WO1996029416A1 - Aspergillus arabinofuranosidase - Google Patents

Abstract

An enzyme capable of degrading arabinoxylan is described. In addition, there is described a nucleotide sequence coding for the same and a promoter for controlling the expression of the same.

Description

ASPERGILLUS ARABINOFURANOSIDASE

The present invention relates to an enzyme. In addition, the present invention relates to a nucleotide sequence coding for the enzyme. Also, the present invention relates to a promoter, wherein the promoter can be used to control the expression of the nucleotide sequence coding for the enzyme.

In particular, the enzyme of the present invention is an arabinofuranosidase enzyme having arabinoxylan degrading activity.

It is known that it is desirable to direct expression of a gene of interest ("GOI") in certain tissues of an organism - such as a filamentous fungus (such as Aspergillus Niger) or even a plant crop. The resultant protein or enzyme may be useful for the organism itself. For example, it may be desirable to produce crop protein products with an optimised amino acid composition and so increase the nutritive value of a crop. For example, the crop may be made more useful as a feed.

In the alternative, it may be desirable to isolate the resultant protein or enzyme and then use the protein or enzyme to prepare, for example, food compositions. In this regard, the resultant protein or enzyme can be a component of the food composition or it can be used to prepare food compositions, including altering the characteristics or appearance of food compositions. It may even be desirable to use the organism, such as a filamentous fungus or a crop plant, to express non-plant genes, such as for the same purposes.

Also, it may be desirable to use an organism, such as a filamentous fungus or a crop plant, to express mammalian genes. Examples of the latter products include interferons, insulin, blood factors and plasminogen activators. It is also desirable to use microorganisms, such as filamentous fungi, to prepare products from GOIs by use of promoters that are active in the micro-organisms. Fruit and vegetable cell walls largely consist of polysaccharide, the major components being pectin, cellulose and xyloglucan (R.R. Selvendran and J.A. Robertson, IFR Report 1989). Numerous cell wall models have been proposed which attempt to incorporate the essential properties of strength and flexibility (P. Albersheim, Sci. Am. 232, 81-95, 1975; P. Albersheim, Plant Biochem. 3rd Edition (Bonner and Varner), Ac. Press, 1976; T. Hayashi, Ann. Rev. Plant Physiol. & Plant Mol. Biol. , 40, 139-168, 1989).

The composition of the plant cell wall is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising various β-xylan chains) and pectic substances (consisting of galacturonans and rhamnogalacturonans; arabinans; and galactans and arabinogalactans). From the standpoint of the food industry, the pectic substances, arabinans in particular, have become one of the most important constituents of plant cell walls (Whitaker, J.R. (1984) Enzyme Microb. Technol. , 6,341).

One form of plant polysaccharide is arabinan. A review of arabinans may be found in EP-A-0506190. According to this document, arabinans consist of a main chain of α-(1→5) groups linked to one another. Side chains are linked α-(1→3) or sometimes α-(1→2) to the main α-(1→5)-L-arabinan backbone. In apple, for example, one third of the total arabinose is present in the side chains. The molecular weight of arabinan is normally about 15 kDa.

Arabinans are degraded by enzymes collectively called arabinases. In this regard, arabinan-degrading activity is the ability of an enzyme to release arabinose residues, either monomers or oligomers, from arabinan backbones or from arabinan-containing side chains of other hemicellulose backbone structures such as arabinogalactans, or even the release of arabinose monomers via the cleavage of the 1-*6 linkage between the terminal arabinofuranosyl unit and the intermediate glucosyl unit of monoterpenyl α-L-arabinofuranosyl glucosides. The activity of the arabinan degrading enzymes of EP-A-0506190 include: a) the ability to cleave (1→2)-α-L-arabinosidic linkages; b) the ability to cleave (1→3)-α-L-arabinosidic linkages; c) the ability to cleave (1→5)-α-L-arabinosidic linkages; d) the ability to cleave the 1→6 linkage between the terminal arabinofuranosyl unit and the intermediate glucosyl unit of monoterpenyl α-L-arabinofuranosyl glucosides.

Arabinan-degrading enzymes are known to be produced by a variety of plants and microorganisms, among these, fungi such as those of the genera Aspergillus, Corticiwn, Rhodotorula (Kaji, A. (1984) Adv. Carbohydr. Chem. Biochem. , 42, 383), Dichotomitus (Brillouet et al. (1985) Carbohydrate Research, 144, 113), Ascomycetes and Basidomycetes (Sydow, G. (1977) DDR Patent Application No. 124,812).

Another plant polysaccharide is xylan, whose major monosaccharide unit is xylose. Xylans are abundant components of the hemicelluloses. In monocotyledonous plants the dominant hemicellulose is an arabinoxylan, in which arabinose side chains are attached to a backbone of xylose residues.

Arabinoxylans are carbohydrates found in the cell wall of cereals. A review of arabinoxylans and the enzymatic degradation thereof may be found in Voragen et al (1992 Characterisation of Cereal Arabinoxylans, Xylans and Xylanases pages 51-67, edited by J. Visser published by Elsevier Science Publishers).

Typically, arabinoxylans comprise a xylose backbone linked together via 0-1 ,4- bonds. The xylose backbone is substituted with L-arabinose residues which are linked via α-1 bonds to the 2 or 3 position of the xylose residues. The xylose residues can be single or double substituted. In addition to substitution with arabinose the xylose residues can be substituted with acetyl groups, glucuronic acid and various other carbohydrates. The arabinose residues can be further substituted with phenolic acids such as ferulic acid and coumaric acid. The degree and kind of substitution depends on the source of the particular arabinoxylan. Arabinoxylans are found in cereal cell wall where they are part of the secondary cell wall. Arabinoxylans form about 3 % of wheat flour - part of it is water soluble (WSP), pan of it is water insoluble (WIP). Despite the fact that the arabinoxylans amount to only about 3 % of wheat the importance of the arabinoxylan fraction is much higher. This is because the arabinoxylans of cereals act as hydrocoUoids, as they form a gel like structure with water. For example, the arabinoxylans of wheat flour bind up to 30% of the water in a dough despite the fact that they amount to only 3 % of the dry matter. When arabinoxylans bind water they increase the viscosity of the ground cereals and to such an extent that the cereals can become difficult to manage.

The rheological properties of several systems where ground cereals are used can be manipulated using enzymes that degrade arabinoxylans. In modern bakery it is advantageous to reduce the viscosity of the dough in order to reduce the energy needed to process the doughs and also to get a higher volume of the bread. This is usually achieved by using enzymes that can degrade the xylose backbone of arabinoxylans.

Enzymes that only cleave the arabinose side chains from the xylan backbone of arabinoxylan are, for the purposes of this application, collectively called arabinoxylan degrading enzymes.

In feeds based on cereals, arabinoxylans in the cereals can increase the viscosity of the fluids in the intestines of the animals after the feeds have been ingested. This is a problem as it causes discomfort, such as indigestion, to the animals. Also, the nutritive value of the feeds is reduced. These problems can be avoided by addition of enzymes that degrade the arabinoxylan (such as xylanases) to the feed to avoid indigestion and to increase the nutritive value of the feed. However, some enzymes that degrade the arabinoxylans (especially some of the xylanases) require the presence of unsubstituted backbones and so their activity can be limited. Further discussions on arabinoxylans can be found in Xylans and Xylanases (1992, edited by J. Visser published by Elsevier Science Publishers).

An arbinoxylan degrading enzyme is (1 ,4)-β-D-arabinoxylan arabinofuranohydrolase (AXH), as described by Kormelink et al 1991 (Kormelink, F.J.M. , Searle-Van Leeuwen M.J.F. , Wood. T.M. , Voragen, A.G.J.(1991) Purification and characterization of a (1 ,4)- β-D-arabinoxylan arabinofuranohydrolase from Aspergillus awamori. Appl. Micro-biol. Biotechnol. 25:753-758). However, this document provides no sequence data for the enzyme or the nucleotide sequence coding for same or for the promoter for the same.

Clearly, it would be useful to be able to degrade arabinoxylans, preferably by use of recombinant DNA techniques.

The present invention seeks to provide an enzyme having arabinoxylan degrading activity; preferably wherein the enzyme can be prepared in certain or specific cells or tissues, such as in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant.

Also, the present invention seeks to provide a GOI coding for the enzyme that can be expressed preferably in specific cells or tissues, such as in certain or specific cells or tissues, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant.

In addition, the present invention seeks to provide a promoter that is capable of directing expression of a GOI, such as a nucleotide sequence coding for the enzyme according to the present invention, preferably in certain specific cells or tissues, such as in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant. Preferably, the promoter is used in Aspergillus wherein the product encoded by the GOI is excreted from the host organism into the surrounding medium. Furthermore, the present invention seeks to provide constructs, vectors, plasmids, cells, tissues, organs and organisms comprising the GOI and/or the promoter, and methods of expressing the same, preferably in specific cells or tissues, such as expression in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, or even a plant.

According to a first aspect of the present invention there is provided an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 33,270 D ± 50 D; a pI value of about 3.7; arabinoxylan degrading activity; a pH optima of from about 2.5 to about 7.0 (more especially from about 3.3 to about 4.6, more especially about 4); a temperature optima of from about 40°C to about 60°C (more especially from about 45°C to about 55°C, more especially about 50°C); and wherein the enzyme is capable of cleaving arabinose from the xylose backbone of an arabinoxylan. According to a second aspect of the present invention there is provided an enzyme having the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof.

According to a third aspect of the present invention there is provided an enzyme coded by the nucleotide sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.

According to a fourth aspect of the present invention there is provided a nucleotide sequence coding for the enzyme according to the present invention. According to a fifth aspect of the present invention there is provided a nucleotide sequence having the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.

According to a sixth aspect of the present invention there is provided a promoter having the sequence shown as SEQ. I.D. No. 3 or a variant, homologue or fragment thereof or a sequence complementary thereto. According to a seventh aspect of the present invention there is provided a terminator having the nucleotide sequence shown as SEQ. I.D. No. 13 or a variant, homologue or fragment thereof or a sequence complementary thereto. According to an eighth aspect of the present invention there is provided a signal sequence having the nucleotide sequence shown as SEQ. I.D. No. 14 or a variant, homologue or fragment thereof or a sequence complementary thereto.

According to a ninth aspect of the present invention there is provided a process for expressing a GOI by use of a promoter, wherein the promoter is the promoter according to the present invention.

According to a tenth aspect of the present invention there is provided the use of an enzyme according to the present invention to degrade an arabinoxylan.

According to an eleventh aspect of the present invention there is provided a combination of enzymes to degrade an arabinoxylan, the combination comprising an enzyme according to the present invention and a xylanase. According to a twelfth aspect of the present invention there is provided plasmid NCIMB 40703, or a nucleotide sequence obtainable therefrom for expressing an enzyme capable of degrading arabinoxylan or for controlling the expression thereof or for controlling the expression of another GOI. According to a thirteenth aspect of the present invention there is provided a signal sequence having the sequence shown as SEQ. I.D. No. 15 or a variant, homologue or fragment thereof.

According to a fourteenth aspect of the present invention there is provided the use of the enzyme according to the present invention in the manufacture of a medicament or foodstuff to reduce or prevent indigestion and/or increase digestibility and/or increase nutrient absorption. According to a fifteenth aspect of the present invention there is provided an arabinofuranosidase enzyme having arabinoxylan degrading activity, which is immunologicaily reactive with an antibody raised against a purified arabinofuranosidase enzyme having the sequence shown as SEQ. I.D. No. 1.

According to a sixteenth aspect of the present invention there is provided an arabinofuranosidase promoter wherein the promoter is inducible by an intermediate in xylose metabolism. According to a seventeenth aspect of the present invention there is provided a process of reducing the viscosity of a branched substrate wherein the enzyme degrades the branches of the substrate but not the backbone of the substrate.

According to a further aspect of the present invention there is provided the use of the enzyme of the present invention as a viscosity modifier.

According to a further aspect of the present invention there is provided the use of the enzyme of the present invention to reduce the viscosity of pectin. Other aspects of the present invention include constructs, vectors, plasmids, cells, tissues, organs and transgenic organisms comprising the aforementioned aspects of the present invention.

Other aspects of the present invention include methods of expressing or allowing expression or transforming any one of the nucleotide sequence, the construct, the plasmid, the vector, the cell, the tissue, the organ or the organism, as well as the products thereof.

Additional aspects of the present invention include uses of the promoter for expressing GOIs in culture media such as a broth or in a transgenic organism. Further aspects of the present invention include uses of the enzyme for preparing or treating foodstuffs, including animal feed.

Preferably the enzyme is coded by the nucleotide sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.

Preferably the nucleotide sequence has the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto. Preferably the nucleotide sequence is operatively linked to a promoter.

Preferably the promoter comprises the sequence CCAAT.

Preferably the promoter is the promoter having the sequence shown as SEQ. I.D. No. 3 or a variant, homologue or fragment thereof or a sequence complementary thereto.

Preferably, the promoter comprises the 100 bps sequence from the Xma 111 to the BamH1 sites. Preferably the promoter of the present invention is operatively linked to a GOI.

Preferably the GOI comprises a nucleotide sequence according to the present invention.

Preferably the transgenic organism is a fungus.

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

Preferably the transgenic organism is a plant.

Preferably, in the use, the enzyme is used in combination with a xylanase, preferably an endoxylanase. Highly preferred embodiments of each of the aspects of the present invention do not include any one of the native enzyme, the native promoter or the native nucleotide sequence in its natural environment. Preferably, in any one of the plasmid, the vector such as an expression vector or a transformation vector, the cell, the tissue, the organ, the organism or the transgenic organism, the promoter is present in combination with at least one GOI.

Preferably the promoter and the GOI are stably incorporated within the transgenic organism's genome.

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

A highly preferred embodiment is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 33,270 D ± 50 D; a pi value of about 3.7; arabinoxylan degrading activity; a pH optima of from about 2.5 to about 7.0 (more especially from about 3.3 to about 4.6, more especially about 4); a temperature optima of from about 40°C to about 60°C (more especially from about 45°C to about 55°C, more especially about 50°C); and wherein the enzyme is capable of cleaving arabinose from the xylose backbone of an arabinoxylan; wherein the enzyme has the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof. Another highly preferred embodiment is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 33,270 D ± 50 D; a pi value of about 3.7; arabinoxylan degrading activity; a pH optima of from about 2.5 to about 7.0 (more especially from about 3.3 to about 4.6, more especially about 4); a temperature optima of from about 40°C to about 60°C (more especially from about 45°C to about 55°C, more especially about 50°C); and wherein the enzyme is capable of cleaving arabinose from the xylose backbone of an arabinoxylan; wherein the enzyme is coded by the nucleotide sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.

The advantages of the present invention are that it provides a means for preparing an arabinofuranosidase enzyme having arabinoxylan degrading activity and the nucleotide sequence coding for the same. In addition, it provides a promoter that can control the expression of that, or another, nucleotide sequence.

Other advantages are that the enzyme of the present invention can affect the viscosity of ground cereals, such as dough, to ease the handling thereof and for example to get a higher volume of the bread.

The enzyme of the present invention is also advantageous for feed because it degrades arabinoxylan and thus increases the nutritive value of the feed. In addition, it reduces the viscosity of the arabinoxylan in the intestine of the animals and so reduces or prevents indigestion.

The combination of the use of the enzyme of the present invention with a xylanase is particularly advantageous because the enzyme of the present invention and the xylanase have a surprising and unexpected synergistic effect with each other.

In this regard, the enzyme of the present invention increases the degradative effect of the xylanase, and the xylanase increases the degradative effect of the enzyme of the present invention. It is believed that the activity of the xylanase is increased because the enzyme of the present invention provides a polysaccharide substrate having fewer substituted groups.

The present invention therefore provides an enzyme having arabinoxylan degrading activity wherein the enzyme can be prepared in certain or specific cells or tissues, such as in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger. The enzyme may even be prepared in a plant. More in particular, the enzyme of the present invention is capable of specifically cleaving arabinose from the xylose backbone of arabinoxylan.

The arabinofuranosidase of the present invention is different from the arabinofuranosidases previously known. In this regard, the previous described arabinofuranosidases - such as those of EP-A-0506190 - are characterised by their ability to degrade unbranched arabinan, and are assayed using p-nitrophenyl-arabinoside.

The arabinofuranosidase of the present invention does not degrade unbranched arabinan, and only a minor activity is seen on nitrophenyl-arabinoside. In contrast, the arabinofuranosidase of the present invention is useful for degrading arabinoxylan. Therefore, the arabinofuranosidase of the present invention is quite different from the previous isolated arabinofuranosidases. Also, the present invention provides a GOI coding for the enzyme that can be expressed preferably in specific cells or tissues, such as in certain or specific cells or tissues, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger. The GOI may even be expressed in a plant. In addition, the present invention provides a promoter that is capable of directing expression of a GOI, such as a nucleotide sequence coding for the enzyme according to the present invention, preferably in certain specific cells or tissues, such as in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant. Preferably, the promoter is used in Aspergillus wherein the product encoded by the GOI is excreted from the host organism into the surrounding medium. The promoter may even be tailored (if necessary) to express a GOI in a plant.

The present invention also provides constructs, vectors, plasmids, cells, tissues, organs and organisms comprising the GOI and/or the promoter, and methods of expressing the same, preferably in specific cells or tissues, such as expression in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, or even a plant.

The terms "variant" , "homologue" or "fragment" in relation to the enzyme include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant amino acid sequence has arabinoxylan degrading activity, preferably having at least the same activity of the enzyme shown in the sequence listings (SEQ I.D. No. 1 or 12). In particular, the term "homologue" covers homology with respect to structure and/or function providing the resultant enzyme has arabinoxylan degrading activity. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85% , more preferably at least 90% homology to SEQ ID NO. 1 shown in the attached sequence listings. More preferably there is at least 95%, more preferably at least 98%, homology to SEQ ID NO. 1 shown in the attached sequence listings. The terms "variant", "homologue" or "fragment" in relation to the nucleotide sequence coding for the enzyme include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for an enzyme having arabinoxylan degrading activity, preferably having at least the same activity of the enzyme shown in the sequence listings (SEQ I.D. No. 2 or 12). In particular, the term "homologue" covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for an enzyme having arabinoxylan degrading activity. With respect to sequence homology, preferably there is at least 75 %, more preferably at least 85%, more preferably at least 90% homology to SEQ ID NO. 2 shown in the attached sequence listings. More preferably there is at least 95%, more preferably at least 98%, homology to SEQ ID NO. 2 shown in the attached sequence listings.

The terms "variant", "homologue" or "fragment" in relation to the promoter include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence has the ability to act as a promoter in an expression system - such as the transformed cell or the transgenic organism according to the present invention. In particular, the term "homologue" covers homology with respect to structure and/or function providing the resultant nucleotide sequence has the ability to act as a promoter. With respect to sequence homology, preferably there is at least 75 % , more preferably at least 85 % , more preferably at least 90% homology to SEQ ID NO. 3 shown in the attached sequence listings. More preferably there is at least 95% , more preferably at least 98%, homology to SEQ ID NO. 3 shown in the attached sequence listings.

The terms "variant" , "homologue" or "fragment" in relation to the terminator or signal nucleotide sequences include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence has the ability to act as a terminator or codes for an amino acid sequence that has the ability to act as a signal sequence respectively in an expression system - such as the transformed cell or the transgenic organism according to the present invention. In particular, the term "homologue" covers homology with respect to structure and/or function providing the resultant nucleotide sequence has the ability to act as or code for a terminator or signal respectively. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85 %, more preferably at least 90% homology to SEQ ID NO.s 13 and 14 (respectively) shown in the attached sequence listings. More preferably there is at least 95 % , more preferably at least 98%, homology to SEQ ID NO.s 13 and 14 (respectively) shown in the attached sequence listings.

The terms "variant" , "homologue" or "fragment" in relation to the signal amino acid sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant sequence has the ability to act as a signal sequence in an expression system - such as the transformed cell or the transgenic organism according to the present invention. In particular, the term "homologue" covers homology with respect to structure and/or function providing the resultant nucleotide sequence has the ability to act as or code for a signal respectively. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to SEQ ID NO 15 shown in the attached sequence listings. More preferably there is at least 95 %, more preferably at least 98%, homology to SEQ ID NO 15 shown in the attached sequence listings.

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

The term "complementary" means that the present invention also covers nucleotide sequences that can hybridise to the nucleotide sequences of the coding sequence or the promoter sequence, respectively. The term "nucleotide" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence of the present invention.

The term "construct" - which is synonymous with terms such as "conjugate" , "cassette" and "hybrid" - includes a GOI directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron or the ADH intron, intermediate the promoter and the GOI. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In each case, it is highly preferred that the terms do not cover the natural combination of the gene coding for the enzyme ordinarily associated with the wild type gene promoter and when they are both in their natural environment. A highly preferred embodiment is the or a GOI being operatively linked to a or the promoter. The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or plants, preferably cereals, such as maize, rice, barley etc. , into which it has been transferred. Various markers exist which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin. The term "vector" includes expression vectors and transformation vectors.

The term "expression vector" means a construct capable of in vivo or in vitro expression. The term "transformation vector" means a construct capable of being transferred from one species to another - such as from an E.coli plasmid to a filamentous fungus, preferably of the genus Aspergillus. It may even be a construct capable of being transferred from an E.coli plasmid to an Agrobacterium to a plant. The term "tissue" includes tissue per se and organ.

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

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

The term "transgenic organism" in relation to the present invention includes any organism that comprises the promoter according to the present invention and/or the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, wherein the promoter can allow expression of a GOI and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism. Preferably the promoter and/or the nucleotide sequence is (are) incorporated in the genome of the organism. Preferably the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger. Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the promoter according to the present invention, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention or the products thereof. For example the transgenic organism can comprise a GOI, preferably an exogenous nucleotide sequence, under the control of the promoter according to the present invention. The transgenic organism can also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a promoter, which may be the promoter according to the present invention.

In a highly preferred embodiment, the transgenic organism does not comprise the combination of the promoter according to the present invention and the nucleotide sequence coding for the enzyme according to the present invention, wherein both the promoter and the nucleotide sequence are native to that organism and are in their natural environment. Thus, in these highly preferred embodiments, the present invention does not cover the native nucleotide coding sequence according to the present invention in its natural environment when it is under the control of its native promoter which is also in its natural environment. In addition, in this highly preferred embodiment, the present invention does not cover the native enzyme according to the present invention when it is in its natural environment and when it has been expressed by its native nucleotide coding sequence which is also in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment.

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

In one aspect, the promoter of the present invention is capable of expressing a GOI, which can be the nucleotide sequence coding for the enzyme of the present invention. In another aspect, the nucleotide sequence according to the present invention is under the control of a promoter that allows expression of the nucleotide sequence. In this regard, the promoter need not necessarily be the same promoter as that of the present invention. In this aspect, the promoter may be a cell or tissue specific promoter. If, for example, the organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of stem, sprout, root and leaf tissues.

By way of example, the promoter for the nucleotide sequence of the present invention can be the α-Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 promoter or the α-Amy 637 promoter) as described in our co-pending UK patent application No. 9421292.5 filed 21 October 1994. That promoter comprises the sequence shown in Figure 1.

Alternatively, the promoter for the nucleotide sequence of the present invention can be the α-Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the α-Amy 351 promoter) as described in our co-pending UK patent application No. 9421286.7 filed 21 October 1994. That promoter comprises the sequence shown in Figure 2. Preferably, the promoter is the promoter of the present invention.

In addition to the nucleotide sequences described above, the promoters, particularly that of the present invention, could additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a Pribnow Box or a TATA box. The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the GOI. For example, suitable other sequences include the Shl-intron or an ADH intron. Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.

Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' signal sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).

In addition the present invention also encompasses combinations of promoters and/or nucleotide sequences coding for proteins or enzymes and/or elements. For example, the present invention encompasses the combination of a promoter according to the present invention operatively linked to a GOI, which could be a nucleotide sequence according to the present invention, and another promoter such as a tissue specific promoter operatively linked to the same or a different GOI. The present invention also encompasses the use of promoters to express a nucleotide sequence coding for the enzyme according to the present invention, wherein a part of the promoter is inactivated but wherein the promoter can still function as a promoter. Partial inactivation of a promoter in some instances is advantageous. In particular, with the Amy 351 promoter mentioned earlier it is possible to inactivate a part of it so that the partially inactivated promoter expresses GOIs in a more specific manner such as in just one specific tissue type or organ.

The term "inactivated" means partial inactivation in the sense that the expression pattern of the promoter is modified but wherein the partially inactivated promoter still functions as a promoter. However, as mentioned above, the modified promoter is capable of expressing a GOI in at least one (but not all) specific tissue of the original promoter. One such promoter is the Amy 351 promoter described above. Examples of partial inactivation include altering the folding pattern of the promoter sequence, or binding species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognised by, for example, RNA polymerase. Another, and preferable, way of partially inactivating the promoter is to truncate it to form fragments thereof. Another way would be to mutate at least a part of the sequence so that the RNA polymerase can not bind to that part or another part. Another modification is to mutate the binding sites for regulatory proteins for example the CreA protein known from filamentous fungi to exert carbon catabolite repression, and thus abolish the catabolite repression of the native promoter. The term "GOI" with reference to the present invention means any gene of interest. A GOI can be any nucleotide that is either foreign or natural to the organism (e.g. filamentous fungus, preferably of the genus Aspergillus, or a plant) in question. Typical examples of a GOI include genes encoding for proteins and enzymes that modify metabolic and catabolic processes. The GOI may code for an agent for introducing or increasing pathogen resistance. The GOI may even be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues. The GOI may even code for a non- natural protein of a filamentous fungus, preferably of the genus Aspergillus, or a compound that is of benefit to animals or humans. For example, the GOI could code for a pharmaceutically active protein or enzyme such as any one of the therapeutic compounds insulin, interferon, human serum albumin, human growth factor and blood clotting factors. In this regard, the transformed cell or organism could prepare acceptable quantities of the desired compound which would be easily retrievable from, the cell or organism. The GOI may even be a protein giving nutritional value to a food or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non-transgenic plant). The GOI may even code for an enzyme that can be used in food processing such as chymosin, thaumatin and α-galactosidase. The GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for patatin or α-amylase, ADP-glucose pyrophosphorylase (e.g. see EP-A-0455316), a protease antisense or a glucanase.

The GOI can be the nucleotide sequence coding for the α-amylase enzyme which is the subject of our co-pending UK patent application 9413439.2 filed on 4 July 1994, the sequence of which is shown in Figure 3. The GOI can be the nucleotide sequence coding for the α-amylase enzyme which is the subject of our co-pending UK patent application 9421290.9 filed on 21 October 1994, the sequence of which is shown in Figure 4. The GOI can be any of the nucleotide sequences coding for the ADP-glucose pyrophosphorylase enzymes which are the subject of our co-pending PCT patent application PCT/EP94/01082 filed 7 April 1994, the sequences of which are shown in Figures 5 and 6. The GOI can be any of the nucleotide sequences coding for the α-glucan lyase enzyme which are described in our co-pending PCT patent application PCT/EP94/03397 filed 15 October 1994, the sequences of which are shown in Figures 7-10.

In one preferred embodiment, the GOI is a nucleotide sequence coding for the enzyme according to the present invention.

As mentioned above, a preferred host organism is of the genus Aspergillus, such as Aspergillus niger. The transgenic Aspergillus according to the present invention can be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357- 393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In: Martinelli S.D., Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology voi 29. Elsevier Amsterdam 1994. pp 525-560), Ballance,D.J. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure. In :Leong,S.A. , Berka R.M. (Editors) Molecular Industrial Mycology. Systems and Applications for Filamentous Fungi. Marcel Dekker Inc. New York 1991. pp 1-29) and Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S.D. , Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666). However, the following commentary provides a summary of those teachings for producing transgenic Aspergillus according to the present invention.

Filamentous fungi have during almost a century been widely used in industry for production of organic compounds and enzymes. Traditional japanese koji and soy fermentations have used Aspergillus sp for hundreds of years. In this century Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry. There are two major reasons for that filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracellular products, for example enzymes and organic compounds such as antibiotics or organic acids. Second filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc. The same reasons have made filamentous fungi attractive organisms as hosts for heterologous expression according to the present invention.

In order to prepare the transgenic Aspergillus, expression constructs are prepared by inserting a GOI (such as an amylase or SEQ. I.D. No. 2) into a construct designed for expression in filamentous fungi.

Several types of constructs used for heterologous expression have been developed. The constructs contain the promoter according to the present invention (or if desired another promoter if the GOI codes for the enzyme according to the present invention) which is active in fungi. Examples of promoters other than that of the present invention include a fungal promoter for a highly expressed extracellular enzyme, such as the glucoamylase promoter or the α-amylase promoter. The GOI can be fused to a signal sequence (such as that of the present invention or another suitable sequence) which directs the protein encoded by the GOI to be secreted. Usually a signal sequence of fungal origin is used, such as diat of the present invention. A terminator active in fungi ends the expression system, such as that of the present invention.

Another type of expression system has been developed in fungi where the GOI is fused to a smaller or a larger part of a fungal gene encoding a stable protein. This can stabilize the protein encoded by the GOI. In such a system a cleavage site, recognized by a specific protease, can be introduced between the fungal protein and the protein encoded by the GOI, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein encoded by the GOI ("POI"). By way of example, one can introduce a site which is recognized by a KEX-2 like peptidase found in at least some Aspergilli. Such a fusion leads to cleavage in vivo resulting in protection of the POI and production of POI and not a larger fusion protein. Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins. The proteins can be deposited intracellularly if the GOI is not fused to a signal sequence. Such proteins will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins. If the GOI is equipped with a signal sequence the protein will accumulate extracellulary.

With regard to product stability and host strain modifications, some heterologous proteins are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracellular proteases which degrade heterologous proteins. To avoid this problem special fungal strains with reduced protease production have been used as host for heterologous production.

For the transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi (Ballance 1991, ibid). Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca²⁺ ions. The transformed protoplasts then regenerate and the transformed fungi are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A very common used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source. Even though the enzyme, the nucleotide sequence coding for same and the promoter of the present invention are not disclosed in EP-B-0470145 and CA-A-2006454, those two documents do provide some useful background commentary on the types of techniques that may be employed to prepare transgenic plants according to the present invention. Some of these background teachings are now included in the following commentary.

The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.

Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/ April 1994 17-27).

Thus, in one aspect, the present invention relates to a vector system which carries a promoter or nucleotide sequence or construct according to the present invention and which is capable of introducing the promoter or nucleotide sequence or construct into the genome of an organism, such as a plant.

The vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system. Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.

One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81 , 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds. : D.S. Ingrams and J.P. Helgeson, 203-208. Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above. A non-limiting example of such a Ti plasmid is pGV3850.

The promoter or nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential for insertion of modified T-DNA into the plant genome.

As will be understood from the above explanation, if the organism is a plant, then the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border pan of a T-DNA sequence, the border part being located on the same vector as the genetic construct.

Furthermore, the vector system is preferably an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.

In the construction of a transgenic plant the promoter or nucleotide sequence or construct of the present invention may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant. An example of a useful microorganism is E. coli, but other microorganisms having the above properties may be used. When a vector of a vector system as defined above has been constructed in E. coli, it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens. The Ti-plasmid harbouring the promoter or nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the promoter or nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified. As reported in CA-A-2006454, a large amount of cloning vectors are available which contain a replication system in E. coli and a marker which allows a selection of the transformed cells. The vectors contain for example pBR 322, pUC series, M13 mp series, pACYC 184 etc. In this way, the nucleotide or construct or promoter of the present invention can be introduced into a suitable restriction position in the vector. The contained plasmid is used for the transformation in E. coli. The E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered. As a method of analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.

After each introduction method of the desired promoter or construct or nucleotide sequence according to the present invention in the plants the presence and/or insertion of further DNA sequences may be necessary. If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected. The use of T-DNA for the transformatron of plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B. , Alblasserdam, 1985, Chapter V; Fraley, et al. , Crit. Rev. Plant Sci. , 4: 1-46; and An et al. , EMBO J. (1985) 4:277-284.

Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds. : D.S. Ingrams and J.P. Helgeson, 203-208. For further teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). With this technique, infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.

Typically, with direct infection of plant tissues by Agrobacterium carrying the promoter and/or the GOI, a plant to be infected is wounded, e.g. by cutting the plant widi a razor or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobacterium. The inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants. When plant cells are constructed, these cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.

Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.

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

In summation, the present invention provides an arabinofuranosidase enzyme having arabinoxylan degrading activity and the nucleotide sequence coding for the same. In addition, it provides a promoter that can control the expression of that, or another, nucleotide sequence. In addition it includes terminator and signal sequences for the same. The following sample was deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1RY on 16 January 1995: E.coli containing plasmid pB53.1 {i.e. E.coli DH5α- pB53.1} . The deposit number is NCIMB 40703.

The present invention will now be described by way of example. In the following Examples reference is made to the accompanying figures in which: Figures 1-10 are sequences of promoters and GOIs of earlier patent applications that are useful for use with the aspects of the present invention;

Figure 11 is a plasmid map of the plasmid pB53.1 , which is the subject of deposit NCIMB 40703;

Figure 12 is a schematic diagram of deletions made to the promoter of the present invention; Figure 13 is a plasmid map of pXP-AMY;

Figure 14 is a plasmid map of pXP-XssAMY;

Figure 15 is a graph;

Figure 16 is an HP-TLC profile;

Figure 17 is an HP-TLC profile; Figure 18 is an HPLC profile;

Figure 19 is a viscosity plot;

Figure 20 is an activity plot;

Figure 21 is an activity plot; and

Figure 22 is an activity plot. The following text discusses the use of inter alia recombinant DNA techniques. General teachings of recombinant DNA techniques may be found in Sambrook, J. , Fritsch, E.F. , Maniatis T. (Editors) Molecular Cloning. A laboratory manual. Second edition. Cold Spring Harbour Laboratory Press. New York 1989.

In these Examples, the enzyme of the present invention is sometimes referred to as AbfC. In addition, the promoter of the present invention is sometimes referred to as the AbfC promoter.

Purification of the arabinofuranosidase

Aspergillus niger 3M43 was grown in medium containing wheat bran and beet pulp. The fermentation broth was separated from the solid part of the broth by filtration. Concentrated fermentation broth was loaded on a 25X 100mm Q-SEPHAROSE (Pharmacia) high Performance column, equilibrated with 20 mM Tris, HCl pH 7.5, and a linear gradient from 0-500 Mm NaCl was performed and fractions of the eluate was collected. The Arabinofuranosidase was eluted at 130-150 Mm NaCl.

The fractions containing the arabinofuranosidase were combined and desalted using a 50×200 mm G-25 SEPHAROSE Superfine (Pharmacia). The column was eluted with distilled water. After desalting the enzyme was concentrated using High-Trap spin columns. Next the concentrated and desalted fractions were subjected to gel filtration on a 50×600 mm SUPERDEX 50 column. The sample was loaded and the column was eluted widi 0.2 M Phosphate buffer pH 7.0 plus 0.2 M NaCl, and fractions of the eluate were collected. The fractions containing arabinofuranosidase were combined and desalted and concentrated as described above. The combined fractions were loaded on a 16X100 mm Phenylsepharose High Performance column (Pharmacia), equilibrated with 50 mM Phosphate buffer pH 6.0, containing 1.5 M (NH₄)₂SO₄. A gradient where the (NH₄)₂SO₄ concentration was varied from 1.5 - 0 M was applied and the eluate collected in fractions. The fractions containing Arabinofuranosidase were combined. The purity of the arabinofuranosidase was evaluated by SDS-PAGE using the Phast system gel (Pharmacia). Characterization

The molecular weight of the purified arabinofuranosidase was determined by mass spectrometry using laser desorption technology. The MW of the arabinofuranosidase was found to be 33,270 D ± 50 D.

The pi value was determined by use of a Broad pi Kit (Pharmacia). The arabinofuranosidase has a pI value of about 3.7. After SDS-PAGE analysis, treatment PAS reagent showed that the arabinofuranosidase was glycosylated. The PAS staining was done according to the procedure of I. Van-Seuningen and M. Davril (1992) Electrophoresis 13 pp 97-99.

Activity Studies Activity of AbfC as a function of water soluble pentosan (WSP) concentrations (mg/ml) was determined. The results are shown in Figure 21. The results show that AbfC activity reached maximum at substrate concentration of 8 mg/ml WSP. pH Activity Studies

The effect of pH on the activity of the arabinofuranosidase of the present invention was investigated using water soluble pentosan (10 mg/ml) from wheat as a substrate in 50 mM citric acid sodium phosphate buffer. The incubation time was 15 minutes. The arabinofuranosidase of the present invention was observed to have a wide pH optima range of from about 2.5 to about 7.0 (see Figure 20), more especially from about 3.3 to about 4.6, more especially about 4.

Temperature Activity Studies The effect of temperature on the activity of the arabinofuranosidase of the present invention was investigated using water soluble pentosan (10 mg/ml) from wheat as a substrate in 50 mM sodium acetate at a pH of 5.0. The incubation time was 15 minutes. The arabinofuranosidase of the present invention was observed to have an optimal activity at a temperature of from about 40°C to about 60°C, more especially from about 45°C to about 55°C, more especially about 50°C (Figure 22). The enzyme is still active at about 10°C and showed residual activity at 70°C and 80°C.

Amino acid sequencing of the arabinofuranosidase

The enzyme was digested with endoproteinase Lys-C sequencing grade from Boehringer Mannheim using a modification of the method described by Stone & Williams 1993 (Stone, K.L. and Williams, K.R. (1993). Enzymatic digestion of Proteins and HPLC Peptide Isolation. In : Matsudaira P. (Editor). A practical Guide to Protein and Peptide Purification for Microsequencing. Second Edition. Academic Press, San Diego 1993. pp 45-73). Freeze dried β-arabinofuranosidase (0.4 mg) was dissolved in 50 μl of 8M urea, 0.4 M NH₄HCO₃, pH 8.4. After overlay with N₂ and addition of 5 μl of 45 Mm DTT, the protein was denatured and reduced for 15 min at 50°C under N₂. After cooling to RT, 5 μl of 100 Mm iodoacetamide was added for the cysteines to be derivatised for 15 min at RT in the dark under N₂. Subsequently, 90 μl of water and 5 μg of endoproteinase Lys-C in 50 μl of 50 Mm Tricine and 10 mM EDTA, pH 8.0, was added and the digestion was carried out for 24h at 37°C under N₂. The resulting peptides were separated by reversed phase HPLC on a VYDAC C18 column (0.46 × 15 cm; 10 μm; The Separations Group; California) using solvent A: 0.1 % TFA in water and solvent B: 0.1 % TFA in acetonitrile. Selected peptides were rechromatographed on a Develosil C18 column (0.46 × 10 cm; 3μm) using the same solvent system prior to sequencing on an Applied Biosystems 476A sequencer using pulsed-liquid fast cycles.

The following peptide sequences were found: SEQ I.D. No. 4

SEQ I.D. No. 5

SEQ I.D. No. 6 SEQ I.D. No. 7

SEQ I.D. No. 8

Isolation of a PCR clone of a fragment of the gene

PCR primers were synthesised using an Applied Biosystems DNA synthesiser model 392. In this regard, PCR primers were synthesized from one of the found peptide sequences, namely SEQ ID No. 5. The primers were: One primer from EMTAQA (reversed)

One primer from MIVEAIG

PCR amplification was performed with 100 pmol of each of these primers in 100 μl reactions using Amplitaq polymerase (PERKIN ELMER). The following program was:

Steps 2-4 were repeated for 40 cycles. PCR reactions were performed on a PERKIN ELMER DNA Thermal Cycler.

A 100 bp amplified fragment was isolated and cloned into a pT7-Blue T-vector, according to the manufacturers instructions (Novagen).

Isolation of A. niger genomic DNA lg. of frozen A. niger mycelium was ground in a mortar under liquid nitrogen. Following evaporation of the nitrogen cover, the ground mycelium was extracted with 15ml of an extraction buffer (100mM Tris-Hcl, pH 8.0, O.50mM EDTA, 500mM NaCl, 10mM β-mercaptoethanol) containing 1ml 20% sodium dodecyl sulphate. After incubation at 65°C for 10 min. 5ml 5M KAc. pH 5.0, was added and the mixture further incubated, after mixing, on ice for 20 mins. After extraction, the mixture was centrifuged for 20 mins. and the supernatant mixed with 0.6 vol. isopropanol to precipitate the extracted DNA. After further centrifugation for 15 mins. the DNA pellet was dissolved in 0.7 ml TE (10mM Tris, HCl pH 8.0, ImM EDTA) and precipitated with 75 μl 3M NaAc, pH 4.8, and 500 μl isopropanol.

After centrifugation the pellet was washed with 70% ETOH and dried under vacuum. The DNA was dissolved in 200 μl TE and stored at -20°C.

Construction of a library

20 μg genomic DNA was partly digested with Tsp509I, which gives ends which are compatible with EcoRI ends. The digested DNA was separated on a 1 % agarose gel and fragments of 4-10 kb was purified. A λZAPII Ec oRI/CIAP kit from Stratagene was used for library construction according to the manufacturers instructions. 2 μl of the ligation (totally 5 μl) was packed with Gigapack Gold II packing extract from Stratagene. The library contained 650,000 independent clones. Screening of the library

2 X 50,000 pfu was plated on NZY plates and plaquelifts were done on Hybond N sheets (Amersham). Plaquelifts were done in duplicates. The sheets were hybridized with the PCR clone labelled with ³²P dCTP (Amersham) using Ready-to-go labelling kit from Pharmacia. Positive clones were reckoned only when hybridization was detected on both sheets. The gene was sequenced, and the found sequence showed that all of the peptides sequenced were coded by the found sequence. Sequence information

SEQ. ID. No. 12 presents the promoter sequence, the enzyme coding sequence, the terminator sequence and the signal sequence and the amino acid sequence of the enzyme of the present invention.

Arabinofuranosidase assay

Two different arabinoxylan preparations from wheat flour, Wheat Insoluble Pentosan (WIP) and Wheat soluble Pentosan (WSP), were degraded with the arabinofuranosidase enzyme of the present invention alone and in combination with an endoxylanase purified from A. niger. The assays were done on 1 % substrate in 50 Mm 50 Mm Na-acetate buffer at pH 5.0. The reactions were performed at 30 °C for 2.5 hours. The reactions were stopped by addition of 3 vol. edianol which precipitates the high molecular weight material. The samples were centrifuged and the supernatants were collected, dried under vacuum and resuspended in 0.5 ml distilled water. The samples were diluted 1 : 1 in water and analysed on a Chromopack Carbohydrate Pb column (300X7.8 mm, cat. 29010) using Shimadzu C-R4A Chromatopac HPLC system using a Shimadzu RI D-6A refractive index detector in accordance with the suppliers instructions. The column was calibrated using a standard composed of 0.48 mg/ml xylotriose, 0.48 mg/ml xylobiose, 0.60 mg/ml xylose and 0.58 mg/ml L-arabinose. The peaks were identified and quantified using the software supplied with the equipment. Results - Liberated saccharides from Wheat Insoluble Pentosan

Substrate 1 % WIP in 50 Mm Na-acetate buffer pH 5.0. Values are expressed in mg/ml.

abfC denotes the enzyme according to the present invention; and xyl denotes the xylanase described before.

Results - Saccharides liberated from Wheat Soluble Pentosan

Substrate 1 % WSP in 50 Mm Na-acetate buffer pH 5.0. Values are expressed in mg/ml.

abfC denotes the enzyme according to the present invention; and xyl denotes the xylanase described before. Figure 17 shows HP-TLC profiles of the AbfC enzyme acting synergistically with Xylanase A. In this Figure, the following abbreviations are used: water-soluble pentosan (WSP); water-insoluble pentosan (WIP); and oat xylan as substrate. The standards were: X- xylose; X₂- xylobiose; X₃- xylotriose; A- arabinose.

Figure 18 shows the HPLC analysis of hydrolysis products using 1 % oat spelt xylan as the substrate. Figure 18(a) and Figure 18(b) show the products when the AbfC enzyme and the xylanase enzyme respectively were used alone. Figure 18(c) show the products when the AbfC enzyme and the xylanase enzyme when combined.

The results of these experiments provide two important findings.

First the enzyme of the present invention liberates arabinose, in particular L-arabinose, from arabinoxylan.

Second the combined actions of the enzyme according to the present invention with the endoxylanase is significantly higher than the sum of their individual action. Accordingly, the two enzymes affect each others enzymatic activities in a synergistic fashion. Induction of the AbfC gene: Identification of inducers

The regulation of transcription of the AbfC encoding gene of Aspergillus niger was studied using a strain containing a fusion of the AbfC promoter to the β-glucuronidase encoding gene (uid A) of E coli.

GUS producing transformants were grown on different carbon sources and assayed both qualitatively and quantitatively for the ability to hydrolyse p-nitrophenol glucuronide.

The results are shown below: CARBON SOURCE GUS ACTIVITY AFTER 24 HOURS INDUCTION

(1 %) (units/mg) xylose 12.37

xylitol 1.49

arabinose 6.66

arabitol 5.30

glucose 0.70

cellubiose 0.95

xylo-oligomer 70 17.26

glucopyranoside 0.40

methyl-xylopyranoside 24.20

xyloglucan 1.00

pectin 0.27

arabinogalactan 2.60

arabitol -1- glucose 2.20

The results show that the AbfC promoter is switched on after 24 hours when grown in the presence of xylose, xylo-oligomer 70, methyl-xylopyranoside, arabinose and arabitol. These studies also suggest that mediyl-xylopyranoside is the natural and strongest inducer of this promoter.

The AbfC promoter is strongly repressed by glucose and is therefore under carbon catabolite repression. However, unlike all the published promoters for arabinofuranosidases, which are induced by arabinose and arabitol, the AbfC promoter of the present invention is regulated strongly by the intermediates in xylose metabolism. Accordingly, the present invention also covers an arabinofuranosidase promoter wherein the promoter is inducible by an intermediate in xylose metabolism. Effects of different promoter deletions on the regulation of the expression of the AbfC gene

To study the regulation at the molecular level, experiments were set up to detect possible upstream regulating sequences required for expression of the AbfC gene. A series of plasmids widi deletions in the 5' upstream region of the gene was constructed (see Figure 12). The E coli uid A gene was used as the reporter gene and a qualitative GUS assay was performed. The results indicated that the truncated AbfC promoter of 590 bp contains sufficient information for the inducibility of the AbfC gene and its regulation. Deletion of 100 bps sequence from the Xma 111 to the BamH1 sites of the promoter led to a reduction in activity of this promoter. Therefore, this 100 bps area is important for good levels of gene expression. Deletion of 290 bps before the ATG identified this region to be important but not sufficient to abolish the activity of this promoter. All the transformants analysed containing diis promoter construct showed very pale blue when tested (+-GUS). This region is as follows:

-170 T C A T C C A A T A T

As seen, this region contains the CCAAT element and is a putative target for a general transcriptional activator. This sequence is similar to the nuclear protein binding sites found in two starch inducible promoters: the Aspergillus niger glucoamylase gene and the Aspergillus oryzae amylase gene as well as the amdS gene of Aspergillus nidulans.

HETEROLOGOUS PROTEIN PRODUCTION USING ASPERGILLUS NIGER TRANSFORMED WITH THE AbfC PROMOTER AND THE AbfC SIGNAL SEQUENCE Transformation of Aspergillus Niger

The protocol for transformation of A. niger was based on the teachings of Buxton,F.P. , Gwynne D.I. , Davis, R.W. 1985 (Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans. Gene 37:207-214), Daboussi,M.J. , Djeballi,A. , Gerlinger, C , Blaiseau, P.L. , Cassan, M. , Lebrun, M.H. , Parisot, D. , Brygoo,Y. 1989 (Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans. Curr. Genet. 15:453-456) and Punt, P.J. , van den Hondel, C.A.M.J.J. 1992 (Transformation of filamentous fungi based on hygromycin B and Phleomycin resistance markers. Meth. Enzym. 216:447-457).

For the purification of protoplasts, spores from one PDA (Potato Dextrose Agar - from Difco Lab. Detroit) plate of fresh sporulated N400 (CBS 120.49, Centraalbureau voor Schimmelcultures, Baarn) (7 days old) are washed off in 5-10 ml water. A shake flask with 200 ml PDC (Potato Dextrose Broth, Difco 0549-17-9, Difco Lab. Detroit) is inoculated with this spore suspension and shaken (250 rpm) for 16-20 hours at 30 °C.

The mycelium is harvested using Miracloth paper and 3-4 g wet mycelium are transferred to a sterile petri dish with 10 ml STC (1.2 M sorbitol, 10 mM Tris Hcl pH 7,5, 50 Mm CaCl₂) with 75 mg lysing enzymes (Sigma L-2265) and 4500 units lyticase (Sigma L-8012).

The mycelium is incubated with the enzyme until the mycelium is degraded and the protoplasts are released. The degraded mycelium is then filtered through a sterile 60 μm mesh filter. The protoplasts are harvested by centrifugation 10 min at 2000 rpm in a swing out rotor. The supernatant is discarded and the pellet is dissolved in 8 ml 1.5 M MgSO₄, and then centrifuged at 3000 rpm for 10 min. The upper band, containing the protoplasts is transferred to another tube, using a transfer pipette and 2 ml 0.6 M K Cl is added. Carefully 5 ml 30% sucrose is added on the top and the tube is centrifuged 15 min at 3000 rpm. The protoplasts, lying in the interface band, are transferred to a new tube and diluted with 1 vol. STC. The solution is centrifuged 10 min at 3000 rpm. The pellet is washed twice with STC, and finally solubilized in 1 ml STC. The protoplasts are counted and eventually concentrated before transformation. For the transformation, 100 μl protoplast solution (10⁶-10⁷ protoplasts) are mixed with 10 μl DNA solution containing 5- 10 μg DNA and incubated 25 min at room temperature. Then 60 % PEG-4000 is carefully added in portions of 200 μl, 200 μl and 800 μl. The mixture is incubated 20 min at room temperature. 3 ml STC is added to the mixture and carefully mixed. The mixture is centrifuged 3000 rpm for 10 min.

The supernatant is removed and the protoplasts are solubilized in the remaining of the supernatant. 3-5 ml topagarose is added and the protoplasts are quickly spread on selective plates. AbfC promoter and heterologous gene expression

The expression vector pXP-Amy (Figure 13) contains the 2.1 kb α-amylase encoding gene from Thermomyces lanuginosus cloned downstream of the AbfC promoter (2.1 kb) and upstream of the Xylanase A terminator. This vector together with the hygromycin gene as a selectable marker was used for co-transformation experiments to test the functionality of the AbfC promoter.

The best transformant was accumulated in shake flask experiments at least 1 gram per litre of α-amylase in the culture media. Starch degrading activity was then detected widiin 48 hours and a peak of enzyme activity is observed at 4 days of growth on sugar beet pulp and wheat bran (Figure 15). AbfC signal sequence functions in protein secretion

An expression construct containing the signal peptide of the AbfC gene translationally fused to the mature α-amylase from T. lanuginosus was prepared and expression of this construct in the production strains was observed. In this regard, the translational fusion construct pXPXss-Amy (Figure 14) was placed under the transcriptional control of the AbfC promoter and the xylanase A termination signal. The incorporation of an endogenous signal peptide resulted in increased detectability of co-transformants expressing both amylase and the hygromycin resistance marker. The endogenous signal peptide directed the secretion of amylase out of the cell.

Substrate Specificity of AbfC Protein

The substrate specificity of the purified AbfC was determined using arabinose containing hemicelluloses: arabinoxylans from wheat, oat and larch, branched and debranched arabinans; arabinogalactan, sugar beet pectin, and xyloglucan.

The HPLC and HP-TLC results are shown in Figure 16, in which the following abbreviations are used: WSP - water-soluble pentosan, WIP - water-insoluble pentosan, AG - arabinogalactan, deB-A - debranched arabinan. The standards used were: A- arabinose, X- xylose.

The results indicate diat arabinose is the hydrolysis product from arabinoxylans. No hydrolysis products were released from arabinogalactan, debranched arabinan or xyloglucan. Arabinose was released as a hydrolysis product from branched arabinan. AbfC is therefore a 1,2/1,2 debranching enzyme and it has no activity towards linear 1 ,5 α-linked L-arabinofuranose residues found in debranched arabinans and arabinogalactan. This enzyme also releases a product when pectin is used as the substrate. It is believed that diis product is an arabinose containing ferulic acid or an arabinobiose. Reduction of Viscosity By AbfC

The results for the substrate specificity studies also suggest that the enzyme of the present invention could be used to reduce the viscosity of feeds. In this regard, the enzyme would reduce the viscosity of branched substrates by removing the branches but not the backbone of that substrate. This is in contrast to the known viscosity modifiers which degrade the substrate backbone.

Accordingly, the present invention covers a process of reducing the viscosity of a branched substrate wherein the enzyme degrades the branches of the substrate but not the backbone of the substrate.

In particular, the present invention covers the use of the enzyme of the present invention as a viscosity modifier.

In this regard, an experiment was carried out to investigate the reduction of viscosity of the water-soluble pentosan fraction from wheat flour by arabinofuranosidase. In this experiment, 6 ml water-soluble pentosan was incubated with 100 μl of AbfC for 20 hours, 20°C at pH 5.5.

The results (see Figure 19) show that the enzyme of the present invention can be used to reduce the viscosity of pectins, especially pectins that are used in beverages - such as fruit juices. Accordingly, the present invention covers the use of the enzyme of the present invention to reduce the viscosity of pectin.

ANTIBODY PRODUCTION Antibodies were raised against the enzyme of the present invention by injecting rabbits with the purified enzyme and isolating the immunoglobulins from antiserum according to procedures described according to N Harboe and A Ingild ("Immunization, Isolation of Immunoglobulins, Estimation of Antibody Titre" In A Manual of Quantitative Immunoelectrophoresis, Methods and Applications, N H Axelsen. et al (eds.), Universitetsforlaget, Oslo, 1973) and by T G Cooper ("The Tools of Biochemistry" , John Wiley & Sons, New York, 1977).

SUMMARY

Even though it is known that Aspergillus niger produces arabinofuranosidases, the present invention provides a novel and inventive arabinofuranosidase, as well as the coding sequence therefor and the promoter for that sequence. An important advantage of the present invention is that the enzyme can be produced in high amounts.

In addition, the promoter and the regulatory sequences (such as the signal sequence and the terminator) can be used to express or can be used in the expression of GOIs in organisms, such as in A. niger.

The arabinofuranosidase of the present invention is different from the arabinofuranosidases previously known. In this regard, the previous described arabinofuranosidases - such as those of EP-A-0506190 - are characterised by their ability to degrade arabinan, and are assayed using p-nitrophenyl-arabinoside.

The arabinofuranosidase of the present invention does not degrade arabinan, and only a minor activity is seen on p-nitrophenyl-arabinoside. In contrast, the arabinofuranosidase of the present invention is useful for degrading arabinoxylan. Therefore, the arabinofuranosidase of the present invention is quite different from the previous isolated arabinofuranosidases.

More in particular, the enzyme of the present invention is capable of specifically cleaving arabinose from the xylose backbone of arabinoxylan. The enzyme of the present invention is useful as it can improve processes for preparing foodstuffs and feeds as well as the foodstuffs and feeds themselves. For example, the enzyme of the present invention may be added to animal feeds which are rich in arabinoxylans. When added to feeds (including silage) for monogastic 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, arabinoxylan-degrading enzymes may be used to reduce the viscosity of feeds containing arabinans. The arabinoxylan-degrading enzyme may be added beforehand to the feed or silage if pre-soaking or wet diets are preferred.

Of particular benefit is the use of the enzyme according to the present invention in combination with a xylanase, especially an endoxylanase.

A possible further application for the enzyme according to the present invention is in the pulp and paper industry. The application of xylanases is often reported to be beneficial in the removal of lignins and terpenoids from the cellulose and hemicellulose residues of a hemicellulose backbone, an essential step in the processing of wood, wood pulp or wood derivative product for the production of paper. The addition of arabinoxylan-degrading enzymes, produced according to the present invention, to the xylanase treatment step should assist in the degradation of an arabinan-containing hemicellulose backbone and thus facilitate an improved, more efficient removal of both lignins and terpenoids. The application of arabinoxylan-degrading enzymes should be particularly advantageous in the processing of soft woods in which the hemicellulose backbone contains glucuronic acid.

The enzyme according to the present invention is also useful as it acts in a synergistic manner with endoxylanase (see results presented above).

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

Claims

1. An enzyme that is obtainable from Aspergillus, wherein the enzyme has the following characteristics: a. a MW of 33,270 D ± 50 D

b. a pI value of about 3.7

c. arabinoxylan degrading activity

d. a pH optima of from about 2.5 to about 7.0 (more especially from about 3.3 to about 4.6, more especially about 4)

e. a temperature optima of from about 40°C to about 60°C (more especially from about 45°C to about 55°C, more especially about 50°C); wherein the enzyme is capable of cleaving arabinose from the xylose backbone of an arabinoxylan.

2. An enzyme having the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof.

3. An enzyme coded by the nucleotide sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.

4. A nucleotide sequence coding for the enzyme according to claim 1.

5. A nucleotide sequence coding for the enzyme according to claim 2.

6. A nucleotide sequence having the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.

7. A nucleotide sequence according to any one of claims 4 to 6 operatively linked to a promoter.

8. A nucleotide sequence according to claim 7 wherein the promoter is the promoter having the sequence shown as SEQ. I.D. No. 3 or a variant, homologue or fragment thereof or a sequence complementary thereto.

9. A promoter having the sequence shown as SEQ. I.D. No. 3 or a variant, homologue or fragment thereof or a sequence complementary thereto.

10. A promoter according to claim 9 operatively linked to a GOI.

11. A promoter according to claim 10 wherein the promoter is operatively linked to a GOI, wherein the GOI comprises a nucleotide sequence according to any one of claims 4-6.

12. A terminator having the nucleotide sequence shown as SEQ. I.D. No. 13 or a variant, homologue or fragment thereof or a sequence complementary thereto.

13. A signal sequence having the nucleotide sequence shown as SEQ. I.D. No. 14 or a variant, homologue or fragment thereof or a sequence complementary thereto.

14. A construct comprising or expressing the invention according to any one of claims 1 to 13.

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

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

17. A transgenic organism comprising or expressing the invention according to any one of claims 1 to 16.

18. A transgenic organism according to claim 17 wherein the organism is a fungus.

19. A transgenic organism according to claim 18 wherein the organism is a filamentous fungus, preferably of the genus Aspergillus.

20. A transgenic organism according to claim 17 wherein the organism is a plant.

21. A process of preparing an enzyme according to any one of claims 1 to 3 comprising expressing a nucleotide sequence according to any one of claims 4-8.

22. A process according to claim 21 wherein the enzyme has the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof, and the nucleotide sequence has the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.

23. A process according to claim 21 or claim 22 wherein the expression is controlled (partially or completely) by use of a promoter according to claim 9.

24. A process for expressing a GOI by use of a promoter, wherein the promoter is the promoter according to claim 9.

25. Use of an enzyme according to any one of claims 1 to 3 or prepared by a process according to any one of claims 21 to 24 to degrade an arabinoxylan.

26. Use according to claim 24 wherein the enzyme is used in combination with a xylanase, preferably an endoxylanase.

27. A combination of enzymes to degrade an arabinoxylan, the combination comprising an enzyme according to any one of claims 1 to 3 or prepared by a process according to any one of claims 21 to 24 claims; and a xylanase.

28. Plasmid NCIMB 40703, or a nucleotide sequence obtainable therefrom for expressing an enzyme capable of degrading arabinoxylan or for controlling the expression thereof or for controlling the expression of another GOI.

29. A signal sequence having the sequence shown as SEQ. I.D. No. 15 or a variant, homologue or fragment thereof.

30. The use of the enzyme according to any one of claims 1 to 3 or prepared by a process according to any one of claims 21 to 24 claims, in the manufacture of a medicament or foodstuff to reduce or prevent indigestion and/or increase nutrient absorption.

31. An arabinofuranosidase enzyme having arabinoxylan degrading activity, which is immunologicaily reactive with an antibody raised against a purified arabinofuranosidase enzyme having the sequence shown as SEQ. I.D. No. 1.