WO2006012899A1 - Maltogenic alpha-amylase variants - Google Patents

Maltogenic alpha-amylase variants Download PDF

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Publication number
WO2006012899A1
WO2006012899A1 PCT/DK2005/000506 DK2005000506W WO2006012899A1 WO 2006012899 A1 WO2006012899 A1 WO 2006012899A1 DK 2005000506 W DK2005000506 W DK 2005000506W WO 2006012899 A1 WO2006012899 A1 WO 2006012899A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
substitution
amino acid
maltose
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Application number
PCT/DK2005/000506
Other languages
French (fr)
Inventor
Lars Beier
Allan Svendsen
Torben Vedel Borchert
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Novozymes A/S
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Publication date
Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to US11/572,272 priority Critical patent/US20080032024A1/en
Priority to EP05762147A priority patent/EP1797179A1/en
Priority to CA002575875A priority patent/CA2575875A1/en
Publication of WO2006012899A1 publication Critical patent/WO2006012899A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01133—Glucan 1,4-alpha-maltohydrolase (3.2.1.133), i.e. maltogenic alpha-amylase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to the preparation of variants of a parent maltogenic alpha- amylase, where hydrolysis products of said variants having a modified of maltose-to-glucose ratio as compared to hydrolysis products of the parent maltogenic alpha-amylase. It also re ⁇ lates to a polynucleotide encoding such variants and to the use of the variants in the produc ⁇ tion of ethanol, beer, dough, maltose syrup and baked products.
  • Maltogenic alpha-amylase (EC 3.2.1.1) is known to be useful, e.g., for production of ethanol from granular starch by fermentation (WO 2003068976) and for retarding the staling of bread (WO 9104669).
  • One maltogenic alpha-amylase is the commercial product Novamyl ® de ⁇ scribed in EP 120693 B1. Variants of Novamyl are known from WO 9943794.
  • Maltogenic al- pha-amylases are known to hydrolyze starch with formation of maltose as the main product together with a minor amount glucose.
  • the inventors realized that in some applications the control of the maltose-to-glucose ratio is of great importance. Particularly for ethanol production from granular starch by fermen ⁇ tation, it may be an advantage to form a larger amount of glucose which is more readily fermentable than maltose. Particularly for production of maltose syrups glucose is an undesired product, and hence it of interest to increase the maltose-to-glucose ratio. They then developed a method of constructing such variants of based on the three-dimensional structure of a parent maltogenic alpha-amylase.
  • the invention provides a method of constructing a variant polypeptide, comprising: a) providing a parent maltogenic alpha-amylase having an amino acid sequence and a three-dimensional structure which includes a cleavage point and a substrate with at least three monosaccharide moieties at the reducing side of the cleavage point, b) selecting an amino acid residue having a C-alpha atom located ⁇ 10 A from an atom in the substrate, c) substituting or deleting the selected residue to obtain a modified amino acid se ⁇ quence, d) preparing a polypeptide having the modified sequence, e) testing the modified polypeptide by incubating it with starch and analyzing the reac ⁇ tion product, and f) selecting a modified polypeptide which has the ability to hydrolyze starch and wherein the hydrolysis product has a modified maltose-to-glucose ratio compared to an hy ⁇ drolysis product made with the parent maltogenic alpha-amy
  • the parent maltogenic alpha-amylase and the substrate may for the purpose of steps a), b), and c) be provided in the form of a computer model.
  • the invention also provides a variant polypeptide which a) has an amino acid sequence having more than 80 % identity to SEQ ID NO: 1 , b) compared to SEQ ID NO: 1 has a different amino acid residue at a position corre ⁇ sponding to W93, T134, G172, N176, D178, F188, D190, D198, I227 V230, K231 , H232, F233, Y258, G259, D260, D261 , P262, T264, N266, F284, T288 or M330 or a deletion corre ⁇ sponding to 191-195, and c) has the ability to hydrolyze starch to form an product having a modified maltose-to- glucose ratio than a product made with the polypeptide of SEQ ID NO: 1.
  • the invention provides a polynucleotide encoding the polypeptide and uses of the polypeptide in production of ethanol from granular starch by fermentation, in production of maltose syrup, and in the production of dough and baked products.
  • the maltogenic alpha-amylase (EC 3.2.1.133) may have the amino acid sequence shown in SEQ ID NO: 1 (in the following referred to as Novamyl) with a 3D structure including a substrate as described in US 6162628 and found in the Protein Data Bank with the identifier
  • the maltogenic alpha-amylase may be a Novamyl variant described in US
  • a 3D structure of such 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
  • An amino acid residue is selected which has a C-alpha atom located ⁇ 10 A from an atom of the substrate.
  • the following residues are selected by this criterion: 13, 15, 18, 43-44, 70, 72-73, 77-78, 82, 86-94, 97, 127-136, 143, 174-180, 183-184, 187-198, 226-233, 255-267, 270, 282-289, 291-292, 299, 307, 324, 327-331 , 360, 370-376.
  • the selection may in particular be for residues ⁇ 10 A from an atom in monosaccha ⁇ ride (glucose) moieties +1 , +2 and +3 at the reducing side of the cleavage point.
  • the moieties are denoted j, k and I, and this lead to selection of the following residues: 13, 70, 73, 90, 92-93, 127-132, 174-180, 183-184, 187-191 , 196, 226-233, 255-267, 270, 282-289, 291- 292, 299, 307, 324, 327-331 , 371-372, 375-376.
  • the selected residue may be substituted so as to push the substrate away or block for it presents in position +1, +2 and +3 etc by making the residues larger at a position corre ⁇ sponding to G172, D178, T189, K231 , H232, Y258, G259, D260, T264, N266 or T288 in No- vamyl (SEQ ID NO: 1), e.g. a substitution corresponding to G172V, T189M, K231R, H232Y, Y258W, G259A/H/Y, T264Y/Q/F, N266Y or T288Y/Q/F/P.
  • the substitution may serve to remove hydrogen bonding or van der Waals contact to the substrate at position +1 , +2 and +3. This may be done by substituting with a smaller resi ⁇ due at a position corresponding to W93, T134, D178, D190, D198, [227, K231, H232, F233, Y258, D260, D261 , T264 or T288 of SEQ ID NO: 1 , particularly a substitution corresponding to W93S/G/V/T/M/E, T134A, D178L/M/T/V, D190G, D198G, I227V, K231L/M, H232L/M, F233S, Y258L/M/T/V, D260L/M/T/V, D261G, T264A ⁇ / or T288A/V.
  • a hydrophilic or electrically charged (positive or negative) residue may be substituted with a hydrophobic residue, particularly at a position corresponding to T134, D178, D190, D198, K231 , H232 or D261 , more particularly a substitution corresponding to T134A, D178V, D190G, D198G, K231 L/M, H232L/M or D261G.
  • substitution or deletion may serve to change indirectly the contact by changing the residues next to the substrate contact residues, particularly a residue corre ⁇ sponding to W93, N176, 191 , 192, 193, 194,195, V230, P262, F284 or M330 in Novamyl, e.g. a substitution corresponding to W93E/G/M/V/T/S, N176L, V230G, F284Y or M330I or a dele ⁇ tion of residues corresponding to 191 , 192, 193, 194, and 195.
  • a substitution corresponding to W93E/G/M/V/T/S, N176L, V230G, F284Y or M330I or a dele ⁇ tion of residues corresponding to 191 , 192, 193, 194, and 195.
  • Amino acid residues are ranked as follows from smallest to largest: (an equal sign in- dicates residues with sizes that are practically indistinguishable):
  • amino acid residues are considered to be hydrophobic: G, A, V, L, I, P, F, W and C as part of a disulfide bridge.
  • the variant of the invention is able to hydrolyze starch to form a product having a modified maltose-to-glucose ratio as compared to a product made with the polypeptide of SEQ ID NO: 1.
  • the starch hydrolysis may be carried out by the following procedures described in the examples.
  • the variants of the invention may show an increased ratio of glucose to maltose (DP1/DP2) or an increased ratio of DP1/(DP1-4) or an increased ratio of maltose to glucose (DP2/DP1) or an increased ratio of (DP1-4)/DP1.
  • Starch is in the context of the present invention intended to include starch as well as breakdown products of starch, such as amylopectin, or amylose, or maltooligosaccharides.
  • the polypeptide of the invention may have identities to the disclosed sequences of at least 80 %, particularly at least 85 % or at least 90 %, e.g. at least 91%, or 92%, or 93%, or 94%, or at least 95 %, such as 96%, or 97%, or 98%, or 99%.
  • alignments of sequences and calculation of identity scores may be done using a Needleman-Wunsch alignment (i.e. global alignment), useful for both protein and DNA alignments.
  • the default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively.
  • the penalty for the first residue in a gap is -12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for proteins and -4 for DNA.
  • Alignment is from the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology, 183:63-98).
  • the variant of the invention may be used in various known applications for amylases, e.g. production of ethanol, beer, dough, maltose syrup and baked products.
  • the variant may be used in a process comprising treating granular starch with the variant and fermentation into ethanol.
  • the treatment of the granular starch serves to produce a hydrolysis product which includes a significant amount of glucose.
  • the fermentation to produce ethanol may be simultaneous with the granular starch treatment, or the starch may first be hy- drolyzed followed by fermentation of the hydrolysate.
  • the process may be performed as de ⁇ scribed in WO 2003068976.
  • the variant may be used in mashing, i.e. in the process of converting starch from milled malt and solid adjuncts into fermentable and unfermentable sugars to produce wort.
  • mashing involves incubating the variant with milled malt and solid adjuncts in water to hydro- lyze the starch.
  • the variant may be added to dough for making baked products such as bread. Addi ⁇ tion of the variant may serve to retard staling of the baked product.
  • the addition to dough may be done as described in WO 9104669.
  • the variant may be used for commercial production of maltose, which today starts from liquefied starch (DE ⁇ 10), which is subsequently treated simultaneously with debranching enzymes (pullulanase or isoamylase) and maltose-forming enzymes (maltogenic ⁇ -amylase or ⁇ -amylase) at a temperature around 60 0 C.
  • debranching enzymes pulseulanase or isoamylase
  • maltose-forming enzymes maltogenic ⁇ -amylase or ⁇ -amylase
  • Maltose is used in large quantities as syrups in e.g. the confectionary industry and as a sweetening agent in the food industry.
  • Mal ⁇ tose syrups have among other capacities reduced browning capacity, a resistance to moisture absorption and to crystallization making maltose syrups suited for e.g. frozen dessert formula ⁇ tions, hard candy, jams, and jellies.
  • a maltogenic alpha-amylase with an increased mal ⁇ tose-to-glucose ratio would be an advantage in the production of maltose syrups.
  • variants were prepared, each having the sequence of SEQ ID NO: 1 with the indicated substitutions. Each variant was tested by incubating it with maltodextrin (DE 11) by application of the following procedure: - Prepare a 30% (w/w) maltodextrin solution (DE 11) in 5OmM Na-acetate, 1mM
  • thermomixer 1 ml substrate is added to 1.5 ml tubes with lid and membrane, and samples are pre ⁇ heated to 6O 0 C on a thermomixer.
  • the carbohydrate profile was determined by chromatography by applying standard procedures, e.g. as described in Norman, B. E. in James N.Bemiller, David J. Manners, and Robert J. Sturgeon (eds), Methods in Carbohydrate Chemistry, Volume X. John Wiley & Sons, Inc., New York, pp. 231-239, 1994.
  • d (191-195) indicates a deletion of the amino acids corresponding to position 191 , 192, 193, 194, and 195.
  • a number of purified variants (each having the sequence of SEQ ID NO: 1 with the in ⁇ dicated substitutions) were prepared by standard purification techniques, see e.g. Beier et al.: "Conversion of the maltogenic alpha-amylase Novamyl into a CGTase” in Protein Engineering, vol. 13 no. 7 pp. 509-513, 2000.
  • Each variant was tested by incubating it with maltodextrin (DE 11) at 60 0 C and pH 5.5 for 42 hours as described in Example 1. Either an amount of 0.81 micro g (variants marked with [1]) or 1.62 micro g (variants marked with [2]) of the variant was added, and further 1.2 mg/g DS of the commercially available pullulanase Promozyme® (EP 63909) was added. Novamyl without substitutions was included as reference. The results were as follows:
  • a number of purified variants (each having the sequence of SEQ ID NO: 1 with the in ⁇ dicated substitutions) were prepared by standard purification techniques, see e.g. Beier et al.: "Conversion of the maltogenic alpha-amylase Novamyl into a CGTase” in Protein Engineering, vol. 13 no. 7 pp. 509-513, 2000.
  • a single variant was dosed at a higher amount, namely 38.2 micro g (variant marked with [3]).
  • a number of purified variants (each having the sequence of SEQ ID NO: 1 with the in ⁇ dicated substitutions) were prepared by standard purification techniques, see e.g. Beier et al.: "Conversion of the maltogenic alpha-amylase Novamyl into a CGTase” in Protein Engineering, vol. 13 no. 7 pp. 509-513, 2000.
  • thermomixer 1 mL substrate is added to 1.5 mL tubes with lid and membrane, and samples are preheated to 6O 0 C on a thermomixer.
  • MiIIi-Q is added with 1-2 drops of 1 M HCI (pH must be less than 3 to inactive the amylase).
  • the carbohydrate profile was determined by chromatography by applying standard procedures, e.g. as described in Norman, B. E. in James N.Bemiller, David J. Manners, and Robert J. Sturgeon (eds), Methods in Carbohydrate Chemistry, Volume X. John Wiley & Sons, Inc., New York, pp. 231-239, 1994. Novamyl without substitutions was included as reference. The results were as follows:
  • Y258W was dosed 5 mg enzyme protein/kg flour for all three tests.
  • W93S was dosed 3 mg enzyme protein/kg flour for all three tests.

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Abstract

The inventors realized that in some applications the control of the maltose-to-glucose ratio is of great importance. Particularly for ethanol production from granular starch by fermen-tation, it may be an advantage to form a larger amount of glucose which is more readily fermentable than maltose. Particularly for production of maltose syrups glucose is an undesired product, and hence it of interest to increase the maltose-to-glucose ratio. They then developed a method of constructing such variants of based on the three-dimensional structure of a parent maltogenic alpha-amylase.

Description

MALTOGENIC ALPHA-AMYLASE VARIANTS
FIELD OF THE INVENTION
The invention relates to the preparation of variants of a parent maltogenic alpha- amylase, where hydrolysis products of said variants having a modified of maltose-to-glucose ratio as compared to hydrolysis products of the parent maltogenic alpha-amylase. It also re¬ lates to a polynucleotide encoding such variants and to the use of the variants in the produc¬ tion of ethanol, beer, dough, maltose syrup and baked products.
BACKGROUND OF THE INVENTION
Maltogenic alpha-amylase (EC 3.2.1.1) is known to be useful, e.g., for production of ethanol from granular starch by fermentation (WO 2003068976) and for retarding the staling of bread (WO 9104669). One maltogenic alpha-amylase is the commercial product Novamyl® de¬ scribed in EP 120693 B1. Variants of Novamyl are known from WO 9943794. Maltogenic al- pha-amylases are known to hydrolyze starch with formation of maltose as the main product together with a minor amount glucose.
SUMMARY OF THE INVENTION
The inventors realized that in some applications the control of the maltose-to-glucose ratio is of great importance. Particularly for ethanol production from granular starch by fermen¬ tation, it may be an advantage to form a larger amount of glucose which is more readily fermentable than maltose. Particularly for production of maltose syrups glucose is an undesired product, and hence it of interest to increase the maltose-to-glucose ratio. They then developed a method of constructing such variants of based on the three-dimensional structure of a parent maltogenic alpha-amylase.
Accordingly, the invention provides a method of constructing a variant polypeptide, comprising: a) providing a parent maltogenic alpha-amylase having an amino acid sequence and a three-dimensional structure which includes a cleavage point and a substrate with at least three monosaccharide moieties at the reducing side of the cleavage point, b) selecting an amino acid residue having a C-alpha atom located <10 A from an atom in the substrate, c) substituting or deleting the selected residue to obtain a modified amino acid se¬ quence, d) preparing a polypeptide having the modified sequence, e) testing the modified polypeptide by incubating it with starch and analyzing the reac¬ tion product, and f) selecting a modified polypeptide which has the ability to hydrolyze starch and wherein the hydrolysis product has a modified maltose-to-glucose ratio compared to an hy¬ drolysis product made with the parent maltogenic alpha-amylase.
The parent maltogenic alpha-amylase and the substrate may for the purpose of steps a), b), and c) be provided in the form of a computer model.
The invention also provides a variant polypeptide which a) has an amino acid sequence having more than 80 % identity to SEQ ID NO: 1 , b) compared to SEQ ID NO: 1 has a different amino acid residue at a position corre¬ sponding to W93, T134, G172, N176, D178, F188, D190, D198, I227 V230, K231 , H232, F233, Y258, G259, D260, D261 , P262, T264, N266, F284, T288 or M330 or a deletion corre¬ sponding to 191-195, and c) has the ability to hydrolyze starch to form an product having a modified maltose-to- glucose ratio than a product made with the polypeptide of SEQ ID NO: 1.
Finally, the invention provides a polynucleotide encoding the polypeptide and uses of the polypeptide in production of ethanol from granular starch by fermentation, in production of maltose syrup, and in the production of dough and baked products.
DETAILED DESCRIPTION OF THE INVENTION
Maltogenic alpha-amylase
The maltogenic alpha-amylase (EC 3.2.1.133) may have the amino acid sequence shown in SEQ ID NO: 1 (in the following referred to as Novamyl) with a 3D structure including a substrate as described in US 6162628 and found in the Protein Data Bank with the identifier
1 QHO. Alternatively, the maltogenic alpha-amylase may be a Novamyl variant described in US
6162628. A 3D structure of such 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
WO 9623874.
Selection of residues
An amino acid residue is selected which has a C-alpha atom located <10 A from an atom of the substrate. In 1qho, the following residues are selected by this criterion: 13, 15, 18, 43-44, 70, 72-73, 77-78, 82, 86-94, 97, 127-136, 143, 174-180, 183-184, 187-198, 226-233, 255-267, 270, 282-289, 291-292, 299, 307, 324, 327-331 , 360, 370-376.
The selection may in particular be for residues <10 A from an atom in monosaccha¬ ride (glucose) moieties +1 , +2 and +3 at the reducing side of the cleavage point. In 1qho, the moieties are denoted j, k and I, and this lead to selection of the following residues: 13, 70, 73, 90, 92-93, 127-132, 174-180, 183-184, 187-191 , 196, 226-233, 255-267, 270, 282-289, 291- 292, 299, 307, 324, 327-331 , 371-372, 375-376.
Amino acid substitutions
The selected residue may be substituted so as to push the substrate away or block for it presents in position +1, +2 and +3 etc by making the residues larger at a position corre¬ sponding to G172, D178, T189, K231 , H232, Y258, G259, D260, T264, N266 or T288 in No- vamyl (SEQ ID NO: 1), e.g. a substitution corresponding to G172V, T189M, K231R, H232Y, Y258W, G259A/H/Y, T264Y/Q/F, N266Y or T288Y/Q/F/P.
The substitution may serve to remove hydrogen bonding or van der Waals contact to the substrate at position +1 , +2 and +3. This may be done by substituting with a smaller resi¬ due at a position corresponding to W93, T134, D178, D190, D198, [227, K231, H232, F233, Y258, D260, D261 , T264 or T288 of SEQ ID NO: 1 , particularly a substitution corresponding to W93S/G/V/T/M/E, T134A, D178L/M/T/V, D190G, D198G, I227V, K231L/M, H232L/M, F233S, Y258L/M/T/V, D260L/M/T/V, D261G, T264AΛ/ or T288A/V. Alternatively, a hydrophilic or electrically charged (positive or negative) residue may be substituted with a hydrophobic residue, particularly at a position corresponding to T134, D178, D190, D198, K231 , H232 or D261 , more particularly a substitution corresponding to T134A, D178V, D190G, D198G, K231 L/M, H232L/M or D261G.
Finally, the substitution or deletion may serve to change indirectly the contact by changing the residues next to the substrate contact residues, particularly a residue corre¬ sponding to W93, N176, 191 , 192, 193, 194,195, V230, P262, F284 or M330 in Novamyl, e.g. a substitution corresponding to W93E/G/M/V/T/S, N176L, V230G, F284Y or M330I or a dele¬ tion of residues corresponding to 191 , 192, 193, 194, and 195.
Amino acid residues are ranked as follows from smallest to largest: (an equal sign in- dicates 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 following amino acid residues are considered to be hydrophobic: G, A, V, L, I, P, F, W and C as part of a disulfide bridge.
Variants Some particular variants according to the invention have the sequence of SEQ ID NO:
1 with the following substitutions:
W93M
W93E
W93M, V230G
Y258W Y258W , F284Y
H232M
F188T
F188G
F188V
W93G
W93V
W93T
W93S
N176L
D178V
F188G, W93M
F188G, W93E
F188G, W93S
F188G, W93T
F188V, W93M
F188V, W93E
F188V, W93S
F188V, W93T
Ability to hydrolyze starch
The variant of the invention is able to hydrolyze starch to form a product having a modified maltose-to-glucose ratio as compared to a product made with the polypeptide of SEQ ID NO: 1. The starch hydrolysis may be carried out by the following procedures described in the examples. The variants of the invention may show an increased ratio of glucose to maltose (DP1/DP2) or an increased ratio of DP1/(DP1-4) or an increased ratio of maltose to glucose (DP2/DP1) or an increased ratio of (DP1-4)/DP1.
Starch is in the context of the present invention intended to include starch as well as breakdown products of starch, such as amylopectin, or amylose, or maltooligosaccharides.
Amino acid identity
The polypeptide of the invention may have identities to the disclosed sequences of at least 80 %, particularly at least 85 % or at least 90 %, e.g. at least 91%, or 92%, or 93%, or 94%, or at least 95 %, such as 96%, or 97%, or 98%, or 99%.
For purposes of the present invention, alignments of sequences and calculation of identity scores may be done using a Needleman-Wunsch alignment (i.e. global alignment), useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is -12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for proteins and -4 for DNA. Alignment is from the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology, 183:63-98).
Industrial uses
The variant of the invention may be used in various known applications for amylases, e.g. production of ethanol, beer, dough, maltose syrup and baked products.
Ethanol production
The variant may be used in a process comprising treating granular starch with the variant and fermentation into ethanol. The treatment of the granular starch serves to produce a hydrolysis product which includes a significant amount of glucose. The fermentation to produce ethanol may be simultaneous with the granular starch treatment, or the starch may first be hy- drolyzed followed by fermentation of the hydrolysate. The process may be performed as de¬ scribed in WO 2003068976.
Beer production
The variant may be used in mashing, i.e. in the process of converting starch from milled malt and solid adjuncts into fermentable and unfermentable sugars to produce wort. The mashing involves incubating the variant with milled malt and solid adjuncts in water to hydro- lyze the starch.
Dough and baked products
The variant may be added to dough for making baked products such as bread. Addi¬ tion of the variant may serve to retard staling of the baked product. The addition to dough may be done as described in WO 9104669.
Maltose syrup
The variant may be used for commercial production of maltose, which today starts from liquefied starch (DE<10), which is subsequently treated simultaneously with debranching enzymes (pullulanase or isoamylase) and maltose-forming enzymes (maltogenic α-amylase or β-amylase) at a temperature around 600C. Glucose is an undesired side product in maltose syrups because it impacts the crystallization of maltose. Maltose is used in large quantities as syrups in e.g. the confectionary industry and as a sweetening agent in the food industry. Mal¬ tose syrups have among other capacities reduced browning capacity, a resistance to moisture absorption and to crystallization making maltose syrups suited for e.g. frozen dessert formula¬ tions, hard candy, jams, and jellies. Thus, a maltogenic alpha-amylase with an increased mal¬ tose-to-glucose ratio would be an advantage in the production of maltose syrups.
EXAMPLES
Example 1 : Starch hydrolysis with variants
A number of variants were prepared, each having the sequence of SEQ ID NO: 1 with the indicated substitutions. Each variant was tested by incubating it with maltodextrin (DE 11) by application of the following procedure: - Prepare a 30% (w/w) maltodextrin solution (DE 11) in 5OmM Na-acetate, 1mM
CaCI2 pH 5.5. Is heated to 6O0C for dissolving the maltodextrins.
- 1 ml substrate is added to 1.5 ml tubes with lid and membrane, and samples are pre¬ heated to 6O0C on a thermomixer.
- 1-100 microliter fermentation broth was added to 1ml preheated substrate. The fer- mentation broth volumes were adjusted thus, that they all contained the same amount of amylase activity measured by the Phadebas amylase assay.
- Samples are incubated for 42h at 6O0C.
- Make a small hole in the lid with e.g. a needle.
- Samples are boiled for 15 minutes (or 990C at the thermomixer). - Add ImI MiIIi-Q.
- After cooling the samples are filtered through a 0.2 micro-m filter.
The carbohydrate profile was determined by chromatography by applying standard procedures, e.g. as described in Norman, B. E. in James N.Bemiller, David J. Manners, and Robert J. Sturgeon (eds), Methods in Carbohydrate Chemistry, Volume X. John Wiley & Sons, Inc., New York, pp. 231-239, 1994.
Novamyl without substitutions was included as reference. The results were as follows:
Figure imgf000007_0001
Figure imgf000008_0001
1) d (191-195) indicates a deletion of the amino acids corresponding to position 191 , 192, 193, 194, and 195.
Example 2: Starch hydrolysis with variants and pullulanase
Further a number of variants were tested applying the same procedure as described in Example 1 , except that 1.2 mg/g DS of the commercially available pullulanase Promozyme® (EP 63909) was added.
The following results were obtained:
Figure imgf000009_0001
Example 3: Starch hydrolysis with purified variants and pullulanase
A number of purified variants (each having the sequence of SEQ ID NO: 1 with the in¬ dicated substitutions) were prepared by standard purification techniques, see e.g. Beier et al.: "Conversion of the maltogenic alpha-amylase Novamyl into a CGTase" in Protein Engineering, vol. 13 no. 7 pp. 509-513, 2000.
Each variant was tested by incubating it with maltodextrin (DE 11) at 600C and pH 5.5 for 42 hours as described in Example 1. Either an amount of 0.81 micro g (variants marked with [1]) or 1.62 micro g (variants marked with [2]) of the variant was added, and further 1.2 mg/g DS of the commercially available pullulanase Promozyme® (EP 63909) was added. Novamyl without substitutions was included as reference. The results were as follows:
Figure imgf000009_0002
Figure imgf000010_0001
Example 4: Starch hydrolysis with purified variants without addition of pullulanase
A number of purified variants (each having the sequence of SEQ ID NO: 1 with the in¬ dicated substitutions) were prepared by standard purification techniques, see e.g. Beier et al.: "Conversion of the maltogenic alpha-amylase Novamyl into a CGTase" in Protein Engineering, vol. 13 no. 7 pp. 509-513, 2000.
Each variant was tested by incubating it with maltodextrin (DE 11 ) at 600C and pH 5.5 for 42 hours as described in Example 1. An amount 1.62 micro g (variants marked with [2]) of the variant was added.
A single variant was dosed at a higher amount, namely 38.2 micro g (variant marked with [3]).
Figure imgf000010_0002
Figure imgf000011_0001
Example 5: Amylopectin hydrolysis with variants
A number of purified variants (each having the sequence of SEQ ID NO: 1 with the in¬ dicated substitutions) were prepared by standard purification techniques, see e.g. Beier et al.: "Conversion of the maltogenic alpha-amylase Novamyl into a CGTase" in Protein Engineering, vol. 13 no. 7 pp. 509-513, 2000.
Each variant was tested by incubating it with amylopectin (waxy maize starch) by ap¬ plication of the following procedure:
- Prepare a 5% (w/w) amylopectin solution in 5OmM Na-acetate, 1mM CaCI2, pH 5.5. The solution is boiled for 2 minutes or until it is dissolved.
- 1 mL substrate is added to 1.5 mL tubes with lid and membrane, and samples are preheated to 6O0C on a thermomixer.
- Enzyme is dosed at a dose corresponding to the amylase activity of 0.81 micro g No¬ vamyl measured by the Phadesbas amylase assay (which is 0.045 PSU/mL substrate, when Novamyl activity is 56.8PSU/mg)
- Samples are incubated for1 hour or 24 hours at 6O0C.
- 1ml MiIIi-Q is added with 1-2 drops of 1 M HCI (pH must be less than 3 to inactive the amylase).
- Make a small hole in the lid with e.g. a needle. - Samples are boiled for 15 minutes.
- After cooling the samples are filtered through a 0.2 micro m filter.
Samples incubated for 1 hour are marked [1], samples incubated for 24 hours are marked [24].
The carbohydrate profile was determined by chromatography by applying standard procedures, e.g. as described in Norman, B. E. in James N.Bemiller, David J. Manners, and Robert J. Sturgeon (eds), Methods in Carbohydrate Chemistry, Volume X. John Wiley & Sons, Inc., New York, pp. 231-239, 1994. Novamyl without substitutions was included as reference. The results were as follows:
Figure imgf000011_0002
Figure imgf000012_0001
Example 6: Baking with variants
Two variants were tested for baking, namely Y258W and W93S. Bread was made by the European Straight Dough method with and without addition of enzymes. The texture was evaluated using standard AACC procedures, and the following results were obtained after 7 days storage:
Figure imgf000012_0002
Y258W was dosed 5 mg enzyme protein/kg flour for all three tests. W93S was dosed 3 mg enzyme protein/kg flour for all three tests.

Claims

1. A method of constructing a polypeptide, comprising: a) providing a parent maltogenic alpha-amylase having an amino acid sequence and a three-dimensional structure which includes a cleavage point and a substrate with at least three monosaccharide moieties at the reducing side of the cleavage point, b) selecting an amino acid residue having a C-alpha atom located <10 A from an atom in the substrate, c) substituting or deleting the selected residue to obtain a modified amino acid se¬ quence, d) preparing a polypeptide having the modified sequence, e) testing the modified polypeptide by incubating it with starch and analyzing the reac¬ tion product, and f) selecting a modified polypeptide which has the ability to hydrolyze starch and wherein the hydrolysis product has a modified maltose-to-glucose ratio compared to a hy- drolysis product made with the parent maltogenic alpha-amylase.
2. The method of claim 1 , wherein the selected residue has a C-alpha atom located <10 A from an atom in the three monosaccharide moieties at the reducing side of the cleavage point.
3. The method of claim 1 or 2, wherein the substitution is with a larger residue, particularly at a position corresponding to Y258, G 172, D 178, T189, K231 , H232, G259, D260, T264, N266 or T288 of SEQ ID NO: 1 , more particularly a substitution corresponding to Y258W, G172V, T189M, K231R, H232Y, G259A/H/Y, T264Y/Q/F, N266Y or T288Y/Q/F/P.
4. The method of claim 1 or 2, wherein the substitution is with a smaller residue, particularly at a position corresponding to W93, T134, D178, D190, D198, I227, K231 , H232, F233, Y258, D260, D261 , T264 or T288 of SEQ ID NO: 1 , more particularly a substitution corresponding to W93S/G/V7T/M/E, T134A, D178L/M/TΛ/, D190G, D198G, I227V, K231 L/M, H232L/M, F233S, Y258L/M/T/V, D260L/M/T/V, D261G, T264A/V, or T288AΛ/.
5. The method of claim 1 or 2, wherein the substitution is with a hydrophobic residue, particu¬ larly at a position corresponding to T134, D178, D190, D198, K231 , H232 or D261 , more par¬ ticularly a substitution corresponding to T134A, D178V, D190G, D198G, K231 L/M, H232L/M or D261G.
6. The method of claim 1 or 2, wherein the selected residue corresponds to W93, N176, 191 , 192, 193, 194,195, V230, P262, F284 or M330 of SEQ ID NO: 1 , particularly a substitution cor- responding to W93E/G/MΛ//T/S, N176L, V230G, F284Y or M330I or a deletion of residues cor¬ responding to 191-195.
7. A polypeptide which a) has an amino acid sequence having more than 80 % identity to SEQ ID NO: 1 , b) compared to SEQ ID NO: 1 has a different amino acid residue at a position corre¬ sponding to W93, T134, G172, N176, D178, F188, D190, D198, I227, V230, K231 , H232, F233, Y258, G259, D260, D261 , P262, T264, N266, F284, T288 or M330 or a deletion corresponding to 191-195, and c) has the ability to hydrolyze starch to form an product having a modified maltose-to- glucose ratio than a product made with the polypeptide of SEQ ID NO: 1.
8. The polypeptide of the preceding claim, which compared to SEQ ID NO: 1 comprises a substitution corresponding to W93E/G/M/V/T/S, T134A, G172V, D178L/M/T/V, F188G/T/V, D190G, D198G, I227V, V230G, K231R/L/M, 232Y/L/M, F233S, Y258W/L/M/T/V, G259A/H/Y, D260L/M/T/V, D261G, T264Y/Q/F/AΛ/, N266Y, F284Y, T288Y/Q/F/AΛ//P or M330I.
9. The polypeptide of claim 7 or 8, which has the amino acid sequence of SEQ ID NO: 1 with the following alterations:
W93M
W93E
W93M, V230G
Y258W
Y258W , F284Y
H232M
F188T
F188G
F188V
W93G
W93V
W93T
W93S
N176L
D 178V
F188G, W93M
F188G, W93E
F188G, W93S F188G. W93T
F188V, W93M
F188V, W93E
F188V, W93S
F188V, W93T
10. A polynucleotide encoding the polypeptide of any preceding claim.
11. A process for the production of ethanol, comprising treating granular starch with the poly¬ peptide of any preceding claim and fermentation into ethanol.
12. A dough comprising the polypeptide of any preceding claim.
13. A method of producing dough or a baked product from dough, comprising adding the poly¬ peptide of any preceding claim.
14. A process for the production of maltose syrup comprising treating liquefied starch with the polypeptide of any preceding claim.
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