WO2000058447A1 - Exoamylase non maltogene issue de b. clausii et son utilisation permettant de freiner la retrogradation d'un produit d'amidon - Google Patents

Exoamylase non maltogene issue de b. clausii et son utilisation permettant de freiner la retrogradation d'un produit d'amidon Download PDF

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Publication number
WO2000058447A1
WO2000058447A1 PCT/IB2000/000433 IB0000433W WO0058447A1 WO 2000058447 A1 WO2000058447 A1 WO 2000058447A1 IB 0000433 W IB0000433 W IB 0000433W WO 0058447 A1 WO0058447 A1 WO 0058447A1
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Prior art keywords
starch
present
maltogenic exoamylase
sequence
enzyme
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PCT/IB2000/000433
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English (en)
Inventor
Karsten Mathias Kragh
Bjarne Larsen
Lene Duedahl-Olesen
Wolfgang Erwin Karl Zimmermann
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Danisco A/S
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Priority claimed from GBGB9921952.9A external-priority patent/GB9921952D0/en
Priority claimed from PCT/IB1999/000649 external-priority patent/WO1999050399A2/fr
Application filed by Danisco A/S filed Critical Danisco A/S
Priority to AU34502/00A priority Critical patent/AU3450200A/en
Publication of WO2000058447A1 publication Critical patent/WO2000058447A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/24—Organic nitrogen compounds
    • A21D2/26—Proteins
    • A21D2/267—Microbial proteins
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A23L29/35—Degradation products of starch, e.g. hydrolysates, dextrins; Enzymatically modified starches
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
    • A23L7/107—Addition or treatment with enzymes not combined with fermentation with microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)

Definitions

  • the present invention relates to an enzyme (otherwise referred to as a protein).
  • the enzyme (protein) is capable of degrading starch.
  • the present invention relates to the use of a protein that is capable of retarding the detrimental retrogradation of starch.
  • Detrimental retrogradation processes such as staling, typically occur after the heating and cooling of starch media, in particular aqueous starch suspensions, 5 and are due to transformation of gelatinised starch to an increasingly ordered state.
  • the present invention relates to the use of a protein that is capable of retarding the detrimental retrogradation of amylopectin.
  • the present invention relates to the use of a proteins to prepare baked bread products, as well as to the baked bread products themselves.
  • the present invention relates to retardation of staling in baked farinaceous bread products.
  • the present invention relates to a process for making a baked farinaceous bread product having retarded or reduced staling, comprising adding a non-maltogenic exoamylase to the bread dough.
  • the present invention also relates to an improver composition for dough and baked farinaceous bread products comprising a non-maltogenic exoamylase.
  • Starch comprises amylopectin and amylose.
  • Amylopectin is a highly branched carbohydrate polymer with short ⁇ -(1 ⁇ 4)-D-glucan chains which are joined together at branch points through ⁇ -(1 ⁇ 6) linkages forming a branched and bushlike structure. On average, there is one branch point for every 20-25 ⁇ -(1 ⁇ 4) linked glucose residues.
  • amylose is a linear structure mainly consisting of unbranched ⁇ -(1 ⁇ 4)-D-glucan units. Typically, starches contain about 75% amylopectin molecules and about 25% amylose molecules.
  • linear malto-oligosaccharides are composed of 2-10 units of ⁇ - D-glucopyranose linked by an ⁇ -(1- ⁇ 4) bond. Due to their properties such as low sweetness, high waterholding capacity, and prevention of sucrose crystallisation [1] these compounds have potential applications in the food industry.
  • the preparation of malto-oligosaccharides with a degree of polymerisation (DP) above 3 (i.e. DP > 3) in larger amounts is however tedious and expensive.
  • DP1 glucose
  • DP2 maltose
  • DP3 maltotriose
  • DP4 maltotetraose
  • DP5 maltopentaose
  • DP6 maltohexaose
  • DP7 maltoheptaose
  • DP8 maltooctaose
  • DP9 maltononaose
  • DP10 maltodecaose.
  • Amylases are starch-degrading enzymes, classified as hydrolases, which cleave ⁇ -D-(1 ⁇ 4) O-glycosidic linkages in starch.
  • ⁇ -amylases (E.C. 3.2.1.1 , ⁇ -D-(1- ⁇ )-glucan glucanohydrolase) are defined as endo-acting enzymes cleaving ⁇ -D-(1 ⁇ 4) O-glycosidic linkages within the starch molecule in a random fashion [3].
  • the exo-acting amylolytic enzymes such as ⁇ -amylases (E.C.
  • ⁇ -Amylases, ⁇ -glucosidases (E.C. 3.2J .20, -D-glucoside glucohydrolase), giucoamylase (E.C. 3.2.1.3, ⁇ -D-(1 ⁇ 4)-glucan glucohydrolase), and product-specific amylases can produce malto-oligosaccharides of a specific length from starch.
  • amylases producing malto-oligosaccharides of a specific DP have been identified previously including maltohexaose-producing amylases from Klebsiella pneumonia [5, 6], Bacillus subtilis [7], B. circulans G-6 [8], B. circulans F-2 [9, 10], and B. caldovelox [11 , 12]. Maltopentaose-producing amylases have been detected in B. licheniformis 584 [13] and Pseudomonas spp. [14, 15]. Furthermore, maltotetraose-producing amylases have been reported from Pseudomonas stutzeri NRRL B-3389 [16, 17], Bacillus sp. MG-4 [18] and Pseudomonas sp. IMD353 [19] and maltotriose-producing amylases from Streptomyces griseus NA-468 [20] and B. subtilis [21].
  • EP-B 1-298,645 describes a process for preparing exo-maltotetraohydrolase of Pseudomonas stutzeri or P. saccharophila using genetic engineering techniques.
  • US-5,204,254 describes a native and a genetically modified exo-maltopentao- hydrolase of an alkalophilic bacterium (DSM 5853).
  • amylose fraction Upon cooling of freshly baked bread the amylose fraction, within hours, retrogrades to develop a network.
  • This process is beneficial in that it creates a desirable crumb structure with a low degree of firmness and improved slicing properties. More gradually crystallisation of amylopectin takes place within the gelatinised starch granules during the days after baking. In this process amylopectin is believed to reinforce the amylose network in which the starch granules are embedded. This reinforcement leads to increased firmness of the bead crumb. This reinforcement is one of the main causes of bread staling.
  • the rate of detrimental retrogradation or crystallisation of amylopectin depends on the length of the side chains of amylopectin. In accordance with this, cereal amylopectin retrogrades at a slower rate than amylopectin from pea or potato, which has a longer average chain length than cereal amylopectin.
  • amylopectin retrogradation is directly proportional to the mole fraction of side chains with DP 14-24 and inversely proportional to the mole fraction of side chains with DP 6-9.
  • amylopectin In wheat and other cereals the external side chains in amylopectin are in the range of DP 12-19. Thus, enzymatic hydrolysis of the amylopectin side chains can markedly reduce their crystallisation tendencies.
  • glucogenic and maltogenic exo-amylases - such as amylogycosidases which hydrolyse starch by releasing glucose - and maltogenic exoamylases or ⁇ -amylases - which hydrolyse starch by releasing maltose from the non-reducing chain ends.
  • JP-62-79745 and JP-62-79746 state that the use of a ⁇ -amylase produced by Bacillus stearothermophilus and Bacillus megaterium, respectively may be effective in retarding staling of starchy foods, including bread.
  • EP-A-412,607 discloses a process for the production of a bread product having retarded staling properties by the addition to the dough of a thermostable exoamylase, which is not inactivated before gelatinization. Only amyloglycosidases and ⁇ -amylases are listed as suitable exoamylases to be used.
  • the exoamylase is in an amount which is able to modify selectively the crystallisation properties of the amylopectin component during baking by splitting off glucose or maltose from the non-reducing ends of amylose and amylopectin.
  • the exoamylase selectively reduces the crystallisation properties of amylopectin, without substantially effecting the crystallisation properties of amylose.
  • EP-A-494,233 discloses the use of a maltogenic exoamylase to release maltose in the ⁇ -configuration and which is not inactivated before gelatinization in a process for the production of a baked product having retarded staling properties. Only a maltogenic ⁇ -amyiase from Bacillus strain NCIB 11837 is specifically disclosed. Apparently, the maltogenic exoamylase hydrolyses (1 ⁇ 4)- ⁇ -glucosidic linkages in starch (and related polysaccharides) by removing ⁇ -maltose units from the non- reducing ends of the polysaccharide chains in a stepwise manner.
  • Japanese patent application No. 4-27392 discloses a G6 amylase gene obtained from Bacillus H-167. No uses are disclosed for the G6 amylase encoded by said gene.
  • glucogenic exoamylases and maltogenic exoamylases can provide an antistaling effect by selectively reducing the detrimental retrogradation tendencies of amylopectin through shortening of the amylopectin side chains.
  • the present invention relates to a novel protein that is capable of retarding the detrimental retrogradation of starch media, in particular starch gels.
  • the present invention relates to the use of the protein to retard the staling of starch.
  • the present invention relates to the use of the protein to retard the detrimental retrogradation of starch media, such as starch gels.
  • nucleotide sequence of the present invention refers respectively to any one or more of the nucleotide sequences and to any one or more of the amino acid sequences present herein.
  • amino acid sequence refers to peptide or protein sequences and may refer to portions thereof.
  • amino acid sequence of the present invention is synonymous with the phrase “polypeptide sequence of the present invention”.
  • nucleotide sequence of the present invention is synonymous with the phrase “polynucleotide sequence of the present invention”.
  • One aspect of the present invention relates to a non-maltogenic exoamylase obtainable from Bacillus clausii, or a functional equivalent thereof; wherein the enzyme has a molecular weight of about 101 ,000 Da (as estimated by sodium dodecyl sulphate polyacrylamide electrophoresis) and/or the enzyme has an optimum of activity at pH 9.5 and 55°C.
  • the non-maltogenic exoamylase can be further characterised in that it has the ability in a waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto- oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D- glucopyranosyl units.
  • the non-maltogenic exoamylase can be further characterised in that it has an endoamylase activity of less than 0.5 endoamylase units (EAU) per unit of exoamylase activity, preferably wherein the non-maltogenic exoamylase has an endoamylase activity of less than 0.05 endoamylase units (EAU) per unit of exoamylase activity, more preferably wherein the non-maltogenic exoamylase has an endoamylase activity of less than 0.01 endoamylase units (EAU) per unit of exoamylase activity.
  • EAU endoamylase units
  • the exoamylase comprises one or more of the sequences presented as SEQ ID No.s 1 to 5, or a variant, homologue or derivative of any thereof.
  • the exoamylase comprises SEQ ID No. 11 , or a variant, homologue or derivative thereof.
  • Another aspect of the present invention relates to a process for making a starch product comprising adding to a starch medium a non-maltogenic exoamylase according to the present invention.
  • Another aspect of the present invention relates to a baked product obtained by the process according to the present invention.
  • Another aspect of the present invention relates to an improver composition for a dough; wherein the composition comprises a non-maltogenic exoamylase according to the present invention, and at least one further dough ingredient or dough additive.
  • Another aspect of the present invention relates to the use of a non-maltogenic exoamylase according to the present invention in a starch product to retard the staling of the starch product.
  • the starch medium comprises flour, wherein the flour is wheat flour or rye flour or mixtures thereof.
  • the non-maltogenic exoamylase is added in an amount which is in the range of 50 to 100,000 units per kg flour, preferably 100 to 50,000 units per kg flour, more preferably in an amount which is in the range of 200 to 20,000 units per kg flour.
  • At least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 85% of the hydrolysis product is maltotetraose, maltopentaose, maltohexaose, maltoheptaose or maltooctaose.
  • At least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 85% of the hydrolysis product is maltotetraose.
  • At least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 85% of the hydrolysis product is maltohexaose.
  • the starch product is a dough.
  • the starch product is a baked dough.
  • the starch product is for the preparation of a baked farinaceous bread product.
  • the starch product is baked.
  • Another aspect of the present invention relates to a nucleotide sequence encoding a non-maltogenic exoamylase obtainable from Bacillus clausii, or a functional equivalent thereof; wherein the enzyme has a molecular weight of about 101 ,000 Da (as estimated by sodium dodecyl sulphate polyacrylamide electrophoresis) and/or the enzyme has an optimum of activity at pH 9.5 and 55°C.
  • the nucleotide sequence comprises one or more of the sequences presented as SEQ ID No.s 6 to 10, or a variant, homologue or derivative of any thereof.
  • the present invention provides an enzyme that is useful in a process for making a starch product that has a retarded detrimental retrogradation property.
  • the enzyme of the present invention is an amylase enzyme. More in particular, the enzyme of the present invention is a non-maltogenic exoamylase enzyme.
  • a process for making a starch product comprising adding to a starch medium a non- maltogenic exoamylase that is capable of hydroiysing starch by cleaving off linear maltooligosaccharides, predominantly consisting of from four to eight D- glucopyranosyl units, from the non-reducing ends of the side chains of amylopectin.
  • Addition of the non-maltogenic exoamylase to the starch medium may occur prior to and/or during and/or after heating of the starch product.
  • the present invention also provides a baked product obtained by the process according to the present invention.
  • the present invention also provides an improver composition for a dough; wherein the composition comprises a non-maltogenic exoamylase, and at least one further dough ingredient or dough additive.
  • the present invention also provides the use of a non-maltogenic exoamylase in a starch product to retard the detrimental retrogradation of the starch product.
  • starch means starch per se or a component thereof, especially amylopectin.
  • starch medium means any suitable medium comprising starch.
  • starch product means any product that contains or is based on or is derived from starch.
  • the starch product contains or is based on or is derived from starch obtained from wheat flour.
  • the starch product is a bakery product.
  • the starch product is a bread product.
  • the starch product is a baked farinaceous bread product.
  • wheat flour as used herein is a synonym for the finely-ground meal of wheat or other grain. Preferably, however, the term means flour obtained from wheat per se and not from another grain. Thus, and unless otherwise expressed, references to “wheat flour” as used herein preferably mean references to wheat flour per se as well as to wheat flour when present in a medium, such as a dough.
  • a preferred flour is wheat flour or rye flour or mixtures of wheat and rye flour.
  • dough comprising flour derived from other types of cereals such as for example from rice, maize, barley, and durra are also contemplated.
  • baked farinaceous bread product is understood to refer to any baked product based on ground cereals and baked on a dough obtainable by mixing flour, water, and a leavening agent under dough forming conditions. It is, however, within the scope of the present invention that further components can be added to the dough mixture.
  • amylase is used in its normal sense - e.g. an enzyme that is inter alia capable of catalysing the degradation of starch.
  • hydrolases which are capable of cleaving ⁇ -D-(1 ⁇ 4) O-glycosidic linkages in starch.
  • a suitable assay for determining amylase activity in accordance with the present invention is presented herein.
  • this assay is called the "Amylase Assay Protocol”.
  • waxy maize amylopectin (obtainable as WAXILYS 200 from Roquette, France) is a starch with a very high amylopectin content (above 90%).
  • One unit of the non-maltogenic exoamylase is defined as the amount of enzyme which releases hydrolysis products equivalent to 1 ⁇ mol of reducing sugar per min. when incubated at 50° C in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calcium chloride, pH 6.0 prepared as described above.
  • Reducing sugars are measured using maltose as standard and using the dinitrosalicylic acid method of Bernfeld, Methods Enzymol., (1954), 1, 149-158 or another method known in the art for quantifying reducing sugars.
  • the hydrolysis product pattern of the non-maltogenic exoamylase is determined by incubating 0.7 units of non-maltogenic exoamylase for 15 or 300 min. at 50 °C in a test tube with 4 ml of 10 mg/ml waxy maize starch in the buffer prepared as described above. The reaction is stopped by immersing the test tube for 3 min. in a boiling water bath.
  • the hydrolysis products are analyzed and quantified by anion exchange HPLC using a Dionex PA 100 column with sodium acetate, sodium hydroxide and water as eluents, with pulsed amperometric detection and with known linear maltooligosaccharides of from glucose to maltoheptaose as standards.
  • the response factor used for maltooctaose to maltodecaose is the response factor found for maltoheptaose.
  • non-maltogenic exoamylase enzyme means the enzyme does not initially degrade starch to substantial amounts of maltose. In a highly preferred aspect, the term also means the enzyme does not initially degrade starch to substantial amounts of maltose and glucose.
  • non-maltogenic exoamylase enzyme means that the enzyme does not initially degrade starch to substantial amounts of maltose as analysed in accordance with the product determination procedure as described in the "Amylase Assay Protocol" presented herein.
  • the non-maltogenic exoamylase can be characterised in that if an amount of 0.7 units of said non-maltogenic exoamylase were to incubated for
  • hydrolysis product(s) that would consist of one or more linear malto- oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis products would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D- glucopyranosyl units.
  • the feature of incubating an amount of 0.7 units of the non-maltogenic exoamylase for 15 minutes at a temperature of 50°C at pH 6.0 in 4 ml of an aqueous solution of 10 mg preboiled waxy maize starch per ml buffered solution containing 50 mM 2-(N- morpholino)ethane sulfonic acid and 2 mM calcium chloride may be referred to as the "waxy maize starch incubation test".
  • a preferred non-maltogenic exoamylase is characterised as having the ability in the waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear maltooligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D- glucopyranosyl units.
  • the hydrolysis products in the waxy maize starch incubation test include one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose.
  • the hydrolysis products in the waxy maize starch incubation test may also include other hydrolytic products. Nevertheless, the % weight amounts of linear maltooligosaccharides of from three to ten D-glucopyranosyl units are based on the amount of the hydrolysis product that consists of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose.
  • the % weight amounts of linear maltooligosaccharides of from three to ten D-glucopyranosyl units are not based on the amount of hydrolysis products other than one or more linear maltooligosaccharides of from two to ten D-glucopyranosyl units and glucose.
  • the hydrolysis products can be analysed by any suitable means.
  • the hydrolysis products may be analysed by anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and with, for example, known linear maltooligosaccharides of from glucose to maltoheptaose as standards.
  • the feature of analysing the hydrolysis product(s) using anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and with known linear maltooligosaccharides of from glucose to maltoheptaose used as standards can be referred to as "analysing by anion exchange”.
  • anion exchange the feature of analysing the hydrolysis product(s) using anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and with known linear maltooligosaccharides of from glucose to maltoheptaose used as standards.
  • a preferred non-maltogenic exoamylase is characterised as having the ability in a waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto- oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose, said hydrolysis products being capable of being analysed by anion exchange; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D- glucopyranosyl units.
  • linear malto- oligosaccha de is used in the normal sense as meaning 2-10 units of ⁇ -D- glucopyranose linked by an ⁇ -(1->4) bond.
  • the term "obtainable from Bacillus clausii" means that the enzyme need not necessarily be obtained from Bacillus clausii. Instead, the enzyme could be prepared by use of recombinant DNA techniques.
  • the term "functional equivalent thereof in relation to the enzyme being obtainable from Bacillus clausii” 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 non-maltogenic exoamylase activity.
  • the functionally equivalent enzyme may have a different chemical structure and/or formula but will have non-maltogenic exoamylase activity.
  • the functionally equivalent enzyme need not necessarily have exactly the same non-maltogenic exoamylase activity as the non-maltogenic exoamylase enzyme obtained from Bacillus clausii.
  • the functionally equivalent enzyme has at least the same activity profile as the enzyme obtained from Bacillus clausii (such as the reactivity profile shown in the attached Figures).
  • 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.
  • 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 al., 1996) or recombinantly, as described below.
  • the amino acid sequence per se the present invention does not cover the native non-maltogenic exoamylase according to the present invention when it is in its natural environment and when it has been expressed by its native nucleotide coding sequence which is also in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment.
  • the "non-native amino acid sequence” we have called this preferred embodiment the "non-native amino acid sequence".
  • variant 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 non- maltogenic exoamylase activity, preferably being at least as biologically active as the enzyme shown in the attached sequence listings.
  • 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.
  • 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 a non-maltogenic exoamylase enzyme in accordance with present invention.
  • Amino acid substitutions may include the use of non-naturally occurring analogues.
  • amino acid sequence of the present invention may be produced by expression of a nucleotide sequence coding for same in a suitable expression system.
  • the protein itself could be produced using chemical methods to synthesize a non-maltogenic exoamylase amino acid sequence, in whole or in part.
  • 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 non-maltogenic exoamylase, 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.
  • a non-maltogenic exoamylase natural, modified or recombinant sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a fusion protein may also be engineered to contain a cleavage site located between a non-maltogenic exoamylase sequence and the heterologous protein sequence, so that the non- maltogenic exoamylase may be cleaved and purified away from the heterologous moiety.
  • the non-maltogenic exoamylase may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • 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 non-maltogenic exoamylase is useful to facilitate purification.
  • non-maltogenic exoamylase Specific amino acid sequences of the non-maltogenic exoamylase are shown as SEQ ID No. 1 - 5. However, the present invention encompasses amino acid sequences encoding other members from the non-maltogenic exoamylase 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. Preferably the non-maltogenic exoamylase has amino acid sequence according to SEQ ID No. 11.
  • 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.
  • the variant enzyme according to the present invention may have a pH optimum less than pH 9.5.
  • nucleotide sequence 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.
  • nucleotide sequence means DNA.
  • nucleotide sequence means DNA prepared by use of recombinant DNA techniques (i.e. recombinant DNA).
  • the nucleotide sequence per se 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.
  • 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.
  • 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.
  • variant also encompasses sequences that are complementary to sequences that are capable of hydridising 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).
  • polynucleotide sequences that are capable of hybridizing to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.
  • 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 OJxSSC).
  • nucleic acids can alternatively be characterised as those nucleotide sequences which encode a non-maltogenic exoamylase protein and hybridise to any one or more of the DNA sequences shown in the attached sequence listings. Preferred are such sequences encoding non-maltogenic exoamylase which hybridise under high-stringency conditions to any one of the sequences shown in the attached sequence listings or the complement thereof.
  • 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.
  • the fragment is between 15 and 50 bases in length.
  • it is about 25 bases in length.
  • variant 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 non- maltogenic exoamylase activity, preferably being at least as biologically active as the enzyme encoded by any one of the sequences shown in the attached sequence listings.
  • 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 non-maltogenic exoamylase activity.
  • 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.
  • 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.
  • the present invention relates to a DNA sequence (preferably a cDNA sequence) encoding non-maltogenic exoamylase.
  • the present invention relates to cDNA sequences encoding non-maltogenic exoamylase.
  • 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.
  • nucleic acid sequences such as cDNA and/or genomic clones that encode the polypeptides of the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • non-maltogenic exoamylase polynucleotide sequences which encode non-maltogenic exoamylase, fragments of the polypeptide, fusion proteins or functional equivalents thereof may be used to generate recombinant DNA molecules that direct the expression of non- maltogenic exoamylase 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 non-maltogenic exoamylase. As will be understood by those of skill in the art, it may be advantageous to produce non-maltogenic exoamylase-encoding nucleotide sequences possessing non-naturally occurring codons.
  • Codons preferred by a particular prokaryotic or eukaryotic host can be selected, for example, to increase the rate of non- maltogenic exoamylase 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 non-maltogenic exoamylase 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 non-maltogenic exoamylase.
  • the protein may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent non-maltogenic exoamylase.
  • 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 non-maltogenic exoamylase is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • an "allele” or “allelic sequence” is an alternative form of non-maltogenic exoamylase. 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.
  • nucleotide sequences of the present invention may be engineered in order to alter a non-maltogenic exoamylase 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.
  • 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.
  • 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 32 P or 35 S, 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.
  • 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.
  • 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 non-maltogenic exoamylase expression.
  • 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.
  • the coding sequence of non- maltogenic exoamylase 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 refers to a non-maltogenic exoamylase with an amino acid sequence found in nature.
  • 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 refers to a non-maltogenic exoamylase according to the present invention - such as a recombinant non-maltogenic exoamylase - 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 non-maltogenic exoamylase.
  • 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.
  • 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 non- maltogenic exoamylase.
  • substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
  • 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.
  • 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
  • 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.
  • ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
  • 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.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • sequence homology may be determined using any suitable homology algorithm, using for example default parameters.
  • BLAST Basic Local Alignment Search Tool
  • blastp, blastn, biastx, 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.
  • 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.
  • substantially 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.
  • 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 non-maltogenic exoamylase activity, preferably having at least the same activity as that comprising the polypeptides presented in the sequence listings.
  • sequences of the present invention may be modified for use in the present invention. Typically, modifications are made that maintain the non-maltogenic exoamylase 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 non-maltogenic exoamylase activity.
  • 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.
  • fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteoiytic 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.
  • variants or derivatives 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 non-maltogenic exoamylase activity, preferably having at least the same activity as that comprising the sequences presented in the sequence listings.
  • 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.
  • variant As used herein, the terms “variant”, “homologue”, “fragment” and “derivative” embrace allelic variations of the sequences.
  • variant also encompasses sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein.
  • hybridization 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.
  • 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), 0J Na+ pyrophosphate and 0J mg/ml denatured salmon sperm DNA as non specific competitor.
  • high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - OJx 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.
  • 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, 0J % 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.
  • background implies a level of signal generated by interaction between the probe and a non ⁇ specific 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 32 P.
  • both strands of the duplex are encompassed by the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • regulatory sequences includes promoters and enhancers and other expression regulation signals.
  • 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
  • heterologous regulatory regions e.g. promoter, secretion leader and terminator regions
  • the nucleotide sequence of the present invention may be operably linked to at least a promoter.
  • 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.
  • 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.
  • 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 (xlnA), phytase, ATP-synthetase, subunit 9 (o/.C), those phosphate isomerase (tpi), alcohol dehydrogenase (AdhA), ⁇ -amylase (amy), amyloglucosidase (AG - from the g/aA gene), acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) promoters.
  • strong yeast promoters are those obtainable from the genes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase and triosephosphate isomerase.
  • strong bacterial promoters are the ⁇ -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.
  • 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.
  • 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.
  • 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).
  • the polypeptide of the present invention may be secreted from the expression host into the culture medium from where the polypeptide of the present invention may be more easily recovered.
  • 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.
  • heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces and Kluyveromyces) or the ⁇ -amylase gene (Bacillus).
  • AG fungal amyloglucosidase
  • a-factor gene e.g. Saccharomyces and Kluyveromyces
  • ⁇ -amylase gene Bacillus
  • 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.
  • 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, preferably of the genus Bacillus, such as Bacillus subtilis, or plants into which it has been transferred.
  • a marker which allows for the selection of the genetic construct in, for example, a bacterium, preferably of the genus Bacillus, such as Bacillus subtilis, or plants into which it has been transferred.
  • markers which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
  • the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter.
  • vector includes expression vectors and transformation vectors and shuttle vectors.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • transformation vector means a construct capable of being transferred from one 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.
  • 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.
  • fungal selection markers are the genes for acetamidase (amdS), ATP synthetase, subunit 9 (o/.C), orotidine-5'-phosphate- decarboxylase (pvrA), phleomycin and benomyl resistance (benA).
  • 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 (E. coli), the neomycin resistance gene (Bacillus) and the E.coli uidA gene, coding for ⁇ -glucuronidase (GUS).
  • Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • 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.
  • 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 non-maltogenic exoamylase or variant, homologue, fragment or derivative thereof for the in vivo or in vitro production of non-maltogenic exoamylase protein or to screen for agents that can affect non- maltogenic exoamylase expression or activity.
  • tissue as used herein includes tissue perse and organ.
  • 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.
  • a further embodiment of the present invention provides host cells transformed or transfected with a polynucleotide of the present invention.
  • a polynucleotide of the present invention 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.
  • E. coli 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.
  • eukaryotic hosts such as yeasts or other fungi may be preferred.
  • yeast cells are preferred over fungal cells because they are easier to manipulate.
  • 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.
  • 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.
  • typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var.
  • 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 phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
  • post-translational modifications e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
  • 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.
  • organisms may include a fungus, yeast or a plant.
  • 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.
  • the nucleotide sequence is incorporated in the genome of the organism.
  • 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.
  • 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.
  • 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.
  • the host organism can be a prokaryotic or a eukaryotic organism.
  • 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.
  • nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
  • the transgenic organism can be a yeast.
  • 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).
  • 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.
  • yeast vectors include integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
  • expression constructs are prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast.
  • 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.
  • a promoter of yeast origin such as the GAL1 promoter
  • 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.
  • transgenic Saccharomyces 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.
  • 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.
  • 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.
  • 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 a non-maltogenic exoamylase 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.
  • expression vectors containing non-maltogenic exoamylase coding sequences can be designed with signal sequences which direct secretion of non- maltogenic exoamylase coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • 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.
  • the present invention also provides a method for producing a polypeptide having non-maltogenic exoamylase 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 non-maltogenic exoamylase 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 non-maltogenic exoamylase 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 non-maltogenic exoamylase 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 non- maltogenic exoamylase coding sequence to detect transformants containing non- maltogenic exoamylase DNA or RNA.
  • oligonucleotides 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.
  • 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 non- maltogenic exoamylase polypeptide are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on non-maltogenic exoamylase 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 er al (1983, J Exp Med 15 8:121 1).
  • Means for producing labelled hybridization or PCR probes for detecting non-maltogenic exoamylase polynucleotide sequences include oligolabelling, nick translation, end- labelling or PCR amplification using a labelled nucleotide.
  • the non- maltogenic exoamylase 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.
  • 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.
  • 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.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed.
  • the non-maltogenic exoamylase coding sequence is inserted within a marker gene sequence, recombinant cells containing non-maltogenic exoamylase coding regions can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a non- maltogenic exoamylase coding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of non-maltogenic exoamylase as well.
  • host cells which contain the coding sequence for non-maltogenic exoamylase and express non-maltogenic exoamylase 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.
  • one aspect of the present invention relates to an improver composition for a starch product, in particular a dough and/or a baked farinaceous bread product made from the dough.
  • the improver composition comprises a non-maltogenic exoamylase according to the present invention and at least one further dough ingredient or dough additive.
  • the further dough ingredient or dough additive can be any of the dough ingredients and dough additives which are described above.
  • the improver composition is a dry pulverulent composition comprising the non-maltogenic exoamylase according to the invention admixed with at least one further ingredient or additive.
  • the improver composition may also be a liquid preparation comprising the non-maltogenic exoamylase according to the invention and at least one further ingredient or additive dissolved or dispersed in water or other liquid. It will be understood that the amount of enzyme activity in the improver composition will depend on the amounts and types of the further ingredients and additives which form part of the improver composition.
  • the improver composition may be in the form of a complete mixture, a so-called pre-mixture, containing all of the dry ingredients and additives for making a particular baked product.
  • the process comprises forming the starch product by adding the novel non-maltogenic exoamylase enzyme presented herein, to a starch medium.
  • the starch medium is a dough
  • the dough is prepared by mixing together flour, water, the non-maltogenic exoamylase according to the invention and other possible ingredients and additives.
  • the process comprises mixing - in any suitable order - flour, water, and a leavening agent under dough forming conditions and further adding a suitable non-maltogenic exoamylase enzyme.
  • the leavening agent may be a chemical leavening agent such as sodium bicarbonate or any strain of Saccharomyces cerevisiae (Baker's Yeast).
  • the non-maltogenic exoamylase can be added together with any dough ingredient including the water or dough ingredient mixture or with any additive or additive mixture.
  • the dough can be prepared by any conventional dough preparation method common in the baking industry or in any other industry making flour dough based products.
  • Baking of farinaceous bread products such as for example white bread, bread made from bolted rye flour and wheat flour, rolls and the like is typically accomplished by baking the bread dough at oven temperatures in the range of from 180 to 250°C for about 15 to 60 minutes.
  • a steep temperature gradient 200->120°C
  • the temperature in the crumb is only close to 100°C at the end of the baking process.
  • the non-maltogenic exoamylase can be added as a liquid preparation or as a dry pulverulent composition either comprising the enzyme as the sole active component or in admixture with one or more additional dough ingredient or dough additive.
  • dough ingredients and/or dough additives may be incorporated into the dough.
  • further added components may include dough ingredients such as salt, grains, fats and oils, sugar, dietary fibre substances, milk powder, gluten and dough additives such as emulsifiers, other enzymes, hydrocolloids, flavouring agents, oxidising agents, minerals and vitamins.
  • the emulsifiers are useful as dough strengtheners and crumb softeners. As dough strengtheners, the emulsifiers can provide tolerance with regard to resting time and tolerance to shock during the proofing. Furthermore, dough strengthe- ners will improve the tolerance of a given dough to variations in the fermentation time. Most dough strengtheners also improve on the oven spring which means the increase in volume from the proofed to the baked goods. Lastly, dough strengtheners will emulsify any fats present in the recipe mixture.
  • the crumb softening which is mainly a characteristic of the monoglycerides, is attributed to an interaction between the emulsifier and the amylose fraction of the starch leading to formation of insoluble inclusion complexes with the amylose which will not recrystallize upon cooling and which will not therefore contribute to firmness of the bread crumb.
  • Suitable emulsifiers which may be used as further dough additives include lecithin, polyoxyethylene stearat, mono- and diglycerides of edible fatty acids, acetic acid esters of mono- and diglycerides of edible fatty acids, lactic acid esters of mono- and diglycerides of edible fatty acids, citric acid esters of mono- and diglycerides of edible fatty acids, diacetyl tartaric acid esters of mono- and diglycerides of edible fatty acids, sucrose esters of edible fatty acids, sodium stearoyl-2-lactylate, and calcium stearoyl-2-lactylate.
  • oxidoreductases such as glucose oxidase, hexose oxidase, and ascorbate oxidase
  • hydrolases such as lipases and esterases as well as glycosidases like a- amylase, pullulanase, and xyianase.
  • Oxidoreductases such as for example glucose oxidase and hexose oxidase, can be used for dough strengthening and control of volume of the baked products and xylanases and other hemicellulases may be added to improve dough handling properties, crumb softness and bread volume.
  • Lipases are useful as dough strengtheners and crumb softeners and a- amylases and other amylolytic enzymes may be incorporated into the dough to control bread volume and further reduce crumb firmness.
  • the amount of the non-maltogenic exoamylase according to the present invention that is added is normally in an amount which results in the presence in the finished dough of 50 to 100,000 units per kg of flour, preferably 100 to 50,000 units per kg of flour. In useful embodiments of the present invention, the amount is in the range of 200 to 20,000 units per kg of flour.
  • 1 unit of the non-maltogenic exoamylase is defined as the amount of enzyme which releases hydrolysis products equivalent to 1 ⁇ mol of reducing sugar per min. when incubated at 50° C in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calcium chloride, pH 6.0 as described hereinafter.
  • the present invention provides suitable amylases for use in the manufacture of a foodstuff.
  • Typical foodstuffs which also include animal feed, include dairy products, meat products, poultry products, fish products and bakery products.
  • the foodstuff is a bakery product, such as the bakery products described above.
  • Typical bakery (baked) products incorporated within the scope of the present invention include bread - such as loaves, rolls, buns, pizza bases etc. - pretzels, tortillas, cakes, cookies, biscuits, krackers etc.
  • 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 amylase enzymes according to the present invention.
  • the or each antibody may be used to isolate amounts of the enzyme of the present invention.
  • 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 oiigopeptide which retains immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • 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
  • Corynebacterium 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).
  • 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).
  • the present invention provides a novel amylase that is suitable for preparing starch products according to the present invention, such as bakery products.
  • the novel amylase of the present invention is a non-maltogenic exoamylase.
  • the present invention is based on the surprising finding that it is possible to isolate an enzyme from Bacillus - which enzyme is a non-maltogenic exoamylase that is effective in retarding or reducing detrimental retrogradation, such as staling, in starch products, in particular baked products.
  • non-maltogenic exoamylase according to the present invention may be more effective in retarding detrimental retrogradation, such as staling, in bread than the glucogenic and maltogenic exoamylases.
  • the present invention also provides a non-maltogenic exoamylase, wherein the non-maltogenic exoamylase is further characterised in that it has the ability in a waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D- glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units; wherein the enzyme is obtainable from Bacillus clausii or is a functional equivalent thereof; and wherein the enzyme has a molecular weight of about 101 ,000 Da (as estimated by sodium dodecyl
  • the amylase is in an isolated form and/or in a substantially pure form.
  • isolated means that the enzyme is not in its natural environment.
  • the present invention provides the use of the amylase of the present invention to prepare starch products, such as bakery products.
  • the amylases - which are non-maltogenic exoamylases - retard or reduce the staling properties (i.e. lower the rate of staling) of the starch product, in particular a baked farinaceous bread product.
  • the non-maltogenic exoamylase enzyme of the present invention does not initially degrade starch to substantial amounts of maltose.
  • the non-maltogenic exoamylase is capable of cleaving off linear maltooligosaccharides, predominantly consisting of from four to eight D-glucopyranosyl units, from the non-reducing ends of the side chains of amylopectin.
  • Non-maltogenic exoamylases having this characteristic and which are suitable for use in the present invention are identified by their ability to hydrolyse gelatinised waxy maize starch in the model system presented in the Amylase Assay Protocol (infra).
  • the non-maltogenic exoamylases which are suitable for use according to the present invention would yield a hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose, such that the product pattern of that hydrolysis product would consist of at least 60%, in particular at least 70%, more preferably at least 80% and most preferably at least 90% by weight of starch hydrolysis degradation products other than maltose and glucose.
  • the non-maltogenic exoamylases which are suitable for use according to the present invention would provide when incubated 15 min. under the described conditions for the waxy maize starch incubation test the said hydrolysis product, such that the hydrolysis product would have a product pattern of at least 60%, in particular at least 70%, more preferably at least 80% and most preferably at least 90% by weight of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, in particular linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units.
  • the said hydrolysis product in said test would have a product pattern of at least 60%, in particular at least 70%, more preferably at least 80% and most preferably at least 85% by weight of linear maltooligosaccharides of 4 or 6 D-glucopyranosyl units.
  • the said hydrolysis product in said test would have a product pattern of at least 60%, in particular at least 70%, more preferably at least 80% and most preferably at least 85% by weight of linear maltooligosaccharides of 6 D-glucopyranosyl units.
  • the non-maltogenic exoamylase does not substantially hydrolyze its primary products to convert them to glucose, maltose and maltotriose. If that were the case, the primary products would compete as substrates with the amylopectin non-reducing chain ends for the enzyme, so that its anti-retrogradation efficiency would be reduced.
  • the non-maltogenic exoamylase useful in the process of the present invention can be characterised in that it has the ability in a waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85%o by weight of the said hydrolysis product(s) would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units; wherein the enzyme is obtainable from Bacillus clausii or is a functional equivalent thereof; and wherein the enzyme has a molecular weight of about 101 ,000 Da (as estimated by sodium dodecyl s
  • the non-maltogenic exoamylases which are suitable for use according to the present invention are active during baking and hydrolyse starch during and after the gelatinization of the starch granules which starts at temperatures of about 55°C.
  • the non-maltogenic exoamylase is preferentially gradually inactivated so that there is substantially no activity after the baking process in the final bread. Therefore preferentially the non-maltogenic exoamylases suitable for use according to the present invention have an optimum temperature above 45°C and below 98°C when incubated for 15 min.
  • the optimum temperature of the non-maltogenic exoamylase is above 55°C and below 95°C and even more preferably it is above 60°C and below 90°C.
  • Non-maltogenic exoamylases which may be found to be less thermostable can be improved by using protein engineering to become more thermostable and thus better suited for use according to present the invention.
  • the use of non- maltogenic exoamylases modified to become more thermostable by protein engineering is encompassed by the present invention.
  • non-maltogenic exoamylases can have some degree of endoamylase activity. In some cases, this type of activity may need to be reduced or eliminated since endoamylase activity can possibly negatively effect the quality of the final bread product by producing a sticky or gummy crumb due to the accumulation of branched dexthns.
  • the non-maltogenic exoamylase of the present invention will have less than 0.5 endoamylase units (EAU) per unit of exoamylase activity.
  • EAU endoamylase units
  • non-maltogenic exoamylases which are suitable for use according to the present invention have less than 0.05 EAU per unit of exoamylase activity and more preferably less than 0.01 EAU per unit of exoamylase activity.
  • the endoamylase units can be determined by use of the Endoamylase Assay Protocol presented below.
  • 0.75 ml of enzyme solution is incubated with 6.75 ml of 0.5% (w/v) of AZCL- amylose (azurine cross-linked amylose available from Megazyme, Ireland) in 50 mM MES (2-(N-morpholino)ethanesulfonic acid), 2 mM calcium chloride, pH 6.0 at 50 °C. After 5, 10, 15, 20 and 25 minutes, respectively 1.0 ml of reaction mix is transferred to 4.0 ml of stop solution consisting of 4% (w/v) TRIS (Tris(hydroxy- methyl)aminomethane).
  • the stopped sample is filtered through a Whatman No. 1 filter and its optical density at 590 nm is measured against distilled water.
  • the enzyme solution assayed should be diluted so that the optical density obtained is a linear function of time.
  • the slope of the line for optical density versus time is used to calculate the endoamylase activity relative to the standard GRINDAMYLTM A1000 (available from Danisco Ingredients), which is defined to have 1000 endoamylase units (EAU) per g.
  • the enzyme of the present invention may be used in conjunction with any other suitable enzyme. If that other enzyme is also a non-maltogenic exoamylase, then examples of such non-maltogenic exoamylases include exo-maltotetraohydrolase (E.C.3.2J .60), exo-maltopentaohydrolase and exo-maltohexaohydrolase (E.C.3.2J .98) which hydrolyze 1 ,4- ⁇ -glucosidic linkages in amylaceous polysaccharides so as to remove successive residues of maltotetraose, maltopentaose or maltohexaose, respectively, from the non-reducing chain ends.
  • exo-maltotetraohydrolase E.C.3.2J .60
  • exo-maltopentaohydrolase E.C.3.2J .98
  • exo-maltotetraohydrolases of Pseudomonas saccharophila and P. stutzeri EP- 0 298 645 B1
  • exo-maltopentaohydrolases of an alkaliphilic Gram-positive bacterium US-5,204,254
  • Pseudomonas sp. Shida er al., Biosci. Biotechnol. Biochem., 1992, 56, 76-80
  • exo-maltohexaohydrolases of Bacillus sp. #707 Tsukamoto et al., Biochem. Biophys. Res. Commun., 1988, 151, 25-31
  • non-maltogenic exoamylase is the exoamylase from an alkalophilic Bacillus strain, GM8901 (28). This is a non-maltogenic exoamylase which produces maltotetraose as well as maltopentaose and maltohexaose from starch.
  • exo-maltoheptaohydrolase or exo-maltooctaohydrolase which hydrolyze 1 ,4- ⁇ - glucosidic linkages in amylaceous polysaccharides so as to remove residues of maltoheptaose or maltooctaose, respectively, from the non-reducing chain ends.
  • Exo-maltoheptaohydrolase and exo-maltooctaohydrolase can be found either by screening wild type strains or can be developed from other amylolytic enzymes by protein engineering.
  • the crumb firmness can be measured 1 , 3 and 7 days after baking by means of an instron 4301 Universal Food Texture Analyzer or similar equipment known in the art.
  • Another method used traditionally in the art and which is used to evaluate the effect on starch retrogradation of a non-maltogenic exoamylase according to the present invention is based on DSC (differential scanning calorimetry).
  • DSC differential scanning calorimetry
  • the DSC equipment applied in the described examples is a Mettler-Toledo DSC 820 run with a temperature gradient of 10°C per min. from 20 to 95°C.
  • For preparation of the samples 10-20 mg of crumb are weighed and transferred into Mettler- Toledo aluminium pans which then are hermetically sealed.
  • the model system doughs used in the described examples contain standard wheat flour and optimal amounts of water or buffer with or without the non- maltogenic exoamylase according to the present invention. They are mixed in a 10 or 50 g Brabender Farinograph for 6 or 7 min., respectively. Samples of the doughs are placed in glass test tubes (15*0.8 cm) with a lid. These test tubes are subjected to a baking process in a water bath starting with 30 min. incubation at 33°C followed by heating from 33 to 95°C with a gradient of 1.1 °C per min. and finally a 5 min. incubation at 95°C. Subsequently, the tubes are stored in a thermostat at 20°C prior to DSC analysis.
  • the present invention is based on the surprising finding that it is possible to isolate a non-maltogenic exoamylase from Bacillus - which enzyme can hydrolyse starch by cleaving off linear maltooligosaccharides in the range of four to eight D-glucopyranosyl units from the non-reducing chain ends of amylopectin and which preferably have a sufficient degree of thermostability.
  • the enzyme is highly effective in retarding or reducing detrimental retrogradation in baked products.
  • the present invention also encompasses sequences derivable and/or expressable from those deposits and embodiments comprising the same, as well as active fragments thereof.
  • Figure 1 shows a graph
  • Figure 2 shows a graph
  • Figure 3 shows a graph
  • Figure 4 shows a graph
  • Figure 5 shows a graph
  • Figure 6 shows a trace
  • Figure 7 shows a graph.
  • Figure 3 Effect of temperature on the activity of the product-specific amylase. Effect of temperature at pH 9.5 ⁇ with 5 mM CaCI 2 ⁇ without CaCI 2 .
  • Figure 4 Thermostability tested as residual activity of the product-specific amylase after incubation at increasing temperatures at pH 9.5 with 5 mM CaCI 2 .
  • Figure 5 Products (in mM) formed by incubating the product-specific amylase (505 mU/mL) with 1 % soluble starch and 5 mM CaCI 2 at 55°C and pH 9.5. O glucose, * maltose, Q maltotriose, ⁇ maltotetraose, • maltopentaose, ⁇ maltohexaose.
  • Figure 6. HPAEC-PAD trace obtained by incubating the product-specific amylase (505 mU7 mL) with 1 % soluble starch at pH 9.5 and 55°C.
  • Figure 7 Determination of endo-and exo-activity of B. clausii BT-21 product- specific amylase compared to amylases of known starch cleavage action. The blue colour formation (% of maximum) is plotted against the production of mM maltose. The slope of the curves indicate the prevalence of endo- or exo-activity
  • Amylopectin and amylose from corn, corn starch, carboxymethylcellulose (CMC), bovine serum albumine (BSA), dextran, pullulan, maltose, maltotriose, and a mixture of maltotetraose to maltodecaose were obtained from Sigma Chemical Co., St. Louis, U.S.A. Soluble starch was obtained from Merck KGaA, Darmstadt, Germany. Yeast extract and tryptone were obtained from Difco Laboratories, Detroit, USA. Whole brown rice from Neue defeatede Reis GmbH mbH, Hamburg, Germany was used.
  • ⁇ -, ⁇ -, and ⁇ -cyclodextrin were obtained from Wacker Chemie Danmark Aps, Glostrup, Denmark. Maltotetraose was prepared as described previously [32]. All chemicals were, unless stated otherwise, of analytical grade.
  • the product-specific amylase After growth of B. clausii BT-21 on whole brown rice for 52 h, the cells and the whole rice grains were removed from the extracellular fluid (1000 mL) by centrifugation at 9600 rpm for 15 min at 4°C.
  • the product-specific amylase was purified using an affinity gel prepared by covalently binding ⁇ -cyclodextrin to an epoxy-activated sepharose 6B matrix (Pharmacia Biotech, Uppsala, Sweden) [33].
  • the extracellular cell-free supernatant was incubated with 12 g of gel while shaking for 1 h at 4°C. The supernatant was then removed by centrifugation at 9600 rpm for 10 min at 4°C.
  • Unbound protein was removed by washing the gel with 75 mL 50 mM phosphate buffer pH 8.0 followed by centrifugation. The washing step was repeated 7 times. Bound protein was eluted with 45mL of 50 mM phosphate buffer pH 8.0 containing 10 mM ⁇ -cyclodextrin followed by centrifugation. The elution step was repeated 4 times. ⁇ -Cyclodextrin was used for elution of the enzyme, since ⁇ - and ⁇ - cyclodextrin interfered with the protein determination method of Bradford (1976) [34].
  • the ⁇ -cyclodextrin was then removed by dialysis (6-8 kDa Spectra/Por dialysis membrane, The Spectrum Companies, Gardena, CA, U.S.A.) against 5 L 10 mM triethanolamin pH 7.5 while stirring at 4°C.
  • the buffer was changed after 2 h followed by an additional 12 h of dialysis.
  • the dialysis bags were placed in CMC to concentrate the sample.
  • Ten mL were applied to a HiTrap Q column (5 mL prepacked, Pharmacia Biotech, Uppsala, Sweden) using a FPLC-system (Pharmacia, Uppsala, Sweden).
  • the proteins were eluted at the rate of 1.0 mL/min with 25 mL 10 mM triethanolamin pH 7.5 followed by a gradient of 20 mM NaCl/ min in 10 mM triethanolamin pH 7.5.
  • the enzyme was eluted at 0.5 M NaCl.
  • the protein content was estimated by the method of Bradford, (1976) [34] using the BIO-RAD Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). BSA was used as standard.
  • SDS-PAGE (10%) was performed according to [36] followed by silver staining [37].
  • a SDS-PAGE broad range molecular weight standard (Bio-Rad laboratories, Hercules, CA, U.S.A.) was used.
  • Enzyme assay Two ml soluble starch solution (1.25%) in 0.1 M borate buffer pH 10.0 was incubated with 0.5 mL enzyme solution for 2 h at 45°C. The reaction was stopped by boiling the mixture for 10 min. The formation of reducing sugars was determined with the CuS0 4 /bicinchonate assay [38] and calculated as mM maltose equivalent formed. One unit of activity corresponded to the amount of enzyme that produced 1 ⁇ mol maltose equivalent min at pH 10.0 and 45°C.
  • Enzyme characterisation For the determination of the temperature optimum, the purified enzyme was incubated in a final concentration of 1 % soluble starch in 0J M borate buffer pH 10.0 (with or without the addition of 5 mM CaCI 2 ) for 15 min at temperatures from 30°C to 90°C. Determination of the temperature stability was performed by incubation of the purified enzyme in 50 mM glycine-NaOH buffer pH 9.5 containing 5 mM CaCI 2 for 30 min at 30, 40, 50, 55, 60, 70, 80, and 90°C.
  • Residual activity was determined by incubation of the heat-treated enzyme in a final concentration of 1% soluble starch in 50 mM glycine-NaOH buffer pH 9.5 at 55°C for 15 min.
  • the pH optimum was determined by incubation of the purified enzyme in a final concentration of 1 % soluble starch in different buffers at 55°C for 15 min.
  • the buffers used were 50 mM citrate (pH 4.0 to 6.0), 50 mM tris- maleate (pH 6.5 to 8.5), and 50 mM glycine-NaOH (pH 9.0 to 11.0).
  • the hydrolysis of different substrates by the purified enzyme was tested with soluble starch from potato, amylopectin from corn, dextran, pullulan (1 %), amylose (0J%>), and 10 mM ⁇ -, ⁇ -, and ⁇ -cyclodextrin.
  • the substrates were dissolved in 50 mM glycine-NaOH buffer with 5 mM CaCI 2 at pH 9.5 and the purified enzyme was added (505 mU/ mL).
  • the various substrates were incubated at 55°C and samples were withdrawn at different time intervals. The reaction was stopped by boiling for 10 min and the samples were analysed as described below.
  • malto-oligosaccharides The hydrolysis of malto-oligosaccharides by the purified enzyme was tested with maltose, maltotriose, and maltotetraose in a final concentration of 2 mM and a mixture of maltotetraose to maltodecaose (5mM).
  • the malto-oligosaccharides were dissolved in 50 mM borate buffer with 5 mM CaCI 2 at pH 9.5 and the purified enzyme was added (147 mU/mL).
  • the substrates were incubated at 55°C and samples were withdrawn at different time intervals. The reaction was stopped by boiling for 10 min and the samples were analysed as described below.
  • Hydrolysis products were detected using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
  • HPAEC-PAD pulsed amperometric detection
  • a CarboPac PA-1 column (Dionex Corporation, Sunnyvale, CA, U.S.A) was used with a gradient of 1.0 M Na-acetate from 0 to 60% over 30 min in 100 mM NaOH and a flow rate of 1.0 mL/min on a Dionex DX-300 or DX-500 system.
  • Starch hydrolysis products were identified by comparison of their retention times with glucose, maltose, maltothose, maltotetraose, maltopentaose, and maltohexaose. Since the retention times of homologous linear maltooligosaccharides increases with the degree of polymerisation, linear malto- oligosaccharides of intermediate DP could be easily identified [40, 41].
  • Activity stained native PAGE indicated the presence of 3 amylolytic activities in the extracellular fluid.
  • the product-specific enzyme was completely separated from the other amylolytic activities after ⁇ -CD affinity chromatography followed by anion-exchange chromatography.
  • SDS-PAGE of the purified enzyme preparation indicated that the product-specific amylase has been purified to homogeneity and has an apparent molecular weight of approximately 101 kDa.
  • Cyclodextrin sepharose 6B affinity chromatography has been previously used for the purification of an ⁇ -amylase as a final purification step after removal of other amylases by anion-exchange chromatography [29].
  • the enzyme recovery of 8.7% and the purification factor of 18.5 obtained for the product-specific amylase were similar to the values reported by these authors.
  • the molecular weight of these enzymes were estimated to be 59, 73 and 80 kDa [23], 180 kDa [24], and 97 kDa [26] while the product-specific amylase from ⁇ . clausii BT-21 showed an estimated molecular weight of 101 kDa.
  • Most of the product-specific amylases and ⁇ -amylases show a lower molecular weight in the range of 50-65 kDa [3, 16, 17, 19, and 21].
  • the temperature optimum of about 55°C was similar to the ones reported for the above product-specific amylases [23, 24, and 26].
  • malto-oligosaccharides After 20 h, the composition of malto-oligosaccharides had changed to 0.6% maltohexaose, 1.3% maltopentaose, 53.2% maltotetraose, 8.3% maltotriose, 27.6% maltose and 9% glucose.
  • Data are indicated as wt % glucose formed compared to the initial amount of substrate
  • Substrate time (min) DP1 DP2 DP3 DP4 DP5 DP6
  • Starches are composed of amylose (20-30%) and amylopectin (80-70%).
  • Amylose is an ⁇ -D-(1 ⁇ 4) O-glycosidically linked linear glucan, while amylopectin is a branched glucan due to the presence of ⁇ -D-(1 ⁇ 6) O-glycosidic linkages in the molecule.
  • the product-specific amylase most readily hydrolysed amylopectin indicated by the formation of maltopentaose (9.2%) and maltohexaose (24.8%) compared to soluble starch (7% and 17.8%) and amylose (6.3%) and 20%).
  • the enzyme did not hydrolyse pullulan, an ⁇ -(1 ⁇ 6) O-glycosidic linked glucan composed of a maltotriose backbone, or dextran, an ⁇ -(1 ⁇ 6) O-glycosidically linked glucan with branches attached to 0-3 of the backbone chain units, ⁇ -, ⁇ -, And ⁇ -cyclodextrins, cyclic malto-oligosaccharides composed of 6, 7, and 8 glucose units were also not hydrolysed even after 24 h incubation.
  • the product-specific amylase activity on a mixture of malto-oligosaccharides from DP4 to DP10 was studied by a time course experiment.
  • the change of the peak areas obtained by the HPAEC-PAD corresponded to the formation or a hydrolysis of malto-oligosaccharides.
  • the formation of maltohexaose (DP6) and the simultaneous decrease in the amount of DP7, DP8, DP9, and DP10 confirmed the maltohexaose forming ability of the enzyme.
  • steady state conditions were reached and the further degradation of DP6 as found by starch hydrolysis was not detected even after 7 days of hydrolysis.
  • the concentration of DP6 was much lower than the one obtained at the starch hydrolysis and indicated that a certain amount of maltohexaose was required for the formation of maltotetraose and maltose to proceed.
  • the starch hydrolysis by the B. clausii BT-21 product specific amylase was found to resemble a two step procedure. This procedure included an initial hydrolysis of starch to mainly maltohexaose and small amounts of maltopentaose, which were further hydrolysed to mainly maltotetraose and maltose accumulating after extensive hydrolysis.
  • the second hydrolysis step to maltotetraose and maltose seemed to be limited by the preliminary hydrolysis of the larger substrate to maltohexaose, since a concentration dependence seemed to a regulator for the second step to proceed.
  • a baking experiment was performed with the product-specific amylase. Doughs were prepared with 10 g of standard Danish wheat flour (Danisco 98078) and 6.2 ml 0.2 M NaOH-glycine buffer, pH 10 without (control) or with 40 units of the enzyme (assayed at 45°C and pH 10 as described in Materials and Methods of Section B), baked and analysed by DSC after storage according to "Assays for measurement of retrogradation and staling". As shown in Table 3 the enzyme significantly reduces the amount of retrograded amylopectin found day 7 after baking which indicates that it has a significant antistaling effect.
  • Extracellular cell-free supernatant from fermentation of B. clausii BT-21 was produced as described herein before in Section A.
  • the product-specific amylase was initially purified using hydrophobic interaction chromatography by loading 100 mL supernatant adjusted to 0.5 M sodium sulphate onto a 70 mL column of Phenyl Sepharose low sub (Pharmacia Biotech, Uppsala, Sweden) at 5 mL/min. Unbound protein was washed off the column with 220 mL 50 mM triethanolamine 2 mM calcium chloride, 0.5 M sodium sulphate, pH 7.2 and bound protein was eluted with a gradient of 0.5 to 0 M sodium sulphate in 50 mM triethanolamine, 2 mM calcium chloride, pH 7.2 over 30 min at 5 mL/min.
  • the amylase containing fractions were pooled (80 mL) and loaded onto a 16 mL column of ⁇ -cyclodextrin coupled Sepharose 6B prepared as described above.
  • the column was washed with 48 mL of 50 mM triethanolamine, 2 mM calcium chloride, pH 7.2 and eluted with 56 mL of the same buffer with 20 mM ⁇ -cyclo- dextrin.
  • the amylase containing fractions of the elute were pooled and characterised subsequently.
  • amylase preparation was subjected to direct amino acid sequencing of the N- terminus as well as to amino acid sequencing after tryptic digestion.
  • Amino acid sequencing was done using a Pulsed Liquid Phase Protein/Peptide Sequencer model 477 and a HPLC On-line PTH-Amino Acid Analyser mode I 120 A from Applied Biosystems (California) according to manufacturers instructions.
  • the amylase pool Prior to tryptic digestion the amylase pool was desalted and concentrated by absorption to a Prosorb (Applied Biosystems) cartridge.
  • the protein absorbed to the membrane was reduced at 60°C for 1 h in 500 ⁇ l 6M guanidine hydrochloride, 2% acetonitrile, 0.3% EDTA and 0.2% DTT in 0.5 M T s-HCI, pH 8.6. It was then carboxymethylated by adding 2.4 mg of iodoacetamide dissolved in 10 ⁇ l 0.5 NaOH and stirring for 15 min. in the dark. Thereafter the membrane was washed for five times with 2% acetonitrile and sonicated once in 0.1 % SDS for 5 min.
  • the carboxymethylated protein was then incubated for 20 min. at room temperature in 0.5% polyvinylpyrrolidone (PVP-40) in 100 mM acetic acid. After thorough washing of the membrane with the digestion buffer 1 %o Triton-X (reduced; Sigma), 5 mM CaCI 2 , 10% acetonitrile in 0.1 M Tris-HCI, pH 8.0 the membrane was cut into small pieces and incubated overnight at 37°C with 10 ⁇ g of trypsin (Sigma, sequencing grade from bovine pancreas). Thereafter the mixture was sonicated for 5 min. and the supernatant transferred to a fresh tube.
  • PVP-40 polyvinylpyrrolidone
  • the purified peptides were analysed using a Voyager DE (Perspective Biosys- terns) mass spectrometer applying 1 ⁇ l samples co-crystallized with 10 ⁇ g/ ⁇ l ⁇ - cyano-4-hydroxycinnamic acid in 0.1% TFA and 60% acetonitrile to check their purity and to verify the amino acids sequences obtained.
  • Voyager DE Perspective Biosys- terns
  • the purified amylase preparation showed a broad band at 101 kD as found before.
  • the product profile of this amylase pool was found to be identical to the one found before suggesting that the two amylases detected are isoforms.
  • SQNSDQKLFSWENATVYFAIT SEQ ID No. 1
  • the finding of only one N-terminal sequence is in agreement with the two amylase bands observed by native PAGE being isoforms with either minor differences in the sequence or with the one being formed by proteolytic processing at the C- terminus of the other.
  • SEQ ID No. 11 The full length sequence is presented as SEQ ID No. 11.
  • the coding sequence is presented as SEQ ID No. 12.
  • SEQ ID No. 13 presents additional sequences at the 5' and 3' ends.
  • Ths indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e g . "Accession Number of Deposit")

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Abstract

L'invention concerne une enzyme qui est une exoamylase non maltogène de Bacillus clausii ou son équivalent fonctionnel. L'enzyme présente un poids moléculaire d'environ 101.000 Da ( calculé par électrophorèse de polyacrylamide de dodécyl sulfate de sodium) et/ou l'enzyme présente une activité optimale à pH 9.5 et 55 °C.
PCT/IB2000/000433 1999-03-30 2000-03-29 Exoamylase non maltogene issue de b. clausii et son utilisation permettant de freiner la retrogradation d'un produit d'amidon WO2000058447A1 (fr)

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WO2005007867A2 (fr) * 2003-07-07 2005-01-27 Genencor International, Inc. Polypeptides d'amylase thermostables, acides nucleiques codant pour lesdits polypeptides et utilisations
WO2006063594A1 (fr) * 2004-12-15 2006-06-22 Novozymes A/S Amylase de bacille alcaline
US7858352B2 (en) 2003-06-13 2010-12-28 Danisco A/S Polypeptide
US8030050B2 (en) 2005-07-07 2011-10-04 Danisco A/S Modified amylases from Pseudomonas species
US8137944B2 (en) 2004-07-07 2012-03-20 Danisco A/S Modified amylases from pseudomonas species, methods of making and uses thereof
US8143048B2 (en) 2003-07-07 2012-03-27 Danisco A/S Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof
US8178336B2 (en) 2006-06-19 2012-05-15 Danisco A/S Polypeptide
CN110157688A (zh) * 2019-05-31 2019-08-23 江南大学 一种产麦芽五糖能力提高的直链麦芽低聚糖生成酶突变体
WO2021219793A1 (fr) 2020-04-30 2021-11-04 Dupont Nutrition Biosciences Aps Système d'ingrédient pour produits de boulangerie

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