WO2011114251A1 - Foodstuff - Google Patents

Foodstuff Download PDF

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Publication number
WO2011114251A1
WO2011114251A1 PCT/IB2011/050869 IB2011050869W WO2011114251A1 WO 2011114251 A1 WO2011114251 A1 WO 2011114251A1 IB 2011050869 W IB2011050869 W IB 2011050869W WO 2011114251 A1 WO2011114251 A1 WO 2011114251A1
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WO
WIPO (PCT)
Prior art keywords
cyclodextrin
enzyme
foodstuff
present
dough
Prior art date
Application number
PCT/IB2011/050869
Other languages
French (fr)
Inventor
Jørn Borch SØE
Jens Frisbæk SØRENSEN
Lene Bojsen Jensen
Original Assignee
Danisco A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Danisco A/S filed Critical Danisco A/S
Publication of WO2011114251A1 publication Critical patent/WO2011114251A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
    • A23D7/005—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by ingredients other than fatty acid triglycerides
    • 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
    • A21D2/14—Organic oxygen compounds
    • A21D2/18—Carbohydrates
    • A21D2/183—Natural gums
    • 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
    • A21D2/14—Organic oxygen compounds
    • A21D2/18—Carbohydrates
    • A21D2/186—Starches; Derivatives thereof
    • 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
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • 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
    • A23L29/06—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
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01004—Phospholipase A2 (3.1.1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01026—Galactolipase (3.1.1.26)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01032—Phospholipase A1 (3.1.1.32)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • This invention relates to a foodstuff. It also relates to a method for producing the foodstuff and uses of certain ingredients of the foodstuff to provide novel technical effects.
  • lipolytic enzymes are capable of hydrolysing ester bonds a range of lipids present in wheat flour (in particular, triglycerides, glycolipids and phospholipids), either to liberate free fatty acid molecules or to transfer fatty acyl groups to other acceptor molecules.
  • WO 2005/087918 describes a lipolytic enzyme and its use in baking.
  • the enzyme described therein and having the sequence listing of SEQ !D No. 1 of this specification is commercially available from Danisco A/S under the trade mark GRINDA YL POWERBAKE 4070. Lipolytic enzymes provide a number of advantageous effects in baking applications.
  • lipolytic enzymes can act on triglycerides present in flour to generate in situ mono- and diglycerides, which are useful as emulsifiers.
  • Lipolytic enzymes can also act on glycolipids present in flour to generate in situ mono- and diglycosyl mono- and diglycerides, and on phospholipids present in flour to generate in situ lysophospholipids.
  • the 5-membered macrocycle is not natural.
  • Typical cyclodextrins contain a number of glucose monomers ranging from six to eight units in a ring, creating a cone shape.
  • Examples of known cyclodextrins are a-cyclodextrin (6- membered glucose ring molecule), ⁇ -cyclodextrin (7-membered glucose ring molecule) and ⁇ -cyclodextrin (8-membered glucose ring molecule).
  • Cyclodextrins are known to interact with free fatty acids by formation of inclusion compounds, where the fatty acid is included in the hydrophobic torus of the cyclodextrin - see Bru e ai, Colloids and Surfaces A: Physicochemical and Engineering Aspects 97 (1995), 263-269.
  • Cyclodextrins may be generated in situ from starch or other polysaccharides present in a dough mixture, for example by the action of a cyclodextrin glycosyltransferase enzyme (also known as cyclodextrin glucosyltransferase or CGTase) (E.G. 2.4.1.19), the enzyme acting firstly to cleave the glycosidic bonds in the polysaccharide followed by bonding the glucose units together to form the ring. Examples of CGTases and their use in baking are described in WO 02/06508 and EP 0687414A.
  • EP 0 493 045 A1 describes a foodstuff to which a ⁇ -cyclodextrin and a phospholipase D is added. This differs from the present invention in that phospholipase D cleaves phospholipids at the phosphate group to liberate phosphatidic acid and an alkanolamine, rather than a free fatty acid.
  • a foodstuff comprising an effective amount of:
  • a foodstuff comprising an effective amount of:
  • a method of producing the above foodstuff comprising adding the lipolytic enzyme and the cyciodextrin to one or more other ingredients of the foodstuff and, if necessary, treating the ingredients to produce the foodstuff.
  • a cyclodextrin for decreasing the uncompiexed free fatty acid content of a foodstuff incorporating a lipolytic enzyme.
  • a cyclodextrin for decreasing the uncompiexed free fatty acid content of a foodstuff incorporating a lipolytic enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule.
  • a cyclodextrin into a foodstuff (particularly although not exclusively a dough and a baked product prepared from dough) enables the amounts of free fatty acid present in the foodstuff to be reduced.
  • this effect is caused by the free fatty acid molecules forming an inclusion complex with the cyclodextrin in which the free fatty acid molecule is included in the hydrophobic interior of the cyclodextrin: the free fatty acids do not exhibit the deleterious effects described above when in the form of an inclusion complex.
  • Inclusion complex' or 'inclusion compound' is understood in the art as meaning a complex in which one component (the host) forms a cavity in which molecular entities of a second component (the guest) are located. There is no covalent bonding between guest and host, the attraction being generally due to van der VVaals forces.
  • the term 'compiexed' in relation to free fatty acids means that the free fatty acid is present as a component of an inclusion complex (as defined above) in which at least part of the fatty acid molecule is located within the cavity of the cyclodextrin molecule.
  • the term 'uncomplexed' means that the free fatty acid molecule is present in the foodstuff as a separate entity from the cyclodextrin molecule.
  • foodstuff' as used herein means a substance which is suitable for human and/or animal consumption.
  • the term "foodstuff ' as used herein may mean a foodstuff in a form which is ready for consumption.
  • the term foodstuff as used herein may mean one or more food materials which are used in the preparation of a foodstuff.
  • the term foodstuff encompasses both baked goods produced from dough as well as the dough used in the preparation of said baked goods.
  • the foodstuff may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
  • composition of the present invention may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient.
  • the present invention provides a foodstuff as defined above wherein the foodstuff is selected from one or more of the following: eggs, egg-based products, including but not limited to mayonnaise, salad dressings, sauces, ice creams, egg powder, modified egg yolk and products made therefrom; baked goods, including breads, cakes, sweet dough products, laminated doughs, liquid batters, muffins, doughnuts, biscuits, crackers and cookies; confectionery, including chocolate, candies, caramels, halawa, gums, including sugar free and sugar sweetened gums, bubble gum, soft bubble gum, chewing gum and puddings; frozen products including sorbets, preferably frozen dairy products, including ice cream and ice milk; dairy products, including cheese, butter, milk, coffee cream, whipped cream, custard cream, milk drinks and yoghurts; mousses, whipped vegetable creams, meat products, including processed meat products; edible oiis and fats, aerated and non- aerated whipped products, oil-in-water
  • the foodstuff in accordance with the present invention may be a "fine food", including cakes, pastry, confectionery, chocolates, fudge and the like.
  • the foodstuff in accordance with the present invention may be a dough product or a baked product, such as bread, a fried product, a snack, cakes, pies, brownies, cookies, noodles, snack items such as crackers, graham crackers, pretzels, and potato chips, and pasta.
  • the foodstuff in accordance with the present invention may be a convenience food, such as a part-baked or part-cooked product.
  • a convenience food such as a part-baked or part-cooked product.
  • Examples of such part-baked or part-cooked product include part-baked versions of the dough and baked products described above.
  • the foodstuff in accordance with the present invention may be a plant derived food product such as flours, pre-mixes, oils, fats, cocoa butter, coffee whitener, salad dressings, margarine, spreads, peanut butter, shortenings, ice cream, cooking oils.
  • the foodstuff in accordance with the present invention may be a dairy product, including butter, milk, cream, cheese such as natural, processed, and imitation cheeses in a variety of forms (including shredded, block, slices or grated), cream cheese, ice cream, frozen desserts, yoghurt, yoghurt drinks, butter fat, anhydrous milk fat, other dairy products.
  • the enzyme according to the present invention may improve fat stability in dairy products.
  • the foodstuff in accordance with the present invention may be a food product containing animal derived ingredients, such as processed meat products, cooking oils, shortenings.
  • the foodstuff in accordance with the present invention may be a beverage, a fruit, mixed fruit, a vegetable, a marinade or wine.
  • the foodstuff in accordance with the present invention is a plant derived oil (i.e. a vegetable oil), such as olive oil, sunflower oil, peanut oil or rapeseed oil.
  • the oil may be a degummed oil.
  • the maximum free fatty acid content of the foodstuff is 5%, preferably 2%, more preferably 1 %, even more preferably 0.5%, yet more preferably 0.2%, still more preferably 0.1 %, and most preferably 0.05%.
  • 'cyclodextrin' means a cyclic oligosaccharide composed of 5 or more glucose units linked in a 1 ->4 manner so as to form a ring.
  • the cyclodextrin comprises a cyclic oligosaccharide containing 5 to 10, preferably 6 to 8, glucose units.
  • the cyclodextrin may optionally be substituted with a side chain comprising 1 to 3 monosaccharide, preferably glucose, units.
  • suitable cyclodextrins include a-cyclodextrin (6-membered glucose ring molecule), ⁇ - cyciodextrin (7-membered glucose ring molecule) and y -cyciodextrin (8-memoered glucose ring molecule).
  • the cyciodextrin is ⁇ -cyclodextrin.
  • the amount of cyciodextrin present is 0.05 to 50 g per kg of the total weight of the foodstuff.
  • the amount of cyciodextrin present is 0.1 to 20 g per kg of the total weight of the foodsiuff. More preferably, the amount of cyciodextrin present is 0.5 to 10 g per kg of the total weight of the foodstuff.
  • the amount of cyciodextrin present is 0.05 to 50 g per kg of the flour used to form the dough.
  • the amount of cyciodextrin present is 0.1 to 20 g per kg flour. More preferably, the amount of cyciodextrin present is 0.5 to 10 g per kg flour.
  • the cyciodextrin is present as an initial component of the foodstuff (i.e. the cyciodextrin is produced separately of the foodstuff and is added as a formed product to the foodstuff during preparation of the same).
  • the cyciodextrin is generated in situ during preparation of the foodstuff, typically by the action of a cyciodextrin glycosyltransferase enzyme (E.G. 2.4.1.19) on polysaccharides present in the foodstuff.
  • 'cyciodextrin glycosyltransferase', 'cyciodextrin glucosyltransferase' and 'CGTase' all mean an enzyme capable of acting on a polysaccharide so as to produce a cyciodextrin (as defined above, either in its broadest aspect or a preferred aspect).
  • the polysaccharide on which the CGTase enzyme (if present) acts is selected from starch, amylose or amylopectin.
  • Suitable CGTase enzymes are described in WO 02/06508 and EP 0687414A.
  • a particular example of a suitable CGTase enzyme is the cyciodextrin glucanotransferase (Bacillus macerans) enzyme available from Amano Enzymes, Japan.
  • the amount of CGTase enzyme present is suitably 0.01 g to 10 g per kg of the total weight of the foodstuff.
  • the amount of CGTase enzyme present is 0.05 g to 1 g per kg of the total weight of the foodstuff.
  • the amount of CGTase enzyme present is suitably 0.01 mg to 2 mg per kg of the total weight of the foodstuff.
  • the amount of CGTase enzyme present is 0.05 mg to 1 mg per kg of the total weight of the foodstuff.
  • the amount of CGTase enzyme present is suitably 6 to 1200 units of enzyme activity (U) per kg of the total weight of the foodstuff.
  • the activity can be measured according to the Tilden-Hudson method described in Tilden E.B. and Hudson C.S. J. Bacterial. (1942), 43, 527-544.
  • the CGTase enzyme may be incorporated into the foodstuff before addition of the lipolytic enzyme.
  • the CGTase enzyme may be incorporated into the foodstuff at the same time as the lipolytic enzyme.
  • the amount of CGTase enzyme, where present is 0.01 to 10 g per kg of the flour used to form the dough.
  • the amount of CGTase enzyme, where present is 0.05 to 1 g per kg flour.
  • the amount of CGTase enzyme, where present is suitably 0.01 mg to 2 mg per kg of the flour used to form the dough.
  • the amount of CGTase enzyme, where present is 0.05 mg to 1 mg per kg of flour.
  • the amount of CGTase enzyme present is suitably 6 to 1200 units of enzyme activity (U) per kg of the flour used to form the dough.
  • the activity can be measured according to the Tilden-Hudson method referred to above.
  • the term lipolytic enzyme' is defined as an enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule.
  • the lipolytic enzyme is an enzyme capable of hydro!ysing an ester bond in a lipid substrate (particularly although not exclusively a triglyceride, a giycolipid and/or a phospholipid) to liberate a free fatty acid molecule. Examples of possible lipid substrate are described below.
  • the lipolytic enzyme used in the present invention has activity on both non-polar and polar lipids.
  • polar lipids as used herein means phospholipids and/or glycolipids.
  • polar lipids as used herein means both phospholipids and glycolipids.
  • Polar and non-polar lipids are discussed in Eliasson and Larsson, "Cereals in Breadmaking: A Molecular Colloidal Approach", publ. Marcel Dekker, 1993.
  • the lipolytic enzyme used in the present invention has activity on the following classes of lipids: triglycerides; phospholipids, particularly but not exclusively phosphatidylcholine (PC) and/or N-acylphosphatidylethanolamine (APE); and glycolipids, particularly although not exclusively digalactosyl diglyceride (DGDG).
  • lipids particularly but not exclusively phosphatidylcholine (PC) and/or N-acylphosphatidylethanolamine (APE); and glycolipids, particularly although not exclusively digalactosyl diglyceride (DGDG).
  • such an acyl group is an alkanoyl group.
  • such an acyl group comprises an alkenoyl group, which may have, for example, 1 to 5 double bonds, preferably 1 , 2 or 3 double bonds.
  • the lipolytic enzyme for use in the present invention may have one or more of the following activities selected from the group consisting of: phospholipase activity (such as phospholipase A1 activity (E.G. 3.1.1.32) or phospholipase A2 activity (E.G. 3.1 .1.4); glycolipase activity (E.G. 3.1.1.26), triacylglycerol hydrolysing activity (E.G. 3.1.1 ,3), lipid acy!transferase activity (generally classified as E.G. 2.3.1.x in accordance with the Enzyme Nomenclature Recommendations (1992) of the Nomenciature Committee of the International Union of Biochemistry and Molecular Biology), and any combination thereof.
  • phospholipase activity such as phospholipase A1 activity (E.G. 3.1.1.32) or phospholipase A2 activity (E.G. 3.1 .1.4
  • glycolipase activity E.G. 3.1.1.26
  • the lipolytic enzyme for use in the present invention may be a phospholipase (such as a phospholipase A1 (E.G. 3.1.1.32) or phospholipase A2 (E.G. 3.1.1.4)); glycolipase or galactolipase (E.G. 3.1.1 .26), triacylglyceride lipase (E.G. 3.1.1.3).
  • a phospholipase such as a phospholipase A1 (E.G. 3.1.1.32) or phospholipase A2 (E.G. 3.1.1.4)
  • glycolipase or galactolipase E.G. 3.1.1 .26
  • triacylglyceride lipase E.G. 3.1.1.3
  • Such enzyme may exhibit additional side activities such as lipid acyltransferase side activity.
  • the lipolytic enzyme for use in the present invention may have the activity of a phospholipase (such as
  • the lipolytic enzyme may be any commercially available lipolytic enzyme.
  • the lipolytic enzyme may be any one or more of: Lecitase UltraTM, Novozymes, Denmark; Lecitase 10TM; a phospholipase A1 from Fusarium spp e.g. Lipopan FTM, LipopanTM LipopanXtraTM, YieldMaxTM; a phospholipase A2 from Aspergillus niger; a phospholiapse A2 from Streptomyces violaceruber e.g.
  • LysoMax PLA2TM a phospholipase A2 from Tuber borchii; or a phospholipase B from Aspergillus niger, Lipase 3, Grindamyl EXEL 16TM, and GRINDA YL POWERBake 4070, PanamoreTM or GRINDAMYL POWERBake 4100.
  • the lipolytic enzyme is GRINDAMYL POWERBake 4070TM, Lipopan FTM, LipopanTM' LipopanXtraTM, or PanamoreTM.
  • the lipolytic enzyme is obtainable from a microorganism, examples of which include bacteria and fungi (such as yeasts).
  • the lipolytic enzyme is obtainable from a filamentous fungus.
  • the lipolytic enzyme is obtainable from a fungus of the genus Fusarium, Aspergillus or Streptomyces, especially from a fungus of the genus Fusarium.
  • the lipolytic enzyme is obtainable from Fusarium heterosporum. !n a particularly preferred embodiment, the lipolytic enzyme is obtainable from Fusarium heterosporum CBS 782.83.
  • prepro-polypeptides which when post-translationally processed in a host organism produces a polypeptide which has hydrolytic activity towards an ester bond in a polar lipid, wherein the prepropolypeptide comprises an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 and SEQ ID No. 25.
  • the lipolytic enzyme for use in the present invention is a polypeptide which has hydrolytic activity towards an ester bond in a polar lipid, which polypeptide is obtainable from a prepro-polypeptide comprising an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 or SEQ ID No. 25.
  • prepro-sequences often go through post- translational modification.
  • the present enzymes it is relatively common for the organism to remove the N-terminal region of the prepro sequence, i.e. remove all or part of the amino acids 1 -30 of SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 or SEQ ID No. 25.
  • the host organism may remove slightly more amino acids than those shown as amino acids 1 -30 of SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No.
  • the host organism may introduce an alternative N-terminal sequence which may encompass all or part of the amino acids shown as amino acids 1 -30 or may comprise a completely different N-terminal sequence (such as EAEA or EA for instance).
  • the mature enzyme produced from the prepro-sequence by the host organism may be a heterogen at its N-terminus end.
  • the post- translational modification may mean modification in the C-terminal region of the prepro sequence. For example, all or part of the amino acids 306-348 may be removed from SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No.
  • the mature enzyme produced from the prepro-sequence by the host organism may be a heterogen at its C-terminus end.
  • the present invention encompasses the use of all mature forms of the protein obtainable from a prepro-polypeptide comprising an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ SD No. 23 or SEQ ID No. 25, particularly those obtained from the host organism Trichoderma reesei.
  • the lipolytic enzyme for use in the present invention may comprise one of the following amino acid sequences:
  • n an amino acid sequence as set forth in SEQ ID No. 26;
  • the lipolytic enzyme for use in the present invention may have at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% identity with any of the sequences shown in a) to n).
  • the lipolytic enzyme for use in the present invention may have an amino acid sequence as set forth in SEQ ID No. 1 or an amino acid sequence having at least 70% identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% identity thereto.
  • the lipolytic enzyme for use in the present invention may be a lipolytic enzyme encoded by any one of the following nucleic acid sequences:
  • n a nucleic acid sequence encoding a lipolytic enzyme having at least 70% identity to any of the sequences in a) to I);
  • the lipolytic enzyme for use in the present invention may be a lipolytic enzyme encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably af least 95%, preferably at least 98%, preferably at least 99% identity with any of the sequences shown in a) to I).
  • the lipolytic enzyme for use in the present invention may be encoded by a nucleic acid sequence as set forth in SEQ ID No. 3 or a nucleic acid sequence having at least 70% identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% identity thereto.
  • Lipolytic enzymes have a number of advantageous effects in baking applications.
  • the lipolytic enzyme for use in the present invention may confer one or more of the following effects on dough and/or a baked product prepared from dough: reducing stickiness; improving machinability; reducing blistering during baking; improved volume; improved softness; prolonged shelf life; reduced staling; improved crumb structure; improved pore homogeneity; reducing mean pore size; enhancing the gluten index; improved flavour; improved colour of the crust. These effects remain substantially unchanged by treatment with cyclodextrin.
  • a cyclodextrin in combination with a lipolytic enzyme allows any or all of the above advantageous effects to be exhibited while reducing the content of free fatty acid in the dough and/or baked product prepared from dough and avoiding the deleterious effects associated therewith.
  • the lipolytic enzyme is in a delayed release form.
  • delayed release also known as “slow release” when used in its broadest sense means that the lipolytic enzyme is released into the foodstuff a time later than that immediately following its introduction.
  • the term “delayed release” means that 50% of the lipolytic enzyme is released into the foodstuff at a time at least 5 minutes after its introduction and 100% of the lipolytic enzyme is released into the foodstuff after at a time at least 10 minutes after its introduction. More preferably, the term “delayed release” means that 50% of the lipolytic enzyme is released into the foodstuff at a time between 5 minutes and 3 hours after its introduction and 100% of the lipolytic enzyme is released into the foodstuff after at a time between 10 minutes and 5 hours after its introduction.
  • the provision of the lipolytic enzyme in delayed release form confers the advantage that the rate of generation of free fatty acid by the lipolytic enzyme can be slowed down.
  • the provision of the lipolytic enzyme in delayed release form allows sufficient time for the CGTase enzyme to generate the cyclodextrin before significant quantities of free fatty acid are generated by the lipolytic enzyme.
  • the lipolytic enzyme is encapsulated. Encapsulation is a process of surrounding or coating an ingredient with a substance in order to prevent or delay the release of the ingredient until a certain time or set of conditions is achieved.
  • capsules Various techniques are employed to form the capsules, including spray drying, spray chilling or spray cooling, extrusion coating, fluidized bed coating, liposome entrapment, coacervation, inclusion complexation, centrifugal extrusion and rotational suspension separation.
  • Fats, emulsifiers, starches, dextrins, alginates, protein and lipid materials can be employed as encapsulating materials.
  • Release can be site-specific, stage-specific or signalled by changes in pH, temperature, irradiation or osmotic shock.
  • the lipolytic enzyme used in the present invention is capable of hydrolysing an ester bond in a lipid substrate.
  • the term 'lipid substrate' is defined as meaning a fat-soluble (lipophilic), naturally-occurring or synthetic molecule, having an ester group capable of being hydrolysed to liberate a free fatty acid molecule (as defined above).
  • suitable lipids include triglycerides, mono- and polyglycerolipids (especially monog!ycerides and diglyce rides), giycolipids, phospholipids, and the like.
  • the lipid substrate may comprise a mixture of lipids.
  • lipophilic' means soluble in non-polar organic solvents.
  • such non-polar organic solvents have one or more of the following properties:
  • a low dielectric constant for example, a dielectric constant less than 20, preferably less than 10.
  • a weak or zero dipole moment for example, a dipoie moment of less than 1 D, preferably less than 0.5 D
  • non-polar organic solvents examples include aliphatic hydrocarbons such as pentane, hexane, heptane, aiicylic hydrocarbons such as cyclohexane, aromatic hydrocarbons such as benzene, toluene or xylene, ethers such as diethyl ether, and halogenated hydrocarbons such as dichloromethane, trichloromethane (chloroform) and 1 ,2-dichloroethane.
  • such acyl groups have a total of 4 to 40 carbon atoms, preferably 6 to 40 carbon atoms, such as at least 10 to 40 carbon atoms, for example 12 to 40, such as 14 to 40, 16 to 40, 18 to 40, 20 to 40 or 22 to 40 carbon atoms, more preferably 10 to 24, especially 12 to 22, particularly 14 to 18, for example 16 or 18 carbon atoms.
  • such an acyl group is an alkanoyl group.
  • such an acyl group comprises an alkenoyl group, which may have, for example, 1 to 5 double bonds, preferably 1 , 2 or 3 double bonds.
  • acyl groups include saturated acyl groups such as butanoyl (butyryl), hexanoyl (caproyl), octanoyl (capryl), decanoyl (caprinyl), dodecanoyl (lauroyl), tetradecanoyl, (myristoyl), hexadecanoyl (palmitoyl), octadecanoyl (stearoyl), eicosanoyl (arachidonyl), docosanoyl (behenoyl) and tetracosanoyl (lignoceroyl) groups, and unsaturated acyl groups such as c/s-tetradec-9-enoyl (myristoleyl), c/s- hexadec-9-enoyl (palmitoleyl), c s-octadec-9-enoyl (oleyl), cis c/s-9, 12-
  • lipids suitable as substrates in the present invention are described below.
  • the lipid substrate is a triglyceride.
  • 'triglyceride' also known as triacylglycerol
  • triacylglycerol means a compound comprising three acyl groups (which may be the same or different) covalently bonded to a single glycerol moiety via ester linkages.
  • the lipid substrate is a glycoglycerolipid (also known as a glycosylglyceride).
  • glycoglycerolipid also known as a glycosylglyceride
  • 'glycoglycerolipid' when used to define the lipid substrate molecule, means a lipid comprising a single glyceroi moiety covalently bound via ester linkages to one or more acyl groups (the typical and preferred lengths of which are defined and exemplified above) and having one or more monosaccharide moieties attached to the glycerol moiety via a glycosidic linkage, provided it contains at least one free hydroxy! group to enable transfer to take place.
  • the monosaccharides may be bonded to different oxygen atoms on the glycerol backbone, may be bonded to each other to comprise a di-, oligo- or polysaccharide moiety attached to one oxygen atom on the glycerol moiety, or any combination thereof.
  • Glycerophospholipids also referred to as phospholipids, are ubiquitous in nature and are key components of the lipid bilayer of cells, as well as being involved in metabolism and signaling.
  • Glycerophospholipids may be subdivided into distinct classes, based on the nature of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria or the sn-1 position in the case of archaebacteria.
  • glycerophospholipids found in biological membranes are phosphatidylcholines (also known as PC or GPCho, and lecithin), phosphatidylethanolamines (PE or GPEtn), phosphatidylserine (PS or GPSer), phosphadityl inositol, lysophosphatidylcholines, lysophosphatidylethanolamines, N- acyl phosphatidylethanolamines and N-acyl lysophosphatidylethanolamines.
  • phosphatidylcholines also known as PC or GPCho, and lecithin
  • PE or GPEtn phosphatidylethanolamines
  • PS or GPSer phosphatidylserine
  • phosphadityl inositol lysophosphatidylcholines
  • lysophosphatidylethanolamines lysophosphatidylethanolamines
  • some g!ycerophospholipids in eukaryotic cells such as phosphatidylinositois and phosphatide acids are either precursors of, or are themselves, membrane-derived second messengers.
  • phosphatidylinositois and phosphatide acids are either precursors of, or are themselves, membrane-derived second messengers.
  • these hydroxyl groups are acylated with long-chain fatty acids (the number of carbon atoms in the chains typically as set out above), but there are also alkyl-linked and 1 Z- alkenyl-linked (plasmalogen) glycerophospholipids, as well as dialkylether variants in prokaryotes.
  • Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.
  • a sugar substitutes for the glycerol backbone that is present in glycerolipids and glycerophospholipids.
  • the most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria.
  • Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E.
  • the lipid substrate is a lysophospholipid.
  • a lysophospholipid comprises a glycerol moiety having only one acyl group (as defined and exemplified above) covalent!y bonded to a glycerol oxygen atom via an ester linkage and a phosphate group covalently bonded to another glycerol oxygen atom to form a phosphate ester: the said lysophospholipids therefore possess a free OH group on the remaining glycerol carbon atom.
  • Suitable lysophospholipids include lysophosphatidylcholines (also known as lyso-PC), lysophosphatidylethanolamines (PE or GPEtn), phosphatidylserine (PS or GPSer), phosphadityl inositol, , N-acyl phosphatidyl-ethanolamines and N-acyl lysophosphatidyl-ethanolamines.
  • the lysophospholipid may be formed in situ by hydrolysis of one of the ester linkages on the corresponding phospholipid.
  • the amount of lipolytic enzyme present is 0.001 g to 2 g per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 0.005 g to 1 g per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 0.001 g to 2 g per kg of the flour used to form the dough.
  • the amount of lipolytic enzyme present is 0.005 g to 0.5 g per kg of the flour.
  • the amount of lipolytic enzyme present is 0.001 mg to 2 mg per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 0.005 mg to 1 mg per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 0.001 mg to 2 g per kg of the flour used to form the dough.
  • the amount of lipolytic enzyme present is 0.005 to 0.5 mg per kg of the flour.
  • the amount of lipolytic enzyme present is 50 to 15,000 TIPU per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 100 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 TIPU per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 50 to 15,000 TIPU per kg of the flour used to form the dough.
  • the amount of lipolytic enzyme present is 100 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 TIPU per kg of the flour.
  • the TIPU unit of activity and the assay used to determine it is described in more detail below.
  • the amount of lipolytic enzyme present is 35 to 10,000 LIPU-NEFA per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 50 to 5,000, preferably 200 to 1 ,000 LIPU-NEFA per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 35 to 10,000 LIPU-NEFA per kg of the f!our used to form the dough.
  • the amount of lipolytic enzyme present is 50 to 5,000, preferably 200 to 1 ,000 LIPU-NEFA per kg of the flour.
  • the LIPU-NEFA unit of activity and the assay used to determine it is described in more detail below.
  • the amount of lipolytic enzyme present is 15 to 15,000 GLU per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 50 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 GLU per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 15 to 15,000 GLU per kg of the flour used to form the dough.
  • the amount of lipolytic enzyme present is 50 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 GLU per kg of the flour.
  • the GLU unit of activity and the assay used to determine it is described in more detail below.
  • glycolipase activity, phospholipase activity and triacyiglyceride lipase activity of the lipolytic enzyme as used in the present invention can be determined using the assays presented hereinbelow.
  • Phospholipase activity was measured using two different methods which give comparable results. Either of these methods can be used to determine phospholipase activity in accordance with the present invention. Preferably, the PLU assay is used for determining the phospholipase activity of any enzyme.
  • Free fatty acid in the samples was analyzed by using the NEFA HR(2) kit from WAKO GmbH. Enzyme activity PLU-7 at pH 7 was calculated as micromoles of fatty acid produced per minute under assay conditions.
  • TIPU Tiration Phospholipase Unit
  • Phospholipase A1 and A2 catalyse the conversion of lecithin to lyso-lecithin with release of the free fatty acid from position 1 and 2, respectively.
  • Phospholipase activity can be determined by continuous titration of the fatty acids liberated from lecithin during enzymation, since the consumption of alkali equals the amount of fatty acid liberated.
  • An enzyme solution was prepared to give a slope on the titration curve between 0.06 and 0.18 ml/min with an addition of 300 ⁇ _ enzyme.
  • a control sample of known activity is included. The samples were dissolved in demineralised water and stirred for 15 min. at 300 rpm. 25.00 ml substrate was thermostatted to 37.0°C for 10-15 minutes before pH was adjusted to 7.0 with 0.05 M NaOH. 300 ⁇ _ enzyme solution was added to the substrate and the continuous titration with 0.05 M NaOH was carried out using a pH-Stat titrator (Phm 290, Mettler Toledo). Two activity determinations are made on each scaling. After 8 minutes the titration is stopped and the slope of the titration curve is calculated between 5 and 7 minutes. The detection limit is 3 TIPU/ml enzyme solution.
  • TIPU/g enzyme The phospholipase activity (TIPU/g enzyme) was calculated in the following way:
  • a is the slope of the titration curve between 5 and 7 minutes of reaction time (ml/min); N is the normality of the NaOH used (mol/l);
  • ⁇ / ⁇ is the volume in which the enzyme is dissolved (ml);
  • Free fatty acid in the samples was analyzed by using the NEFA HR(2) kit from WAKO GmbH. Enzyme activity LSPU-NEFA at pH 5.5 was calculated as micromoles of fatty aid produced per minute under assays conditions.
  • one or more further food grade enzymes may be used, for example added to the food, dough preparation, or foodstuff.
  • At least one further enzyme may be added to the baked product and/or the dough.
  • Such further enzymes include starch degrading enzymes such as endo- or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g.
  • glucose oxidase pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as one which oxidises maltose, for example hexose oxidase (HOX), lipases, phospholipases and hexose oxidase, proteases, and acyltransferases (such as those described in WO 2004/064987 for instance).
  • HOX hexose oxidase
  • lipases phospholipases and hexose oxidase
  • proteases and acyltransferases
  • the amylase may be a non-maltogenic amylase, such as a polypeptide having non- maltogenic exoamylase activity, in particular, giucan 1 ,4-alpha-maltotetrahydrolase (EC 3.2.1.60) activity (as disclosed in WO 2005/003339).
  • a suitable non-maltogenic amylase is commercially available as PowersoftTM (available from Danisco A/S, Denmark).
  • the combined use of alpha amylases and the lipolytic enzyme of the invention may be used in a dough, and/or the production of a baked product, such as bread, cakes, doughnuts, cake doughnuts or bagels.
  • the combination of alpha amylases and the lipolytic enzyme of the invention is also considered as preferable for use in methods of production of tortillas, such as wheat and/or maize tortillas.
  • the lipolytic enzyme according to the present invention may be used in combination with a xylanase in producing food products.
  • GR!NDAMYLTM and POWERBake 7000 are examples of commercially available xylanase enzymes available from Danisco A/S. Other examples of xylanase enzymes may be found in WO 03/020923 and WO 01/42433.
  • the lipolytic enzyme according to the present invention may be used in combination with a xylanase and an alpha amylase.
  • the alpha amylase may be a maltogenic, or a non-maltogenic alpha amylase (such as GRINDAMYLTM or POWERSoft, commercially available from Danisco A/S), or a combination thereof.
  • the lipolytic enzyme of the invention can also preferably be used in combination with an oxidising enzyme, such as a maltose oxidising enzyme (MOX), for example hexose oxidase (HOX). Suitable methods are described in WO 03/099016.
  • MOX maltose oxidising enzyme
  • HOX hexose oxidase
  • Suitable methods are described in WO 03/099016.
  • Commercially available maltose oxidising enzymes GRINDAMYLTM and SUREBake are available from Danisco A/S.
  • an alpha-amylase such as a non-maltogenic exoamylase and/or a maltogenic amylases, and/or a maltose oxidising enzyme (MOX) in combination with the enzyme according to the present invention may be used in methods of preparing a dough, a baked product, tortilla, cake, instant noodle/fried snack food, or a dairy product such as cheese.
  • a non-maltogenic exoamylase and/or a maltogenic amylases and/or a maltose oxidising enzyme (MOX) in combination with the enzyme according to the present invention
  • MOX maltose oxidising enzyme
  • the invention further comprises methods for including the lipolytic enzyme in the foodstuff or other composition.
  • the invention provides in a further aspect a method of producing a foodstuff according to the invention, comprising adding the lipolytic enzyme and the cyclodextrin to one or more other ingredients of the foodstuff or other composition and, if necessary, treating the ingredients to produce the foodstuff.
  • the method further comprises adding a cyclodextrin glycosyltransferase enzyme and, if necessary, a polysaccharide substrate to generate cyclodextrin in situ.
  • the cyclodextrin glycosyltransferase enzyme may be added to the foodstuff before addition of the lipolytic enzyme.
  • the cyclodextrin glycosyltransferase enzyme is added to the foodstuff or other composition at least 10 minutes (more preferably between 10 minutes and 3 hours) before the lipolytic enzyme.
  • the cyclodextrin glycosyltransferase enzyme may be added to the foodstuff or other composition after addition of the lipolytic enzyme.
  • the cyclodextrin glycosyltransferase enzyme is added to the foodstuff or other composition at least 10 minutes (more preferably between 10 minutes and 3 hours) after the lipolytic enzyme.
  • the cyclodextrin glycosyltransferase enzyme is added at the same time as the lipolytic enzyme.
  • Such methods are generally known to the skilled person, and include adding the lipolytic enzyme directly to the foodstuff or composition, addition of the lipolytic enzyme in combination with a stabilizer and/or carrier, and addition of a mixture comprising the lipolytic enzyme and a stabilizer and/or carrier.
  • Suitable stabilizers for use with the present invention include but is not limited to inorganic salts (such as NaCL ammonium sulphate), sorbitol, emulsifiers and detergents (such as Tween 20, Tween 80, Panodan AB100 without triglycerides, polyglycerol ester, sorbitan monoo!eate), oil (such as rape seed oil, sunflower seed oil and soy oil), pectin, trehalose and glycerol.
  • inorganic salts such as NaCL ammonium sulphate
  • sorbitol such as sorbitol
  • emulsifiers and detergents such as Tween 20, Tween 80, Panodan AB100 without triglycerides, polyglycerol ester, sorbitan monoo!eate
  • oil such as rape seed oil, sunflower seed oil and soy oil
  • pectin such as rape seed oil, sunflower seed oil and soy oil
  • Suitable carriers for use with the present invention include but is not limited to starch, ground wheat, wheat flour, NaCS and citrate.
  • the bread and/or dough improving composition may further comprise another enzyme, such as one or more other suitable food grade enzymes, including starch degrading enzymes such as endo- or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g.
  • glucose oxidase pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as one which oxidises maltose, for example hexose oxidase (HOX), lipases, phosphoiipases and hexose oxidase, proteases and acyltransferases (such as those described in WO 2004/064987 for instance).
  • HOX hexose oxidase
  • lipases phosphoiipases and hexose oxidase
  • proteases acyltransferases
  • 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.
  • Further enzymes may be selected from the group consisting of a cellulase, a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase.
  • oxidases such as a glucose oxidase (EC 1.1 .3.4), carbohydrate oxidase, glycerol oxidase, pyranose oxidase, galactose oxidase (EC 1 .1.3.10), a maltose oxidising enzyme such as hexose oxidase (EC 1.1 .3.5).
  • the further enzyme is at least a xyianase and/or at least an antistaling amylase.
  • xyianase refers to xylanases (EC 3.2.1 .32) which hydrolyse xylosidic linkages.
  • amylase refers to amylases such as a-amylases (EC 3.2.1.1 ), ⁇ -amylases (EC 3.2.1.2) and ⁇ -amylases (EC 3.2.1.3).
  • the further enzyme can be added together with any dough ingredient including the flour, water or optional other ingredients or additives, or a dough improving composition.
  • the further enzyme can be added before the flour, water, and optionally other ingredients and additives or the dough improving composition.
  • the further enzyme can be added after the flour, water, and optionally other ingredients and additives or the dough improving composition.
  • the further enzyme may conveniently be a liquid preparation. However, the composition may be conveniently in the form of a dry composition.
  • host cell in relation to the present invention includes any cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of an enzyme having the specific properties as defined herein.
  • a further embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence that expresses the enzyme of the present invention.
  • the cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
  • the host cells are not human cells. Examples of suitable bacterial host organisms are gram positive or gram negative bacterial species.
  • eukaryotic hosts such as yeasts or other fungi may be preferred.
  • some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast).
  • a different fungal host organism should be selected.
  • 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.
  • the host cell may be a protease deficient or protease minus strain.
  • the genotype of the host cell may be modified to improve expression.
  • host cell modifications include protease deficiency, supplementation of rare tRNA's, and modification of the reductive potential in the cytoplasm to enhance disulphide bond formation.
  • the host cell may be deficient in a number of reducing enzymes thus favouring formation of stable disulphide bonds as exemplified/described in Bessette (Proc Natl Acad Sci USA (1999), 96, 13703-13708 "Efficient folding of proteins with multiple disulphide bonds in the Escherichia coli cytoplasm").
  • the enzymes for use in the present invention may be in an isolated form.
  • isolated means that the sequence or protein is at least substantially free from at least one other component with which the sequence or protein is naturally associated in nature and as found in nature.
  • the enzymes for use in the present invention may be used in a purified form.
  • purified means that the sequence is in a relatively pure state - e.g. at least about 51 % pure, or at least about 75%, or at least about 80%, or at least about 90% pure, or at least about 95% pure or at least about 98% pure.
  • a nucleotide sequence encoding either a polypeptide which has the specific properties as defined herein or a polypeptide which is suitable for modification may be isolated from any cell or organism producing said polypeptide.
  • Various methods are well known within the art for the isolation of nucleotide sequences.
  • a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the polypeptide.
  • labelled oligonucleotide probes may be synthesised and used to identify polypeptide-encoding clones from the genomic library prepared from the organism.
  • a labelled oligonucleotide probe containing sequences homologous to another known polypeptide gene could be used to identify polypeptide-encoding clones. Sn the latter case, hybridisation and washing conditions of lower stringency are used.
  • polypeptide-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing an enzyme inhibited by the polypeptide, thereby allowing clones expressing the polypeptide to be identified.
  • an expression vector such as a plasmid, transforming enzyme- negative bacteria with the resulting genomic DNA library
  • the nucleotide sequence encoding the polypeptide may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al (1981 ) Tetrahedron Letters 22, 1859-1869, or the method described by Matthes et al (1984) EMBO J. 3, 801 -805.
  • the phosphoroamidite method oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.
  • the nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence.
  • the DNA sequence may also be prepared by polymerase chain reaction (PGR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al (Science (1988) 239, 487-491 ).
  • nucleotide sequences encoding polypeptides having the specific properties as defined herein.
  • nucleotide sequence refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homoiogues, fragments and derivatives thereof (such as portions thereof).
  • the nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be doubie-stranded or single-stranded whether representing the sense or antisense strand.
  • nucleotide sequence in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence.
  • the nucleotide sequence per se encoding a polypeptide having the specific properties as defined herein does not cover the native nucleotide sequence in its natural environment when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment.
  • the term "non-native nucleotide sequence” means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment.
  • amino acid sequence encompassed by scope the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism.
  • amino acid sequence encompassed by scope of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
  • the polypeptide is not a native polypeptide.
  • native polypeptide means an entire polypeptide that is in its native environment and when it has been expressed by its native nucleotide sequence.
  • nucleotide sequence encoding polypeptides having the specific properties as defined herein is prepared using recombinant DNA techniques (i.e. recombinant DNA).
  • recombinant DNA i.e. recombinant DNA
  • the nucleotide sequence could be synthesised, in whole or in part, using chemical methods wel! known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et a/ (1980) Nuc Acids Res Symp Ser 225-232).
  • an enzyme-encoding nucleotide sequence has been isolated, or a putative enzyme-encoding nucleotide sequence has been identified, it may be desirable to modify the selected nucleotide sequence, for example it may be desirable to mutate the sequence in order to prepare an enzyme in accordance with the present invention.
  • Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
  • EP 0 583 265 refers to methods of optimising PGR based mutagenesis, which can also be combined with the use of mutagenic DNA analogues such as those described in EP 0 866 796.
  • Error prone PGR technologies are suitable for the production of variants of lipid acyl transferases with preferred characteristics.
  • WO 02/06457 refers to molecular evolution of lipases.
  • a third method to obtain novel sequences is to fragment non-identical nucleotide sequences, either by using any number of restriction enzymes or an enzyme such as Dnase I, and reassembling full nucleotide sequences coding for functional proteins. Alternatively one can use one or multiple non-identical nucleotide sequences and introduce mutations during the reassembly of the full nucleotide sequence.
  • DNA shuffling and family shuffling technologies are suitable for the production of variants of lipid acyl transferases with preferred characteristics. Suitable methods for performing 'shuffling' can be found in EP 0 752 008, EP 1 138 763, EP 1 103 606. Shuffling can a!so be combined with other forms of DNA mutagenesis as described in US 6, 180,406 and WO 01/34835.
  • mutations or natural variants of a polynucleotide sequence can be recombined with either the wild type or other mutations or natural variants to produce new variants.
  • Such new variants can also be screened for improved functionality of the encoded polypeptide.
  • the nucleotide sequence encoding a lipolytic enzyme used in the invention may encode a variant, i.e. the lipolytic enzyme may contain at least one amino acid substitution, deletion or addition, when compared to a parental enzyme.
  • Variant enzymes retain at least 70%, 80%, 90%, 95%, 97%, 99% homology with the parent enzyme.
  • Variant lipolytic enzymes may have decreased activity on triglycerides, and/or monoglycerides and/or digiycerides compared with the parent enzyme.
  • the variant enzyme may have no activity on triglycerides and/or monoglycerides and/or digiycerides. Alternatively, the variant enzyme may have increased thermostability.
  • the variant enzyme may have increased activity on one or more of the following, polar lipids, phospholipids, lecithin, phosphatidylcholine, glycolipids, digalactosyl monoglyceride, monogalactosyl monoglyceride.
  • variants of lipid acyltransferases are known, and one or more of such variants may be suitable for use in the methods and uses according to the present invention and/or in the enzyme compositions according to the present invention.
  • variants of lipid acyltransferases are described in the following references may be used in accordance with the present invention: Hilton & Buckley J Biol. Chem. 1991 Jan 15: 266 (2): 997-1000; Robertson et al J. Biol. Chem. 1994 Jan 21 ; 269(3):2146-50; Brumlik et al J. Bacteriol 1996 Apr; 178 (7): 2060-4; Peelman et al Protein Sci. 1998 Mar; 7(3):587-99.
  • AMINO ACID SEQUENCES AMINO ACID SEQUENCES
  • the present invention also encompasses the use of amino acid sequences encoded by a nucleotide sequence which encodes an enzyme for use in any one of the methods and/or uses of the present invention.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”.
  • amino acid sequence is synonymous with the term “peptide”.
  • amino acid sequence is synonymous with "enzyme”.
  • the amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
  • amino acid sequences may be obtained from the isolated polypeptides taught herein by standard techniques.
  • One suitable method for determining amino acid sequences from isolated polypeptides is as follows: Purified polypeptide may be freeze-dried and 100 pg of the freeze-dried material may be dissolved in 50 ⁇ of a mixture of 8 M urea and 0.4 M ammonium hydrogen carbonate, pH 8.4. The dissolved protein may be denatured and reduced for 15 minutes at 50°C following overlay with nitrogen and addition of 5 ⁇ of 45 mM dithiothreitol. After cooling to room temperature, 5 pi of 100 mM iodoacetamide may be added for the cysteine residues to be derivatized for 15 minutes at room temperature in the dark under nitrogen.
  • the resulting peptides may be separated by reverse phase HPLC on a VYDAC C18 column (0.46x15cm; 10pm; The Separation Group, California, USA) using solvent A: 0.1 % TFA in water and solvent B: 0.1 % TFA in acetonitrile.
  • Selected peptides may be re-chromatographed on a Develosil C18 column using the same solvent system, prior to N-terminal sequencing. Sequencing may be done using an Applied Biosystems 476A sequencer using pulsed liquid fast cycles according to the manufacturer's instructions (Applied Biosystems, California, USA).
  • homologue means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences.
  • the term “homology” can be equated with “identity”.
  • the homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the enzyme.
  • a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 60%, 70%, 71 %, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or 98% identical, preferably at least 95 or 98% identical to the subject sequence.
  • the homologues will comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence).
  • the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties.
  • a suitable computer program for carrying out such an alignment is the Vector NTI (Invitrogen Corp.).
  • Other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4 th Ed - Chapter 18), and FASTA (Altschul et al 1990 J. Mol. Biol. 403-410). Both BLAST and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60). However, for some applications, it is preferred to use the Vector NTI program.
  • BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1 ): 187-8 and tafiana@ncbi.nlm.nih.gov).
  • % homology can be measured in terms of identity
  • the alignment process itself is typically not based on an all-or-nothing pair comparison.
  • 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.
  • Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI Advance ' 10 package.
  • percentage homologies may be calculated using the multiple alignment feature in Vector NT! AdvanceTM 10 (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1 ), 237-244).
  • the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, preferably over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 100 contiguous nucleotides.
  • the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
  • the default parameters for the programme are used for pairwise alignment.
  • the following parameters are the current default parameters for pairwise alignment for BLAST 2:
  • the sequence identity for the nucleotide sequences and/or amino acid sequences may be determined using BLAST2 (blastn) with the scoring parameters set as defined above.
  • the degree of identity is based on the number of sequence elements which are the same.
  • the degree of identity in accordance with the present invention for amino acid sequences may be suitably determined by means of computer programs known in the art such as Vector NTI AdvanceTM 1 1 (Invitrogen Corp.).
  • the scoring parameters used are preferably BLOSUM62 with Gap existence penalty of 1 1 and Gap extension penalty of 1.
  • the degree of identity with regard to an amino acid sequence is determined over at least 20 contiguous amino acids, preferably over at least 30 contiguous amino acids, preferably over at least 40 contiguous amino acids, preferably over at least 50 contiguous amino acids, preferably over at least 60 contiguous amino acids, preferably over at least 100 contiguous amino acids.
  • the degree of identity with regard to an amino acid sequence may be determined over the whole sequence.
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • 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 secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present invention aiso encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • Non-homologous substitution may also occur i.e.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine
  • pyridylalanine thieny!alanine
  • naphthylalanine phenylglycine
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including a Iky I groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ -alanine residues.
  • Iky I groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or ⁇ -alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue's nitrogen atom rather than the ⁇ -carbon.
  • Nucleotide sequences for use in the present invention or encoding a polypeptide having the specific properties defined herein 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 and/or the 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 order to enhance the in vivo activity or life span of nucleotide sequences.
  • the present invention also encompasses the use of nucleotide sequences that are complementary to the sequences discussed herein, or any derivative, fragment or derivative thereof, !f the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
  • 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 made from a range of individuals, for example individuals from different populations.
  • other viral/bacterial, or cellular homoiogues particularly cellular homoiogues found in mammalian cells (e.g.
  • rat, mouse, bovine and primate cells may be obtained and such homoiogues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes 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 homoiogues and allelic variants of the polypeptide or nucleotide sequences of the invention.
  • Variants and strain/species homoiogues may also be obtained using degenerate PGR which will use primers designed to target sequences within the variants and homoiogues 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 PGR 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 sequence changes are required 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 polypeptide recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
  • Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PGR primer, a primer for an alternative amplification reaction, a probe e.g.
  • 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 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 stepwise 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 PGR (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 mPxNA or cDNA obtained from an animal or human 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 present invention also encompasses the use of sequences that are complementary to the sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.
  • hybridisation shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PGR) technologies.
  • the present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the subject sequences discussed herein, or any derivative, fragment or derivative thereof.
  • the present invention also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences discussed herein.
  • Hybridisation conditions are based on the melting temperature (Tm) of the nucleotide 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.
  • 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 Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • a maximum stringency hybridisation can be used to identify or detect identical nucleotide sequences while an intermediate (or low/) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences.
  • the present invention also relates to the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein).
  • the present invention also relates to the use of nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein).
  • polynucleotide sequences that are capable of hybridising to the nucleotide sequences discussed herein under conditions of intermediate to maximal stringency.
  • the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under stringent conditions (e.g. 50°C and 0.2 x SSC).
  • stringent conditions e.g. 50°C and 0.2 x SSC.
  • the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under high stringency conditions (e.g. 65°C and 0.1 x SSC).
  • the variant sequences etc. are at least as biologically active as the sequences presented herein.
  • biologically active refers to a sequence 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) of the naturally occurring sequence.
  • sequence for use in the present invention is a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques.
  • sequence for use in the present invention is a synthetic sequence - i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms - such as the methylotrophic yeasts Pichia and Hansenula.
  • a nucleotide sequence for use in the present invention or for encoding a polypeptide having the specific properties as defined herein can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate and express the nucleotide sequence, in polypeptide form, in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • the polypeptide produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intrace!lularly depending on the sequence and/or the vector used.
  • the coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • the expression vector is incorporated into the genome of a suitable host organism.
  • the term "incorporated” preferably covers stable incorporation into the genome.
  • the nucleotide sequence encoding an enzyme for use in the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host organism.
  • the vectors for use in 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.
  • vector e.g. a plasmid, cosmid, or phage vector
  • the choice of vector e.g. a plasmid, cosmid, or phage vector will often depend on the host cell into which it is to be introduced.
  • the vectors for use in the present invention may contain one or more selectable marker genes such as a gene which confers antibiotic resistance e.g. ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Alternatively, the selection may be accomplished by co-transformation (as described in W091/17243).
  • Vectors may be used in vitro, for example for the production of RNA or used to transfect, transform, transduce or infect a host cell.
  • the vector may further comprise a nucleotide sequence enabling the vector to replicate in the host eel! in question.
  • a nucleotide sequence enabling the vector to replicate in the host eel! in question.
  • sequences are the origins of replication of plasmids pUC19, pACYC177, pUB1 10, pE194, pAMB1 and plJ702.
  • the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell.
  • the present invention covers a vector comprising the nucleotide sequence 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 nucleotide sequence encoding the enzyme of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions.
  • the nucleotide sequence according to the present invention is operably linked to at least a promoter.
  • Suitable promoters for directing the transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.
  • construct which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - includes a nucleotide sequence encoding a polypeptide having the specific properties as defined herein for use 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 some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wiid 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.
  • the construct comprises at least a nucleotide sequence of the present invention or a nucleotide sequence encoding a polypeptide having the specific properties as defined herein operably linked to a promoter.
  • organism in relation to the present invention includes any organism that could comprise a nucleotide sequence according to the present invention or a nucleotide sequence encoding for a polypeptide having the specific properties as defined herein and/or products obtained therefrom.
  • transgenic organism in relation to the present invention includes any organism that comprises a nucleotide sequence coding for a polypeptide having the specific properties as defined herein and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence coding for a polypeptide having the specific properties as defined herein within the organism.
  • a promoter can allow expression of the nucleotide sequence coding for a polypeptide having the specific properties as defined herein within the organism.
  • the nucleotide sequence is incorporated in the genome of the organism.
  • Suitable organisms include a prokaryote, fungus yeast or a plant.
  • transgenic organism does not cover native nucleotide coding sequences in their natural environment when they are under the control of their 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, a nucleotide sequence coding for a polypeptide having the specific properties as defined herein, constructs as defined herein, vectors as defined herein, plasmids as defined herein, cells as defined herein, or the products thereof.
  • the transgenic organism can also comprise a nucleotide sequence coding for a polypeptide having the specific properties as defined herein under the control of a promoter not associated with a sequence encoding a lipid acyltransferase in nature.
  • the host organism can be a prokaryotic or a eukaryotic organism.
  • suitable prokaryotic hosts include bacteria such as E. coli and Bacillus licheniformis, preferably B. licheniformis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et a! (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
  • the transgenic organism can be a yeast.
  • Filamentous fungi cells may be transformed using various methods known in the art - such as a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the ceil wall in a manner known.
  • Aspergillus as a host microorganism is described in EP 0 238 023.
  • T. reesei is the host organism.
  • Another host organism can be a plant.
  • a review of the general techniques used for transforming plants may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol (1991 ) 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.
  • a host organism may be a fungus - such as a filamentous fungus.
  • suitable such hosts include any member belonging to the genera Fusarium, Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma and the like.
  • Trichoderma is the host organism, preferably T. reesei.
  • the host organism can be of the genus Aspergillus, such as Aspergillus niger.
  • a transgenic Aspergillus according to the present invention can also be prepared by following, for example, the teachings of Turner G. 1994 (Vectors for genetic manipulation, in: Martinelli S.D., Kinghorn J R. (Editors) "Aspergillus: 50 years on”; Progress in industrial microbiology vol 29; Elsevier Amsterdam 1994; pp. 641 -666).
  • the transgenic organism can be a yeast.
  • yeast - such as the species Saccharomyces cerevisi or Pichia pastoris or Hansenula polymorpha (see FEMS Microbiol Rev (2000 24(1 ):45-66), may be used as a vehicle for heterologous gene expression.
  • transgenic Saccharomyces can be prepared by following the teachings of Hinnen et a/., (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H ef al (1983, J Bacteriology 153, 163-168).
  • the transformed yeast cells may be selected using various selective markers - such as auxotrophic markers dominant antibiotic resistance markers.
  • a suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as, but not limited to, yeast species selected from Pichia spp., Hansenula spp., Kluyveromyces, Yarrowinia spp., Saccharomyces spp., including S. cerevisiae, or Schizosaccharomyce spp. including Schizosaccharomyce pom be.
  • a strain of the methylotrophic yeast species Pichia pastoris may be used as the host organism.
  • the host organism may be a Hansenula species, such as H. polymorpha (as described in WO 01/39544).
  • a host organism suitable for the present invention may be a plant.
  • a review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol ( 991 ) 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27), or in WO 01/16308.
  • the transgenic plant may produce enhanced levels of phytosterol esters and phytostanol esters, for example.
  • a host organism suitable for the present invention may be a plant.
  • a review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994
  • Host cells transformed with the nucleotide sequence of the present invention may be cultured under conditions conducive to the production of the encoded enzyme and which facilitate recovery of the enzyme from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in questions and obtaining expression of the enzyme.
  • the protein produced by a recombinant cell may be displayed on the surface of the cell
  • the enzyme may be secreted from the host cells and may conveniently be recovered from the culture medium using well-known procedures.
  • the polypeptide may be secreted from the expression host into the culture medium from where the enzyme 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.
  • Typical examples of secretion leader sequences not associated with a nucleotide sequence encoding a lipid acyltransferase in nature are those originating from the fungal amyloglucosidase (AG) gene (g/aA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and Hansenula) or the a-amylase gene (Bacillus).
  • AG fungal amyloglucosidase
  • a-factor gene e.g. Saccharomyces, Kluyveromyces and Hansenula
  • Bacillus e.g. Saccharomyces, Kluyveromyces and Hansenula
  • a variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiiuminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3,817,837; US-A-3,850,752; US-A-3,939,350; US-A-3,996,345; US-A-4,277,437; US-A-4,275,149 and US-A-4,366,241.
  • recombinant immunoglobulins may be produced as shown in US-A-4, 816,567.
  • An enzyme for use in the present invention may be produced as a fusion protein, for example to aid in extraction and purification thereof.
  • 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 proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the protein sequence.
  • amino acid sequence of a polypeptide having the specific properties as defined herein may be ligated to a non-native sequence to encode a fusion protein.
  • a non-native sequence For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a non- native epitope that is recognised by a commercially available antibody.
  • sequences for use according to the present invention may also be used in conjunction with one or more additional proteins of interest (POIs) or nucleotide sequences of interest (NO!s).
  • POIs proteins of interest
  • NO!s nucleotide sequences of interest
  • Non-limiting examples of POIs include: proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, a-galactosidases, ⁇ -ga!actosidases, a-glucanases, glucan lysases, endo- ⁇ - glucanases, glucoamylases, glucose oxidases, a-glucosidases, ⁇ -glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases
  • the POI may even be a fusion protein, for example to aid in extraction and purification.
  • the POI may even be fused to a secretion sequence.
  • sequences can also facilitate secretion or increase the yield of secreted POI.
  • sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821 198.0.
  • the NOI may be engineered in order to alter their activity for a number of reasons, including but not limited to, alterations which modify the processing and/or expression of the expression product thereof.
  • the NOI may also be modified to optimise expression in a particular host cell.
  • Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
  • the NO! may include within it synthetic or modified nucleotides- such as methylphosphonate and phosphorothi emerge backbones.
  • the NOI may be modified to increase intracellular stability and half-life.
  • composition of the present invention may be used as a food ingredient.
  • the term "food ingredient” includes a formulation which is or can be added to functional foods or foodstuffs as a nutritional supplement and/or fiber supplement.
  • the term food ingredient as used here also refers to formulations which can be used at low levels in a wide variety of products that require gelling, texturising, stabilising, suspending, film-forming and structuring, retention of juiciness and improved mouthfeel, without adding viscosity.
  • the food ingredient may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
  • the amino acid sequence is used for large scale applications.
  • the amino acid sequence is produced in a quantity of from 1g per litre to about 25g/litre, preferably from above 2.5g/litres to about 18 g/litre, preferably above 8 g per litre of the total cell culture volume after cultivation of the host organism.
  • Figure 1 illustrates mini breads made in Baking Trial 1 to demonstrate the effect of generating increasing levels of free fatty acids in situ using the recipe described in Table 2;
  • Figure 2 illustrates the relative volume (as compared with blank), % of triglyceride and % of free fatty acids as a function of lipase (Exel 16) dose on top of fixed GRINDAMYL POWERBAKE 4070 dose (1000 TIPU/kg flour) from Baking Trial 1 using the recipe described in Table 2;
  • Figures 3A and 3B illustrate mini breads made in Baking Trial 2 to illustrate the effect of adding increasing levels of ⁇ -cyclodextrins together with a high dose of the lipolytic enzyme Grindamyl POWERBAKE 4070 using the recipe described in Table 3 ( Figure 3A showing bread in tins and Figure 3B showing bread made without tins);
  • Figure 4 illustrates the relative volume (as compared with blank) as a function of added amount of ⁇ -cyclodextrin on top of fixed high Grindamyl POWERBAKE 4070 dose (5000 TIPU/kg flour) made in Baking Trial 2 using the recipe described in Table 3B;
  • Figure 5 illustrates a scheme for extraction of free fatty acids from lyophilized dough
  • Figure 7 illustrates mini breads made in Baking Trial 3 to compare the baking effect of ⁇ -cyclodextrin and pectin using the recipe described in Table 4 (no tins);
  • Figure 8 illustrates the relative volume (as compared with blank) as a function of added amount of p-cyclodextrin and pectin on top of increasing dosages of Exel 16 in Baking Trial 3;
  • Figure 9 illustrates mini breads made in Baking Trial 4 to compare the performance of a combination of Grindamyl POWERBAKE 4070 and CGTase compared with a combination of Grindamyl POWERBAKE 4070 and ⁇ -cyclodextrin using the recipe described in Table 5 (in tins);
  • Figure 10 illustrates mini breads with added starch made in Baking Trial 5 to compare the performance of a combination of Grindamyl POWERBAKE 4070 and CGTase compared with a combination of Grindamyl POWERBAKE 4070 and ⁇ -
  • Figure 13 shows an amino acid sequence of a fungal lipolytic enzyme derived from Fusarium heterosporum comprising an N terminal signal sequence (underlined) (SEQ ID No. 2);
  • Figure 14 shows a nucleotide sequence (SEQ ID No. 3) encoding a fungal lipolytic enzyme derived from Fusarium heterosporum in accordance with the present invention
  • Figure 15 shows an amino acid sequence (SEQ ID No. 4) of a lipolytic enzyme derived from Fusarium semitectum;
  • Figure 16 shows a nucleotide sequence (SEQ ID NO. 5) encoding a lipolytic enzyme derived from Fusarium se itectum;
  • Figure 17 shows SEQ ID No. 6 a Humicola lanuginosa lipase produced by Humicola lanuginosa DSM 4109, sold commercially as GrindamyS Exel 16;
  • Figure 18 shows a nucleotide sequence (SEQ ID No.7) of a lipolytic enzyme derived from Fusarium heterosporum which includes a a- factor signal sequence;
  • Figure 19 shows SEQ ID No. 8 which is the DNA sequence for the polypeptide variant designated "mut 3";
  • FIG. 9 shows SEQ ID No. 9 which is the protein preprosequence for the polypeptide variant designated "mut 3";
  • FIG. 21 shows SEQ ID No. 10 which is the DNA sequence for the polypeptide variant designated "mut 4";
  • FIG. 22 shows SEQ ID No. 1 1 which is the protein preprosequence for the polypeptide variant designated "mut 4";
  • FIG. 23 shows SEQ ID No. 12 which is the DNA sequence for the polypeptide variant designated "mut 5";
  • Figure 24 shows SEQ ID No. 13 which is the protein preprosequence for the polypeptide variant designated "mut 5";
  • FIG. 14 shows SEQ ID No. 14 which is the DNA sequence for the polypeptide variant designated "mut 345";
  • FIG. 26 shows SEQ ID No. 15 which is the protein preprosequence for the polypeptide variant designated "mut 345";
  • FIG. 27 shows SEQ ID No. 16 which is the DNA sequence for the polypeptide variant designated "mut 3459”;
  • Figure 28 shows SEQ ID No. 17 which is the protein preprosequence for the polypeptide variant designated "mut 3459”;
  • Figure 29 shows SEQ ID No. 18 which is the DNA sequence for the polypeptide variant designated "mut 9";
  • FIG. 30 shows SEQ ID No. 19 which is the protein preprosequence for the polypeptide variant designated "mut 9";
  • FIG. 31 shows SEQ ID No. 20 which is the DNA sequence for the polypeptide variant designated "mut 10";
  • FIG. 32 shows SEQ ID No. 21 which is the protein preprosequence for the polypeptide variant designated "mut 10";
  • Figure 33 shows SEQ ID No. 22 which is the DNA sequence for the polypeptide variant designated "mut 1 1 ";
  • Figure 34 shows SEQ ID No. 23 which is the protein preprosequence for the polypeptide variant designated "mut 1 1";
  • Figure 35 shows SEQ ID No. 24 which is the DNA sequence for the polypeptide variant designated "mut 12 " ;
  • FIG. 36 shows SEQ ID No. 25 which is the protein preprosequence for the polypeptide variant designated "mut 12".
  • Figure 37 shows SEQ ID No. 26 which is a lipolytic enzyme sold commercially as Lipopan FTM.
  • Lipopan F the enzyme having SEQ ID No. 26 as described herein
  • Novozymes A/S, Denmark 1204468, lot 4010629964 holding 12900 TIPU/g was used as a positive control.
  • Phospholipase Grindamyi POWERBAKE 4070 (the enzyme having SEQ ID No. 1 as described herein) (available from Danisco A S, Denmark) (EDS 129, 5830 TIPU/g) was used in combination with either Lipase Exel 16, ⁇ -cyclodextrin, CGTase or CGTase and starch.
  • Lipase Exel 16 (available from Danisco A/S, Denmark) (Control sample for protocol A770, 37350 LIPU/g) was used in combination with Grindamyi POWERBAKE 4070, ⁇ -cyclodextrin and pectin.
  • CGTase (Amano) was used in combination with either Grindamyi POWERBAKE 4070 or Grindamyi POWERBAKE 4070 and starch
  • ⁇ -cyclodextrin (Sigma C4767) was used in combination with Grindamyi POWERBAKE 4070 or Exel 16.
  • the water absorption of the flour and composite flours was determined using a Farinograph (Brabender, Germany) according to AACC 54-21 .
  • Baking performance was evaluated in small scale baking trials (50 gram mixer and 10 gram loaves) using the recipe shown in Table 1 below.
  • the amount of flour was adjusted according to the amount of ⁇ -cyclodextrin or pectin added to give a total of 50 g.
  • the same was the case for the trial described in Table 1 , where the amount of flour was adjusted so the total of added starch and flour summed up to 50 g.
  • Table 1 shows the recipe used for evaluation of baking performance.
  • Salt/sugar is a 1 :1 (w/w) mixture.
  • 'Water' is the water absorption determined by Farinograph analysis.
  • the flour and dry ingredients were mixed for one minute, water (with or without enzyme) was added and mixing was continued for another five minutes.
  • the aim of Baking Trial 1 was to test the effect of increasing levels of in situ generated free fatty acid (FFA).
  • the aim of Baking Trial 2 was to test the effect of adding increasing amounts of ⁇ -cyclodextrin for complexing of in situ generated FFA. In both cases mini bread in tins as well as without tins were made.
  • the setup of Baking Trial 2 the high amount of ⁇ -cyclodextrin added to the dough was evaluated by including a ⁇ -cyclodextrin blank.
  • the experimental set-up is listed in Tables 2 and 3.
  • Baking Trial 3 tested the baking effect of ⁇ -cyclodextrin versus the baking effect of the hydrocolloid pectin, which was used as a control. Only mini breads without tins were made. In this trial setup both a ⁇ -cyclodextrin blank and pectin blank was included. The experimental setup is listed in Table 4. Cyclodextrin glycosyltransferase (CGTase) is closely related to a-amylases but has the unique ability to produce cyclodextrins from linear a(1 ⁇ 4)-linked glucans via an intramolecular transg!ycosyiation reaction known as cyclization.
  • CCTase Cyclodextrin glycosyltransferase
  • Baking Trials 4 and 5 The aim of Baking Trials 4 and 5 was to test whether the same baking effect could be obtained by combining Grindamyl POWERBAKE 4070 and CGTase as with combining Grindamyl POWERBAKE 4070 and added ⁇ -cyclodextrin. In Baking Trial 5 additional substrate (starch) for the CGTase was added. In Baking Trial 5 a starch blank was included. In both trials only mini breads without tins were made. The experimental setup is listed in Tables 5 and 6.
  • the aim of Baking Trial 6 was to evaluate the effect of accumulating cyclodextrins in the dough before generating free fatty acids.
  • Table 2 Baking Trial 1 : Trial to test the effect of increasing dosage of Exei 16 in combination with a fixed dose of Grindamyl POWERBAKE 4070 (1000 TIPU/kg flour).
  • Baking Trial 2 Trial to test the effect of increasing amount of ⁇ -cyclodextrin in combination with a fixed dose of Grindamyl POWERbake 4070 (5000 TIPU/kg flour).
  • Baking Trial 4 Trial to test if combination of Grindamyl POWERbake 4070 and CGTase performed on level with combination of Grindamyl POWERbake 4070 and added ⁇ -cyclodextrin.
  • Lyophilized dough samples were milled in a coffee mill before extraction of the lipid fraction applying one of the following procedures.
  • Oven temperature °C. 80 200 240 360
  • Sample preparation Evaporated sample was dissolved in 1 ml pyridine containing internal standard heptadecane, 0.5 mg/ml. 500 ⁇ sample solution was transferred to a crimp vial, 100 pi BSTFA ( ⁇ /, ⁇ -bis-trimethylsilyl-trifluoroacetamide) + TMCS (trimethylchlorosilane) (99+1 ) was added and reacted for 15 minutes at 60°C.
  • the amount of free fatty acid was determined on Konelab Autoanaiyser (Thermo, Finland) using the NEFA HR(2) kit (WAKO GmbH, Germany). Assay was run at 30°C. 150 ⁇ _ solution A and 15 ⁇ _ redispersed extracted lipids were incubated for 3 minutes. 75 ⁇ solution B was added and incubated for 4.5 minutes. The absorbance at 520 nm was measured. The amount of free fatty acid was determined, using the read absorbance and a standard curve based on oleic acid (0.05 mM to 1.0 mM).
  • Table 7 Water absorption relative to fiour at 400 BLJ determined for each p- cyclodextrin concentration tested in Baking Thai 2.
  • This baking trial was conducted to evaluate the effect of adding increasing levels of ⁇ - cyclodextrin together with a high dose of Grindamyl POWERbake 4070 enzyme.
  • the addition of ⁇ -cyclodextrin should serve to lower the concentration of free fatty acids by formation of inclusion compounds and thereby eliminate/minimize the negative baking effect of increasing levels of free fatty acid in terms of reduced bread voiume.
  • Figure 5 shows the flow of the dough extraction made to differentiate between fatty acids incorporated in ⁇ -cyclodextrin inclusion compounds and free fatty acid.
  • extract 1 a small amount of free fatty acid will be extracted.
  • butanohethanol (85:15 (v/v)) to the watery extract will cause the fatty acid-p- cyclodextrin inclusion compounds to break down into free fatty acid and ⁇ - cyclodextrin.
  • the free fatty acid will be in the organic phase (extract 3) and the ⁇ - cyclodextrin will be in the water phase (extract 2).
  • the precipitate from the watery extract will contain the fatty acid not incorporated in inclusion compounds and by adding butanohethanol (85: 15 (v/v)) to the precipitate this fatty acid will be extracted (extract 4).
  • butanohethanol 85: 15 (v/v)
  • the results of the dough extractions depicted in Figure 5 are summarized in Table 8.
  • Table 8 For dough with no enzyme added (b!ank and ⁇ -cyclodextrin) the total amount of free fatty acid was in the proximity of 6 ⁇ /g dough, and for dough with enzyme added the total !evel of free fatty acid was in the range 1 1 -13 rnmol/ g dough.
  • Extract 3 was to be a number for the amount of free fatty acid released from ⁇ - cyclodextrin inclusion compounds upon addition of organic solvent.
  • Extract 2 As expected only a very small amount of free fatty acid was found in Extract 2: see Table 8. Presumably the rather varying free fatty acid numbers determined for mini bread made in tins was caused by inadequate centrifugation of the sample resulting in poor phase separation.
  • the amount of free fatty acid in extract 2, 3 and 4 should add up to the amount determined for the total extract. But as is evident from Table 8 this was not the case as the recovery (sum extract 2, 3 and 4 relative to the total extract) was in the range 40-60%, meaning only close to half the free fatty acid was extracted. The low recovery could be due to gluten agglumeration during water extraction of the iyophilized dough. Agglumerated gluten could trap free fatty acids and render them inaccessible for analysis. In fact a gum-like lump was observed in the precipitate of the water extracted Iyophilized dough indicating gluten agglumeration taking place. To verify if this was the case papain (a plant protease) was added during water extraction of selected Iyophilized doughs.
  • papain a plant protease
  • Table 9 shows the levels of free fatty acids in papain treated Iyophilized dough from baking trial conducted to illustrate the effect of increasing levels of added ⁇ - cyclodextrin (no tins).
  • the Iyophilized doughs were extracted as depicted in Figure 5 with the exception that 500 ppm papain was added during the water extraction. The amount of free fatty acid was determined applying the NEFA method.
  • Extract 1 Extract 2 Extract 3 Extract 4 (Extract (2+3+4) extraction / total)
  • Baking Trial 1 indicated increasing amounts of in situ generated free fatty acids had a negative baking effect in terms of reduced bread volume. Complexing free fatty acids with ⁇ -cyclodextrin had a significant positive effect on bread volume, 40% increase compared to blank, !n combination with Grindamyl POWERbake 4070, ⁇ -cyciodextrin had an additive effect, as an additional 20% volume increase was obtained, giving a total of 60% volume increase compared to blank.
  • Baking Trials 4 and 5 comparing the baking performance of Grindamyl POWERbake 4070 in combination with either ⁇ -cyclodextrin or CGTase showed the two combinations to perform on level. Further results observed in relation to the use of lipases include improved crumb structure, improved pore homogeneity and reduced mean pore size. These effects remain substantially unchanged by the inclusion of cyclodextrin.
  • CGTase was added to a fraction of the flour (20% of flour comprising the dough), together with the water needed to form the dough. Brews were stirred gently and left at 30°C for 1 hour. Afterwards the brew was added the residual amount of flour (80% of flour comprising the dough), mixed to form a dough, proofed, scaled and baked using the below baking procedure. 20% of flour is added to water required for dough (with or without CGTase) and the mixture is stirred gently at 30 °C for 1 hour. At this point, mixing of the dry ingredients (residual flour, yeast, salt, sugar) begins.

Abstract

A foodstuff containing an effective amount of: (a) a lipolytic enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule; and (b) a cyclodextrin; is disclosed. Methods of producing the foodstuff, as well as uses of a cyclodextrin for decreasing the free fatty acid content of a foodstuff incorporating a lipolytic enzyme, are also disclosed.

Description

Foodstuff
Field of the Invention
This invention relates to a foodstuff. It also relates to a method for producing the foodstuff and uses of certain ingredients of the foodstuff to provide novel technical effects.
Background to the Invention The use of lipolytic enzymes in baking is well established in the art. Lipolytic enzymes are capable of hydrolysing ester bonds a range of lipids present in wheat flour (in particular, triglycerides, glycolipids and phospholipids), either to liberate free fatty acid molecules or to transfer fatty acyl groups to other acceptor molecules. In particular, WO 2005/087918 describes a lipolytic enzyme and its use in baking. The enzyme described therein and having the sequence listing of SEQ !D No. 1 of this specification is commercially available from Danisco A/S under the trade mark GRINDA YL POWERBAKE 4070. Lipolytic enzymes provide a number of advantageous effects in baking applications. In particular, lipolytic enzymes can act on triglycerides present in flour to generate in situ mono- and diglycerides, which are useful as emulsifiers. Lipolytic enzymes can also act on glycolipids present in flour to generate in situ mono- and diglycosyl mono- and diglycerides, and on phospholipids present in flour to generate in situ lysophospholipids.
However, a number of problems are associated with the use of lipolytic enzymes in baking. Specifically, the addition of too much lipolytic enzyme to the dough mixture can cause an excess of free fatty acids to be present in the dough. The presence of high levels of free fatty acids (FFA) in raw materials or food products is generally recognised as a quality defect and food processors and customers will usually include a maximum FFA level in the food specifications. The resulting effects of excess FFA levels can be in organoleptic and/or functional defects. Cyclodextrins (sometimes called cycloamyloses) make up a family of cyclic oligosaccharides, composed of 5 or more glucose units linked 1 ->4, as in amylose (a fragment of starch). The 5-membered macrocycle is not natural. Typical cyclodextrins contain a number of glucose monomers ranging from six to eight units in a ring, creating a cone shape. Examples of known cyclodextrins are a-cyclodextrin (6- membered glucose ring molecule), β-cyclodextrin (7-membered glucose ring molecule) and γ -cyclodextrin (8-membered glucose ring molecule).
Cyclodextrins are known to interact with free fatty acids by formation of inclusion compounds, where the fatty acid is included in the hydrophobic torus of the cyclodextrin - see Bru e ai, Colloids and Surfaces A: Physicochemical and Engineering Aspects 97 (1995), 263-269.
Cyclodextrins may be generated in situ from starch or other polysaccharides present in a dough mixture, for example by the action of a cyclodextrin glycosyltransferase enzyme (also known as cyclodextrin glucosyltransferase or CGTase) (E.G. 2.4.1.19), the enzyme acting firstly to cleave the glycosidic bonds in the polysaccharide followed by bonding the glucose units together to form the ring. Examples of CGTases and their use in baking are described in WO 02/06508 and EP 0687414A. EP 0 493 045 A1 describes a foodstuff to which a β-cyclodextrin and a phospholipase D is added. This differs from the present invention in that phospholipase D cleaves phospholipids at the phosphate group to liberate phosphatidic acid and an alkanolamine, rather than a free fatty acid. Summary of the Invention
According to one aspect of the invention, there is provided a foodstuff comprising an effective amount of:
(a) a lipolytic enzyme; and
(b) a cyclodextrin. In one embodiment, there is provided a foodstuff comprising an effective amount of:
(a) a lipolytic enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule; and
(b) a cyclodextrin.
According to another aspect of the invention, there is provided a method of producing the above foodstuff, comprising adding the lipolytic enzyme and the cyciodextrin to one or more other ingredients of the foodstuff and, if necessary, treating the ingredients to produce the foodstuff.
According to a further aspect of the invention, there is provided use of a cyclodextrin for decreasing the uncompiexed free fatty acid content of a foodstuff incorporating a lipolytic enzyme. In one embodiment, there is provided use of a cyclodextrin for decreasing the uncompiexed free fatty acid content of a foodstuff incorporating a lipolytic enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule.
Advantages of the invention
It has surprisingly been found according to the present invention that the incorporation of a cyclodextrin into a foodstuff (particularly although not exclusively a dough and a baked product prepared from dough) enables the amounts of free fatty acid present in the foodstuff to be reduced. Without wishing to be bound by theory, it is believed that this effect is caused by the free fatty acid molecules forming an inclusion complex with the cyclodextrin in which the free fatty acid molecule is included in the hydrophobic interior of the cyclodextrin: the free fatty acids do not exhibit the deleterious effects described above when in the form of an inclusion complex. In particular, the incorporation of a cyclodextrin into a foodstuff (particularly although not exclusively a dough and a baked product prepared from dough) in which one or more lipolytic enzymes are present enables the amounts of free fatty acid liberated by the lipolytic enzyme to be reduced. The term Inclusion complex' or 'inclusion compound' is understood in the art as meaning a complex in which one component (the host) forms a cavity in which molecular entities of a second component (the guest) are located. There is no covalent bonding between guest and host, the attraction being generally due to van der VVaals forces.
Accordingly, in this specification the term 'compiexed' in relation to free fatty acids means that the free fatty acid is present as a component of an inclusion complex (as defined above) in which at least part of the fatty acid molecule is located within the cavity of the cyclodextrin molecule. Conversely, the term 'uncomplexed' means that the free fatty acid molecule is present in the foodstuff as a separate entity from the cyclodextrin molecule.
Detailed Description
Foodstuff
The term "foodstuff' as used herein means a substance which is suitable for human and/or animal consumption.
Suitably, the term "foodstuff' as used herein may mean a foodstuff in a form which is ready for consumption. Alternatively or in addition, however, the term foodstuff as used herein may mean one or more food materials which are used in the preparation of a foodstuff. By way of example only, the term foodstuff encompasses both baked goods produced from dough as well as the dough used in the preparation of said baked goods.
The foodstuff may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
When used as - or in the preparation of - a food - such as functional food - the composition of the present invention may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient. Sn a preferred aspect the present invention provides a foodstuff as defined above wherein the foodstuff is selected from one or more of the following: eggs, egg-based products, including but not limited to mayonnaise, salad dressings, sauces, ice creams, egg powder, modified egg yolk and products made therefrom; baked goods, including breads, cakes, sweet dough products, laminated doughs, liquid batters, muffins, doughnuts, biscuits, crackers and cookies; confectionery, including chocolate, candies, caramels, halawa, gums, including sugar free and sugar sweetened gums, bubble gum, soft bubble gum, chewing gum and puddings; frozen products including sorbets, preferably frozen dairy products, including ice cream and ice milk; dairy products, including cheese, butter, milk, coffee cream, whipped cream, custard cream, milk drinks and yoghurts; mousses, whipped vegetable creams, meat products, including processed meat products; edible oiis and fats, aerated and non- aerated whipped products, oil-in-water emulsions, water-in-oil emulsions, margarine, shortening and spreads including low fat and very low fat spreads; dressings, mayonnaise, dips, cream based sauces, cream based soups, beverages, spice emulsions and sauces.
Suitably the foodstuff in accordance with the present invention may be a "fine food", including cakes, pastry, confectionery, chocolates, fudge and the like.
In one aspect the foodstuff in accordance with the present invention may be a dough product or a baked product, such as bread, a fried product, a snack, cakes, pies, brownies, cookies, noodles, snack items such as crackers, graham crackers, pretzels, and potato chips, and pasta.
In another aspect the foodstuff in accordance with the present invention may be a convenience food, such as a part-baked or part-cooked product. Examples of such part-baked or part-cooked product include part-baked versions of the dough and baked products described above.
In a further aspect, the foodstuff in accordance with the present invention may be a plant derived food product such as flours, pre-mixes, oils, fats, cocoa butter, coffee whitener, salad dressings, margarine, spreads, peanut butter, shortenings, ice cream, cooking oils. In another aspect, the foodstuff in accordance with the present invention may be a dairy product, including butter, milk, cream, cheese such as natural, processed, and imitation cheeses in a variety of forms (including shredded, block, slices or grated), cream cheese, ice cream, frozen desserts, yoghurt, yoghurt drinks, butter fat, anhydrous milk fat, other dairy products. The enzyme according to the present invention may improve fat stability in dairy products.
In another aspect, the foodstuff in accordance with the present invention may be a food product containing animal derived ingredients, such as processed meat products, cooking oils, shortenings.
In a further aspect, the foodstuff in accordance with the present invention may be a beverage, a fruit, mixed fruit, a vegetable, a marinade or wine.
In one aspect, the foodstuff in accordance with the present invention is a plant derived oil (i.e. a vegetable oil), such as olive oil, sunflower oil, peanut oil or rapeseed oil. The oil may be a degummed oil. The claims of the present invention are to be construed to include each of the foodstuffs listed above.
Suitably, the maximum free fatty acid content of the foodstuff is 5%, preferably 2%, more preferably 1 %, even more preferably 0.5%, yet more preferably 0.2%, still more preferably 0.1 %, and most preferably 0.05%.
Cyclodextrin
In this specification the term 'cyclodextrin' means a cyclic oligosaccharide composed of 5 or more glucose units linked in a 1 ->4 manner so as to form a ring.
Suitably, the cyclodextrin comprises a cyclic oligosaccharide containing 5 to 10, preferably 6 to 8, glucose units. The cyclodextrin may optionally be substituted with a side chain comprising 1 to 3 monosaccharide, preferably glucose, units. Examples of suitable cyclodextrins include a-cyclodextrin (6-membered glucose ring molecule), β- cyciodextrin (7-membered glucose ring molecule) and y -cyciodextrin (8-memoered glucose ring molecule). Preferably, the cyciodextrin is β-cyclodextrin.
Suitably, the amount of cyciodextrin present is 0.05 to 50 g per kg of the total weight of the foodstuff. Preferably, the amount of cyciodextrin present is 0.1 to 20 g per kg of the total weight of the foodsiuff. More preferably, the amount of cyciodextrin present is 0.5 to 10 g per kg of the total weight of the foodstuff.
In one embodiment, where the foodstuff is a dough, the amount of cyciodextrin present is 0.05 to 50 g per kg of the flour used to form the dough. Preferably, the amount of cyciodextrin present is 0.1 to 20 g per kg flour. More preferably, the amount of cyciodextrin present is 0.5 to 10 g per kg flour.
In one embodiment, the cyciodextrin is present as an initial component of the foodstuff (i.e. the cyciodextrin is produced separately of the foodstuff and is added as a formed product to the foodstuff during preparation of the same). However, it is preferred according to the present invention that the cyciodextrin is generated in situ during preparation of the foodstuff, typically by the action of a cyciodextrin glycosyltransferase enzyme (E.G. 2.4.1.19) on polysaccharides present in the foodstuff.
In this specification the terms 'cyciodextrin glycosyltransferase', 'cyciodextrin glucosyltransferase' and 'CGTase' all mean an enzyme capable of acting on a polysaccharide so as to produce a cyciodextrin (as defined above, either in its broadest aspect or a preferred aspect). Typically, the polysaccharide on which the CGTase enzyme (if present) acts is selected from starch, amylose or amylopectin.
Suitable CGTase enzymes are described in WO 02/06508 and EP 0687414A. A particular example of a suitable CGTase enzyme is the cyciodextrin glucanotransferase (Bacillus macerans) enzyme available from Amano Enzymes, Japan.
Where present, the amount of CGTase enzyme present (measured by weight of the formulated enzyme) is suitably 0.01 g to 10 g per kg of the total weight of the foodstuff. Preferably, the amount of CGTase enzyme present (measured by weight of the formulated enzyme) is 0.05 g to 1 g per kg of the total weight of the foodstuff.
Where present, the amount of CGTase enzyme present (measured by weight of the pure enzyme protein) is suitably 0.01 mg to 2 mg per kg of the total weight of the foodstuff. Preferably, the amount of CGTase enzyme present (measured by weight of the pure enzyme protein) is 0.05 mg to 1 mg per kg of the total weight of the foodstuff.
Where present, the amount of CGTase enzyme present is suitably 6 to 1200 units of enzyme activity (U) per kg of the total weight of the foodstuff. The activity can be measured according to the Tilden-Hudson method described in Tilden E.B. and Hudson C.S. J. Bacterial. (1942), 43, 527-544.
Where present, the CGTase enzyme may be incorporated into the foodstuff before addition of the lipolytic enzyme. Alternatively, the CGTase enzyme may be incorporated into the foodstuff at the same time as the lipolytic enzyme.
In one embodiment, where the foodstuff is a dough, the amount of CGTase enzyme, where present (measured by weight of the formulated enzyme) is 0.01 to 10 g per kg of the flour used to form the dough. Preferably, the amount of CGTase enzyme, where present (measured by weight of the formulated enzyme) is 0.05 to 1 g per kg flour.
In this embodiment, the amount of CGTase enzyme, where present (measured by weight of the pure enzyme protein) is suitably 0.01 mg to 2 mg per kg of the flour used to form the dough. Preferably, the amount of CGTase enzyme, where present (measured by weight of the pure enzyme protein) is 0.05 mg to 1 mg per kg of flour.
In this embodiment, where present, the amount of CGTase enzyme present is suitably 6 to 1200 units of enzyme activity (U) per kg of the flour used to form the dough. The activity can be measured according to the Tilden-Hudson method referred to above. Lipolytic Enzyme
In this specification the term lipolytic enzyme' is defined as an enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule. Preferably, the lipolytic enzyme is an enzyme capable of hydro!ysing an ester bond in a lipid substrate (particularly although not exclusively a triglyceride, a giycolipid and/or a phospholipid) to liberate a free fatty acid molecule. Examples of possible lipid substrate are described below. The lipolytic enzyme used in the present invention has activity on both non-polar and polar lipids. The term "polar lipids" as used herein means phospholipids and/or glycolipids. Preferably, the term "polar lipids" as used herein means both phospholipids and glycolipids. Polar and non-polar lipids are discussed in Eliasson and Larsson, "Cereals in Breadmaking: A Molecular Colloidal Approach", publ. Marcel Dekker, 1993.
In particular, the lipolytic enzyme used in the present invention has activity on the following classes of lipids: triglycerides; phospholipids, particularly but not exclusively phosphatidylcholine (PC) and/or N-acylphosphatidylethanolamine (APE); and glycolipids, particularly although not exclusively digalactosyl diglyceride (DGDG).
In this specification the term 'free fatty acid' means a compound of the formula R-C(=0)-OH wherein R is a straight- or branched chain, saturated or unsaturated, hydrocarbyl group, the compound having a total of 4 to 40 carbon atoms, preferably 6 to 40 carbon atoms, such as at least 10 to 40 carbon atoms, for example 12 to 40, such as 14 to 40, 16 to 40, 18 to 40, 20 to 40 or 22 to 40 carbon atoms, more preferably 10 to 24, especially 12 to 22, particularly 14 to 18, for example 16 or 18 carbon atoms. In one particular embodiment, such an acyl group is an alkanoyl group. Alternatively, such an acyl group comprises an alkenoyl group, which may have, for example, 1 to 5 double bonds, preferably 1 , 2 or 3 double bonds.
Suitably, the lipolytic enzyme for use in the present invention may have one or more of the following activities selected from the group consisting of: phospholipase activity (such as phospholipase A1 activity (E.G. 3.1.1.32) or phospholipase A2 activity (E.G. 3.1 .1.4); glycolipase activity (E.G. 3.1.1.26), triacylglycerol hydrolysing activity (E.G. 3.1.1 ,3), lipid acy!transferase activity (generally classified as E.G. 2.3.1.x in accordance with the Enzyme Nomenclature Recommendations (1992) of the Nomenciature Committee of the International Union of Biochemistry and Molecular Biology), and any combination thereof. Such lipolytic enzymes are well known within the art.
Suitably, the lipolytic enzyme for use in the present invention may be a phospholipase (such as a phospholipase A1 (E.G. 3.1.1.32) or phospholipase A2 (E.G. 3.1.1.4)); glycolipase or galactolipase (E.G. 3.1.1 .26), triacylglyceride lipase (E.G. 3.1.1.3). Such enzyme may exhibit additional side activities such as lipid acyltransferase side activity. Suitably, the lipolytic enzyme for use in the present invention may have the activity of a phospholipase (such as a phospholipase A1 (E.G. 3.1.1.32) or phospholipase A2 (E.G. 3.1.1.4)); glycolipase or galactolipase (E.G. 3.1.1.26), triacylglyceride lipase (E.G. 3.1.1.3) under dough conditions.
Suitably, the lipolytic enzyme may be any commercially available lipolytic enzyme. For instance, the lipolytic enzyme may be any one or more of: Lecitase Ultra™, Novozymes, Denmark; Lecitase 10™; a phospholipase A1 from Fusarium spp e.g. Lipopan F™, Lipopan™ LipopanXtra™, YieldMax™; a phospholipase A2 from Aspergillus niger; a phospholiapse A2 from Streptomyces violaceruber e.g. LysoMax PLA2™; a phospholipase A2 from Tuber borchii; or a phospholipase B from Aspergillus niger, Lipase 3, Grindamyl EXEL 16™, and GRINDA YL POWERBake 4070, Panamore™ or GRINDAMYL POWERBake 4100. Preferably, the lipolytic enzyme is GRINDAMYL POWERBake 4070™, Lipopan F™, Lipopan™' LipopanXtra™, or Panamore™.
SOURCE
Suitably, the lipolytic enzyme is obtainable from a microorganism, examples of which include bacteria and fungi (such as yeasts). Preferably, the lipolytic enzyme is obtainable from a filamentous fungus. In particular, the lipolytic enzyme is obtainable from a fungus of the genus Fusarium, Aspergillus or Streptomyces, especially from a fungus of the genus Fusarium. In preferred embodiments, the lipolytic enzyme is obtainable from Fusarium heterosporum. !n a particularly preferred embodiment, the lipolytic enzyme is obtainable from Fusarium heterosporum CBS 782.83. Disclosed herein are prepro-polypeptides which when post-translationally processed in a host organism produces a polypeptide which has hydrolytic activity towards an ester bond in a polar lipid, wherein the prepropolypeptide comprises an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 and SEQ ID No. 25.
In one aspect the present invention the lipolytic enzyme for use in the present invention is a polypeptide which has hydrolytic activity towards an ester bond in a polar lipid, which polypeptide is obtainable from a prepro-polypeptide comprising an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 or SEQ ID No. 25.
Depending on the host organism prepro-sequences often go through post- translational modification. With the present enzymes it is relatively common for the organism to remove the N-terminal region of the prepro sequence, i.e. remove all or part of the amino acids 1 -30 of SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 or SEQ ID No. 25. In some embodiments the host organism may remove slightly more amino acids than those shown as amino acids 1 -30 of SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 or SEQ ID No. 25 such as removing amino acids 1-31 or 1 -32 or 1 -33 for instance. In some instances the host organism may introduce an alternative N-terminal sequence which may encompass all or part of the amino acids shown as amino acids 1 -30 or may comprise a completely different N-terminal sequence (such as EAEA or EA for instance). In some cases the mature enzyme produced from the prepro-sequence by the host organism may be a heterogen at its N-terminus end. In some embodiments the post- translational modification may mean modification in the C-terminal region of the prepro sequence. For example, all or part of the amino acids 306-348 may be removed from SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 or SEQ ID No. 25 in the mature form. In some embodiments, the host organism may remove slightly more amino acids than those shown as amino acids 306-348 of SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 or SEQ ID No. 25, such as removing amino acids 305-348 or 304-348 or 303=348 for instance. In some cases the mature enzyme produced from the prepro-sequence by the host organism may be a heterogen at its C-terminus end. It is envisaged that the present invention encompasses the use of all mature forms of the protein obtainable from a prepro-polypeptide comprising an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ SD No. 23 or SEQ ID No. 25, particularly those obtained from the host organism Trichoderma reesei.
Suitably the lipolytic enzyme for use in the present invention may comprise one of the following amino acid sequences:
a) an amino acid sequence as set forth in SEQ ID No. 1 ;
b) an amino acid sequence as set forth in SEQ ID No. 2;
c) an amino acid sequence as set forth in SEQ ID No. 4;
d) an amino acid sequence as set forth in SEQ ID No.6;
e) positions 31 to 348 of SEQ ID No. 9;
f) positions 31 to 348 of SEQ ID No. 1 1 ;
g) positions 31 to 348 of SEQ ID No. 13;
h) positions 31 to 348 of SEQ ID No. 15;
i) positions 31 to 348 of SEQ ID No. 17;
j) positions 31 to 348 of SEQ ID No. 19;
k) positions 31 to 348 of SEQ ID No. 21 ;
I) positions 31 to 348 of SEQ ID No. 23;
m) positions 31 to 348 of SEQ ID No. 25;
n) an amino acid sequence as set forth in SEQ ID No. 26;
o) an amino acid sequence encoding a lipolytic enzyme having at least 70% identity to any of the sequences in a) to n);
p) a sub-sequence of any of the sequences in a) to o) or
q) an amino acid sequence derived from any one of the amino acid sequences shown as a) to n) by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, amino acids, or more amino acids, such as 10 or more than 10 amino acids; and having lipolytic enzyme activity. Suitably, the lipolytic enzyme for use in the present invention may have at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% identity with any of the sequences shown in a) to n).
Suitably, the lipolytic enzyme for use in the present invention may have an amino acid sequence as set forth in SEQ ID No. 1 or an amino acid sequence having at least 70% identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% identity thereto.
Suitably the lipolytic enzyme for use in the present invention may be a lipolytic enzyme encoded by any one of the following nucleic acid sequences:
a) a nucleic acid sequence as set forth in SEQ ID No. 3;
b) a nucleic acid sequence as set forth in SEQ ID No. 5;
c) a nucleic acid sequence as set forth in SEQ ID No. 7;
d) a nucleic acid sequence as set forth in SEQ ID No. 8;
e) a nucleic acid sequence as set forth in SEQ ID No. 10;
f) a nucleic acid sequence as set forth in SEQ ID No. 12;
g) a nucleic acid sequence as set forth in SEQ ID No. 14;
h) a nucleic acid sequence as set forth in SEQ ID No. 16;
i) a nucleic acid sequence as set forth in SEQ ID No. 18;
j) a nucleic acid sequence as set forth in SEQ ID No. 20;
k) a nucleic acid sequence as set forth in SEQ ID No. 22;
1) a nucleic acid sequence as set forth in SEQ ID No. 24;
m) a nucleic acid sequence encoding a lipolytic enzyme having at least 70% identity to any of the sequences in a) to I);
n) a DNA molecule which hybridizes under stringent conditions (or high stringency conditions) to the nucleic acid sequence defined in any one of a) to
I) and encodes a protein having lipolytic enzyme activity; or
o) a nucleic acid sequence derived from the nucleic acid sequence as set forth in any one of a) to I) by substitution, deletion or addition of one or several nucleic acids, such as 2, 3, 4, 5, 6, 7, 8, 9, nucleic acids, or more nucleic acids, such as 10 or more than 10 nucleic acids; and having lipolytic enzyme activity. Suitably, the lipolytic enzyme for use in the present invention may be a lipolytic enzyme encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably af least 95%, preferably at least 98%, preferably at least 99% identity with any of the sequences shown in a) to I).
Suitably, the lipolytic enzyme for use in the present invention may be encoded by a nucleic acid sequence as set forth in SEQ ID No. 3 or a nucleic acid sequence having at least 70% identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% identity thereto.
Lipolytic enzymes have a number of advantageous effects in baking applications. Suitably, the lipolytic enzyme for use in the present invention may confer one or more of the following effects on dough and/or a baked product prepared from dough: reducing stickiness; improving machinability; reducing blistering during baking; improved volume; improved softness; prolonged shelf life; reduced staling; improved crumb structure; improved pore homogeneity; reducing mean pore size; enhancing the gluten index; improved flavour; improved colour of the crust. These effects remain substantially unchanged by treatment with cyclodextrin. Accordingly, the use of a cyclodextrin in combination with a lipolytic enzyme allows any or all of the above advantageous effects to be exhibited while reducing the content of free fatty acid in the dough and/or baked product prepared from dough and avoiding the deleterious effects associated therewith.
DELAYED RELEASE / ENCAPSULATION
In one embodiment, the lipolytic enzyme is in a delayed release form. In this specification the term "delayed release" (also known as "slow release") when used in its broadest sense means that the lipolytic enzyme is released into the foodstuff a time later than that immediately following its introduction.
Preferably, the term "delayed release" means that 50% of the lipolytic enzyme is released into the foodstuff at a time at least 5 minutes after its introduction and 100% of the lipolytic enzyme is released into the foodstuff after at a time at least 10 minutes after its introduction. More preferably, the term "delayed release" means that 50% of the lipolytic enzyme is released into the foodstuff at a time between 5 minutes and 3 hours after its introduction and 100% of the lipolytic enzyme is released into the foodstuff after at a time between 10 minutes and 5 hours after its introduction.
The provision of the lipolytic enzyme in delayed release form confers the advantage that the rate of generation of free fatty acid by the lipolytic enzyme can be slowed down. In particular, when the cyclodextrin is generated in situ by the action of a CGTase enzyme on a polysaccharide, the provision of the lipolytic enzyme in delayed release form allows sufficient time for the CGTase enzyme to generate the cyclodextrin before significant quantities of free fatty acid are generated by the lipolytic enzyme. in a particular embodiment, the lipolytic enzyme is encapsulated. Encapsulation is a process of surrounding or coating an ingredient with a substance in order to prevent or delay the release of the ingredient until a certain time or set of conditions is achieved. Various techniques are employed to form the capsules, including spray drying, spray chilling or spray cooling, extrusion coating, fluidized bed coating, liposome entrapment, coacervation, inclusion complexation, centrifugal extrusion and rotational suspension separation. Fats, emulsifiers, starches, dextrins, alginates, protein and lipid materials can be employed as encapsulating materials.
Various methods exist to release the ingredients from the capsules. Release can be site-specific, stage-specific or signalled by changes in pH, temperature, irradiation or osmotic shock.
Lipid Substrate The lipolytic enzyme used in the present invention is capable of hydrolysing an ester bond in a lipid substrate. In this specification the term 'lipid substrate' is defined as meaning a fat-soluble (lipophilic), naturally-occurring or synthetic molecule, having an ester group capable of being hydrolysed to liberate a free fatty acid molecule (as defined above). Examples of suitable lipids include triglycerides, mono- and polyglycerolipids (especially monog!ycerides and diglyce rides), giycolipids, phospholipids, and the like. It is intended within the scope of the present invention that the lipid substrate may comprise a mixture of lipids. By lipophilic' means soluble in non-polar organic solvents. Preferably, such non-polar organic solvents have one or more of the following properties:
(a) a low dielectric constant (for example, a dielectric constant less than 20, preferably less than 10) and/or
(b) a weak or zero dipole moment (for example, a dipoie moment of less than 1 D, preferably less than 0.5 D); and/or
(c) an absence of or substantially no hydrogen-bonding groups (OH and/or N-H).
Examples of such non-polar organic solvents include aliphatic hydrocarbons such as pentane, hexane, heptane, aiicylic hydrocarbons such as cyclohexane, aromatic hydrocarbons such as benzene, toluene or xylene, ethers such as diethyl ether, and halogenated hydrocarbons such as dichloromethane, trichloromethane (chloroform) and 1 ,2-dichloroethane. The lipid substrate includes one or more fatty acyl groups, ie groups of the formula R-C{=O wherein R is a straight- or branched chain, saturated or unsaturated hydrocarbyl group. Typically, such acyl groups have a total of 4 to 40 carbon atoms, preferably 6 to 40 carbon atoms, such as at least 10 to 40 carbon atoms, for example 12 to 40, such as 14 to 40, 16 to 40, 18 to 40, 20 to 40 or 22 to 40 carbon atoms, more preferably 10 to 24, especially 12 to 22, particularly 14 to 18, for example 16 or 18 carbon atoms. In one particular embodiment, such an acyl group is an alkanoyl group. Alternatively, such an acyl group comprises an alkenoyl group, which may have, for example, 1 to 5 double bonds, preferably 1 , 2 or 3 double bonds. Examples of acyl groups include saturated acyl groups such as butanoyl (butyryl), hexanoyl (caproyl), octanoyl (capryl), decanoyl (caprinyl), dodecanoyl (lauroyl), tetradecanoyl, (myristoyl), hexadecanoyl (palmitoyl), octadecanoyl (stearoyl), eicosanoyl (arachidonyl), docosanoyl (behenoyl) and tetracosanoyl (lignoceroyl) groups, and unsaturated acyl groups such as c/s-tetradec-9-enoyl (myristoleyl), c/s- hexadec-9-enoyl (palmitoleyl), c s-octadec-9-enoyl (oleyl), cis c/s-9, 12- octadecadienoyi (lino!eyl), cis, cis. c/s-9, 12, 15- octadecatrienoyl (linolenyl), and cis,cis,cis,cis-5,8, 1 1 ,14-eicosa-tetraenoyl (arachidonyl) groups.
Some typical classes of lipids suitable as substrates in the present invention are described below.
In a preferred embodiment, the lipid substrate is a triglyceride. The term 'triglyceride' (also known as triacylglycerol) means a compound comprising three acyl groups (which may be the same or different) covalently bonded to a single glycerol moiety via ester linkages.
In another embodiment, the lipid substrate is a glycoglycerolipid (also known as a glycosylglyceride). In this specification the term 'glycoglycerolipid', when used to define the lipid substrate molecule, means a lipid comprising a single glyceroi moiety covalently bound via ester linkages to one or more acyl groups (the typical and preferred lengths of which are defined and exemplified above) and having one or more monosaccharide moieties attached to the glycerol moiety via a glycosidic linkage, provided it contains at least one free hydroxy! group to enable transfer to take place. When more than one monosaccharide moiety is present on the glycoglycerolipid product, the monosaccharides may be bonded to different oxygen atoms on the glycerol backbone, may be bonded to each other to comprise a di-, oligo- or polysaccharide moiety attached to one oxygen atom on the glycerol moiety, or any combination thereof. Glycerophospholipids, also referred to as phospholipids, are ubiquitous in nature and are key components of the lipid bilayer of cells, as well as being involved in metabolism and signaling. Glycerophospholipids may be subdivided into distinct classes, based on the nature of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria or the sn-1 position in the case of archaebacteria. Examples of glycerophospholipids found in biological membranes are phosphatidylcholines (also known as PC or GPCho, and lecithin), phosphatidylethanolamines (PE or GPEtn), phosphatidylserine (PS or GPSer), phosphadityl inositol, lysophosphatidylcholines, lysophosphatidylethanolamines, N- acyl phosphatidylethanolamines and N-acyl lysophosphatidylethanolamines. In addition to serving as a primary component of cellular membranes and binding sites for intra- and intercellular proteins, some g!ycerophospholipids in eukaryotic cells, such as phosphatidylinositois and phosphatide acids are either precursors of, or are themselves, membrane-derived second messengers. Typically one or both of these hydroxyl groups are acylated with long-chain fatty acids (the number of carbon atoms in the chains typically as set out above), but there are also alkyl-linked and 1 Z- alkenyl-linked (plasmalogen) glycerophospholipids, as well as dialkylether variants in prokaryotes.
Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a sugar substitutes for the glycerol backbone that is present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa- acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno- octulosonic acid (Kdo) residues. in an alternative embodiment, the lipid substrate is a lysophospholipid. A lysophospholipid comprises a glycerol moiety having only one acyl group (as defined and exemplified above) covalent!y bonded to a glycerol oxygen atom via an ester linkage and a phosphate group covalently bonded to another glycerol oxygen atom to form a phosphate ester: the said lysophospholipids therefore possess a free OH group on the remaining glycerol carbon atom. Suitable lysophospholipids include lysophosphatidylcholines (also known as lyso-PC), lysophosphatidylethanolamines (PE or GPEtn), phosphatidylserine (PS or GPSer), phosphadityl inositol, , N-acyl phosphatidyl-ethanolamines and N-acyl lysophosphatidyl-ethanolamines. The lysophospholipid may be formed in situ by hydrolysis of one of the ester linkages on the corresponding phospholipid.
Dosage
Suitably, the amount of lipolytic enzyme present (measured by weight of the formulated enzyme) is 0.001 g to 2 g per kg of the total weight of the foodstuff. Preferably, the amount of lipolytic enzyme present (measured by weight of the formulated enzyme) is 0.005 g to 1 g per kg of the total weight of the foodstuff.
In one embodiment, where the foodstuff is a dough, the amount of lipolytic enzyme present (measured by weight of the formulated enzyme) is 0.001 g to 2 g per kg of the flour used to form the dough. Preferably, the amount of lipolytic enzyme present (measured by weight of the formulated enzyme) is 0.005 g to 0.5 g per kg of the flour.
Suitably, the amount of lipolytic enzyme present (measured by weight of pure enzyme protein) is 0.001 mg to 2 mg per kg of the total weight of the foodstuff. Preferably, the amount of lipolytic enzyme present (measured by weight of pure enzyme protein) is 0.005 mg to 1 mg per kg of the total weight of the foodstuff.
In one embodiment, where the foodstuff is a dough, the amount of lipolytic enzyme present (measured by weight of pure enzyme protein) is 0.001 mg to 2 g per kg of the flour used to form the dough. Preferably, the amount of lipolytic enzyme present (measured by weight of pure enzyme protein) is 0.005 to 0.5 mg per kg of the flour.
Suitably, the amount of lipolytic enzyme present is 50 to 15,000 TIPU per kg of the total weight of the foodstuff. Preferably, the amount of lipolytic enzyme present is 100 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 TIPU per kg of the total weight of the foodstuff.
!n one embodiment, where the foodstuff is a dough, the amount of lipolytic enzyme present is 50 to 15,000 TIPU per kg of the flour used to form the dough. Preferably, the amount of lipolytic enzyme present is 100 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 TIPU per kg of the flour. The TIPU unit of activity and the assay used to determine it is described in more detail below. Suitably, the amount of lipolytic enzyme present is 35 to 10,000 LIPU-NEFA per kg of the total weight of the foodstuff. Preferably, the amount of lipolytic enzyme present is 50 to 5,000, preferably 200 to 1 ,000 LIPU-NEFA per kg of the total weight of the foodstuff. In one embodiment, where the foodstuff is a dough, the amount of lipolytic enzyme present is 35 to 10,000 LIPU-NEFA per kg of the f!our used to form the dough. Preferably, the amount of lipolytic enzyme present is 50 to 5,000, preferably 200 to 1 ,000 LIPU-NEFA per kg of the flour. The LIPU-NEFA unit of activity and the assay used to determine it is described in more detail below.
Suitably, the amount of lipolytic enzyme present is 15 to 15,000 GLU per kg of the total weight of the foodstuff. Preferably, the amount of lipolytic enzyme present is 50 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 GLU per kg of the total weight of the foodstuff.
In one embodiment, where the foodstuff is a dough, the amount of lipolytic enzyme present is 15 to 15,000 GLU per kg of the flour used to form the dough. Preferably, the amount of lipolytic enzyme present is 50 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 GLU per kg of the flour. The GLU unit of activity and the assay used to determine it is described in more detail below.
The glycolipase activity, phospholipase activity and triacyiglyceride lipase activity of the lipolytic enzyme as used in the present invention can be determined using the assays presented hereinbelow.
Determination of galactolipase activity (glycolipase activity assay):
Substrate:
0.6% digalactosyldiglyceride (Sigma D 4651 ), 0.4% Triton-X 100 (Sigma X-100) and 5 mM CaCI2 was dissolved in 0.05M 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) buffer pH 7.
Assay procedure:
400 pL substrate was added to an 1.5 mL Eppendorf tube and placed in an Eppendorf Thermomixer at 37°C for 5 minutes. At time t= 0 min, 50 pL enzyme solution was added. Also a blank with water instead of enzyme was analyzed. The sample was mixed at 10*100 rpm in an Eppendorf Thermomixer at 37°C for 10 minutes. At time t=10 min the Eppendorf tube was placed in another thermomixer at 99°C for 10 minutes to stop the reaction. Free fatty acid in the samples was analyzed by using the NEFA HR(2) kit from WAKO GmbH. Enzyme activity GLU at pH 7 was calculated as micromoles of fatty acid produced per minute under assay conditions. Determination of phospholipase activity (phospholipase activity assay):
Phospholipase activity was measured using two different methods which give comparable results. Either of these methods can be used to determine phospholipase activity in accordance with the present invention. Preferably, the PLU assay is used for determining the phospholipase activity of any enzyme.
"PLU assay" for determination of phospholipase activity
Substrate:
0.6% L-a Phosphatidylcholine 95% Plant (Avanti #441601 ), 0.4% Triton-X 100 (Sigma X-100) and 5 m CaCI2 was dissolved in 0.05M HEPES buffer pH 7.
Assay procedure:
400 μΐ_ substrate was added to an 1.5 mL Eppendorf tube and placed in an Eppendorf Thermomixer at 37°C for 5 minutes. At time t= 0 min, 50 μί. enzyme solution was added. Also a blank with water instead of enzyme was analyzed. The sample was mixed at 10*100 rpm in an Eppendorf Thermomixer at 37°C for 10 minutes. At time t=10 min the Eppendorf tube was placed in another thermomixer at 99°C for 10 minutes to stop the reaction.
Free fatty acid in the samples was analyzed by using the NEFA HR(2) kit from WAKO GmbH. Enzyme activity PLU-7 at pH 7 was calculated as micromoles of fatty acid produced per minute under assay conditions.
"TIPU assay" for determination of phospholipase activity 1 TIPU (Titration Phospholipase Unit) is defined as the amount of enzyme which liberates Ι μΐτιοΙ free fatty acid per minute at the assay conditions.
Phospholipase A1 and A2 catalyse the conversion of lecithin to lyso-lecithin with release of the free fatty acid from position 1 and 2, respectively. Phospholipase activity can be determined by continuous titration of the fatty acids liberated from lecithin during enzymation, since the consumption of alkali equals the amount of fatty acid liberated.
Substrate:
4% lecithin, 4% Triton-X 100, and 6 mM C-aCS2: 12 g lecithin powder (Avanti Po!ar Lipids #44160) and 12 g Triton-X 100 (Merck 108643) was dispersed in approx. 200 mi demineralised water during magnetic stirring. 3.0 ml 0.6 M CaCI2 (p. a. Merck 1 .02382) was added. The volume was adjusted to 300 mL with demineralised water and the emulsion was homogenised using an Ultra Thurax. The substrate was prepared freshly every day.
Assay procedure:
An enzyme solution was prepared to give a slope on the titration curve between 0.06 and 0.18 ml/min with an addition of 300 μΐ_ enzyme. A control sample of known activity is included. The samples were dissolved in demineralised water and stirred for 15 min. at 300 rpm. 25.00 ml substrate was thermostatted to 37.0°C for 10-15 minutes before pH was adjusted to 7.0 with 0.05 M NaOH. 300 μΐ_ enzyme solution was added to the substrate and the continuous titration with 0.05 M NaOH was carried out using a pH-Stat titrator (Phm 290, Mettler Toledo). Two activity determinations are made on each scaling. After 8 minutes the titration is stopped and the slope of the titration curve is calculated between 5 and 7 minutes. The detection limit is 3 TIPU/ml enzyme solution.
Calculations:
The phospholipase activity (TIPU/g enzyme) was calculated in the following way:
Figure imgf000023_0001
In the formula above:
a is the slope of the titration curve between 5 and 7 minutes of reaction time (ml/min); N is the normality of the NaOH used (mol/l);
\/Ί is the volume in which the enzyme is dissolved (ml);
m is the amount of enzyme added to V! (g); and V2 is the volume of enzyme solution added to the substrate (m!). "LIPU-NEFA assay" for determination of triacylgiycerol lipase activity Substrate:
0.2% tris(octanoyl)giycero! (Sigma T9126), 13% Triton X-100 (Sigma X-100) and 0.3% NaCI was dissolved in 0.120 M sodium acetate buffer, pH 5.5.
Assay procedure:
400 μΙ_ substrate was added to a 1 .5 mL Eppendorf tube and placed in an Eppendorf Thermomixer at 37°C for 5 minutes. At time t= 0 min, 50 pL enzyme solution was added. Also a blank with water instead of enzyme was analysed. The sample was mixed at 10000 rpm in an Eppendorf Thermomixer at 37°C for 10 minutes. At time t= 10 mi the Eppendorf tube was placed in another thermomixer at 99°Cfor 10 minutes to stop the reaction.
Free fatty acid in the samples was analyzed by using the NEFA HR(2) kit from WAKO GmbH. Enzyme activity LSPU-NEFA at pH 5.5 was calculated as micromoles of fatty aid produced per minute under assays conditions.
FURTHER ENZYMES
In addition to the lipolytic enzyme one or more further food grade enzymes may be used, for example added to the food, dough preparation, or foodstuff.
Thus, it is within the scope of the present invention that, in addition to the lipolytic enzyme of the present invention, at least one further enzyme may be added to the baked product and/or the dough. Such further enzymes include starch degrading enzymes such as endo- or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g. glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as one which oxidises maltose, for example hexose oxidase (HOX), lipases, phospholipases and hexose oxidase, proteases, and acyltransferases (such as those described in WO 2004/064987 for instance). St is particularly preferred that the lipolytic enzyme of the invention is used in combination with alpha amylases in producing food products. In particular, the amylase may be a non-maltogenic amylase, such as a polypeptide having non- maltogenic exoamylase activity, in particular, giucan 1 ,4-alpha-maltotetrahydrolase (EC 3.2.1.60) activity (as disclosed in WO 2005/003339). A suitable non-maltogenic amylase is commercially available as Powersoft™ (available from Danisco A/S, Denmark). Maltogenic amylases such as Novamyl™ (Novozymes A/S, Denmark) may also be used, !n one embodiment, the combined use of alpha amylases and the lipolytic enzyme of the invention may be used in a dough, and/or the production of a baked product, such as bread, cakes, doughnuts, cake doughnuts or bagels. The combination of alpha amylases and the lipolytic enzyme of the invention is also considered as preferable for use in methods of production of tortillas, such as wheat and/or maize tortillas. In another preferred embodiment, the lipolytic enzyme according to the present invention may be used in combination with a xylanase in producing food products. GR!NDAMYL™ and POWERBake 7000 are examples of commercially available xylanase enzymes available from Danisco A/S. Other examples of xylanase enzymes may be found in WO 03/020923 and WO 01/42433.
Preferably, the lipolytic enzyme according to the present invention may be used in combination with a xylanase and an alpha amylase. Suitably the alpha amylase may be a maltogenic, or a non-maltogenic alpha amylase (such as GRINDAMYL™ or POWERSoft, commercially available from Danisco A/S), or a combination thereof.
The lipolytic enzyme of the invention can also preferably be used in combination with an oxidising enzyme, such as a maltose oxidising enzyme (MOX), for example hexose oxidase (HOX). Suitable methods are described in WO 03/099016. Commercially available maltose oxidising enzymes GRINDAMYL™ and SUREBake are available from Danisco A/S.
Optionally an alpha-amylase, such as a non-maltogenic exoamylase and/or a maltogenic amylases, and/or a maltose oxidising enzyme (MOX) in combination with the enzyme according to the present invention may be used in methods of preparing a dough, a baked product, tortilla, cake, instant noodle/fried snack food, or a dairy product such as cheese.
The invention further comprises methods for including the lipolytic enzyme in the foodstuff or other composition.
Therefore, the invention provides in a further aspect a method of producing a foodstuff according to the invention, comprising adding the lipolytic enzyme and the cyclodextrin to one or more other ingredients of the foodstuff or other composition and, if necessary, treating the ingredients to produce the foodstuff.
In a preferred embodiment, the method further comprises adding a cyclodextrin glycosyltransferase enzyme and, if necessary, a polysaccharide substrate to generate cyclodextrin in situ.
In this embodiment, the cyclodextrin glycosyltransferase enzyme may be added to the foodstuff before addition of the lipolytic enzyme. Preferably, the cyclodextrin glycosyltransferase enzyme is added to the foodstuff or other composition at least 10 minutes (more preferably between 10 minutes and 3 hours) before the lipolytic enzyme.
Alternatively, the cyclodextrin glycosyltransferase enzyme may be added to the foodstuff or other composition after addition of the lipolytic enzyme. Preferably, the cyclodextrin glycosyltransferase enzyme is added to the foodstuff or other composition at least 10 minutes (more preferably between 10 minutes and 3 hours) after the lipolytic enzyme.
In a further alternative, the cyclodextrin glycosyltransferase enzyme is added at the same time as the lipolytic enzyme.
Such methods are generally known to the skilled person, and include adding the lipolytic enzyme directly to the foodstuff or composition, addition of the lipolytic enzyme in combination with a stabilizer and/or carrier, and addition of a mixture comprising the lipolytic enzyme and a stabilizer and/or carrier. Suitable stabilizers for use with the present invention include but is not limited to inorganic salts (such as NaCL ammonium sulphate), sorbitol, emulsifiers and detergents (such as Tween 20, Tween 80, Panodan AB100 without triglycerides, polyglycerol ester, sorbitan monoo!eate), oil (such as rape seed oil, sunflower seed oil and soy oil), pectin, trehalose and glycerol.
Suitable carriers for use with the present invention include but is not limited to starch, ground wheat, wheat flour, NaCS and citrate. The bread and/or dough improving composition may further comprise another enzyme, such as one or more other suitable food grade enzymes, including starch degrading enzymes such as endo- or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g. glucose oxidase, pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as one which oxidises maltose, for example hexose oxidase (HOX), lipases, phosphoiipases and hexose oxidase, proteases and acyltransferases (such as those described in WO 2004/064987 for instance).
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.
Further enzymes that may be used may be selected from the group consisting of a cellulase, a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase.
Examples of useful oxidoreductases include oxidases such as a glucose oxidase (EC 1.1 .3.4), carbohydrate oxidase, glycerol oxidase, pyranose oxidase, galactose oxidase (EC 1 .1.3.10), a maltose oxidising enzyme such as hexose oxidase (EC 1.1 .3.5).
Other useful starch degrading enzymes which may be added to a dough composition include glucoamylases and pullulanases. Preferably, the further enzyme is at least a xyianase and/or at least an antistaling amylase. The term "xyianase" as used herein refers to xylanases (EC 3.2.1 .32) which hydrolyse xylosidic linkages.
The term "amylase" as used herein refers to amylases such as a-amylases (EC 3.2.1.1 ), β-amylases (EC 3.2.1.2) and γ-amylases (EC 3.2.1.3).
The further enzyme can be added together with any dough ingredient including the flour, water or optional other ingredients or additives, or a dough improving composition. The further enzyme can be added before the flour, water, and optionally other ingredients and additives or the dough improving composition. The further enzyme can be added after the flour, water, and optionally other ingredients and additives or the dough improving composition. The further enzyme may conveniently be a liquid preparation. However, the composition may be conveniently in the form of a dry composition. Some enzymes of the dough improving composition are capable of interacting with each other under the dough conditions to an extent where the effect on improvement of the rheological and/or machineability properties of a flour dough and/or the quality of the product made from dough by the enzymes is not only additive, but the effect is synergistic.
In relation to improvement of the product made from dough (finished product), it may be found that the combination results in a substantial synergistic effect in respect to crumb structure. Also, with respect to the specific volume of baked product a synergistic effect may be found.
HOST CELL
The term "host cell" - in relation to the present invention includes any cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of an enzyme having the specific properties as defined herein.
Thus, a further embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence that expresses the enzyme of the present invention. 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. Preferably, the host cells are not human cells. Examples of suitable bacterial host organisms are gram positive or gram negative bacterial species.
Depending on the nature of the nucleotide sequence encoding the enzyme of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected. The use of suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
The host cell may be a protease deficient or protease minus strain.
The genotype of the host cell may be modified to improve expression. Examples of host cell modifications include protease deficiency, supplementation of rare tRNA's, and modification of the reductive potential in the cytoplasm to enhance disulphide bond formation. For example, the host cell £. coli may overexpress rare tRNA's to improve expression of heterologous proteins as exemplified/described in Kane [Curr Opin Biotechnol (1995), 6, 494=500 "Effects of rare codon clusters on high-level expression of heterologous proteins in E.coi ). The host cell may be deficient in a number of reducing enzymes thus favouring formation of stable disulphide bonds as exemplified/described in Bessette (Proc Natl Acad Sci USA (1999), 96, 13703-13708 "Efficient folding of proteins with multiple disulphide bonds in the Escherichia coli cytoplasm"). ISOLATED
In one aspect, the enzymes for use in the present invention may be in an isolated form. The term "isolated" means that the sequence or protein is at least substantially free from at least one other component with which the sequence or protein is naturally associated in nature and as found in nature.
PURIFIED
In one aspect, the enzymes for use in the present invention may be used in a purified form.
The term "purified" means that the sequence is in a relatively pure state - e.g. at least about 51 % pure, or at least about 75%, or at least about 80%, or at least about 90% pure, or at least about 95% pure or at least about 98% pure.
CLONING A NUCLEOTIDE SEQUENCE ENCODING A POLYPEPTIDE ACCORDING TO THE PRESENT INVENTION
A nucleotide sequence encoding either a polypeptide which has the specific properties as defined herein or a polypeptide which is suitable for modification may be isolated from any cell or organism producing said polypeptide. Various methods are well known within the art for the isolation of nucleotide sequences. For example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the polypeptide. If the amino acid sequence of the polypeptide is known, labelled oligonucleotide probes may be synthesised and used to identify polypeptide-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known polypeptide gene could be used to identify polypeptide-encoding clones. Sn the latter case, hybridisation and washing conditions of lower stringency are used.
Alternatively, polypeptide-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing an enzyme inhibited by the polypeptide, thereby allowing clones expressing the polypeptide to be identified.
In a yet further alternative, the nucleotide sequence encoding the polypeptide may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al (1981 ) Tetrahedron Letters 22, 1859-1869, or the method described by Matthes et al (1984) EMBO J. 3, 801 -805. In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.
The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA sequence may also be prepared by polymerase chain reaction (PGR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al (Science (1988) 239, 487-491 ).
NUCLEOTIDE SEQUENCES
The present invention also encompasses nucleotide sequences encoding polypeptides having the specific properties as defined herein. The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homoiogues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be doubie-stranded or single-stranded whether representing the sense or antisense strand.
The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence.
In a preferred embodiment, the nucleotide sequence per se encoding a polypeptide having the specific properties as defined herein does not cover the native nucleotide sequence in its natural environment when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, we shall call this preferred embodiment the "non-native nucleotide sequence". In this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. However, the amino acid sequence encompassed by scope the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism. Preferably, however, the amino acid sequence encompassed by scope of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
Preferably the polypeptide is not a native polypeptide. In this regard, the term "native polypeptide" means an entire polypeptide that is in its native environment and when it has been expressed by its native nucleotide sequence.
Typically, the nucleotide sequence encoding polypeptides having the specific properties as defined herein is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods wel! known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et a/ (1980) Nuc Acids Res Symp Ser 225-232).
MOLECULAR EVOLUTION
Once an enzyme-encoding nucleotide sequence has been isolated, or a putative enzyme-encoding nucleotide sequence has been identified, it may be desirable to modify the selected nucleotide sequence, for example it may be desirable to mutate the sequence in order to prepare an enzyme in accordance with the present invention.
Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
A suitable method is disclosed in Morinaga et al (Biotechnology (1984) 2, 646-649). Another method of introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, 147-151 ).
Instead of site directed mutagenesis, such as described above, one can introduce mutations randomly for instance using a commercial kit such as the GeneMorph PGR mutagenesis kit from Stratagene, or the Diversify PGR random mutagenesis kit from Clontech. EP 0 583 265 refers to methods of optimising PGR based mutagenesis, which can also be combined with the use of mutagenic DNA analogues such as those described in EP 0 866 796. Error prone PGR technologies are suitable for the production of variants of lipid acyl transferases with preferred characteristics. WO 02/06457 refers to molecular evolution of lipases.
A third method to obtain novel sequences is to fragment non-identical nucleotide sequences, either by using any number of restriction enzymes or an enzyme such as Dnase I, and reassembling full nucleotide sequences coding for functional proteins. Alternatively one can use one or multiple non-identical nucleotide sequences and introduce mutations during the reassembly of the full nucleotide sequence. DNA shuffling and family shuffling technologies are suitable for the production of variants of lipid acyl transferases with preferred characteristics. Suitable methods for performing 'shuffling' can be found in EP 0 752 008, EP 1 138 763, EP 1 103 606. Shuffling can a!so be combined with other forms of DNA mutagenesis as described in US 6, 180,406 and WO 01/34835.
Thus, it is possible to produce numerous site directed or random mutations into a nucleotide sequence, either in vivo or in vitro, and to subsequently screen for improved functionality of the encoded polypeptide by various means. Using in silico and exo mediated recombination methods (see WO 00/58517, US 6,344,328, US 6,361 ,974), for example, molecular evolution can be performed where the variant produced retains very low homology to known enzymes or proteins. Such variants thereby obtained may have significant structural analogy to known transferase enzymes, but have very low amino acid sequence homology.
As a non-limiting example, In addition, mutations or natural variants of a polynucleotide sequence can be recombined with either the wild type or other mutations or natural variants to produce new variants. Such new variants can also be screened for improved functionality of the encoded polypeptide.
The application of the above-mentioned and similar molecular evolution methods allows the identification and selection of variants of the enzymes of the present invention which have preferred characteristics without any prior knowledge of protein structure or function, and allows the production of non-predictable but beneficial mutations or variants. There are numerous examples of the application of molecular evolution in the art for the optimisation or alteration of enzyme activity, such examples include, but are not limited to one or more of the following: optimised expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in preferred environmental conditions, e.g. temperature, pH, substrate. As will be apparent to a person skilled in the art, using molecular evolution tools an enzyme may be altered to improve the functionality of the enzyme.
Suitably, the nucleotide sequence encoding a lipolytic enzyme used in the invention may encode a variant, i.e. the lipolytic enzyme may contain at least one amino acid substitution, deletion or addition, when compared to a parental enzyme. Variant enzymes retain at least 70%, 80%, 90%, 95%, 97%, 99% homology with the parent enzyme.
Variant lipolytic enzymes may have decreased activity on triglycerides, and/or monoglycerides and/or digiycerides compared with the parent enzyme.
Suitably the variant enzyme may have no activity on triglycerides and/or monoglycerides and/or digiycerides. Alternatively, the variant enzyme may have increased thermostability.
The variant enzyme may have increased activity on one or more of the following, polar lipids, phospholipids, lecithin, phosphatidylcholine, glycolipids, digalactosyl monoglyceride, monogalactosyl monoglyceride.
Variants of lipid acyltransferases are known, and one or more of such variants may be suitable for use in the methods and uses according to the present invention and/or in the enzyme compositions according to the present invention. By way of example only, variants of lipid acyltransferases are described in the following references may be used in accordance with the present invention: Hilton & Buckley J Biol. Chem. 1991 Jan 15: 266 (2): 997-1000; Robertson et al J. Biol. Chem. 1994 Jan 21 ; 269(3):2146-50; Brumlik et al J. Bacteriol 1996 Apr; 178 (7): 2060-4; Peelman et al Protein Sci. 1998 Mar; 7(3):587-99. AMINO ACID SEQUENCES
The present invention also encompasses the use of amino acid sequences encoded by a nucleotide sequence which encodes an enzyme for use in any one of the methods and/or uses of the present invention.
As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with "enzyme". The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
Suitably, the amino acid sequences may be obtained from the isolated polypeptides taught herein by standard techniques.
One suitable method for determining amino acid sequences from isolated polypeptides is as follows: Purified polypeptide may be freeze-dried and 100 pg of the freeze-dried material may be dissolved in 50 μΙ of a mixture of 8 M urea and 0.4 M ammonium hydrogen carbonate, pH 8.4. The dissolved protein may be denatured and reduced for 15 minutes at 50°C following overlay with nitrogen and addition of 5 μΐ of 45 mM dithiothreitol. After cooling to room temperature, 5 pi of 100 mM iodoacetamide may be added for the cysteine residues to be derivatized for 15 minutes at room temperature in the dark under nitrogen.
135 μΙ of water and 5 pg of endoproteinase Lys-C in 5 pi of water may be added to the above reaction mixture and the digestion may be carried out at 37°C under nitrogen for 24 hours.
The resulting peptides may be separated by reverse phase HPLC on a VYDAC C18 column (0.46x15cm; 10pm; The Separation Group, California, USA) using solvent A: 0.1 % TFA in water and solvent B: 0.1 % TFA in acetonitrile. Selected peptides may be re-chromatographed on a Develosil C18 column using the same solvent system, prior to N-terminal sequencing. Sequencing may be done using an Applied Biosystems 476A sequencer using pulsed liquid fast cycles according to the manufacturer's instructions (Applied Biosystems, California, USA). SEQUENCE IDENTITY OR SEQUENCE HOMOLOGY
Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity". The homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the enzyme. In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 60%, 70%, 71 %, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or 98% identical, preferably at least 95 or 98% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher re!atedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the Vector NTI (Invitrogen Corp.). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), and FASTA (Altschul et al 1990 J. Mol. Biol. 403-410). Both BLAST and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60). However, for some applications, it is preferred to use the Vector NTI program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1 ): 187-8 and tafiana@ncbi.nlm.nih.gov).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI Advance ' 10 package.
Alternatively, percentage homologies may be calculated using the multiple alignment feature in Vector NT! Advance™ 10 (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1 ), 237-244).
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. Suitably, the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, preferably over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 100 contiguous nucleotides.
Suitably, the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
Should Gap Penalties be used when determining sequence identity, then preferably the default parameters for the programme are used for pairwise alignment. For example, the following parameters are the current default parameters for pairwise alignment for BLAST 2:
FOR BLAST2 DNA PROTEIN
EXPECT THRESHOLD 10 10
WORD SIZE 1 1 3
SCORING PARAMETERS
Match/Mismatch Scores 2, -3 n/a
Matrix n/a BLOSUM62
Gap Costs Existence: 5 Existence: 11
Extension: 2 Extension: 1 in one embodiment, preferably the sequence identity for the nucleotide sequences and/or amino acid sequences may be determined using BLAST2 (blastn) with the scoring parameters set as defined above. For the purposes of the present invention, the degree of identity is based on the number of sequence elements which are the same. The degree of identity in accordance with the present invention for amino acid sequences may be suitably determined by means of computer programs known in the art such as Vector NTI Advance™ 1 1 (Invitrogen Corp.). For pairwise alignment the scoring parameters used are preferably BLOSUM62 with Gap existence penalty of 1 1 and Gap extension penalty of 1.
Suitably, the degree of identity with regard to an amino acid sequence is determined over at least 20 contiguous amino acids, preferably over at least 30 contiguous amino acids, preferably over at least 40 contiguous amino acids, preferably over at least 50 contiguous amino acids, preferably over at least 60 contiguous amino acids, preferably over at least 100 contiguous amino acids.
Suitably, the degree of identity with regard to an amino acid sequence may be determined over the whole sequence.
The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. 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 secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar G A P
1 L V
Poiar - uncharged C S T
N Q
Polar - charged D E
K R
AROMATIC H F W Y
The present invention aiso encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine, ornithine (hereinafter referred to as O), pyridylalanine, thieny!alanine, naphthylalanine and phenylglycine.
Replacements may also be made by unnatural amino acids. Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including a Iky I groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.
Nucleotide sequences for use in the present invention or encoding a polypeptide having the specific properties defined herein 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 and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood thai the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences.
The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences discussed herein, or any derivative, fragment or derivative thereof, !f the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc. 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 made from a range of individuals, for example individuals from different populations. In addition, other viral/bacterial, or cellular homoiogues particularly cellular homoiogues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homoiogues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes 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 homoiogues and allelic variants of the polypeptide or nucleotide sequences of the invention. Variants and strain/species homoiogues may also be obtained using degenerate PGR which will use primers designed to target sequences within the variants and homoiogues 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 PGR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required 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 polypeptide recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PGR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or nonradioactive 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 DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
In general, primers will be produced by synthetic means, involving a stepwise 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 PGR (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 mPxNA or cDNA obtained from an animal or human 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.
HYBRIDISATION
The present invention also encompasses the use of sequences that are complementary to the sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.
The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PGR) technologies.
The present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the subject sequences discussed herein, or any derivative, fragment or derivative thereof.
The present invention also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences discussed herein.
Hybridisation conditions are based on the melting temperature (Tm) of the nucleotide 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.
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 Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridisation can be used to identify or detect identical nucleotide sequences while an intermediate (or low/) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences. Preferably, the present invention encompasses the use of sequences that are complementary to sequences that are capable of hybridising under high stringency conditions or intermediate stringency conditions to nucleotide sequences encoding polypeptides having the specific properties as defined herein. More preferably, the present invention encompasses the use of sequences that are complementary to sequences that are capable of hybridising under high stringency conditions (e.g. 65°C and O.lxSSC {ixSSC = 0.15 M NaCI, 0.015 M Na-citrate pH 7.0}) to nucleotide sequences encoding polypeptides having the specific properties as defined herein.
The present invention also relates to the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein). The present invention also relates to the use of nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein).
Also included within the scope of the present invention are the use of polynucleotide sequences that are capable of hybridising to the nucleotide sequences discussed herein under conditions of intermediate to maximal stringency.
In a preferred aspect, the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under stringent conditions (e.g. 50°C and 0.2 x SSC).
In a more preferred aspect, the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under high stringency conditions (e.g. 65°C and 0.1 x SSC). BIOLOGICALLY ACTIVE
Preferably, the variant sequences etc. are at least as biologically active as the sequences presented herein.
As used herein "biologically active" refers to a sequence 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) of the naturally occurring sequence.
RECOMBINANT
In one aspect the sequence for use in the present invention is a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques.
These recombinant DNA techniques are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Co!d Spring Harbor Laboratory Press.
SYNTHETIC
In one aspect the sequence for use in the present invention is a synthetic sequence - i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms - such as the methylotrophic yeasts Pichia and Hansenula.
EXPRESSION OF POLYPEPTIDES
A nucleotide sequence for use in the present invention or for encoding a polypeptide having the specific properties as defined herein can be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence, in polypeptide form, in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
The polypeptide produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intrace!lularly depending on the sequence and/or the vector used. The coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
EXPRESSION VECTOR
The term "expression vector" means a construct capable of in vivo or in vitro expression.
Preferably, the expression vector is incorporated into the genome of a suitable host organism. The term "incorporated" preferably covers stable incorporation into the genome.
The nucleotide sequence encoding an enzyme for use in the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host organism.
The vectors for use in 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 choice of vector e.g. a plasmid, cosmid, or phage vector will often depend on the host cell into which it is to be introduced. The vectors for use in the present invention may contain one or more selectable marker genes such as a gene which confers antibiotic resistance e.g. ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Alternatively, the selection may be accomplished by co-transformation (as described in W091/17243).
Vectors may be used in vitro, for example for the production of RNA or used to transfect, transform, transduce or infect a host cell.
The vector may further comprise a nucleotide sequence enabling the vector to replicate in the host eel! in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB1 10, pE194, pAMB1 and plJ702.
REGULATORY SEQUENCES In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell. By way of example, the present invention covers a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.
The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals.
The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site. Enhanced expression of the nucleotide sequence encoding the enzyme of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions. Preferably, the nucleotide sequence according to the present invention is operably linked to at least a promoter.
Examples of suitable promoters for directing the transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.
CONSTRUCTS
The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes a nucleotide sequence encoding a polypeptide having the specific properties as defined herein for use according to the present invention directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Sh1-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wiid 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.
For some applications, preferably the construct comprises at least a nucleotide sequence of the present invention or a nucleotide sequence encoding a polypeptide having the specific properties as defined herein operably linked to a promoter.
ORGANISM
The term "organism" in relation to the present invention includes any organism that could comprise a nucleotide sequence according to the present invention or a nucleotide sequence encoding for a polypeptide having the specific properties as defined herein and/or products obtained therefrom.
The term "transgenic organism" in relation to the present invention includes any organism that comprises a nucleotide sequence coding for a polypeptide having the specific properties as defined herein and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence coding for a polypeptide having the specific properties as defined herein within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism.
Suitable organisms include a prokaryote, fungus yeast or a plant.
The term "transgenic organism" does not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment.
Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, a nucleotide sequence coding for a polypeptide having the specific properties as defined herein, constructs as defined herein, vectors as defined herein, plasmids as defined herein, cells as defined herein, or the products thereof. For example the transgenic organism can also comprise a nucleotide sequence coding for a polypeptide having the specific properties as defined herein under the control of a promoter not associated with a sequence encoding a lipid acyltransferase in nature.
TRANSFORMATION OF HOST CELLS/ORGANISM
The host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include bacteria such as E. coli and Bacillus licheniformis, preferably B. licheniformis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et a! (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
In another embodiment the transgenic organism can be a yeast.
Filamentous fungi cells may be transformed using various methods known in the art - such as a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the ceil wall in a manner known. The use of Aspergillus as a host microorganism is described in EP 0 238 023. In one embodiment, preferably T. reesei is the host organism. Another host organism can be a plant. A review of the general techniques used for transforming plants may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol (1991 ) 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.
General teachings on the transformation of fungi, yeasts and plants are presented in following sections.
TRANSFORMED FUNGUS
A host organism may be a fungus - such as a filamentous fungus. Examples of suitable such hosts include any member belonging to the genera Fusarium, Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma and the like. In one embodiment, Trichoderma is the host organism, preferably T. reesei.
Teachings on transforming filamentous fungi are reviewed in US-A-5741665 which states that standard techniques for transformation of filamentous fungi and culturing the fungi are well known in the art. An extensive review of techniques as applied to N. crassa is found, for example in Davis and de Serres, Methods Enzymol (1971 ) 17 A: 79-143. Further teachings on transforming filamentous fungi are reviewed in US-A-5674707.
In one aspect, the host organism can be of the genus Aspergillus, such as Aspergillus niger.
A transgenic Aspergillus according to the present invention can also be prepared by following, for example, the teachings of Turner G. 1994 (Vectors for genetic manipulation, in: Martinelli S.D., Kinghorn J R. (Editors) "Aspergillus: 50 years on"; Progress in industrial microbiology vol 29; Elsevier Amsterdam 1994; pp. 641 -666).
Gene expression in filamentous fungi has been reviewed in Punt et a/. Trends Biotechnol. (2002); 20(5):20Q-6, Archer & Peberdy Cr/'f. Rev. Biotechnol. (1997) 77(4):273-306.
TRANSFORMED YEAST
In another embodiment, the transgenic organism can be a yeast.
A review of the principles of heterologous gene expression in yeast are provided in, for example, Methods Mol Biol (1995), 49:341-54, and Curr Opin Biotechnol (1997); 8(5):554-60.
In this regard, yeast - such as the species Saccharomyces cerevisi or Pichia pastoris or Hansenula polymorpha (see FEMS Microbiol Rev (2000 24(1 ):45-66), may be used as a vehicle for heterologous gene expression.
A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J. Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
For the transformation of yeast, several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et a/., (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H ef al (1983, J Bacteriology 153, 163-168).
The transformed yeast cells may be selected using various selective markers - such as auxotrophic markers dominant antibiotic resistance markers.
A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as, but not limited to, yeast species selected from Pichia spp., Hansenula spp., Kluyveromyces, Yarrowinia spp., Saccharomyces spp., including S. cerevisiae, or Schizosaccharomyce spp. including Schizosaccharomyce pom be.
A strain of the methylotrophic yeast species Pichia pastoris may be used as the host organism. In one embodiment, the host organism may be a Hansenula species, such as H. polymorpha (as described in WO 01/39544).
TRANSFORMED PLANTS/PLANT CELLS A host organism suitable for the present invention may be a plant. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol ( 991 ) 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27), or in WO 01/16308. The transgenic plant may produce enhanced levels of phytosterol esters and phytostanol esters, for example.
TRANSFORMED PLANTS/PLANT CELLS
A host organism suitable for the present invention may be a plant. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994
17-27).
CULTURING AND PRODUCTION Host cells transformed with the nucleotide sequence of the present invention may be cultured under conditions conducive to the production of the encoded enzyme and which facilitate recovery of the enzyme from the cells and/or culture medium. The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in questions and obtaining expression of the enzyme.
The protein produced by a recombinant cell may be displayed on the surface of the cell
The enzyme may be secreted from the host cells and may conveniently be recovered from the culture medium using well-known procedures.
SECRETION
Often, it is desirable for the polypeptide to be secreted from the expression host into the culture medium from where the enzyme may be more easily recovered. According to the present invention, the secretion leader sequence may be selected on the basis of the desired expression host. Hybrid signal sequences may also be used with the context of the present invention.
Typical examples of secretion leader sequences not associated with a nucleotide sequence encoding a lipid acyltransferase in nature are those originating from the fungal amyloglucosidase (AG) gene (g/aA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and Hansenula) or the a-amylase gene (Bacillus).
DETECTION A variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays.
A number of companies such as Pharmacia Biotech (Piscataway, NJ, USA), Promega (Madison, Wl, USA), and US Biochemical Corp (Cleveland, OH, USA) supply commercial kits and protocols for these procedures.
Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiiuminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3,817,837; US-A-3,850,752; US-A-3,939,350; US-A-3,996,345; US-A-4,277,437; US-A-4,275,149 and US-A-4,366,241.
Also, recombinant immunoglobulins may be produced as shown in US-A-4, 816,567.
FUSION PROTEINS
An enzyme for use in the present invention may be produced as a fusion protein, for example to aid in extraction and purification thereof. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the protein sequence.
Gene fusion expression systems in E. coli have been reviewed in Curr. Opin. Biotechnol. (1995) 6(5):501 -6.
The amino acid sequence of a polypeptide having the specific properties as defined herein may be ligated to a non-native sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a non- native epitope that is recognised by a commercially available antibody. ADDITIONAL POIs
The sequences for use according to the present invention may also be used in conjunction with one or more additional proteins of interest (POIs) or nucleotide sequences of interest (NO!s).
Non-limiting examples of POIs include: proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, a-galactosidases, β-ga!actosidases, a-glucanases, glucan lysases, endo-β- glucanases, glucoamylases, glucose oxidases, a-glucosidases, β-glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: 02-oxidoreductase, EC 1.1.3.5) or combinations thereof. The NOI may even be an antisense sequence for any of those sequences.
The POI may even be a fusion protein, for example to aid in extraction and purification. The POI may even be fused to a secretion sequence.
Other sequences can also facilitate secretion or increase the yield of secreted POI. Such sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821 198.0.
The NOI may be engineered in order to alter their activity for a number of reasons, including but not limited to, alterations which modify the processing and/or expression of the expression product thereof. By way of further example, the NOI may also be modified to optimise expression in a particular host cell. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites. The NO! may include within it synthetic or modified nucleotides- such as methylphosphonate and phosphorothioaie backbones. The NOI 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 phosphorothioaie or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. FOOD INGREDIENT
The composition of the present invention may be used as a food ingredient.
As used herein the term "food ingredient" includes a formulation which is or can be added to functional foods or foodstuffs as a nutritional supplement and/or fiber supplement. The term food ingredient as used here also refers to formulations which can be used at low levels in a wide variety of products that require gelling, texturising, stabilising, suspending, film-forming and structuring, retention of juiciness and improved mouthfeel, without adding viscosity.
The food ingredient may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
LARGE SCALE APPLICATION
In one preferred embodiment of the present invention, the amino acid sequence is used for large scale applications.
Preferably the amino acid sequence is produced in a quantity of from 1g per litre to about 25g/litre, preferably from above 2.5g/litres to about 18 g/litre, preferably above 8 g per litre of the total cell culture volume after cultivation of the host organism. Brief Description of the Figures
Figure 1 illustrates mini breads made in Baking Trial 1 to demonstrate the effect of generating increasing levels of free fatty acids in situ using the recipe described in Table 2;
Figure 2 illustrates the relative volume (as compared with blank), % of triglyceride and % of free fatty acids as a function of lipase (Exel 16) dose on top of fixed GRINDAMYL POWERBAKE 4070 dose (1000 TIPU/kg flour) from Baking Trial 1 using the recipe described in Table 2;
Figures 3A and 3B illustrate mini breads made in Baking Trial 2 to illustrate the effect of adding increasing levels of β-cyclodextrins together with a high dose of the lipolytic enzyme Grindamyl POWERBAKE 4070 using the recipe described in Table 3 (Figure 3A showing bread in tins and Figure 3B showing bread made without tins);
Figure 4 illustrates the relative volume (as compared with blank) as a function of added amount of β-cyclodextrin on top of fixed high Grindamyl POWERBAKE 4070 dose (5000 TIPU/kg flour) made in Baking Trial 2 using the recipe described in Table 3B;
Figure 5 illustrates a scheme for extraction of free fatty acids from lyophilized dough; Figure 6 illustrates the relative volume (as compared with blank) and amount of free fatty acids in extract 1 and 3 as a function of added amount of β-cyclodextrin on top of fixed high Grindamyl POWERBAKE 4070 dose (5000 TIPU/kg flour) made in Baking Trial 2 using the recipe described in Table 3B (n=3, one extract per dough, triple determination on each extract);
Figure 7 illustrates mini breads made in Baking Trial 3 to compare the baking effect of β-cyclodextrin and pectin using the recipe described in Table 4 (no tins); Figure 8 illustrates the relative volume (as compared with blank) as a function of added amount of p-cyclodextrin and pectin on top of increasing dosages of Exel 16 in Baking Trial 3; Figure 9 illustrates mini breads made in Baking Trial 4 to compare the performance of a combination of Grindamyl POWERBAKE 4070 and CGTase compared with a combination of Grindamyl POWERBAKE 4070 and β-cyclodextrin using the recipe described in Table 5 (in tins); Figure 10 illustrates mini breads with added starch made in Baking Trial 5 to compare the performance of a combination of Grindamyl POWERBAKE 4070 and CGTase compared with a combination of Grindamyl POWERBAKE 4070 and β-cyclodextrin using the recipe described in Table 6 (in tins); Figure 1 1 illustrates the relative volume (as compared with blank) as a function of added amount CGTase on top of a fixed medium dose Grindamyl POWERBAKE 4070 (750 TIPU/kg flour) made in Baking Trials 4 and 5 using the Recipe described in Tables 5 and 6 (in tins); Figure 12 shows an amino acid sequence (SEQ ID No. 1 ) of a fungal lipolytic enzyme derived from Fusarium heterosporum sold commercially as GRINDAMYL POWERbake 4070;
Figure 13 shows an amino acid sequence of a fungal lipolytic enzyme derived from Fusarium heterosporum comprising an N terminal signal sequence (underlined) (SEQ ID No. 2);
Figure 14 shows a nucleotide sequence (SEQ ID No. 3) encoding a fungal lipolytic enzyme derived from Fusarium heterosporum in accordance with the present invention;
Figure 15 shows an amino acid sequence (SEQ ID No. 4) of a lipolytic enzyme derived from Fusarium semitectum; Figure 16 shows a nucleotide sequence (SEQ ID NO. 5) encoding a lipolytic enzyme derived from Fusarium se itectum;
Figure 17 shows SEQ ID No. 6 a Humicola lanuginosa lipase produced by Humicola lanuginosa DSM 4109, sold commercially as GrindamyS Exel 16;
Figure 18 shows a nucleotide sequence (SEQ ID No.7) of a lipolytic enzyme derived from Fusarium heterosporum which includes a a- factor signal sequence; Figure 19 shows SEQ ID No. 8 which is the DNA sequence for the polypeptide variant designated "mut 3";
Figure 20 shows SEQ ID No. 9 which is the protein preprosequence for the polypeptide variant designated "mut 3";
Figure 21 shows SEQ ID No. 10 which is the DNA sequence for the polypeptide variant designated "mut 4";
Figure 22 shows SEQ ID No. 1 1 which is the protein preprosequence for the polypeptide variant designated "mut 4";
Figure 23 shows SEQ ID No. 12 which is the DNA sequence for the polypeptide variant designated "mut 5"; Figure 24 shows SEQ ID No. 13 which is the protein preprosequence for the polypeptide variant designated "mut 5";
Figure 25 shows SEQ ID No. 14 which is the DNA sequence for the polypeptide variant designated "mut 345";
Figure 26 shows SEQ ID No. 15 which is the protein preprosequence for the polypeptide variant designated "mut 345";
Figure 27 shows SEQ ID No. 16 which is the DNA sequence for the polypeptide variant designated "mut 3459"; Figure 28 shows SEQ ID No. 17 which is the protein preprosequence for the polypeptide variant designated "mut 3459"; Figure 29 shows SEQ ID No. 18 which is the DNA sequence for the polypeptide variant designated "mut 9";
Figure 30 shows SEQ ID No. 19 which is the protein preprosequence for the polypeptide variant designated "mut 9";
Figure 31 shows SEQ ID No. 20 which is the DNA sequence for the polypeptide variant designated "mut 10";
Figure 32 shows SEQ ID No. 21 which is the protein preprosequence for the polypeptide variant designated "mut 10";
Figure 33 shows SEQ ID No. 22 which is the DNA sequence for the polypeptide variant designated "mut 1 1 "; Figure 34 shows SEQ ID No. 23 which is the protein preprosequence for the polypeptide variant designated "mut 1 1";
Figure 35 shows SEQ ID No. 24 which is the DNA sequence for the polypeptide variant designated "mut 12";
Figure 36 shows SEQ ID No. 25 which is the protein preprosequence for the polypeptide variant designated "mut 12"; and
Figure 37 shows SEQ ID No. 26 which is a lipolytic enzyme sold commercially as Lipopan F™. EXAMPLES
Enzymes
Lipopan F (the enzyme having SEQ ID No. 26 as described herein) (obtained from Novozymes A/S, Denmark) (1204468, lot 4010629964 holding 12900 TIPU/g) was used as a positive control.
Phospholipase Grindamyi POWERBAKE 4070 (the enzyme having SEQ ID No. 1 as described herein) (available from Danisco A S, Denmark) (EDS 129, 5830 TIPU/g) was used in combination with either Lipase Exel 16, β-cyclodextrin, CGTase or CGTase and starch.
Lipase Exel 16 (available from Danisco A/S, Denmark) (Control sample for protocol A770, 37350 LIPU/g) was used in combination with Grindamyi POWERBAKE 4070, β-cyclodextrin and pectin.
CGTase (Amano) was used in combination with either Grindamyi POWERBAKE 4070 or Grindamyi POWERBAKE 4070 and starch
Papain (Extrakt Chemie, #2317)
Additives
β-cyclodextrin (Sigma C4767) was used in combination with Grindamyi POWERBAKE 4070 or Exel 16.
Pectin (1388) was used in combination with Exel 16.
Starch (pregelatinised)
Flour
Danish Reform flour (internal Danisco flour database reference: 2008-00145) supplied by Havnem0llerne, Vejle, Denmark was used for the tests.
Flour water absorption
The water absorption of the flour and composite flours was determined using a Farinograph (Brabender, Germany) according to AACC 54-21 .
Baking Recipe
Baking performance was evaluated in small scale baking trials (50 gram mixer and 10 gram loaves) using the recipe shown in Table 1 below. For the baking trial described in Table 1 , the amount of flour was adjusted according to the amount of β-cyclodextrin or pectin added to give a total of 50 g. The same was the case for the trial described in Table 1 , where the amount of flour was adjusted so the total of added starch and flour summed up to 50 g. Table 1 shows the recipe used for evaluation of baking performance. Salt/sugar is a 1 :1 (w/w) mixture. 'Water' is the water absorption determined by Farinograph analysis.
Figure imgf000063_0001
Table 1
BU = Brabender Units Dough making and baking
The flour and dry ingredients were mixed for one minute, water (with or without enzyme) was added and mixing was continued for another five minutes.
After mixing, four dough lumps were weighed out, each containing 10 grams flour. These were moulded into bread using a hand moulder. Loaves were put into baking pans and placed in a sealed container (with a lid) and left on the table for 10 minutes. Hereafter, bread was proofed at 34°C and 85% RH for 45 minutes and finally baked at 220°C for 5.5 minutes in a Bago oven (Bago-line, Faborg, Denmark). The bread was cooled for 20 minutes before evaluation (weighing, volume measurement, crumb and crust evaluation).
Baking Trials
Six series of trials were carried out. The aim of Baking Trial 1 was to test the effect of increasing levels of in situ generated free fatty acid (FFA). The aim of Baking Trial 2 was to test the effect of adding increasing amounts of β-cyclodextrin for complexing of in situ generated FFA. In both cases mini bread in tins as well as without tins were made. In the setup of Baking Trial 2, the high amount of β-cyclodextrin added to the dough was evaluated by including a β-cyclodextrin blank. The experimental set-up is listed in Tables 2 and 3.
Baking Trial 3 tested the baking effect of β-cyclodextrin versus the baking effect of the hydrocolloid pectin, which was used as a control. Only mini breads without tins were made. In this trial setup both a β-cyclodextrin blank and pectin blank was included. The experimental setup is listed in Table 4. Cyclodextrin glycosyltransferase (CGTase) is closely related to a-amylases but has the unique ability to produce cyclodextrins from linear a(1→4)-linked glucans via an intramolecular transg!ycosyiation reaction known as cyclization.
The aim of Baking Trials 4 and 5 was to test whether the same baking effect could be obtained by combining Grindamyl POWERBAKE 4070 and CGTase as with combining Grindamyl POWERBAKE 4070 and added β-cyclodextrin. In Baking Trial 5 additional substrate (starch) for the CGTase was added. In Baking Trial 5 a starch blank was included. In both trials only mini breads without tins were made. The experimental setup is listed in Tables 5 and 6.
The aim of Baking Trial 6 was to evaluate the effect of accumulating cyclodextrins in the dough before generating free fatty acids.
Table 2: Baking Trial 1 : Trial to test the effect of increasing dosage of Exei 16 in combination with a fixed dose of Grindamyl POWERBAKE 4070 (1000 TIPU/kg flour).
Baking Mutant ID TIPU/kg LIPU/kg ml Grindamyl ml Exel ml flour flour POWERbake 16 water
4070 sample* sample"
1 Blank - - - - 29.00
2 Lipopan F 650 - 1.0* - 28.00
3 Grindamyl 1000 - 1.0 - 28.00
POWERbake 4070
4 Grindamyl 1000 4000 1.0 0.45 27.55
POWERbake 4070 +
Exel 16 4000U
5 Grindamyl 1000 12000 1.0 1.36 26.64
POWERbake 4070 +
Exel 16 12000U
6 Grindamyl 1000 20000 1.0 2.27 25.73
POWERbake 4070
+ Exel 16 20000U
* 1.0 mL Lipopan F sample was added holding 32.5 TIPU/mL
# 50 TIPU/mL
n 440 LIPU/mL
Table 3: Baking Trial 2: Trial to test the effect of increasing amount of β-cyclodextrin in combination with a fixed dose of Grindamyl POWERbake 4070 (5000 TIPU/kg flour).
A: Bread in tins
Baking Mutant ID TIPU/kg 9 β- ml ml β- ml
flour cyclodextrin/ Grindamyl cyclodextrin water kg flour POWERbake sample"
4070
sample*
1 Blank - - - - 27.5
2 Lipopan F 650 - 1.0* - 26.5
3 β-cyclodextrin 8.4 10.30 18.5 blank
4 Grindamyl 5000 5.0 0.00 22.5
POWERbake
4070
5 Grindamyl 5000 0.84 5.0 1.00 21.9 POWERbake
4070 + very low
β-cyclodextrin
6 Grindamyl 5000 2.1 5.0 2.50 21.0
POWERbake
4070 + low
β-cyclodextrin
7 Grindamyl 5000 4.2 5.0 5.40 18.3 POWERbake
4070 + medium
β-cyclodextrin
8 Grindamyl 5000 8.4 5.0 10.30 13.5 POWERbake
4070 + high β- cyclodextrin B: Bread without tins
Baking Mutant ID TIPU/kg g β- ml ml β- ml water flour cyclodextrin/ Grindamyi cyclodextrin
kg flour POWERbake sample0
4070
sample
1 Blank - - - 29.00
2 Lipopan F 650 - 1.0* - 28.00
3 β-cyclodextrin 8.4 10.30 20.00 blank
4 Grindamyi 5000 - 5.0 0.00 24.00
POWERbake
4070
5 Grindamyi 5000 0.84 5.0 1.00 23 40
POWERbake
4070 + very
low β- cyclodextrin
6 Grindamyi 5000 2.1 5.0 2.50 22.50
POWERbake
4070 + low β- cyclodextrin
7 Grindamyi 5000 4.2 5.0 5.40 19.80
POWERbake
4070 +
medium β- cyclodextrin
8 Grindamyi 5000 8.4 5.0 10.30 15.00
POWERbake
4070 + high
β-cyclodextrin
*1.0 mL Lipopan F sample was added holding 32.2 TIPU/mL
# 50 TIPU/mL
n 0.042 g β-cyclodextrin/mL
Table 4: Baking Triai 3: Trial to test the effect baking effect of β-cyclodextrin versus the baking effect of pectin (used as a control).
Baking Mutant ID LIPU/ g g ml mL mL ml kg P-CD/ Pectin/ Exel P-CD* Pectin" water flour kg flour kg flour 16*
1 Blank - - - 0.00 0.00 0.00 28.00
2 Exel 16_4000U 4000 - - 0.45 0.00 0.00 27.55
3 Exel 16_12000U 12000 - - 1.36 0.00 0.00 26.64
4 Exel 16_2QQ00U 20000 - - 2.27 0.00 0.00 25.73
5 β-cyclodextrin - 9.0 - 0.00 10.0 0.00 18.00 blank
6 β-cyclodextrin + 4000 9.0 - 0.45 10.0 0.00 17.55 Exel 16 4000U
7 β-cyclodextrin + 12000 9.0 - 1.36 10.0 0.00 16.64 Exel 16 12000U
8 β-cyclodextrin + 20000 9.0 - 2.27 10.0 0.00 15.73
Exel 16 20000U
9 Pectin blank - - 2.5 0.00 0.00 12.5 15.50
(control)
10 Pectin + Exel 4000 - 2.5 0.45 0.00 12.5 15.05 (control) 16 4000U
11 Pectin + Exel 12000 - 2.5 1.36 0.00 12.5 14.14
(control) 16 12000U
12 Pectin + Exel 20000 - 2.5 2.27 0.00 12.5 13.23 (control) 16 20000U
* 440 LIPU/ml
# 0.045 g β-cyclodextrin/mL
n 0.01 g pectin/mL
Table 5: Baking Trial 4: Trial to test if combination of Grindamyl POWERbake 4070 and CGTase performed on level with combination of Grindamyl POWERbake 4070 and added β-cyclodextrin.
Baking Mutant ID TIPU/kg g ml ml β- ml flour CGTase/ Grindamyl cyclodextrin water kg flour POWERbake sample"
4070
sample*
1 Blank - - - - 27.5
2 CGTase medium - 0.2 - 1.0 26.5
3 CGTase high - 1.0 5.0 22.5
4 Grindamyl 750 - 5.0 - 22.5
POWERbake 4070
5 Grindamyl 750 0.2 5.0 1.0 21.5
POWERbake 4070 +
CGTase medium
6 Grindamyl 750 1.0 5.0 5.0 17.5
POWERbake 4070 +
CGTase high
# 7.5 TIPU/mL
n 0.01 g CGTase/mL Table 6; Baking Trial 5: Trial to test if addition of substrate for CGTase can make the combination Grindamyl POWERbake 4070 and CGTase perform on level with combination of Grindamyl POWERbake 4070 and added [3-cyc!odextrin. For samples added starch 10 g starch was added to 49 g flour. For samples with no added starch 50 g flour was used, see Table 1.
Baking Mutant ID TIPU/ g CGTase/ ml ml p- ml
kg flour kg flour Grindamyl cyclodextrin water
POWERb sample"
ake 4070
sample*
1 Blank - - - - 27.5
2 Starch blank
3 Starch + CGTase - 0.2 - 1.0 26.5 medium
4 Starch + CGTase - 1.0 - 5.0 22.5 high
5 Starch + 750 5.0 22.5 Grindamyl
POWERbake 4070
6 Starch + 750 0.2 5.0 1.0 21.5 Grindamyl
POWERbake 4070
+ CGTase medium
7 Starch + 750 1.0 5.0 5.0 17.5 Grindamyl
POWERbake 4070
+ CGTase high
# 7.5 TIPU7mL
H 0.01 g CGTase/mL Dough for analysis
During scaling of the bread 5.0 grams were scaled, put into a vial and proofed at room temperature for 10 minutes before being proofed at 34°C for 45 minutes. After proofing, the vial was placed in liquid nitrogen and sample was lyophilized and kept frozen until further analysis.
Extraction of lipid
Lyophilized dough samples were milled in a coffee mill before extraction of the lipid fraction applying one of the following procedures.
A: Warm butanol extraction
1 .0 g lyophilized milled dough sample was added 7.5 ml water saturated (WST) butanol and placed on a rotor mixer for 5 min. The sample was then placed in a water bath at 97°C for 10 minutes. Then the sample was placed on a rotor mixer for 30 minutes before being placed in a water bath at 97°C for 10 minutes followed by another 30 minutes on a rotor mixer. Finally the sample was centrifuged at 3500 rpm for 10 minutes before the organic phase was transferred to a new tube and kept at -18°C until analysis.
B: Cold butanol extraction
1 .0 g lyophilized milled dough sample was added 7.5 ml WST butanol and placed on a rotor mixer for 60 min. Then the sample was centrifuged at 3500 rpm for 10 minutes before the organic phase was transferred to a new tube and kept at -18°C until analysis.
C: Water extraction
1.0 g lyophilized milled dough sample was added 7.5 ml water and placed on a rotor mixer for 60 min. Then the sample was centrifuged at 3500 rpm for 10 . minutes. The supernatant was transferred to a new tube and 4 mL hereof was added 4 mL butanokethanol (85:15 (v/v)). To the precipitate 7.5 mL butanokethanol (85:15 (v/v)) was added. Both samples were then placed on a rotor mixer for 60 minutes before being centrifuged at 3500 rpm for 10 minutes. For the supernatant sample the two phases were separated and both kept at -18°C until analysis. For the precipitate sample the organic phase was kept at -18°C until analysis.
D: Water extraction with added papain
1.0 g lyophilized milled dough sample was added 500 ppm papain and 7.5 ml water and placed at 30°C for 90 minutes with stirring. Then the sample was centrifuged at 3500 rpm for 10 minutes. The supernatant was transferred to a new tube and 4 mL hereof was added 4 mL butanohethanol (85:15 (v/v)). To the precipitate 7.5 mL butanohethanol (85: 15 (v/v)) was added. Both samples were then placed on a rotor mixer for 60 minutes before being centrifuged at 3500 rpm for 10 minutes. For the supernatant sample the two phases were separated and both kept at -18°C until analysis. For the precipitate sample the organic phase was kept at -18°C until analysis.
Free fatty acid determination applying GC
Perkin Elmer Autosystem 9000 Capillary Gas Chromatograph equipped with WCOT fused silica column 12.5 m x 0.25 mm ID x 0.1 μ film thickness 5% phenyl-methyl- silicone (CP Sil 8 CB from Chrompack). Carrier gas: Helium.
injector. PSSi cold split injection (initial temp 90°C heated to 400°C), volume 1 .0μΙ Detector FID: 400°C
Oven program: 1 2 3 4
Oven temperature, °C. 80 200 240 360
isothermal, time, min. 2 0 0 20
Temperature rate, °C/min. 20 10 12
Sample preparation: Evaporated sample was dissolved in 1 ml pyridine containing internal standard heptadecane, 0.5 mg/ml. 500μΙ sample solution was transferred to a crimp vial, 100 pi BSTFA (Λ/,Ο-bis-trimethylsilyl-trifluoroacetamide) + TMCS (trimethylchlorosilane) (99+1 ) was added and reacted for 15 minutes at 60°C.
Calculation: Response factors for triglyceride was determined from wheat lipid extract, whereas it was fixed for FFA.
Free fatty acid determination applying NEFA HR(2)
Sample preparation:
For samples dissolved in organic solvent 1 .0 mL extracted lipid sample was evaporated at 70°C under nitrogen cover and then redispersed in 1.0 ml 0.1 % Triton X-100.
Assay conditions:
The amount of free fatty acid was determined on Konelab Autoanaiyser (Thermo, Finland) using the NEFA HR(2) kit (WAKO GmbH, Germany). Assay was run at 30°C. 150 μΙ_ solution A and 15 μΐ_ redispersed extracted lipids were incubated for 3 minutes. 75 μΐ solution B was added and incubated for 4.5 minutes. The absorbance at 520 nm was measured. The amount of free fatty acid was determined, using the read absorbance and a standard curve based on oleic acid (0.05 mM to 1.0 mM).
Baking Trials - Results
Flour Water absorption determination
For the flour used in the baking trials described in Tables 2 and 3B analysis of the flour water absorption resulted in a water absorption of 60% relative to flour at 400BU
(Brabender Units), meaning that 50 g flour had to be added: Water addition to dough, ml = (flour, g x (water abs - 2), %)/100% Water addition to dough, ml = (50 g x (60 - 2)%)/100% = 29.0 mL
For the flour used in the baking trials described in Tables 3A, 5 and 6 analysis of the flour water absorption resulted in a water absorption of 57% relative to flour at 400BU, meaning that 50 g fiour had to be added:
Water addition to dough, ml = (50 g x (57 - 2)%)/100% = 27.5 mL
As p-cyclodextrin influenced the water absorption of the flour, analysis of the water absorption relative to flour at 400 BU was made for each β-cyclodextrin concentration tested: see Table 7.
For the flour used in Baking Trial 3 (β-cyclodextrin and pectin) analysis of the flour water absorption resulted in a water absorption of 56% relative to flour at 400BU, meaning that 50 g fiour had to be added:
Water addition to dough, ml = (50 g x (56 - 2)%)/100% = 27.0 mL
As opposed to Baking Trial 2 the amount of water/liquid added in this trial was kept constant instead of keeping the BU constant. Hence to keep the BU below 700, 28.0 mL water was added in all instances instead of the standard 27.0 mL.
Table 7: Water absorption relative to fiour at 400 BLJ determined for each p- cyclodextrin concentration tested in Baking Thai 2.
A: Bread in tins
Figure imgf000072_0001
Baking Trial 1 - Effect of increasing levels of FFA generated in situ This trial was conducted to evaluate the effect of generating increasing levels of FFA in situ in mini breads the recipes being described in Table 2. Grindamyl POWERbake 4070 was dosed at 1000 T!PU/kg flour based on data in TN6526, as this dose had a positive effect on bread volume and still was close to the dose which had a negative effect on bread volume. In addition to the fixed dose of Grindamyl POWERbake 4070 (1000 TIPU/kg flour), Exel 16 was dosed stepwise from 4000 LIPU/kg flour to 44000 LIPU/kg flour to generate increasing levels of FFA in situ.
Mini breads from this baking trial are depicted in Figure 1 , the samples being follows: Lane Sample
1 Blank
2 Control-Lipopan F
3 Grindamy! POWERbake 4070
4 Grindamyl POWERbake 4070 +
Exel 16 4000 U
5 Grindamy] POWERbake 4070 +
Exe! 16 12000 U
6 Grindamyl POWERbake 4070 +
Exel 16 20000 U
It is evident that increasing the dose of Exei 16 from 4000 to 20000 LIPU/kg fiour on top of a fixed dose of Grindamyl POWERbake 4070 (1000 TIPU/ kg flour) had a negative effect on the bread volume as compared to Grindamyl POWERbake 4070 alone. The relative volume compared to the blank (based on specific volumes) is illustrated in Figure 2. Also the relative volume compared to the blank (based on specific volumes) decreased when Exel 16 dosages between 4000-20000 LIPU/ kg flour on top of a fixed dose of Grindamyl POWERbake 4070 was used. Also shown in Figure 2 is the amount of triglyceride and free fatty acid in dough determined by applying GC (doughs being extracted with warm butanol). As a function of increasing dose of Exel 16 the amount of triglyceride diminished concomitant with the FFA amount increased. Compared to Grindamyl POWERbake 4070 alone (1000 TIPU/ kg flour) increased dose of Exel 16 (4000-20000 LIPU/kg flour) on top of Grindamyl POWERbake 4070 (1000 TIPU/ kg flour) led to decreased relative volume vs. blank and increased amounts of free fatty acid. Thus the results in Figure 2 gave a strong indication of a negative correlation between the level of free fatty acid in dough and specific bread volume, and hence the negative baking effect of free fatty acid in terms of reduced bread volume.
A baking trial as described in Table 2 but with no tins was also conducted. The trend in relative volume compared to the blank (data not shown) was in line with results described for mini breads baked in tins. Baking Trial 2 - Effect of increasing levels of added β-cyclodextrin
This baking trial was conducted to evaluate the effect of adding increasing levels of β- cyclodextrin together with a high dose of Grindamyl POWERbake 4070 enzyme. The addition of β-cyclodextrin should serve to lower the concentration of free fatty acids by formation of inclusion compounds and thereby eliminate/minimize the negative baking effect of increasing levels of free fatty acid in terms of reduced bread voiume.
Mini breads from this baking trial are depicted in Figures 3A and 3B (Figure 3A showing bread in tins and Figure 3B showing bread without tins), the samples being as follows:
Figure imgf000074_0001
It is evident from these Figures that just by adding β-cyclodextrin (high dose) the bread volume increased significantly as compared to the blank both for mini bread baked with and without tins. By visual judgement addition of only β-cyclodextrin (high dose) made the flour perform on level with flour added Lipopan F as the control- Lipopan F (Bread 2 in Figures 3A and 3B) and β-cyclodextrin blank (Bread 3 in Figures 3A and 3B) mini breads were of equal size. The expected increase in bread volume with increasing level of added β-cyclodextrin was not very evident for mini breads neither with nor without tins. For mini breads baked in tins the increase in bread volume with increasing amount of added β-cyclodextrin was not consistent over the range of β-cyclodextrin doses tested. For mini breads baked without tins the visual judgement of the bread volume was troubled by the change in shape of the breads from more bread-like to more bun-like as the amount of added β-cyclodextrin was increased.
Further results observed in relation to the use of lipases include improved crumb structure, improved pore homogeneity and reduced mean pore size. These effects remain substantially unchanged by the inclusion of cyclodextrin. It was observed that cyclodextrin when used alone increased the stickiness of the dough. In Figure 4 the relative volume compared to the blank is shown for mini breads baked without tins. As mentioned above addition of simply β-cyclodextrin (high dose) had a significant positive effect and the data in Figure 4 shows thai the bread volume increased by 40% compared to blank. When increasing levels of β-cyclodextrin was combined with a fixed high dose of Grindamyl POWERbake 4070 the volume increased additionally as much as 20%, giving up to 60% total volume increase compared to blank, see Figure 4. Further results observed in relation to the use of lipases include improved crumb structure, improved pore homogeneity and reduced mean pore size. These effects remain substantially unchanged by the inclusion of cyclodextrin. It was observed that cyclodextrin when used alone increased the stickiness of the dough. To verify if the observed volume increase was an effect of a lower level of free fatty acid due to the formation of fatty acid-p-cyclodextrin inclusion compounds or to an effect of β-cyciodextrin on its own, the level of free fatty acid in the lyophilized dough was determined. Figure 5 shows the flow of the dough extraction made to differentiate between fatty acids incorporated in β-cyclodextrin inclusion compounds and free fatty acid. By adding WST butanol to lyophilized dough the total fatty acids will be extracted, both the free fatty acid and the fatty acid incorporated in inclusion compounds. By adding water to lyophilized dough the fatty acid-p-cyclodextrin inclusion compounds will be extracted, as these are water soluble. Along with the free fatty acid-p-cyclodextrin inclusion compounds, a small amount of free fatty acid will be extracted (extract 1 ). Adding butanohethanol (85:15 (v/v)) to the watery extract will cause the fatty acid-p- cyclodextrin inclusion compounds to break down into free fatty acid and β- cyclodextrin. The free fatty acid will be in the organic phase (extract 3) and the β- cyclodextrin will be in the water phase (extract 2). The precipitate from the watery extract will contain the fatty acid not incorporated in inclusion compounds and by adding butanohethanol (85: 15 (v/v)) to the precipitate this fatty acid will be extracted (extract 4). The results of the dough extractions depicted in Figure 5 are summarized in Table 8. For dough with no enzyme added (b!ank and β-cyclodextrin) the total amount of free fatty acid was in the proximity of 6 μιηοΐ/g dough, and for dough with enzyme added the total !evel of free fatty acid was in the range 1 1 -13 rnmol/ g dough.
The 6 pmol free fatty acid / g dough in the blank was determined by applying the NEFA method and this amount of free fatty acid corresponded to 0.17% free fatty acid (w/w) ((6x10"6 mol/g dough) x 280g/mol x 100%=0.168%). This number correlated very well with the 0.18% free fatty acid (w/w) determined by GC analysis for the blank in the baking trial conducted to illustrate the effect of generating increasing amounts of FFA in situ: see Figure 2.
Even though Grindamyl POWERbake 4070 in this baking trial was dosed very high (5000 TIPU/kg flour as opposed to 750-1000 TIPU/kg flour being optimal) the total amount of free fatty acid (1 1 -13 pmol/g flour corresponding to 0.31 -0.36 % (w/w) free fatty acid) was not elevated as compared to the Grindamyl POWERbake 4070 mini bread (1000 TIPU/kg flour and 0.40 % (w/w) free fatty acid (w/w)) in the trial made to illustrate the effect of generating increasing amounts of free fatty acid in situ, see Figure 2.
Water extraction of the lyophilized doughs was made to be able to distinguish between free fatty acid (Extract 1 ) and FA incorporated in β-cyclodextrin inclusion compounds (Extract 3) assuming the later not being detectable by the NEFA method. However fatty acid incorporated in β-cyclodextrin, forming inclusion compounds were measurable by the NEFA method, as the amount of free fatty acid in Extract 1 from Grindamyl POWERbake 4070 treated samples increased with increasing addition of β-cyclodextrin, while the total amount of free fatty acid in Grindamyl POWERbake 4070 treated samples did not change, see Table 8. Thus the addition of β-cyclodextrin enabled a larger part of the free fatty acid to become water soluble, due to the formation of inclusion compounds. However, from the experiments conducted here, it could not be concluded whether the fatty acid^-cyclodextrin inclusion compounds were formed in the dough or if the formation took place during extraction of the dough. Extract 3 was to be a number for the amount of free fatty acid released from β- cyclodextrin inclusion compounds upon addition of organic solvent. As evident from Table 8 the amount of free fatty acid did increase with increasing addition of p-cyciodextrin (For Grindamyi POWERbake 4070 treated samples from 0.70 up to 0.97 pmol/g dough in tins and from 0.50 up to 0.67 pmol/g dough with no tins) indicating that free fatty acid had indeed been incorporated in β-cycSodextrin forming inclusion compounds. Again, it was uncertain whether the inclusion compounds were formed in the dough or during extraction of the dough. Comparing the amount of free fatty acid in extracts 1 and 3 and the relative volume vs blank showed a trend towards a positive correlation between increased bread volume and increased amount of free fatty acid in extract 1 and 3 as function of amount added β- cyclodextrin: see Figure 6.
As expected only a very small amount of free fatty acid was found in Extract 2: see Table 8. Presumably the rather varying free fatty acid numbers determined for mini bread made in tins was caused by inadequate centrifugation of the sample resulting in poor phase separation.
In all instances approximately 90% of all free fatty acid extracted in the water extraction (sum of Extracts 2, 3 and 4) was found in the precipitate (Extract 4) implying pronounced interaction between gluten and starch and the free fatty acid. This high percentage of the free fatty acid in the precipitate also indicated that only a small amount of free fatty acid was available for incorporation in β-cyclodextrin, forming inclusion compounds. Table 8: Level of free fatty acid in lyophilized dough from baking trial conducted to illustrate the effect of increasing levels of added β-cyclodextrin. The lyophilized doughs were extracted as depicted in Figure 5 and the amount of free fatty acid was determined applying the NEFA method. A: In tins
Figure imgf000078_0001
In theory the amount of free fatty acid in extract 2, 3 and 4 should add up to the amount determined for the total extract. But as is evident from Table 8 this was not the case as the recovery (sum extract 2, 3 and 4 relative to the total extract) was in the range 40-60%, meaning only close to half the free fatty acid was extracted. The low recovery could be due to gluten agglumeration during water extraction of the iyophilized dough. Agglumerated gluten could trap free fatty acids and render them inaccessible for analysis. In fact a gum-like lump was observed in the precipitate of the water extracted Iyophilized dough indicating gluten agglumeration taking place. To verify if this was the case papain (a plant protease) was added during water extraction of selected Iyophilized doughs. Addition of papain would degrade the gluten and thereby hinder agglomeration and the concurrent trapping of free fatty acids. Table 9 shows the levels of free fatty acids in papain treated Iyophilized dough from baking trial conducted to illustrate the effect of increasing levels of added β- cyclodextrin (no tins). The Iyophilized doughs were extracted as depicted in Figure 5 with the exception that 500 ppm papain was added during the water extraction. The amount of free fatty acid was determined applying the NEFA method.
As is evident from Table 9, the addition of papain released additional free fatty acids from the precipitate as compared to extraction made without papain (Table 8) adding up to a total recovery of more than 75%. This additional release of free fatty acids from the precipitate upon addition of papain further highlighted the observation made above of a high percentage of free fatty acids tightly interacting with gluten. This interaction between free fatty acids and gluten could very well restrict the stretchability of the gluten network as the amount of free fatty acid increase resulting in a reduced bread volume. Possibly the positive baking effect of β-cyclodextrin in terms of increased bread volume is caused by less restriction of the gluten network as incorporation of free fatty acids in β-cyclodextrin, forming inclusion compounds hinders/reduces free fatty acid interaction with gluten.
Table 9
Sample ID μπιοΙ FFA/ g dough Recovery
Total Extract 1 Extract 2 Extract 3 Extract 4 (Extract (2+3+4) extraction / total)
Grindamyl 13.07 0.80 0.02 0.53 9.45 77% POWERbake
4070
Grindamyl 13.04 1.01 0.05 0.37 9.48 76% POWERbake
4070+high
cyclodextrin Summarizing, all the data from the extractions of free fatty acids from lyophiiized dough (Tables 8 and 9) and the baking results (Figures 4 and 6) did indeed indicate a positive correlation between the amount of added β-cyciodextrin, level of FFA and relative volume. Yet it could not be ruled out that the effect of the added cyclodextrin was not a hydrocolloid effect. Therefore a baking trial was performed comparing the baking effect of β-cyclodextrin to the baking effect of the hydrocolloid pectin.
Baking Trial 3 - Comparison of baking effect of β-cyclodextrin and pectin
As described above (Table 7), the addition of β -cyclodextrin did influence the water absorption of the flour so the observed positive baking effect of β-cyclodextrin in terms of increased bread volume could be due to a hydrocolloid effect rather than to minimizing the negative effect of FFA by formation of inclusion compounds. To evaluate whether the observed positive baking effect of β-cyclodextrin was a hydrocolloid effect or not, a baking trial comparing the baking effect of β-cyclodextrin to the baking effect of pectin was conducted. To further investigate this aspect a lipase (Exel 16) generating increasing amount of FFA in situ was added on top of β- cyclodextrin and pectin.
The water absorption of the flour used in this trial was determined to 56% implying 27.0 mL water had to be added to 50 g flour. But as both β-cyclodextrin and pectin influenced the water absorption and the amount of added water was to be kept constant and BU in all instances had to be below 700 it was necessary to add 28.0 mL water/liquid as opposed to the standard 27.0 mL. To have comparable hydrocolloid effects with the same volume water/liquid added the BU had to be the same with addition of β-cyclodextrin and with addition of pectin. To obtain a BU of 625 as for 0.9% β-cyclodextrin 0.25% pectin had to be added, see Table 4.
Mini breads are illustrated in Figure 7 and the relative volume in comparison with blank is depicted in Figure 8. In Figure 7, the samples are as follows:
Lane Sample Lane Sample
1 Blank 7 |<-cyclodextrin+ Exel 16_12000U
2 Exel 16 4000U 8 .-cyclodextrin+ Exel 16 20000U
3 Exel 16 12000U 9 Pectin blank
4 Exel 16 20000U 10 Pectin+ Exel 16 4000U
5 β -cyclodextrin blank 11 Pectin + Exel 16 12000U
6 -cyclodextrin+ Exel 16_4000U 12 Pectin+ Exel 16 20000U As in Baking Trial 2 (see Figure 6) the addition of only β-cyclodextrin again resulted in 40% volume increase as compared to blank. In comparison addition of pectin gave a 10% volume increase. Thus the positive baking effect of β-cyclodextrin was not solely due to hydrocoiloid effect as pectin only gave a limited effect as compared to β- cyciodextrin. For mini breads with Exel 16 the highest dose gave a negative baking effect as the bread volume decreased compared to the medium Exel 16 dose. Most likely this was caused by an elevated free fatty acids level due to the high Exel 16 dose added. Combination of Exel 16 and β-cyclodextrin hindered the negative baking effect at high Exel 16 dosages as the bread volume was constant through out the Exel 16 dosage range tested. Thus β-cyclodextrin minimized the negative effect of the elevated free fatty acid level presumably by formation of inclusion compounds. In addition, the combination of Exel 16 and pectin also hindered the negative baking effect at high Exel 16 dosages as the bread volume was constant through out the Exe! 16 dosage range tested. Presuming β -cyciodextrin did not influence the viscosity of the dough fluid/liquid contrary to pectin that is known to increase the viscosity of a fluid/liquid, the positive effect of the combination of Exel 16 and pectin is expected to be caused by an overall stabilization of the dough due to increased dough fluid/liquid viscosity. Combining the above results strongly indicated that the positive baking effect of β- cyclodextrin was due to the incorporation of free fatty acids in inclusion compounds hereby minimizing the negative baking effect of free fatty acids and not due to a hydrocoiloid effect. Further results observed in relation to the use of lipases include improved crumb structure, improved pore homogeneity and reduced mean pore size. These effects remain substantially unchanged by the inclusion of cyciodextrin. It was observed that cyciodextrin when used alone increased the stickiness of the dough. Baking Trials 4 and 5 - Comparison of performance of a combination of Grindamyl POWERbake 4070 and CGTase with a combination of Grindamyl POWERbake 4070 and added B-cyciodextrin As described above, CGTase is closely related to oc-amylases, but has the unique ability to produce cyclodextrins. Hence by addition of CGTase to fiour it should be possible to generate cyclodextrin in situ in the dough. To evaluate if Grindamyl POWERbake 4070 and in situ generated cyclodextrin by CGTase performed on level with Grindamyl POWERbake 4070 and added β-cyclodextrin, the baking trials described in Table 5 and Table 6 were conducted. According to manufacturer the CGTase used for these trials generates 45% a-cyclodextrin, 45% β-cyclodextrin and 10% cyclodextrin DP>7. To supplement substrate for CGTase starch was added in the trial described in Table 6. Mini breads with no starch added can be viewed in Figure 9 and mini breads with added starch can be viewed in Figure 10 (notice bread placed opposite order in the two figures), showing an effect was obtained.
Figure imgf000082_0001
In Figure 10, the samples are as follows:
Lane Sample
1 Grindamyl POWERbake 4070 + CGTase high + starch
2 Grindamyl POWERbake 4070 + CGTase low + starch
3 Grindamyl POWERbake 4070 + starch
4 CGTase high + starch
5 CGTase low + starch
6 Blank starch
7 Blank Baking Trials 1 to 5 - Conclusion
Baking Trial 1 indicated increasing amounts of in situ generated free fatty acids had a negative baking effect in terms of reduced bread volume. Complexing free fatty acids with β-cyclodextrin had a significant positive effect on bread volume, 40% increase compared to blank, !n combination with Grindamyl POWERbake 4070, β-cyciodextrin had an additive effect, as an additional 20% volume increase was obtained, giving a total of 60% volume increase compared to blank.
From Baking Trial 3 it was evident that the positive baking effect of β -cyclodextrin was not solely due to a hydrocolloid effect as pectin only gave a 10% bread volume increase vs 40% for β -cyclodextrin. Rather the positive baking effect of β-cyclodextrin was due to complexing of free fatty acids.
Baking Trials 4 and 5 comparing the baking performance of Grindamyl POWERbake 4070 in combination with either β-cyclodextrin or CGTase showed the two combinations to perform on level. Further results observed in relation to the use of lipases include improved crumb structure, improved pore homogeneity and reduced mean pore size. These effects remain substantially unchanged by the inclusion of cyclodextrin.
Baking Trial 6
As could be seen from Baking Trials 4 and 5, little additive effect was seen from combining a CGTase producing cyclodextrins in the dough, with a lipase (phospholipase, glycolipase or triglyceride lipase). To evaluate whether this was due to the fact that the cyclodextrin concentration will need to be at a certain level before the free fatty acids (FFA) generation starts, this further baking trial was conducted.
To accumulate a certain level of cyclodextrins before generation of FFA, CGTase was added to a fraction of the flour (20% of flour comprising the dough), together with the water needed to form the dough. Brews were stirred gently and left at 30°C for 1 hour. Afterwards the brew was added the residual amount of flour (80% of flour comprising the dough), mixed to form a dough, proofed, scaled and baked using the below baking procedure. 20% of flour is added to water required for dough (with or without CGTase) and the mixture is stirred gently at 30 °C for 1 hour. At this point, mixing of the dry ingredients (residual flour, yeast, salt, sugar) begins. After 1 minute, liquid (or brew) is added (with or without lipase) and mixing carried out for a further 5 minutes, after which the dough is placed in a proofing cabinet at 34°C for 10 minutes. The dough is then scaled to four 15.5 g portions, moulded and placed in tins before being rested for 10 minutes. It is then reintroduced into the proofing cabinet at 34°C for 45 minutes before being baked in an oven at 220°C for 5½ minutes. After 20 minutes the bread is removed from the tin and its volume and weight measured. To enable the evaluation the effect of the experimental setup, the baking experiments described in Table 10 below were conducted. The results are shown in Table 10 and in Figure 1 1.
Figure imgf000084_0001
Table 10
As can be seen from the results shown in Table 10 and Figure 1 1 , allowing the CGTase to accumulate cyclodextrins before adding a lipase generating free fatty acids has a positive effect. It is also clear from the results, that an additive affect can be obtained by combining a lipase (phospholipase, glycolipase or triglyceride lipase) with a CGTase. The latter is especially clear if results from Baking Trials 4 to 6 above are compared. Finally it can be concluded that the effect of lipases in baking can be improved by complexing the free fatty acids (and hereby controlling the detrimental effect of free fatty acids generated) using a CGTase.
Ail publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduiy limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Claims

1. A foodstuff comprising an effective amount of:
(a) a lipolytic enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule; and
(b) a cyclodextrin.
2. A foodstuff according to claim 1 , wherein the cyclodextrin is a β-cyclodextrin.
3. A foodstuff according to claim 1 or claim 2, wherein the cyclodextrin is present as an initial component of the foodstuff.
4. A foodstuff according to claim 1 or claim 2, wherein the cyclodextrin is generated in situ.
5. A foodstuff according to claim 4, wherein the cyclodextrin is generated in situ by the action of a cyclodextrin glycosyltransferase enzyme on a polysaccharide substrate.
6. A foodstuff according to any preceding claim, selected from a dough and a baked product prepared from dough.
7. A foodstuff according to any preceding claim, wherein the lipolytic enzyme is in a delayed release form.
8. A foodstuff according to any preceding claim, wherein the amount of lipolytic enzyme present, by weight of pure enzyme protein, is 0.001 mg to 2 mg per kg of the total weight of the foodstuff.
9. A foodstuff according to any preceding claim, wherein the amount of cyclodextrin present is 0.05 to 50 g per kg of the total weight of the foodstuff.
10. A foodstuff according to claim 8, wherein the amount of cyclodextrin present is 0.5 to 10 g per kg of the total weight of the foodstuff.
1 1 . A method of producing a foodstuff according to any one of claims 1 to 9,
comprising adding the lipolytic enzyme and the cyclodextrin to one or more other ingredients of the foodstuff and, if necessary, treating the ingredients to produce the foodstuff.
12. A method according to claim 1 1 , further comprising adding a cyclodextrin
glycosyltransferase enzyme and, if necessary, a polysaccharide substrate to generate cyclodextrin in situ.
13. A method according to claim 1 1 , wherein the cyclodextrin glycosyltransferase enzyme is added before addition of the lipolytic enzyme.
14. A method according to claim 1 1 , wherein the cyclodextrin glycosyltransferase enzyme is added at the same time as the lipolytic enzyme.
15. Use of a cyclodextrin for decreasing the uncomplexed free fatty acid content of a foodstuff incorporating a lipolytic enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule.
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2019121585A1 (en) * 2017-12-19 2019-06-27 Dupont Nutrition Biosciences Aps Improved enzymatic modification of phospholipids in food

Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3817837A (en) 1971-05-14 1974-06-18 Syva Corp Enzyme amplification assay
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3939350A (en) 1974-04-29 1976-02-17 Board Of Trustees Of The Leland Stanford Junior University Fluorescent immunoassay employing total reflection for activation
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4277437A (en) 1978-04-05 1981-07-07 Syva Company Kit for carrying out chemically induced fluorescence immunoassay
US4366241A (en) 1980-08-07 1982-12-28 Syva Company Concentrating zone method in heterogeneous immunoassays
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
EP0238023A2 (en) 1986-03-17 1987-09-23 Novo Nordisk A/S Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0449375A2 (en) 1990-03-23 1991-10-02 Gist-Brocades N.V. The expression of phytase in plants
WO1991017243A1 (en) 1990-05-09 1991-11-14 Novo Nordisk A/S A cellulase preparation comprising an endoglucanase enzyme
EP0493045A1 (en) 1990-12-21 1992-07-01 Kyowa Hakko Kogyo Co., Ltd. Method of reducing cholesterol concentration in food
EP0583265A1 (en) 1991-04-16 1994-02-23 Evotec BioSystems GmbH Method for preparing new biopolymers
US5310679A (en) * 1985-05-13 1994-05-10 Artiss Joseph D Composition for reducing turbidity in samples of biological fluids
EP0687414A1 (en) 1994-06-17 1995-12-20 Gist-Brocades B.V. Bread improving composition
EP0752008A1 (en) 1994-02-17 1997-01-08 Affymax Technologies N.V. Dna mutagenesis by random fragmentation and reassembly
US5674707A (en) 1992-12-10 1997-10-07 Gist-Brocades N.V. Production of heterologous proteins in filamentous fungi
WO1998013479A1 (en) * 1996-09-26 1998-04-02 Novo Nordisk A/S An enzyme with cyclomaltodextrin glucanotransferase (cgtase) activity
US5741665A (en) 1994-05-10 1998-04-21 University Of Hawaii Light-regulated promoters for production of heterologous proteins in filamentous fungi
EP0866796A1 (en) 1995-09-22 1998-09-30 Medical Research Council Improvements in or relating to mutagenesis of nucleic acids
US5824354A (en) * 1995-02-22 1998-10-20 Cerestar Usa, Inc. Process for reducing sterols and free fatty acids from animal fat
WO2000058517A1 (en) 1999-03-26 2000-10-05 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
US6180406B1 (en) 1994-02-17 2001-01-30 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
WO2001016308A2 (en) 1999-08-30 2001-03-08 Monsanto Technology Llc Plant sterol acyltransferases
WO2001034835A2 (en) 1999-11-09 2001-05-17 Max-Planck Gesellschaft zur Förderung der Wissenschaften e.V. Method for the production of biopolymers with modified properties
WO2001039544A1 (en) 1999-11-25 2001-05-31 Natural Colour Kari Kirjavainen Oy Electromechanic film and acoustic element
WO2001042433A2 (en) 1999-12-07 2001-06-14 Danisco A/S Talaromyces emersonii xylanase
WO2002006457A2 (en) 2000-07-13 2002-01-24 Maxygen, Inc. Novel lipase genes
WO2002006508A2 (en) 2000-07-19 2002-01-24 Novozymes A/S CYCLOMALTODEXTRIN GLUCANOTRANSFERASE (CGTase9, DNA SEQUENCE ENCODING IT AND USES THEREOF
US6344328B1 (en) 1995-12-07 2002-02-05 Diversa Corporation Method for screening for enzyme activity
US6361974B1 (en) 1995-12-07 2002-03-26 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
WO2003020923A1 (en) 2001-09-04 2003-03-13 Danisco A/S Xylanase variants
WO2003099016A2 (en) 2002-05-24 2003-12-04 Danisco A/S Method
WO2004064987A2 (en) 2003-01-17 2004-08-05 Danisco A/S Method
WO2004084638A1 (en) * 2003-03-28 2004-10-07 Consejo Superior De Investigaciones Científicas Enzymatic composition for improving the quality of bread and pastry doughs
WO2005003339A1 (en) 2003-07-07 2005-01-13 Danisco A/S Food additive comprising pseudomonas non-maltogenic exoamylase variants
WO2005087918A2 (en) 2004-03-12 2005-09-22 Danisco A/S Fungal lipolytic enzymes

Patent Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3817837A (en) 1971-05-14 1974-06-18 Syva Corp Enzyme amplification assay
US3939350A (en) 1974-04-29 1976-02-17 Board Of Trustees Of The Leland Stanford Junior University Fluorescent immunoassay employing total reflection for activation
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4277437A (en) 1978-04-05 1981-07-07 Syva Company Kit for carrying out chemically induced fluorescence immunoassay
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4366241A (en) 1980-08-07 1982-12-28 Syva Company Concentrating zone method in heterogeneous immunoassays
US4366241B1 (en) 1980-08-07 1988-10-18
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (en) 1985-03-28 1990-11-27 Cetus Corp
US5310679A (en) * 1985-05-13 1994-05-10 Artiss Joseph D Composition for reducing turbidity in samples of biological fluids
EP0238023A2 (en) 1986-03-17 1987-09-23 Novo Nordisk A/S Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus
EP0449375A2 (en) 1990-03-23 1991-10-02 Gist-Brocades N.V. The expression of phytase in plants
WO1991017243A1 (en) 1990-05-09 1991-11-14 Novo Nordisk A/S A cellulase preparation comprising an endoglucanase enzyme
EP0493045A1 (en) 1990-12-21 1992-07-01 Kyowa Hakko Kogyo Co., Ltd. Method of reducing cholesterol concentration in food
EP0583265A1 (en) 1991-04-16 1994-02-23 Evotec BioSystems GmbH Method for preparing new biopolymers
US5674707A (en) 1992-12-10 1997-10-07 Gist-Brocades N.V. Production of heterologous proteins in filamentous fungi
EP0752008A1 (en) 1994-02-17 1997-01-08 Affymax Technologies N.V. Dna mutagenesis by random fragmentation and reassembly
US6180406B1 (en) 1994-02-17 2001-01-30 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5741665A (en) 1994-05-10 1998-04-21 University Of Hawaii Light-regulated promoters for production of heterologous proteins in filamentous fungi
EP0687414A1 (en) 1994-06-17 1995-12-20 Gist-Brocades B.V. Bread improving composition
US5824354A (en) * 1995-02-22 1998-10-20 Cerestar Usa, Inc. Process for reducing sterols and free fatty acids from animal fat
EP0866796A1 (en) 1995-09-22 1998-09-30 Medical Research Council Improvements in or relating to mutagenesis of nucleic acids
EP1103606A2 (en) 1995-11-30 2001-05-30 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
EP1138763A2 (en) 1995-11-30 2001-10-04 Maxygen, Inc. Method for generating polynucleotides having desired characteristics by iterative selection and recombination
US6361974B1 (en) 1995-12-07 2002-03-26 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
US6344328B1 (en) 1995-12-07 2002-02-05 Diversa Corporation Method for screening for enzyme activity
WO1998013479A1 (en) * 1996-09-26 1998-04-02 Novo Nordisk A/S An enzyme with cyclomaltodextrin glucanotransferase (cgtase) activity
WO2000058517A1 (en) 1999-03-26 2000-10-05 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
WO2001016308A2 (en) 1999-08-30 2001-03-08 Monsanto Technology Llc Plant sterol acyltransferases
WO2001034835A2 (en) 1999-11-09 2001-05-17 Max-Planck Gesellschaft zur Förderung der Wissenschaften e.V. Method for the production of biopolymers with modified properties
WO2001039544A1 (en) 1999-11-25 2001-05-31 Natural Colour Kari Kirjavainen Oy Electromechanic film and acoustic element
WO2001042433A2 (en) 1999-12-07 2001-06-14 Danisco A/S Talaromyces emersonii xylanase
WO2002006457A2 (en) 2000-07-13 2002-01-24 Maxygen, Inc. Novel lipase genes
WO2002006508A2 (en) 2000-07-19 2002-01-24 Novozymes A/S CYCLOMALTODEXTRIN GLUCANOTRANSFERASE (CGTase9, DNA SEQUENCE ENCODING IT AND USES THEREOF
WO2003020923A1 (en) 2001-09-04 2003-03-13 Danisco A/S Xylanase variants
WO2003099016A2 (en) 2002-05-24 2003-12-04 Danisco A/S Method
WO2004064987A2 (en) 2003-01-17 2004-08-05 Danisco A/S Method
WO2004084638A1 (en) * 2003-03-28 2004-10-07 Consejo Superior De Investigaciones Científicas Enzymatic composition for improving the quality of bread and pastry doughs
WO2005003339A1 (en) 2003-07-07 2005-01-13 Danisco A/S Food additive comprising pseudomonas non-maltogenic exoamylase variants
WO2005087918A2 (en) 2004-03-12 2005-09-22 Danisco A/S Fungal lipolytic enzymes

Non-Patent Citations (42)

* Cited by examiner, † Cited by third party
Title
"Progress in industrial microbiology", vol. 29, 1994, ELSEVIER, pages: 641 - 666
ALTSCHUL ET AL., J. MOL. BIOL., 1990, pages 403 - 410
ARCHER, PEBERDY, CRIT. REV. BIOTECHNOL., vol. 17, no. 4, 1997, pages 273 - 306
AUSUBEL: "Short Protocols in Molecular Biology", 1999
BEGGS, J D, NATURE, vol. 275, 1978, pages 104
BERGER, KIMMEL: "Guide to Molecular Cloning Techniques, Methods in Enzymology", vol. 152, 1987, ACADEMIC PRESS
BESSETTE, PROC NATL ACAD SCI USA, vol. 96, 1999, pages 13703 - 13708
BEUCAGE S.L. ET AL., TETRAHEDRON LETTERS, vol. 22, 1981, pages 1859 - 1869
BRU ET AL., COLLOIDS AND SURFACES A: PHYSICOCHEMICAL AND ENGINEERING ASPECTS, vol. 97, 1995, pages 263 - 269
BRUMLIK ET AL., J. BACTERIOL, vol. 178, no. 7, April 1996 (1996-04-01), pages 2060 - 4
CARUTHERS MH ET AL., NUC ACIDS RES SYMP SER, 1980, pages 215 - 23
CHRISTOU, AGRO-FOOD-INDUSTRY HI-TECH, 17 March 1994 (1994-03-17)
CHRISTOU, AGRO-FOOD-INDUSTRY HI-TECH, March 1994 (1994-03-01), pages 17 - 27
CURR OPIN BIOTECHNOL, vol. 8, no. 5, 1997, pages 554 - 60
CURR. OPIN. BIOTECHNOL., vol. 6, no. 5, 1995, pages 501 - 6
DAVIS, DE SERRES, METHODS ENZYMOL, vol. 17A, 1971, pages 79 - 143
E HINCHCLIFFE, E KENNY: "Yeasts", vol. 5, 1993, ACADEMIC PRESS LTD., article "Yeast as a vehicle for the expression of heterologous genes"
ELIASSON, LARSSON: "Cereals in Breadmaking: A Molecular Colloidal Approach", 1993, MARCEL DEKKER
FEMS MICROBIOL LETT, vol. 174, no. 2, 1999, pages 247 - 50
FEMS MICROBIOL LETT, vol. 177, no. 1, 1999, pages 187 - 8
FEMS MICROBIOL REV, vol. 24, no. 1, 2000, pages 45 - 66
HIGGINS DG, SHARP PM, GENE, vol. 73, no. 1, 1988, pages 237 - 244
HILTON, BUCKLEY, J BIOL. CHEM., vol. 266, no. 2, 15 January 1991 (1991-01-15), pages 997 - 1000
HINNEN, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 75, 1978, pages 1929
HORN T ET AL., NUC ACIDS RES SYMP SER, 1980, pages 225 - 232
HORWELL DC, TRENDS BIOTECHNOL., vol. 13, no. 4, 1995, pages 132 - 134
ITO, H ET AL., J BACTERIOLOGY, vol. 153, 1983, pages 163 - 168
J. SAMBROOK, E. F. FRITSCH, T. MANIATIS: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS, pages: 1 - 3
KANE, CURR OPIN BIOTECHNOL, vol. 6, 1995, pages 494 - 500
MATTHES ET AL., EMBO J., vol. 3, 1984, pages 801 - 805
METHODS MOL BIOL, vol. 49, 1995, pages 341 - 54
MORINAGA ET AL., BIOTECHNOLOGY, vol. 2, 1984, pages 646 - 649
NELSON, LONG, ANALYTICAL BIOCHEMISTRY, vol. 180, 1989, pages 147 - 151
PEELMAN ET AL., PROTEIN SCI., vol. 7, no. 3, March 1998 (1998-03-01), pages 587 - 99
POTRYKUS, ANNU REV PLANT PHYSIOL PLANT MOL BIOL, vol. 42, 1991, pages 205 - 225
PUNT ET AL., TRENDS BIOTECHNOL., vol. 20, no. 5, 2002, pages 200 - 6
ROBERTSON ET AL., J. BIOL. CHEM., vol. 269, no. 3, 21 January 1994 (1994-01-21), pages 2146 - 50
SAIKI R K ET AL., SCIENCE, vol. 239, 1988, pages 487 - 491
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SIMON RJ ET AL., PNAS, vol. 89, no. 20, 1992, pages 9367 - 9371
TILDEN E.B., HUDSON C.S., J. BACTERIOL., vol. 43, 1942, pages 527 - 544
TURNER G.: "Aspergillus: 50 years on", 1994, article "Vectors for genetic manipulation"

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