IE84033B1 - Cloning and expression of xylanase genes from fungal origin - Google Patents

Description

PATENTS ACT 1964 COMPLETE SPECIFICATION "CLONING AND EXPRESSION OF XYLANASE GENES FROM FUNGAL ORIGIN" GIST—BROCADES N.V., a Dutch Body Corporate, of wateringseweg I, P.O. Box 1, 2600 MA DeTft, The Netherlands Gist—brocades N.V. ]A Series 2566 Cloning and Expression of Xylanase Genes From Fungal Origin The present invention relates to the field of molecular biology. In particular, invention the present relates to the cloning and overexpression of a fungal DNA sequence encoding a protein having the activity of a xylanase. The present invention also provides methods for the production and use of a single xylanase which is obtainable in a form which is free of other xylanases, and indeed from other enzymes in general.

Background of the Invention The composition of a plant cell wall is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising various B- xylan chains) and pectin. The occurrence, distribution and structural features of plant cell wall polysaccharides are determined. by (1) plant species; (2) variety; (3) tissue type, (4) growth conditions; (5) ageing and (6) processing of plant material prior to feeding.

Basic differences exist between monocotyledons (e.g. cereals and grasses) and dicotyledons (e.g. clover, rapeseed and soybean) and between the seed and vegetative parts of the plant (Chesson, 1987; 1986).

Carré and Brillouet, Monocotyledons are characterized by the presence of an arabinoxylan complex as the major hemicellulose backbone.

The main structure of hemicellulose in dicotyledons is a xyloglucan complex. Moreover, higher pectin concentrations are found in dicotyledons than in monocotyledons. Seeds are generally very high in pectic substances but relatively low in cellulosic material.

A cross-sectional diagram of a plant cell is depicted in Figure 1. Three more or less interacting polysaccharide structures can be distinguished in the cell wall: (1) The middle lamella forms the exterior cell wall.

It also serves as the point of attachment for the individual cells to one another within the plant middle primarily «of calcium salts «of highly esterified tissue matrix. The lamella consists pectins; (2) The primary wall is situated just inside the middle lamella. It is a well—organized structure embedded in an pectin, of cellulose microfibrils amorphous matrix of hemicellulose, phenolic esters and proteins; (3) The secondary wall is formed as the plant matures. During" the plant's growth and ageing phase, cellulose microfibrils, hemicellulose and lignin are deposited.

The primary cell wall of mature, metabolically active cells (e.g. susceptible to enzymatic hydrolysis than the secondary cell plant mesophyll and epidermis) is more wall, which by this stage, has become highly lignified.

There is a high degree of interaction between cellulose, hemicellulose and pectin in the cell wall. The enzymatic degradation of these rather intensively cross- linked polysaccharide structures is not a simple process. At least five different enzymes are needed to completely break down an arabinoxylan, for example. The endo—cleavage is effected by the use of an endo—B(l+4)~D—xylanase. Exo~(l»4)~ D—xylanase liberates xylose units at the non—reducing end of the polysaccharide. Three other enzymes (a—glucuronidase, a- L-arabinofuranosidase and acetyl esterase) are used to attack substituents on the xylan backbone. The choice of the specific enzymes is of course dependent on the specific hemicellulose to be degraded (McCleary and Matheson, 1986).

For certain applications, however, complete degradation of the entire hemicellulose into monomers is not In the arabinoxylan, for example, one needs simply to cleave the necessary or is not desirable. liquefaction of main xylan backbone into shorter units. This may be achieved by the action of an endo—xylanase, which ultimately results in a mixture of xylose monomer units and oligomers such as xylobiose and xylotriose. These shorter subunits are then sufficiently soluble for the desired use.

Filamentous fungi are widely known for their capacity to secrete large amounts of a variety of hydrolytic enzymes such as a—amylases, proteases and amyloglucosidases and various plant cell wall degrading enzymes such as cellulases, hemicellulases, and pectinases. Among these, multiple xylan-degrading enzymes have been recognized, which have been shown to possess a ‘variety’ of biochemical and physical properties. This heterogeneity in xylanase function allows for the selection of a xylanase of interest which is best suited for’ a desired application (see Wong gt al. (1988), Woodward (1984) and Dekker and Richards (1977)).

Multiple Xylanases of ‘various ‘molecular' weights are known to be produced by micro-organisms such as Aspergillus niqer, Clostridium Trichoderma thermocellum, reesei, Penicillium ianthinellum, as well as species of Bacillus and Streptomyces.

On the contrary, in yeast no xylanase multiplicity has Trichosporon, Cryptococcus and Aureobasidium, only a single xylanase could be detected. been observed. In three east enera I In nature, microbial Xylanases are always produced together with other enzymes having polysaccharide-degrading activities, such. as exo—arabinanase, acetyl esterase and cellulases. For some applications, these enzyme activities are not needed or are unwanted.

It is known that fermentation conditions may be varied to favor the production of an enzyme of interest. It is also known that the cloning’ of the gene encoding" the desired enzyme and overexpressing it in its natural host, or other compatible expression host will specifically enhance the production of the enzyme of interest. This latter method is }.a U] particularly useful if the enzyme of interest is to be obtained in a form which is free of undesired enzyme activity.

The expression of recombinant bacterial xylanase has been previously described in European Patent Application .138. The gene encoding the bacterial xylanase was isolated from Bacillus chromosomal DNA and brought to expression in an E. coli host. However, E. coli expression hosts are, in some instances, considered to be unsafe for the production of proteins by recombinant DNA methods due to their production of unacceptable by—products such as toxins.

Since bacterial genes contain no introns, one is confronted with few problems in cloning and expressing such hosts. On the hand, the eukaryotic always so straightforward. It is well known that genes isolated from eukaryotic genes in prokaryotic other expression of genes is not strains contain introns. This inherently introduces complications in the cloning and expression of these genes, should a prokaryotic host be preferred.

Furthermore, certain differences exist, in general, between the physical characteristics of xylanases of fungal origin and those from bacteria. In general, fungal xylanases have a pH optimum in the range of between pH 3.5 - 5.5 as compared to bacterial xylanases which generally have a pH optimum in the range of pH 5.0 - 7.0. Fungal xylanases also generally have a broader pH stability range (pH 3 — 10) than (pH 5.0 — 7.5). xylanases generally have a temperature optimum of about °C. do their bacterial counterparts Fungal Bacterial xylanases generally have a temperature optimum between 50°C and 70°C. For a further discussion of the physical characteristics of xylanases see Wong et gl. (1988), Woodward (1984) and Dekker and Richards (1977).

Thus, it is clear that bacterial xylanases are less suitable for use in, for example, processes requiring lower pH conditions. In other instances, bacterial xylanases are too thermostable for certain applications such as the lagering of beer (see European Patent No. 227.159).

Accordingly, it would be of great importance to obtain genes encoding xylan-degrading enzymes of fungal origin which may be brought to expression in other, high—producing microbial expression hosts.

Summary of the Invention The present invention provides purified and isolated DNA sequences of fungal ongnrasdefinedincknniL\Nmchencodepnndnshavmg xylan-degrading activity. These DNA sequences include the xylanase encoding sequence and preferably the adjacent 5' and 3' regulatory sequences as well.

It is also an object of the present invention to provide constructs for the microbial overexpression of the xylanase—encoding sequences using either their native regulatory sequences or, in an alternative embodiment, the xylanase—encoding sequence operably linked to selected regulatory regions such as promoter, secretion leader and terminator signals which are of the capable of directing the overexpression xylanase protein in a suitable expression host.

It is a further object of the present invention to provide microbial expression hosts, transformed with the expression constructs of the present invention, which are capable of the overexpression and, if desired, the secretion of a xylanase of fungal origin.

It is yet a further object of the present invention to provide methods for the production of a xylanase of interest which may, in turn, advantageously be used in an industrial process. Typically, such an industrial process requires xylanase activity at a lower pH than that at which xylanases of bacterial origin optimally function.

Brief Description of the Figures Figure 1: A cross—sectional diagram of a plant cell.

Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure HPLC elution profile of a culture filtrate obtained from Asperqillus nicer DS16813 (CBS 323.90). This strain was later reclassified as more likely belonging to the species Aspergillug tubigensis.

Oligonucleotide probes AB80l - AB806, designed from the N—terminal amino acid sequence of the Aspergillus tubiqensis XYL A protein (Formula 1).

Oligonucleotide probe AB1255, designed from the N—terminal amino acid sequence of an internal 19 kDa fragment of the asperqilius tubigensis XYL A protein, digested with the peptidase (Formula 2).

S. aureus V8 endo~ Restriction map of the genomic region containing the xln A gene, as derived from Southern blot analysis of bacteriophage lambdaflfl. Indicated are the hybridizing fragments and their corresponding lengths.

Strategy employed to sequence the gspergillus tupigensis xln A gene. The arrows indicate the direction and number of bp sequenced.

Restriction map of pIMlOO containing the 6.9 kb Sall fragment containing the gspergillus tubigensis xlg A gene. In addition to the two HinDIII sites indicated, two further HinDIII sites are present in the plasmid insert.

Nucleotide sequence of the Aspergillus tubigensis gin A gene. The positions of the intron and the propeptide are putative.

Representation of a zymogram exhibiting the XYL A protein expressed by_transformants TrX2 and TrX9.

SDS—polyacrylamide gel electrophoresis showing the expression of the XYL A protein in A. niger CBS 513.88 (A) and A. niger N593 (B).

: Native gradient PAGE exhibiting the XYL A protein expressed. by A. niqer CBS 513.88 transformants Figure 12: Figure 13: Figure 14: Figure 15: Figure 16: Figure 17: Figure 18: Figure 19: numbers 10, with an RBB-xylan overlay (B).

Physical map of pAB 6—1. The 14.5 kbp fiigdIII DNA on a fragment in [Abbreviations: insert -in pUC19 contains the entire amyloglucosidase (AG) locus from A} niger.

A schematic View of the generation of AG promoter/xylanase gene fusions performed by the polymerase chain reaction.

Construction pathway of the intermediate plasmid pXYL2AG.

Construction pathway of the intermediate pXYL2.

Construction pathway of the intermediate pXYL3AG.

[Abbreviationsz see Figure 12] plasmid [Abbreviations: see Figure 12] plasmid [Abbreviations: see Figure 12] Construction pathway of the intermediate pXYL3. plasmid [Abbreviations: see Figure 12] Schematic representation of the constructs made in the the gpal site used in of the A. niger pyr by creating deletions Xln A promoter region. For orientation, the cloning A gene is indicated.

Detailed Description of the Invention The present invention describes purified and isolated DNA sequences of fungal origin which encode xylanases and genetic variants thereof, as defined in claim 1. The DNA sequence preferably includes the xylanase—encoding sequence and adjacent 5' and ' regulatory sequences. Genetic variants include hybrid DNA sequences containing the xylanase-encoding sequence coupled such as secretion and to regulatory regions, promoter, terminator signals, originating from homologous or heterologous organisms. Genetic variants also include DNA sequences encoding mutant xylanase proteins and degenerate DNA sequences wherein the xylan—degrading activity of the enzyme is retained. The present invention also includes DNA sequences which are capable of hybridizing to the xylanase- encoding DNA sequences and genetic ‘variants thereof, as described above, but which may differ in codon sequence due to the degeneracy of the genetic code or cross—species wnmUonTHmsemeab0spedfiednidamiL The present invention also provides DNA constructs vectors in the claims for . in a desired the expression of a xylanase of interest expression host. These include hybrid DNA sequences containing the xylanase—encoding region operably linked to regulatory regions, such as promoter, secretion and terminator signals originating from homologous or heterologous organisms, these regulatory regions being capable of directing the overexpression of the enzyme encoded by the xylanase—encoding DNA sequence in an appropriate host. Preferably, the expression construct will 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 microbial hosts via the introduction into the microbial host of the DNA constructs for the expression of the xylanese of interest. Vectors within theinvennonaredefinedinthe<flanns,asarenncnfinaihogs In addition, the present invention concerns homologous hosts transformed by DNA or heterologous constructs described above. Microbial expression hosts may be selected from bacteria, yeasts or fungi.

Within the context of the present invention, the term "homologous" is Aunderstood to intend all that which is native to the DNA sequence encoding the xylanase of interest, including its regulatory regions. A homologous host is defined as the species from which such DNA sequence may be isolated.

The term "heterologous" is thus defined as all that which is not native to the DNA sequence encoding the xylanase of interest itself, including regulatory regions. A "heterologous" host is defined as any microbial species other than that from which the Xylanase-encoding gene has been isolated.

Within the scope of the present invention, a xylanase of interest is ‘understood. to include a xylan-degrading enzyme which is naturally produced by a filamentous fungus. interest are those which are Xylanases of particular naturally produced by filamentous Aspergillus. fungi of the gums Especially preferred Xylanases are those originating from Aspergillus tubiqensis.

An endo—xylanase of interest. may be identified Via assay methods not critical to the present invention, such as a spot test assay. .According to this method, a filtrate obtained from the culturing of a microorganism induced (e.g. with oat spelts xylan) to produce an endo—xylanase may be tested for the presence of endo—xylanase activity. Drops of the elution fractions are placed individually onto an agar film containing a citrate—phosphate buffer (see Example 1.1, below) and oat spelt xylan. The film is then incubated. If .._'LO.. endo~xy1anase activity is present, the location of the individual drops on the agar film are visibly clear.

Once a xylanase of interest has been identified, the DNA sequence encoding such xylanase may be obtained from the filamentous fungus which naturally produces it by culturing the fungus in a xylan-containing medium, isolating the desired xylanase using known methods such as column chromatography (e.g. HPLC — see Figure 2) and determining at least a portion of the amino acid sequence of the purified protein. A, DNA probes may thereafter be designed by synthesizing oligonucleotide sequences based (N1 the partial amino acid sequence. Amino acid sequences may be determined from the N- terminus of the complete protein and/or from the N—termini of internal peptide fragments obtained via proteolytic or chemical digestion of the complete protein. Once obtained, the DNA probe(s) are then used to screen a genomic or CDNA library.

If this method is unsuccessful, the genomic library may’ be differentially‘ screened. with CDNA. probes obtained from mRNA from non~induced and induced cells. Induced mRNA is prepared from cells grown on media containing xylan as a carbon source, while non-induced mRNA must be isolated from cells grown on a carbon source other than xylan, e.g. glucose. Among the clones which only hybridize with the induced cDNA probe, a clone containing the desired Xylanase gene may be recovered. Alternatively, a xylanase gene may be identified by cross—hybridization with a related xylanase sequence .

A genomic library may be prepared by partially digesting the fungal chromosomal DNA with a suitable may be screened with a suitable DNA probe.

Alternatively, a cDNA library may be prepared by synthesized. frtmi mRNA. isolated from fungal induced for the cloning CDNA, cells synthesis of xylanase, into an appropriate phage vector, e.g. lambda gt 10 or lambda gt 11.

The CDNA library may then be screened with a DNA probe, or alternatively using immunological means or via a plate assay.

In 21 preferred embodiment of the present invention, oligonucleotide probes are designed from the N-terminal amino acid sequence (see Figure 3, formula 1) of a xylanase having an apparent molecular weight of 25 kDa purified from an Asperqillus tubiqensis culture filtrate and/or from the amino acid sequence of an internal peptide fragment (see Figure 4, formula 2) obtained by digestion of the xylanase with Staphylococcus aureus endoprotease V8. The oligo- DNA isolated from the four phage clones hybridized with the N—terminal oligo mixture as well as with the oligo mixture derived from the amino acid sequence of the internal fragment (see Figure 4). Restriction enzyme analysis revealed that all four clones contained DNA from the same genomic region of A. tubigensis.

A region of approximately 2.1 kb which hybridizes with both oligo mixtures has been sequenced. The nucleotide sequence, as depicted in Figure 8, comprises a xylanase ...l2_ coding sequence of 681 bp (which is interrupted by one small intron of’ 49 bp from: position 1179 to 1230), as well_ as sequences of 949 and 423 nucleotides of the 5' and 3' flanking regions, respectively.

Variants among the purified xylanase proteins have also been discovered. It has been determined that the corresponding xylanases have three different N-termini, possibly as a conditions.

Approximately one—third of these xylanases have serine as the N—termina1 amino acid (Figure 8, position 1), another result of fermentation approximately one—third have alanine as the N—termina1 amino acid (Figure 8, position 2) and the remaining proteins have glycine as the N—terminal amino acid (Figure 8, position 3).

The availability of a DNA sequence encoding a xylanase protein enables the construction of mutant xylanases by site—directed mutagenesis. If the tertiary structure of the xylanase is known, and its catalytic and substrate binding domains are localized, amino acids may be selected for mutagenesis (for example with the aid of‘computer modelling) which most likely affect catalytic and/or substrate binding functions. If the tertiary structure of the protein is not available, random mutants may be either generated along with the entire coding sequence, or the tertiary structure of the protein may be predicted by comparison with similar known xylanases isolated from another microorganism.

To facilitate the insertion of the DNA fragment containing the xylanase—encoding sequence into expression constructs comprising one or more heterologous regulatory regions, the polymerase chain reaction (PCR) (Ehrlich, H.A. (editor), 1989) may be used for introduction of appropriate ends of the xylanase coding sequence. The choice of restriction sites restriction enzyme sites in the 5' and 3' depends on the DNA sequence of the expression vector, i.e. the presence of other restriction sites within the DNA molecule.

To obtain overexpression of the xylanase protein in the original (homologous) production species, or 6.9 kb SalI fragment (see Figure 5) comprising the complete gene with its 5' alternatively in another fungal strain, a and. 3' regulatory regions, or alternatively, the complete gene fused to the regulatory regions of other genes, is introduced into the selected expression host to increase the copy number of ‘the gene and, consequently, protein expression. A If a heterologous expression host is preferred, and a yeast or a bacterial strain is selected, an uninterrupted (intronless) DNA sequence is used for the construction of a heterologous avoid the expression vector in order to possibility that splice signals residing on the genomic fragment are not recognized by the heterologous host. This uninterrupted DNA sequence may be obtained from a CDNA library constructed from mRNA isolated from cells, induced for the synthesis of xylanases. This library may be screened with an oligonucleotide or CDNA probe obtained as described before. Alternatively, an uninterrupted DNA sequence may be obtained by applying a polymerase chain reaction using appropriate 5' and 3‘ oligonucleotides on the first strand CDNA synthesized from the RNA of xylan-induced cells.

Within the overexpression is defined as the expression of the xylanase context of the present invention, of interest at levels above that which are ordinarily In the same context, overexpression also intends the expression of encountered in the homologous wild—type organism. the xylanase of interest in a heterologous organism which does not normally produce such xylanase except for the introduction of the DNA sequence encoding the xylanase of interest into the heterologous expression host. Progeny of these expression hosts are, of course, also to be understood to be embraced by the present invention. overexpression of the xylanase of interest may also be achieved by the selection of heterologous regulatory regions, e.g promoter, secretion leader and terminator regions, which serve to increase expression and, if desired, secretion levels of the protein of interest from the chosen expression host and/or to provide for the inducible control of the expression of the xylanase of interest.

Aside from the xylanase of interest's native promoter, other promoters may be used to direct its expression. The promoter may be selected for its efficiency in directing the expression of the xylanase of interest in the desired expression host.

In another embodiment, a constitutive promoter may be selected to direct the expression of the desired xylanase, relatively" free from other xylanases. Such. an expression construct is furthermore advantageous since it circumvents the need to culture the expression hosts on a medium containing solid xylans as an inducing substrate.

Examples of strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are the ATP-synthetase, subunit; 9 (oliC), triose phosphate isomerase (tpi), alcohol dehydrogenase (adhA), a- amylase (amy), amyloglucosidase (AG), acetamidase (amds) and glyceraldehyde—3—phosphate dehydrogenase (gpd) promoters.

Examples of strong yeast promoters are the alcohol dehydrogenase, lactase, kinase and —phosphoglycerate triosephosphate isomerase promoters.

Examples of strong bacterial promoters are the a- amylase and gpgz promoters as well as promoters from extracellular protease genes.

Hybrid promoters may also advantageously be used to improve inducible regulation of the expression construct.

Preferred promoters according to the present invention are those originating from the amyloglucosidase (AG) gene and native xylanase promoters.

It is often desirable for the xylanase of interest to be secreted from the expression host into the culture medium from where the xylanase may be more easily recovered.

According to the present invention, the xylanase of interest's native secretion leader sequence may be used to effect the secretion of the expressed xylanase.

However, an increase in the expression of the xylanase sometimes results in the production of the protein in levels beyond that which the processing‘ and secreting, expression host‘ is capable of creating" a build—up of protein product within the cell due to a bottleneck in the transport of the protein through the cell wall. Accordingly, the present invention also provides heterologous leader sequences to provide for the most efficient secretion of the xylanase from the chosen expression host.

According to the present invention, the secretion leader may be selected on the basis of the desired expression host. A heterologous secretion leader may be chosen which is homologous to the other regulatory regions of the expression construct. For example, the leader of the highly secreted amyloglucosidase protein may be used in combination with the amyloglucosidase promoter itself, as well as in combination with other promoters. Hybrid signal sequences may also advantageously be used within the context of the present invention.

Examples of preferred heterologous secretion leader sequences are those originating from the amyloglucosidase gene (fungi), the a—factor gene (yeasts) or the a-amylase gene (Bacillus).

Most preferred secretion leader sequences according to the present invention are the those originating from the amyloglucosidase (AG) gene and the native xylanase leader sequence. considered to be In general, terminators are not critical elements for the overexpression of genes. If desired, a terminator may be selected from the same genes as .._l6_. the promoters, 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 which have incorporated the desired gene from the bulk of untransformed cells. This selection marker, provided with the appropriate 5' and 3' regulatory sequences, may reside on the same DNA molecule containing the desired gene or be present on a separate molecule. In the latter case, a co—transformation must be performed. The ratio of the expression vector/selection vector must be adjusted in such a manner that a high percentage of the selected transformants also have incorporated the vector containing the expression construct of the xylanase of interest.

The most suitable selection systems for industrial micro—organisms are those formed by the group of selection markers which 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 benomyl resistance (benA). Exemplary of non-fungal selection markers are the G418 resistance gene (yeast), the ampicillin resistance gene (E. coli) and the neomycin resistance gene (Bacillus).

Once the desired expression construct has been assembled, it is transformed into a suitable cloning host such as E. Coli to propagate the construct. Afterwards, the expression construct is introduced into a suitable expression host wherein the expression construct is preferably integrated into the genome. Certain hosts such as Bacillus species may be used as both cloning and expression hosts, thus avoiding an extra transformation step.

According to the present invention, a variety of expression hosts may be used to overexpress the xylanase of interest. In one embodiment, a homologous expression host may be used. This involves the introduction of the desired expression construct back into the strain from which the xylanase encoding DNA sequence was isolated either in increased gene copy numbers, or under the control of heterologous regulatory regions as described above, or both.

In another embodiment, a xylanase of interest may be overexpressed by introducing and expressing the DNA construct encoding the xylanase of interest under the control of the appropriate regulatory regions in heterologous hosts such as bacteria, yeasts or fungi. For that purpose, the DNA sequence encoding’ the xylanase of interest is preferably expressed under the control of promoter and terminator sequences originating from the heterologous host. In addition, it may be necessary to replace the native secretion leader sequence of the xylanase of interest with a leader sequence homologous to the expression host in order to achieve the most efficient expression and secretion of the product.

Factors such as the size (molecular weight), the possible need for glycosylation or the desirability of the extracellular secretion of the xylanase of interest play an important role in the selection of the expression host.

The gram—negative bacterium E. coli is widely used as a host for heterologous gene expression, but mostly accumulates large amounts of heterologous protein inside the cell. Subsequent purification of the desired protein from the bulk of E. coli intracellular proteins can sometimes be difficult.

In contrast to E. coli, bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium.

Alternatively, a heterologous host selected from the group of yeasts or fungi may be preferred. In general, yeast cells are preferred over fungal cells because they are easier to manipulate.

However, some proteins are either poorly secreted from the yeast cell, or in some cases are _..l8.. not processed properly (e.g. hyperglycosylation in yeast).

In these instances, a fungal host organism should be selected.

A heterologous host may also be chosen to express the xylanase of interest substantially free from other polysaccharide-degrading enzymes by choosing 21 host which does not normally produce such enzymes such as Kluyyeromyces lactis.

Examples of preferred expression hosts within the scope of the present invention are fungi such as Aspergillus in EP 184.438 and EP 284.603) and Bacillus species (described Trichoderma species, bacteria such as species (described in EP 134.048) and yeasts such as Kluyveromyces species (described in EP 96.430 and EP 301.670) and Saccharomyces species.

Particularly" preferred expression hosts may be selected from Asperqillus niqer, Asperqillus awamori, Asperqillus aculeatus, Asperqillus oryzae, Asperqillus tubiqensis, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Kluyveromyces lactis and Saccharomyces cerevisiae.

The overexpression of the xylanase of interest is effected. by the culturing of the expression. hosts, which have been transformed with the xylanase expression construct, in a conventional nutrient fermentation medium.

The fermentation medium consists of an ordinary culture medium containing a carbon source (e.g. glucose, maltose, molasses, etc.), a nitrogen source (e.g. ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g. yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (e.g. phosphate, magnesium, potassium, zinc, iron, etc.).

Optionally, an inducer (e.g. oat spelts xylan) may be included.

The selection of the appropriate medium may be based on the choice of expression hosts and/or based on the _.. _. regulatory requirements of the expression construct. Such media are well—known to those skilled in the art. The medium may, if desired, contain additional components favoring the expression transformed hosts over other potentially contaminating microorganisms.

The fermentation is performed over a period of 0.5—20 days in a batch or fed—batch process at a temperature in the range of between 0 and 45 °C and a pH between 2 and 10.

Preferred fermentation conditions are a temperature in the range of between 20 and 37 "C and a pH 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 xylanase of interest may then be recovered and, if desired, purified and isolated by conventional means.

The product is stably formulated either in liquid or dry form. For certain applications, immobilization of the enzyme on a solid matrix may be preferred. xylanases of interest, produced by means of the present invention, may be applied either alone, or together with other selected enzymes in a variety of processes requiring the action of a xylan-degrading enzyme. Moreover, the fungal xylanases of the present invention, which generally have lower pH optima than xylanases of bacterial origin, are particularly well suited for use in industrial processes which are performed at low pH.

In accordance with the present invention, it has been found that the xylanases produced via the present invention may be used in the baking of breads. The incorporation of small amounts of xylanase to the flour imparts favorable characteristics to the dough and thus to the bread itself such as increased loaf better characteristics such as break and shred quality and crumb quality. volume and textural _20__ added to rich in When added to feeds Xylanases may also be animal feed compositions which are arabinoxylans and glucoxylans. (including silage) for monogastric animals (e.g. poultry or swine) which contain cereals such as barley, wheat, maize, rye or oats or cereal by—products such as wheat bran or maize bran, the enzyme significantly improves the break—down of plant cell walls which leads to better utilization of the plant nutrients by the animal. As a consequence, growth rate and/or feed conversion are improved. Moreover, Xylanases may be used to the reduce the viscosity of feeds containing xylans.

Xylanase may be added beforehand to the feed or silage if pre—soaking or wet diets are preferred. More advantageously, however, the Xylanases produced via the present invention when added to feed continue to hydrolyze xylans in the feed in vivo. Fungal Xylanases, which generally’ have lower pH optima, are capable of releasing important nutrients in acidic environments as the such stomach of the animal ingesting such xylanase—supplemented feed.

The Xylanases produced via the present invention are also effective in filtration and improving removing dissolved organic substances from the broth in processes wherein apple microbial biomass. distillery waste is bioconverted into Xylanases originating from filamentous fungi may be advantageously used in this process.

Also according to the present invention, glucose syrups having improved filterability and/or lower viscosity are produced from impure cereal starch by subjecting the impure starch first to the action of an a-amylase, then to fungal Xylanases produced via the present invention and finally" to a hydrolysis. Similarly, the Xylanases of the present invention may be used in beer brewing to improve the filterability of the wort.

Xylanases may also be used. to remove lignins from and thus facilitate bleaching by reducing the amount of chlorine needed in the preparation of paper products. kraft pulp In addition, the xylanases produced via the present invention may be used in other processes such as to increase yield in the preparation of fruit or vegetable juices, the enzymatic hydrolysis of sugar beet pulp, the resulting hydrolyzed fraction being capable of use in microorganism culture medium; of agricultural residues such as corn cobs, wheat—straw and ground nutshell; and of certain recyclable materials such as waste paper.

The following examples are provided so as to give those of ordinary skill in the art a complete disclosure and description of how to make and use the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, pH, etc.) but some experimental errors and deviation should be accounted for. Unless indicated otherwise, temperature is in degrees Celsius and pressure is at or near atmospheric.

EXAMPLE 1 Purification and characterization of Aspergillus tubigensis endo—xy1anase XYL A.

Example 1.1 Purification of Asperqillus tubiqensis endo—xylanase XYL A. approximately 35 ml which was then was ultrafiltrated on a Diaflo PM 10 filter in a 50 ml Amicon :module to remove salts.

The supernatant was then concentrated to a volume of ml and the retentate was washed twice with 25 ml 25 mM Tris—HCl buffer (pH 7.0). the retentate volume was brought to 25 ml.

After washing, This retentate was injected in 1 ml quantities onto a Syn Chropak. AX 300 column eluted in the following HPLC regime: (dimensions 10 x 250 mm) and elution rate: 2 ml/min. elution buffer A: 25 mM Tris-HCl pH 7.0 elution buffer B: 25 mM Tris—HCl pH 7.0 + 1 M NaCl elution gradient: time (min) %A %B O 99 1 12 97 3 80 20 50 50 50 70 O 100 90 O 100 95 99 1 Fractions of 1 ml each were collected. Detection of the eluted protein was performed by continuous measurement of the UV absorption at 280 nm. The elution profile is shown in Figure 2.

The fractions were tested for the presence of endo- xylanase activity by a spot test. This spot test consists of adding 12 ml citrate—phosphate buffer (prepared by mixing 900 ml 0.2 M Na2HPO, and 125 ml 0.5 M citric acid, followed by an adjustment of the pH of the solution to pH 5.6 using 0.5 M citric acid or 0.2 M Na2HPOfl containing 0.5% oat spelt xylan (Sigma) to 180 mg agar (Difco) and heating the mixture to 100 "C to dissolve the agar. After cooling to 60 "C, the agar mixture is poured evenly onto Agarose gel—bond film. Drops of the elution fractions are placed individually onto the film and incubated for 30 min. at 30 °C. If endo- xylanase activity is present, the location of the individual drops on the agar film is clear.

Total Xylanase activity in the collected fractions was quantitatively determined by amount of measuring the reducing sugars produced over a predetermined time period in the microassay as described by Leathers gt gl. (1984), using oat spelt xylan in 50 mM sodium acetate at pH 5.0 as a substrate. Activity units are also as defined by leathers (supra).

Exo—xylanase activity in the eluted fractions was determined by the method. described by Poutanen and Puls (1988), using p-nitro—pheny1—B—D—xylopyranoside (0.3 mM, Sigma) as a substrate at pH 5.0 and 30 °C.

The spot test revealed that the elution fractions corresponding to peaks B, F and K (see Figure 2) contain endo—xylanase activity. The total xylanase assay showed activity in the elution fractions of peaks B, F, H and K.

The elution fractions of peaks B and H were determined to contain exo-xylanase activity.

The elution fractions of peaks F (XYL2 protein) and K (XYL A protein) exchange were further purified by repeated ion chromatography. The endo—xylanases contained therein were characterized by SDS/PAGE (Moonen gg gl., 1982) and. Iso Electric Focussing (3.5 according to the manufacturer's instructions. The apparent molecular weight of endo—xylanase F, as determined by SD3- PAGE, was approximately 22 kDa: the apparent molecular weight of endo—xylanase K was approximately 24 kDa. The iso- electric point (IEP) of endo—xylanase F was approximately pH 4.0, while the IEP of endo—xylanase K was determined to be lower than pH 3.5.

Example 1.2 Amino acid sequencing of the N—terminus of Aspergillus tubigensis endo-xylanase XYL A.

Approximately 5 pg of endo-xylanase, purified as described in Example 1.1, was subjected to electrophoresis on a 12% SDS-polyacrylamide gel, followed by electroblotting onto 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 25 kDa is subjected to sequence analysis in a gas-phase sequenator (Eurosequence, Groningen). The following N-terminal sequence has been determined: 1 5 ’ 10 Ala—Gly—I1e—Asn—Tyr—Val—Gln—Asn-Tyr—Asn (Figure 3, Formula 1) However, roughly equal amounts of two other variants were also discovered wherein either a serine (Figure 8, position 1) or a glycine (Figure 8, position 3) were determined to be the N-terminal amino acid.

Example 1.3 Amino acid sequence determination of endo-proteinase Glu—C released peptides of endo—xylanase XYL A.

Approximately 260 pg of endo—xylanase, described in Example 1.1, purified as was dissolved in 110 pl of a solution containing 50 mM ammonium bicarbonate buffer pH 7.5 and 2 mg/ml SDS.

After heating the solution for three minutes at 100°C and cooling to room. temperature, endo~ proteinase Glu-C (Staphylococcus aureus protease V8) was added in an 18—fold molar excess. Protein digestion was performed for 20 minutes at room temperature, after which the reaction mixture was heated for three minutes at 100°C.

Approximately one—fifth. of the reaction xnixture was subjected to electrophoresis on a 15% SDS—polyacrylamide gel, followed by blotting onto Immobilon—P membrane (Millipore) according to the method described by Matsudaira (1987). Three fragments were observed with a molecular mass of 19, 16 and 4 kDa respectively. The two largest fragments (19 and 16 kDa) were used in gas—phase sequencing (Applied 470A protein Groningen). Membrane fragments containing 2-3 nmol of the Biosystems model se encer Eurose ence, I particular peptide were washed and subjected to sequence analysis, (1987). according to the program described by Amons The following N-terminal amino acid sequence has been determined from the 19 kDa fragment: 14 Tyr—Tyr-Ile—Val—Glu~Asp—Tyr~Gly— X -Tyr—Asn-Pro—Cys—(Ser) (Figure 4, Formula 2) The identity" of the amino acid at position 9 (X), could not be determined. At position 14, only a trace of Ser is found as indicated by brackets.

The following amino acid sequence has been determined from the N—terminus of the 16 kDa fragment: 14 Tyr—Tyr-I1e—Val-Glu—Asp—Tyr—Gly~(Ser)— X —Asn—Pro—Cys-Ser (Figure 4, Formula 3) The identity of amino acid (X) at position 10 could not be determined. The sequence found for this fragment is almost identical to the sequence of the 19 kDa fragment.

Both peptides share the same N—terminal sequence, which is not identical to the amino acid N—termina1 sequence determined for the intact protein (Example 1.2, Formula 1).

It has been determined that these two internal fragments correspond to the sequence beginning with position 79 as illustrated in Figure 8.

EXAMPLE 2 Construction of a genomic library of Aspergillus niger DSl6813 (CBS 323.90; tubigensis). strain later reclassified as A; Example 2.1 Isolation of DNA from Aspergillus niger DS16813 (CBS 323.90; later reclassified as A. tubigensis).

Fungal DNA was isolated via the procedure described by de Graaff et a1. (1988). Mycelium, grown overnight in liquid minimal medium (per 1000 ml: 6.0 g NaNO3; 1.5 g KH2PO,; 0.5 g MgSO,*HgO; 0.5 g KC1; 1 ml Visniac solution [Visniac and Santer, 1957: 10 g EDTA; 4.4 g ZnSO4*NgO; 1.0 g Mnclzwugo; 0.32 g CoC12-6H2O: 0.32 g cuso,-5H2o; 0.22 g (NH,)6Mo,02,-4H20; 1.47 g Caclz-ZHZO; 1.0 g Peso,-7H20; pH 4.0]; pH 6.0) supplemented with 0.2 % casamino acids and 0.5 % yeast extract, was harvested, washed with cold saline, frozen in liquid. nitrogen and stored at —80‘C. Nucleic acids were isolated by disrupting 0.5 g frozen mycelium using a microdismembrator (Braun). The mycelial powder obtained was extracted with freshly prepared extraction buffer.

The extraction buffer was prepared as follows: 1 ml tri—isopropylnaphtalene sulfonic acid (TNS) (20 mg/ml) was thoroughly mixed with 1 ml p—aminosalicylic acid (PAS) (120 mg/ml) and 0.5 nd.ES x RNB buffer (per 1000 ml: 121.10 g Tris; 73.04 g NaCl: 95.10 g EGTA; adjusted to pH 8.5 with Hcl) was added. After the addition of 1.5 ml phenol, the extraction buffer was equilibrated for 10 minutes at 55 °C.

The warm buffer was then added to the mycelial powder, and the suspension was thoroughly mixed for 1 minute using a vortex mixer. After the addition of 21 ml chloroform, the suspension was remixed for 1 min. After centrifugation at 104 x g for 10 min. using a Sorvall high speed centrifuge, the aqueous phase was extracted once more with an equal volume of phenol/chloroformx (1:1) and was then extracted twice with chloroform. DNA was isolated from the aqueous phase using the following procedure; the DNA was immediately precipitated with 2 volumes ethanol at room temperature and was subsequently collected by centrifugation using a Sorvall high speed centrifuge at 104 x g for 10 min., washed twice by redissolving the DNA in distilled, sterile water and precipitating it again with ethanol. RNA was removed by adding RNase A (20 g pg/ml) to the final solution.

Example 2 Partial digestion of Asperqillus tubiqensis DNA with Sau 3A and isolation of DNA fragments after agarose gel electrophoresis.

DNA (30 pg), isolated from Aspergillus niger DSl6813 (recently reclassified as A. tubigensis) as described in Example 2.1, was partially digested by incubation of the DNA with 0.1 U Sau 3A during 30 minutes at 37°C. The resulting fragments were size fractionated by electrophoresis on 0.4% agarose in TAE buffer containing 0.5 pg/ml ethidiumbromide.

Fragments of 14 kb to 22 kb in size, compared to fragments of bacteriophage lambda DNA. digested with fig; II (22.0, 13.3, 9.7, 2.4, 0.65 and 0.44 kb) as size markers, were recovered from the gel by cutting" the appropriate. region from the gel.

These fragments were recovered from the piece of agarose by electro—elution using ISCO cups. A dialysis both the and the small containers of this cup, the cup was filled with 0.005 x TAE (diluted from 50 x TAE stock solution (per 1000 ml): 242.0 g 57.1 ml glacial 100 ml 0.5 M EDTA; adjusted to pH 8.0 with HCl) and the piece of agarose was placed in the large container of the cup. Subsequently, the cup was placed in the electro—elution apparatus, with the large container in the cathode chamber containing TAE and membrane was mounted on large Tris: acetic acid; the small container at the anode chamber containing TAE/3 M NaCl. The fragments were electro—eluted at 100 V for a period of 2 hours. Afterwards, the cup was taken from the electro—elution apparatus and the buffer was removed from the large container, while the buffer was removed only from the upper part of the small container. The remaining buffer (200 pl) containing the DNA fragments was dialyzed in the cup against distilled water for a period. of 30 minutes.

Finally, the DNA was precipitated by the addition of 0.1 volume 3 M NaAc, pH 5.6 and 2 volumes cold (—20°C) ethanol.

The DNA was collected by centrifugation (Eppendorf centrifu- ge) for 30 min. at 14,000 x g. at 4°C. After removal of the DNA pellet was Speedvac vacuum centrifuge. Following ethanol precipitation, supernatant, the dried using a Savant the DNA was dissolved in 10 pl TE buffer (10 mM Tris—HCl pH 8.0; 1 mM EDTA; pH 8.0) and the concentration was determined by agarose electrophoresis, using lambda DNA with a known concentration as a reference and ethidiumbromide staining to detect the DNA.

Example 2.3 Cloning of Asperqillus tubiqensis DNA fragments into bacteriophage lambda EMBL 3.

Fragments obtained by partial digestion of genomic DNA, as Example 2.2 were bacteriophage lambda EMBL 3 Bam HI arms, The ligated DNA was packaged in vitro using Gigapack II Gold packaging extract (Stratagene) and plated on E. coli LE392 (Murray, 1977) using NZYCM medium (per 1000 ml: 10 g NZ amine; 5 g Nacl; 5 g yeast extract; 1 g casamino acids; 2 g Mgso,-7H20; pH 7.5; according to the manufacturer's instructions. for plates 12 g agar is added), The complete reaction described above was repeated once, using 3 pl genomic DNA fragments in a final volume of pl.

Example 2.4 Titration and amplification of the Aspergillus tubigensis genomic library.

Dilutions of the primary genomic library were made in SM buffer (per 1000 ml: 5.8 g NaCl; 2.0 g MgS04*HgO; 50 ml Tris—HCl; pH 7.5; 5 ml 20% gelatin) and plated on E.coli LE392 as a host as described by Maniatis et al. (1982, pp. 64) using NZYCM medium. After incubation overnight at 37°C, the resulting plaques were counted and the amount of phages was calculated. The first ligation and packaging resulted in about '7 x 104 pfu (plaque—forming ‘units), the second in about 4 X 105 pfu, resulting in a total of about 5 x 105 pfu. " pfu/ml. centrifugation at 4,000 g for 10 min.

This phage stock contained approximately EXAMPLE 3 Screening of the Asperqillus tubiqensis genomic library for the endo—xylanase A gene (Xln A) and isolation of the gene.

Example 3.1 RP-labelling of synthetic oligonucleotides.

The amino acid sequence derived in Example 1.2 (Formula 1) was used to synthesize oligonucleotide mixes corresponding 1x) the Neterminal amino acid sequence. The oligonucleotides were synthesized. by the phosphoramidite method, synthesizer. using an Applied Biosystems oligonucleotide The oligonucleotide mixes AB801 to AB806 (Figure 3) were mixed. in equal amounts, hereinafter referred to as oligonucleotide mix AB800, to give a final concentration of 37 pmol oligonucleotides per pl. This oligonucleotide mixture was labelled in a reaction mixture of the following composition: 37 pmol oligonucleotide mixture, 66 mM Tris-HC1 pH 7.6, 1 mM ATP, 10 mM MgCl2, 15 mM dithiothreitol, 200 pg/ml BSA, 34 pmol gamma—"P ATP (NEN, 6000 Ci/mMo1) and 30 U T1 polynucleotide kinase (BRL) in a final volume of 50 ul. The reaction mixture was incubated mM spermidine, for 60 min. at 37°C, after which the reaction was terminated by the addition of 4 pl 0.5 M EDTA: pH 8.0.

Oligonucleotide mixture AB1255, derived from the amino acid sequence obtained in Example 1.3 (Formulas 2 and 3) (Figure 4), was labelled via the same procedure as described above. The oligonucleotide mixtures were used in the screening of the genomic library (Example 3.2) and in Southern blot analysis (Example 3.4 and 3.5) without further purification.

Example 3.2 Screening of the Asperqillus tubiqensis genomic library for xln A gene.

To screen for the xln A gene in an Aspergillus tubigensis genomic library, 3 x 103 pfu per plate were plated in NZYCM top agarose containing 0.7% agarose (NZYCM medium plus 7 g agarose) on four 85 mm diameter NZYCM (1.2% agar) plates as described by Maniatis et gl. (1982, pp. 64).

E. coli LE392 were used as plating bacteria.

After incubation of the plates overnight at 37°C, two replicas of each plate were made on nitrocellulose filters (Schleicher and Schull BA85) as described by Maniatis gt gl. (1982, pp. 32o~321).

After baking the filters for 2 hours at 80°C, the filters were wetted and washed for 60 minutes at room temperature in 3 x SSC (diluted from 20 X SSC stock solution (per 1000 ml): 175.3 g Nacl; 107.1 g sodium citrate~5.5 H53; pH 7.0). The filters were prehybridized. at 65°C for two 6 X SSC (diluted from the 20 x SSC stock solution (see above)), 0.5 % SDS, 10 X Denhardt's solution (per 5000 ml: 10 g Ficoll- hours in a prehybridization buffer containing: ; 10 g polyvinylpyrrolidone; 10 g Bovine Serum Albumin (Pentax Fraction V)) and 100 pg/ml heat denatured herring After two prehybridization, the prehybridization buffer was replaced buffer identical to the prehybridization buffer, except that this buffer did not sperm DNA, but RP-labelled oligonucleotide mix AB800, prepared as described in Example sperm DNA (Boerhinger Mannheim). hours by hybridization which was contain herring contained 3.1. The filters were hybridized for 18 hours at an final temperature of 38°C, achieved by slow, controlled cooling from the initial temperature of 65°C.

After hybridization, the filters were first washed in 2 x SSC, after which the filters were washed in prewarmed hybridization buffer at 38°C for the same period of time.

Finally, the filters were washed for 30 minutes at 38°C in 6 x SSC, 0.05% sodium pyrophosphate. The air dried filters were taped onto a sheet of Whatman 3MM paper, keying marks were made with radioactive ink and the Whatman paper and filters were covered with Saran Wrap". Hybridizing plaques were identified by exposure of Kodak XAR X—ray film for 72 hours at —70°C using an intensifying screen.

Four of the oligonucleotide mixture hybridizing plaques, appearing in duplicate on the replica filters, were 1 to lambdaflm. Each positive plaque was removed from the plate using a Pasteur identified and were designated lambdafln pipette and the phages were eluted from the agar plug in 1 ml of SM buffer containing 20 ul chloroform, as described by Maniatis gt; al. (1982, p. 64). The phages obtained. were purified by repeating the procedure described above using filter replicas from plates containing 50-100 plaques of the isolated phages.

After purification, the phages were propagated by plating 55 X 103 phages on NZYCM medium. After incubation overnight at 37°C, confluent. plates were obtained, from which the phages were eluted by adding 5 ml SM buffer and storing" the plate for 2 hours at 4°C with intermittent shaking. After removal of the supernatant, the bacteria were removed from the solution by centrifugation at 4,000 X g for minutes at 4°C. (0.3%) was added to the supernatent and the number of pfu. was determined. These Chloroform phage stocks contained approximately 10w pfu/ml.

,E,>ia.I_rp_le 3 - 3 Isolation of DNA from bacteriophage lambda.

Each of the isolated phages lambdaflfl to lambdafifl were propagated as described in Example 3.2 using five plates for each of the phages. The phages were precipitated from the thus—obtained supernatant (25 ml) by addition of an equal volume of a solution containing 20% PEG~6000 (w/v) and 2 M Nacl, followed by thorough mixing and incubation on ice for collected by centrifugation at 14,000 x g at 4°C for 20 minutes. The supernatant was removed by aspiration, while the last traces minutes. The precipitated phages were of liquid were removed using a paper towel. The phages were carefully resuspended in 4 ml SM buffer and extracted once with chloroform.

Prior to extracting the DNA from the phage particles, DNA and RNA originating from the lysed bacteria were removed by incubation of the phage suspension with DNase I and RNase A (both 100 pg/ml) for 30 minutes at 37°C. The phage DNA was subsequently released from the phages by the addition of SDS and EDTA to a final concentration of 0.1% and mM respectively, followed by incubation at 65°C for 10 minutes.

Protein was removed from the solution by extracting twice alcohol (25:24:1). After separation of the phases by centrifugation (14,000 X g, 10 min.), the extracted once with an equal volume with an equal volume phenol/chloroform/isoamyl in an Eppendorf centrifuge aqueous phase was chloroform/isoamylalcohol (24:1). The phases were separated by centrifugation 14,000 X g, 10 after which the DNA was precipitated from the by the addition 0.1 perchlorate and 0.1 volume isopropanol and incubation on ice (Eppendorf centrifuge, minutes), aqueous phase volume 5 M sodium for 30 min. The DNA was recovered by centrifugation for 10 minutes at 4°C (14,000 X g). The supernatant was removed by aspiration, after which the DNA was resuspended in 400 pl TE buffer. The ethanol: DNA was once again precipitated Wlfll The DNA was centrifugation for 10 minutes at 4°C collected by (14,000 X g). The supernatant was removed by aspiration, the remaining pellet after" which the DNA. was resuspended in 125 pl TE buffer containing 0.1 pg/ml RNase A. This purification procedure resulted in the isolation of approximately 40-50 pg DNA from each phage. was briefly dried under vacuum, Example 3.4 Restriction analysis of xln A containing phages.

The isolated DNA of phages lambdaxm1 to lambdaxm, was analyzed by Southern analysis using the following restriction enzymes; BamHI; BqlII; EcoRI; HinDIII; Kpnl; following solutions; 3 pl DNA give a final volume of 50 pl. After digestion, the DNA was precipitated by the addition of 0.1 volume 3 M NaAc and 2 volumes ethanol. The DNA was collected by centrifugation for minutes 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 addition of :1 pl DNA loading buffer (0.25 % (w/V) bromophenol blue; 0.25 % (w/V) xylene cyanol; 15 % (w/V) Ficoll type 400 in Inc), the samples were incubated for 10 minutes at 65°C and rapidly cooled on ice. The samples were then loaded on a 0.6% agarose gel in 1 X TAE buffer. The DNA fragments were separated by electrophoresis at 25 V for 15-18 hours.

After electrophoresis, the DNA was denatured and transferred. to 21 nitrocellulose ‘membrane as described. by Maniatis et gl. (1982, pp. 383-386), followed by subsequent prehybridization and labelled described in hybridization using the AB8OO and AB1255 as Example 3.1 and. hybridization conditions as described in Example 3.2. The oligonucleotide mixes hybridization pattern for each oligonucleotide mixture was obtained by exposure of Kodak XAR—5 X-ray film for 18 hours at —70°C using an intensifying screen.

From the results, it was concluded that the DNA of all four isolated clones hybridized with the oligonucleotide mixture derived from the N—terminal amino acid sequence (mix AB800), as well as with the oligonucleotide mixture derived from the amino acid sequence obtained from the peptide isolated after S. aureus V8 digestion (AB1255). In all four clones, fragments originating from the same genomic region were found.

The restriction fragment patterns and the hybridization patterns were used to construct an approximate restriction map of the genomic region where the gig A gene is located (Figure 5).

Example 3.5 subcloning of the xln A gene. isolated from phage vector pUC9 dephosphorylated with alkaline minutes. The linearized vector‘ was isolated from a 0.6% agarose gel as described in Example 2.2. sterile water. 10 pl of the diluted mixture was used to transform E. coli JMIO1 (Yanisch-Perron gt gl., 1985) competent cells, prepared by the CM1, CM2 method as described in the Pharmacia Manual for the M13 cloning/sequencing system. E. coli JM101 containing plasmid pIMl00 was deposited at the Schimmelcultures, Baarn, The Netherlands on July 19, 1990 Centraal Bureau voor and was assigned the designation CBS 322.90.

A selection of six of the resulting colonies was grown overnight in LB medium (per 1000 ml: 10 g trypticase peptone (BBL); 5 g yeast extract (BBL); 10 g Nacl; 0.5 mM Tris—HCl; pH 7.5) containing 100 pg/ml ampicillin.

Plasmid DNA was isolated from the cultures by the alkaline lysis method as described by Maniatis et al. (1982, pp. 368—369). analysis, This plasmid DNA was used in restriction as described in Example 3.4 to select £1 clone harboring the desired plasmid. Plasmid DNA was isolated on a large scale from 500 ml cultures E. coli JM101 containing the plasmid pIM100 grown in LB medium containing 100 pg/ml A 1982, p.86) The plasmid was ethanol The yield was ampicillin (Maniatis gt_ al., purified by CsCl centrifugation, phenolized, precipitated and dissolved. in 400 pl TE. approximately 500 pg.

The plasmid pIM100 was further analyzed by restriction enzymes resulting in the restriction map shown in Figure 7.

The orientation of the gene, as indicated, was determined from hybridization experiments under conditions described in Example 3.2 using the oligonucleotide mixes AB8OO and ABl255 as probes.

EXAMPLE 4 Characterization of the Asperqillus tubiqensis xln A gene.

Example 4.1 Sequence determination of the A. tubigensis xln A gene The sequence of the Asperqillus tubigensis xln A gene, which comprises its promoter/regulation region, the structural gene and the termination region, was determined by subcloning fragments from pIM100 in Ml3mp18/mp19, in combination with the use of specific oligonucleotides as primers in the sequencing reactions.

For nucleotide sequence analysis, restriction fragments were isolated as described in Example 2.2 and were then cloned in bacteriophage M13 mp18/19 RF DNA ‘vectors (Messing, 1983: Norrander gt al., 1983), digested with the appropriate restriction enzymes. The nucleotide sequences Example 4.2 The A. tubigensis gin A gene The sequence obtained comprises 2054 bp, 949 bp in the ' non~coding region and 420 bp in the 3' non-coding region.

In the 5' upstream region, a putative TATA box (TATAAAT) was found at 854, before the (position. 950). A triplicate (5'GTCCATTTAGCCA3') was in the region 190 to 350 bp from the translation initiation site (positions; 618 to 632: 636 to 650; 656 to 670). position 848 to position translation initiation site repeating sequence found The structural section of the zlg A gene is 681 bp long and is interrupted by a single putative intron 48 bp long. The polypeptide derived from the sequence is 211 AA in length. A 17 AA long hydrophobic signal sequence is found at the N-terminus of this polypeptide, which is followed by a propeptide which is 12 residues long. The mature protein is 184 AA in size with an predicted molecular weight of 19 kDa and has a theoretical IEP of 3.6.

EXAMPLE 5 Expression of the xln A gene in an Aspergillus niger N593.

Example 5.1 Introduction of the xln A gene into Aspergillus niger N593 by co—transformation.

The plasmid pIM100, obtained in Example 3.5 was introduced in Asperqillus niqer by co—transformation of Aspergillus nigg; N593 (py;' mutant of A. niger N402; Goosen et al., 1987) using the Aspergillus giggr py; A gene as a selective marker on the plasmid pGW635 (Goosen gt al., 1989) and the plasmid pIM100 as the co—transforming plasmid.

Protoplasts were prepared from mycelium by growing Aspergillus giggr N593 on minimal medium supplemented with 0.5% yeast extract, 0.2% casamino acids, 50 mM glucose and mM uridine for 20 hours at 30°C. The preparation of protoplasts of Aspergillus Qiggr N593 and the transformation procedure was performed as described by Goosen gt al. (1987). The resulting PYR+ transformants were then analyzed for the expression of the xln A gene.

Example 5.2 Screening of transformants for the expression of the xln A gene.

The transformants obtained in filtrate was analyzed. by SDS—polyacrylamide gel electrophoresis, using a gel containing 12% acrylamide. The XYL A after protein was detected on nitrocellulose electroblotting and incubation with polyclonal antibodies raised against the XYI. A protein, which. was purified as described in Example 1.1. The antibody bound, was detected after incubation with goat—anti—rabbit antibody conjugated to alkaline phosphatase, according to the Biorad instruction manual.

Sixteen of the twenty transformants analyzed produced the XYL A protein as detected by this procedure. The protein was secreted into the medium. Of the transformants analyzed, transformant TrX9 was selected by I.E.F. analysis, using a pH gradient of pH 3 to 7 and. subsequent staining of a dilution series of transformants TrX2 and TrX9, using the method as described by Biely gt al. (1985 a and b).

Figure 9 is a zymogram exhibiting the XYL A protein expressed by transformants TrX2 and TrX9.

SDS-PAGE analysis was performed using 4 pl supernatant samples of individual transformants and the A. niger control strain, adjusted to pH 7 with 3 N NaOH and subsequently brought to a final volume of 20 pl with 1 X SB buffer, as described by Laemmli (1970). After heating for 5 100°C, total polyacrylamide gel first minutes at sos/12.5% mixtures were subjected to a electrophoresis and subsequently stained with coomassie brilliant blue. As shown in Figure 10 B, a protein band having an apparent molecular weight of 25 kDa (comparable with purified xylanase (lane 2) could be detected in transformants TrX2 (lane 4) and TrX9 (lane 5), which is absent from the supernatant of the control strain (lane 3). Molecular weight markers (lane 1) represent 92, 68, 46 and 30 kDa.

Example 5.3 Deletion analysis of the xln A promoter region tubiqensis xln A Regulatory elements in the A. promoter were studied by promoter deletion analysis. A series of five constructs of the xln A gene were made and cloned in combination with the A. niger py; A gene. The pyr A gene allows selection in transformations experiments as described in Example 5.1. In addition, the pyr A gene permits the selection of transformants having a single copy of the plasmid integrated at the pyr A locus.

The plasmids pIMl12, pIM113, pIM1l4, pIM116 and pIM117 were used to transform A. niger N593 as described in Example .1. The resulting PYR* transformants of each plasmid were cultivated and DNA was isolated as described in Example 2.1.

The resulting DNA was digested with fipal and single copy integrations were selected by Southern analysis using a y labeled 3.8 kb XbaI fragment P (labelled as described in Example 7.2), which contained the Q1; A gene as a probe.

Transformants having a hybridizing flpal fragment (the size of which was increased by a unit plasmid length as compared to the size of the flpgl fragment in A. niger N593) were selected as single copy integrations at the pyr A locus.

Single copy transformants of each of the plasmids, selected as described. above, were grown for 36 hours as described in Example 5.2. The expression of the xln A gene was analyzed by IEF analysis as described in Example 5.2 and of total RNA as described by de Graaff _e3:_ a_1. (1988), using the 32:» labelled by Northern analysis after isolation bp Xhgl/gamfll fragment of the xln A gene as a probe.

In transformants originating from the plasmids pIM1l2, pIM113 and pIMll4, expression of the gin A gene was found as detected by IEF and Northern analysis. from the However, the transformants originating plasmids pIM116 and pIMl17 did not express the x13 A gene, since neither XYL A protein nor hybridizing RNA were found. From these results it was concluded that the 158 bp ggtl/ghgl fragment, the essential difference between pIM114 and pIM116, contains an element necessary for the induction of the xln A gene in A; giggr, which were grown on medium using xylan as a carbon SOUICB .

EXAMPLE 6 Expression in A. niger of the xln A gene fused to the promoter and/or signal sequence of the A. niger amyloglucosidase (AG) gene.

Example 6.1 Xylanase expression vectors To obtain expression of xylanase in the strain A; gigg; CBS 513.88, additional expression cassettes (pXYL3 and pXYL3AG) were created in which the xln A gene is under the control of the A. niger amyloglucosidase (AG) promoter in combination with different signal sequences.

In expression cassette pXYL3, the AG-promoter sequence was fused. to the gin A encoding" sequence including the xylanase leader.

In the expression cassette pXYL3AG, the AG-promoter sequence, as well as the 18 amino acid (aa) leader sequence of the AG—gene were fused to the xln A gene fragment encoding solely the mature protein.

Example 6.2 Construction of intermediate plasmids. a) Subcloninq the X1nA locus.

To reduce the length of the genomic xln A locus, the 2 kb Pstl pIM1OO comprising the entire xln A gene including the 5'— and 3' fragment of (described in Example 3.5) flanking sequences, was subcloned. into the Pstl site of pTZ18R (Promega). The plasmid containing the xln A gene in the proper orientation (indicated in Figure 12) was designated pXYL1. b) Basic selection Vector pAmdSH To serve as a selection marker for the transformation of Aspergillus, the EcoRI/KpnI DNA fragment of plasmid pGW325 (Wernars, K. (1986)) containing the homologous Asperqillus nidulans amds gene, was inserted into the EcoRI/KpnI sites of pTZ18R (Promega). -In the resulting vector (pAmds), an additional HindIII restriction site was introduced by insertion of the synthetic fragment: ' AATTCAAGCTTG 3‘ 3‘ GTTCGAACTTAA 5' into the EcoRI-site. The designated. pAmdSH. thus-obtained plasmid was In this basic vector, the AG/xylanase fusion DNA fragments will be inserted. c) Isolation of the qenomic AG locus: QAB6-l.

Plasmid pAB6-1 contains the entire AG locus from A; construction of niger, isolated from an A. niger plasmid library containing the 13-15 kb HindIII fragments, inserted into pUC19.

For this oligonucleotides were used: isolation, the following AG-specific AG-1: 5'-GACAATGGCTACACCAGCACCGCAACGGACATTGTTTGGCCC-3' AG-2: 5'-AAGCAGCCATTGCCCGAAGCCGAT-3', both based on the nucleotide sequence published for A. niger (Boel, gt gl. (1984a); (1984b)). The derived from the Boel, et al. oligonucleotide probes were sequence surrounding intron 2: oligo AG—1 is located downstream this intron and has a polarity identical to the AGmRNA; oligo AG- is found upstream of intron 2 and is chosen antiparallel to the AGmRNA. Plasmid pAB6—1 contains the entire AG locus on a 14.5 kb HindIII fragment (see Figure 13). d) The intermediate plasmids pXYLAG and pXYL2AG.

Fusion of the AG-promoter and the 18 aa AG—leader se- quence to the 313 A gene encoding the mature protein (lacking the serine from position 1) was performed by the Polymerase Chain Reaction (PCR) method.

In the PCR reactions, two templates were used: pXYL1, containing the gln A gene and pAB6—1, containing the entire AG genomic locus.

As primers for the PCR DNA—amplifications, four synthetic oligo nucleotides were designed having the following sequence: Oligo AB 177125'-CTCTGCAGGAATTCAAGCTAG-3' (an AG specific sequence around the EQQRI site approx; 250 bp upstream from the ATG initiation codon).

Oligo AB 1985:5'-GTAGTTGATACCGGCACTTGCCAACCCTGTGCAGAC-3' mature xylanase -—-—i~—+- 18 aa AG~leader Oligo AB 1986:5'-GTCTGCACAGGGTTGGCAAGTGCCGGTATCAACTAC-3' 18 aa AG—leader -—i~—- mature xylanase Oligo AB 1984:5'-CCGGGATCCGATCATCACACC-3' (a gln A specific sequence located at the figmhl site on position 1701 as shown in Figure 8).

The PCR was performed as described by Saiki g; 1. (1988) and according to the supplier of TAQ~polymerase (Cetus). Twenty-five amplification cycles (each: 2 minutes at 55°C; 3 minutes at 72°C; 1 minute at 94°C) were carried out in a DNA—amplifier (Perkin—Elmer/Cetus).

To fuse the AG sequences to the gin A coding sequence, two separate polymerase chain reactions were performed: the pAB6~l as the template and oligonucleotides AB 1771 and AB 1985 as primers to amplify a 300 bp DNA fragment which contained the 3‘ piece of the AG- promoter and the 18 aa AG—leader sequence, flanked at the first reaction with ‘—border by the first 18 nucleotides of the coding sequence -44.. of gin A and the second reaction with pXYLl as the template and oligonucleotides AB 1986 and 1984 as primers to amplify Kim A DNA sequences encoding the mature xylanase protein, flanked at the 5’—border by the last 18 nucleotides of the AG-signal peptide. A schematic view of these amplifications is presented in Figure 14.

The two DNA fragments generated were purified by agarose gel electrophoresis and ethanol precipitation and subsequently used as templates in the third PCR with oligo nucleotides AB 1771 and 1984 as primers to generate the AG- xylanase fusion. The thus—obtained DNA fragment was digested with EQQRI and fiamfil and subcloned into the appropriate sites of pTZl8R. The resultant fusion was sequenced and designated pXYLAG (see Figures 14 and 15).

The remaining wherefrom the thus-obtained plasmid pXYL2AG is shown in Figure 15. e) The intermediate plasmids pXYL and pXYL2.

Fusion of the AG—promoter sequence to the gin A gene including the xylanase leader was performed as described in part d), above. As primers, two additional oligonucleotides were designed having the following sequence: Oligo AB 1982: 5'-AGCCGCAGTGACCTTCATTGCTGAGGTGTAATGATG'3' Xylanase gene ———-i*——- AG—promoter 01190 AB 1983! 5'-CATCATTACACCTCAGCAATGAAGGTCACTGCGGCT‘3' AG—promoter ———~L——*- Xylanase gene -45..

To fuse the AG promoter sequence to the xylanase gene (including the xylanase chain signal sequence), two separate performed: the first reaction with pAB6—1 as template and oligonucleotides AB 1771 and AB 1982 as primers to amplify a 282 bp fragment containing the 3'-part of the AG promoter flanked at the 3'- border by 18 nucleotides of the xylanase leader and the second with pXYLl as template and the oligonucleotides AB 1983 and AB 1984 as primers to amplify a DNA fragment containing the entire xylanase gene (including the xylanase leader) and flanked at. the 5'—border by 18 nucleotides of the AG—promoter.

The two polymerase reactions were reaction DNA fragments generated were purified by agarose gel electrophoresis and ethanol precipitation and subsequently used as templates in a third PCR with oligonucleotides AB 1771 and 1984 as primers to generate the AG~Xy1anase fusion. The thus~obtained DNA fragment was digen sted with EQQRI and fiamhl and subcloned into the appropriate sites of pTZl8R. The resultant fusion was sequenced and designated pXYL (see Figures 14 and 16). (3.5 kb) upstream region of the AG promoter was inserted into pXYLl as described in part d), above. The thus—obtained plasmid was designated pXYL2.

The remaining Exampleiéii Construction of the pXYL3AG and pXYL3. xylanase expression cassettes Both expression cassettes were created by insertion of the AG/xylanase fusions of pXYL2AC or pXYL2 into the basic A, niger vector pAmdSH. For this final construction, pAmdSH was digested with gpnl and fiindlll (partially) and pXYL2AG and pXYL2 with fiindxir with gpgl. All fragments were isolated and purified by gel electrophoresis and partially and ethanol precipitation. To the 6.8 kb Kpnl/HindIII DNA fragment of pAmdSH, either the 5.3 kb Kpnl/HindIII DNA of pXYL2AG or pXYL2 was added, subsequently molecular cloned by transferring both ligation fragment ligated and mixtures to E.coli. The thus—derived expression cassettes were designated pXYL3AG (containing the AG-leader) and pXYL3 (containing the xylanase leader), as shown in Figures 17 and 18, respectively.

Example 6.4 Expression of the xln A gene under the control of the AG promoter in A. niger. a) Transformation of A. niger (CBS 513.88).

Before transferring both expression cassettes pXYL3AG and pXYL3 to A. niger, the E. coli sequences were removed by HindIII ethanol digestion, gel electrophoresis and precipitation. Transformation of the strain A. niger (CBS 513.88, deposited October 10, 1988) was performed with 10 pg linearized DNA fragment by procedures as Tilburn, J. gt gl. described by (1983) and Kelly and Hynes (1985) with the following modifications: — Mycelium was minimal medium Aspergillus (Cove, D. (1966)) supplemented with 10 mM arginine and mM proline for 16 hours at 30%: in a rotary shaker grown on at 300 rpm.

— Only Novozym 234, and no helicase, was used for formation of protoplasts.

~ After 90 minutes of protoplast formation, 1 volume of STC buffer (1.2 M sorbitol, 10 mM Tris—HCl pH 7.5, 50 mM CaCl2) was added to the protoplast suspension and centrifuged. at 2500 rpnx at. 4°C for 10 Ininutes in a swing—out rotor. The protoplasts were washed and resuspended in STC—buffer at a concentration of 108 cells/ml.

— Plasmid DNA was added. in a volume of 10 pl in TE buffer (10 mM Tris—HCl pH 7.5, 0.1 mM EDTA) to 100 pl of the protoplast suspension.

— After incubation of the DNA-protoplast suspension at 0°C for 25 minutes, 200 pl PEG solution was added dropwise (25% PEG 4000 (Merck), 10 mM Tris—HCL pH 7.5, mM Caclz). Subsequently, 1 ml of PEG solution ( 60% PEG 4000 in 10 mM Tris-HCl pH 7.5, 50 mM CaCl?) was added slowly, with repeated mixing of the tubes. After incubation at room temperature, the suspensions were diluted with STC—buffer, centrifuged at 2000 rpm at 4%} for 10 minutes. The mixed by inversion and protoplasts were resuspended. gently in 200 pl STC- buffer and plated on Aspergillus minimal medium with mM acetamide as sole nitrogen source, 15 mM Cscl, I M sucrose, solidified with 0.75% bacteriological agar #1 (Oxoid). Growth. was performed at 33°C for 6-10 days. b) Growth of transformants in shake flasks.

Single A. niger transformants from each expression cassette were isolated, and the spores were streaked on selective acetamide-agar plates. Spores of each transformant were collected from cells grown for 3 days at 37°C on 0.4% (Oxoid, production was tested in shake flasks under the following potato—dextrose England) agar plates. Xylanase growth conditions: — About 1.108 spores were inoculated in 100 ml pre- 1 g KHZPO4; 30 g maltose; 5 g yeast—extract; 10 g casein—hydrolysate; 0.5 g MgSO,JNg0 and 3 g Tween 80. The pH was adjusted to 5.5. culture medium containing (per litre): - After growing overnight at 34 °C in a rotary shaker, 1 ml of the growing culture was inoculated in a 100 ml main- 2 g 1 dextrin (Maldex MDO3, Amylum); 12.5 g yeast—extract; 25 g casein—hydrolysate; 2 g Kgxr; 0.5 g MgSO,.7Hg3; 0.03 g Znclfi culture containing (per litre): 70 g malto- .02 g CaCl2: 0.05 g MnSO,.4 I50 and FeSO,. ‘The pH was adjusted to 5.6. The mycelium was grown for at least 140 hours. c) Analyses of transformants.

Xylanase analyses of individual transformants were by sDs— polyacrylamide gel electrophoresis stained with Coomassie Brilliant Blue A Remazolbrilliant blue R. performed by measuring the Xylanase activity; and by a zymogram stained with Xylan- Xylanase activities were determined as described by Leathers gt al. (1984), with some substrate concentration was increased from 1% to 5% oat xylan, dissolved in 100 mM NaAc at pH 3.5 and heated to modification. The °C for 10 minutes. In addition, enzyme reactions were carried out at 39°C instead of 30°C.

Xylanase measured in the production levels were supernatant of 6 day-shake flask fermentations of several, randomly chosen transformants obtained from each expression cassette. The results are shown in Table 1.

Table l Xylanase production of several A. niqer CBS 513.88 strains transformed with plasmids containing the xlnA gene under the control of the A. niqer AG-promoter in combination with different leaders.

Expression cassette Transformant # Xylanase activity (U/ml) .1 2400 pXYL3 1.2 1700 (AG—promoter/ 10 3600 xln—leader) 29 3500 pXYL3AG (AG~promoter/ 3.1 2400 AG—leader) A. niger CBS 513.88 (control strain) — O sDs—PAGE analysis was performed as follows: after 5 days of growth as described in part b) above, 4 pl supernatant samples from individual transformants and from the . er control strain were first adjusted to pH 7 with 3 N NaOH and subsequently brought to a final volume of 20 pl with 1 x SB buffer, as described by Laemmli (1970). After heating for 5 l00'C, total mixtures were subjected to a SDS/12.5% polyacrylamide gel electrophoresis and subsequently stained with coomassie brilliant blue. As shown in Figure 10 A, a proteirx band having" an apparent molecular weight of 25 kDa (lane 1) minutes at (comparable with purified could. be detected in transformants 10 (lane 4), 29 (lane 5) and 1.1 (lane 6). This protein band was absent from the supernatant of the control strain (lane 3). Molecular weight markers (lane 2) represent 94, 67, 43 , 20 and 14.5 kDa. xylanase Zymogram analysis was performed as follows. The same samples as used above were also applied two times to a native 8~25% PAA (BRL). Following electrophoresis, gel A was stained with Coomassie brillant blue (see Fioure 11A) Blue—xylan phast system gel and gel R with a Remazol Brillant overlay at pH 3.5 as described by (see Figure 11B), to visualize the supplied to this RBB—xylan overlay gel contained 5 times less protein as compared to the samples provided to the gels as shown in Figures 10 and 11A. The identification of the lanes in Figures 11 A and B Samples are the same as Figures 10 A and B.

These analyses clearly show the expression and secretion of active endo-xylanase in A, niger CBS 513.88 transformed with an expression cassette wherein the xln A gene is under the control of the A. nigcr amyloglucosidase promoter. Expression and secretion were also observed with different 5, njge; protein lacking the signal sequences. Furthermore, the serine residue from position 1 nevertheless retained xylan-deoradinc activity.

EXAMPLE 7 Application of the XYL A protein in animal feed compositions.

The efficacy’ of endo-xylanase supplementation to a diet rich in wheat by—products on nutrient digestibility and zootechnical performance is demonstrated using the following experimental protocol.

One—day old female chicks were housed in battery cages with wire floors and fed on a commercial starter diet until the start of the experimental period. At day 13, the birds were allocated at random to equal live—weight treatment groups. Three different experimental diets were assigned to 18 groups of birds with 8 birds per group. The diets were pelleted under very mild conditions and the chicks were fed ad libitum during days 13 to 34 (post—hatching).

Feed consumption and growth were monitored weekly for each cage. Digestibility was measured by a :3 day excreta collection period. A semi—quantitative collection of the excreta was performed. Using a marker (HCL—insoluble ash), individual digestibility coefficients for protein, fat, crude fibre and Nitrogen Free Extract were calculated. The (AME) of calculated from the following equation: apparent metabolisable energy each diet was AME (MJ/kg D.M.) = 17.46 a,-+ 38.81 a2-+ 8.0 as + 16.5 a, a1 = crude protein (gram per kg D.M.) X d.c."/ a2 = crude fat (gram per kg D.M.) x d.c. a3 = crude fibre (gram per kg D;M) x d.c. a, = Nitrogen Free Extract (gram per kg D.M.) X d.c.

U d.c. = digestibility coefficient The basal diet was based on wheat bran, maize starch and protein-rich animal by—products (Table 3). To this diet, endo—xylanase was supplemented at two different levels, ,000 U/kg and 174,000 U/kg (specific activity 300,000 U/g).

Table 3 Composition of the basal diet purified endo-xylanase Ingredient [o\° wheat bran maize starch maize 4.4 animal by—products (meat meal, La)-$5 00 fish meal, feather meal) 9.7 soy isolate (81% cp) 6.0 soy oil 6.0 fat blend 0.7 ground limestone 0.32 mono calcium phosphate 0.13 premix (including DL—methionine & Lysine - HCL) 1.35 SiO2—marker (Diamole) 1 Calculated content AME (MJ/kg) 12.66 crude protein, % 18.0 lysine, % 1.2 methionine + cystine, % 0.9 Analyzed content crude protein, % 18.2 crude fat, % 9.7 crude fibre,% 4.1 sioz - marker, % 1.07 Ca, % 0.8 P, % 0.85 ash, % 5.9 The most important results are summarized in Table 4 and Table 5 (digestibility data). The performance data refer to the entire experimental period, days 13 to 34 while vthe digestibility figures are the average Values derived from analyses in (performance data) (post-hatching), excreta collected during days 21 to 24 and days 28 to 31.

Table 4 The effect of endoxvlanase addition on chick performance from 13 to 34 days of aqe. diet 1 diet 2 diet 3 36.000 U/kg 174.000 U/kg Parameter basal endoxvlanase endoxylanase —growth (g per day) 50.5 50.80 51.9 —feed consumption (g/bird/day) 95.6 89.7 92.6 —feed: gain (gzg) 1.89 1.77 1.79 Table 5 The effect of endoxvlanase addition on diqestibility coefficients (d.c.) of the orqanic nutrients and calculated enerqy value of the diet.

Digestibility diet 1 diet 2 diet 3 36,000 174,000 coefficient zbasal units/kq XYL A units/kq XYL A crude protein‘, % 79 82 82 crude fat, % 85 91 82 crude fibre, % O 11 11 Nitrogen Free Extract, % 73 75 75 AME (MJ/kg D.M.) 13.81 14.28 14.

L/I The nitrogen measured in the excreta was corrected for the uric acid content in the urine.

This experiment (demonstrates the efficacy of endo- xylanase addition to feed compositions for broilers which Both the performance of the chicks and the energy value of the diets contain a large proportion of wheat bran. were affected positively.

Regarding the performance feed conversion, efficiency which reflected the influence of enzyme addition. There was a tendency towards a was the most sensitive parameter slightly reduced feed consumption in the enzyme supplemented groups associated with a similar or better growth.

Consequently, the feed: gain ratio was decreased substantially.

The improvement in performance can be explained by the effects from enzyme addition on the digestibility coefficients. All nutrient digestibility figures were affected positively, although the increase in fat which led to ‘a 3.5% increase in the energy value of the enzyme—supplemented digestibility was most pronounced, diets.

No dose—response relationship ‘was noticed at these levels of enzyme inclusion.

EXAMPLE 8 The use of endo—xylanase in breadmaking Pup—loaves were baked from 150 g dough pieces obtained by mixing 200 g wheat flour (100%), 106 ml water (53%), 1.2 g instant dry baker's yeast (0.6%: Gist—brocades N.V., Delft, The Netherlands), 4 g Nacl (2%), 400 mg caclzcngo (0.2%), 10 mg fungal SKB/kg flour) xylanase (xyl A) activity. After mixing for 6 minutes and 15 a—amylase Pmo (Gist—brocades, 2250 and a variable number of units of endo- seconds at 52 r.p.m. in a pin mixer, the dough was divided, proofed for 45 minutes at 31°C, punched, proofed- for an additional 25 minutes, molded. and panned. After‘ a final proof of 70 ndnutes at 31°C, the dough was baked for 20 minutes in an oven at 250°C. Loaf volume was determined by the rapeseed displacement method. The results are summarized in Table 6, below.

Table 6 Characteristics of bread prepared with various amounts of endo-xylanase (xvl A) activity Endo- xylanase Loaf activity volume Break{ Crumb * (units) (ml) Shred structure 0 546 6 6 32 560 7 6 128 579 7.5 7 320 609 8 6-5 640 621 7.5 6.5 960 624 7.5 7 2560 618 7.5 7.5 = Score from 1 (lowest quality) to 10 (highest quality) From these results, it is clear" that an increasing amount of endo—xylanase activity added to the dough leads to an increase in loaf volume and. an improvement. of bread quality in terms of break and shred and crumb structure.

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Claims (1)

1. A DNA sequence comprising a sequence encoding a polypeptide having fungal xylanase activity which is: (a) a DNA sequence encoding the xylanase amino acid sequence of