Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01055—Alpha-N-arabinofuranosidase (3.2.1.55)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to an enzyme.
• the present invention relates to a nucleotide sequence coding for the enzyme.
• 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.
• the enzyme of the present invention is an arabinofuranosidase enzyme having arabinoxylan degrading activity.
• a gene of interest (“GOI")
• GOI gene of interest
• tissue 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.
• the crop may be made more useful as a feed.
• 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.
• an organism such as a filamentous fungus or a crop plant
• mammalian genes include interferons, insulin, blood factors and plasminogen activators.
• micro ⁇ organisms such as filamentous fungi
• 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.
• 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 ⁇ -xylan chains) and pectic substances (consisting of galacturonans and rhamnogalacturonans; arabinans; and galactans and arabinogalactans).
• pectic substances consisting of galacturonans and rhamnogalacturonans; arabinans; and galactans and arabinogalactans.
• arabinan One form of plant polysaccharide is arabinan.
• a review of arabinans may be found in EP-A-0506190.
• arabinans consist of a main chain of ⁇ - (1 ⁇ 5) groups linked to one another. Side chains are linked -(l ⁇ 3) or sometimes a- (l-*2) to the main -(l-*5)-L-arabinan backbone.
• l-*2 the main -(l-*5)-L-arabinan backbone.
• the molecular weight of arabinan is normally about 15 kDa.
• Arabinans are degraded by enzymes collectively called arabinases.
• 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 l-*6 linkage between the terminal arabinofuranosyl unit and the intermediate glucosyl unit of monoterpenyl ⁇ -L- arabinofuranosyl glucosides.
• the activity of the arabinan degrading enzymes of EP-A-0506190 include: a) the ability to cleave (l ⁇ 2)-o_-L-arabinosidic linkages; b) the ability to cleave (l ⁇ 3)-o.-L-arabinosidic linkages; c) the ability to cleave (l-»5)- ⁇ -L-arabinosidic linkages; d) the ability to cleave the 1 ⁇ 6 linkage between the terminal arabinofuranosyl unit and the intermediate glucosyl unit of monoterpenyl ⁇ -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).
• 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 Basidom
• xylan Another plant polysaccharide is xylan, whose major monosaccharide unit is xylose.
• Xylans are abundant components of the hemicelluloses.
• 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).
• 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 -1 bonds to the 2 or 3 position of the xylose residues.
• the xylose residues can be single or double substituted.
• 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) .
• the arabinoxylans amount to only about 3 % of wheat the importance of the arabinoxylan fraction is much higher.
• the arabinoxylans of cereals act as hydrocoUoids, as they form a gel like structure with water.
• 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.
• 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.
• 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.
• 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.
• enzymes that degrade the arabinoxylan such as xylanases
• some enzymes that degrade the arabinoxylans 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 (l ,4)- ⁇ -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)-/3-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.
• 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.
• 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.
• 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.
• a promoter is used in Aspergillus wherein the product encoded by the GOI is excreted from the host organism into the surrounding medium.
• 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.
• 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.
• an enzyme having the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof.
• nucleotide sequence coding for the enzyme according to the present invention there is provided a nucleotide sequence coding for the enzyme according to the present invention.
• nucleotide sequence having die sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.
• a promoter having the sequence shown as SEQ. I.D. No. 3 or a variant, homologue or fragment thereof or a sequence complementary thereto.
• 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.
• 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.
• 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.
• a combination of enzymes to degrade an arabinoxylan comprising an enzyme according to the present invention and a xylanase.
• 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.
• a signal sequence having the sequence shown as SEQ. I.D. No. 15 or a variant, homologue or fragment thereof.
• 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.
• an arabinofuranosidase promoter wherein the promoter is inducible by an intermediate in xylose metabolism.
• 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.
• aspects of the present invention include constructs, vectors, plasmids, cells, tissues, organs and transgenic organisms comprising the aforementioned aspects of the present invention.
• 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.
• 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.
• nucleotide sequence has the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto.
• nucleotide sequence is operatively linked to a promoter.
• the promoter comprises the sequence CCAAT.
• 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.
• the promoter comprises the 100 bps sequence from the Xma 111 to the BamHl sites.
• the promoter of the present invention is operatively linked to a GOI.
• the GOI comprises a nucleotide sequence according to the present invention.
• the transgenic organism is a fungus.
• the transgenic organism is a filamentous fungus, more preferably of the genus Aspergillus.
• the transgenic organism is a plant.
• the enzyme is used in combination with a xylanase, preferably an endoxylanase.
• 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.
• the vector such as an expression vector or a transformation vector
• the cell, the tissue, the organ, the organism or the transgenic organism is present in combination with at least one GOI.
• the promoter and the GOI are stably incorporated within the transgenic organism's genome.
• 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.
• 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.
• 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.
• arabinofuranosidase of the present invention is different from the arabinofuranosidases previously known.
• 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.
• 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.
• 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.
• 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.
• a GOI such as a nucleotide sequence coding for the enzyme according to the present invention
• 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.
• variant 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).
• homologue covers homology with respect to structure and/or function providing the resultant enzyme has arabinoxylan degrading activity.
• 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.
• variant 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).
• homologue covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for an enzyme having arabinoxylan degrading activity.
• 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.
• variant 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.
• 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.
• 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.
• variants 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.
• 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.
• 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.
• variant 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.
• 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.
• 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.
• nucleotide sequences that can hybridise to the nucleotide sequences of the coding sequence or the promoter sequence, respectively.
• 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.
• 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 SAi-intron or the ADH intron, intermediate the promoter and the GOI.
• fused in relation to the present invention which includes direct or indirect attachment.
• 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.
• 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.
• expression vector means a construct capable of in vivo or in vitro expression.
• 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.
• tissue includes tissue per se and organ.
• 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.
• the organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.
• 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.
• the promoter and/or the nucleotide sequence is (are) incorporated in the genome of the organism.
• 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.
• the transgenic organism can comprise a GOI, preferably an exogenous nucleotide sequence, under the control of the promoter according to the present invention.
• the transgenic organism can also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a promoter, which may be the promoter according to the present invention.
• the 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.
• 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.
• 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.
• 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.
• 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.
• the nucleotide sequence according to the present invention is under the control of a promoter that allows expression of the nucleotide sequence.
• the promoter need not necessarily be the same promoter as that of the present invention.
• 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.
• the promoter for the nucleotide sequence of the present invention can be the ⁇ -Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 promoter or the ⁇ -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.
• the promoter for the nucleotide sequence of the present invention can be the ⁇ -Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the ⁇ -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.
• the promoter is the promoter of the present invention.
• the promoters could additionally include features to ensure or to increase expression in a suitable host.
• 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.
• suitable other sequences include the S ⁇ i-intron or an ADH intron.
• Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.
• TMV 5' signal sequence see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
• the present invention also encompasses combinations of promoters and/or nucleotide sequences coding for proteins or enzymes and/or elements.
• 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.
• Amy 351 promoter 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.
• 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.
• 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.
• partial inactivation examples 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.
• a 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.
• 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.
• 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 ⁇ -galactosidase.
• the GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for patatin or ⁇ -amylase, ADP-glucose pyrophosphorylase (e.g. see EP-A-0455316), a protease antisense or a glucanase.
• an antisense transcript such as that for patatin or ⁇ -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 ⁇ -amylase enzyme which is the subject of our co-pending UK patent application 9413439.2 filed on 4 July 1994, d e sequence of which is shown in Figure 3.
• the GOI can be the nucleotide sequence coding for the ⁇ -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 ⁇ - 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.
• the GOI is a nucleotide sequence coding for the enzyme according to the present invention.
• 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 . 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 Asperg ⁇ illus. In: Martinelli S.D., Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology voi 29. Elsevier Amsterdam 1994.
• Filamentous fungi have during almost a century been widely used in industry for produc ⁇ tion 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.
• 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.
• a GOI such as an amylase or SEQ. I.D. No. 2
• 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.
• 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 ⁇ -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.
• 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.
• 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").
• POI protein encoded by the GOI
• 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.
• 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.
• filamentous fungi For the transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi (Ballance 1991, ibid). Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca 2+ ions. The transformed protoplasts then regenerate and the transformed fungi are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as argB, ⁇ rpC, 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.
• auxotrophic markers such as argB, ⁇ rpC, niaD and pyrG
• antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance.
• 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.
• 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 Agrobacte ⁇ um tumefaciens or a Ri plasmid from Agrobacte ⁇ um 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.
• 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.
• 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.
• the vector system is preferably an Agrobacte ⁇ um 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.
• the promoter or nucleotide sequence or construct of die 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.
• a vector of a vector system as defined above has been constructed in £. 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 Agrobacte ⁇ um strain, e.g. A. tumefaciens, so as to obtain an Agrobacte ⁇ um cell harbouring the promoter or nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
• cloning vectors 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, Ml 3 mp series, pACYC 184 etc.
• 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.
• sequence 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 die same or different plasmid.
• 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 die 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.
• 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.
• a plant to be infected is wounded, e.g. by cutting the plant wid 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.
• 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.
• the present invention provides an arabinofuranosidase enzyme having arabinoxylan degrading activity and die nucleotide sequence coding for the same.
• it provides a promoter that can control the expression of that, or another, nucleotide sequence.
• it includes terminator and signal sequences for the same.
• NCIMB National Collections of Industrial and Marine Bacteria Limited
• the deposit number is NCIMB 40703.
• 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
• Figure 22 is an activity plot.
• the enzyme of the present invention is sometimes referred to as AbfC.
• the promoter of the present invention is sometimes referred to as the AbfC promoter.
• 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, HCI 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 enzyme was concentrated using High-Trap spin columns. Next the concentrated and desalted fractions were subjected to gel filtration on a 50x600 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 (NH 4 ) 2 SO 4 .
• a gradient where the (NH 4 ) 2 SO 4 concentration was varied from 1.5 - 0 M was applied and e 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.
• 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.
• 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 die 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.
• 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 ⁇ -arabinofuranosidase (0.4 mg) was dissolved in 50 ⁇ l of 8M urea, 0.4 M NH 4 HCO 3 , pH 8.4. After overlay with N 2 and addition of 5 ⁇ l of 45 Mm DTT, the protein was denatured and reduced for 15 min at 50°C under N 2 . After cooling to RT, 5 ⁇ l of 100 Mm iodoacetamide was added for the cysteines to be derivatised for 15 min at RT in the dark under N 2 .
• PCR primers were synthesised using an Applied Biosystems DNA synthesiser model 392.
• PCR primers were synthesized from one of the found peptide sequences, namely SEQ ID No. 5.
• the primers were:
• PCR amplification was performed with 100 pmol of each of these primers in 100 ⁇ l 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).
• isopropanol to precipitate the extracted DNA. After further centrifugation for 15 mins. the DNA pellet was dissolved in 0.7 ml TE (lOmM Tris, HCI pH 8.0, ImM EDTA) and precipitated with 75 ⁇ l 3M NaAc, pH 4.8, and 500 ⁇ l isopropanol.
• TE lOmM Tris, HCI pH 8.0, ImM EDTA
• SEQ. ID. No. 12 presents die promoter sequence, the enzyme coding sequence, die terminator sequence and d e signal sequence and die amino acid sequence of the enzyme of the present invention.
• 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 die software supplied with the equipment.
• abfC denotes the enzyme according to the present invention
• xyl denotes the xylanase described before.
• abfC denotes d e enzyme according to the present invention
• xyl denotes the xylanase described before
• Figure 17 shows HP-TLC profiles of the AbfC enzyme acting synergistically with Xylanase A.
• WSP water-soluble pentosan
• WIP water-insoluble pentosan
• oat xylan as substrate.
• the standards were: X- xylose; X 2 - xylobiose; X 3 - xylotriose; A- arabinose.
• Figure 18 shows the HPLC analysis of hydrolysis products using 1 % oat spelt xylan as d e 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 enzyme of the present invention liberates arabinose, in particular L- arabinose, from arabinoxylan.
• the combined actions of die enzyme according to die 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.
• 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 j3-glucuronidase encoding gene (uid A) of E coli.
• GUS producing transformants were grown on different carbon sources and assayed bodi qualitatively and quantitatively for the ability to hydrolyse p-nitrophenol glucuronide.
• the AbfC promoter is strongly repressed by glucose and is dierefore under carbon catabolite repression.
• the AbfC promoter of the present invention is regulated strongly by the intermediates in xylose metabolism.
• 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
• 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 die Aspergillus oryzae amylase gene as well as the amdS gene of Aspergillus nidulans.
• 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 CaCl 2 ) with 75 mg lysing enzymes (Sigma L-2265) and 4500 units lyticase (Sigma L- 8012).
• the mycelium is incubated with the enzyme until die mycelium is degraded and die protoplasts are released.
• the degraded mycelium is then filtered through a sterile 60 ⁇ m 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 MgSO 4 , and d en centrifuged at 3000 rpm for 10 min.
• the upper band, containing die protoplasts is transferred to another tube, using a transfer pipette and 2 ml 0.6 M KC1 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.
• 100 ⁇ l protoplast solution (10 6 -10 7 protoplasts) are mixed with 10 ⁇ l DNA solution containing 5- 10 ⁇ g DNA and incubated 25 min at room temperature. Then 60 % PEG-4000 is carefully added in portions of 200 ⁇ l, 200 ⁇ and 800 ⁇ l. 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.
• the expression vector pXP-Amy ( Figure 13) contains the 2.1 kb ⁇ -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 die AbfC promoter.
• 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.
• 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.
• 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 ⁇ -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 enzyme of the present invention could be used to reduce the viscosity of feeds.
• 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.
• 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.
• the present invention covers the use of the enzyme of the present invention as a viscosity modifier.
• the results 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.
• d e present invention covers the use of the enzyme of the present invention to reduce the viscosity of pectin.
• Antibodies were raised against the enzyme of the present invention by injecting rabbits with the purified enzyme and isolating die 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).
• the present invention provides a novel and inventive arabinofuranosidase, as well as the coding sequence dierefor and the promoter for that sequence.
• An important advantage of the present invention is that the enzyme can be produced in high amounts.
• d e promoter and die regulatory sequences can be used to express or can be used in the expression of GOIs in organisms, such as in A. niger.
• arabinofuranosidase of die present invention is different from the arabinofuranosidases previously known.
• 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.
• arabinofuranosidase of the present invention does not degrade arabinan, and only a minor activity is seen on p-nitrophenyl-arabinoside.
• arabinofuranosidase of die present invention is useful for degrading arabinoxylan. Therefore, the arabinofuranosidase of the present invention is quite different from the previous isolated arabinofuranosidases.
• 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 die foodstuffs and feeds themselves.
• the enzyme of the present invention may be added to animal feeds which are rich in arabinoxylans.
• feeds including silage
• monogastic animals e.g. poultry or swine
• 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.
• 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.
• a possible further application for the enzyme according to d e 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 die 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 die processing of soft woods in which the hemicellulose backbone contains glucuronic acid.
• the enzyme according to d e present invention is also useful as it acts in a synergistic manner with endoxylanase (see results presented above).
• GTC TCT GAC GGC AAA CAT ATC GTC TAT GCG TCC ACT ACT GAT GAA GCG

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• 1995
  • 1995-03-17 GB GBGB9505479.7A patent/GB9505479D0/en active Pending
• 1996
  • 1996-03-11 EP EP96907403A patent/EP0871745A1/de not_active Withdrawn
  • 1996-03-11 WO PCT/EP1996/001009 patent/WO1996029416A1/en not_active Application Discontinuation
  • 1996-03-11 NZ NZ303970A patent/NZ303970A/en unknown
  • 1996-03-11 MX MX9707072A patent/MX9707072A/es unknown
  • 1996-03-11 CN CN96193916A patent/CN1198778A/zh active Pending
  • 1996-03-11 JP JP8528034A patent/JPH11502113A/ja active Pending
  • 1996-03-11 BR BR9607535A patent/BR9607535A/pt not_active Application Discontinuation
  • 1996-03-11 CA CA002214591A patent/CA2214591A1/en not_active Abandoned
  • 1996-03-11 AU AU51043/96A patent/AU5104396A/en not_active Abandoned

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