WO2000017368A1

WO2000017368A1 - Orange fruit pectinacetylesterase

Info

Publication number: WO2000017368A1
Application number: PCT/IB1999/001601
Authority: WO
Inventors: Tove Martel Ida Else Christensen; Anette Amstrup Pedersen; Janne Brunstedt
Original assignee: Danisco A/S
Priority date: 1998-09-24
Filing date: 1999-09-21
Publication date: 2000-03-30
Also published as: EP1115873A1; AU5754899A; GB9820817D0

Abstract

An acetyl esterase is described. The enzyme comprises the amino acid sequence shown as SEQ.I.D. No. 2, or a variant, derivative or homologue thereof, including combinations thereof.

Description

ORANGE FRUIT PECTINACETYLESTERASE

FIELD OF THE PRESENT INVENTION

The present invention relates to an enzyme and a process using same.

In particular, the present invention relates to the use of an enzyme for enzymatically modifying vegetable and/or fruit material - such as pectin.

BACKGROUND OF THE PRESENT INVENTION

Pectin is a structural polysaccharide commonly found in the form of protopectin in plant cell walls. The backbone of pectin comprises α-1-4 linked galacturonic acid residues which are interrupted with a small number of 1,2 linked α-L-rhamnose units.

In addition, pectin comprises highly branched regions with an almost alternating rhamno-galacturonan chain. These highly branched regions also contain other sugar units (such as D-galactose, L-arabinose and xylose) attached by glycosidic linkages to the C3 or C4 atoms of the rhamnose units or the C2 or C3 atoms of the galacturonic acid units. The long chains of α-1-4 linked galacturonic acid residues are commonly referred to as "smooth" regions, whereas the highly branched regions are commonly referred to as the "hairy regions".

Some of the carboxyl groups of the galacturonic residues are esterified (e.g. the carboxyl groups are methylated). Typically esterification of the carboxyl groups occurs after polymerisation of the galacturonic acid residues. However, it is extremely rare for all of the carboxyl groups to be esterified (e.g. methylated). Usually, the degree of esterification will vary from 0-90%. If 50% or more of the carboxyl groups are esterified then the resultant pectin is referred to as a "high ester pectin" ("HE pectin" for short) or a "high methoxyl pectin". If less than 50% of the carboxyl groups are esterified then the resultant pectin is referred to as a "low ester pectin" ("LE pectin" for short) or a "low methoxyl pectin". If 50% of the carboxyl groups are esterified then the resultant pectin is referred to as a "medium ester pectin" ("ME pectin" for short) or a "medium methoxyl pectin". If the pectin does not contain any - or only a few - esterified groups it is usually referred to as pectic acid.

The source of pectin will also govern to some extent whether or not other ester groups are present in the pectin structure. In this respect, it is known that some pectins comprise acetyl groups. Here, typically the hydroxyl groups on C₂ or C₃ may be acetylated. By way of example, sugar beet pectin is highly acetylated at C2 and/or C3 of the galacturonic acid residues. If acetyl groups are present, then it is known that acetyl esterases can act on those groups.

According to WO-A-95/02689 "many polysaccharides can exist in acetylated forms in various biological plant materials (mainly in xylan, mantan and pectin polymers). The biological significance of the acetyl groups is not fully understood. It is known that the acetyl group often protects the polysaccharide from degradation by hydrolytic enzymes. Hence, deacetylation of these polysaccharides is necessary in order to achieve partial or complete enzymatic breakdown of the acetylated polysaccharide. Accordingly, it is contemplated that acetyl esterases are important enzymes for the food industry, primarily in fruit and vegetable processing such as fruit juice production, wine making or pectin extraction, where their ability to modify acetylated polysaccharides to a readily degradable form may be utilised. It is known that many fungi contain enzymes capable of deacetylating acetylated polysaccharides, which enzymes are commonly designated acetyl esterases. Some fungal acetyl esterases have been purified. However, the study of these enzymes have been hampered by the lack of well-characterized homogeneous sdbstrates, and by the difficult and time consuming assays for measuring acetate release. Any industrial use of these enzymes has not been j described. WO 92/19728 describes a rhamnogalacturonan acetyl esterase isolated from the fungal species Aspergillus aculeatus. This enzyme is specific for acetylated galacturonic acid residues in hairy regions of pectin. EP 507 369 discloses a DNA sequence encoding an acetyl xylan esterase isolated from Aspergillus. For many purposes, it would be desirable to provide acetyl esterases in a form essentially free from other components. In this way, it would be possible to produce enzyme preparations adapted for a specific purposes, such preparations either containing a single acetyl esterase or arbitrary combinations thereof, and optionally containing other polysaccharide degrading enzymes. To serve this end, it is convenient to provide single-component acetyl esterases by recombinant DNA techniques."

According to Fogarty and Ward (1974 Progress In Industrial Microbiology pp 59- 64), the acetyl groups can be significant in the sense that they effect gelation properties. The authors present the following Table of degree of acetylation for pectins obtained from various sources.

TABLE TYPICAL ACETYL CONTENT OF SOME PECTIC POLYSACCHARIDES

Source Acetyl content (%)

Apricot 1.36

Cherry 0.18

Citrus 0.24

Raspberry 0.25

Strawberry 1.48

Sugar beet 2.50

In more detail with respect to sugar beet pectin, Thibault (1993 Winter Congress, Int. Inst. Beet Research vol 56 pp 325-335) reports that "sugar-beet pulp is composed mainly of cellulose (-20%), "hemicelluloses" (-25%) and pectins (-25%) together with small amounts of proteins, lignin and ashes. This composition shows that sugar-beet pulp could be used as a source of pectins or dietary fibres. Dietary fibre can be obtained directly from the pulp by processes which tend to eliminate ash, colour and odour. The polysaccharide structure and some physico- chemical properties (cation-exchange capacity and water-holding capacity) showed that the resulting fibres may have satisfactory functionality as a dietary ingredient in food products. Sugar-beet pectins have poor gelling properties as compared to those of citrus or apple because of the presence of acetylester groups."

Thibault (ibid) further reports that "Apple pomace and citrus peels are until now the only sources of commercial pectins. Substitutes for these pectins have been investigated, especially from sugar-beet pulp because this by-product is very rich in pectins. However, attempts to commercialise sugar-beet pectins have failed, because they have poor gelling properties compared to those from citrus and apple."

Thibault (ibid) further reports that "Sugar-beet pectins have the distinctive feature of containing acetyl groups. This acetylation was recognized as the critical factor for the gelling power of beet pectins because the presence of acetyl groups on the C2/C3 of the galacturonic units may sterically hinder the intermolecular association leading to the gel formation either in sugar-acid systems or in calcium gels. Deacetylation is therefore necessary in order to obtain pectin with gelling properties; however the treatment must not change significantly the molecular weight."

Thus, the structure of the pectin, in particular the degree of esterification (e.g. methylation and/or acetylation), dictates many of the resultant physical and/or chemical properties of the pectin. For example, pectin gelation depends on the chemical nature of the pectin, especially the degree of esterification. In addition, however, pectin gelation also depends on the soluble-solids content, the pH and calcium ion concentration. With respect to the latter, it is believed that the calcium ions form complexes with free carboxyl groups, particularly those on a LE pectin. Thibault (ibid) also reports that "Several chemical treatments have been studied, particularly acidic deacetylation during or after pectin extraction; as the demethoxylation rate is much slower than the deacetylation rate and as the pectic backbone is very acid-resistant. It is possible to produce beet pectins in which the acetyl groups have been markedly reduced and the degree of methylesterifation within suitable limits. Alkaline deacetylation is also possible but the side-reactions (demethoxylation and depolymerisation) cannot be controlled."

Thibault (ibid) then reports that "Another way to deacetylate pectins is to use enzymes."

Pectic enzymes are classified according to their mode of attack on the galacturonan part of the pectin molecule. A review of some pectic enzymes has been prepared by Pilnik and Voragen (Food Enzymology, Ed.: P.F.Fox; Elsevier; (1991); pp: SOS- 337). By way of example, pectin methylesterases (EC 3.1.1.11), otherwise referred to as PMEs, de-esterify HE pectins to LE pectins or pectic acids. In contrast, and by way of further example, pectin depolymerases split the glycosidic linkages between galacturonosyl methylester residues.

With specific reference to PME, PME activity produces free carboxyl groups and free methanol. The increase in free carboxyl groups can be easily monitored by automatic titration. In this regard, earlier studies have shown that some PMEs de- esterify pectins in a random manner, in the sense that they de-esterify any of the esterified (e.g. methylated) galacturonic acid residues on one or more than one of the pectin chains. Examples of PMEs that randomly de-esterify pectins may be obtained from fungal sources such as Aspergillus aculeatus (see WO 94/25575) and Aspergillus japonicus (Ishii et al 1980 J Food Sci 44 pp 611-14). Baron et al (1980 Lebensm. Wiss. M-Technol 13 pp 330-333) apparently have isolated a fungal PME from Aspergillus niger. This fungal PME is reported to have a molecular weight of 39000 D, an isoelectric point of 3.9, an optimum pH of 4.5 and a K_m value (mg/ml) of 3. In contrast, some PMEs are known to de-esterify pectins in a block-wise manner, in the sense that it is believed they attack pectins either at non-reducing ends or next to free carboxyl groups and then proceed along the pectin molecules by a single- chain mechanism, thereby creating blocks of un-esterified galacturonic acid units which can be calcium sensitive. Examples of such enzymes that block-wise enzymatically de-esterify pectin are plant PMEs. Up to 12 isoforms of PME have been suggested to exist in citrus (Pilnik W. and Voragen A.G.J. (Food Enzymology (Ed.: P.F.Fox); Elsevier; (1991); pp: 303-337). These isoforms have different properties.

Random or biockwise distribution of free carboxyl groups can be distinguished by high performance ion exchange chromatography (Schols er al Food Hydrocolloids 1989 6 pp 115-121). These tests are often used to check for undesirable, residual PME activity in citrus juices after pasteurisation because residual PME can cause, what is called, "cloud loss" in orange juice in addition to a build up of methanol in the juice.

PME substrates, such as pectins obtained from natural plant sources, are generally in the form of a high ester pectin having a DE of about 70%. Sugar must be added to extracts containing these high ester PME substrates to provide sufficient soluble solids to induce gelling. Usually a minimum of 55% soluble solids is required. Syneresis tends to occur more frequently when the percentage soluble solids is less than 55%. When syneresis does occur, expensive additives must be used to induce gelling.

With reference to enzymatic de-acetylation reference may be made to Searle-van Leeuwen et al (1996 Pectin and Pectinases, pages 793-798, J Visser and A. Voragen (Eds), Elsevier Science BV) who reports on acetyl esterases with different specificity occuring in one Aspergillus niger preparation. In this respect, three acetyl esterases were purified and characterised: pectin acetyl esterase (PAE), feruloyl acetyl esterase (FAE) and rhamnogalacturonan acetyl esterase (RGAE). In addition, Bordenave et al (1995 Phytochemistry vol 38 No. 2, pp 315-319) report on the purification of an acetyl esterase from cell walls isolated from mung bean hypocotyls. According to the authors "the purified enzyme had an apparent Mw, of 43,300 and an apparent pi > 9. It rapidly deesterified triacetin and p- nitrophenylacetate and slowly released acetate from beet and flax pectins, the deesterification rate being increased by previous demethylation of the pectins."

WO-A-95/02689 itself discloses an enzyme with acetyl esterase activity. The enzyme comprises the sequence lle-X-Phe-Gly-Asp-X-Tyr-Tyr-Thr; where X= any amino acid. According to WO-A-95/02689 the enzyme is a single-component ACE and has activity towards acetylated xylan and mannan. It is useful in the degradation or modification of plant material, e.g. in the preparation of feed or food, such as in fruit and vegetable processing, wine making and the modification or degradation of gums, e.g. guar gum and gum arabic. According to WO-A-95/02689, it is also useful in the paper or pulp industry, e.g. to improve the bleachability or drainability of lignocellulosic pulp. The enzyme can also be used in combination with other enzymes to improve different kinds of processing, facilitate the purification of different components like carbohydrate, improve feed value, decrease water binding capacity, improve degradability in waste water plants, improve the conversion of, e.g. corn and grass to silane, or to hydrolyse various plant cell wall-derived materials or waste materials, e.g. from paper production, or agricultural residues, e.g. wheat straw, corn cobs or nut shells. According to WO-A-95/02689, the enzyme can be used to deacetylate carbohydrates to change their properties, e.g. rheology, stabilising ability or hydrophobicity. In systems with low water activity the enzyme can also be used to esterify, e.g. acetylate carbohydrates like xylans and mannans. According to WO-A-95/02689 this confers increased hydrophobicity, and so leads to improved ability to emulsify and/or stabilise fat-containing emulsions. According to WO-A-95/02689, the term "With acetyl esterase activity" as used therein is used to define a group of enzymes, the members of which have the common characteristic of being capable of cleaving the acetyl esterase substrate p- nitrophenol-acetate (PNP-acetate). WO-A-93/20190 discloses a rhamnogalacturonan acetyl esterase (which they call RGAE) that is immunologically reactive with an antibody raised against purified RGAE from Aspergillus aculeatus CBS 101.43. The RGAE has the following partial sequence: Asp-Arg-Val-Tyr-Leu-Ala-Gly-Asp-Ser-Thr-Met-Thr-Lys-Asn-Gly-Gly-X- Ser-Gly-Thr-Asn-Gly-Trp-Gly-Glu-Tyr-Leu-Ala. The enzyme is said to be highly specific for the deacyetylation of the enzyme-resistant polysaccharide 'modified hairy region' (MHR), but does not show any activity towards triacetin and beet pectin.

WO-A-97/10726 discloses pectinaceous homogenates or slurries are treated with (a) a mixture of galactanase, arabinase, chi-arabinofuranosidase, rhamnogalacturonan acetyl esterase (RGAE), endoglucanase, mannasase, xylanase and/or proteolytic enzymes and (b) with a pectinesterase (PE) free of pectic depolymerising enzymes, and then (c) inactivating enzymes. Apparently, the process increases the viscosity or gel strength of foods. The homogenate or slurry can be a juice, puree, concentrate, ketchup, condiment, sauce, soup, salsa, chutney, yoghurt or desert.

EP-A-0507369 discloses a recombinant DNA fragment which encodes a protein having acetyl xylan esterase (AXE) activity. Apparently the acetyl xylan esterase can be used to deacetylate xylan, preferably using the enzyme in conjunction with other xylan degrading enzymes e.g. xylanases, arabinofuranosidases, xylosidases and glucoronidases. Specific applications of acetyl xylan esterases include (i) the pretreatment of animal feed to increase the digestibility, (ii) addn. of these enzymes to feed ^'treatment in situ, (iii) treatment of fruit juices and beer in order to improve rheological characteristics and clarity; and (iv) pulp and (waste-) paper processing in order to improve the process of bleaching and de-watering, by releasing lignin from kraft pulp. In general, this enzyme can be used to degrade biological cell walls to increase digestibility or flow characteristics in industrial applications relating to the preparation of fruit juices or beers. Deacylation of xylan decreases the solubility of feed components and decreases the viscosity, leading to increased ease of handling and reduced anti-nutritional effect of pentosanes. Other teachings on acetyl esterases - such as the uses thereof and activities thereof - may be found in Leeuwen et al (1996 Pectin and Pectinases, pages 793-798, J Visser and A. Voragen (Eds), Elsevier Science BV); Christensen et al (1996 Pectin and Pectinases, pages 723-730, J Visser and A. Voragen (Eds), Elsevier Science BV); Williamson (1991 Phytochemistry vol 30 No.2, pp 445-449); Faulds and Williamson (Gums and Stabilisers For The Food Industry 5, pages 227-280 Eds: Phillips, Williams and Wedlock, IRL Press); Bordenave et al (1995 Phytochemistry vol 38 No. 2, pp 315-319); Thibault (1993 Winter Congress, Int. Inst. Beet Research vol 56 pp 325-335); and Fogarty and Ward (1974 Progress In Industrial Microbiology pp 59-64).

Acetyl esterases also have found use in the pharmaceutical industry. For example, WO-A-92/17600 reports on the production of cephalosporins by the enzymatic hydrolysis of O-acyl derivatives using an orange peel acetyl esterase.

Thus, pectins, de-esterified pectins, and de-acetylated pectins - in addition to pectin enzymes (such as PMEs and acetyl eterases) - have an industrial importance.

However, any benefit derived from the use of an enzyme in the preparation of, for example, a foodstuff will depend to some extent on the quality and quantity and type of the enzyme used and on the quality and quantity and type of the enzymatic substrates - in particular pectin - that may be present in the material used to prepare the foodstuff. For example, if the substrate is a fruit material or a vegetable material then the amount and/or structure of natural pectin in that substrate will be different for different types of fruit material or vegetable material. This is also borne out by the data presented in WO-A-94/25575, especially Figure 7 where it is clear to see that its system for treating pectin is not ideal. SUMMARY ASPECTS OF THE PRESENT INVENTION

In accordance with the present invention there is provided an acetyl esterase enzyme - as well as the coding sequence for same - that is useful in treating acetyl esterase substrates, such as pectin. For convenience, the acetyl esterase of the present invention can be written as ACE (viz - acetyl esterase).

This and other aspects of the present invention are presented in the claims and in the following commentary.

For ease of reference, aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.

In the following commentary references to "nucleotide sequence of the present invention" and "amino acid sequence of the present invention" refer respectively to any one or more of the nucleotide sequences and to any one or more of the amino acid sequences present herein. Also, and as used herein, "amino acid sequence" refers to peptide or protein sequences and may refer to portions thereof. In addition, the term "amino acid sequence of the present invention" is synonymous with the phrase "polypeptide sequence of the present invention". Also, the term "nucleotide sequence of the present invention" is synonymous with the phrase "polynucleotide sequence of the present invention".

DETAILED ASPECTS OF THE PRESENT INVENTION

According to a first aspect of the present invention there is provided an acetyl esterase (ACE) comprising the amino acid sequence shown as SEQ.I.D. No.2, or a variant, derivative or homologue thereof, including combinations thereof According to a second aspect of the present invention there is provided a nucleotide sequence comprising the nucleotide sequence shown as SEQ.I.D. No. 1 , or a variant, derivative or homologue thereof.

According to a third aspect of the present invention there is provided a process comprising contacting an acetyl esterase substrate with the enzyme of the present invention or a nucleotide sequence of the present invention or the expression product thereof.

According to a fourth aspect of the present invention there is provided a foodstuff comprising an ACE treated pectin prepared by the process according to the present invention.

According to a fifth aspect of the present invention there is provided a transformed cell or transfomed organism comprising the enzyme of the present invention or a nucleotide sequence of the present invention or the expression product thereof.

The present invention also relates to any one or more of:

a construct expressing or comprising the ACE as defined herein or the nucleotide sequence as defined herein.

a vector expressing or comprising a construct of the present invention or the ACE as defined herein or the nucleotide sequence as defined herein.

a combination of constructs comprising at least a first construct expressing or comprising the ACE enzyme as defined herein or the nucleotide sequence as defined herein; and a second construct comprising a gene of interest (GOI) and a promoter.

a cell, tissue or organ expressing or comprising a vector according to the present invention or a construct according to the present invention or the ACE as defined herein or the nucleotide sequence as defined herein or the combination of constructs according to the present invention.

a transgenic organism expressing or comprising a cell, tissue or organ expressing or comprising a vector according to the present invention or a construct according to the present invention or the ACE as defined herein or the nucleotide sequence as defined herein or the combination of constructs according to the present invention.

a recombinant ACE enzyme which is immunologicaily reactive with an antibody raised against an ACE enzyme as defined herein.

In addition to the sequences presented in the attached sequence listings (as well as fragments, derivatives or homologues thereof), the present invention also covers sequences that are complementary to the aforementioned sequence listings (as well as fragments, derivatives or homologues thereof). The present invention also covers sequences that can hybridise to the aforementioned sequence listings (as well as fragments, derivatives or homologues thereof). The present invention also covers sequences that are complementary to sequences that can hybridise to the aforementioned sequence listings (as well as fragments, derivatives or homologues thereof).

The process of the present invention can occur ex vivo or even in vivo - such as in planta.

In the latter respect, the plant may be a transgenic plant, such as a plant that has been genetically engineered to produce different levels and/or types of pectin. The plant may also be plant material, rather than a whole plant. Here, the plant material may be obtained from a transgenic plant, such as a plant that has been genetically engineered to produce different levels and/or types of pectin. The plant or plant material may be or may be derived from a vegetable, a fruit, or other type of pectin containing or producing plant. Here, the vegetable material and/or the fruit material can be a mash.

PREFERABLE ASPECTS

In a highly preferred aspect, the present invention relates to a process for treating a pectin with an ACE.

Preferably, the ACE has the amino acid sequence shown as SEQ.I.D. No.2, or a variant, derivative or homologue thereof.

Preferably, the ACE has the amino acid sequence shown as SEQ.I.D. No.2.

Preferably, the ACE has been expressed by a nucleotide sequence comprising the nucleotide sequence shown as SEQ.I.D. No. 1 , or a variant, derivative or homologue thereof, or combinations thereof.

Preferably, the ACE has been expressed by a nucleotide sequence having the nucleotide sequence shown as SEQ.I.D. No. 1 , or a variant, derivative or homologue thereof.

Preferably, the ACE has been expressed by a nucleotide sequence having the nucleotide sequence shown as SEQ.I.D. No. 1.

Preferably, the ACE has been prepared by use of DNA techniques.

Preferably, the ACE is obtainable from a plant.

Preferably, the nucleotide sequence is a cDNA.

Preferably, the nucleotide sequence is obtainable from a plant. Preferably, the acetyl esterase substrate is or is obtainable from a plant or a plant material.

Preferably, the acetyl esterase substrate is or is obtainable from a fruit or a vegetable.

Preferably, the acetyl esterase substrate is pectin.

Preferably, the treated acetyl esterase substrate is suitable for consumption.

Preferably, the transformed cell or transformed organism is a transformed plant cell or a transformed plant or a transformed micro-organism.

Preferably, the transformed plant cell or the transformed plant is a transformed sugar beet cell or a transformed sugar beet.

Preferably, the process includes the further step of isolating the ACE treated pectin from the active ACE.

Preferably, the process includes the further step of adding the ACE treated pectin to a medium that is suitable for consumption.

Preferably, the process includes the further step of adding the ACE treated pectin to a medium that is for subsequent consumption.

ACE

Thus, the present invention relates to an ACE. Preferably, the ACE comprises the amino acid sequence shown as SEQ.I.D. No.2, or a variant, derivative or homologue thereof, including combinations thereof. In addition, the present invention covers the amino acid sequence shown as SEQ.I.D. No.2, or a functional equivalent thereof. Preferably the ACE is or is derived from or is based on an ACE obtainable from a plant.

Preferably the ACE is or is derived from or is based on an ACE obtainable from an orange.

FUNCTIONAL EQUIVALENT THEREOF

The term "functional equivalent thereof in relation to the enzyme of the present invention means that the functional equivalent could be obtained from other sources. The functionally equivalent enzyme may have a different amino acid sequence but will have ACE activity. The functionally equivalent enzyme may have a different chemical structure and/or formula but will have ACE activity. The functionally equivalent enzyme need not necessarily have exactly the same ACE activity as the ACE enzyme as presented in the sequence listings. For some applications, preferably, the functionally equivalent enzyme has at least the same activity profile as the ACE enzyme as presented in the sequence listings.

DERIVED FROM AN ACE OBTAINABLE FROM A PLANT

The term "derived from an ACE obtainable from a plant" means that the ACE has a sequence similar to that of an ACE that is obtainable from a plant, providing the ACE can de-acetylate pectin acetyl groups.

DERIVED FROM AN ACE OBTAINABLE FROM AN ORANGE

The term "derived from an ACE obtainable from an orange" means that the ACE has a sequence similar to that of an ACE that is obtainable from a orange, providing the ACE can de-acetylate pectin acetyl groups. PECTIN

The term "pectin" includes pectin in its normal sense, as well as fractionates and derivatives thereof, as well as modified pectins (e.g. chemically modified pectins and enzymatically modified pectins).

By way of example, the pectin can be a derivatised pectin, a degraded (such as partially degraded) pectin or a modified pectin. An example of a modified pectin is pectin that has been prior treated with an enzyme such as an ACE - which may be the same as the ACE of the present invention or a different ACE or a combination thereof. An example of a pectin derivative is pectin that has been chemically treated - eg. amidated.

Preferably, the pectin is not a pectin that has been prior treated with the enzyme polygalacturonase to substantially reduce the length of the pectin backbone.

MEDIUM

The medium can be an aqueous solution, such as a beverage. The beverage can be a drinking yoghurt, a fruit juice or a beverage comprising whey protein.

For some applications, preferably the medium comprises a protein. Here, preferably, the protein is derived from or is derivable from or is in a dairy product, such as milk or cheese. Preferably, the protein is casein or whey protein.

PURITY OF ACE

The purity of the ACE can be investigated by SDS-PAGE using Pharmacia PhastSystem™ with 10 - 15% SDS-gradient gels. Electrophoresis and silver staining of the proteins can be done as described by the manuals from Pharmacia. For determination of pi IEF 3-9 PhastSystem™ gels can be used. Immuno gel electrophoresis can be used for characterisation of antibodies (see later section) - such as polyclonal antibodies - raised against ACE. The enzyme fractions are then separated on SDS-PAGE and transferred to NC-paper by semi-dry blotting technique on a Semidry transfer unit of the PhastSystem™. The NC-paper is incubated with the primer antibody diluted 1 :500 and stained with the second antibody coupled to alkaline phosphatase (Dako A/S Glsotrup, Denmark) used in a dilution of 1:1000.

Further studies that can be performed on the ACE include peptide mapping. In this respect, ACE can be digested with either trypsin or endo-proteinase Lys-C from Lysobacter enzymogenes (both enzyme preparations should be are sequencing grade) - which can be purchased from Boerhinger Mannheim, Germany.

Typically, 100 mg purified ACE is carboxy methylated with iodoacetamide to protect the reduced SH-groups. Then the protein is cleaved with trypsin (4 mg/20-100 ml). The hydrolytic cleavage is performed at 40°C for 2 x 3 hrs. The reaction is stopped with addition of 20 ml TFA. After centrifugation at 15,000 rpm for 5 min the peptides are purified on a reverse-phase HPLC column (Vydac 10 C18 column). 2 x 500 ml samples are applied. The peptides are eluted and separated with an increasing acetonitrile gradient from 0.05 - 0.35% in 60 min in 0.1% TFA. The peptides are collected manually in Eppendorf tubes.

For digestion with endo-proteinase Lys-C, freeze dried ACE (0.1 mg) is dissolved in 50 ml of 8 M urea, 0.4 M NH₄HC0₃, pH 8.4. After overlay with N₂ and addition of 5 ml of 45 mM DTT, the protein is denatured and reduced for 15 min at 50°C under N₂. After cooling to room temperature, 5 ml of 100 mM iodoacetamide is added for the cysteines to be derivatised for 15 min at room temperature in the dark under N₂. Subsequently, 90 ml of water and 5 mg of endo-proteinase Lys-C in 50 ml 50 mM tricine and 10 mM EDTA, pH 8.0, are added and the digestion was carried out for 24 hrs at 37°C under N₂. The resulting peptides are then separated as described for trypsin digested peptides.

Selected peptides can be further purified on a Devosil 3 C₁₈ RP-HPLC column 0.46x10 cm (Novo Nordisk, Denmark). The purified peptides are then applied on an amino acid sequencer, Applied Biosystems 476A, using puised-liquid fast cycles.

USES OF ACE

The ACE of the present invention is advantageous as it deacetylates pectin. We believe that the enzyme is specific for de-acetylating the homogalacturonan. We beiieve that the ACE might not deacetylate the hairy region.

Hence, a further aspect of the present invention is the use of the ACE to deacetylate homogalacturonan.

It has been reported that a specific rhamnogalacturonan acetylesterase has been isolated from Aspergillus. This enzyme is specific for the hairy region of the pectin molecule and cannot deacetylate the homogalactumonan. We believe that this is in contrast to the ACE of the present invention. Moreover, a pectin acetylesterase has also been purified from Aspergillus but not cloned. That enzyme is very unstable in contrast to ACE of the present invention, which is very stable.

Sugar beet pectin is more hydrophobic than citrus pectin due to the acetylgroups. It has been found that the sugar beet pectin therefore is a good stabilizing agent for emulsions.

Sugar beet pectin has poor gelling properties compared with citrus pectin which is due to steric hindrance of the acetylgroups. Improved gelling properties are obtained by de-acetylating the sugar beet pectin with the ACE of the present invention. Acetylation of pectin or other carbohydrates hinders the degradation of the polymer. Polygalacturonase or pectate lyase or pectin lyase can degrade sugar beet pectin but deacetylation (and optionally also de-esterifcation) by the ACE of the present invention (optionally also by a PME) of the pectin prior to de- polymerization increases the suitability of the substrate for the subsequent action of polygalacturonase, pectate lyase, pectin lyase.

The ACE of the present invention enzyme may be used in fruit and vegetable processing in the fruit juice preparation, wine production. Here, the use of enzyme will increase the yield of the fruit juice and make the residual of cell wall polymers more degradable in order to extract more juice. Examples of sources of juices or extracts thereof include: nectars, bases or concentrates, preferably from vegetables, such as carrots, celery or onions, or from fruits, such as apples, pears, citrus fruits, tomatoes, grapes, blackcurrants, redcurrants, raspberries, strawberries, cranberries, prunes, cherries, pineapples or tropical fruits such as apples.

Furthermore, treatment of plant material with the ACE of the present invention will give a better degradability of animal fodder meaning that the animal will get higher yield of the fodder. We believe that treatment of sugar beet pulp which is used for animal feed with the ACE of the present invention will increase the feed yield.

The ACE of the present invention could also be used in combination with other pectolytic enzymes - such as PME, polygalacturonase, pectin lyase etc. - and/or other enzymes - such as glucanase, xylanase etc.

By removing the water in the reaction the ACE of the present invention could acetylate e.g. citrus pectin which might give new application areas for citrus pectin.

It is known that strawberry pectin has a high content of acetylgroups. Thus, treatment of strawberry fruit with the ACE of the present invention will result in improved gelling properties in e.g. jam, marmalade. The modification of the pectin could even be in the fruit - namely in situ. This latter aspect could be achieved by preparing transformed plants or cells thereof.

The use of the ACE of the present invention to modify acetylated pectin also yields the possibility of preparing new pectin raw material e.g. potato pectin.

In accordance with the present invention, plant material may be treated with the ACE of the present invention in combination with other enzymes in order to improve different kinds of processing, facilitate purification or extraction of different component like carbohydrates, improve the feed value, decrease the water binding capacity, improve the degradability in waste water plants, improve the conversion of e.g. grass and corn to ensilage, or to hydrolyse various plant cell wall-derived materials or waste materials, e.g. from paper production, or agricultural residues such as wheat-straw, corn cobs, whole corn plants, nut shells, grass, vegetable hulls, bean hulls, spent grains, sugar beet pulp, and the like.

The ACE of the present invention may even be used to deacetylate carbohydrates in order to change properties thereof, such as the rheology, the stabilizing ability of the hydrophobicity of the carbohydrates. Examples of which include the deacetylation of xylans and mannans. The carbohydrate acetyl esterase preparation that is to be used for the above purpose is preferable essentially free from activities with can depolymerize said carbohydrates.

Furthermore the ACE of the present invention may in some cases be able to act on other acetylated non-saccharide substrates.

FOODSTUFF

In accordance with the present invention, the de-acetylated pectin of the present invention is advantageous for the preparation of a foodstuff. Preferably, the foodstuff is food for human and/or animal consumption. Typical preferred foodstuffs include jams, marmalades, jellies, dairy products (such as milk or cheese), meat products, poultry products, fish products, bakery products and feed. The foodstuff may even be a beverage. The beverage can be an yone or more of: an acidified milk drink, a drinking yoghurt, a fruit juice comprising whey protein, a beverage comprising plant protein - such as soya - or whey, or combinations of such beverages.

In addition to the foodstuff comprising the ACE treated pectin, the foodstuff may comprise more other components, such as one or more suitable food ingredients. Typical food ingredients include any one or more of an acid - such as citric acid - or a sugar - such as sucrose, glucose or invert sugar - or fruit - or other enzymes, preservatives, colourings and other suitable components.

In one preferred embodiment, the foodstuff of the present invention comprises fruit. Here, fruit imparts taste, colour and structure to the gel, as well as pectin, acid and a small amount of solids. Depending on the level of natural flavour and colour in the fruit, fruit dosages are normally from 25% to 60% of the jam. The solids content of ordinary fruit is around 10% Brix, but fruit concentrate, which is typically 65-70% Brix, can also be used. The pH in fruit varies widely, depending on the fruit in question, but most fruits have a pH between 3.0 and 3.5.

The pectin content also varies, depending on the fruit in question. For example, redcurrants, blackcurrants and oranges have a high pectin content, and satisfactory gels from these fruits can be obtained by adding only a small amount of extra pectin. The choice of GRINDSTED™ Pectin depends on the type of jam in question. For example, GRINDSTED™ Pectin SS 200 is used in jams containing no fruit pieces or jam containing only very small fruit pieces. Fruit separation in such jams is not a problem, and consequently a slow-setting pectin and lower filling temperature can be used. By way of example, GRINDSTED™ Pectin RS 400 is used in jams containing large fruit pieces or whole fruit, for instance cherries or strawberries. In jams containing whole fruit it may be difficult to avoid fruit separation, and it is therefore necessary to use a rapid-set pectin such as GRINDSTED™ Pectin RS 400.

The choice of pectin type may also depend on the container size in question. When standard jars are used, the filling temperature is less critical with regard to the stability of pectin, as the jars will cool down relatively quickly after filling and the pectin will not degrade. However, if the jam is filled into large containers, eg 500 or 1 ,000 kg, the cooling time will be very long. In the centre of such a large container the pectin will be especially subject to degradation, and the gel will be weaker at the centre than at the sides. Consequently, a more slow-setting pectin is generally used for large containers, allowing filling at lower temperatures and thereby avoiding degradation of the pectin.

Sugar is added to jam for various reasons, such as:

1. To provide soluble solids

2. To provide sweetness 3. To provide increased physical, chemical and microbiological stability

4. To provide an improved mouthfeel

5. To provide improved colour and gloss

Sucrose is the sugar normally used, but other sugars may well be used depending on the taste, sweetening effect, crystallisation or structure required. Price may also influence which type of sugar is used.

Invert sugar has the same sweetening effect as sucrose, whereas glucose syrup, glucose and sorbitol have a reduced sweetening effect. High fructose corn syrup and fructose will have a greater sweetening effect than sucrose. The structure and strength of the gel as well as the gelling temperature will, to some extent, be influenced by changes in sugar composition.

Acid is added for two reasons: 1) partly to reduce the pH level to 3.0-3.2 to obtain a satisfactory gel with the pectin, and 2) partly to enhance the flavour of the fruit. The optimum pH for gelation using the HE pectins depends on the type of pectin and solids content in question.

If GRINDSTED™ Pectin SS 200 is used in jam with 65-68% Brix, the optimum pH is 3.0-3.2. If the solids content is higher than this, the optimum pH is 3.1-3.3. Conversely, if the solids content is lower the optimum pH is 2.8-3.0. If GRINDSTED™ Pectin RS 400 is used, the optimum pH is approximately 0.2 units higher than for GRINDSTED™ Pectin SS 200.

The acid most commonly used is citric acid, monohydrate, in a 50% w/v solution.

Other acids (such as malic acid, tartaric acid or phosphoric acid) may be used but must always be in solution.

The choice of acid depends on legislation, price, and the tartness of sweetness required in the finished product.

Citric acid imparts a relatively strong acid taste to the finished product, whereas malic acid results in a softer but longer-lasting taste.

Tartaric acid may result in a slightly bitter taste, and phosphoric acid results in a sweeter taste.

Enzymatically treated pectin can prevent syneresis which can often occur in the manufacture of marmalades and jams with low soluble solids contents. In some instances, the de-acetylated pectin of the present invention is also advantageous for use as a stabiliser and/or viscosity modifier in the preparation of pharmaceuticals, pharmaceutical appliances, cosmetics and cosmetic appliances.

POLYPEPTIDE OF THE PRESENT INVENTION

The term "polypeptide" - which is interchangeabe with the term "protein" - includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means.

Preferably, the polypeptide of the present invention is a single-chain polypeptide.

Polypeptides of the present invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the present invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is a polypeptide of the present invention. Polypeptides of the present invention may be modified for example by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote their secretion from a cell as discussed below.

Polypeptides of the present invention may be produced by synthetic means (e.g. as described by Geysen et ai, 1996) or recombinantly, as described below.

In a preferred embodiment, the amino acid sequence per se the present invention does not cover the native ACE 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. For ease of reference, we have called this preferred embodiment the "non-native amino acid sequence".

The terms "variant", "homologue" or "fragment" in relation to the amino acid sequence for the enzyme of the present invention 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 enzyme has ACE activity, preferably being at least as biologically active as the enzyme shown in the attached sequence listings. In particular, the term "homologue" covers homology with respect to structure and/or function. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to any one of the sequences shown in the attached sequence listings. More preferably there is at least 95%, more preferably at least 98%, homology to any one of the sequence showns as shown in the attached sequence listings.

Typically, the types of amino acid substitutions that could be made should maintain the hydrophobicity/hydrophilicity of the amino acid sequence. Amino acid substitutions may be made, for example from 1 , 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the ability to act as an ACE enzyme in accordance with present invention. Amino acid substitutions may include the use of non-naturally occurring analogues.

The amino acid sequence of the present invention may be produced by expression of a nucleotide sequence coding for same in a suitable expression system.

In addition, or in the alternative, the protein itself could be produced using chemical methods to synthesize an ACE amino acid sequence, in whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure).

Direct peptide synthesis can be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequence of ACE, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant polypeptide.

In another embodiment of the invention, an ACE natural, modified or recombinant sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for inhibitors of ACE activity, it may be useful to encode a chimeric ACE protein expressing a heterologous epitope that is recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between an ACE sequence and the heterologous protein sequence, so that the ACE may be cleaved and purified away from the heterologous moiety.

The ACE may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3 -.26328 1), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and ACE is useful to facilitate purification. Specific amino acid sequences of the ACE are shown as SEQ ID No. 1 - 5. However, the present invention encompasses amino acid sequences encoding other members from the ACE family which would include amino acid sequences having at least 60% identity (more preferably at least 75% identity) to any one of the amino acid sequences.

Polypeptides of the present invention also include fragments of the presented amino acid sequence and variants thereof. Suitable fragments will be at least 5, e.g. at least 10, 12, 15 or 20 amino acids in size.

Polypeptides of the present invention may also be modified to contain one or more (e.g. at least 2, 3, 5, or 10) substitutions, deletions or insertions, including conserved substitutions. These aspects are discussed in a later section.

A variant enzyme according to the present invention may have a pH optimum different to pH 9.5. By way of example, the variant enzyme according to the present invention may have a pH optimum less than pH 9.5.

NUCLEOTIDE SEQUENCE OF THE PRESENT INVENTION

The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be DNA or RNA which may be of genomic or synthetic or recombinant origin which may be double-stranded or single-stranded whether representing the sense or antisense strand.

Preferably, the term "nucleotide sequence" means DNA.

More preferably, the term "nucleotide sequence" means DNA prepared by use of recombinant DNA techniques (i.e. recombinant DNA). In a preferred embodiment, the nucleotide sequence perse of 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. For ease of reference, we have called this preferred embodiment the "non-native nucleotide sequence".

The nucleotide sequences of the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in to enhance the in vivo activity or life span of nucleotide sequences of the present invention.

The present invention also encompasses nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used a probe to identify similar coding sequences in other organisms etc.

The present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences presented herein, or any derivative, fragment or derivative thereof.

The present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. The term "variant" also encompasses sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein.

Preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hydridising under stringent conditions (e.g. 65°C and O.lxSSC {1xSSC = 0.15 M NaCl, 0.015 Na₃ citrate pH 7.0}) to the nucleotide sequences presented herein.

The present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).

The present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).

Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridizing to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.

In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under stringent conditions (e.g. 65°C and O.lxSSC).

Exemplary nucleic acids can alternatively be characterised as those nucleotide sequences which encode an ACE protein and hybridise to any one or more of the DNA sequences shown in the attached sequence listings. Preferred are such sequences encoding ACE which hybridise under high-stringency conditions to any one of the sequences shown in the attached sequence listings or the complement thereof. Advantageously, the invention provides nucleic acid sequences which are capable of hybridising, under stringent conditions, to a fragment of any one of the sequences shown in the attached sequence listings or the complement thereof. Preferably, the fragment is between 15 and 50 bases in length. Advantageously, it is about 25 bases in length.

The terms "variant", "homologue" or "fragment" in relation to the nucleotide sequence coding for the preferred enzyme of the present invention 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 or is capable of coding for an enzyme having ACE activity, preferably being at least as biologically active as the enzyme encoded by any one of the sequences shown in the attached sequence listings. In particular, the term "homologue" covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for or is capable of coding for an enzyme having ACE activity. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to a nucleotide sequence coding for the amino acid sequences presented herein. More preferably there is at least 95%, more preferably at least 98% homology. Preferably, with respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to any one of the sequences shown in the attached sequence listings. More preferably there is at least 95%, more preferably at least 98%, homology to any one of the sequences shown in the attached sequence listings.

As indicated, the present invention relates to a DNA sequence (preferably a cDNA sequence) encoding ACE. In particular, the present invention relates to cDNA sequences encoding ACE.

The present invention also relates to DNA segments comprising the DNA sequence of any one of the sequences shown in the attached sequence listings or allelic variations of such sequences. The present invention also relates to polypeptides produced by expression in a host cell into which has been incorporated the foregoing DNA sequences or allelic variations thereof.

The present invention also relates provides DNA comprising the DNA sequence of any one of the sequences shown in the attached sequence listings or an allelic variation thereof.

The present invention also relates to non-native DNA comprising the DNA sequence of any one of the sequences shown in the attached sequence listings or an allelic variation thereof.

A highly preferred aspect of the present invention relates to recombinant DNA comprising the DNA sequence of any one of the sequences shown in the attached sequence listings or an allelic variation thereof.

Polynucleotides of the present invention include nucleotide acid sequences encoding the polypeptides of the present invention. It will appreciated that a range of different polynucleotides encode a given amino acid sequence as a consequence of the degeneracy of the genetic code.

By knowledge of the amino acid sequences set out herein it is possible to devise partial and full-length nucleic acid sequences such as cDNA and/or genomic clones that encode the polypeptides of the present invention. For example, polynucleotides of the present invention may be obtained using degenerate PCR which will use primers designed to target sequences encoding the amino acid sequences presented herein. The primers will typically contain multiple degenerate positions. However, to minimise degeneracy, sequences will be chosen that encode regions of the amino acid sequences presented herein containing amino acids such as methionine which are coded for by only one triplet. In addition, sequences will be chosen to take into account codon usage in the organism whose nucleic acid is used as the template DNA for the PCR procedure. PCR will be used at stringency conditions lower than those used for cloning sequences with single sequence (non-denegerate) primers against known sequences.

Nucleic acid sequences obtained by PCR that encode polypeptide fragments of the present invention may then be used to obtain larger sequences using hybridization library screening techniques. For example a PCR clone may be labelled with radioactive atoms and used to screen a cDNA or genomic library from other species, preferably other mammalian species. Hybridization conditions will typically be conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C).

Degenerate nucleic acid probes encoding all or part of the amino acid sequence may also be used to probe cDNA and/or genomic libraries from other species, preferably other mammalian species. However, it is preferred to carry out PCR techniques initially to obtain a single sequence for use in further screening procedures.

In accordance with the present invention, ACE polynucleotide sequences which encode ACE, fragments of the polypeptide, fusion proteins or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of ACE in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used to clone and express ACE. As will be understood by those of skill in the art, it may be advantageous to produce ACE-encoding nucleotide sequences possessing non-naturally occurring codons. Codons preferred by a particular prokaryotic or eukaryotic host (Murray E et al (1989) Nuc Acids Res 17:477-508) can be selected, for example, to increase the rate of ACE expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence. Polynucleotide sequences of the present invention obtained using the techniques described above may be used to obtain further homologous sequences and variants using the techniques described above. They may also be modified for use in expressing the polypeptides of the present invention in a variety of host cells systems, for example to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

Altered ACE polynucleotide sequences which may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotide residues resulting in a polynucleotide that encodes the same or a functionally equivalent ACE. The protein may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent ACE. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of ACE is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Included within the scope of the present invention are alleles of ACE. As used herein, an "allele" or "allelic sequence" is an alternative form of ACE. Alleles result from a mutation, i.e., a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

The nucleotide sequences of the present invention may be engineered in order to alter an ACE coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns or to change codon preference.

Polynucleotides of the present invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non- radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the present invention as used herein.

Polynucleotides or primers of the present invention may carry a revealing label. Suitable labels include radioisotopes such as ³²P or ³⁵S, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers of the present invention and may be detected using by techniques known per se.

Polynucleotides such as a DNA polynucleotide and primers according to the present invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the nucleotide sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from a fungal, plant or prokaryotic cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

DNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.

As mentioned earlier, the present invention also relates to nucleotide sequences that are capable of hybridising to all or part of any one of the sequences shown in the attached sequence listings or an allelic variation thereof. These nucleotide sequences may be used in anti-sense techniques to modify ACE expression. Alternatively, these sequences (or portions thereof) can be used as a probe, or for amplifying all or part of such sequence when used as a polymerase chain reaction primer. In an alternative embodiment of the invention, the coding sequence of ACE could be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).

NATURALLY OCCURRING

As used herein "naturally occurring" refers to an ACE with an amino acid sequence found in nature.

ISOLATED/PURIFIED

As used herein, the terms "isolated" and "purified" refer to molecules, either nucleic or amino acid sequences, that are removed from their natural environment and isolated or separated from at least one other component with which they are naturally associated.

BIOLOGICALLY ACTIVE

As used herein "biologically active" refers to an ACE according to the present invention - such as a recombinant ACE - having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) and/or immunological activity (but not necessarily to the same degree) of the naturally occurring ACE.

DERIVATIVE

The term "derivative" as used herein in relation to the amino acid sequence includes chemical modification of an ACE. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. DELETION

As used herein a "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.

INSERTION/ADDITION

As used herein an "insertion" or "addition" is a change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring ACE.

SUBSTITUTION

As used herein "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

HOMOLOGUE

The term "homologue" with respect to the nucleotide sequence of the present invention and the amino acid sequence of the present invention may be synonymous with allelic variations of the sequences.

In particular, the term "homology" as used herein may be equated with the term "identity". Here, sequence homology with respect to the nucleotide sequence of the present invention and the amino acid sequence of the present invention can be determined by a simple "eyeball" comparison (i.e. a strict comparison) of any one or more of the sequences with another sequence to see if that other sequence has at least 75% identity to the sequence(s). Relative sequence homology (i.e. sequence identity) can also be determined by commercially available computer programs that can calculate % homology between two or more sequences. A typical example of such a computer program is CLUSTAL. % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et ai, 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel ef al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel ef al., 1999 ibid, pages 7-58 to 7-60). However, for soem applications it is preferred to use the GCG Bestfit program.

Although the final % homology can be measured in terms of identity, in some cases, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

As indicated, for some applications, sequence homology (or identity) may be determined using any suitable homology algorithm, using for example default parameters. BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (see http://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements. The BLAST programs were tailored for sequence similarity searching, for example to identify homologues to a query sequence. The programs are not generally useful for motif-style searching. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994) Nature Genetics 6:119-129.

Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html.

Advantageously, "substantial homology" when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more. The default threshold for EXPECT in BLAST searching is usually 10.

Should Gap Penalties be used when determining sequence identity, then preferably the following parameters are used:

Other computer program methods to determine identify and similarity between the two sequences include but are not limited to the GCG program package (Devereux et al 1984 Nucleic Acids Research 12: 387 and FASTA (Atschul et al 1990 J Molec Biol 403-410).

POLYPEPTIDE VARIANTS AND DERIVATIVES

The terms "variant" or "derivative" in relation to the amino acid sequences of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence has ACE activity, preferably having at least the same activity as that comprising the polypeptides presented in the sequence listings.

The sequences of the present invention may be modified for use in the present invention. Typically, modifications are made that maintain the ACE activity of the sequence. Amino acid substitutions may be made, for example from 1 , 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the ACE activity.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

As indicated above, proteins of the invention are typically made by recombinant means, for example as described herein, and/or by using synthetic means using techniques well known to skilled persons such as solid phase synthesis. Varaiants and derivatives of such sequences include fusion proteins, wherein the fusion proteins comprise at least the amino acid sequence of the present invention being linked (directly or indirectly) to another amino acid sequence. These other amino acid sequences - which are sometimes referred to as fusion protein partners - will typically impart a favourable functionality - such as to aid extraction and purification of the amino acid sequence of the present invention. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of the present invention so as to allow removal of the latter. Preferably the fusion protein partner will not hinder the function of the protein of the present invention.

POLYNUCLEOTIDE VARIANTS AND DERIVATIVES

The terms "variant" or "derivative" in relation to the nucleotide sequence of the present invention 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 a polypeptide having ACE activity, preferably having at least the same activity as that comprising the sequences presented in the sequence listings.

As indicated above, with respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequences shown in the sequence listing herein. More preferably there is at least 95%, more preferably at least 98%, homology. Nucleotide homology comparisons may be conducted as described above. For some applications, a preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.

As used herein, the terms "variant", "homologue", "fragment" and "derivative" embrace allelic variations of the sequences.

The term "variant" also encompasses sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein.

HYBRIDISATION

The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY) as well as the process of amplification as carried out in polymerase chain reaction technologies as described in Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY).

Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below.

Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the field. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrid which decreases approximately 1 to 1.5°C with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency. As used herein, high stringency refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68 °C

Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe).

High stringency at about 5°C to 10°C below the Tm of the probe. High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 5x Denhardt's, 1 % SDS (sodium dodecyl sulphate), 0.1 Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor. Following hybridisation, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - O.lx SSC, 0.1 % SDS.

Moderate, or intermediate, stringency typically occurs at about 10°C to 20°C below the Tm of the probe.

Low stringency typically occurs at about 20°C to 25°C below the Tm of the probe.

As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.

Moderate stringency refers to conditions equivalent to hybridisation in the above described solution but at about 60-62°C. In that case the final wash is performed at the hybridisation temperature in 1x SSC, 0.1 % SDS.

Low stringency refers to conditions equivalent to hybridisation in the above described solution at about 50-52°C. In that case, the final wash is performed at the hybridisation temperature in 2x SSC, 0.1 % SDS. It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g. formamide-based buffers, and temperatures. Denhardt's solution and SSC are well known to those of skill in the art as are other suitable hybridisation buffers (see, e.g. Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds. (1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridisation conditions have to be determined empirically, as the length and the GC content of the probe also play a role.

Polynucleotides of the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.

The term "selectively hybridizable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide of the invention is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening. In this event, background implies a level of signal generated by interaction between the probe and a nonspecific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³²P.

In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to any one or more of the nucleotide sequences of the present invention under stringent conditions (e.g. 65°C and O.lxSSC {1xSSC = 0.15 M NaCl, 0.015 M Na₃Citrate pH 7.0). Where the polynucleotide of the present invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present invention. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention.

Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.

Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.

Polynucleotides such as a DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from a suitable cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. REGULATORY SEQUENCES

Preferably, the polynucleotide of the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the coding sequence, such as by the chosen host cell. By way of example, the present invention covers a vector comprising the polynucleotide of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals.

The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site.

Enhanced expression of the polynucleotide encoding the polypeptide of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions, which serve to increase expression and, if desired, secretion levels of the protein of interest from the chosen expression host and/or to provide for the inducible control of the expression of the polypeptide of the present invention

Preferably, the nucleotide sequence of the present invention may be operably linked to at least a promoter. Aside from the promoter native to the gene encoding the polypeptide of the present invention, other promoters may be used to direct expression of the polypeptide of the present invention. The promoter may be selected for its efficiency in directing the expression of the polypeptide of the present invention in the desired expression host.

In another embodiment, a constitutive promoter may be selected to direct the expression of the desired polypeptide of the present invention. Such an expression construct may provide additional advantages since it circumvents the need to culture the expression hosts on a medium containing an inducing substrate.

Examples of strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are those which are obtainable from the fungal genes for xylanase (x/πA), phytase, ATP-synthetase, subunit 9 (o//C), those phosphate isomerase (tpi), alcohol dehydrogenase (AdhA), α-amylase (amy), amyloglucosidase (AG - from the glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) promoters.

Examples of strong yeast promoters are those obtainable from the genes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase and triosephosphate isomerase.

Examples of strong bacterial promoters are the α-amylase and SP02 promoters as well as promoters from extracellular protease genes.

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

The promoter can 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 promoter may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention. For example, suitable other sequences include the Sh1-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 one aspect, the nucleotide sequence according to the present invention is under the control of a promoter that 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 tuber, stem, sprout, root and leaf tissues.

In addition the present invention also encompasses combinations of promoters and/or nucleotide sequences coding for proteins or recombinant enzymes and/or elements.

SECRETION

Often, it is desirable for the polypeptide of the present invention to be secreted from the expression host into the culture medium from where the polypeptide of the present invention may be more easily recovered. According to the present invention, the secretion leader sequence may be selected on the basis of the desired expression host. Hybrid signal sequences may also be used with the context of the present invention.

Typical examples of heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (g/aA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces and Kluyveromyces) or the α-amylase gene (Bacillus). CONSTRUCTS

The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes the nucleotide sequence according to the present invention 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 Sh1 -intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In each case, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.

The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a bacterium, such as Bacillus subtilis, or plants 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.

Preferably the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter.

VECTORS

The term "vector" includes expression vectors and transformation vectors and shuttle 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 entity to another entity - which may be of the species or may be of a different species. If the construct is capable of being transferred from one species to another - such as from an E.coli plasmid to a bacterium, such as of the genus Bacillus, then the transformation vector is sometimes called a "shuttle vector". It may even be a construct capable of being transferred from an E.coli plasmid to an Agrobacterium to a plant.

The vectors of the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide of the present invention. Thus, in a further aspect the invention provides a process for preparing polypeptides according to the present invention which comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.

The vectors of the present invention may contain one or more selectable marker genes. The most suitable selection systems for industrial micro-organisms are those formed by the group of selection markers which do not require a mutation in the host organism. Examples of fungal selection markers are the genes for acetamidase (am S), ATP synthetase, subunit 9 (o//C), orotidine-5'-phosphate- decarboxylase (pvrA), phleomycin and benomyl resistance (benA). Examples of non-fungal selection markers are the bacterial G418 resistance gene (this may also be used in yeast, but not in filamentous fungi), the ampicillin resistance gene (£. coli), the neomycin resistance gene (Bacillus) and the E.coli uidA gene, coding for β-glucuronidase (GUS). Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

Thus, polynucleotides of the present invention can be incorporated into a recombinant vector (typically a replicable vector), for example a cloning or expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the present invention by introducing a polynucleotide of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.

The present invention also relates to expression vectors and host cells comprising polynucleotide sequences encoding ACE or variant, homologue, fragment or derivative thereof for the in vivo or in vitro production of ACE protein or to screen for agents that can affect ACE expression or activity.

TISSUE

The term "tissue" as used herein includes tissue perse and organ.

HOST CELLS

The term "host cell" - in relation to the present invention includes any cell that could comprise the nucleotide sequence coding for the recombinant protein according to the present invention and/or products obtained therefrom, wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the host cell. Thus, a further embodiment of the present invention provides host cells transformed or transfected with a polynucleotide of the present invention. Preferably said polynucleotide is carried in a vector for the replication and expression of said polynucleotides. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.

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

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

Depending on the nature of the polynucleotide encoding the polypeptide of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.

Examples of suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species. By way of example, typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae.

The use of suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphoryiation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.

ORGANISM

The term "organism" in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the recombinant protein according to the present invention and/or products obtained therefrom, wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the organism. Examples of organisms may include a fungus, yeast or a plant.

The term "transgenic organism" in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the protein according to the present invention and/or products obtained therefrom, wherein the promoter can allow expression of the nucleotide sequence according to the present invention within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism.

The term "transgenic organism" 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, the present invention does not cover the native protein 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.

Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the amino acid sequence according to the present invention, constructs according to the present invention (including combinations thereof), 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 transformed cell or organism could prepare acceptable quantities of the desired compound which would be easily retrievable from, the cell or organism.

TRANSFORMATION OF HOST CELLS/HOST ORGANISMS

As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.

If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.

In one aspect, preferably the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.

In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression.

The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).

TRANSFORMATION OF ASPERGILLUS

As mentioned above, a preferred host organism is of the genus Aspergillus, such as Aspergillus niger.

A 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 Asperg- illus. In: Martinelli S.D., Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 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.

For almost a century, filamentous fungi have been widely used in many types of industry for the production of organic compounds and enzymes. For example, traditional Japanese koji and soy fermentations have used Aspergillus sp. Also, 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 why filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracelluar 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 the nucleotide sequence according to the present invention (or even the GOI) into a construct designed for expression in filamentous fungi.

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

Another type of expression system has been developed in fungi where the nucleotide sequence according to the present invention (or even the GOI) can be fused to a smaller or a larger part of a fungal gene encoding a stable protein. This can stabilize the protein encoded by the nucleotide sequence according to the present invention (or even 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 nucleotide sequence according to the present invention (or even the GOI), so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein encoded by the nucleotide sequence according to the present invention (or even the GOI). 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 expressed product 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 nucleotide sequence according to the present invention (or even 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 nucleotide sequence according to the present invention (or even the GOI) is equipped with a signal sequence the protein will accumulate extracelluarly.

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 extracelluar 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 commonly 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. TRANSFORMATION OF SACCHAROMYCES CEREVISIAE

In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).

For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.

A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).

Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.

In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. The constructs contain a promoter active in yeast fused to the nucleotide sequence of the present invention, usually a promoter of yeast origin, such as the GAL1 promoter, is used. Usually a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system.

For the transformation of yeast several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163- 168).

The transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1 , and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.

PLANT TRANSFORMATION

Another host organism is a plant. 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). Further teachings on plant transformation may be found in EP-A-0449375.

Even though the enzyme and the nucleotide sequence coding therefor 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 nucleotide sequence or construct according to the present invention and which is capable of introducing the 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 Agrobactehum tumefaciens or a Ri plasmid from Agrobacteήum 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 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 wr region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct. Preferably, the vector system is an Agrobactehum tumefaciens Ti-plasmid or an Agrobactehum 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 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 Agrobactehum strain, e.g. Agrobactehum tumefaciens. The Ti-plasmid harbouring the nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobactehum strain, e.g. A. tumefaciens, so as to obtain an Agrobactehum cell harbouring the 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, the pUC series, the M13 mp series, pACYC 184 etc.

In this way, the nucleotide or construct 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 transformation 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 Agrobactehum 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 Agrobactehum carrying the promoter and/or the GOI, a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobactehum. 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.

CULTURING HOST CELLS

Thus, the present invention also provides a method of transforming a host cell with a nucleotide sequence shown as any one of the sequences shown in the attached sequence listings or a derivative, homologue, variant or fragment thereof.

Host cells transformed with an ACE nucleotide coding sequence may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing ACE coding sequences can be designed with signal sequences which direct secretion of ACE coding sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join ACE coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53, see also above discussion of vectors containing fusion proteins).

PRODUCTION OF THE POLYPEPTIDE

According to the present invention, the production of the polypeptide of the present invention can be effected by the culturing of, for example, microbial expression hosts, which have been transformed with one or more polynucleotides of the present invention, in a conventional nutrient fermentation medium. The selection of the appropriate medium may be based on the choice of expression hosts and/or based on the regulatory requirements of the expression construct. Such media are well-known to those skilled in the art. The medium may, if desired, contain additional components favouring the transformed expression hosts over other potentially contaminating microorganisms.

Thus, the present invention also provides a method for producing a polypeptide having ACE activity, the method comprising the steps of a) transforming a host cell with a nucleotide sequence shown as any one of the sequences shown in the attached sequence listings or a derivative, homologue, variant or fragment thereof; and b) culturing the transformed host cell under conditions suitable for the expression of said polypeptide.

The present invention also provides a method for producing a polypeptide having ACE activity, the method comprising the steps of a) culturing a host cell that has been transformed with a nucleotide sequence shown as any one of the sequences shown in the attached sequence listings or a derivative, homologue, variant or fragment thereof under conditions suitable for the expression of said polypeptide; and b) recovering said polypeptide from the host cell culture. The present invention also provides a method for producing a polypeptide having ACE activity, the method comprising the steps of a) transforming a host cell with a nucleotide sequence shown as any one of the sequences shown in the attached sequence listings or a derivative, homologue, variant or fragment thereof; b) culturing the transformed host cell under conditions suitable for the expression of said polypeptide; and c) recovering said polypeptide from the host cell culture.

DETECTION

The presence of the ACE polynucleotide coding sequence can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, portions or fragments of the sequence presented as any one of the sequences shown in the attached sequence listings. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the ACE coding sequence to detect transformants containing ACE DNA or RNA. As used herein "oligonucleotides" or "oligomers" may refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides which can be used as a probe or amplimer. Preferably, oligonucleotides are derived from the 3' region of the nucleotide sequence shown as any one of the sequences shown in the attached sequence listings.

A variety of protocols for detecting and measuring the expression of ACE polypeptide, such as by using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on ACE polypeptides is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE ef a/ (1983, J Exp Med 15 8:121 1). A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays. Means for producing labelled hybridization or PCR probes for detecting ACE polynucleotide sequences include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled nucleotide. Alternatively, the ACE coding sequence, or any portion of it, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, Wl), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3817837; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A- 4366241. Also, recombinant immunoglobulins may be produced as shown in US- A-4816567.

Additional methods to quantitate the expression of a particular molecule include radiolabeling (Melby PC et al 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. Quantitation of multiple samples may be speeded up by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or calorimetric response gives rapid quantitation.

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed. For example, if the ACE coding sequence is inserted within a marker gene sequence, recombinant cells containing ACE coding regions can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with an ACE coding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of ACE as well.

Alternatively, host cells which contain the coding sequence for ACE and express ACE coding regions may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane-based, solution-based, or chip-based technologies for the detection and/or quantification of the nucleic acid or protein.

ANTIBODIES

The enzyme of present invention can also be used to generate antibodies - such as by use of standard techniques. Thus, antibodies to the enzyme according to the present invention may be raised. The or each antibody can be used to screen for other suitable enzymes according to the present invention. In addition, the or each antibody may be used to isolate amounts of the enzyme of the present invention.

For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc. may be immunized by injection with the inhibitor or any portion, variant, homologue, fragment or derivative thereof or oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacilli Calmette- Guerin) and Corynebactehum parvum are potentially useful human adjuvants which may be employed.

Monoclonal antibodies to the enzyme may be even prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81 :6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies (US-A-4946779) can be adapted to produce inhibitor specific single chain antibodies.

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991 ; Nature 349:293-299).

GOI

The term "GOI" with reference to the combination of constructs according 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-native protein of a filamentous fungus, preferably of the genus Aspergillus, or a compound that is of benefit to animals or humans.

Examples of GOIs include nucleotide sequences encoding pectinases, PMEs, pectin depolymerases, polygalacturonases, pectate lyases, pectin lyases, rhamno- galacturonases, hemicellulases, endo-β-glucanases, arabinases, or acetyl esterases, or combinations thereof, as well as antisense sequences thereof.

By way of example, the GOI can be a PME as disclosed in WO-A-97/03574 or the PME disclosed in either WO-A-94/25575 or WO-A-97/31102 as well as variants, derivatives or homologues of the sequences disclosed in those patent applications.

The GOI may 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, a glucanase or genomic PME.

The GOI may even code for an intron of a particular enzyme but wherein the intron can be in sense or antisense orientation. In the latter instance, the particular enzyme could be genomic ACE. Antisense expression of genomic exon or intron sequences as the GOI would mean that the natural ACE expression would be reduced or eliminated but wherein the recombinant ACE expression would not be affected. This is particularly true for antisense intron or sense intron expression. COMBINATION S OF ENZYMES

The enzyme of the present invention may be used in conjunction with any other suitable enzyme.

Examples of other enzymes include one or more of: pectinases, PMEs, pectin depolymerases, polygalacturonases, pectate lyases, pectin lyases, rhamno- galacturonases, hemicellulases, endo-β-giucanases, arabinases, or acetyl esterases.

These other types of enzymes can be added at the same time as the ACE or, alternatively, prior to or after the addition of the ACE.

GENERAL METHODOLOGY

Although in general the recombinant DNA techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^th Ed, John Wiley & Sons, Inc.

INTRODUCTION TO THE EXAMPLES SECTION

The present invention will now be described by way of example only.

EXPERIMENTAL SECTION

Acetylesterase from orange.

Acetylesterase (ACE) was purified from orange peels by the method published by Christensen et al (1996 Pectin and Pectinases, pages 723-730, J Visser and A. Voragen (Eds), Elsevier Science BV).

In this respect, Spanish Navelina oranges were used for isolation of ACE. The oranges were peeled manually and the peels were stored at -80°C. For extraction of ACE from orange peel, 600g frozen orange peels were thawed and cut into minor pieces. They were homogenized in a Waring blender for 2 min. in 1200 ml buffer (100 mM Na-succinate pH 6.2, 1 mM DTT). Solid NaC1 was added to the homogenate to reach an end-concentration of 3% (w/v) in order to isolate ionically bound proteins. After two hours of incubation with gently stirring at 4°C the suspension was filtered through a nylon mesh and the filtrate was centrifuged at

15,000 x g for 20 min, to remove insoluble material. The supernatant was then fractionated using (NH₄)₂S0 precipitation. The activity was precipitated between

30% and 60% (NH₄)₂S0₄ saturation. The precipitate was resuspended in 50 ml 50 mM MES pH 6.8 1 mM DTT and dialysed against the same buffer over night.

All operations were performed at 4°C.

For chromatographic studies, the dialysed sample was fractionated by cation exchange chromatography. A 40-50 ml sample was applied to a CM-Sepharose CL-6B (1.5 x 15 cm). Unbound proteins were removed with 50 mM MES pH 6.8, 1mM DTT, and the bound proteins were eluted with an increasing NaC1 gradient from 0 - 0.4 M NaC1 in a total volume of 500 ml. The flow was 25 ml/h and fractions of 8.33 ml were collected. The protein profile was measured at 280 nm.

All fractions were then analysed for ACE activity and protein content. The protein content was measured spectrophotometrically according to Bradford using the BioRad protein assay kit with γ-globulin as standard. The fractions containing ACE activity were pooled and concentrated by ultrafiltration using Amicon filter system (YM 10). Desalting of the sample was performed by dialysis against 50mM MES pH 6.8. The ACE preparation (10 ml) was applied to a prepacked Mono S HR 10/10 FPLC column (Pharmacia). The column was equilibrated with 50 mM MES pH 6.8 with a flow of 4 ml/min. ACE was eluted with an increasing NaC1 gradient (0 - 0.3M). Fractions were collected manually according to the protein profile measured at 280 nm. Active fractions were concentrated as described above and buffer exchange to 50 mM Tris pH 7, 1mM DTT, 0.1 M NaC1 was done on the same system as above. The concentrated ACE sample (9 ml) was then applied to a Sephacryl S-200 (2.6 x 70 cm) gel filtration column. The column was equilibrated with the Tris buffer described above. The flow was 40 ml/h and fractions of 5.33 ml were collected. The fractions containing ACE activity were pooled and concentrated.

SDS-Polyacrylamide gelelectrophoresis

The purity of the ACE fraction was investigated by SDS-PAGE using Pharmacia PhastSystem with 10 - 15% SDS-gradient gels. Electrophoresis and silver staining of the proteins were performed as described by the manuals from Pharmacia. For determination of pi IEF 3-9 PhastSystem gels were used.

Sequence Analysis

N-terminal sequence

The mature protein has been N-terminal sequenced and the N-terminal sequence is:

FNVGITYVENAVVKGAV-LDGSPPAYHFDKGFGAGINN Internal peptide sequences

Internal peptide sequences have been obtained by endo-proteinase LysC or trypsin as described in the other applications.

The internal sequences are:

AVGDWYYDR

DVSGASHIEQFY

YEDGASFTGDVEAVNPANNLHFR

ALFPVYT

Cloning of pectin acetyl esterase cDNA from orange.

The amino acid sequences of the acetyl esterase peptides were used to design PCR primers for the isolation of a partial cDNA clone of orange acetyl esterase.

The amino acid sequence V E A V N P was used to generate Primer 1 :

5'-GT(ACGT)GA(AG)GC(ACGT)GT(ACGT)AA(CT)CC

The amino acid sequence H I E Q F Y was used to generate Primer 2:

5'-CA(CT)AT(ACT)GA(AG)CA(AG)TT(CT)TA

The amino acid sequence D W Y Y D R was used to generate Primer 3:

5'-C(GT)(AG)TC(AG)TA(AG)TACCA(AG)TC The amino acid sequence G D W Y Y D was used to generate Primer 4:

5'-TC(AG)TA(AG)TACCA(AG)TC(ACGT)CC

A cDNA library in lambda Zapll prepared from mRNA isolated from the flesh and albedo layer of the orange fruit was used as template for PCR. The first PCR was performed with primer 1 and primer 3, followed by a second nested PCR using primer 2 and primer 4. The PCR product was cloned and sequenced and contained a DNA fragment of 456 bp encoding a 152 amino acid sequence containing some of the peptide sequences and showing homology to a mung bean acetyl esterase.

The cloned PCR fragment was isolated and radiolabelled using the Ready to Go DNA labelling kit (Pharmacia), and used to screen the orange cDNA library. Several hybridizing clones were identified and plasmid DNA was excised in vitro according the suppliers instructions. One of the clones pPAE 11 contained a insert of 1540 bp and was selected for further analysis.

The nucleotide sequence was determined and is shown as SEQ.I.D. NO 1. The sequence comprises 1542 bp with an open reading frame starting at nucleotide 131 and ending at nucleotide 1327 encoding an acetyl esterase of 399 amino acids shown as SEQ.I.D. NO 2.

Enzyme activity

ACE catalyses the cleavage of acetyl groups from different substrates. The enzyme activity was determined by measuring the release of acetic acid. The amount of acetic acid was measured spectrophotometrically using an acetic acid analysis kit (Boehringer, Maunheim). The activity of ACE was measured in 0.6% sugar beet pectin solubilised in 25 mM Na-succinate pH 6.2 and incubated with enzyme fraction in total 500 μl assay. The samples were incubated at 40°C and aliquots were examined after 0, 1 , 2 and 3 hours of incubation. The enzyme reaction was stopped by incubating the samples at 100°C for 5 min. Precipitated protein was removed by centrifugation and the amount of acetic acid in the supernatant was determined.

ACE was also detected by using the substrate triacetin. The enzyme fraction was incubated with 80 mM triacetin in 25 mM Na-succinate pH 6.2. The samples were incubated at 40°C for 30 min. After boiling for 5 min. the samples were analysed for released acetic acid. During purification triacetin was used as substrate.

Antibody production

Antibodies are 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).

Immunohistochemistry

Antibodies raised against the purified ACE were produced. They were then used for th e immunolocaiization of ACE.

ACE had the same overall localisation in the fruits of all the Citrus species investigated. A striking difference comparing the result of ACE and PME histo immuno labelling, was that the staining for ACE was a lot stronger in the juice vesicles. Although there were a tendency for the same stronger labelling of the outermost part of the juice sacs and somewhat less labelling of the juice vesicles. The labelling of orange peel and core had the same intensity as the fruit boats, whereas the peels and core of the other citrus fruits was a little less intensely stained compared with their respective fruit boats. Again we must conclude that the antibody recognize PME^'s or serological similar proteins in the other Citrus fruits.

In tomato, ACE was found throughout the cell wall containing parts of both mature and mature green berries. Judged by the intensity of the staining there is more ACE in the green mature berries than in the mature ones. As with PME there is an elevated staining for ACE in the integument surrounding the seeds in the green mature fruits.

Both apple and potato had intensive staining for ACE throughout the fruits and tubers. Sugar beet leaf bases, hypocotyl and root showed intensive staining for ACE.

Controls with preimmune serum were all negative.

ACE has also been investigated in Mexican lime from flower to small fruits. Here, ACE is found in high amounts in all parts of lime flowers except xylem, the lining of the stylar canals. ACE is found in elevated amounts in papilla of stigma and abcision layers of stigma, pollen and the entire embryosac apart from the embryo itself which is negative. Generally the level of ACE is high in all parts of small fruits (¹ cm long) and gradually with increasing size (5 cm d. fruit) the distribution of ACE resembles that of mature Orange fruits, in the sense that the endocarp is weaker in staining for ACE than the excocarp and innermost endocarp. The inner epidermis of the endocarp and the outermost layer of lamella are initially negative, but become positive in 2 and 5 cm fruits respectively. The immunolocalizations are found in the cell wall and intracellular. The vegetative leaf (young and old) are positive in all cell except xylem. Applications

1. The ACE of the present invention is added to pectin obtained from sugar beet. The ACE de-acetylated the pectin.

The de-acetylated pectin is then used to prepare a foodstuff.

2. Transformed sugar beet was prepared (such as by adapting the teachings of EP-A-0517833) by inserting the ACE coding sequence.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry or related fields are intended to be within the scope of the following claims.

Claims

1. An acetyl esterase (ACE) comprising the amino acid sequence shown as SEQ.I.D. No.2, or a variant, derivative or homologue thereof, including combinations thereof.

2. An ACE according to claim 1 wherein the ACE has the amino acid sequence shown as SEQ.I.D. No.2, or a variant, derivative or homologue thereof.

3. An ACE according to claim 1 or claim 2 wherein the ACE has the amino acid sequence shown as SEQ.I.D. No.2.

4. An ACE according to any one of the preceding claims wherein the ACE has been expressed by a nucleotide sequence comprising the nucleotide sequence shown as SEQ.I.D. No. 1 , or a variant, derivative or homologue thereof, or combinations thereof.

5. An ACE according to any one of the preceding claims wherein the ACE has been expressed by a nucleotide sequence having the nucleotide sequence shown as SEQ.I.D. No. 1 , or a variant, derivative or homologue thereof.

6. An ACE according to any one of the preceding claims according to any one of the preceding claims wherein the ACE has been expressed by a nucleotide sequence having the nucleotide sequence shown as SEQ.I.D. No. 1.

7. An ACE according to any one of the preceding claims wherein the ACE has been prepared by use of DNA techniques.

8. An ACE according to any one of the preceding claims, wherein the ACE is obtainable from a plant.

9. A nucleotide sequence comprising the nucleotide sequence shown as SEQ.I.D. No. 1 , or a variant, derivative or homologue thereof.

10. A nucleotide sequence according to claim 9 wherein the nucleotide sequence is a cDNA.

11. A nucleotide sequence according to claim 9 or claim 10 wherein the nucleotide sequence is obtainable from a plant.

12. A process comprising contacting an acetyl esterase substrate with the enzyme according to any one of claims 1 to 8 or a nucleotide sequence according to any one of claims 9 to 11 or the expression product thereof.

13. A process according to claim 12 wherein the acetyl esterase substrate is or is obtainable from a plant or a plant material.

14. A process according to claim 12 or claim 13 wherein the acetyl esterase substrate is or is obtainable from a fruit or a vegetable.

15. A process according to any one of claims 12 to 14 wherein the acetyl esterase substrate is pectin.

16. A process according to any one of claims 12 to 15 wherein the treated acetyl esterase substrate is suitable for consumption.

17. A foodstuff comprising an ACE treated pectin prepared by the process according to any one of claims 12 to 16.

18. A transformed cell or transfomed organism comprising the enzyme according to any one of claims 1 to 8 or a nucleotide sequence according to any one of claims

9 to 11 or the expression product thereof.

19. A transformed cell or transformed organism according to claim 18 wherein the transformed cell or transformed organism is a transformed plant cell or a transformed plant.

20. A transformed cell or transformed organism according to claim 19 wherein the transformed plant cell or the transformed plant is a transformed sugar beet cell or a transformed sugar beet.

21. Use of the enzyme according to any one of claims 1 to 8, or the expression product of the nucleotide sequence according to any one of claims 9 to 11 , to deacetylate homogalacturonan.