# Patent

## Title
Method of preparing a dough-based product

## Abstract

Dough with a high sucrose content (such as cake dough) tends to inhibit the activity of an anti-staling amylase such as Novamyl®, making it less effective to prevent the staling of dough-based products with high sucrose content such as cakes. A good anti-staling effect in cakes can be achieved by using a carefully selected anti-staling amylase with certain properties. Analysis of a 3D structure of Novamyl® shows that sucrose may inhibit by binding in the active site. Sucrose docks into the active site of Novamyl® differently from the substrate or inhibitor in published models 1QHO and 1QHP. This finding is used to design sucrose-tolerant variants.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/EP2008/056999 filed Jun. 5, 2008, which claims priority or the benefit under 35 U.S.C. 119 of European application no. 07109793.5 filed Jun. 7, 2007 and U.S. provisional application No. 60/944,543 filed Jun. 18, 2007, the contents of which are fully incorporated herein by reference.
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
U.S. Pat. No. 3,026,205 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). U.S. Pat. No. 6,162,628 describes Novamyl® variants and their use for the same purpose. Three-dimensional structures of Novamyl® are published in U.S. Pat. No. 6,162,628 and in the Protein Data Bank (available at http://www.rcsb.org/pdb/) with identifiers 1QHO and 1QHP, 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 D261G 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 D261G 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 D261G 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 1EZ, UK and the protein part of the x-ray structure 1QHO.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 1QHO.pdb are given in FIG. 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 U.S. Pat. No. 6,162,628.
For Novamyl® (SEQ ID NO: 1), a 3D structure including a substrate or inhibitor as described in U.S. Pat. No. 6,162,628 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 U.S. Pat. No. 6,162,628 or in this specification, e.g. the variant F188L+D261G+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 if (26 Mar. 1987); J. Greer, Proteins: Structure, Function and Genetics, 7:317-334 (1990); or Example 1 of WO 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 1EZ, UK and the protein part of the x-ray structure 1 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 1QHO.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° 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 1QHO and the sucrose docking in FIG. 1, the following residues are within 4 Å: 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 D261G 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/S/V/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/GN/T/S, F188H/L/I/T/G/V, D190E/Q/G, P191S/N, A192S/T, F194S/L/Y, L196F, Y360F/I/N, N371K/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, I, 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 U.S. Pat. No. 6,162,628.
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, D261G, T288P, I290V, N375S
F194Y, D261G, T288P, N375S
I15T, P191S, D261G, T288P, N375S, S640I
Y89F, P191S, D261G, T288P
I15T, Y89F, P191S, D261G, T288P
Y89F, F194Y, D261G, T288P
Y89F, D261G, T288P, N375S
Y89F, P1915, F194Y, D261G, T288P
Y89F, P1915, D261G, T288P, N3755
Y89F, P191S, D261G, T288P, Y360N
Y89F, P191S, D261G, T288P, Y360F
Y89F, W93Y, P191S, D261G, T288P
Y89F, W93F, P191S, D261G, T288P
Y89F, P1915, F194Y, D261G, T288P, N3755
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, D261G, 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 60° 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° A activity at 10% sugar, more than 10% activity at 20% sucrose, or more than 4% activity at 40° A 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° C. at pH 5.7 (50 mM Na-acetate, 1 mM CaCl2) 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








Variant
Substitutions





Ref
F188L, D261G, T288P


A
Y89F, D261G, T288P, I290V, N375S


B
F194Y, D261G, T288P, N375S


C
I15T, P191S, D261G, T288P, N375S, S640I


D
Y89F, P191S, D261G, T288P


E
I15T, Y89F, P191S, D261G, T288P


F
Y89F, F194Y, D261G, T288P


G
Y89F, D261G, T288P, N375S


H
Y89F, P191S, F194Y, D261G, T288P


I
Y89F, P191S, D261G, T288P, N375S


J
Y89F, P191S, D261G, T288P, Y360N


K
Y89F, P191S, D261G, T288P, Y360F


L
Y89F, W93Y, P191S, D261G, T288P


M
Y89F, W93F, P191S, D261G, T288P


N
Y89F, P191S, F194Y, D261G, T288P, N375S









Recipe

The following recipe was used:














%



















Tegral
100 



Allegro mix*



Pasteurized
50



whole egg



Butter
50



Enzymes
According to trial. In the




range 0-25 mg protein




enzyme/kg flour.







*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° 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 and 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.







TABLE 2





Volume of cakes as a function of added enzyme






















0 mg/kg
5 mg/kg
10 mg/kg
25 mg/kg







Ref
100

98
97



A
100

95
97



C
100
96
96



E
100
98
100


















0 mg/kg
3 mg/kg
4 mg/kg
5 mg/kg







M
100
94
97
95



B
100
95
94
93



D
100
95
96
96



















0 mg/kg
3 mg/kg
6 mg/kg
9 mg/kg
12 mg/kg







F
100
99
99
101
103



















0 mg/kg
2 mg/kg
4 mg/kg
6 mg/kg
8 mg/kg







D
100
96
97
94



H
100
97
93
94



N
100
95
95
95



J
100
93
93
92



K
100
94
92
90



I
100
94
97
96



L
100
99
98
97



G
100

93
92
94










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










Dosage



















Day 1
Day 7
Day 14







No enzyme
—
0.46
0.36
0.32



Ref
10 mg/kg
0.45
0.42
0.39




25 mg/kg
0.45
0.42
0.42



A
10 mg/kg
0.44
0.40
0.38




25 mg/kg
0.43
0.42
0.40



No enzyme
—
0.52
0.43
0.39



Ref
10 mg/kg
0.56
0.49
0.44




25 mg/kg
0.57
0.52
0.49



H
 2 mg/kg
0.56
0.49
0.45




 4 mg/kg
0.58
0.51
0.48




 6 mg/kg
0.56
0.51
0.48



N
 2 mg/kg
0.54
0.47
0.45




 4 mg/kg
0.57
0.53
0.47




 6 mg/kg
0.56
0.52
0.48



D
 2 mg/kg
0.57
0.48
0.45




 4 mg/kg
0.55
0.50
0.46




 6 mg/kg
0.55
0.49
0.46









Day 1
Day 7
Day 15







No enzyme
—
0.52
0.42
0.36



Ref
10 mg/kg
0.54
0.48
0.44




25 mg/kg
0.56
0.50
0.46



J
 2 mg/kg
0.54
0.46
0.41




 4 mg/kg
0.53
0.47
0.42




 6 mg/kg
0.54
0.49
0.44



K
 2 mg/kg
0.54
0.46
0.40




 4 mg/kg
0.53
0.48
0.43




 6 mg/kg
0.53
0.48
0.43









Day 1
Day 7
Day 14







No enzyme
—
0.50
0.47
0.43



Ref
10 mg/kg
0.54
0.51
0.48




25 mg/kg
0.55
0.55
0.52



I
 2 mg/kg
0.53
0.51
0.48




 4 mg/kg
0.54
0.53
0.50




 6 mg/kg
0.51
0.50
0.49



L
 2 mg/kg
0.52
0.49
0.47




 4 mg/kg
0.54
0.51
0.49




 6 mg/kg
0.53
0.52
0.51









Day 1
Day 6
Day 14







No enzyme
—
0.46
0.38
0.35



Ref
10 mg/kg
0.49
0.43
0.42




25 mg/kg
0.48
0.44
0.44



E
 5 mg/kg
0.47
0.42
0.39




10 mg/kg
0.47
0.41
0.41









Day 1
Day 7
Day 14







No enzyme
—
0.43
0.33
0.28



Ref
10 mg/kg
0.45
0.38
0.35




25 mg/kg
0.45
0.40
0.37



G
 4 mg/kg
0.46
0.36
0.34




 6 mg/kg
0.45
0.40
0.37




 8 mg/kg
0.44
0.38
0.37









Day 3
Day 7
Day 14







No enzyme
—
0.45
0.41
0.37



Ref
10 mg/kg
0.50
0.47
0.45




25 mg/kg
0.51
0.49
0.48



D
 3 mg/kg
0.48
0.46
0.43




 4 mg/kg
0.50
0.48
0.45




 5 mg/kg
0.50
0.47
0.45



B
 3 mg/kg
0.50
0.48
0.45




 4 mg/kg
0.51
0.49
0.47




 5 mg/kg
0.52
0.49
0.47



M
 3 mg/kg
0.50
0.48
0.45




 4 mg/kg
0.50
0.47
0.45




 5 mg/kg
0.51
0.49
0.46









Day 1
Day 7
Day 14







No enzyme
—
0.42
0.34
0.31



Ref
10 mg/kg
0.44
0.40
0.38




25 mg/kg
0.44
0.43
0.40



F
 3 mg/kg
0.43
0.39
0.34




 6 mg/kg
0.43
0.40
0.35




 9 mg/kg
0.43
0.40
0.35




12 mg/kg
0.43
0.43
0.36



No enzyme
—
0.40
0.32
0.28



Ref
10 mg/kg
0.42
0.38
0.35




25 mg/kg
0.44
0.40
0.40



C
 5 mg/kg
0.43
0.36
0.33




10 mg/kg
0.42
0.36
0.34










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










Dosage



















Day 1
Day 7
Day 14







No enzyme
—
586
875
1093



Ref
10 mg/kg
597
768
955




25 mg/kg
603
732
957



A
10 mg/kg
612
801
953




25 mg/kg
602
731
827



No enzyme
—
515
811
1046



Ref
10 mg/kg
438
651
762




25 mg/kg
530
687
869



H
 2 mg/kg
521
809
892




 4 mg/kg
573
773
908




 6 mg/kg
523
702
797



N
 2 mg/kg
516
751
898




 4 mg/kg
484
668
872




 6 mg/kg
565
753
821



D
 2 mg/kg
556
794
1015




 4 mg/kg
524
715
953




 6 mg/kg
521
716
876









Day 1
Day 7
Day 15







No enzyme
—
484
738
956



Ref
10 mg/kg
518
753
976




25 mg/kg
559
714
868



J
 2 mg/kg
576
823
1058




 4 mg/kg
549
761
908




 6 mg/kg
601
798
1092



K
 2 mg/kg
584
889
1081




 4 mg/kg
551
811
993




 6 mg/kg
564
782
935









Day 1
Day 7
Day 14







No enzyme
—
760
1064
1397



Ref
10 mg/kg
748
943
1175




25 mg/kg
747
937
1101



I
 2 mg/kg
821
1085
1394




 4 mg/kg
829
1089
1315




 6 mg/kg
756
947
1071



L
 2 mg/kg
795
1033
1363




 4 mg/kg
808
1062
1164




 6 mg/kg
770
1020
1160









Day 1
Day 6
Day 14







No enzyme
—
602
984
1172



Ref
10 mg/kg
636
897
1035




25 mg/kg
627
851
1018



E
 5 mg/kg
660
987
1176




10 mg/kg
644
894
1023









Day 1
Day 7
Day 14







No enzyme
—
480
966
1284



Ref
10 mg/kg
593
961
1106




25 mg/kg
535
843
1079



G
 4 mg/kg
692
985
1336




 6 mg/kg
637
1095
1287




 8 mg/kg
616
1074
1214









Day 3
Day 7
Day 14







No enzyme
—
595
827
981



Ref
10 mg/kg
641
829
1020




25 mg/kg
589
657
809



D
 3 mg/kg
599
766
987




 4 mg/kg
678
801
1009




 5 mg/kg
652
847
1018



B
 3 mg/kg
627
767
973




 4 mg/kg
639
780
923




 5 mg/kg
578
768
960



M
 3 mg/kg
679
772
1014




 4 mg/kg
601
814
1005




 5 mg/kg
646
789
957









Day 1
Day 7
Day 14







No enzyme
—
616
1038
1255



Ref
10 mg/kg
682
912
1155




25 mg/kg
640
844
1077



F
 3 mg/kg
683
843
1131




 6 mg/kg
626
831
1131




 9 mg/kg
644
785
947




12 mg/kg
571
903
992



No enzyme
—
469
724
952



Ref
10 mg/kg
474
672
956




25 mg/kg
515
661
873



C
 5 mg/kg
546
800
1050




10 mg/kg
546
753
993










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










Dosage



















Day 1
Day 7
Day 14







No enzyme
—
7703
5339
4138



Ref
10 mg/kg
7755
5670
4434




25 mg/kg
7502
5751
4366



A
10 mg/kg
7809
6124
4839




25 mg/kg
7753
6175
4811



No enzyme
—
6464
5577
4618



Ref
10 mg/kg
7009
5507
4831




25 mg/kg
7109
5897
5004



H
 2 mg/kg
6819
5521
4743




 4 mg/kg
7171
5906
5157




 6 mg/kg
7340
6196
5131



N
 2 mg/kg
7034
5697
4955




 4 mg/kg
6897
6024
5067




 6 mg/kg
7196
6184
5368



D
 2 mg/kg
6936
5753
4910




 4 mg/kg
7169
5787
5031




 6 mg/kg
6876
5983
5228









Day 1
Day 7
Day 15







No enzyme
—
7358
5537
4385



Ref
10 mg/kg
7606
5735
4830




25 mg/kg
7456
5648
4977



J
 2 mg/kg
7502
5638
4818




 4 mg/kg
7575
5663
5081




 6 mg/kg
7853
6093
5193



K
 2 mg/kg
7714
5649
4845




 4 mg/kg
7999
5766
5022




 6 mg/kg
8029
6291
5312









Day 1
Day 7
Day 14







No enzyme
—
7004
4887
4266



Ref
10 mg/kg
7354
5331
4567




25 mg/kg
7489
5486
4856



I
 2 mg/kg
7569
5374
4766




 4 mg/kg
7596
5241
4925




 6 mg/kg
7710
5673
5110



L
 2 mg/kg
7495
5101
4647




 4 mg/kg
7433
5228
4821




 6 mg/kg
7577
5342
4988









Day 1
Day 6
Day 14







No enzyme
—
7137
5308
4130



Ref
10 mg/kg
7523
6554
4751




25 mg/kg
7449
5808
4778



E
 5 mg/kg
7387
6227
4725




10 mg/kg
7288
5670
4786









Day 1
Day 7
Day 14







No enzyme
—
6954
5037
4152



Ref
10 mg/kg
7307
5384
4278




25 mg/kg
7461
5323
4440



G
 4 mg/kg
7240
5254
4435




 6 mg/kg
7077
5371
4659




 8 mg/kg
7207
5634
4893









Day 3
Day 7
Day 14







No enzyme
—
6322
5101
4458



Ref
10 mg/kg
6037
5452
4769




25 mg/kg
6882
5515
5304



D
 3 mg/kg
6604
5446
4952




 4 mg/kg
6373
5466
4550




 5 mg/kg
6460
5507
4891



B
 3 mg/kg
7126
5896
5334




 4 mg/kg
7134
5774
5341




 5 mg/kg
7117
6014
5203



M
 3 mg/kg
7054
5917
5189




 4 mg/kg
6449
5362
4833




 5 mg/kg
7057
5501
5173









Day 1
Day 7
Day 14







No enzyme
—
7285
4791
3766



Ref
10 mg/kg
7410
5437
4542




25 mg/kg
7367
4959
4511



F
 3 mg/kg
7114
5462
4593




 6 mg/kg
7457
5698
4648




 9 mg/kg
7298
5309
4293




12 mg/kg
7528
5938
4599



No enzyme
—
6722
4344
3477



Ref
10 mg/kg
6816
4761
3769




25 mg/kg
7008
5109
3923



C
 5 mg/kg
6606
5623
4265




10 mg/kg
6746
4782
3690

## Claims

1. 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 D261G 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.

2. The method of claim 1, wherein the at least one additional amino acid alteration comprises at least two amino acid alterations.

3. The method of claim 1, wherein the at least one additional amino acid alteration comprises at least four amino acid alterations.

4. The method of claim 1, wherein the at least one additional amino acid alteration comprises at least six amino acid alterations.

5. The method of claim 1, wherein the at least one additional amino acid alteration comprises a combination of two or more amino acid alterations in positions compared to SEQ ID NO: 1, selected from the group of position combinations consisting of:
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.

6. The method of claim 1, wherein the at least one amino acid alteration comprises one or more of the following substitutions, alone or in combination:
Y89F,W
W93F,Y
P191S,N
F194Y,S,L
Y360F,I,N
N375S,T.

7. The method of claim 1, wherein the amino acid alteration is substitution with a larger or smaller amino acid residue.

8. The method of claim 1, wherein the alteration is insertion of 1-4 amino acid residues at the N- or C-side of the specified residue.

9. The method of claim 1, wherein the polypeptide comprises a substitution I15T/S/V/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/G/V/T/S, F188H/L/I/T/G/V, D190E/Q/G, A192G/S/T/Q/R, F194S/L/Y, L196F, N371K/R/F/Y/Q or D372E/Q/S/T/A, a deletion of 191 or 192 or an insertion of Ala after 192.

10. The method of claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1 with one of the following combinations of amino acid alterations:
Y89F, D261G, T288P, I290V, N375S
F194Y, D261G, T288P, N375S
I15T, P191S, D261G, T288P, N375S, S6401
Y89F, P191S, D261G, T288P
I15T, Y89F, P191S, D261G, T288P
Y89F, F194Y, D261G, T288P
Y89F, D261G, T288P, N375S
Y89F, P191S, F194Y, D261G, T288P
Y89F, P191S, D261G, T288P, N3755
Y89F, P191S, D261G, T288P, Y360N
Y89F, P191S, D261G, T288P, Y360F
Y89F, W93Y, P191S, D261G, T288P
Y89F, W93F, P191S, D261G, T288P
Y89F, P191S, F194Y, D261G, T288P, N375S.

11. An isolated polypeptide which:
has amylase activity which is less inhibited by sucrose than the amylase activity of SEQ ID NO: 1,
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 D261G 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.

12. The polypeptide of claim 11, wherein the at least one additional amino acid alteration comprises at least two amino acid alterations.

13. The polypeptide of claim 11, wherein the at least one additional amino acid alteration comprises at least four amino acid alterations.

14. The polypeptide of claim 11, wherein the at least one additional amino acid alteration comprises at least six amino acid alterations.

15. The polypeptide of claim 11, wherein the at least one additional amino acid alteration comprises a combination of two or more amino acid alterations in positions compared to SEQ ID NO: 1, selected from the group of position combinations consisting of:
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.

16. The polypeptide of claim 11, wherein the at least one amino acid alteration comprises one or more of the following substitutions, alone or in combination:
Y89F,W
W93F,Y
P191S,N
F194Y,S,L
Y360F,I,N
N375S,T.

17. The polypeptide of claim 11, wherein the alteration is substitution with a larger or smaller amino acid residue.

18. The polypeptide of claim 11, which comprises insertion of 1-4 amino acid residues at the N- or C-side of the specified residue.

19. The polypeptide of claim 11, which has the amino acid sequence of SEQ ID NO: 1 with one of the following combinations of amino acid alterations
Y89F, D261G, T288P, 1290V, N375S
F194Y, D261G, T288P, N375S
I15T, P191S, D261G, T288P, N375S, S6401
Y89F, P191S, D261G, T288P
I15T, Y89F, P191S, D261G, T288P
Y89F, F194Y, D261G, T288P
Y89F, D261G, T288P, N375S
Y89F, P191S, F194Y, D261G, T288P
Y89F, P191S, D261G, T288P, N3755
Y89F, P191S, D261G, T288P, Y360N
Y89F, P191S, D261G, T288P, Y360F
Y89F, W93Y, P191S, D261G, T288P
Y89F, W93F, P191S, D261G, T288P
Y89F, P191S, F194Y, D261G, T288P, N375S.

20. A method of preparing a polypeptide, comprising
providing a parent polypeptide having an amino acid sequence and having maltogenic alpha-amylase activity,
selecting at least one amino acid residue in the sequence corresponding to Y360 in SEQ ID NO: 1 or selecting a combination of two or more amino acid residues in positions corresponding to SEQ ID NO: 1, selected from the group of combinations consisting of:

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
substituting or deleting the selected residue(s) or inserting one or more residues adjacent to the selected residue(s) to obtain an altered amino acid sequence,
preparing an altered polypeptide having the altered amino acid sequence,
testing the amylase activity and the sugar tolerance of the altered polypeptide, and selecting a polypeptide which has amylase activity and has higher sucrose tolerance than the parent polypeptide.