed by the group of selection markers that do not require a mutation in the host organism. Examples of fungal selection markers are the genes for acetamidase (amdS), ATP synthetase, subunit 9 (oliC) and domain resistance (benA). Examples of selection markers that do not belong to fungi are the bacterial resistance gene G418 (this can also be used in yeast, but not fungi), the amplicillin resistance gene (E. coli) and the gene for resistance to neomycin. { Bacill us). Once the desired expression construct has been assembled, this is transformed into a suitable cloning host such as E. coli to propagate the construct. Later, the expression construct is introduced into a suitable expression host in which the expression construct is preferably integrated into the genome. Certain hosts such as the Bacill species can be used as hosts for both cloning and expression, thus avoiding an extra transformation step. In accordance with the present invention, a variety of organisms can be used as hosts for the production of the AXDA of interest. In one embodiment, a homologous expression host can be used. This involves introducing the expression construct back into the strain from which the DNA sequence encoding the AXDA was isolated either in increased gene copy numbers, or under the control of the heterologous regulatory regions as described above, or both In another embodiment, an AXDA of interest can be produced by introducing and expressing the DNA construct encoding for enzyme that degrades the arabinoxylan of interest under the control of the appropriate regulatory regions in heterologous hosts such as bacteria, yeast or fungi. For that purpose, the DNA sequences encoding the AXDA of interest are preferably expressed under the control of the promoter and terminator sequences that originate from the heterologous host. In addition, it may be necessary to replace the leader sequence of the native secretion with a homologous leader sequence to that of the expression host in order to achieve the most efficient expression and secretion of the product. Factors such as size (molecular weight), the need for appropriate glycosylation or the convenience of extracellular expression of the AXDA of interest play an important role in the selection of the expression host. The negative bacteria E. coli is widely used as a host for the expression of heterologous genes. However, large amounts of heterologous protein tend to accumulate within the cell. The subsequent purification of the desired protein from the mass of intracellular proteins of E. coli can sometimes be difficult. In contrast to E. coli, bacteria of the genus Bacillus are very suitable as heterologous hosts due to their ability to secrete proteins in the culture medium. Depending on the nature of the DNA molecule encoding the AXDA itself, and / or the convenience of further processing of the expressed protein, eukaryotic hosts such as yeast or fungi may be preferred. In general, wash cells are preferred over fungal cells because they are easier to handle. However, some proteins are either poorly secreted from the yeast cells or in some cases are not processed properly (eg hyperglycosylation in the yeast). In those cases, a fungal host organism should be selected. A heterologous host may also be chosen to express the AXDA of interest substantially free of other enzymes that degrade polysaccharides by choosing a host that does not normally produce such enzymes such as Kl uyveromyces lactis. Examples of preferred expression hosts within the scope of the present invention are fungi such as Aspergillus species (described in European Patent 184,438 and European Patent 284,603) and Trichoderma species, bacteria such as Bacill species. (described in European Patent 134,048) and yeasts such as Kluyveromyces species (described in European Patent 96,430 and European Patent 301,670) and Saccharomyces species. Particularly preferred expression hosts can be selected from Aspergillus niger, Aspergillus niger variant awamori, Aspergillus aculeatis, Aspergillus oryzae, Trichoderma reesei, Bacillus subtilis, Bacillus lincheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae. The expression of the AXDA enzyme of interest is effected by culturing the expression hosts, which have been transformed with the appropriate expression construct, into a conventional nutrient fermentation medium. The fermentation medium consists of an ordinary culture medium containing a carbon source (for example glucose, maltose, badges, etc.), a nitrogen source (for example, ammonium sulfate, ammonium nitrate, ammonium chloride, etc.). ), a source of organic nitrogen (eg yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (for example phosphate, magnesium, potassium, zinc, iron, etc.). Optionally, an inducer (arabinoxylan) may be included. The selection of the appropriate medium can be based on the choice of expression hosts and / or be based on the regulatory requirements in the expression construct. Such means are well known to those skilled in the art. The medium can, if desired, contain additional components that favor transformed expression hosts on other potentially contaminating microorganisms. The fermentation takes place during a period of 0.5-20 days in a batch process or of batches fed at a temperature in the range between 0 and 45 ° C and a pH between 2 and 10. The preferred fermentation conditions are a temperature in the range of between 20 and 37 ° C and a pH of between 3 and 9. The appropriate conditions are selected based on the choice of the expression host. After fermentation, the cells are removed from the fermentation broth by means of centrifugation or filtration. After removal of the cells, the enzyme can then be recovered and, if desired, purified and isolated by conventional means. The product is formulated in a stable manner in either liquid or dry form. For certain applications, the immobilization of the enzyme on a solid matrix may be preferred. Enzymes produced by means of the present invention can be applied either alone, or together with other selected enzymes in a variety of processes that require the action of an enzyme that degrades arabinoxylan. The enzyme that degrades the arabinoxylan of the present invention is used in the treatment or preparation of foods (eg, bread), fodder and beverages (eg, beer and fruit juices). The enzyme that degrades arabinoxylan is also used advantageously in the preparation and treatment of paper and pulp especially in combination with endoxylanases. As illustrated in the present invention, the enzyme that degrades arabinoxylan is added to animal fodder that is rich in arabinoxylans. When added to forages (including silage) for monogastric animals (eg birds or pigs) that contain cereals such as barley, wheat, corn, rye or oats or by-products of cereals such as wheat bran or corn bran, the enzyme it significantly improves the decomposition of the cell walls of the plant which leads to a better utilization of plant nutrients by the animal. As a consequence, the growth rate and / or conversion of the forage are improved. In addition, enzymes that degrade arabinoxylans can be used to reduce the viscosity of forages containing arabinoxylans. An enzyme that degrades arabinoxylan before the forage or silage can be added if pre-wet or wet diets are preferred. More advantageously, the AXDA produced via the present invention continues to hydrolyze the arabinoxylans in the forage in vivo. Enzymes that degrade arabinoxylan are also useful in the preparation of bread as illustrated in Example 8.

Experimental Part Strains E. coli BB4 (Silvay et al., 1984) el4 (mcrA) hsdR514, supE44, supF58, lacYl, or Q (laclZY) 6, galK2, galT22, metBl, trpR55, Q (argF-lac) U169 [f, proAB , lacI < lzClM15, TnlO, (tetr)] E. coli DH5a (Hanahan, 1983): supE44, ülacU169, (F80IacZÜM15),? SdR17, recAl, endAl, gyrA96, thi-1, relAl E. coli LE 392 (Murray, 1977): el4 (mcrA) hsdR514, supE44, supF58, lacYl, or 0 (laclZY) 6, galK2, galT22, metBl, trpR55 E. coli XLl-Blue MRF '(Jerpseth et al., 1992): O (mcrA) 183, Q (mcrCB-hsdSMR-mrr) 173, endAl, supE44, thi-1, recAl, gyrA96, relAl, lac, [ F ', proAB, lad ^ zOílS, TnlO, (tetr)] Aspergillus niger N400 (CBS 120.49) Aspergillus niger N402. { cspAl) Goosen et al. , 1987) Aspergillus niger NW219 (leuAl, nicAl pyrA6) Vectors: pBluescript SK +/- (Short, J.M., et al., 1988) pUC19 (Yanish-Perron et al., 1985) The following solutions were prepared according to Sambrook et al. (1989): Shock absorbers: TE, 50 * TAE, 20 * SSC; Hybridization, 100 * Denhardts, SM, DNA loading Medium: NZYCM, LB and minimal medium. the solution of Visniac was prepared in agreement with Visniac and Santer (1957).

Examples Example 1: Isolation of the enzyme Example 1.1: Purification and characterization of the degradation activity of arabinoxylan A (AXDA) from A. niger variant tubigensis A. The niger variant tubigensis DS 16813 was grown as described in European Patent Application 0 463 706 without yeast extract and 2% a fraction of crude wheat arabinoxylan instead of oat substitute xylan. The cells were removed by filtration and the supernatants were dried by ultrafiltration. For the purification of AXDA, 5 grams of the crude enzyme preparation was diluted in 100 ml of 10 mM Tris / HCl pH 8.0. The enzyme solution was filtered (Sietz EKS from Sietz / supro and 250 from Sietz / supro) and diluted to a final protein concentration (Biorad protein assay) of 5.2 g / 1. The crude enzyme was fractionated by CLAP (Waters Preparative 650 Advanced Protein Purification System) on an anion exchange column of DEAE TSK 650 (M) (600 ml) (speed 25 ml / min). The column was equilibrated with Tris / HCl (pH 8.0). The mixture (3 g of protein) was applied to the column and eluted with a gradient of the equilibrium buffer containing 100 mM NaCl. The AXDA enzyme was eluted at a NaCl concentration of 53 mM as shown in Figure 1.

Example 1.2: Optimum pH For the determination of the optimum pH of the crude enzyme preparation AXDA, a fraction of water-insoluble solid polymers (SIA) of corn with a substrate was used. 1.6 mg of the crude enzyme preparation was added to 0.5 g of SIA and diluted to 5 ml with buffer (100 mM NaAC) with a pH ranging from 3.40 to 7.65. The samples were incubated for 60 minutes at 39 ° C and centrifuged for 15 minutes (2800 g). In the supernatant, the reducing sugars were measured using the Sumner reagent. Preparation of the Sumner reagent: 10 g of phenol were added to 22 ml of 10% NaOH and the volume was then adjusted to 100 ml with demineralized water. 10 g of NaHS? 3 were added. To 70 ml of this solution were added 300 ml of 4.5% NaOH followed by 255 g of K / Na tartrate and 800 ml of 3% 5-dinitrosalicylic acid. The mixture was then stirred at room temperature until the chemical reagents dissolved. 0.5 ml of Sumner's reagent was added to 200 ul of sample and boiled for 10 minutes. After cooling, 5 ml of demineralized water were added. The extinction was measured at 515 nm. The reducing sugar content of the samples was calculated using a calibration curve of xylose.

The optimum pH for the enzyme was 5.0 as shown in Figure 2.

Example 1.3: Composition of Amino Acids From the AXDA fractions of the DEAE column, the amino acid compositions were measured on a 150 MM PICO-TAG column (detection at 254 nm). The following composition was found: Table 1: Amino acid composition of the CLAP pump AXDA: CM4000; detection M440, 254 nm; injection 4 ul, Gilson 232; PICO-TAG column 150 MM; Maximum data system.

Example 1.4 Biochemical characterization (IEP and molecular weight) The IEP of the AXDA was determined on a Pharmacia minigel of pH 3-9. The protein was stained with a silver staining solution from Pharmacia. It was estimated that the AXDA IEP was 3.6. The molecular weight of the AXDA was determined on a SDS-gel minigradient from Pharmacia (10-15%) and the proteins were detected with silver staining (Pharmacia). It was estimated that the molecular weight of the AXDA was 32 kDa.

Example 1.5: Sequencing of the protein of activity A that degrades arabinoxylan (AXDA) of A. niger variant tubigensis Example 1.5.1: Sequencing of N-terminal amino acids from an arabinoxylan A (AXDA) activity of A. niger variant tubigensis Approximately 1 nmol ("30 μg) of AXDA was subjected to electrophoresis on a 15% SDS-polyacrylamide gel, followed by by electrospinning on an Immobilon-P membrane (Millipore), according to the method described by Matsudaira (1987). The membrane fragment containing the main band having an apparent molecular weight (SDS-page) of 32 Kda was submitted to the analysis of the sequence in the gas phase sequencing (Amons, 1987) (SON facility, Leiden). The following sequence was determined: Lys -? - Ala-Leu-Pro-Ser-Ser-Tyr (IDSECNO :!) Example 1.5.2: Determination of the amino acid sequence of a CNBr peptide of activity A that degrades arabinoxylan (AXDA) Approximately 2 nmol of AXDA was subjected to chemical precision by CNBr. Approximately 2 nmol ("60 μg) of AXDA was leofilized and resuspended in 60 μl of 70% formic acid containing 125 μg of CNBr (2.5 mg / ml). This reaction mixture was incubated in the dark at room temperature for 48 hours. The liquid was evaporated in a Speedvac, washed with sterile bidistilled water and evaporated again. This washing step was repeated twice. The reaction mixture was then subjected to electrophoresis on a 15% SDS-polyacrylamide gel, followed by electrospinning on an Immobilon-P membrane (Millipore), according to the method described by Matsudaira (1987). The membrane fragment containing the major band having an apparent molecular weight (SDS-page) of approximately 9 kDa was subjected to sequence analysis in the gas phase sequencing (Amons, 1987) (SON facility, Leiden). The following sequence was determined: CNBr peptide: Ile-Val-Glu-Ala-Ile-Gly-Ser-Thr-Gly-His-Arg-Tyr-Phe- (Arg / Asn) - (Ser) - (Phe) - (Thr) (IDSECNO: 2) Ambiguous amino acids are given in parentheses.

Example 2: Enzymatic profile of the AXDA The activity of a-arabinofuranosidase was measured on a substrate of para-nitro phenyl arabinofuranoside (Sigma). To a 1 ml of substrate, 300 μl of enzyme solution was added. 15 minutes after the incubation at 30 ° C the reaction was stopped with 5 ml of Na 2 C 3 (5M). The extinction is medium at 402 nm. The activity of a-arabinofuranosidase (volume * extinction) / (E * time) was calculated. The crude enzyme preparation containing an a-arabinofuranosidase activity of 0.30 pNPAF U / mg. The activity of the fractions of the DEAE column was tested. The combined active arabinofuranosidase had 3.0 pNPAF U / mg (see Figure 1). They did not find other fractions with a-furanosidase activity. The activity of the pooled a-arabinofuranosidase was later tested on a water-insoluble corn polymer fraction and activity on this substrate was not measured. This indicated that AXDA does not have a-arabinofuranosidase activity on the para-nitrophenyl substrate. In a further attempt to identify the enzymatic activity of AXDA, filtrates were selected from an Aspergillus niger culture transformed with the axperA gene from Aspergillus tubigensis to determine activity on a substrate consisting of wheat arabinoxylan. The tubigensis gene was placed under the control of the pyruvate kinase promoter (a glycolytic) and growth conditions were chosen that favored the expression of the transgene., avoiding the expression of enzymes that degrade endogenous arabinose. Accordingly, it was determined that AXDA had arabinose-releasing activity on high molecular weight arabinoxylan substrates. The protein content was determined using the Sedmak method. The activity of the AXDA was determined by adding 100 μl of different solutions (10 mM Na acetate buffer pH 5.0) of culture filtrates to 150 μl of 50 mM Na acetate (pH 5.0) and 250 μl of wheat arabinoxylan to 0.4 % (Megazyme), and incubation at 40 ° C for one hour. The mixture was stopped by heating the mixtures for 10 minutes at 100 ° C. The degradation products of the arabinoxylan were checked using CIAAR (high performance ion exchange chromatography) using the heat inactivated incubations as samples.

The results are the following: Table O Activity of AXDA of Aspergillus Tubigensis on wheat arabinoxylan ® One unit of arabinose furan hydrolase is defined as the amount of enzyme capable of releasing 1 μmol of arabinoxylan arabinose per minute.

It was concluded that the AXDA enzyme from Aspergillus tubigensis has an activity of arabi nofuranohydrolase (AXH). The arabinase activity was measured with a Megazyme arabinase test kit, and executed according to the instructions. In the experiment, the AXDA collected from the DEAE column did not show activity on the linear arabinano. This indicated that AXDA is not an arabinase.

The activity of the endoxylanase was measured on oat substitute xylan. 10 ml of 5% xylan suspension (100 mM NaAc pH 3.5) was boiled for 10 minutes. After cooling, the suspension was incubated at 39 ° C for 10 minutes. The reaction began with the addition of 0.5 ml of enzyme solution. After several periods of incubation (5, 10, 15, 20, and 25 minutes), 1.5 ml of sample was added, from the incubation at 39 ° C, to 1.5 ml of demineralized water and boiled for 10 minutes. The samples were centrifuged for 15 minutes (2800 g). In the supernatant, the reducing sugars were detected with the Sumner reagent (see Example 1.2). In the assembled AXDA fraction an endoxylanase activity of 399.0 U / mg was found. The endoxylanase and AXDA were later tested in an in vitro digestion system (see Example 6). From this it was concluded that the endoxylanase activity in the pooled AXDA fraction was an activity associated with a different polypeptide activity. An additional indication that a polypeptide having endoxylanase coelute with an enzyme similar to AXDA was found by Kormelink (1992, Thesis of Doctorate, Chapter 6, P. 107, last two paragraphs and page 108). It was concluded that AXDA does not have endoxylanase activity by itself.

Example 3: Construction of the expression library of CDNA Example 3.1: Induction and isolation of mRNA A culture of A. niger N400 was grown for 69 and 81 h respectively, as described in European Patent Application 0 463 706 without yeast extract and 2% of a fraction of crude wheat arabinoxylan instead of substitute xylan. Oats, after which the mycelia were harvested by filtration and then washed in sterile saline. The mycelia were subsequently frozen in liquid nitrogen after which they were pulverized using a Microdisperser (Braun). All RNA was isolated from the mycelial powder according to the guanidinium thiocyanate / CsCl protocol described in Sambrook et al. (1989), except that the RNA was centrifuged twice using a CsCl gradient. Poly A * mRNA of 5 mg of total RNA was isolated by chromatography on oligo (dT) -cellulose (Aviv and Leder, 1972, Sambrook et al., 1989) with the following modifications: the SDS was omitted from all solutions and the charge buffer was supplemented with 9% (volume / volume) of dimethylsulfoxide.

Example 3.2: Construction of the cDNA library The cDNA was synthesized from 7 μg of poly A + mRNA and ligated to the bacteriophage lamda? Uni-ZAP XR using the ZAPMR cDNA synthesis kit (Stratagene) according to the manufacturer's instructions. After ligation of cDNA to the arms of the Uni-ZAP XR vector, the phage DNA was packaged using Packagene1 ^ (Promega) extracts according to the manufacturer's instructions. The ligation of 120 mg of cDNA into 1.2 μg of vector arms and the subsequent packing of the reaction mixture resulted in a primary library consisting of 3.5 * 104 recombinant phages. This first library was amplified using XLl-Blue MRF 'from E. coli, tritiated and stored at 4 ° C. example 4 Selection of A. niger N400 cDNA library for (axdA) with antibodies produced against AXDA and isolation of cDNA clones.

Example 4.1: Preparation of antibodies produced against AXDA in a rabbit. 500 μg of AXDA was dialyzed against 1 mM phosphate buffer pH 7.0 and lyophilized. The protein was resuspended in 1 ml of sterile PBS (0.136 M NaCl, 2.7 mM KCl, 8 mM Na2HP04, 1.75 mM KH2PO4, pH 7.4). To this protein mixture, 1 ml of Freund's complete adjuvants was added and stirred mortally for 30 minutes to obtain a stable emulsion. This mixture was injected into the rabbit subcutaneously. In week 6, reinforcement was given by injecting 250 μg of AXDA in 0.5 ml of sterile PBS to which 0.5 ml of incomplete Freund's adjuvants had been added. The rabbit was bled in week 7 and the serum tested. In week 13 the rabbit was given a second 250 μg booster followed by a bleed at week 14. This reinforcement procedure with a 6 week interval followed by a bleed may be repeated several times.

The collected blood was incubated for 30 minutes at 37 ° C and then stored at 4 ° C for 16 hours. After centrifugation at 5000 rpm in a high-speed Sorvall centrifuge, the serum was collected.

Example 4.2: Immunoselection of the A. niger N400 cDNA library with antibodies produced against the AXDA-JUB To select the A. niger N400 cDNA library, constructed as described in Example 3.2, 5 * 103 pfu clones of axdA cDNA per plate in NZYCM topagarose containing 0.7% agarose were plated on NZYCM plates. (1.5% agar) of 85 mm diameter as described (Maniatis et al., 1982, pp. 64), using E. coli BB4 as a plate culture bacterium. Two duplicates of each plate were made on nitrocellulose filters (Schleicher and Schüll BA85) as described by Sambrook et al. (1989, pp. 12.16-12.17). AXDANIG was visualized using antibodies raised against purified AXDATUB (Example 4.1) after immunostaining as described in European Patent Application 91205944.5 (Publication No. 0 463 706 Al). The immunostained plates, which appeared replicated in the duplication filters, were identified; 8 positive plates were selected. Each positive plate was detached from the plate using a Pasteur pipette and the phages were eluted from the agar plug in 1 ml of SM buffer containing 20 μl of chloroform, as described in Maniatis et al. (1982, pp. 64). The obtained phages were purified by repeating the procedure described above using filter duplicates of the plates containing 50-100 plates of the isolated phages. After purification the phage were propagated by growing 5xl03 phage on NZYCM medium. After incubation overnight at 37 ° C, the confluent plates were obtained, from which the phages were eluted by adding 5 ml of SM buffer and storing the plate for 2 h. at 4 ° C with intermittent agitation. After collecting the supernatant using a pipette, the bacteria were removed from the solution by centrifugation at 4000 x g for 10 min. at 4 ° C. 0.3% chloroform was added and the ufe number was determined. These phage patterns contain approximately 1010 cfu / ml.

Example 4.3: Restriction analysis on axdA cDNA clones Recombinant Uni-ZAP XR clones containing axdA cDNAs were converted to the Bluescript phagemids using superinfection with the filamentous helper phage ExAssist ™, which is included in Stratagene's ZAP1 ^ cDNA synthesis kit, according to the instructions of The manufacturers. The phagemid DNA was subsequently isolated as described in Sambrook et al. (1989, pp. 1.25-1.28). DNA isolated from the 8 cDNA clones of axdA was subjected to restriction analysis using the following restriction enzymes: £ coRI and Xhol. The DNA was digested for 2 hours at 37 ° C in a reaction mixture composed of the following solutions; 2 μl («1 μg) of DNA solution; 2 μl of buffer 10 * Appropriate reagent (BRL); 10 U of each enzyme of Restriction (BRL) and sterile distilled water to give a final volume of 20 μl. After the addition of 4 μl of DNA loading buffer the samples were loaded onto a 0.7% TAE agarose gel. The DNA fragments were separated by electrophoresis at 80 V for 1.5 hours.

The restriction analysis revealed that the axdA cDNA clones had a molecular size of approximately 1.2 kb.

Example 4.4: Sequence analysis on A. niger axdA cDNA clones The primary structure of the cDNA clones was determined by sequence analysis combined with the use of specific oligonucleotides as primers in the sequencing reactions.

The nucleotide sequences were determined by the idesoxynucleotide chain termination method (Sanger et al., 1977) using the T7 DNA polymerase sequencing kit from Pharmacia. The computer analysis was carried out using the PC / GENE program.

Example 4.5: Marking the fragment with 32P The fragment of the A. niger axdA cDNA clone was isolated and labeled as described in European Patent Application 91205944.5 (Publication No. 0 463 706 Al, Examples 2.2 and 7.1, incorporated herein by reference).

Example 4.6: Selection of the genomic library of A. niger variant niger for the axdA gene and isolation of the gene.

For the selection of the genomic library of A. niger variant niger, constructed as described by Harmsen et al. (1990), 3 x 10 3 pfu of axdA gene were plated per plate in agarose coated with NZYCM containing 0.7% agarose in plates of NZYCM (1.5% agar) of 85 mm diameter as described (Maniatis et al., 1982, pp. 64), using E. coli LE392 as a plate culture bacterium. After overnight incubation of the plates at 37 ° C, two duplicates of each plate were made on HybondN filters (Amersham) as described in Maniatis et al. (1982, pp. 320-311).

After being wetted in 3xSSC the filters were washed for 60 min. at room temperature in 3xSSC. The filters were prehybridized at 65 ° C for two hours in a prehybridization buffer containing; 6xSSC, 0.5% SDS, lOxDenhardt solution, 0.01 M EDTA and 100 μg / ml thermally denatured herring sperm DNA (Boerhinger Mannheim). After two hours of prehybridization the prehybridization buffer was replaced by the hybridization buffer which was identical to the prehybridization buffer, but which contained the 1.2 kb fragment labeled with 32P containing an A. niger axdA cDNA clone. (see Example 4.3) and prepared as described in Example 4.5. The filters were hybridized for 18 h at a temperature of 65 ° C. After hybridization the filters were washed first at 65 ° C for 30 minutes in 5 * SSC / 0.1% SDS followed by a second wash at 65 ° C for 30 minutes in 2 * SSC / 0.1% SDS. The filters were then washed at 65 ° C for 30 minutes with 0.1 * SSC / 0.1% SSC, followed by a final wash at 65 ° C for 30 minutes in 0.1 * SSC. The air-dried filters were wrapped on a sheet of 3MM Whatman paper, the code marks were made with radioactive ink and the Whatman paper and the filters were covered with Saran wrap. Hybridization plates were identified by exposure of the Kodak XAR X-ray film for 72 h at -70 ° C using an intensifying screen. Ten positive hybridization plates were found, which appear in duplicate on the duplication filters. Four positive plates were detached from the plate using a Pasteur pipette and the phages were eluted from the agar plug in 1 ml of SM buffer containing 20 μl of chloroform, as described in Maniatis et al. (1982, pp. 64). The obtained phages were purified by repeating the procedure described above using duplicates of the filters of the plates containing 50-100 plates of the isolated phages. After purification the phage were propagated by growing 5x1O3 phage on NZYCM medium. After incubation overnight at 37 ° C the confluent plates were obtained, of which the phages were eluted by the addition of 5 ml of SM buffer and storing the plate for 2 h. at 4 ° C with intermittent agitation. After collection of the supernatant using a pipette, the bacteria were removed from the solution, by centrifugation at 4,000 x g for 10 min. at 4 ° C. 0.3% chloroform was added to the supernatant and the number of pfu was determined. Those phage patterns contained approximately 107 cfu / ml.

Example 4.7: Isolation of bacteriophage lambda DNA Each of the isolated phages was propagated by combining 5 * 109 E. coli LE392 bacteria in 300 μl of SM buffer with 2 * 10 ^ phages and incubating at 37 ° C for 15 min. After the incubation period, infected bacteria were used to inoculate 100 ml of pre-warmed NZYCM medium (37 ° C) and subsequently incubated for 9-12 hrs at 37 ° C on a New Brunswick rotary shaker at 250 rpm, after which time the baceterias were lysed. The bacterial remnants were removed by centrifugation for 10 min. at 10,000 rpm at 4 ° C in a Sorvall High Speed centrifuge. The phages were precipitated from the obtained supernatant (100 ml) by the addition of 10 g of polyethylene glycol-6000 and 11.7 g of NaCl and storing the solution overnight at 4 ° C. The precipitated phages were harvested by centrifugation at 14,000 x g at 4 ° C for 20 min. The supernatant was removed by aspiration, while the last traces of liquids were removed using a paper towel. The phages were carefully resuspended in 4 ml of SM buffer and extracted once with an equal volume of chloroform. Before the DNA was extracted from the phage particles, the DNA and RNA originating from the lysed bacteria were removed by incubation of the phage suspension with DNase I and RNase A (both 100 μg / ml) for 30 min. at 37 ° C. The phage DNA was subsequently released from the phage by adding EDTA to a final concentration of 20 mM while the protein was removed from the solution by extracting twice with an equal volume of phenol / chloroform / isoamyl alcohol (25:24: 1). After separation of the phage by centrifugation using a Sorvall centrifuge (14,000 x g, 10 min.), The aqueous phase was extracted once with an equal volume of chloroform / isoamyl alcohol (24: 1). The phases were separated by centrifugation after which the DNA was precipitated from the aqueous phase by the addition of 0.1 vol. of sodium perchlorate 5 M and 0.1 vol. of isopropanol and incubating on ice for 30 min. The DNA was removed by centrifugation for 10 min. at 4 ° C (14,000 x g). The supernatant was removed by aspiration after which the DNA was resuspended in 400 μl of TE buffer. The DNA was once again precipitated from this solution by the addition of 0.1 vol. of sodium acetate 3 M and 2 vol. of ethanol. The DNA was harvested by centrifugation for 10 min at 4 ° C (14,000 x g). The supernatant was removed by aspiration, the remaining pellet was briefly dried under vacuum, after which the DNA was resuspended in 125 μl of TE buffer containing 0.1 μg / ml of RNase A. This purification procedure resulted in isolation of approximately 50-100 μg of DNA from each phage.

Example 4.8: Southern analysis of phages containing axdA DNA from A. niger variant niger DNA isolated from phage nigax? A n_gax? 4 was analyzed by Southern analysis using the following restriction enzymes; Kpnl; I left; Sstl; Xbal and Xhol and the Kpnl + Xbal combinations; Kpnl + Sstl and Kpnl + Xhol for 5 hours at 37 ° C in a reaction mixture composed of the following solutions; 5 μl («1 μg) of DNA solution; 2 μl of 10 x Appropriate reaction buffer (BRL); 10 U of restriction enzyme (BRL) and sterile distilled water to give a final volume of 20 μl. After digestion, the DNA was precipitated by the addition of 0.1 vol. of NaAc 3M and 2 vol. of ethanol. The DNA was collected by centrifugation for 10 min. at room temperature (14,000 x g). The supernatant was removed by aspiration, the remaining pellet was briefly dried under vacuum and resuspended in sterile distilled water. After the addition of 4 μl of DNA loading buffer the samples were incubated for 10 min. at 65 ° C and rapidly cooled on ice, before loading the samples on a 0.6% agarose gel in TAE buffer. The DNA fragments were separated by electrophoresis at 25 V for 15-18 hours. After electrophoresis the DNA was transferred and denatured by alkaline vacuum staining (VacuGene XL, Pharmacia LKB) to a nylon membrane (HybondN, Amersham) as described in the instruction manual (pp. 25-26) and subsequently prehybridized and hybrid using A. niger axdA cDNA clones labeled as described in Example 4.6 and the hybridization conditions as described in Example 3.3. The hybridization pattern was obtained by exposure of the Kodak X-ray film XAR-5 for 18 h. to -70CC using an intensifying screen. From the results obtained, it was concluded that the DNA of the four isolated clones was hybridized with the axdA cDNA of A. niger. In the four clones were found original fragments of the same genomic region.

Example 4.9: Subcloning of the axdA gene of A. niger variant niger We isolated the 3.7 kb Xhol fragment from? Nj_gax? 0.7% agarose gel by lyophilization method: After electrophoresis, the appropriate band was cut and stirred gently for 30 minutes in 1 ml of FS1 solution (0.3 M NaAc pH 7.0, 1 mM EDTA). The agarose slice was transferred to a 0.5 ml eppendorf tube, which contained a small plug of sili- nized glass wool and a perforation in the bottom, and was frozen in liquid nitrogen. The 0.5 ml tube was then transferred to a 1.5 ml eppendorf tube and subsequently centrifuged for 10 minutes. To the supernatant were added 1/50 volumes of FS2 solution (0.5 M MgCl2, 5% acetic acid) and 2.5 volumes of 100% ethanol. After 20 minutes of incubation at -70 ° C, the DNA was harvested by centrifugation for 15 minutes at 14,000 * g at 4 ° C. After removal of the supernatant, the DNA pellet was dried using a Savant Speedvac vacuum centrifuge. The DNA was dissolved in 10 μl of TE buffer and the concentration was determined by electrophoresis on agarose, using lambda DNA at a concentration known as reference and staining with ethidium bromide to detect DNA.

The Pbluescript SK ~ vector digested with Xhol and dephosphorylated with alkaline phosphatase was prepared as follows: 1 μl (1 μg / μl) pBluescript was mixed with 2 μl of 10 * Reagent 2 (BRL), 1 μl (lU / μl) of Xhol and 16 μl of sterile distilled water. The DNA was digested for 2 hours at 37 ° C, after which 0.5 μl of alkaline phosphatase (1 U / μl) (Pharmacia) was added, followed by an additional incubation at 37 ° C for an additional 30 minutes. The linearized vector was isolated from 0.7% agarose as described above. The 3.7 kb Xhol fragment was ligated into the dephosphorylated pBluescript SK ~ vector, digested with Xhol via the following procedure: 40 ng of pBluescript fragment was mixed with 300 ng of the 3.7 kb Xhol fragment and 4 μl of 5 * were added. ligation buffer (500 mM Tris-HCl, pH 7.6, 100 mM MgCl, 10 mM ATP, 10 mM dithiothreitol, 25% PEG-6000) and 1 μl (lU / μl) of T4 DNA ligase (BRL) to this mixture, resulting in a final volume of 20 μl. The resulting plasmid was designated pIM3002. After incubating for 16 h at 16 ° C the mixture was diluted to 100 μl with Ca-HEPES buffer pH 7.0. 10 μl of the diluted mixture was used to transform competent E. coli DH5a cells prepared as follows: 200 μl of a culture of E. coli DH5a pre-grown overnight in an LB medium (LB medium per 1000 ml: 10 g of trypticase peptone (BBL), 5 g of yeast extract (BBL), 10 g of NaCl, 0.5 mM Tris-HCl pH 7.5). This culture was incubated in an orbital shaker at 37 ° C until its density corresponded to a D.O.goo of 0.15-0.2. The bacteria were then harvested by centrifugation at 5000 rpm at 4 ° C. After discharging the supernatant, the cells were kept on ice constantly. The Baceterian pellet was washed in 100 ml of 100 mM MgCl 2, 5 mM Tris-HCl pH 7.4 resuspending those cells followed by centrifugation as described above. This was repeated with 100 ml of 100 mM CaCl2, 5 ml Tris-HCl pH 7.4. Finally the cells were resuspended in 2 ml of 100 mM CaCl2, 5 mM Tris.HCl pH 7.4, 14% glycerol. Aliquots (50 μl) were used either to be used immediately for transformation or to freeze at -70 ° C. Competent cells of E. coli DH5a were used in the transformation experiments, combining 50 μl of the cell suspension with 4.5 μl of ligation mixture. After an incubation period of 30 minutes on ice, the cells were incubated for 2 minutes at 42 ° C. Then 1 mg of LB medium was added and the cells incubated at 37 ° for 1 hr. The bacteria were then harvested by centrifugation at 14,000 g for 30 s. After discharging the supernatant, the cells were resuspended in 200 μl of LB medium. The resulting bacterial suspension was plated on LB medium containing 100 μg / ml ampicillin, 50 μg / ml X-gal and 60 μg / ml IPTG.

The selection of twelve of the resulting colonies was grown overnight in LB medium containing 100 μg / ml of ampicillin. From the plasmid cultures, the DNA was isolated by the alkaline lysis method according to that described by Maniatis et al. (1982, pp. 368-369), which was used in the restriction analysis, as described in Example 4.3 to select a clone that would harbor the desired plasmid. The plasmid DNA was isolated on a large scale to 250 ml of E. coli DH5a cultures containing the plasmid pIM3002 grown in LB medium containing 100 μg / ml ampicillin using the Nucleobond PC-500 (Nagel) equipment according to manufacturer's instructions. Plasmid pIM3002 was further analyzed with restriction enzymes resulting in the restriction map shown in Figure 4. A sample of E. coli DH5a containing pIM3002 had been deposited on August 28, 1995 at CBS, Baarn, Holland, under the number CBS 637.95.

Example 4.10: Overexpression of the cloned gene in A. niger NW219 Example 4.10.1: Introduction of axdA of A. niger variant niger in A. niger NW219 by cotransformation Plasmid PIM3002, obtained in Example 4.9 was introduced into A. niger by cotransformation of A. niger NW219 using A. niger pyrA as a selective marker on plasmid pGW635 (Goosen et al., 1987) and plasmid PIM3002 as the plasmid cotransformant. A mixture of A. niger NW219 was deposited on August 25, 1995, at CBS, Baarn, The Netherlands, under number CBS 635.95.

Mycelium protoplasts were prepared by growing A. niger NW219 on minimal medium supplemented with 0.5% yeast extract, 0.2% casamino acids, 50 Mm glucose, 10 Mm nicotinamide and 10 Mm uridine for 18 h at 30 ° C. The preparation of the protoplasts of A. niger NW219 and the transformation procedure were carried out according to what was described by Goosen et al., 1987. The resulting PYR 'transformants were analyzed to determine the expression of the adxA gene of A. niger variant niger by Western spotting analysis.

Example 4.10.2: Selection of transformants for the expression of the axdA gene of A. niger variant niger The transformants obtained in the Example 4. 10.1 were analyzed to determine the product formation of the axdA gene of A. niger variant niger, the AXDAJJIQ protein. Nineteen of these transformants were used in a growth experiment to analyze the expression of AXDANIQ. The strain of A. niger N402 was used as the wild-type control. The transformants were grown, as described in Example 3.1 by 24, 40, 48 and 68 hours. After growth, the mycelia were removed by filtration and the culture filtrate was analyzed by Western blotting using antibodies raised against AXDATUB in a rabbit (Example 4.1). In eight of the nineteen transformants analyzed there was overproduction of the XDA ^ IG protein according to what was detected by this procedure.

Example 4.11: Analysis of the sequence and description of the axdA gene of A. niger variant niger The sequence of the axdA gene of A. niger variant niger, its promoter / regulatory region, the structural part of the gene and the termination region, were determined by subcloning fragments of PIM3002 into pBluescript SK "and pUC19 in combination with the use of specific oligonucleotides. as primers in the sequencing reactions The nucleotide sequence was determined as described in Example 4.4 (IDNOSEC: 5) The sequence obtained comprises 2101 bp, 783 bp in the region with 5 'coding and 322 bp in the non-coding region. encoder 3 'In the non-coding region 5? a TATA box was found at positions 665-670. The coding part of the A. niger axdA gene is 996 bp in length and is not interrupted by introns. for a protein of 332 amino acids in size The N-terminal sequence determined as in Example 1.5.1 on the basis of the A. niger protein variant tubigensis AXD, is preceded by a 26 amino acid peptide. The mature protein is 306 amino acids in size and has an estimated molecular weight of 33,101 kDa and a theoretical isoelectric point of 4.07.

Example 5.1: Cloning of the cDNA fragment containing the axdA sequences of A. niger variant tubigensis obtained by PCR Example 5.1.1: Generation of cDNA fragments by PCR The partial amino acid sequence of the internal CNBr fragment (IDNOSEC: 2) as determined for the AXDATUB was used to design the oligonucleotide mixture.

The following mixtures were derived, 5 '-ATG ATK GTI GAR GCI ATK GG-3' (20-mer) (IDNNOSEC: 3) in which I means inosine; K for an A, T or C and R for an A or G. This oligonucleotide was derived from the internal amino acid sequence of AXDATUB as described above in Example 1.4.2 from amino acid 1 (I) to amino acid 6 G. The ATG it was derived from methionine which is known to be present at the N-terminal end of the peptide fragment due to the CNBr cleavage mechanism. The oligonucleotide mixture was used in the PCR (Saiki et al., 1988) in combination with oligonucleotide T7 (Stratagene) 5 '-AAT ACG ACT CAC TAT AG-3' (17-mer) (IDNOSEC: 4) using the cDNA which was isolated as described in Example 4.7. For a PCR, 2 μl of the resulting α -CDNc combined with 5 μl of 10 * reaction buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl 2, 0.01% gelatin), 4.0 μl 1.25 mM were combined. of each of four deoxynucleotide triphosphates and 0.5 μg of the oligonucleotides in a final volume of 50 μl. The reaction mixture was mixed and 0.5 μl of TAQ polymerase (5 U / μl) was added (HT Biotechnology, Cambridge UK). The DNA was denatured with heat by incubating for 3 minutes at 95 ° C followed by 25 cycles of one minute at 95 ° C, 1 minute at 42 ° C and 1 minute at 72 ° C. After these 25 cycles the mixture was incubated for 5 minutes at 72 ° C. The analysis of the reaction products revealed two discrete products of approximately 500 bp and 600 bp. Based on the apparent molecular weight of 32 kDa for the AXDA and the apparent molecular weight of 9 kDa for the CNBr peptide, the fragment was within the expected limits.

Example 5.1.1: Cloning and analysis of PCR fragments of 500 bp and 600 bp Example 5.1.2.1: Cloning of 500 bp and 600 bp PCR fragments Both 500 bp and 600 bp PCR fragments were ligated into a pGEMMR-T vector (Promega) according to the manufacturer's instructions. After incubating for 16 hours at 14 ° C, 4.5 μl of ligation mixture was added to transform the competent E. coli DH5a cells. A selection of six of the colonies resulting from each ligation was grown overnight in LB medium containing 100 μg / ml ampicillin. From the plasmid cultures, the DNA was isolated by the alkaline lysis method according to that described by Maniatis et al. (1982, pp. 368-369).

Example 5.1.2.2: Analysis of the sequence of the fragments of the PCR of 500 bp and 600 bp The primary structures of both 500 bp and 600 bp PCR fragments were determined by sequencing the fragments as described in Example 4.4. Computer analysis of the nucleotide sequence of both PCR fragments showed that the 600 bp PCR fragment had no similarity to the known arabinoxylan degrading genes and was therefore considered a PCR artifact. The nucleotide sequence of the 500 bp PCR fragment was very similar to the nucleotide sequence of the cDNA clone shown in Figure 4 of nucleotide 706 to 1204.

Example 5.2: Marking with 32P of the 500 bp PCR fragment The 500 bp fragment obtained by the PCR was isolated and labeled as described in the application European Patent No. 91205944.5 (publication number 0 463 706 Al, Examples 2.2 and 7.1, incorporated herein by reference).

Example 5.3: Selection of the genomic library of A. niger variant tubigensis for the axdA gene For the selection of the genome library of A. niger variant tubigensis constructed as described in the case of European Patent Application No. 91205944.5 (publication number 0.463 706 Al), Example 2, for the axdA gene, 3 x 103 ufe were plated per plate as described in Example 4.6, with the 500 bp PCR fragment labeled with 32 P, prepared as described in Example 5.1. 1, as a probe. Three of the hybridization plates, which appear in duplicate in the duplication filters, were identified and designated as ^ u axhl a ^ tu axh3- The phages were isolated and propagated as described in Example 4.6.

Example 5.4: Southern analysis of phages containing axdA DNA from A. niger variant tubigensis The DNA was isolated from lambda bacteriophages as described in Example 4.7. DNA isolated from phages? Axuba ^ ^ ^ ^ ^ ^ ^ ^ ^ analyzed by Southern analysis using the following restriction enzymes; BamHl; £ coRI; HindIII; Kpnl; Sstl and Xhol. The Southern analysis was conducted as described in Example 4.8. From the results obtained it was concluded that the DNA of the three isolated clones was hybridized with the axdA cDNA of A. niger variant tubigensis. In the three clones fragments were found that originated from the same genomic region.

Example 5.5: Subcloning of the axdA gene of A. niger variant tubigensis The 5.5 kB Sstl fragment of ^ tubaxh3 was isolated and ligated into the digested and dephosphorylated pBluescript SK ~ SStI vector as described in Example 4.9, resulting in a plasmid designated pIM3001. Plasmid pIM3001 was further analyzed with restriction enzymes resulting in the restriction map shown in Figure 6. A sample of E. coli DH5a harboring pIM3001 was deposited on August 28, 1995 at CBS, Baarn, The Netherlands. , under the number CBS 636.95.

Example 5.6: Expression of the axdA gene of A. niger variant tubigensis in A. niger NW219 Example 5.6.1: Introduction of the axdA gene of A. niger variant tubigensis in A. niger NW219 by cotransformation Plasmid pIM3001, obtained in Example 5.5, was introduced into an A. niger by cotransformation of A. niger NW219 as described in example 4.10.1. The resulting PYR + transformants were then analyzed to determine the expression of the axdA gene of A. niger variant tubigensis by Western blot analysis.

Example 5.6.2: Selection of transformants for the expression of the axdA gene of A. niger variant tubigensis The transformants obtained in Example 5.6.1 were analyzed to determine the product of the axdA gene of A. niger variant tubigensis, the AXDATUB protein. • Nineteen of these transformants were used in a growth experiment to analyze AD TUB expression. The strain of A. niger N402 was used as a wild-type control. The transformants were grown as described in Example 3.1 for 20, 41, 60 and 86 hours. After growth, the mycelium was removed by filtration and the culture filtrate was analyzed by Western blotting using antibodies raised against AXDATu3 in a rabbit (Example 4.1). Twelve of the nineteen transformants analyzed produced the AXDATUB protein according to what was detected by this procedure.

Example 5.7: Analysis of the sequence and description of the axdA gene of A. niger variant tubigensis The sequence of the axdA gene of A. niger variant tubigensis, its promoter / regulatory region, the structural part of the gene and the termination region, were determined by subcloning fragments of pIM3001 into pBluescript SK ~ and pUC19 in combination with the use of specific oligonucleotides as primers in the sequencing reactions. The nucleotide sequence was determined as described in Example 4.4 (See IDNOSEC: 7).

The obtained sequence comprises 2859 bp, 823 bp in the 5 'non-coding region and 1041 bp in the 3' non-coding region. In the non-coding region 5 * a CAAT box was found at positions 651-655 and the TATA box was found at positions 713-720. The coding part of the axdA gene of A. niger variant tubigensis is 996 bp long and is not interrupted by introns. The genes code for a protein of 332 amino acids in size. The N-terminal sequence, according to what is determined in Example 1.5.1, is preceded by a peptide of 26 amino acids in length. The mature protein is 306 amino acids in size and has an estimated molecular weight of 33,250 kDa and a theoretical isoelectric point of 4.20.

Example 6: Maize digestion with AXDA Example 6.1 Isolation of Water Insoluble Solids (SIA) For the in vitro digestion system, the insoluble polymer fraction of corn was isolated. The corn was ground (3 mm) in a Retsch mill and degassed with hexane (soxlet construction for distillation) for 6 hours. After degreasing, the corn was dried at 105 ° C and ground again (1 m, Retsch mill). To 400 g of defatted corn, 1.5 1, 1.5% SDS / 0.05% ß-mercaptoethanol were added and stirred at room temperature for 1 hour (to break proteins) after which it was centrifuged for 15 minutes at 24,000 g . After denaturation of the protein the pellet was washed 3 times with 1 1 SDS / β-mercaptoethanol, and 2 times with 1 1 of demineralized water. The pellet was resuspended in 4 1 of demineralized water and sieved over a 32 μm mesh. The washing and sieving procedure was repeated. The pellet of the crude SIA fraction (greater than 32 μm) was dissolved in 1 1 of maleate buffer (pH 6.5) and incubated at 90 ° C for 50 minutes. 2 mg of α-amylase (Maxamyl1 ^ Gist-brocades) was added and incubated for 16 hours at 30 ° C, after which it was centrifuged for 15 minutes (24,000 g). The enzymatic digestion was repeated after which the pellet was washed 3 times with demineralized water at 65 ° C. The pellet (SIA) was lyophilized. The AIS contained 30% xylose, 29% arabinose, 23% glucose and 7% galactose. The glucose was not determined to be starch (Boeringer Mannheim, test kit for starch).

Example 6.2 Poultry digestion system The dry matter content of the SIA (95.73%) was determined by determining the weight of the SIA before and after drying at 120 ° C. In the poultry digestion system, 200 μN of NaN3 (Merck) and 1 mL of internal standard (sorbitol, 35 mg / mL, Sigma) were added to 1 g of SIA. For digestion of the enzyme culture (with (200 μg of protein) or without enzyme) the sample was gauged with buffer (NaAc, pH 5.5) to 15 ml and incubated at 39 ° C for 1 hour. For stomach digestion, 5 ml of pepsin (5.28 g / 1, pH 3.0, Merck) were added and incubated for 1.5 hours at 39 ° C. For intestinal digestion, 2.5 ml of pancreatin / bile salts (16 g / 1, 0.1 g / 1 respectively, Merck) were added and incubated for 1.5 hours at 39 ° C. The mixture was centrifuged for 15 minutes (2800 g).

The supernatant was used to measure monoazúcares (CLAP, Spectra Physics; HPX-87P column, Biorad) that were released during enzymatic digestion. The pellet was dried at 120 ° C and the weight was determined. The percentage of dry matter was calculated. As a control, a dose response of crude enzyme preparation was added to the in vi tro system (the results are shown in Figure 3). The AXDA enzyme (200 μg of AXDA protein was added) showed a considerable increase in dry matter digestion compared to the blank. The dry matter digestion percentage of 15.8% over the SIA fraction of corn had an increase of 4.1% in relation to the SIA of corn.

Example 7: Digestibility experiment in vi tro with SIA of corn Example 7.1: Isolation of corn SIA Isolation of the SIAs from the maize was performed as described in Example 6 with some modifications because the incubation with SDS / mercaptoethanol can not be applied to the animal fodder. The isolation procedure was carried out on 1 kg of defatted corn. To 1 kg of defatted corn, 4 g of papain (Gist-brocades) / 100 g of corn protein were added and suspended at 30 ° C for 2 hours. After this, the suspension was digested with Maxamyl ™ (Gist-brocades) (2 ml / l) at 100 ° C and centrifuged (15 minutes 24,000 g). The supernatant was discharged and the enzymatic digestion procedure was repeated on the pellet. The suspension was centrifuged again and washed three times with demineralized water (11) after washing, the pellet (water insoluble solids) was lyophilized.

The content of mono / oligosaccharides in the SIA of corn was determined by the analysis with trifluoroacetic acid (TFA). To 0.3 g of SIA, 1 ml of sorbitol 35 mg / ml (internal standard), 2 ml of demineralised water and 450 μl of TFA were added followed by hydrolysis for 1 hour at 120 ° C. After cooling, 20 ml of demineralized water were added and hydrolysis continued for 1 hour at 120 ° C. After cooling, the samples were lyophilized and resuspended in 3 ml of demineralized water. The monosaccharides were removed on an HPX-87P column (Biorad) in a CLAP system (Spectra Physics). The polymeric fraction of the SIAs of the corn contained: 124.8 mg / g of xylose, 96.6 mg / g of arabinose, 28.12 mg / g of galactose and 156 mg / g of glucose. Due to the high glucose content, the SIA fraction was analyzed to determine the starch content (test equipment for starch, Boehringer Mannheim). It was found that the SIA fraction did not contain any starch.

Example 7.2: Digestibility experiment with broilers An experiment was carried out to detect the influence of the AXDA enzyme on the actual metabolizable energy content of a diet, to which 20% of corn SIA was added. The method used was a modified "sibbald test".

Male broilers (3 weeks old) were housed individually in boxes in a room with controlled environment. The animals were fasted for 48 hours. An exact amount was then fed through a feed cannula. To correct the losses of components containing endogenous energy, a control group was included in the experiment. Those animals were fed 10 g of D-glucose, while the others received 10 g of food. Each food was given to 6 animals. The control group with glucose consisted of 5 animals.

The animals were fasted for another 48 hours, during which period the excreta were collected quantitatively. The excreta was stored at -20 ° C until the analysis was carried out. Water was available to the animals during this period once, while they were also given water once through a tube.

The digestion products collected were lyophilized and weighed after being balanced to air. The nitrogen and energy content were analyzed in the food samples and in the digested dry matter samples.

The results of the control animals were used to correct the results of the animals fed the experimental diets. The applied method is an adaptation of the experimental procedures described by McNab and Blair, 1988. This article gives a description of the methods of analysis and calculation.

Dry matter digestibility measurements were also made in vi tro according to the method described in Example 6.2.

The experimental treatments were the following: I corn basal food (table 2) II corn basal food + 10% corn SIA III corn basal food + 20% corn SIA IV corn basal food + 20% corn SIA + 100 mg / kg of AXDA V corn basal food + 20% of corn SIA + 45 mg / kg of crude enzyme VI corn basal food + 20% of corn SIA + 90 mg / kg of crude enzyme VII corn basal food + 20% of SIA of corn + 200 mg / kg of endoxylanase.

The composition of the basal diet is given in Table 2.

Table 2: Composition of a basal diet used in the experiment.

* The vitamin / mineral premix administered per kg of food or feed: rivoflavin, 4 mg; niacin 40 mg; d-pantothenic acid, 12 mg; choline chloride, 500 mg; B12, 15 μg; E, 15 mg; K3, 5 mg; retinyl acetate, 3.44 mg; cholecalciferol 50 μg; biotin, 0.1 mg; folic acid, 0.75 mg; FeS04-7H20, 300 mg; Mn02, 100 mg; CuS04.5H20, 100 mg; ZnS04.7H20, 150 mg; Na2Se03, 0.15 mg; antioxidant, 100 mg and virginiamycin, 20 mg.

The results (True Metabolizable Energy Corrected for Nitrogen (NME) and the results of the in vi tro test) are given in Table 3.

Table 3: True metabolizable energy (corrected for nitrogen: EMVn) and dry matter digestibility in vi tro.

Residual standard error: 0.42 LDS (5%): 0.76 The values of EMVn with a different letter are significantly different (P <0.05) according to the LSD test.

The level of EMVn decreased significantly with an increase in the addition of corn SIA. All the enzymatic preparations improved the values of EMVn, but only for the endoxylase (+ 7.0%) and for the AXDA (+ 10.7%) this was significant. According to the average values, the AXDA improved the value of the energy to the level with the food or basal forage supplemented with 10% SIA of corn. In the in vi tro model (simulating the digestive process in the chicken), the AXDA improved the digestion of dry matter in an important way (+ 24.5%). The endoxylanase and the crude enzyme preparation in this model were not as effective as the AXDA.

Example 8: AXDA in bread baking The AXDA was tested in a puff pastry baking test using thin loaves. The puff pastries were baked from dough pieces of 150 g obtained by mixing 200 g of flour (RobijnRM / ColumbusRM 80/20), 104 ml of water, 1.4 mg of dry baking yeast (FermipanRM) 4 g of salt, 3 g of sugar, 400 mg of CaCl2.H20, 3 mg (15ppm) of ascorbic acid, and 5 mg (25ppm) of fungal alpha-amylase (FermizymeRM) and 25 ug of purified AXDA. After mixing for 6 min and 15 s at 52 rpm in a premixer, the dough was divided, tested for 45 min at 30 ° C, minced, tested for another 25 min, molded and cooked. After a final test of 70 min at 30 ° C the dough was baked for 20 min at 225 ° C. The volume of the puff pastry was determined by the rapeseed oil displacement method. The volume of the puff pastry was increased from 490 ml (control) to 535 ml for the puff pastries containing AXDA, ie an increase of 9%.

References Amons, R. (1987), FEBS lett., 212: 68-72. Carré B. and Brillouet J.M. (1986), J. Science and Food Agrie. 37: 341-351. Chesson, A. (1987) Recent Advances in Animal Food Nitrition.

Goosen, T. et al. (1987) Curr. Genet 11: 499-503. Hanahan, D. (1983) J. Mol. Biol. 166: 557. Harmsen, J.A.M. et al. , (1990) Curr. Genet 18: 161-166 Haresign, W. and Cole D.J.A., eds. Butterworth, London, 71-89. Jerpseth, B., et al. (1992), Strategies 5: 81-83 Maniatis T., E. F. Fritsch, J. Sambrook (1982): Molecular cloning, a manual laboratory; Cold Spring Harbor Laboratory, New York. Matsudaira, P. (1987), J. Biol. Chem., 262: 10035-10038.

McCleary, B.V. and Matheson, N.K. (1986), Adv. Carb.

Chem. And Biochem. 44: 147-276. McNab, J.M. and Blair J.C. (1988), British Poultry Science 29: 697-707. Murray, N. (1977), Mol. Gen. Genet. 150: 53-58 Saiki R.K. et al. (1988), Science, 239, 487-491 Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). In: Molecular Cloning: a Laboratory Manual, 2nd edn., Cold Spring Harbor Labatory Press, NY. Sanger, F., Nickelen, S. and Coulson, A. R. (1977) Proc.

Nati Acad. Sci. USA, vol. 74: 5463-5467 Silvay, T.J., Berman, M.L., and Enquist, L.W. (1984) Experiments with gene fusions, Cold Spring Harbor Labatory: New York. pp. xi-xii Short, J.M. et al. , (1988) Nucleic Acids Res. 16: 7583-7600. Visniac, W. and Santer, M. (1957), Bact. Rev. 21: 195-213 Yanish perron, C. et al. , (1985) Gene 33, 103-119 LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Gist-brocades B.V. (B) STREET: Wateringseweg 1 (C) CITY: Delft (E) COUNTRY: The Netherlands (F) POSTAL CODE (C.P.): 2611 XT (ii) TITLE OF THE INVENTION: Enzymes degrading arabinoxylan (iii) SEQUENCE NUMBER: 8 (iv) LEGIBLE FORM IN COMPUTER: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent in process # 1.25 version 1.25 (EPO) (2) INFORMATION FOR SEC ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linearF MOLECULE: peptide (iii) HYPOTHETICAL: NO (iii) ANTICIPATION: NO (iv) TYPE OF FRAGMENT: N-terminal (vi) SOURCE OF ORIGIN: (A) ORGANISM: Aspergillus niger variant tubigensis (B) CEPA: DS16813 xaa = cys (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO: 1 Lys Xaa Ala Leu Pro Ser Ser Tyr 1 5 (2) INFORMATION FOR SEC ID NO: 2 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (iü) ANTISENTIDO: NO (iv) TYPE OF FRAGMENT: internal (vi) SOURCE OF ORIGIN: (A) ORGANISM: Aspergillus niger variant tubigensis (B) CEPA: DS16813 (ix) CHARACTERISTICS: (A) NAME / KEY: amino acid residue (B) LOCATION: 14 (D) OTHER INFORMATION: Arg or Asn (ix) CHARACTERISTICS: (A) NAME / KEY: amino acid residue (B) LOCATION: 15 (D) OTHER INFORMATION: residual = uncertain (ix) CHARACTERISTICS: (A) NAME / KEY: amino acid residue (B) LOCATION: 16 (D) OTHER INFORMATION: residual = uncertain (ix) CHARACTERISTICS: (A) NAME / KEY: amino acid residue (B) LOCATION: 17 (D) OTHER INFORMATION: residual = uncertain (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO: 2: He Val Glu Ala He Gly Ser Thr Gly His Arg Tyr 1 5 10 Phe Xaa Ser Phe Thr 25 15 (2) INFORMATION FOR SEC ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (Üi) ANTISENTIDO: NO (vi) SOURCE OF ORIGIN: (A) ORGANISM: Aspergillus niger variant tubigensis (B) CEPA: DS16813 (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO: 3: ATGATKGTIG ARGCIATKGG (2) INFORMATION FOR SEC ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTICIPATION: NO (vi) SOURCE OF ORIGIN: (A) ORGANISM: Aspergillus niger variant tubigensis (B) CEPA: DS16813 (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO: 4: AATACGACTC ACTATAG_2_) INFORMATION FOR SEC ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2101 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTICIPATION: NO (vi) SOURCE OF ORIGIN: (A) ORGANISM: Aspergillus niger (B) CEPA: CBS 120.49 (ix) CHARACTERISTICS: (A) NAME / KEY: signal_TATA (B) LOCATION: 665..670 (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 784..1779 (D) OTHER INFORMATION: / product = "enzymes that degrade arabinoxylan" / gen = "axdA" / standard_name = "enzymes that degrade arabinoxylan " (ix) CHARACTERISTICS: (A) NAME / KEY: sig_peptid? (B) LOCATION: 784. . 861 (ix) CHARACTERISTICS: (A) NAME / KEY: mat_peptid? (B) LOCATION: 862. . 1779 (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO: 5: CTGATTGGGA TTCTGCAGGA ATTCTCTGGG GTATGCCAAA AAAGTATACC GACCTGTAAA 60 AGTCCAACCA GTTCGAAATT ACTAACAATA TGTTTTTGAT CAGGATATCT TTGGCATCTA 120 TGGTGAGAGC CATATCATCA TCTCTTCTTC CGGCAGCTGT CAACTGCCTG CCGAAAGTAC 130 TGGAAGCCAT TGTGTTTTAA GGTGAAACAA GATCAGGGCG GCTATGTGTC AGGGTAGAAC 240 CAGTTTGCTT AGCGCCATCA GGGTCCACGT CTAGACTTTC GATGCCCGGA GTTATTCGCC 300 TTCCCACAGC AGTCATTTCC CCGAATCTAA ACCGATGGAC GGATATTGTG GTGTAATGAT 360 AGAACAACAC GGTGTAGTGT AßTTTTAAGT GCCGTGCTAG? CACGGCAAC GTTCCGGTGG 420 GCGATTGTTT CTGGCTAATG TACTCCßTAG TTTAGGCAAC AGGCCGATCA TCTTCCCCCA 480 TAGGAAAGßA CCCTGAATAG TGCGTCAAAA AGAGCTTGAG GCAAAGGASG ACTGCACTTT S40 CCAAGGCCGA AGTGGGGGGs GGGGATAACC AAGCAGCCCA ACTTTTATCC GAAACCTTTC 600 AGGTGTCÁTC TAATTTGGAT AAATCCGGAT TGTTCTTCGG CATATGTGGA TGTCACCATG 6S0 AGCCATAAAT ACAAATATCT GGACAAGCTG TTGCCCTTTG TTCAAGTTAT TCGTTCTCTG 720 TGGAC ACGA TCCCAACC? T TGATCTCTTT TGTTTGTTCC TCAGCGGATA AAGTCATACG 80 AAA ATG AAA TTC CTC AAA GCC AAG GGT AGC TTG CTG TCG TCT GGC ATA 328 Mee Lys Phe Leu Lys Wing Lys Gly Ser Leu Leu Ser Ser Gly He -25 -20 -15 TAC CTC ATT GCA TTG GCC CCC TTT GTC AAC GCA AAA TGC GCT CTT CCG 376 Tyr Leu He Wing Leu Wing Pro Phe Val Asn Wing Lys Cys Wing Leu Pro -10 -5 1 5 TCG ACÁ TAT AGT TGG ACT TCG ACC GAT GCT CTC GCC ACC CCA AAG TCC 924 Ser Thr .Tyr Ser Trp Thr Ser Thr Aap Wing Leu Wing Thr Pro Lys Ser 10 15 20 GGA TGG ACT GCA CTC AAG GAC TTC ACC GAT GTC GTC TCT AAC GGC AAA 972 Gly Tro Thr Wing Leu Lvs Asp Phe T r Asp Val Val Ser Asn Gly Lys 25 * 30 35 CAT ATT GTC TAT GCG TCC ACT ACC GAC ACA CAG GGA AAT TAC GGC TCC 1020 His lie Val Tyr Wing Ser Thr Thr Aap Thr Gln Gly Asn Tyr Gly Ser 40 45 50 ATG GGC TTT GGC GCC TTT TCG GAC TGG TCG GAC ATG GCA TCC GCT AGT 1068 Met Gly Phe Gly Wing Phe Being Asp Trp Being Asp Mee Wing Being Wing Ser 55 60 65 CAA ACG GCC ACA AGC TTC AGC GCC GTA GCT CCA ACC TTG TTC TTC TTC 1116 Gln Thr Wing Thr Ser Phe Ser Wing Val Wing Pro Thr Leu Phe Tyr Phe 70 75 80 8S CAG CCA AAG AGT ATC TGG GTT CTG GCC TAC CAG TGG GGC TCC AGC ACT 1164 G n Pro Lys Ser He Trp Val Leu Wing Tyr Gln Trp Gly Ser Ser Thr 90 95 100 TC ACC AC CGC ACC TCT AÁ GAT CCC ACC AAT GTC AAC GG C TGG TCA 1212 Phe Thr Tyr Arg Thr Ser Gln Asp Pro Thr Asn Val Asn Gly Trp Ser 105 110 115 TCC GAG CA GCT CTT TTC ACG GGC AAA ATC AGC GGC TCA AGT ACC GGT 1250 Ser Glu Gln Ala Leu Phe Thr Gly Lys Xle Ser Gly Be Ser Thr Gly 120 125 130 GCC ATT GAT CAG ACT GTG ATT GGT GAT GAT GAT ACG AAT ATG TAT CTT TTC 1308 Wing He Asp Gln Thr Val He Gly Asp Asp Thr Asn Mee Tyr Leu Phe 135 140 145 TTT GCC GGC GAC AAT GGC AAG ATC TAC CGA TCC AGC ATG TCT ATC AAT 1356 Phe Wing Gly Asp Handle Gly Lys He Tyr Arg Ser Ser Mee Ser He Asn 150 155 160 165 GAC TTC CCC GGA AGC TTC GGC AGC CAG TAC GAG GAG ATC CTC AGC GGC 1404 Asp Phe Pro Gly Ser Phe Gly Ser Gln Tyr Glu Glu He Leu Ser Gly 170 175 180 GCG ACC AAC GAT TTG TTC GAG GC® GTC CA GTG TAC ACC GTC GAC GGC 1452 Thr Wing Asn Asp Leu Phe Glu Wing Val Gln Val Tyr Thr Val Asp Gly 185 190 195 GGC GAG GGT GAC AGC AAG T? C CTC ATG ATC GTC GAG GCG? TC GGT TCC 1500 Gly Glu Gly? Sp Ser Lys Tyr Leu Mee He Val Glu Wing He Gly Ser 200 205 210 ACC GGA CAT CGT TAT TTC C GC TCC TTC ACG GCC AGC AGT CTC GGC GGA 15 9 Thr Gly His? Rg Tyr Phe Arg Ser Phe Thr Wing Ser Ser Leu Gly Gly 215 220 22S GAG TGG ACÁ GCC CAG GCG GCA AGT GAA GAT CAA CCC TTC GCG GGC AAA 159S G u Trp Thr Ala Gln Wing Wing Ser Glu Asp Gln Pro Phe Wing Gly Lys 230"235 240 245 GCC AAC AGT GGC GCC ACC TGG ACC GAC GAC ATC AGT CAT GGT GAC TTG 1644 Wing Roasting Ser Gly Wing Thr Trp Thr Aap Asp He Ser His Gly Aso Leu 250 - 255 26 * 0 GTT CGC AAC AAC CCT GAT CAA ACC ATG ACG GTC CCT TGC AAC CTC 1S92 Val Arg Asn Asn Pro Asp Gln Thr Mee Thr Val Asp Pro Cvs Asn Leu 265 270 275 CAG CTT CTC TAC CAG GGC CAT GAC CCC AAC AGC AAT AGT GAC TAC AAC 1740 Gln eu Lau Tyr Gln Gly His Asp Pro Asn Ser Asn Ser Asp Tyr Asn 230 235 290 CTC TTG CZZ TGG AAG CCA GGA GTT CTT ACC TTG AAG CAG TGAAAGGCTt 1789 Leu Leu Pro Trp Lys Pro Gly Val Leu Thr Leu Lys Gln 295 300 305 ATCATTTGGT TGCAGACCGG GGTTTTTCTTC CCCTTCCTTG AGTAGTATTG TTGGTGGAAG 1349 ACAGCGGGAT GGGGAGTGAA TACTATCTTG GGCTCAATTG AGGTGGAATC CTGTCAGACT 1909 GTGTACATAG GCTACATGCG AATGATTTGG TTTATTCACA AATAGTATTA ACAGATAGTG 1969 TAGTATACAC CTCTGTATTC ACAGGTGATA GCCTGTCTAC TAGTAGTAGA sATstGGCTC 2029 GAGATGACTG CACGTGATGA TCACATCATC ATCATCGCAG TCGCTCACGC GACAGTCTCA 2089 GACACACACA TA 2101 (2) INFORMATION FOR SEC ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 332 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protelna (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO: 6: Mee Lys Phß Leu Lys Wing Lys Gly Ser Leu Leu Ser Ser Gly He Tyr -25 -20 -is Leu He Ala Leu Wing Pro Phß Val Asn? The Lys Cys? The Leu Pro Ser -10 -5 1 5 Thr Tyr Ser Trp Thr Ser Thr? Sp? The Leu? The Thr Pro Lys Ser Gly 10 15 20 Trp Thr? The Leu Lys? Sp Ph? Thr? Sp Val Val Ser P? T. Gly Lys His 25 30 35 He Val Tyr? The Thr Ser Thr? Sp Thr Gln Gly? Sn Tyr Gly Ser Mee 40 45 50 Gly Phß Gly? The Phe Ser? Sp Trp Ser? Sp Me? The Ser? The Ser Gln 55 60 65 70 Thr? The Thr Ser Ph? Ser? The val ? the Pro Thr Leu Ph? Tyr Ph? Gla 75 30 35 Pro Lys Ser He Trp Val Leu? the Tyr Gln Trp Gly Ser Ser Thr Ph? 90 95? oo Thr Tyr? rg Thr Ser Gln? sp Pro Thr Asn Val Asn Gly Trp Ser Ser 105 110 n5 Glu Gln Ala Leu Phe Thr Gly Lys He Ser Gly Ser Ser Thr Gly Wing 120 125 130 He Asp Gln Thr Val He Gly Aap Asp Thr Asn Met Tyr Leu Phe Phe 135 140 145 lso Wing Gly Asp Asn Gly Lys He Tyr Arg Ser Ser Mee Ser He Asn Asp 1S5 160 155 Phe Pro Gly Ser Phe Gly Ser Gln Tyr Glu Glu He Leu Ser Gly Wing 170 175 130 Thr Asn Asp Leu Phß Glu Wing Val Gln Val Tyr Thr Val Asp Gly Gly 135 190 195 Glu Gly Asp Ser Lys Tyr Leu Met He Val Val Glu Ala He Gly Ser Thr 200 205 210 Gly His Arg Tyr Phe Arg Ser Phe Thr Wing Ser Ser Leu Gly Gly Glu 215 220 225 230 Trp Thr? The Gln? The? The Ser Glu? Sp Gla Pro Ph? The Gly Lys? The 235 240 245 Asn Ser Gly Wing Thr Trp Thr Asp Aso He Ser His Gly Asp Leu Val 250 255 260 Arg Asn Asn Pro Asp Gln Thr Mee Thr Val Asp Pro Cys Asn Leu ßln 265 270 275 Leu Leu Tyr Gln Gly His Aso Pro Asn Ser Asn Ser Aso Tyr Asn Leu 230 235 290 Leu Pro Trp Lys Pro Gly Val Leu Thr Leu Lys Gln 295 300 305 (2) INFORMATION FOR SEC ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2859 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (üi) ANTICIPATE: NO (vi) SOURCE OF ORIGIN: 5 (A) ORGANISM: Aspergillus niger variant tubigensis (B) CEPA: DS16813 (ix) FEATURES: (A) NAME / KEY: signal_CAAT 10 (B) LOCATION : 651..655 (ix) FEATURES: (A) NAME / KEY: signal_TATA (B) LOCATION: 713..720 (ix) CHARACTERISTICS: 15 (A) NAME / KEY: CDS (B) LOCATION: 823..1818 (D) OTHER INFORMATION: / product = "enzymes that degrade arabinoxylan" / gen = "axdA" 20 / standard_name = "enzymes that degrade arabinoxylan" (ix) FEATURES: (A) NAME / KEY: sig_peptide (B) LOCATION: 823 ..901 25 (ix) FEATURES: (A) NAME / KEY: mat_peptid? (B) LOCATION: 901..1818 (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO: 7: CATACTGGGT GTGGT? CT3T AGGGAACCTG CAGATGCTCT GCCGAAGGTC TGCAAAT? GT S O CCCTGGAGTT TGGTAGTAAA GAGTACCAAC TCGTAAAAGT AGTATGTCCA ACCAATTTGA 120 AAGTACAAAC TTTTAGTTTG ATTGATTA ?? ? T? CTTTTGG TGTGTACAGT GACAGCCAAA 130 ATATCATCTC TTCAGCCG? T? G? TGTCAAC TGCCCGCCG? ?? sT? CCGGA AGGTCGTGGT 240 GTTTTAAGGT GA ?? C? CT? T C? GGGCGGCA ATGTGTCAA? GT? G ?? CCAG TTTGCTTAGC 300 GCCATTAGGG TCCACGCCTA GACCCCTCGA TGCCCGGG? ß TCATCCGTCC TGTCACAGCA 360 ATTATTTCCC CGAGTCTACT GCCGAAG ?? G? GCT? TTGTG GCGT ?? TCAT GGAATTACCC 420 CTGTGTAGG GTAGTCTTGA? CGCCGTTCT? G? C? CGGCA? CGTTCCGGT GGACGATCGT 430 TTCTGGCTAA TGTACTCCGT? GTTT? GGC? GCT? GCTG? T CATCTTCCCC CTAGGGAA? G 540 GACCTGAATA- GTGCGCCAAA ATG? GCTTG? GC ?? AGG ?? T GTTCTTTCT? ? GCCAA? GTG 500 GGGGAAATAA CCAAGCAGCC CACTTTTATC CGA? CGTTT CTGGTGTCAT CCAAT? TGG? 660 TAAATCCCG? TTGTTCTTCT GCACGT? TCA GT? TTGCC? T C? CGT ?? CT? CAT? TATTT 720 GAAC? TGGTC TGGTCCTCCG TTCGATTTAT TCGTTCTCC? TGGCC ?? CG? CTTCAGCCAT 730 TGATCTCTTT TGTTTCTTTC CTGCGGCT ?? ? CCC? TTCG? ? G? TG AAG TTC TTC 834 Mee Lys Phe Phe - 2S AAA GCC AAA GGC AGC TTG CTβ TCA TCA GGC ATC TAC CTC ATT GCA TTA 382 Lys? Lys Gly Ser Leu Leu Ser Ser ßly He Tyr Leu He? The Leu - 20 -IS-10? CC CCC TTT 6TC? AC GCC A? A TGT GCT CTT CCG TC? TCC T? T? GT TGG 930 Thr Pro Ph? Val Asa? Lys Cys? The Leu Pro Ser Ser Tyr Ser Trp -5 1 5 10? GT TC? ? CC G? T GCT CTC GC? CT CT? AAG TCA GG? TGG? CC GC? CTG 978 Ser Ser Thr? Sp? The Leu? The Thr Pro Lys Ser ßly Trp Thr? The Leu 15 20 25 ?? ß G? C TTT? CT G? T GTT GTC TCG GAC G? C A? CAT? TT 6TC TAT GCß 1026 Lys Asp Phß Thr Asp Val Val Ser? Sp ßly Lys His He Val Tyr? The 30 35 40 TCC? CT ACT GAT GAA GCG GGA ?? CT? T GGC TCG? TG? CC TTT GGC GCC 1074 Ser Thr Thr? Sp Glu? The Gly? Sa Tyr Gly Ser Mee Thr Ph? Gly? The ^ 5 50 55 TTC GCA TGG TCG AAC ATG GCA TCC GCT AGC CAG AC? GCC ACC CCC 1122 Phe Se "Glu Trp Ser Asa Mee Wing Ser Wing Gla Thr Wing Thr Pro SO 65 70 TTC AAT GCC GTG GCT CCT ACC CTG TTC TAC TTC AAG CCG AAA AGT ATC 1170 Pb- Handle Wing Val Wing Pro Thr Leu Phe Tyr Phe Lys Pro Lys Ser He 75 80 35 90 tss s ~ "CG GCC TAC CAÁ TGG GGC TCC AGC AC? TTC? Cc TAC CGC ACC 121a T-3 Val Leu Wing Tyr Gln Trp Gly Ser Ser Thr Phe Thr Tyr Arg Thr 95 100 105 TCC CAA GAT CCC ACC AAT GTC AAT GGC TGG TCG TCG GAG CAG GCG CTT 1266 Ser Gln Aso Pro Thr Asn Val Asn Gly Trp Ser Ser Glu Gln Ala Leu "no 115 120 TC ACC GGC AAA ATC AGC GAC TCA AGC ACC AAT GCC ATT GAC CAG ACG 1314 Phe Thr Gly Lys He Ser Asp Ser Ser Thr Asn Ala He Asp Gln Thr 12S 130 135 GTG ATT GGC GAT GAT ACG A? T ATG TAT CTC TTC TTC GCC GGC GAC A? C 1362 Val He Gly Aso Asp-Thr Asn Met Tyr Leu Phe Phe Wing Gly Aap Asn 140"145 150 GGC AAG ATC TAC CGA TCC AGC ATG TCC ATC A? T GAC TTC CCC GGA? GC 1410 Glv Lys He Tyr? Rg Ser Ser Mee Ser He? Sn? Sp Phß Pro Gly Ser 155 160 165 170 .., GGC? GC CAG TAC GAG GTG ATC CTG? GT GGC GCC CGC ?? C G? T CT? 1458 Phe Gly Ser G n Tyr Glu Val He Leu Ser Gly? La? Rg? Sn? Sp Leu 175 180 185 TTC GAG GCG GTC CA GTA TAC ACC GTC GAC GGC GGT GAG GGC GAC ACG 1506 Phe Glu Wing Val Gln Val Tyr Thr Val Asp Gly Gly Glu Gly? Sp Thr 190 195 200 AAG TAT CTC ATG ATC GTT G? G GCG? TC GGG TCC? CC GGA CAT CGT TAT 1554 Lys Tyr Leu Mee He Val Glu? He He Gly Ser Thr Gly His? Rg Tyr 205 210 215 TTC CGC TCC TTC? CG GCC? GC? GT CTG GGT GG? G? G TG? C? GCC CAG 1602 Phe Arg Ser Phß Thr Wing Ser Ser Leu Gly Gly Glu Trp Thr Wing Gln 220 225 230 GCG GCA AGT G? G GAT CA GCC CCC TTC GCA GGC AAA GCC A? C? GT GGT GCC 1650? The? Ser Glu ? sp Gln Pro Phß? the ßly Lys? the? sn Ser ßly? the 235 240 245 250 ACC TGG ACC G ?? G? C? TT? GC C? T G? T G? C TTG GTT CGC ?? C ?? C CCT 1698 Thr Trp Thr Glu? Sp He Ser His Gly? Sp Leu Val? Rg? Sn? Sn Pro 255 260 265 G T CAÁ ACß? TG? CT ßTC ß? T CCT TGC? C CTC C? ß TT6 CTC T? TC? G 1746 Asp Gla Thr Mee Thr Val Asp Pro Cys? Sa Leu ßla Leu Leu Tyr ßln 270 275 280 GGC CAT GAC CCC A? C? GC? GT GßC ß? CT? C? CTC TT? CCG TG??? 1794 Gly His? Sp Pro? Sa Ser Ser Gly? Sp Tyr? Sn Leu Leu Pro Trp Lys 285 290 295 CCG GGC GTC CTT? CC TTG? ßC? ß TG? ßßTATT ATAATTAGTT GCAßATTßTG 1848 Pro ßly Val Leu Thr Leu Lys ßln 300 305 TTTTCATTCC TTCTTCAAGA GTGCTTAGTG GTGGAAG? CA ßCAßAAßßTG ßTCACTATCT TAGGCTCSGT TGGGGTGGGC TTGTGTCCAT AGGCTAGTA? TGTGCGCATA ATTC? GTTC? TTGGCAÁGGA GTGCGGTATA AATACCTGTT CTCACAAAAA AAAATAGGCC CGGTGGTCAT 2028 ACTCCGTATT GGGATAGAG? TCTCGTAGTA GTAGGATTGT GGGCCTCAGA GGATGACCGA 2088 CACGTGAGCA GTCTCCTTCT ACGGCTAGTC GCGTTCTACA TAAGAAATAG TCAGCTCAGA 21 a GTTTGTTTTT TGGCTACTTT sAAGGATGGC CTATCGAATC GCACGTCTCC TCAATTGGCC 2208 AGGTATTGGC ATTCACTCTC CGCGCTTTGC GGGTGCCGGC ACGAGATGTC TCCTGGAGA? 2268 ACTGGGCAAC GAGCAGACTA C3G? TATGGG AGATTGTTGA CGACGTTCTT CTTGGTAAAT 2328 TTGAACCCTT CAGGGGCTCT ATAAAGGCGG AAATCTAAAT CTCATGTGCC CTAACGTGTC 2338 C3ACCACGGT GTTGATCAGC ACCTATTAGA TCAG? CAAC? ? CCTTTGGCT CGGAAATTGA 2443 ACAGGTAGCT CTTGAATGAC ACTCTGGATC CTGATTCAAT TTATAATGCG TCACTTGAGC 2508 sTGCAAGGGs T3CTATATTC ACATCTTGCC CCAATCCAAG GGGCGTCGGA TCCCATTGTG 2563 CTCGACAGCC TGGAACTTCG CCGACAGTAT TCTTACG? CG TCG? TACTGA AATAGTCCAC 2628 CTGGTGTGCA TTCGTACGCC GGAAAGACCC TCGTCCGACC GCGTGGCCTT GATTCTGACG 2688 AGATGCTTCA ACAAGCGGCC AATTCGATGC CAGCTGTTCA TCGGTTAGAT GTGCTACACA 2743 GTGACCTGAT TCCAGGA ?? C ATATTCTGG? ? CG ?? GGAA? TGGCCGCGTC AATTTTCATT 2808 GACTTTGAGT GTGCAATAAC CCAA ?? T ?? C G ?? AT ?? TG? ? Cß? CCGCTG T 2859 (2) INFORMATION FOR SEC ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 332 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO: 8: Mee Lys Phß Phß Lys Ala Lys Gly Ser Leu Leu Ser Ser Gly He Tyr -25 -20 -15 Leu He? The Leu Thr Pro Ph? Val? Sa? The Lys Cys? The Leu Pro Ser - 10 -5 1 5 Ser Tvr Ser Trp Ser Ser Thr? Sp? The Leu? The Thr Pro Lys Ser Gly 1 10 15 20 Trp Thr? The Leu Lys? Sp Ph? Thr? Sp Val Val Ser? Sp ßly Lys His 25 30 35 He Val Tyr? The Ser Thr Thr? Sp Glu? The Gly? Sa Tyr ßly Ser Mee 40 45 50 Thr Phß ßly? The Ph? Ser ßlu Trp Ser? Sa Mee? The Ser? The Ser 61a 55 60 65 70 Thr? The Thr Pro Ph?? Sa? the Val? the Pro Thr Leu Pb? Tyr Ph? ly? 75 80 85 Pro Lys Ser He Trp Val Leu? the Tyr Gla Trp Gly Ser Ser Thr Phe 90 95 100 Thr Tyr Arg Thr Ser Gln Asp Pro Thr Aaa Val? sa Gly Trp Ser You will not be 115 Glu Gln? The Leu Phe Thr Gly Lys He Ser? Sp Ser Ser Thr? Sn? The 120 125 130 He? S Gla Thr Val He Gly? Sp Asp Thr Asa Mee Tyr Leu Phe Phe 135 140 145 150 Wing Gly Asp Asn Gly Lys He Tyr Arg Ser Ser Mee Ser He Asn Aso 155 160 155 Phe Pro Gly Ser Phe Gly Ser Gln Tyr Glu Val He Leu Ser Gly Wing 170 175 180 Arg Asn Aso Leu Phe Glu Wing Val Gln Val Tyr Thr Val Asp Gly Gly 185 190 195 Glu Glv Aso Thr Lys Tyr Leu Met He Val Glu Wing He Gly Ser Thr 200"205 210 Gly His Arg Tyr Phe Arg Ser Phe Thr Wing Being Ser Leu Gly Gly Glu 215 220 225 230 Trp Thr Wing Gln Wing Wing Ser Glu Asp Gln Pro Phe Wing Gly Lys? 235 240 245 Asn Ser Gly Wing Thr Trp Thr Glu Aso He Ser His Gly Aso Leu Val 250 255 260 Arg Asn Asn Pro Aso Gln Thr Met Thr Val Asp Pro Cys Asn Leu Gln 265"270 275 Leu Leu Tyr Gln Gly His Asp Pro Asn Ser Ser Gly Asp Tyr Asn Leu 280 235 290 Leu Pro Tro Lys Pro Gly Val Leu Thr Leu Lys Gln 295 * 300 305 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (23)

1. A recombinant DNA comprising a DNA fragment encoding a polypeptide having arabinoxylan degrading activity, or a precursor polypeptide thereof, characterized in that the DNA fragment is selected from: (a) a DNA fragment encoding a polypeptide having the amino acid sequence represented by amino acids 1 to 306, or a polypeptide precursor of the polypeptide represented by amino acids -27 to 306 in IDSECNO: 5; (b) a DNA fragment encoding a polypeptide having the amino acid sequence represented by amino acids 1 to 306, or a precursor of the polypeptide represented by amino acids -27 to 306 in IDSECNO: 7; (c) A DNA fragment encoding a variant or portion of the polypeptides represented by residual amino acids 1 to 306 described in IDSECNO: 5 or 7, which still has arabinoxylan degrading activity, or a precursor polypeptide thereof; (d) A DNA fragment encoding a polypeptide having arabinoxylan degrading activity and having the nucleotide sequence represented by nucleotides 784 through 1779 in the IDSECNO: 5 or nucleotides 823 to 1818 in the IDSECNO: 7; (e) A DNA fragment encoding a polypeptide having arabinoxylan degrading activity, or a portion of such a polypeptide, which DNA fragment is capable of hybridizing to a DNA fragment as represented by nucleotides 784 through 1779 in the IDSECNO: 5 or the nucleotides 823 to 1818 in the IDSECNO: 7.

2. The recombinant DNA according to claim 1, characterized in that the degradation activity of arabinoxylan is obtained from a filamentous fungus.

3. The recombinant DNA according to claim 2, characterized in that the filamentous fungus is of a species of Aspergillus.

4. The recombinant DNA according to claim 3, characterized in that the Aspergillus is niger or tubigensis.

5. The recombinant DNA according to claim 1, characterized in that it further comprises the regulatory DNA sequences required for the expren of DNA fragment in a prokaryotic or eukaryotic host cell when present therein.

6. The recombinant DNA according to claim 6, characterized in that the regulatory DNA sequences are heterologous with respect to the sequence encoding the polypeptide of the DNA fragment.

7. The recombinant DNA according to claim 6, characterized in that the heterologous regulatory DNA sequences are selected to increase the expren of the DNA fragment in a host as compared to the expren of the DNA fragment in such a host when it is linked to its sequences. Regulatory DNA homologous.

8. The recombinant DNA according to any of claims 1 to 7, characterized in that it is in the form of a vector.

9. A transformed eukaryotic or prokaryotic host cell, characterized in that it comprises the recombinant DNA according to any of claims 1 to 8.

10. The transformed eukaryotic host cell according to claim 9, characterized in that the host belongs to the genus Aspergillus.

11. A method for obtaining a host cell that is capable of increasing the expren of an enzyme that degrades arabinoxylan, characterized in that treatment of the host cell is carried out under conditions of transformation with a recombinant DNA according to claim 8 and selected expren increased of such an enzyme that degrades arabinoxylan.

12. The method according to claim 11, characterized in that the host cell is a host cell of the species Aspergillus.

13. A method for obtaining an arabinoxylan degrading enzyme, comprising the steps of growing the host cells capable of producing such an enzyme under the conditions that lead to that and recovering the enzyme, characterized in that the host cells, or their ancestors, have been transformed with a recombinant DNA according to claim 8.

14. A substantially pure polypeptide having arabinoxylan degrading activity, characterized in that the amino acid sequence described in the IDSECNO: 6 or IDSECNO: 8, as well as the variants and genetic portions thereof continue to have such activity.

15. A composition comprising a substantially pure polypeptide according to claim 14, characterized in that it is formulated for use in forages, foods, or paper and pulp processing.

16. The composition according to claim 15, characterized in that the enzyme is immobilized.

17. The use of a polypeptide according to claim 14, to assist in the degradation of arabinoxylan.

18. The use of a polypeptide according to claim 14, as an additive for forages or foods.

19. The use of a polypeptide according to claim 14, in the processing of paper and pulp.

20. The use of a polypeptide according to claim 14, in the baking of bread.

21. A forage or food, characterized in that it contains a polypeptide according to claim 14.

22. A recombinant DNA, characterized in that it comprises a DNA fragment represented by nucleotides 1 to 783 of the IDSECNO: 5, or a subfragment thereof, capable of regulating the expression of a DNA sequence linked to it.

23. A recombinant DNA, characterized in that it comprises a DNA fragment represented by nucleotides 1 to 822 of the IDSECNO: 7, or a subfragment thereof, capable of regulating the expression of a DNA sequence linked to it.