WO2016030448A1 - Variants alpha-amylase d'alicyclobacillus pohliae - Google Patents

Variants alpha-amylase d'alicyclobacillus pohliae Download PDF

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Publication number
WO2016030448A1
WO2016030448A1 PCT/EP2015/069612 EP2015069612W WO2016030448A1 WO 2016030448 A1 WO2016030448 A1 WO 2016030448A1 EP 2015069612 W EP2015069612 W EP 2015069612W WO 2016030448 A1 WO2016030448 A1 WO 2016030448A1
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Prior art keywords
variant
alpha
polypeptide
amino acid
activity
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PCT/EP2015/069612
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English (en)
Inventor
DE René Marcel JONG
Hanna Elisabet ABBAS
Jeroen GODEFROOIJ
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Dsm Ip Assets B.V.
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Publication of WO2016030448A1 publication Critical patent/WO2016030448A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes

Definitions

  • the invention relates to a variant polypeptide having alpha-amylase activity.
  • the invention also relates to a nucleic acid sequence encoding such a variant, to a recombinant expression vector a said nucleic acid construct and to a recombinant host cell comprising a said expression vector. Further, the invention relates to a method for producing an alpha amylase via use of such a host cell. Also, the invention relates to a method of producing an alpha-amylase polypeptide variant.
  • the invention further relates to a composition comprising an alpha-amylase variant, to use of such an alpha-amylase variant or alpha-amylase variant-containing composition in the preparation of a baked product, to a process for the production of a baked product and to the resulting baked product.
  • amylopectin starts to re-crystallize (also called retrogradation).
  • the staling of bread is believed to be a direct reflection of the retrogradation of amylopectin.
  • the starch and thus the breadcrumb become more rigid.
  • the firmness of bread after a certain storage time is depending on the initial softness, which is the softness after cooling down, and the rate of increase of firmness, the rate of staling. Studies on bread staling have indicated that the starch fraction in bread recrystallizes during storage, thus causing an increase in crumb firmness, which may be measured as an increase in hardness of bread slices.
  • the present invention relates to an alpha-amylase.
  • Alpha-amylases have been used in industry for a long time.
  • Alpha-amylases have traditionally been provided through the inclusion of malted wheat or barley flour and give several advantages to the baker. Alpha-amylase is used to give satisfactory gas production and gas retention during dough leavening and to give satisfactory crust color. This means that if this enzyme is not used in sufficient amount, the volume, texture, and appearance of the loaf are substantially impaired. Alpha-amylase occurs naturally within the wheat crop itself, measured routinely by Hagberg Falling Number (ICC method 107), and steps are taken to minimise such variations by the addition of alpha-amylase at the mill and through the use of specialty ingredients at the bakery as the enzyme is of such critical importance.
  • ICC method 107 Hagberg Falling Number
  • alpha-amylase from cereal has been largely replaced with enzymes from microbial sources, including fungal and bacterial sources.
  • enzymes from microbial sources including fungal and bacterial sources.
  • enzymes can be prepared from such microbial sources and this brings advantage over malt flour because the enzyme is of more controlled quality, relatively pure and more cost effective in use.
  • alpha-amylases do however show important differences. Besides giving influence to gas production, gas retention and crust color, alpha-amylase can have bearing on the shelf-life of the baked product.
  • Starch within the wheat flour contains two principal fractions, amylose and amylopectin, and these are organised in the form of starch granules. A proportion of these granules from hard-milling wheat varieties become "damaged", with granules splitting apart as a consequence of the energy of milling.
  • the starch granules gelatinise; this process involves a swelling of the granule by the uptake of water and a loss of the crystalline nature of the granule; in particular amylopectins within the native granule are known to exist as crystallites and these molecules dissociate and lose crystallinity during gelatinisation.
  • amylopectin recrystallises slowly over a numbers of days and it is this recrystallisation, or retrogradation of starch, that is regarded as being the principal cause of bread staling.
  • alpha-amylase from fungal sources, most typically coming from Aspergillus species, acts principally on damaged starch during the mixing of dough and throughout fermentation/proof.
  • the low heat stability of the enzyme means that the enzyme is inactivated during baking and, critically before starch gelatinisation has taken place, such that there is little or no breakdown of the starch from the undamaged fraction.
  • fungal amylase is useful in providing sugars for fermentation and color, but has practically no value in extending shelf-life.
  • Bacterial alpha-amylase most typically from Bacillus amyloliquifaciens, on the other hand does bring extended temperature stability and activity during the baking of bread and while starch is undergoing gelatinisation. Bacterial amylase then leads to more extensive modification of the starch and, in turn, the qualities of the baked bread; in particular the crumb of the baked bread can be perceptibly softer throughout shelf-life and can permit the shelf-life to be increased.
  • Bacterial alpha-amylase can be useful with regard to shelf-life extension, it is difficult to use practically as small over-doses lead to an unacceptable crumb structure of large and open pores, while the texture can become too soft and "gummy".
  • US 4,598,048 describes the preparation of a maltogenic amylase enzyme.
  • US 4,604,355 describes a maltogenic amylase enzyme, preparation and use thereof.
  • US RE38,507 describes an antistaling process and agent.
  • W099/43793 discloses amylolytic enzyme variants.
  • WO2004/081 171 discloses an enzyme.
  • WO2006/012899 discloses maltogenic alpha-amylase variants.
  • US8426182 discloses an alpha amylase.
  • WO2008/148845 discloses a method for preparing a dough.
  • WO2006/032281 discloses a method for preparing a dough.
  • Figure 1 Sets out the plasmid map op pDBC1 , the plasmid is used to construct the expression vectors for alpha-amylase variants.
  • the SsmBI sites are used to exchange the CAP marker for a promoter fragment and gene of interest.
  • the region between the amyE flanking regions integrated integrates in the amyE locus and integrands are selected on spectinomycin.
  • Figure 2. Sets out the plasmid map of pDBC-AM1 containing the G01 expression module and the DSM-AM gene that is used for the production of a reference alpha- amylase.
  • SEQ ID NO: 1 sets out the polynucleotide sequence from Alicyclobacillus pohliae NCIMB14276 encoding the wild type signal sequence (set out in nucleotides 1 to 99), the wild-type alpha-amylase polypeptide (set out in nucleotides 100 to 2157), and a stop codon at the 3'-terminus (set out in nucleotides 2157 to 2160).
  • SEQ ID NO: 2 sets out the amino acid sequence of the mature Alicyclobacillus pohliae NCIMB14276 wild type alpha-amylase polypeptide.
  • SEQ ID NO: 3 sets out a synthetic DNA fragment containing a TthWW site, 340 bp of 5'- amyE, SsmBI site, chloramphenicol selection marker, terminator, BsmBI site, lox 66 site, spectinomycin selection marker, lox71 site, 120 bp of 3'-amyE and Asis ⁇ site.
  • SEQ ID NO: 4 sets out a synthetic DNA fragment of the G01 expression module which contains a P15 promoter, a modified RNA leader sequence as described in EP2186880 (nucleotides 31 -251 of SEQ ID NO: 70 therein) a Nde ⁇ site at the ATG start and two SsmBI sites at the 5' and 3' ends.
  • SEQ ID NO: 5 sets out the nucleic acid sequence that contains a BsmB ⁇ restriction, which is placed at the 5' end of the DNA sequences encoding the alpha-amylase variants.
  • SEQ ID NO 6 sets out the nucleic acid sequence that contains a BsmB ⁇ restriction, which is placed at the 3' end of the DNA sequences encoding the alpha-amylase variants.
  • SEQ ID NO 7 sets out the polynucleotide sequence of a synthetic DNA construct containing a SsmBI site, wild type DSM-AM sequence as set out in SEQ ID NO: 1 , double stop codon and BsmB ⁇ restriction site.
  • the invention relates to variant polypeptides having alpha-amylase activity, i.e. to alpha-amylase variants.
  • An alpha-amylase variant of the invention may have one or more improved properties in comparison with a reference polypeptide, the reference polypeptide typically having alpha-amylase activity.
  • a reference polypeptide may be a wild-type alpha-amylase, such as wild-type alpha-amylase, for example from Alicyclobacillus pohliae, in particular Alicyclobacillus pohliae NCIMB14276 strain.
  • the reference polypeptide having alpha-amylase activity may be the polypeptide as set out in SEQ ID NO: 2.
  • the improved property will typically be a property with relevance to the use of the variant alpha-amylase in the preparation of a baked product.
  • the improved property may include:
  • thermostability a) an increased thermostability
  • polypeptide having alpha-amylase activity as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the improved property may include
  • the ratio of the activity at a temperature of 50 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions is herein also referred to as the Activity at 50°C or higher: Activity at 37°C ratio.
  • a variant polypeptide having alpha- amylase activity wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity.
  • the invention also provides:
  • nucleic acid construct comprising such a nucleic acid sequence operably linked to one or more control sequences capable of directing the expression of an alpha- amylase in a suitable expression host;
  • a recombinant expression vector comprising such a nucleic acid construct; and - a recombinant host cell comprising such an expression vector.
  • the invention also relates to a method for producing an alpha-amylase comprising cultivating the host cell of the invention under conditions conducive to production of the alpha-amylase and recovering the alpha-amylase.
  • composition or pre-mix comprising the variant of the invention or obtainable by a method of the invention
  • a process for the production of a baked product comprises comprising adding an effective amount of a variant polypeptide according to the invention of a composition according to the invention to dough and carrying out appropriate further baking manufacturing steps.
  • the invention relates to a method of producing an alpha-amylase polypeptide variant, which method comprises:
  • Alpha-amylase activity can suitably be determined using the Ceralpha® procedure, which is recommended by the American Association of Cereal Chemists (AACC).
  • a variant of the invention will typically retain alpha-amylase activity. That is to say, a variant of the invention will typically be capable of alpha amylase activity.
  • the reference polypeptide as set out in SEQ ID NO: 2 showed alpha-amylase activity in the CERALPHA assay.
  • a variant of the invention will typically be a starch degrading enzyme.
  • Variant polypeptides of the invention may be referred to as "variant polypeptide having alpha-amylase activity", “alpha-amylase variant”, “improved alpha-amylase”, and the like.
  • a gene or cDNA coding for an alpha-amlyase or pro-alpha-amylase may be cloned and over-expressed in a host organism.
  • Well known host organisms that have been used for alpha amylase over-expression in the past include Aspergillus, Kluyveromyces, Trichoderma, Escherichia coli, Pichia, Saccharomyces, Yarrowia, Neurospora or Bacillus.
  • the alpha-amylase variant may be manufactured industrially using recombinant DNA technology, e.g. using filamentous fungi such as Aspergillus species, yeast strains, e.g. of Kluyveromyces species, or bacterial species, e.g. E. coli, as host organisms.
  • recombinant DNA technology e.g. using filamentous fungi such as Aspergillus species, yeast strains, e.g. of Kluyveromyces species, or bacterial species, e.g. E. coli, as host organisms.
  • Such recombinant microbial production strains are constructed and continuously improved using DNA technology as well as classical strain improvement measures directed towards optimising the expression and secretion of a heterologous protein.
  • an alpha-amylase variant may be provided in the form of prealpha-amylase variant or (mature) alpha-amylase variant.
  • a corresponding nucleic acid sequence may also be provided, i.e. a polynucleotide that encodes a pre-alpha- amylase or a (mature) alpha-amylase may be provided.
  • positions which may be substituted to achieve a variant of the invention are defined with reference to SEQ ID NO: 2 which is a mature alpha-amylase, i.e. it is a sequence which does not include a presequence.
  • the invention concerns variant polypeptides having alpha-amylase activity as compared with a reference polypeptide having alpha-amylase activity.
  • the reference polypeptide may typically be a wild-type polypeptide having alpha-amylase activity, such as the alpha-amylase of SEQ ID NO: 2.
  • the reference polypeptide may also be referred to as a parent polypeptide or comparison polypeptide.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 200, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • variant has one or more altered properties as compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2.
  • the one or more altered properties are selected from an increased thermostability, an increased sucrose tolerance, an increased an increased Activity at pH4 : Activity at pH5 ratio and an increased Activity at pH7 : Activity at pH5 ratio.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to amino acid 200, said positions being defined with reference to SEQ ID NO: 2;
  • variant has one or more altered properties as compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an increased thermostability, an increased sucrose tolerance, an increased an increased Activity at pH4 : Activity at pH5 ratio and an increased Activity at pH7 : Activity at pH5 ratio.
  • a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an increased thermostability, an increased sucrose tolerance, an increased an increased Activity at pH4 : Activity at pH5 ratio and an increased Activity at pH7 : Activity at pH5 ratio.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to amino acid 200, said positions being defined with reference to SEQ ID NO: 2;
  • variant has one or more altered properties as compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an improved crumb softness of the baked product, improved resilience both initial and in particular after storage, reduced hardness after storage and improved anti-staling of the baked product.
  • a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an improved crumb softness of the baked product, improved resilience both initial and in particular after storage, reduced hardness after storage and improved anti-staling of the baked product.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises the amino acid substitutions W70Y and S200N, said positions being defined with reference to SEQ ID NO: 2;
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises the amino acid substitutions W70Y and S200N, said positions being defined with reference to SEQ ID NO: 2;
  • variant has one or more altered properties as compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an improved crumb softness of the baked product, improved resilience both initial and in particular after storage, reduced hardness after storage and improved anti-staling of the baked product.
  • a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an improved crumb softness of the baked product, improved resilience both initial and in particular after storage, reduced hardness after storage and improved anti-staling of the baked product.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to amino acid 200, said positions being defined with reference to SEQ ID NO: 2;
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises the amino acid substitutions W70Y and S200N, said positions being defined with reference to SEQ ID NO: 2;
  • the variant demonstrates a reduced loss of resilience over storage of a baked product comprising at least 2 wt% sugar, in an aspect comprising at least 3 wt% sugar, in an aspect comprising at least 5 wt% sugar, compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to amino acid 200, said positions being defined with reference to SEQ ID NO: 2;
  • the variant demonstrates a reduced hardness after storage of a baked product comprising at least 2 wt% sugar, in an aspect comprising at least 3 wt% sugar, in an aspect comprising at least 5 wt% sugar, compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises the amino acid substitutions W70Y and S200N, said positions being defined with reference to SEQ ID NO: 2;
  • the variant demonstrates a reduced hardness after storage of a baked product comprising at least 2 wt% sugar, in an aspect comprising at least 3 wt% sugar, in an aspect comprising at least 5 wt% sugar, compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2.
  • the invention relates to a variant polypeptide having alpha- amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity.
  • a wild type reference polypeptide may be obtained from any suitable organisms.
  • Suitable wild type reference polypeptides may be obtained from Alicyclobacillus po liae NCI MB14276.
  • the reference polypeptide is the alpha amylase set out in SEQ ID NO: 2.
  • the parent polypeptide having alpha-amylase activity is preferably is the alpha amylase set out in SEQ ID NO: 2.
  • variant polypeptide having alpha-amylase activity wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • An altered property includes an improved property.
  • a variant polypeptide will typically have an improved property as compared to a reference polypeptide, in particular with respect to a property relevant to the use of the variant polypeptide in baked product making.
  • Improved productivity may be demonstrated by an alpha-amylase variant that shows improved expression as compared with a parent polypeptide.
  • the improved property will typically be a property with relevance to the use of the variant alpha-amylase in the preparation of a baked product.
  • An alpha-amylase variant with an improved property relevant for preparation of a baked product may demonstrate reduced hardness after storage of a baked product and/or reduced loss of resilience over storage of a baked product.
  • the improved property may include increased strength of the dough, increased elasticity of the dough, increased stability of the dough, reduced stickiness of the dough, improved extensibility of the dough, improved machineability of the dough, increased volume of the baked product, improved flavour of the baked product, improved crumb structure of the baked product, improved crumb softness of the baked product, reduced blistering of the baked product, improved crispiness, improved resilience both initial and in particular after storage, reduced hardness after storage and/or improved anti-staling of the baked product.
  • the improved property may include an improved oven spring.
  • the improved property may include an improved slice-ability.
  • the improved property may include an improved crumbliness.
  • the improved property may include an improved slice-stickiness.
  • the improved property may include faster dough development time of the dough and/or reduced dough stickiness of the dough.
  • the improved property may include improved foldability of the baked product, such as improved foldability of a tortilla, a pancake, a flat bread, a pizza crust, a roti and/or a slice of bread.
  • the improved property may include improved flexibility of the baked product including improved flexibility of a tortilla, a pancake, a flat bread, a pizza crust, a roti and/or a slice of bread.
  • the improved property may include improved stackability of flat baked products including tortillas, pancakes, flat breads, pizza crusts, roti.
  • the improved property may include reduced stickiness of noodles and/or increased flexibility of noodles.
  • the improved property may include reduced clumping of cooked noodles and/or improved flavor of noodles even after a period of storage.
  • the improved property may include reduction of formation of hairline cracks in a product in crackers as well as creating a leavening effect and improved flavor development.
  • the improved property may include improved mouth feel and /or improved softness on squeeze,
  • the improved property may include reduced damage during transport, including reduced breaking during transport.
  • the improved property may include reduced hardness after storage of gluten-free bread.
  • the improved property may include improved resilience of gluten-free bread.
  • the improved property may include improved resilience both initial and in particular after storage of gluten-free bread.
  • the improved property may include reduced hardness after storage of rye bread.
  • the improved property may include reduced loss of resilience over storage of rye bread.
  • the improved property may include reduced loss of resilience over storage of a baked product comprising at least 2 wt% sugar, in an aspect comprising at least 3 wt% sugar, in an aspect comprising at least 5 wt% sugar, in an aspect at least 8 wt% sugar, in an aspect comprising at least 12 wt% sugar, in an aspect comprising at least 15 wt% sugar based on total recipe weight.
  • at least 18 wt% sugar in an aspect comprising at least 20 wt% sugar, in an aspect comprising at least 25 wt% sugar, in an aspect comprising at least 30 wt% sugar based on total recipe weight.
  • 5 wt% sugar means 50 grams sugar per 1000 grams total recipe weight etc.
  • the improved property may include reduced hardness after storage of a baked product comprising at least 2 wt% sugar, in an aspect comprising at least 3 wt% sugar, in an aspect comprising at least 5 wt% sugar, in an aspect comprising at least 8 wt% sugar, in an aspect comprising at least 12 wt% sugar, in an aspect comprising at least 15 wt% sugar based on total recipe weight.
  • at least 18 wt% sugar in an aspect comprising aspect at least 20 wt% sugar, in an aspect comprising at least 25 wt% sugar, in an aspect comprising at least 30 wt% sugar based on total recipe weight.
  • 5 wt% sugar means 50 grams sugar per 1000 grams of total recipe weight, etc.
  • each of these improvements may be determined as compared with a reference polypeptide.
  • the improved property may be demonstrated by preparing a baked product comprising the alpha-amylase variant and another comprising a parent polypeptide and comparing the results.
  • a variant which exhibits a property which is improved in relation to the parent polypeptide having alpha-amylase activity is one which demonstrates a measurable reduction or increase in the relevant property, typically such that the variant is more suited to use as set out below, for example in a method for the production of a foodstuff.
  • the improved property may be demonstrated in an assay or (bio)chemical analysis.
  • a variant alpha-amylase of the invention may show improved productivity in comparison with a reference polypeptide.
  • a variant alpha-amylase of the invention may show an altered, such as reduced or increased, temperature stability or an altered activity at pH relevant for the baked product making process, such as a lower pH or a higher pH, as compared with a reference polypeptide having alpha-amylase activity.
  • the improved property may include one or more of:
  • thermostability for example at pH 4, pH 5, pH 6 and/or pH 7;
  • alpha-amylase variant an increased productivity in the production of the alpha-amylase variant, as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO :2).
  • the improved property may include one or more of:
  • thermostability a) an increased thermostability
  • the improved property may include an increased ratio of the activity at a temperature of 60 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • the improved property may include an increased ratio of the activity at a temperature of 60 degrees Celsius versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • the improved property may include an increased ratio of the activity at a temperature of 80 degrees Celsius versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • the ratio of the activity at a temperature of 60 degrees Celsius versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions is herein also referred to as the Activity at 60°C: Activity at 37°C ratio.
  • the ratio of the activity at a temperature of 80 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions is herein also referred to as the Activity at 80°C: Activity at 37°C ratio.
  • Table 1 sets out positions that may influence specific properties of the variant alpha- amylases of the invention.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises the amino acid substitution W70Y and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises the amino acid substitution W70Y and comprises a substitution of an amino acid residue
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises the amino acid substitutions W70Y and S200N, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • W70Y and S200N means 2 substitutions: the amino acid at position 70 and the amino acid at position 200 are substituted.
  • the W (Tryptophan) at position 70 is changed into a Y (Tyrosine) and that S (Serine) at position 200 is changed into a N (Asparagine), etc.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises the amino acid substitution L225F and comprises a substitution of an amino acid residue corresponding to one or more of 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • variant has one or more altered properties as compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • variant has one or more altered properties as compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an increased thermostability, an increased sucrose tolerance, an increased an increased Activity at pH4 : Activity at pH5 ratio and an increased Activity at pH7 : Activity at pH5 ratio.
  • a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an increased thermostability, an increased sucrose tolerance, an increased an increased Activity at pH4 : Activity at pH5 ratio and an increased Activity at pH7 : Activity at pH5 ratio.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises the amino acid substitution L225F and comprises a substitution of an amino acid residue corresponding to one or more of 114V, 115V, V124I, S133T, S200N, L282I, L282F and S358A,
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of the amino acid residues corresponding to amino acids 225 and 133 or 200, said positions being defined with reference to SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises the amino acid substitutions L225F and S200N, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2), preferably an improved property selected from a) to j) as described herein.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions: W70Y and S200N
  • the variant has at least one altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2), preferably an improved property selected from a) to j) as described herein.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ I D NO: 2, comprises one of the following sets of substitutions:
  • positions being defined with reference to SEQ ID NO: 2, and wherein the variant has at least one altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2), preferably an improved property selected from a) to j) as described herein.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • positions being defined with reference to SEQ ID NO: 2, and wherein the variant has at least one altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2), preferably an improved property selected from a) to j) as described herein.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358,
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises the amino acid substitution S200N and at least one further substitution of an amino acid residue corresponding to any of amino acids of 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha- amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • variant has one or more altered properties as compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2.
  • the variant polypeptide according to the invention is a variant having alpha-amylase activity, wherein the variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • variant has one or more altered properties as compared with a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an increased thermostability, an increased sucrose tolerance, an increased an increased Activity at pH4 : Activity at pH5 ratio and an increased Activity at pH7 : Activity at pH5 ratio.
  • a reference polypeptide having alpha amylase activity as set out in SEQ ID NO: 2 selected from an increased thermostability, an increased sucrose tolerance, an increased an increased Activity at pH4 : Activity at pH5 ratio and an increased Activity at pH7 : Activity at pH5 ratio.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises the amino acid substitution S200N and one or more of 114V, 115V, W70Y, V124I, S133T, S200N, L282I, L282F and S358A,said positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of the amino acid residues corresponding to amino acids 133 and 200, said positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises the amino acid substitutions S200N and S133T, said positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions: W70Y and S200N;
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • substitution in the context of the present disclosure indicates that a position in the variant which corresponds to one of the positions set out above in SEQ ID NO: 2 comprises an amino acid residue which does not appear at that position in the reference polypeptide (the reference polypeptide may be SEQ ID NO: 2).
  • a variant of the invention may be generated using any combination of substitutions set out in Table 1.
  • Table 1 Preferred substitutions defined in relation to SEQ ID NO: 2 Amino acids are depicted according to the single letter annotation
  • V50 Reference alpha-amylase having an amino acid sequence as set out in SEQ ID NO 2
  • W70Y and S200N means 2 changes: the amino acid at position 70 and the amino acid at position 200 are changed.
  • the W (Tryptophan) at position 70 is changes into a Y (Tyrosine) and that S (Serine) at position 200 is changed into a N (Asparagine), etc.
  • a variant of the invention was generated using any combination of substitutions set out in Table 2. Under the same conditions a reference alpha-amylase polypeptide having an amino acid sequence as set out in SEQ ID NO 2 was generated.
  • W70Y and S200N means 2 changes: the amino acid at position 70 and the amino acid at position 200 are changed.
  • the W (Tryptophan) at position 70 is changes into a Y (Tyrosine) and that S (Serine) at position 200 is changed into a N (Asparagine), etc.
  • a variant alpha-amylase of the invention may also comprise additional modifications in comparison to the parent polypeptide at positions other than those specified above, for example, one or more additional substitutions, additions or deletions.
  • a variant of the invention may comprise a combination of different types of modification of this sort.
  • a variant may comprise one, two, three, four, least 5, at least 10, at least 15, at least 20, at least 25, at least 30 or more such modifications (which may all be of the same type or may be different types of modification).
  • the additional modifications may be substitutions.
  • the invention thus also provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2) and wherein said variant polypeptide comprises at least one additional substitution other than those defined herein.
  • a reference polypeptide having alpha- amylase activity such as the polypeptide of SEQ ID NO: 2
  • said variant polypeptide comprises at least one additional substitution other than those defined herein.
  • the invention thus also provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) and wherein said variant polypeptide comprises at least one additional substitution other than those defined herein.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • said variant polypeptide comprises at least one additional substitution other than those defined herein.
  • the invention thus also provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358,
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2) and wherein said variant polypeptide comprises at least one additional substitution other than those defined herein.
  • a reference polypeptide having alpha- amylase activity such as the polypeptide of SEQ ID NO: 2
  • said variant polypeptide comprises at least one additional substitution other than those defined herein.
  • the alpha-amylase variant of the invention comprises an amino acid sequence having at least 70% identity to the parent polypeptide, such as the alpha- amylase of SEQ ID NO: 2, in an aspect at least 80% identity, in an aspect at least 85% identity, in an aspect at least 90% identity, in an aspect at least 95% identity, in an aspect at least 96% identity, in an aspect at least 97% identity, in an aspect at least 98% identity, in an aspect at least 99% identity to the parent polypeptide.
  • Such a variant will typically have one or more substitutions or sets of substitutions as set out in Table 1 .
  • the variant will have one or more substitutions or sets of substitutions as set out in Table 2.
  • alpha-amylase variant of the invention variant polypeptide having alpha amylase activity and alpha-amylase polypeptide variant are used interchangeably herein.
  • the terms reference polypeptide and parent polypeptide are used interchangeably herein.
  • the variant polypeptide having alpha-amylase activity of the invention comprises an amino acid sequence having at least 70% identity to the parent polypeptide, such as the alpha-amylase of SEQ ID NO: 2, in an aspect at least 75% identity, in an aspect at least 80% identity, in an aspect at least 85% identity, in an aspect at least 90% identity, in an aspect at least 95% identity, in an aspect at least 96% identity, in an aspect at least 97% identity, in an aspect at least 98% identity, in an aspect at least 99% identity to the parent polypeptide.
  • the variant polypeptide having alpha-amylase activity of the invention comprises an amino acid sequence having at least 70% identity to the parent polypeptide, such as the alpha-amylase of SEQ ID NO: 2, in an aspect at least 75% identity, in an aspect at least 80% identity, in an aspect at least 85% identity, in an aspect at least 90% identity, in an aspect at least 95% identity, in an aspect at least 96% identity, in an aspect at least 97% identity, in an aspect at least 98% identity, in an aspect at least 99% identity to the parent polypeptide.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates one or more altered properties as compared with a reference polypeptide having alpha-amylase activity, with the proviso that the variant is not the polypeptide encoded by the wildtype DNA as set out in SEQ ID NO: 1 .
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates one or more altered properties as compared with a reference polypeptide having alpha-amylase activity, with the proviso that the variant is not the polypeptide encoded by the wildtype DNA as set out in SEQ ID NO: 1 .
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates one or more altered properties as compared with a reference polypeptide having alpha-amylase activity, with the proviso that the variant is not the polypeptide encoded by the wildtype DNA as set out in SEQ ID NO: 1 .
  • the variant polypeptide is a variant which is not the polypeptide encoded by the wildtype DNA as set out in SEQ ID NO: 1.
  • a variant polypeptide is not a naturally-occurring polypeptide.
  • the present invention provides a variant polypeptide having alpha-amylase activity, wherein said variant has an amino acid sequence having at least 70% identity to the amino acid sequence as set out in SEQ ID NO: 2 which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid W70Y and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity as set out in SEQ ID NO: 2.
  • the present invention provides a variant polypeptide having alpha-amylase activity, wherein said variant has an amino acid sequence having at least 70% identity to the amino acid sequence as set out in SEQ ID NO: 2 which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid L225F and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2, and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity as set out in SEQ ID NO: 2.
  • the present invention provides a variant polypeptide having alpha-amylase activity, wherein said variant has an amino acid sequence having at least 70% identity to the amino acid sequence as set out in SEQ ID NO: 2 which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid S200N and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity as set out in SEQ ID NO: 2.
  • the alpha-amylase variant of the invention comprises an amino acid sequence having at least one of Asp at position 151 , Ala at position 264, Thr at position 335 and Asn at position 456, said positions being defined with reference to SEQ ID NO: 2.
  • the alpha-amylase variant of the invention comprises an amino acid sequence having all of Asp at position 151 , Ala at position 264, Thr at position 335 and Asn at position 456, said positions being defined with reference to SEQ ID NO: 2.
  • a variant of the invention will typically retain alpha-amylase activity.
  • the alpha-amylase variant according to the invention and the parent polypeptide herein are a starch degrading enzymes.
  • the alpha-amylase variant according to the invention and the parent polypeptide herein have alpha-amylase activity.
  • Alpha-amylase activity can suitably be determined using the Ceralpha® procedure, which is recommended by the American Association of Cereal Chemists (AACC).
  • Enzymatic activity of an alpha-amylase variant and of a parent polypeptide may be expressed as NBAU.
  • NBAU activity can suitably be determined using the NBAU assay as described herein.
  • a variant of the invention will typically exhibit improved properties in comparison with the reference alpha-amylase polypeptide from which it is derived.
  • Such an improved property will typically be one which is relevant if the variant were to be used as set out herein, for example in a method for preparing a baked product.
  • a variant which exhibits a property which is improved in relation to the reference alpha-amylase is one which demonstrates a measurable reduction or increase in the relevant property, typically such that the variant is more suited to use as set out herein, for example in a method for the production of a baked product.
  • the property may thus be decreased by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.
  • the property may be increased by at least 10%, at least 25%, at least 50%, at least 100%, at least, 200%, at least 500% or at least 1000%.
  • the percentage decrease or increase in this context represents the percentage decrease or increase in comparison to the reference alpha-amylase polypeptide. It is well known to the skilled person how such percentage changes may be measured - it is a comparison of the activity of the reference alpha-amylase and the variant alpha- amylase.
  • variants described herein are collectively comprised in the terms "a polypeptide according to the invention” or “a variant according to the invention” or “a variant polypeptide according to the invention”.
  • the one or more altered properties of alpha-amylase variant according to the invention may include without limitation one or more of
  • thermostability a) an increased thermostability
  • the improved property may include
  • the improved property may include an increased ratio of the activity at a temperature of 60 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • the improved property may include an increased ratio of the activity at a temperature of 80 degrees Celsius versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased thermostability as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha- amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased sucrose tolerance as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha- amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased - Activity at pH4 : Activity at pH5 ratio as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased Activity at pH7 : Activity at pH5 ratio as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased specific activity at pH 4 as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased specific activity at pH 5 as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased specific activity at pH 7 as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased ratio of the activity at a temperature of 50 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased ratio of the activity at a temperature of 60 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased ratio of the activity at a temperature of 60 degrees Celsius versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased thermostability as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha- amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased sucrose tolerance as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha- amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased Activity at pH4 : Activity at pH5 ratio as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased Activity at pH7 : Activity at pH5 ratio as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased specific activity at pH 4 as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased specific activity at pH 5 as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased specific activity at pH 7 as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased ratio of the activity at a temperature of 50 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased ratio of the activity at a temperature of 60 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased ratio of the activity at a temperature of 60 degrees Celsius versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased thermostability as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased sucrose tolerance as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased Activity at pH4 : Activity at pH5 ratio as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased Activity at pH7 : Activity at pH5 ratio as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased specific activity at pH 4 as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased specific activity at pH 5 as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased specific activity at pH 7 as compared with a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased ratio of the activity at a temperature of 50 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased ratio of the activity at a temperature of 60 degrees Celsius or higher versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2, and wherein the variant demonstrates an increased ratio of the activity at a temperature of 60 degrees Celsius versus the activity at a temperature of 37 degrees Celsius as compared to a reference polypeptide having alpha-amylase activity (such as the polypeptide of SEQ ID NO: 2) measured under the same conditions.
  • a reference polypeptide having alpha-amylase activity such as the polypeptide of SEQ ID NO: 2
  • the alpha-amylase variant according to the invention has two altered properties, in an aspect three altered properties selected from a) to f) above as compared to a reference polypeptide having alpha-amylase activity.
  • the reference polypeptide having alpha-amylase activity is the polypeptide as set out in SEQ ID NO: 2.
  • the alpha-amylase variant according to the invention has two altered properties, in an aspect three altered properties selected from a) to g) above as compared to a reference polypeptide having alpha-amylase activity.
  • the reference polypeptide having alpha-amylase activity is the polypeptide as set out in SEQ ID NO: 2.
  • the alpha-amylase variant according to the invention has two altered properties, in an aspect three altered properties selected from a) to j) described herein as compared to a reference polypeptide having alpha-amylase activity.
  • the reference polypeptide having alpha-amylase activity is the polypeptide as set out in SEQ ID NO: 2.
  • the alpha-amylase variant according to the invention has both an increased thermostability and an increased specific activity at pH 4 as compared to a reference polypeptide having alpha-amylase activity, wherein the reference polypeptide is the alpha-amylase as set in SEQ ID NO:2.
  • the alpha-amylase variant according to the invention has both an increased thermostability and an increased specific activity at pH 5 as compared to a reference polypeptide having alpha-amylase activity, wherein the reference polypeptide is the alpha-amylase as set in SEQ ID NO:2.
  • the alpha-amylase variant according to the invention has both an increased thermostability and an increased specific activity at pH 7 as compared to a reference polypeptide having alpha-amylase activity, wherein the reference polypeptide is the alpha-amylase as set in SEQ ID NO:2.
  • the alpha-amylase variant according to the invention has both an increased thermostability and an increased sucrose tolerance as compared to a reference polypeptide having alpha-amylase activity, wherein the reference polypeptide is the alpha-amylase as set in SEQ ID NO:2.
  • the alpha-amylase variant according to the invention has both an increased thermostability and an increased sucrose tolerance at pH 7 as compared to a reference polypeptide having alpha-amylase activity, wherein the reference polypeptide is the alpha-amylase as set in SEQ ID NO:2.
  • the alpha-amylase variant according to the invention has both an increased thermostability at pH 7 and an increased sucrose tolerance at pH 7 as compared to a reference polypeptide having alpha-amylase activity, wherein the reference polypeptide is the alpha-amylase as set in SEQ ID NO:2.
  • the alpha-amylase variant according to the invention has an increased sucrose tolerance and an increased thermostability; as compared to a reference polypeptide having alpha-amylase activity, wherein the reference polypeptide is the alpha-amylase as set in SEQ ID NO:2.
  • the alpha-amylase variant according to the invention has an increased sucrose tolerance at pH 7, an increased Activity pH7 : Activity at pH5 ratio and an increased thermostability at pH 7 as compared to a reference polypeptide having alpha- amylase activity, wherein the reference polypeptide is the alpha-amylase as set in SEQ ID NO:2.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • the variant has an increased thermostability compared with the reference polypeptide as set out in SEQ ID NO: 2, while the specific activity at pH 4 of the variant has reduced by at most 20% as compared with the reference polypeptide (such as the polypeptide of SEQ ID NO: 2).
  • the specific activity at pH 4 of the variant has reduced by at most 30%, in an aspect by at most 40%, in an aspect by at most 50% as compared to the reference polypeptide. In a further aspect of this embodiment the specific activity at pH 4 of the variant is at least the same or has increased compared to the reference polypeptide.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2; and wherein the variant has an increased thermostability compared with the reference polypeptide as set out in SEQ ID NO: 2, while the specific activity at pH 5 of the variant has reduced by at most 20% as compared with the reference polypeptide (such as the polypeptide of SEQ ID NO: 2).
  • the specific activity at pH 5 of the variant has reduced by at most 30%, in an aspect by at most 40%, in an aspect by at most 50% as compared to the reference polypeptide. In a further aspect of this embodiment the specific activity at pH 5 of the variant is at least the same or has increased compared to the reference polypeptide.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 225, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • the variant has an increased thermostability compared with the reference polypeptide as set out in SEQ ID NO: 2, while the specific activity at pH 7 of the variant has reduced by at most 20% as compared with the reference polypeptide (such as the polypeptide of SEQ ID NO: 2).
  • the specific activity at pH 7 of the variant has reduced by at most 30%, in an aspect by at most 40%, in an aspect by at most 50% as compared to the reference polypeptide. In a further aspect of this embodiment the specific activity at pH 7 of the variant is at least the same or has increased compared to the reference polypeptide.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2; and wherein the variant has an increased thermostability compared with the reference polypeptide as set out in SEQ ID NO: 2, while the specific activity at pH 4 of the variant has reduced by at most 20% as compared with the reference polypeptide (such as the polypeptide of SEQ ID NO: 2).
  • the specific activity at pH 4 of the variant has reduced by at most 30%, in an aspect by at most 40%, in an aspect by at most 50% as compared to the reference polypeptide. In a further aspect of this embodiment the specific activity at pH 4 of the variant is at least the same or has increased compared to the reference polypeptide.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • the variant has an increased thermostability compared with the reference polypeptide as set out in SEQ ID NO: 2, while the specific activity at pH 5 of the variant has reduced by at most 20% as compared with the reference polypeptide (such as the polypeptide of SEQ ID NO: 2).
  • the specific activity at pH 5 of the variant has reduced by at most 30%, in an aspect by at most 40%, in an aspect by at most 50% as compared to the reference polypeptide. In a further aspect of this embodiment the specific activity at pH 5 of the variant is at least the same or has increased compared to the reference polypeptide.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 133, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2; and wherein the variant has an increased thermostability compared with the reference polypeptide as set out in SEQ ID NO: 2, while the specific activity at pH 7 of the variant has reduced by at most 20% as compared with the reference polypeptide (such as the polypeptide of SEQ ID NO: 2).
  • the specific activity at pH 7 of the variant has reduced by at most 30%, in an aspect by at most 40%, in an aspect by at most 50% as compared to the reference polypeptide. In a further aspect of this embodiment the specific activity at pH 7 of the variant is at least the same or has increased compared to the reference polypeptide.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • the variant has an increased thermostability compared with the reference polypeptide as set out in SEQ ID NO: 2, while the specific activity at pH 4 of the variant has reduced by at most 20% as compared with the reference polypeptide (such as the polypeptide of SEQ ID NO: 2).
  • the specific activity at pH 4 of the variant has reduced by at most 30%, in an aspect by at most 40%, in an aspect by at most 50% as compared to the reference polypeptide. In a further aspect of this embodiment the specific activity at pH 4 of the variant is at least the same or has increased compared to the reference polypeptide.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2; and wherein the variant has an increased thermostability compared with the reference polypeptide as set out in SEQ ID NO: 2, while the specific activity at pH 5 of the variant has reduced by at most 20% as compared with the reference polypeptide (such as the polypeptide of SEQ ID NO: 2).
  • the specific activity at pH 5 of the variant has reduced by at most 30%, in an aspect by at most 40%, in an aspect by at most 50% as compared to the reference polypeptide. In a further aspect of this embodiment the specific activity at pH 5 of the variant is at least the same or has increased compared to the reference polypeptide.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 (preferably comprises the amino acid substitution S200N) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 70, 124, 133, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • the variant has an increased thermostability compared with the reference polypeptide as set out in SEQ ID NO: 2, while the specific activity at pH 7 of the variant has reduced by at most 20% as compared with the reference polypeptide (such as the polypeptide of SEQ ID NO: 2).
  • the specific activity at pH 7 of the variant has reduced by at most 30%, in an aspect by at most 40%, in an aspect by at most 50% as compared to the reference polypeptide. In a further aspect of this embodiment the specific activity at pH 7 of the variant is at least the same or has increased compared to the reference polypeptide.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 15, 124, 200, 225, 358, said positions being defined with reference to SEQ ID NO: 2;
  • the variant has an increased sucrose tolerance at pH 7, an increased Activity pH7 : Activity at pH5 ratio and an increased thermostability at pH 7 compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 15, 124, 200, 225, 358, said positions being defined with reference to SEQ ID NO: 2;
  • the variant has an increased sucrose tolerance at pH 7 and an increased thermostability at pH 7 compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 15, 124, 200, 225, 358, said positions being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to amino acid 225, said position being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 200, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to amino acid 200, said position being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 200, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2; and wherein the variant has an increased Activity pH7 : Activity at pH5 ratio compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 14, 15, 124, 358, said positions being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to amino acid 358, said position being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to amino acid 358, said position being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to amino acid 358, said position being defined with reference to SEQ ID NO: 2;
  • Activity pH4 Activity at pH5 ratio compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to amino acid 358, said position being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to amino acid 358 said position being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution W70Y) and at least one further substitution of an amino acid residue corresponding to amino acid 358 said position being defined with reference to SEQ ID NO: 2;
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 70, 133, 200, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • the variant has an altered property compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • the altered property is one or more of an increased sucrose tolerance; an increased thermostability; and an increased thermostability in the presence of sucrose.
  • said variant has an amino acid sequence having at least 70% identity with the polypeptide sequence as set out in SEQ ID NO: 2 which, when aligned with the sequence as set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 (preferably comprises the amino acid substitution L225F) and at least one further substitution of an amino acid residue corresponding to any of amino acids 70, 133, 200, 225, 282, 358, said positions being defined with reference to SEQ ID NO: 2;
  • the variant has an altered property compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • the altered property is one or more of an increased sucrose tolerance; an increased thermostability; an increased thermostability in the presence of sucrose; Activity at 60°C : Activity at 37°C ratio and an Activity at 80°C : Activity at 37°C ratio.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the variant has an altered property compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • the altered property is one or more of an increased sucrose tolerance; an increased thermostability; and an increased thermostability in the presence of sucrose.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the variant has an altered property compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • the altered property is an increased sucrose tolerance and anincreased thermostability.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • S200N, L282F, S133T, L225F and S358A said positions being defined with reference to SEQ ID NO: 2, and wherein the variant has an increased sucrose tolerance compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • S200N, L282F, S133T, L225F and S358A said positions being defined with reference to SEQ ID NO: 2,
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the variant has an increased sucrose tolerance at pH7 and an increased thermostability at pH7 compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • the variant has an increased sucrose tolerance at pH7, an increased thermostability at pH7 and an increased Activity at pH7 : Activity at pH5 ratio compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions: W70Y and S200N
  • the variant has an increased sucrose tolerance at pH7, an increased thermostability at pH7 and an increased Activity at pH7 : Activity at pH5 ratio compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • the invention further provides a variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha- amylase comprising the sequence set out in SEQ ID NO: 2, comprises one of the following sets of substitutions:
  • variant has an increased sucrose tolerance at pH7, an increased thermostability at pH7 and an increased Activity at pH7 : Activity at pH5 ratio compared with the reference polypeptide as set out in SEQ ID NO: 2.
  • variant can be used interchangeably. They can refer to either polypeptides or nucleic acids. Variants include substitutions, insertions, deletions, truncations, transversions, and/or inversions, at one or more locations relative to a reference sequence.
  • Variants can be made for example by site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombination approaches known to a skilled person in the art.
  • polypeptide' includes proteins.
  • the polypeptides according to the invention comprise only conventional or natural amino acids.
  • the single letter code for amino acids is used, where A stands for Alanine, C for Cysteine, D for Aspartic acid, E for Glutamic acid, F for Phenylalanine, G for Glycine, H for Histidine, I for Isoleucine, K for Lysine, L for Leucine, M for Methionine, N for Asparagine, P for Proline, Q for Glutamine, R for Arginine, S for Serine, T for Threonine, V for Valine, W for Tryptophan, Y for Tyrosine.
  • Such single letter codes are commonly known in the art, see e.g. Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2 nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • peptide and oligopeptide are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages.
  • polypeptide is used herein for chains containing more than about seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one- letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • a polypeptide of the invention may be in isolated form, such as substantially isolated form.
  • isolated polypeptide or protein is intended a polypeptide or protein removed from its native environment.
  • recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are recombinant polypeptides which have been substantially purified by any suitable technique.
  • a polypeptide variant according to the invention can be recovered and purified from recombinant cell cultures by methods known in the art.
  • Polypeptides of the present invention include products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • a polypeptide according to the present invention may be a fusion protein.
  • Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame. Expression of the fused polypeptide is under control of the same promoter (s) and terminator.
  • the hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to a host cell.
  • Such fusion polypeptides from at least two different polypeptides may comprise a binding domain from one polypeptide, such as a starch binding domain or a carbohydrate binding domain, operably linked to a catalytic domain from a second polypeptide. Examples of fusion polypeptides and signal sequence fusions are for example as described in WO2010/121933, WO2013/007820 and WO2013/007821.
  • a polypeptide according to the present invention may comprise a catalytic domain and a binding domain, such as a starch or carbohydrate binding domains.
  • the invention also features biologically active fragments of the polypeptide variants according to the invention. Such fragments are considered to be encompassed within the term "a variant of the invention”.
  • Biologically active fragments of a polypeptide variant of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a variant protein of the invention which include fewer amino acids than the full length protein but which exhibit at least one biological activity of the corresponding full-length protein.
  • biologically active fragments comprise a domain or motif with at least one activity of a variant protein of the invention.
  • a biologically active fragment of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the invention.
  • a protein fragment of the invention will comprise one or more of the substitutions defined herein.
  • the invention also features nucleic acid fragments which encode the above biologically active fragments (which biologically active fragments are themselves variants of the invention).
  • the present invention provides polynucleotides encoding the variant polypeptides of the invention.
  • the invention also relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide variant of the invention. Typically, such a domain will comprise one or more of the substitutions described herein.
  • the nucleic acid sequence according to the invention encodes a polypeptide, wherein the polypeptide is a variant comprising an amino acid sequence that has one or more truncation(s), and/or at least one substitution, deletion and/or insertion of an amino acid as compared to the parent alpha-amylase.
  • a polypeptide will, however, typically comprise one or more of the substitutions described herein.
  • the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a variant as described herein.
  • a gene may include coding sequences, non-coding sequences, introns and regulatory sequences. That is to say, a “gene”, as used herein, may refer to an isolated nucleic acid molecule as defined herein. Accordingly, the term “gene”, in the context of the present application, does not refer only to naturally-occurring sequences.
  • a nucleic acid molecule of the present invention can be generated using standard molecular biology techniques well known to those skilled in the art taken in combination with the sequence information provided herein.
  • a nucleic acid molecule of the present invention can be adapted according to the method described in patent application US090286280.
  • nucleic acid molecule may be synthesized de novo. Such a synthetic process will typically be an automated process.
  • a nucleic acid molecule of the invention may be generated by use of site-directed mutagenesis of an existing nucleic acid molecule, for example a wild-type nucleic acid molecule. Site-directed mutagenesis may be carried out using a number of techniques well known to those skilled in the art.
  • PCR is carried out on a plasmid template using oligonucleotide "primers" encoding the desired substitution.
  • primers are the ends of newly-synthesized strands, should there be a mis-match during the first cycle in binding the template DNA strand, after that first round, the primer-based strand (containing the mutation) would be at equal concentration to the original template. After successive cycles, it would exponentially grow, and after 25, would outnumber the original, unmutated strand in the region of 8 million: 1 , resulting in a nearly homogeneous solution of mutated amplified fragments.
  • the template DNA may then be eliminated by enzymatic digestion with, for example using a restriction enzyme which cleaves only methylated DNA, such as Dpn1.
  • a restriction enzyme which cleaves only methylated DNA, such as Dpn1.
  • the template which is derived from an alkaline lysis plasmid preparation and therefore is methylated, is destroyed in this step, but the mutated plasmid is preserved because it was generated in vitro and is unmethylated as a result.
  • more than one mutation (encoding a substitution as described herein) may be introduced into a nucleic acid molecule in a single PCR reaction, for example by using one or more oligonucleotides, each comprising one or more mis-matches.
  • more than one mutation may be introduced into a nucleic acid molecule by carrying out more than one PCR reaction, each reaction introducing one or more mutations, so that altered nucleic acids are introduced into the nucleic acid in a sequential, iterative fashion.
  • a nucleic acid of the invention can be generated using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate mis-matched oligonucleotide primers according to the site-directed mutagenesis technique described above.
  • a nucleic acid molecule derived in this way can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • a nucleic acid sequence of the invention may comprise one or more deletions, i.e. gaps, in comparison to the parent alpha-amylase. Such deletions/gaps may also be generated using site-directed mutagenesis using appropriate oligonucleotides. Techniques for generating such deletions are well known to those skilled in the art. Furthermore, oligonucleotides corresponding to or hybridizable to nucleotide sequences according to the invention can be prepared by standard synthetic techniques, e.g. using an automated DNA synthesizer.
  • a nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex.
  • One aspect of the invention pertains to isolated nucleic acid molecules that encode a variant of the invention, or a biologically active fragment or domain thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules, such as for the preparation of nucleic acid molecules of the invention.
  • an "isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promotor) sequences that are immediately contiguous to the coding sequence.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
  • nucleic acid molecule As used herein, the terms “polynucleotide” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • the nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a nucleic acid molecule of the invention.
  • the terms “homology” or “percent identity” are used interchangeably herein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • % identity number of identical positions/total number of positions (i.e. overlapping positions) x 100).
  • the two sequences are the same length.
  • a sequence comparison may be carried out over the entire lengths of the two sequences being compared or over fragment of the two sequences. Typically, the comparison will be carried out over the full length of the two sequences being compared. However, sequence identity may be carried out over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.
  • the percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm.
  • the Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. In an aspect the NEEDLE program from the EMBOSS package is used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. LongdenJ. and BleasbyA Trends in Genetics 16, (6) p.
  • EBLOSUM62 is used for the substitution matrix.
  • EDNAFULL is used for the substitution matrix.
  • the optional parameters used are a gap- open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.
  • the identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as "longest-identity".
  • the protein sequences or nucleic acid sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403—10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • vectors preferably expression vectors, containing a nucleic acid encoding a variant alpha-amylase polypeptide of the invention.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably herein as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g. tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. an alpha-amylase variant of SEQ ID NO: 2, for example a functional equivalent or fragment, or a fusion protein comprising one or more of such variants).
  • nucleic acids as described herein (e.g. an alpha-amylase variant of SEQ ID NO: 2, for example a functional equivalent or fragment, or a fusion protein comprising one or more of such variants).
  • the recombinant expression vectors of the invention can be designed for expression of variant proteins of the invention in prokaryotic or eukaryotic cells.
  • a variant protein of the invention can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • Other suitable promoters will be known to the skilled person.
  • promoters are preferred that are capable of directing a high expression level of alpha-amylase in filamentous fungi. Such promoters are known in the art.
  • the expression constructs may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will include a translation initi
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-percipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methatrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a variant protein of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • nucleic acid sequences according to the invention can be cloned in a suitable vector and after introduction in a suitable host, the sequence can be expressed to produce the corresponding alpha-amylase variants according to standard cloning and expression techniques, which are known to the person skilled in the art (e. g., as described in (Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001 ).
  • the invention also relates to such vectors comprising a nucleic acid sequence according to the invention.
  • Suitable vectors are the vectors normally used for cloning and expression and are known to the person skilled in the art. Examples of suitable vectors for expression in E. coli are given e.g. in table 1 in Makrides, S. C, Microbiological Reviews, Vol. 60, No. 3, (1996), 512-538.
  • the vector contains a promoter upstream of the cloning site containing the nucleic acid sequence encoding the polypeptide with alpha-amylase activity, which can be switched on after the host has been grown to express the corresponding polypeptide having alpha-amylase activity.
  • Promoters which can be switched on and off are known to the person skilled in the art and are for example the lac promoter, the aroH promoter, the araBAD promoter, the T7 promoter, the trc promoter, the tac promoter and the trp promoter.
  • Particularly useful in the framework of the invention are for example the vectors as described in WO 00/66751 , e.g. pKAFssECtrp or pKAFssECaro without the insert, the penicillin G acylase gene.
  • Suitable hosts are the hosts normally used for cloning and expression and are known to the person skilled in the art. Examples of suitable host strains are for example Echerichia coli strains, e.g. £.
  • Escherichia coli K- 12 strains e.g. DH1 , HB101 , RV308, RR1 , W31 10, C600 and/or derivatives of these strains.
  • the choice of the vector can sometimes depend on the choice of the host and vice versa. If e.g. a vector with the araBAD promoter is being used, an E. coli host strain that is unable to break down the arabinose inducer (ara-), is strongly preferred.
  • nucleic acid constructs there are several ways of inserting a nucleic acid into a nucleic acid construct or an expression vector which are known to a skilled person in the art. It may be desirable to manipulate a nucleic acid encoding a polypeptide of the present invention with control sequences, such as promoter and terminator sequences.
  • a promoter may be any appropriate promoter sequence suitable for a eukaryotic or prokaryotic host cell, which shows transcriptional activity, including mutant, truncated, and hybrid promoters, and may be obtained from polynucleotides encoding extracellular or intracellular polypeptides either endogenous (native) or heterologous (foreign) to the cell.
  • the promoter may be a constitutive or inducible promoter.
  • the promoter is an inducible promoter, for instance a starch inducible promoter.
  • Promoters suitable in filamentous fungi are promoters which may be selected from the group, which includes but is not limited to promoters obtained from the polynucleotides encoding A.
  • oryzae TAKA amylase Rhizomucor miehei aspartic proteinase, Aspergillus gpdA promoter, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or A. awamori glucoamylase (glaA), A. niger or A. awamori endoxylanase (xlnA) or beta-xylosidase (x/nD), T. reesei cellobiohydrolase I (CBHI), R. miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, A.
  • Trichoderma reesei beta-glucosidase Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesese
  • Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or GAL1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, EN01, TPI1, and AOX1.
  • Other suitable promoters include PDC1, GPD1, PGK1, TEF1, and TDH3. All of the above-mentioned promoters are readily available in the art.
  • Suitable inducible promoters useful in bacteria include: promoters from Gram-positive microorganisms such as, but are not limited to, SP01 -26, SP01 -15, veg, pyc (pyruvate carboxylase promoter), and amyE.
  • promoters from Gram- negative microorganisms include, but are not limited to, tac, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, P15, T7, T5, T3, gal, trc, ara, SP6, ⁇ -PR, and ⁇ -PL.
  • any terminator which is functional in a cell as disclosed herein may be used, which are known to a skilled person in the art.
  • suitable terminator sequences in filamentous fungi include terminator sequences of a filamentous fungal gene, such as from Aspergillus genes, for instance from the gene A. oryzae TAKA amylase, the genes encoding A. niger glucoamylase (glaA), A. nidulans anthranilate synthase, A. niger alpha-glucosidase, trpC and/or Fusarium oxysporum trypsin-like protease
  • Fusion vectors add a number of amino acids to a protein encoded therein, e.g. to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1 ) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • the expression vectors will preferably contain selectable markers.
  • Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracyline or ampicillin resistance for culturing in E. coli and other bacteria.
  • appropriate host include bacterial cells, such as E. coli, Streptomyces Salmonella typhimurium and certain Bacillus species; fungal cells such as Aspergillus species, for example A. niger, A. oryzae and A. nidulans, such as yeast such as Kluyveromyces, for example K. lactis and/or Puchia, for example P.
  • insects such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS and Bowes melanoma
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • Vectors preferred for use in bacteria are for example disclosed in WO-A1 - 2004/074468, which are hereby enclosed by reference. Other suitable vectors will be readily apparent to the skilled artisan.
  • Known bacterial promotors suitable for use in the present invention include the promoters disclosed in WO-A1 -2004/074468, which are hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretation signal may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • a variant of the invention may be expressed in form such that it may include additional heterologous functional regions, for example secretion signals.
  • a variant of the invention may also comprise, for example, a region of additional amino acids, particularly charged amino acids, added to the N-terminus of the polypeptide for instance to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • peptide moieties may be added to a variant of the invention to facilitate purification, for example by the addition of histidine residues or a T7 tag.
  • variants of the invention such as proteins of the present invention or functional equivalents thereof, e.g., biologically active portions and fragments thereof, can be operatively linked to a non-variant polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
  • a non-variant polypeptide e.g., heterologous amino acid sequences
  • a "non-variant polypeptide” in this context refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to a variant alpha-amylase of the invention.
  • the variant of the invention can correspond to a full length sequence or a biologically active fragment of a polypeptide of the invention.
  • a fusion protein of the invention comprises at least two biologically active portions.
  • the term "operatively linked" is intended to indicate that the variant polypeptide and the non-variant polypeptide are fused in-frame to each other.
  • the non-variant polypeptide can be fused to the N- terminus or C-terminus of the variant polypeptide.
  • a variant alpha-amylase may be enhanced by expressing the variant in the form of a fusion protein.
  • a nucleic acid sequence may encode for a fusion protein comprising pre-alpha-amylase or alpha- amylase. More specifically, the fusion partner may be glucoamylase or a fragment thereof.
  • the pre-alpha-amylase or alpha-amylase, or a fusion protein thereof is secreted over the host cell membrane.
  • the fusion protein is a fusion protein in which the variant sequence/s is/are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of a recombinant variant according to the invention.
  • the fusion protein is a variant of the invention containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of a variant of the invention can be increased through use of a hetereologous signal sequence.
  • the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).
  • Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California).
  • useful prokarytic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
  • a signal sequence can be used to facilitate secretion and isolation of a variant of the invention.
  • Signal sequences are typically characterized by a core of hydrophobic amino acids, which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.
  • the signal sequence may direct secretion of the variant, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence may then be subsequently or concurrently cleaved.
  • the variant of the invention may then be readily purified from the extracellular medium by known methods.
  • the signal sequence can be linked to the variant of interest using a sequence, which facilitates purification, such as with a GST domain.
  • the sequence encoding the variant of the invention may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused variant of the invention.
  • the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the HA tag is another peptide useful for purification which corresponds to an epitope derived of influenza hemaglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance.
  • a fusion protein of the invention may be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers, which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide).
  • a variant-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the said variant.
  • Functional equivalents and “functional variants” are used interchangeably herein.
  • Functional equivalents according to the invention are isolated DNA fragments that encode a polypeptide that exhibits a particular function of a variant as defined herein. Functional equivalents therefore also encompass biologically active fragments and are themselves encompassed within the term "a variant" of the invention.
  • a functional equivalent of the invention comprises one or more of the substitutions described herein.
  • a functional equivalent may comprise one or more modifications in addition to the substitutions described above.
  • nucleic acid equivalents may typically contain silent mutations or mutations that do not alter the biological function of encoded polypeptide. Accordingly, the invention provides nucleic acid molecules encoding a variant alpha-amylase protein that contains changes in amino acid residues that are not essential for a particular biological activity. Such variant proteins differ in amino acid sequence from the parent alpha-amylase sequence from which they are derived yet retain at least one biological activity thereof, preferably they retain at least alpha-amylase activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises a substantially homologous amino acid sequence of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the reference amino acid sequence (for example that shown in SEQ ID NO: 2).
  • substantially homologous refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequences have a common domain.
  • amino acid or nucleotide sequences which contain a common domain having about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or more are defined herein as sufficiently identical.
  • an alpha-amylase variant of the invention is preferably a protein which comprises an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the reference amino acid sequence, for example that shown in SEQ ID NO: 2, and typically also retains at least one functional activity of the reference polypeptide.
  • Variants of the invention for example functional equivalents of a protein according to the invention, can also be identified e.g.
  • a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level.
  • a variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display).
  • the recombinant host cell is preferably capable of expressing or overexpressing the nucleotide sequence or nucleic acid molecule according to the invention
  • the mutant microbial host cell may further comprise one or more modifications in its genome such that the mutant microbial host cell is deficient in the production of at least one product selected from glucoamylase (glaA), acid stable alpha-amylase (amyA), neutral alpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, PepA, a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthase npsE, agsE or amyC if compared to a parent host cell and measured under the same conditions.
  • Suitable methods of producing said host cells include the
  • libraries of fragments of the sequence encoding a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
  • REM Recursive ensemble mutagenesis
  • Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction.
  • Nucleic acids according to the invention irrespective of whether they encode functional or non-functional polypeptides can be used as hybridization probes or polymerase chain reaction (PCR) primers.
  • Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having alpha-amylase activity include, inter alia, (1 ) in situ hybridization (e.g.
  • FISH FISH to metaphase chromosomal spreads to provide precise chromosomal location of an alpha-amylase-encoding gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (2) Northern blot analysis for detecting expression of alpha-amylase mRNA in specific tissues and/or cells; and (3) probes and primers that can be used as a diagnostic tool to analyse the presence of a nucleic acid hybridizable to such a probe or primer in a given biological (e.g. tissue) sample.
  • a nucleic acid hybridizable to such a probe or primer in a given biological (e.g. tissue) sample.
  • Variants of a given reference alpha-amylase enzyme can be obtained by the following standard procedure: Mutagenesis (error-prone, doped oligo, spiked oligo) or synthesis of variants
  • the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention.
  • a "transformed cell” or “recombinant cell” is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention.
  • Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from yeasts, for example, K. lactis.
  • Host cells also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.
  • suitable bacterial host organisms are gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium and Bacillus thu- ringiensis, Streptomyces species such as Streptomyces murinus, lactic acid bacterial species including Lactococcus spp. such as Lactococcus lactis, Lactobacillus spp.
  • Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans
  • strains of a gram negative bacterial species such as a species belonging to Enterobacteriaceae, including E. coli or to Pseudomonadaceae may be selected as the host organism.
  • a suitable yeast host organism may advantageously be selected from a species of Saccharomyces including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces.
  • Further useful yeast host organisms include Pichia spp. such as methylotrophic species hereof, including Pichia pastoris, and Klyuveromyces spp. including Klyuveromyces lactis.
  • Suitable host organisms among filamentous fungi include species of Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophtora, Neurospora, Penicillium, Rasamsonia, Thielavia, Tolypocladium or Trichoderma, such as e. g.
  • Aspergillus aculeatus Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus oryzae, Aspergillus nidulans or Aspergillus niger, including Aspergillus nigervar.
  • Fusarium bactridioides Fusa- rium cereals, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichiodes, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola langinosa, Mucor miehei,
  • Myceliophtora thermophila Neurospora crassa, Penicillium chrysogenum, Penicillium camenbertii, Penicillium purpurogenum, Rasamsonia emersonii, Rhizomucor miehei, Thielavia terestris, Tricho- derma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesii or Trochoderma viride.
  • a host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the product encoded by the incorporated nucleic acid sequence in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the encoded protein.
  • Various host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins and gene products.
  • Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.
  • Such host cells are well known in the art.
  • a stably transfected cell line can produce a variant according to the invention.
  • a number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al. (supra).
  • the present invention also provides a nucleic acid sequence encoding the variant polypeptides of the invention.
  • the invention thus also provides a nucleic acid sequence encoding a variant polypeptide having alpha-amylase activity which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 70 and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention thus also provides a nucleic acid sequence encoding a variant polypeptide having alpha-amylase activity which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 225 and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the invention thus also provides a nucleic acid sequence encoding a variant polypeptide having alpha-amylase activity which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises a substitution of an amino acid residue corresponding to amino acid 200 and at least one further substitution of an amino acid residue corresponding to any of amino acids
  • positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having alpha- amylase activity (such as the polypeptide of SEQ ID NO: 2).
  • the present invention also provides a nucleic acid construct comprising the nucleic acid sequence of the invention operably linked to one or more control sequences capable of directing the expression of an alpha-amylase in a suitable expression host.
  • the present invention also provides a recombinant expression vector comprising the nucleic acid construct of the invention.
  • the present invention also provides a recombinant host cell comprising the expression vector of the invention.
  • the present invention also provides a method for producing an alpha-amylase comprising cultivating the host cell of the invention under conditions conducive to production of the alpha-amylase and recovering the alpha-amylase.
  • the present invention also provides a method of producing an alpha-amylase polypeptide variant according to the invention, which method comprises:
  • the reference polypeptide has the sequence as set in SEQ ID NO: 2.
  • the reference polypeptide may have a sequence having at least 70% identity to the sequence as set in SEQ ID NO: 2.
  • the present invention also provides a composition
  • a composition comprising the variant polypeptide according to the invention or obtainable by a method according to the invention and one or more components selected from the group consisting of milk, gluten, granulated fat, an additional enzyme, an amino acid, a salt, oxidants, reducing agents, emulsifiers, gums, flavours, acids, starch, modified starch, humectants and preservatives.
  • a composition according to the invention comprises the variant polypeptide of the invention.
  • the present invention further discloses a composition
  • a composition comprising the alpha- amylase variants according to the invention and one or more components selected from the group consisting of milk (including liquid milk and milk powder), gluten, granulated fat, an additional enzyme, an amino acid, a salt, oxidants (including ascorbic acid, bromate and Azodicarbonamide (ADA), reducing agents (including L-cysteine), emulsifiers (including without limitation mono- and diglycerides, monoglycerides such as glycerol monostearate (GMS), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE) and diacetyl tartaric acid esters of mono- and diglycerides (DATEM), propylene glycol monostearate (PGMS), lecithin), gums (including guargum and xanthangum), flavours, acids (including citric acid, propi
  • the additional enzyme may include including a further alpha-amylase, such as a fungal alpha-amylase (which may be useful for providing sugars fermentable by yeast and retarding staling), beta- amylase, a cyclodextrin glucanotransferase, a protease, a peptidase, in particular, an exopeptidase (which may be useful in flavour enhancement), transglutaminase, triacyl glycerol lipase (which may be useful for the modification of lipids present in the dough or dough constituents so as to soften the dough), galactolipase, phospholipase, cellulase, hemicellulase, in particular a pentosanase such as xylanase (which may be useful for the partial hydrolysis of pentosans, more specifically arabinoxylan, which increases the extensibility of the dough), protease (which may be useful for the fungal alpha-amylase
  • the additional enzyme is a lipolytic enzyme.
  • a lipolytic enzyme also referred to herein as lipase, is an enzyme that hydrolyses triacylglycerol and/or galactolipid and/or phospholipids.
  • the specificity of the lipase can be shown through in vitro assay making use of appropriate substrate, for example triacylglycerol lipid, phosphatidylcholine and digalactosyldiglyceride, or preferably through analysis of the reactions products that are generated in the dough during mixing and fermentation.
  • the triacyl glycerol lipase may be a fungal lipase, preferably from Rhizopus, Aspergillus, Candida, Penicillum, Thermomyces, or Rhizomucor.
  • the triacyl glycerol lipase is from Rhyzopus, in a further embodiment a triacyl glycerol lipase from Rhyzopus oryzae is used.
  • a combination of two or more triacyl glycerol lipases may be used
  • the additional enzyme is a phospholipase.
  • the additional enzyme is a galactolipase.
  • the additional enzyme is an enzyme having both phospholipase and galactolipase activity.
  • a phospholipase is an enzyme that catalyzes the release of fatty acyl groups from a phospholipid. It may be a phospholipase A2 (PLA2, EC 3.1 .1.4) or a phospholipase A1 (EC 3.1 .1.32). It may or may not have other activities such as triacylglycerol lipase (EC 3.1 .1.3) and/or galactolipase (EC 3.1.1.26) activity.
  • the phospholipase may be a native enzyme from mammalian or microbial sources.
  • a mammalian phospholipase is pancreatic PLA2, e.g. bovine or porcine PLA2 such as the commercial product Lecitase 10L (porcine PLA2, product of Novozymes A S).
  • Microbial phospholipases may be from Fusarium, e.g. F. oxysporum phospholipase A1 (WO 1998/026057 ), F. venenatum phospholipase A1 (described in WO 2004/097012 as a phospholipase A2 called FvPLA2), from Tuber, e.g. T borchii phospholipase A2 (called TbPLA2, WO 2004/097012 ).
  • the phospholipase may also be a lipolytic enzyme variant with phospholipase activity, e.g. as described in WO 2000/032758 or WO 2003/0601 12 .
  • the phospholipase may also catalyze the release of fatty acyl groups from other lipids present in the dough, particularly wheat lipids.
  • the phospholipase may have triacylglycerol lipase activity (EC 3.1 .1.3) and/or galactolipase activity (EC 3.1 .1 .26).
  • the phospholipase may be a lipolytic enzyme as described in WO2009/106575, such as the commercial product Panamore®, product of DSM.
  • Panamore®, Lipopan® F, Lipopan® 50 and Lipopan® S are commercialised to standardised lipolytic activity, using a measurement of DLU for Panamore® from DSM and a measurement of LU for the Lipopan® family from Novozymes.
  • DLU is defined as the amount of enzyme needed to produce 1 micromol/min of p-nitrophenol from p- nitrophenyl palmitate at pH 8.5 at 37°C
  • LU is defined as the amount of enzyme needed to produce 1 micromol/min of butyric acid from tributyrin at pH 7 at 30 °C.
  • Lipases are optimally used with the alpha-amylase of the invention at 2-850 DLU/kg flour or at 50-23500 LU/kg flour.
  • the cellulase may be from A. niger or from Trichoderma reesei.
  • the amyloglucosidase may be an amyloglucosidase from Aspergillus such as from A. oryzae or A. niger, preferably from A. niger.
  • the additional enzyme may include without limitation an enzyme as disclosed in any of US 4,598,048; US 4,604,355; US RE38,507; W099/43794; W099/43794, in particular in EP1058724B1 ; WO2004/081 171 ; WO2006/012899; WO2008/148845; WO2006/032281 .
  • the additional enzyme may include without limitation an enzyme as disclosed in any of US8426182; WO20131 13665; non-prepublished European patent application having a filing number EP13183144.8.
  • the additional enzyme may include a G4-forming amylase.
  • a G4-forming amylase is an enzyme that is inter alia capable of catalysing the degradation of starch. In particular it is capable of cleaving a-D-(l— >4) O-glycosidic linkages in starch. It may be referred to as a glucan 1 ,4-alpha-maltotetraohydrolase (EC 3.2.1.60). It may also be referred as a maltotetraohydrolase.
  • Pseudomonas saccharophila (GenBank Acc. No. X16732) expresses a G4-forming amylase.
  • the G-4 forming amylase is capable of producing maltotetraose from either liquefied starch or other source of maltodextrins at a high temperature e.g. about 60 °C to about 75 °C.
  • Suitable G4-forming amylases may be G4-forming amylases described in any one of WO9950399, WO2005007818, WO20041 1 1217, WO2005003339, WO2005007818, WO2005007867, WO2006003461 , WO2007007053, WO2007148224, WO2009083592, WO2009088465.
  • the enzyme composition according to the invention is provided in a dry form, to allow easy addition to the dough, the dough ingredients, but liquid forms are also possible.
  • a liquid form includes without limitation an emulsion, a suspension and a solution. Irrespective of the formulation of the enzyme composition, any additive or additives known to be useful in the art to improve and/or maintain the enzyme's activity, the quality of a dough and/or a baked product may be applied.
  • the present invention also provides a pre-mix comprising flour and the variant polypeptide according to the invention or obtainable by the method of the invention.
  • the alpha-amylase variant according to the invention may be incorporated in a pre-mix, e.g. in the form of a flour composition, for dough and/or baked products made from dough, in which the pre-mix comprises a polypeptide of the present invention.
  • pre-mix is defined herein to be understood in its conventional meaning, i.e. as a mix of baking agents, generally including flour, which may be used not only in industrial bread-baking plants/facilities, but also in retail bakeries.
  • the pre-mix may be prepared by mixing the alpha-amylase polypeptide variant according to the invention or the enzyme composition according to the invention with a suitable carrier such as flour, starch or a salt.
  • the pre-mix may contain additives as mentioned herein.

Abstract

La présente invention concerne un polypeptide variant possédant une activité alpha-amylase, variant possédant une ou plusieurs propriétés modifiées par rapport à un polypeptide de référence ayant une activité alpha-amylase. Un tel polypeptide variant peut être utilisé dans l'industrie alimentaire, comme par exemple, dans la préparation d'un produit cuit au four.
PCT/EP2015/069612 2014-08-28 2015-08-27 Variants alpha-amylase d'alicyclobacillus pohliae WO2016030448A1 (fr)

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EP14182624.8 2014-08-28
EP14182624 2014-08-28
EP15163870.7 2015-04-16
EP15163870 2015-04-16

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WO2016030448A1 true WO2016030448A1 (fr) 2016-03-03

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11653655B2 (en) 2016-07-15 2023-05-23 Novozymes A/S Improving the rollability of flat breads
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008148845A2 (fr) * 2007-06-07 2008-12-11 Novozymes A/S Procédé de préparation d'un produit à base de pâte
WO2011058105A1 (fr) * 2009-11-13 2011-05-19 Novozymes A/S Procédé de brassage
US8426182B1 (en) * 2012-01-30 2013-04-23 Dsm Ip Assets B.V. Alpha-amylase
WO2014131842A1 (fr) * 2013-03-01 2014-09-04 Dsm Ip Assets B.V. Variants d'alpha-amylase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008148845A2 (fr) * 2007-06-07 2008-12-11 Novozymes A/S Procédé de préparation d'un produit à base de pâte
WO2011058105A1 (fr) * 2009-11-13 2011-05-19 Novozymes A/S Procédé de brassage
US8426182B1 (en) * 2012-01-30 2013-04-23 Dsm Ip Assets B.V. Alpha-amylase
WO2014131842A1 (fr) * 2013-03-01 2014-09-04 Dsm Ip Assets B.V. Variants d'alpha-amylase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE UniProt [online] 19 February 2014 (2014-02-19), "SubName: Full=Alpha amylase {ECO:0000313|EMBL:CDK35218.1};", XP002744948, retrieved from EBI accession no. UNIPROT:V6DNK0 Database accession no. V6DNK0 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11653655B2 (en) 2016-07-15 2023-05-23 Novozymes A/S Improving the rollability of flat breads
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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