TITLE: METHOD OF PREPARING A DOUGH-BASED PRODUCT

SEQUENCE LISTING AND DEPOSITED MICROORGANISMS

Sequence listing

The present invention comprises a sequence listing.

Deposit of biological material

None.

FIELD OF THE INVENTION

The present invention relates to the use of anti-staling amylases in the preparation of dough or dough-based edible products with a high sucrose content.

BACKGROUND OF THE INVENTION

US 3026205 describes a process of producing baked confections and the products resulting therefrom by alpha-amylase.

WO 9104669 describes the use of a maltogenic alpha-amylase to retard the staling of baked products such as bread; the maltogenic alpha-amylase described therein is commercially available under the tradename Novamyl ^® (product of Novozymes A/S). US 6162628 describes Novamyl ^® variants and their use for the same purpose. Three-dimensional structures of Novamyl ^® are published in US 6162628 and in the Protein Data Bank (available at http://www.rcsb.org/pdb/) with identifiers 1 QHO and 1 QHP, the structures are included herein by reference.

WO 2006/032281 describes methods of preparing a dough-based product with a high sucrose content using anti-staling amylases.

SUMMARY OF THE INVENTION

The inventors have found that a high sucrose content dough (such as cake dough) tends to inhibit the activity of an anti-staling amylases such as Novamyl ^® , making it less effective to prevent the staling of dough-based products with high sucrose content such as cakes. They have found that a good anti-staling effect in cakes can be achieved by using a carefully selected anti-staling amylase with certain properties, and they have identified such amylases.

By analyzing a 3D structure of Novamyl ^® , the inventors further found that sucrose may inhibit by binding in the active site. They have found that sucrose docks into the active site of Novamyl ^® differently from the substrate or inhibitor in published models 1QHO and 1QHP, and

they have used this finding to design sucrose-tolerant variants. A selection of particularly interesting Novamyl ^® (SEQ ID NO:1 ) variants were identified comprising the two specific substitutions D261 G and T288P in combination with at least one other amino acid alteration, preferably at least two other amino acid alterations, or three other amino acid alterations, or most preferably in combination with at least four other amino acid alterations.

Accordingly, in a first aspect the invention provides a method of preparing a dough or a dough-based edible product, comprising adding a polypeptide to the dough, wherein the dough comprises at least 10 % sucrose by weight, and the polypeptide has an amino acid sequence which is at least 70 % identical to SEQ ID NO: 1 , and compared to SEQ ID NO: 1 comprises the two substitutions D261 G and T288P and at least one additional amino acid alteration which is substitution or deletion of or insertion adjacent to Y89, W93, P191 , F194, Y360, or N375.

In a second aspect, the invention provides polypeptides, which have amylase activity less inhibited by sucrose than the amylase activity of SEQ ID NO: 1 , which have an amino acid sequence which is at least 70 % identical to SEQ ID NO: 1 , and which when compared to SEQ ID NO: 1 comprises the two substitutions D261 G and T288P and at least one additional amino acid alteration, which is substitution or deletion of or insertion adjacent to Y89, W93, P191 , F194, Y360, or N375. The invention also provides methods of producing such novel sucrose tolerant polypeptide variants of a maltogenic alpha-amylase.

BRIEF DESCRIPTION OF DRAWINGS

Docking of sucrose into the active site of Novamyl ^® (using the software GOLD version 2.1.2, Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1 EZ, UK and the protein part of the x-ray structure I QHO.pdb) reveals a specific binding configuration as unique to sucrose. The cartesian coordinates for the sucrose atoms in this binding configuration, using the coordinate system of the x-ray structure IQHO.pdb are given in Figure 1.

DETAILED DESCRIPTION OF THE INVENTION

Maltogenic alpha-amylase and sucrose docking

A maltogenic alpha-amylase (EC 3.2.1.133) having more than 70 % identity, such as, at least 75%, (particularly more than 80 % identity, such as at least 85%, or 90% identity, such as at least 95% or 96% or 97% or 98% or 99%) with the Novamyl ^® sequence shown as SEQ

ID NO: 1 may be used as the parent enzyme for designing sucrose tolerant variants. Amino acid identity may be calculated as described in US 6162828.

For Novamyl ^® (SEQ ID NO: 1 ), a 3D structure including a substrate or inhibitor as described in US 6162628 or in the Protein Data Bank with the identifier 1QHO or 1QHP may be used. Alternatively, a Novamyl ^® variant may be used, such as a variant described in US 6162628 or in this specification, e.g. the variant F188L +D261 G +T288P, which is used as a reference enzyme in the examples below. A 3D structure of a variant may be developed from the Novamyl ^® structure by known methods, e.g. as described in T. L. Blundell et al., Nature, vol. 326, p. 347 ff (26 March 1987); J. Greer, Proteins: Structure, Function and Genetics, 7:317- 334 (1990); or Example 1 of VVO 9623874.

The inventors found that sucrose may inhibit Novamyl ^® by binding in the active site. Docking of sucrose into the active site of Novamyl ^® (using the software GOLD version 2.1.2, Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1 EZ, UK and the protein part of the x-ray structure I QHO.pdb) reveals a specific binding configuration as unique to sucrose. The cartesian coordinates for the sucrose atoms in this binding configuration, using the coordinate system of the x-ray structure IQHO.pdb are given in Fig. 1.

Maltogenic alpha-amylase assay

The activity of a maltogenic alpha-amylase may be determined using an activity assay such as the MANU method. One MANU (Maltogenic Amylase Novo Unit) is defined as the amount of enzyme required to release one micro-mole of maltose per minute at a concentration of 10 mg of maltotriose substrate per ml in 0.1 M citrate buffer at pH 5.0, 37 ⁰ C for 30 minutes.

Amino acid alterations

The amino acid sequence of a maltogenic alpha-amylase may be altered to decrease the sucrose inhibition. The inventors found that the alteration may be made at an amino acid residue having at least one atom within 4 Angstroms from any of the sucrose atoms when the sucrose molecule is docked in the 3D structure of the maltogenic alpha-amylase. Using the Novamyl ^® structure 1 QHO and the sucrose docking in Fig. 1 , the following residues are within 4 A: K44, N86, Y89, H90, Y92, W93, F188, T189, D190, P191 , A192, F194, D372, P373, R376.

Further the following two positions of SEQ ID NO: 1 have been identified as relevant: Y360 and N375.

Particularly preferred embodiments of the invention are amino acid alterations in the following 2 - 6 positions compared to Novamyl ^® (SEQ ID NO: 1 ) in combination with the two specific substitutions D261 G and T288P:

Y89 + W93 Y89 + P191

Y89 + F194

Y89 + Y360 Y89 + N375 W93 + P191 W93 + F194 W93 + Y360

W93 + N375 P191 + F194 P191 + Y360 P191 + N375 F194 + Y360

F194 + N375 Y360 + N375

Y89 + W93 + P191 Y89 + W93 + F194

Y89 + W93 + Y360

Y89 + W93 + N375

Y89 + P191 + F194

Y89 + P191 + Y360 Y89 + P191 + N375

Y89 + F194 + Y360

Y89 + F194 + N375

Y89 + Y360 + N375

W93 + P191 + F194 W93 + P191 + Y360

W93 + P191 + N375

W93 + F194 + Y360

W93 + F194 + N375

W93 + Y360 + N375 P191 + F194 + Y360

P191 + F194 + N375

P191 + Y360 + N375

F194 + Y360 + N375

Y89 + W93 + P191 + F194

Y89 + W93 + P191 + Y360 Y89 + W93 + P191 + N375

Y89 + W93 + F194 + Y360 Y89 + W93 + F194 + N375 Y89 + W93 + Y360 + N375 Y89 + P191 + F194 + Y360 Y89 + P191 + F194 + N375

Y89 + P191 + Y360 + N375 Y89 + F194 + Y360 + N375 W93 + P191 + F194 + Y360 W93 + P191 + F194 + N375 W93 + P191 + Y360 + N375

W93 + F194 + Y360 + N375 P191 + F194 + Y360 + N375

Y89 + W93 + P191 + F194 + Y360 Y89 + W93 + P191 + F194 + N375

Y89 + W93 + P191 + Y360 + N375 Y89 + W93 + F194 + Y360 + N375 Y89 + P191 + F194 + Y360 + N375 W93 + P191 + F194 + Y360 + N375

Y89 + W93 + P191 + F194 + Y360 + N375

The alteration may be a substitution or deletion of one or more of the selected residues, or one or more residues (particularly 1-4 residues or 5-6 residues) can be inserted adjacent to a selected residue. The substitution may be with a smaller or larger residue. A substitution to increase the size of the residue may diminish the space obtained by the docked sucrose molecule thereby preventing the binding of sucrose. Amino acid residues are ranked as follows from smallest to largest: (an equal sign indicates residues with sizes that are practically indistinguishable):

G < A=S=C < V=T < P < L=I=N=D=M < E=Q < K < H < R < F < Y < W The substitution may also be such as to eliminate contacts with the sucrose molecule, in particular by moving or removing potential sites of hydrogen bonding or Van der Waals interactions.

The substitution may particularly be with another residue of the same type where the type is negative, positive, hydrophobic or hydrophilic. The negative residues are D, E, the positive residues are K/R, the hydrophobic residues are A,C,F,G,I,L,M,P,V,W,Y, and the hydrophilic residues are H, N, Q, S, T.

Some particular examples of substitutions are I15T/SA//L, R18K, K44R/S/T/Q/N, N86Q/S/T, T87N/Q/S, G88A/S/T, Y89W/F/H, H90W/F/Y/R/K/N/Q/M, W93Y/F/M/E/GA//T/S, F188H/L/I/T/GA/, D190E/Q/G, P191S/N, A192S/T, F194S/L/Y, L196F, Y360F/I/N, N371 K/R/F/Y/Q, D372E/Q/S/T/A and N375S/T/D/E/Q. Most preferred embodiments of amino acid alterations in the above-listed preferred positions are the following substitutions, alone or in combination: Y89F,W W93F,Y P191S,N F194Y,S,L

Y360F,l,N N375S.T

Examples of deletions are deletion of residue 191 or 192. An example of an insertion is Ala inserted between 192 and 193. The polypeptide may include other alterations compared to Novamyl ^® (SEQ ID NO:

1 ), e.g. alterations to increase the thermostability as described in US 6162628.

Particularly preferred embodiments of the invention are the following amino acid alterations compared to Novamyl ^® (SEQ ID NO: 1 ), all of which are tested in the examples below: Y89F, D261 G, T288P, I290V, N375S

F194Y, D261 G, T288P, N375S I15T, P191S, D261 G, T288P, N375S, S640I Y89F,P191S,D261 G,T288P I15T _! Y89F _! P191S _! D261 G,T288P Y89F,F194Y,D261 G,T288P

Y89F,D261 G,T288P,N375S Y89F _! P191S,F194Y _! D261 G,T288P Y89F _! P191S,D261 G _! T288P,N375S Y89F,P191 S,D261 G,T288P,Y360N Y89F _! P191 S,D261 G _! T288P,Y360F

Y89F,W93Y _! P191S _! D261 G,T288P Y89F,W93F _! P191S _! D261 G,T288P Y89F,P191S _! F194Y _! D261 G _! T288P,N375S

Nomenclature for amino acid alterations In this specification, an amino acid substitution is described by use of one-letter codes, e.g. K44R. Slashes are used to indicate alternatives, e.g. K44R/S/T/Q/N to indicate

substitution of K44 with R or S etc. P191 ^* indicates a deletion of P191. ^* 192aA indicates insertion of one Ala after A192. Commas are used to indicate multiple alterations in the sequence, e.g. F188L, D261 G, T288P to indicate a variant with three substitutions.

Properties of anti-staling amylase for use with sucrose The amylase for use in high-sucrose dough may be selected so as to have mainly exo-amylase activity. More specifically, the amylase hydrolyzes amylose so that the average molecular weight of the amylose after 0.4-4 % hydrolysis is more than 50 % (particularly more than 75 %) of the molecular weight before the hydrolysis.

Thus, the amylase may hydrolyze amylose (e.g. wheat amylose or synthetic amylose) so that the average molecular weight of the amylose after 0.4-4 % hydrolysis (i.e. between 0.4- 4 % hydrolysis of the total number of bonds) is more than 50 % (particularly more than 75 %) of the value before the hydrolysis. The hydrolysis can be conducted in a 1.7 % amylose solution by weight at suitable conditions (e.g. 10 minutes at 6O ⁰ C, pH 5.5), and the molecular weight distribution before and after the hydrolysis can be determined by HPLC. The test may be carried out as described in C. Christophersen et al., Starch 50 (1 ), 39-45 (1998).

An exo-amylase for use in high-sucrose dough may have a specified sugar tolerance. Compared to its activity in the absence of sucrose, the amylase may have more than 20 % activity at 10 % sugar, more than 10 % activity at 20 % sucrose, or more than 4 % activity at 40 % sucrose. The sugar tolerance may be determined as described in the examples. The exo-amylase may have optimum activity in the pH range 4.5-8.5. It may have sufficient thermostability to retain at least 20 % (particularly at least 40 %) activity after 30 minutes incubation at 85 ⁰ C at pH 5.7 (50 mM Na-acetate, 1 mM CaCI ₂ ) without substrate.

The exo-amylase may be added to the dough in an amount corresponding to 1-100 mg enzyme protein per kg of flour, particularly 5-50 mg per kg. The exo-amylase may be non-liquefying. This can be determined by letting the exo- amylase act on a 1 % wheat starch solution until the reaction is complete, i.e. addition of fresh enzyme causes no further degradation, and analyzing the reaction products, e.g. by HPLC. Typical reaction conditions are e.g. 0.01 mg enzyme per ml starch solution for 48 hours. The exo-amylase is considered non-liquefying if the amount of residual starch after the reaction is at least 20 % of the initial amount of starch.

The exo-amylase may have maltogenic alpha-amylase activity (EC 3.2.1.133). The exo-amylase may be the amylase described in WO 2005/066338, or it may be a Novamyl ^® variant described in this specification.

Dough and dough-based edible product The dough may have a sucrose content above 10 % by weight, particularly above 20

% or 30 %, e.g. 30-40 %. The flour content is typically 25-35 % by weight of total ingredients.

The dough may be made by a conventional cake recipe, typically with cake flour, sugar, fat/oil and eggs as the major ingredients. It may include other conventional ingredients such as emulsifiers, humectants, gums, starch and baking powder. It generally contains such ingredients as soft wheat flour, milk or other liquids, sugar, eggs, chemical leaveners, flavor extracts and spices, as well as others that may or may not include shortening.

Examples of emulsifiers include mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxethylene stearates, or lysolecithin. Conventional emulsifiers used in making flour dough products include as examples monoglycerides, diacetyl tartaric acid esters of mono- and diglycerides of fatty acids, and lecithins e.g. obtained from soya. The emulsifying agent may be an emulsifier per se or an agent that generates an emulsifier in situ. Examples of emulsifying agents that can generate an emulsifier in situ include enzymes, such as, lipase or phospholipases.

The dough is generally heat treated, e.g. by baking or deep frying to prepare an edible product such as cakes including pound cake, yellow and white layer cakes, cakes containing chocolate and cocoa products, sponge cakes, angel food cake, fruit cakes and foam-type cakes and doughnuts.

Optionally, one or more additional enzymes may be used together with the anti-staling amylase of the present invention in preparing dough and dough-based edible products. The additional enzyme may be a starch degrading enzyme, such as, another amylase (e.g., an alpha-amylase, beta-amylase and/or a glucoamylase) or pullulanase, a cyclodextrin glucanotransferase, a peptidase, in particular an exopeptidase, a transglutaminase, a lipase, a phospholipase, a cellulase, a hemicelluase, a protease, a glycosyltransferase, a branching enzyme (1 ,4-alpha-glucan branching enzyme), an oxidoreductase or oxidase (e.g., a monosaccharide oxidase, such as, glucose oxidase, hexose oxidase, galactose oxidase or pyranose oxidase). Sources of these additional enzymes are well known in the art.

The additional enzyme may be of any origin, including mammalian and plant, and preferably of microbial (bacterial, yeast or fungal) origin. For example, the amylase may be fungal or bacterial, e.g., an alpha-amylase from Bacillus, e.g. B. lichenifornis or B. amyloliquefaciens, a beta-amylase, e.g. from plant (e.g. soy bean) or from microbial sources (e.g. Bacillus), a glucoamylase, e.g. from A. niger, or a fungal alpha-amylase, e.g. from A. oryzae.

The hemicellulase may be a pentosanase, e.g. a xylanase which may be of microbial origin, e.g. derived from a bacterium or fungus, such as a strain of Aspergillus, in particular of A. aculeatus, A. niger, A. awamori, or A. tubigensis, from a strain of Trichoderma, e.g. T. reesei, or from a strain of Humicola, e.g. H. insolens.

The protease may be from Bacillus, e.g. B. amyloliquefaciens.

The lipase may be derived from a strain of Thermomyces {Humicola), Rhizomucor, Candida, Aspergillus, Rhizopus, or Pseudomonas, in particular from T. lanuginosus (H. ianuginosa), Rhizomucor miehei, C. antarctica, A niger, Rhizopus delemar, Rhizopus arrhizus or P. cepacia.

The phospholipase may have phospholipase A1 or A2 or lysophospholipase activity; it may or may not have lipase activity. It may be of animal origin, e.g. from pancreas, snake venom or bee venom, or it may be of microbial origin, e.g. from filamentous fungi, yeast or bacteria, such as Aspergillus or Fusarium, e.g. A. niger, A. oryzae or F. oxysporum. Also the variants described in WO 0032578 may be used.

The oxidoreductase may be a peroxidase, a laccase or a lipoxygenase. The glucose oxidase may be derived from a strain of Aspergillus or Penicillium, particularly A. niger, P. notatum, P. amagasakiense or P. vitale. The hexose oxidase may be one described in EP 833563. The pyranose oxidase may be one described in WO 9722257, e.g. derived from Trametes, particularly T. hirsuta. The galactose oxidase may be one described in WO 0050606.

EXAMPLES

Example 1. Baking procedure Tegral Allegro cake

Baking examples on the following Novamyl ^® variants are included in this example:

Table 1 Variants, Mutations, Report number and Study number

The following recipe was used:

^* commercially available from Puratos NV/SA, Groot-Bijgaarden, Belgium

Procedure

The ingredients were scaled into a mixing bowl and mixed using an industrial mixer (e.g. Bjørn/Bear AR 5 A Varimixer ^® ) with a suitable paddle speed. 300 g of the dough was poured into forms. The cakes were baked in a suitable oven (e.g. Sveba Dahlin deck oven) for 45 min. at 180 ⁰ C. The cakes were allowed to cool down at room temperature for 1 hour. The volumes of the cakes were determined when the cakes had cooled down, using the rape seed displacement method. The cakes were packed under nitrogen in sealed plastic bags and stored at room temperature until analysis.

The cakes were evaluated on day 1 , 7 and 14, two cakes were used at each occasion, and three slices of cakes were analyzed from each cake.

The cohesiveness and hardness of the cakes was evaluated using the texture profile analysis (TPA) with TA-XTplus texture analyzer and the water mobility was characterized by low field NMR.

The Texture profile analysis (TPA) was performed as described in Bourne M. C. (2002) 2. ed., Food Texture and Viscosity: Concept and Measurement. Academic Press.

The mobility of free water was determined as described by P. L. Chen, Z. Long, R. Ruan and T. P. Labuza, Nuclear Magnetic Resonance Studies of water Mobility in Bread during Storage. Lebensmittel Wissenschaft und Technologie 30, 178-183 (1997). The mobility of free water has been described in literature to correlate to moistness of bread crumb.

Result

The volume of the cakes can be found in table 2, the volume of cakes without any enzyme is set to 100%.

Compared to cakes with no addition of enzymes the volume of the cakes is not affected by the addition of the reference enzyme (SEQ ID NO.: 1 ) nor by the addition of variants hereof, i.e. the cakes did not collapse upon addition of enzyme.

The cohesiveness of the cakes decreased with storage time. The addition of variants of Novamyl ^® delayed this decrease as can be seen in Table 3.

Table 3 Change in Cohesiveness of cakes with time measured with the TPA method

The hardness of the cakes increased with storage time. The addition of variants of Novamyl ^® delayed this increase in hardness as can be seen in Table 4.

Table 4 Change in Hardness [g] of cakes with time measured with the TPA method

The free water mobility is correlated with the moist perception of the cake crumb, it decreases with time. The addition of the Novamyl ^® variants increased the mobility compared to the control, indicating that the amylases were able to keep the cakes more moist. Results are listed in Table 5.

Table 5 Change in Free water mobility [μs] of cakes with time measured with low field NMR