ES2856687T3 - Amylase polypeptides - Google Patents

Abstract

A polypeptide having amylase activity that comprises an amino acid sequence that has at least 78% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises an amino acid substitution at position 311, with reference to the position numbering of the sequence shown as SEQ ID NO: 1, wherein the polypeptide has improved thermostability compared to amylase of SEQ ID NO: 1.

Description

DESCRIPTION

Amylase polypeptides

Field of the invention

This invention relates to polypeptides, specifically amylase polypeptides and nucleic acids encoding these, and their uses for example, as non-maltogenic exoamylases in food production or animal feed products.

Background of the invention

Improved amylases can ameliorate problems inherent in certain processes, such as in the conversion of vegetable starches, or in baking.

Amylopectin crystallization takes place in the starch granules days after baking, which leads to an increase in the firmness of the bread and causes the aging of the bread. When bread ages, the bread loses the softness of the crumb and the moisture of the crumb. As a result, the crumbs become less elastic, and the bread develops a leather crust.

Enzymatic hydrolysis (eg, by amylases) of amylopectin side chains can reduce crystallization and enhance anti-aging. Crystallization depends on the length of the amylopectin side chains: the longer the side chains, the greater the crystallization. Most starch granules are made up of a mixture of two polymers: amylopectin and amylose, of which approximately 75% is amylopectin. Amylopectin is a very large, branched molecule consisting of chains of α-D-glucopyranosyl units units by bonds (1-4), in which the chains are joined by α-D- (1-6) bonds to form branches. Amylose is a straight chain of linked α-D-glucopyranosyl units (1-4) that have few α-D- (1-6) branches.

The baking of farinaceous bakery items such as white bread, bread made from rye flour and sifted wheat flour and rollers is achieved by baking the bread dough at oven temperatures in the range of 180 to 250 ° C during approximately 15 to 60 minutes. During the baking process a pronounced temperature gradient (200 to 120 ° C) prevails over the outer dough layers where the crust of the baked product develops. However, due to the steam, the temperature in the crumb is only about 100 ° C at the end of the baking process. At temperatures above about 85 ° C, enzyme inactivation may occur and the enzyme will not have anti-aging properties. Therefore, only thermostable amylases are able to efficiently modify starch during baking.

Endoamylase activity can adversely affect the quality of the final bread product by producing a sticky or gummy crumb due to the accumulation of branched dextrins. Exoamylase activity is preferred, because it carries out the desired starch modification that leads to retardation of aging, with fewer of the negative effects associated with endo-amylase activity. Reduced endoamylase activity can lead to higher exospecificity, which can reduce branched dextrins and produce better quality bread.

Converting vegetable starches, especially cornstarch, to ethanol is a rapidly expanding industry. Maltotetraose syrup (G4 or DP4) is one of many commercially important products derived from the enzymatic treatment of starch. The conversion of vegetable starches, especially cornstarch, to maltotetraose and lower sugars, such as glucose or maltose, is a rapidly expanding industry. The current process consists of two sequential enzyme catalyzed stages that result in the production of glucose or maltose. Yeast can be used to ferment glucose to ethanol.

The first stage is catalyzed by the enzyme is the liquefaction of starch. Typically, a starch suspension is gelatinized by rapid heating to 85 ° C or higher. • -amylases (EC 3.2.1.1) are used to degrade the viscous liquefied to maltodextrins. • -amylases are endohydrolases that catalyze the random cleavage of internal • -1,4-D-glucosidic bonds. As the • -amylases break down the starch, the viscosity decreases. Because liquefaction is typically carried out at elevated temperatures, thermostable • -amylases, such as a • -amylase from Bacillus sp., Are preferred for this step.

Maltodextrins produced in this way generally cannot be fermented by yeast to form alcohol. Consequently, a second stage of enzymatically catalyzed saccharification is required to break down the maltodextrins. Glucoamylases and / or maltogenic • -amylases are commonly used to catalyze the hydrolysis of non-reducing ends of maltodextrins formed after liquefaction, the release of D-glucose, maltose and isomaltose. Debranching enzymes such as pullulanases can be used to aid saccharification. Saccharification is generally carried out under acidic conditions at elevated temperatures, eg 60 ° C, pH 4.3.

G4 syrup (also referred to as DP4) has numerous advantageous properties compared to sucrose syrups. For example, the partial replacement of sucrose with G4 syrup in a food reduces the sweetness of the food without affecting its taste or aroma. G4 syrup has high moisture retention in foods and exhibits less harmful Maillard reaction products due to lower glucose and maltose content. G4 syrup also has a higher viscosity than sucrose, which improves the texture of foods. G4 syrup lowers the freezing point of water less than sucrose or high fructose syrup, so G4 syrup can better control the freezing points of frozen foods. After ingestion, G4 syrup also affects osmotic pressure less than sucrose. Together these qualities make G4 syrup ideal as an ingredient in food and medical products. G4 syrup is useful in other industries, too. For example, G4 syrup imparts shine and can be used to advantage as a paper meter. See, for example, Kimura et al, "Maltotetraose, a new saccharide of tertiary property", Starch 42: 151-57 (1990).

One of the yeasts used to produce ethanol is Saccharomyces cerevisiae. S. cerevisiae contains • glucosidase which has been shown to use mono-, di-, and tri-saccharides as substrates. Yoon et al., Carbohydrate Res. 338: 1127-32 (2003). The ability of S. cerevisiae to utilize tri-saccharides can be enhanced by Mg2 + supplementation and AGT1 permease overexpression (Stambuck et al, Lett Appl Microbiol 43: 370-76 (2006)), MTT1 and MTT1alt overexpression to increase the uptake of maltotriose (Dietvorst et al., Yeast 22: 775-88 (2005)), or the expression of MAL32 maltase on the cell surface (Dietvorst et al., Yeast 24: 27 38 (2007)). The saccharification step can be omitted entirely, if the liquefaction step produced sufficient levels of mono-, di-, or tri-saccharides and S. cerevisiae or its genetically modified variants were used for the fermentation step.

Pseudomonas saccharophila expresses a maltotetraose-forming maltotetraohydrolase (EC 3.2.1.60; G4-forming amylase; G4-amylase). The nucleotide sequence of the P. saccharophila gene encoding PS4 has been determined. Zhou et al., "Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila", FEBS Lett. 255: 37-41 (1989); GenBank Acc. No. X16732. PS4 is expressed as a precursor protein with an N-terminal signal peptide of 21 residues The mature form of PS4, as set forth in SEQ ID NO: 10, contains 530 amino acid residues with a catalytic domain at the N-terminus and a starch-binding domain at the C-terminus. PS4 it shows both endo and exo-a-amylase activity Endoa-amylase activity is useful in lowering the viscosity of gelatinized starch, and exo-a-amylase activity is useful in breaking down maltodextrins into smaller saccharides.

WO 2007/148224 refers to a variant PS4 polypeptide that can be derived from a parent polypeptide having non-maltogenic exo-amylase activity.

WO 02/068589 refers to alpha amylases and polynucleotides encoding alpha amylases, which have increased activity and stability at acidic and alkaline pH and increased temperature.

Synthesis of the invention

The present invention provides a polypeptide having amylase activity comprising an amino acid sequence that has at least 78% sequence identity to the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises a substitution of amino acid at position 311, with reference to the position numbering of the sequence shown as SEQ ID NO: 1; wherein the polypeptide has improved thermostability compared to amylase of SEQ ID NO: 1.

This invention relates to polypeptides, specifically amylase polypeptides and nucleic acids encoding them, and their uses for example as non-maltogenic exoamylases in the production of food products. The amylases of the present invention have been genetically engineered to have more beneficial qualities. Specifically, the amylases of the current invention show altered exospecificity and / or altered thermostability. In particular, the polypeptides are derived from polypeptides having non-maltogenic exoamylase activity, in particular glucan 1,4-alpha maltotetrahydrolase activity (EC 3.2.1.60). In one aspect, the polypeptides defined herein have a better anti-aging effect, compared to the amylase of SEQ ID NO: 1.

In another aspect, the polypeptides defined herein have better thermostability compared to amylase of SEQ ID NO: 1.

A polypeptide variant is provided which is set forth in the claims. In a further aspect, the use of such a polypeptide variant is provided, which is included in and as food additives, food products, bakery products, improver compositions, food products, including animal food, etc. as stated in the claims. In a further aspect, nucleic acids encoding and relating to polypeptide variants are provided, as set forth in the claims. Methods for producing such polypeptide variants, as well as other aspects, are also set forth in the claims.

Legends of the figures

The accompanying figures are incorporated into and constitute a part of this specification and illustrate various embodiments.

Figure 1 shows the development of firmness of bread baked with pMS1776 (SEQ ID NO: 12), pMS2020 (SEQ ID NO: 14), pMS2022 (SEQ ID NO: 15) and pMS2062 (SEQ ID NO: 16) compared to bread baked with a composition comprising SEQ ID NO: 1.

Figure 2 shows the development of firmness of bread baked with pMS1776 (SEQ ID NO: 12), pMS1934 (SEQ ID NO: 13), pMS2022 (SEQ ID NO: 15) and pMS2062 (SEQ ID NO: 16) compared to baked bread with a composition comprising SEQ ID NO: 1 and Novamil 1500.

Detailed description of the invention

In accordance with this detailed description, the following abbreviations and definitions apply. It should be noted that, as used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "the formulation" includes reference to one or more formulations and equivalents thereof known to those skilled in the art, and the like,

Described herein is a polypeptide having amylase activity comprising an amino acid sequence having

a) at least 65% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises one or more amino acid substitutions at the following positions: 88 or 205, and / or b) at least 78% sequence identity with the amino acid sequence of SEQ ID NO: 1, and where the polypeptide comprises one or more amino acid substitutions at the following positions: 16, 48, 97, 105, 235, 240, 248 , 266, 311, 347, 350, 362, 364, 369, 393, 395, 396, 400, 401,403, 412 or 409, and / or

c) at least 78% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises one or more of the following amino acid substitutions: 42K / A / V / N / I / H / F, 34Q, 100Q / K / N / R, 272D, 392 K / D / E / Y / N / Q / R / T / G or 399C / H, and / or

d) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises one or more amino acid substitutions at the following positions: 44, 96, 204, 354 or 377, and / or

e) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises the following amino acid substitution: 392S

with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

Also described herein is a polypeptide having amylase activity comprising an amino acid sequence that has at least 65% sequence identity to the amino acid sequence of SEQ ID NO: 1; and wherein the polypeptide comprises one or more amino acid substitutions at the following positions: 88 or 205 and / or one or more of the following amino acid substitutions: 42K, 235H / K / R, 240E, 392 K / D / E / Y or 409E with reference to the sequence position numbering shown as SEQ ID NO: 1.

Also described herein is the use of polypeptides having a degree of sequence identity or sequence homology to the amino acid sequence defined herein or to a polypeptide having the specific properties defined herein. In particular, peptides having a degree of sequence identity with SEQ ID NO: 1, defined below, or homologues thereof, are disclosed. Herein, the term "homologous" means an entity sequence identity having the present amino acid sequence or the present nucleotide sequences, where the present amino acid sequence is preferably SEQ ID NO: 1 and the present sequence of nucleotides is preferably SEQ ID NO: 52.

In one aspect, the amino acid sequence and / or homologous nucleotide sequence can provide and / or encode a polypeptide that retains the functional activity and / or enhances the activity of a polypeptide of SEQ ID NO: 1.

In the present context, a homologous sequence is taken to include an amino acid sequence that can be at least 65%, 70%, 75%, 78%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, identical to the present sequence. Typically, homologues will comprise the same active sites etc. than the present amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues that have similar chemical properties / functions), it is preferred herein to express homology in terms of sequence identity.

Sequence identity comparisons can be performed visually, or more typically, with the aid of readily available sequence comparison programs. These commercially available computer programs use complex comparison algorithms to align two or more sequences that better reflect evolutionary events that could have led to the difference) between the two or more sequences. Therefore, these algorithms work with a reward scoring system of identical or similar amino acid alignment and gap insertion penalty, gap extensions, and dissimilar amino acid alignments. The comparison algorithm scoring system includes:

i) assigning a penalty score each time a gap is inserted (gap penalty score),

ii) the allocation of a penalty score each time an existing gap is extended with an extra position (extension penalty score),

iii) assigning high scores after identical amino acid alignment, and

iv) assignment of variable scores after alignment of non-identical amino acids.

Most alignment programs allow you to modify penalties. However, it is preferred to use the default values when using such software for sequence comparisons.

Scores given for the alignment of non-identical amino acids are assigned according to a scoring matrix also called a substitution matrix. The scores provided in such substitution matrices are reflecting the fact that the probability that one amino acid will be substituted for another during evolution varies and depends on the physical / chemical nature of the amino acid to be substituted. For example, the probability that a polar amino acid will be substituted with another polar amino acid is higher compared to that it will be substituted with a hydrophobic amino acid. Therefore, the scoring matrix will assign the highest score for identical amino acids, lowest score for non-identical but similar amino acids, and even lowest score for non-identical non-similar amino acids. The most frequently used scoring matrices are the PAM matrices (Dayhoff et al. (1978), Jones et al. (1992)), the BLOSUM matrices (Henikoff and Henikoff (1992)) and the Gonnet matrix (Gonnet et al. (1992)).

Suitable computer programs to carry out such an alignment include, but are not limited to, Vector NTI (Invitrogen Corp.) and the ClustalV, ClustalW and ClustalW2 programs (Higgins DG and Sharp PM (1988), Higgins et al. (1992), Thompson et al. (1994), Larkin et al. (2007) A selection of different alignment tools are available from the ExPASy Proteomics server at www.expasy.org Another example of software that can perform sequence alignment is BLAST (Basic Local Alignment Lookup Tool), which is available on the National Center for Biotechnology Information web page that can currently be found at http://www.ncbi.nlm.nih.gov/ and which was described in first in Altschul et al. (1990) J. Mol Biol 215: 403-410.

Once the software has produced an alignment, it is possible to calculate% similarity and% sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

In one embodiment, it is preferred to use ClustalW software to perform sequence alignments. Preferably, the ClustalW alignment is performed with the following parameters for pairwise alignment:

ClustalW2 is, for example, available on the Internet by the European Bioinformatics Institute at the www.ebi.ac.uk EMBL-EBIen website under tools - sequence analysis - ClustalW2. Currently, the exact address of the ClustalW2 tool is www.ebi.ac.uk/Tools/clustalw2.

In another embodiment, it is preferred to use Vector NTI's Align X program (Invitrogen) to perform sequence alignments. In one embodiment, Exp10 has been used with the default settings: Gap Squeeze Penalty: 10

Gap extension penalty: 0.05

Gap separation penalty range: 8

Scoring matrix: blosum62mt2

Therefore, the use of variants, homologues and derivatives of any amino acid sequence of a protein or polypeptide as defined herein is described herein, in particular those of SEQ ID NO: 1 or SEQ ID NO: 2, 4, 6, 8 or 10 is defined below.

The sequences, in particular those of the variants, homologues and derivatives of SEQ ID NO: 1, can also have deletions, insertions or substitutions of amino acid residues that produce a silent change and produce a functionally equivalent substance. Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; Positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

The present invention also encompasses a conservative substitution (substitution and replacement are used herein to mean the exchange of an existing amino acid residue, with an alternative residue) that can occur i.e. peer-to-peer substitution, such as basic for basic. , acid for acid, polar for polar, etc. Non-conservative substitution can also occur, i.e. from one class of residue to another, or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), ornithine norleucine (hereinafter referred to as O), pyrilalanine, thienylalanine, naphthylalanine and phenylglycine.

Conservative substitutions that can be made are, for example, within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), aliphatic amino acids (alanine, valine, leucine, isoleucine), polar amino acids (glutamine, asparagine, serine, threonine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), hydroxylamino acids (serine, threonine), large amino acids (phenylalanine and tryptophan), and small amino acids (glycine, alanine).

Replacements can also be made using unnatural amino acids and include; alpha * and alpha-disubstituted amino acids *, N-alkyl amino acids *, lactic acid *, halide derivatives of natural amino acids such as trifluorotyrosine *, p-Cl-phenylalanine *, p-Br-phenylalanine *, pI-phenylalanine *, L -allyl-glycine *, B-alanine *, L- a- amino butyric acid *, L- Y- amino butyric acid *, L- a- amino isobutyric acid *, L- £ -amino caproic acid #, 7-acid amino heptanoic *, L-methionine sulfone # *, L-norleucine *, L-norvaline *, p-nitro-L-phenylalanine *, L-hydroxyproline #, L-thioproline *, methyl derivatives of phenylalanine (Phe) such as 4 -methyl-Phe *, pentamethyl-Phe *, L-Phe (4-amino) #, L-Tyr (methyl) *, L-Phe (4-isopropyl) *, L-Tic (1,2,3 acid, 4-tetrahydroisoquinoline-3-carboxyl) *, L-diaminopropionic acid # and L-Phe (4-benzyl) *. The notation * has been used for the purpose of the above discussion (regarding homologous or non-conservative substitution), to indicate the hydrophobic nature of the derivative while # has been used to indicate the hydrophilic nature of the derivative # * indicates amphipathic characteristics .

Amino acid sequence variants can include suitable spacer groups that can be inserted between any two amino acid residues in the sequence including alkyl groups such as methyl, ethyl, or propyl groups, in addition to amino acid spacers such as glycine or p residues. -to the girl. A further form of variation, involving the presence of one or more amino acid residues in peptoid form, will be well understood by those of skill in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variants of amino acid residues in which the a -carbon substituent group is on the residue's nitrogen atom instead of the a -carbon. Processes for preparing peptides in peptoid form are known in the art, for example Simon RJ et al. (1992), Horwell ^d C. (1995).

Described herein, the polypeptide variant is a Pseudomonas saccharophila (PS4) amylase variant having the sequence shown in SEQ ID NO: 1 and having at least at least 65%, at least 70%, at least 75%, at least 78%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity of the amino acid sequence with it.

In one aspect, preferably the sequence used in the present invention is in a purified form. The term "purified" means that a given component is present at a high level. The component is desirably the predominant active component present in a composition.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood by one of ordinary skill in the art. The following terms are provided below.

"Amylase" means an enzyme that is, among other things, capable of catalyzing the degradation of starch. An endo-acting amylase activity cleaves - ^ D- (1 • 4) O-glycosidic bonds within the starch molecule in a random manner. In contrast, an exo-acting amylolytic activity cleaves a starch molecule from the non-reducing end of the substrate. "Endo-acting amylase activity", "endo-activity", "endospecific activity" and "endo-specificity" are synonymous, when the terms refer to polypeptide variants as defined in the claims. The same is valid for the corresponding terms for exo activity. "Maltotetraose-forming maltotetrahydrolase; EC 3.2.1.60; G4-forming amylase; G4-amylase and glucan 1,4-alpha-maltotetrahydrolase" can be used interchangeably. "

In the present context "PS4" is used to designate Pseudomonas saccharophila maltotetrahydrolase.

A "variant" or "variants" refers to polypeptides or nucleic acids. The term "variant" can be used interchangeably with the term "mutant". Variants include insertions, substitutions, transversions, truncations, and / or inversions at one or more locations in the amino acid or nucleotide sequence, respectively. The phrases "polypeptide variant", "polypeptide", "variant" and "enzyme variant" mean a polypeptide / protein having an amino acid sequence that has been modified from the amino acid sequence of SEQ ID NO: 1. The Polypeptide variants include a polypeptide that has a certain percentage, for example, 65%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. As used herein, "parent enzymes", "parent sequence", "parent polypeptide" means enzymes and polypeptides from which the polypeptide variants are based, eg, SEQ ID NO: 1. An "parent nucleic acid" means a nucleic acid sequence encoding the polypeptide original. The signal sequence of a "variant" may be the same (eg, SEQ ID NO: 11) or it may differ from the signal sequence of wild-type PS4. A variant can be expressed as a fusion protein containing a heterologous polypeptide. For example, the variant may comprise a signal peptide from another protein or a sequence designed to aid in the identification or purification of the expressed fusion protein, such as a His-Tag sequence.

To describe the different variants that are considered to be encompassed by the present description, the following nomenclature will be adopted for ease of reference. When the substitution includes a number and a letter, eg 141P, then it refers to {position according to the numbering system / substituted amino acid}. Accordingly, for example, the substitution of an amino acid to proline at position 141 is designated as 141P. When the substitution includes a letter, a number, and a letter, for example, A141P, then it refers to {original amino acid / position according to the numbering system / substituted amino acid}. Accordingly, for example, the substitution of alanine for proline at position 141 is designated A141P.

When two or more substitutions are possible at a particular position, it will be designated by contiguous letters, which may optionally be separated by slash marks "/", eg G303ED or G303E / D.

The amino acid positions and substitutions mentioned herein are listed with reference to the corresponding position and the corresponding amino acid of SEQ ID NO: 1. Equivalent positions in another sequence can be found by aligning this sequence with SEQ ID NO: 1 to find an alignment with the highest percent identity and determine thereafter which amino acid is aligned to correspond to an amino acid at a specific position of SEQ ID NO: 1. Such alignment and use of a sequence as a First referral is simply a matter of course for one of ordinary skill in the art.

"Nucleic acid variants" can include sequences that are complementary to sequences that are capable of hybridizing to the nucleotide sequences presented herein, in particular, to SEQ ID NO: 52. For example, a sequence variant is complementary to sequences capable of hybridizing under stringent conditions, for example, (50 ° C and 0.2X SSC (1X SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), to the presented nucleotide sequences in this document, in particular, to SEQ ID NO: 52 (DNA pMS 382) More particularly, the term variant encompasses sequences that are complementary to sequences that are capable of hybridizing under very stringent conditions, for example, 65 ° C and 0.1 x SSC, to the nucleotide sequences presented in this document, in particular, to SEQ ID NO: 52 (DNA pMS 382). The melting point (Tm) of a nucleic acid variant can be approximately 1 2, or 3 ° C lower than the Tm of the salv-type nucleic acid aje. Nucleic acid variants include a polynucleotide that has a certain percentage, eg, 80%, 85%, 90%, 95%, or 99%, of sequence identity to the nucleic acid encoding SEQ ID NO: 1.

As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

"Isolated" means that the sequence is at least substantially free of at least one other component than the naturally associated and naturally occurring sequence, eg, genomic sequences.

"Purified" means that the material is in a relatively pure state, eg, at least about 90% pure, at least about 95% pure, or at least about 98% pure.

"Thermostable" means that the enzyme maintains its activity after exposure to elevated temperatures. The thermostability of an enzyme can be measured by its half-life (t1 / 2), where half of the enzyme activity is lost by the half-life. The half-life value is calculated under certain conditions by measuring the residual amylase activity. To determine the half-life of the enzyme, the sample is heated to the test temperature for 1-10 min, and the activity is measured using a standard assay for amylase activity, such as the Betamyl® assay (Megazyme, Ireland ).

As used herein, "optimum pH" means the pH at which the polypeptide variants described herein show activity in a standard assay for amylase activity, measured over a pH range. As used herein, "polypeptide" is used interchangeably with the terms "amino acid sequence", "enzyme", "peptide" and / or "protein". As used herein, "nucleotide sequence" or "nucleic acid sequence" refers to an oligonucleotide sequence or polynucleotide sequence and variants, homologues, fragments, and derivatives thereof. The nucleotide sequence can be of genomic, synthetic or recombinant origin and can be double-stranded or single-stranded, whether it represents the sense or anti-sense strand. As used herein, the term "nucleotide sequence" includes genomic DNA, cDNA, synthetic DNA, and RNA.

"Homologous" means an entity that has a certain degree of identity or "homology" with the amino acid sequences present and the nucleotide sequences present. A "homologous sequence" includes a polynucleotide or polypeptide that has a certain percentage, eg, 80%, 85%, 90%, 95%, or 99%, of sequence identity to another sequence. Percent identity means that, when aligned, that percentage of bases or amino acid residues are the same as when the two sequences are compared. The amino acid sequences are not identical, where an amino acid is substituted, removed or added compared to the present sequence. Percent sequence identity is typically measured relative to the mature protein sequence present, ie, for example after removal of a signal sequence. Typically, homologues will comprise the same active site residues as the present amino acid sequence. Homologs also retain amylase activity, although the homolog may have different enzymatic properties than wild-type PS4.

As used herein, "hybridization" includes the process by which a nucleic acid strand joins with a complementary strand through base pairing, as well as the amplification process that takes place in reaction technologies. polymerase chain (PCR). The nucleic acid variant can exist as single or double stranded DNA or RNA, a DNA / RNA heteroduplex, or an RNA / DNA copolymer. As used herein, "copolymer" refers to a single nucleic acid strand that comprises both ribonucleotides and deoxyribonucleotides. The nucleic acid variant can be codon optimized to further increase expression.

As used herein, a "synthetic" compound is produced by chemical or enzymatic synthesis in vitro. It includes, but is not limited to, nucleic acid variants made with the optimal codon usage for the host organisms, such as a yeast host cell or other expression hosts of choice.

As used herein, "transformed cell" includes cells, including bacterial and fungal cells, that have been transformed using recombinant DNA techniques. Transformation typically occurs by inserting one or more nucleotide sequences into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence, that is, it is a sequence that is not natural to the cell to be transformed, such as a fusion protein.

As used herein, "operably linked" means that the components described are in a relationship that allows them to function as intended. For example, a regulatory sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is obtained under conditions compatible with control sequences.

As used herein, the term "starch" refers to any material composed of the complex polysaccharide carbohydrates of plants, such as corn, amylose compound and amylopectin with the formula (C6H105) x, where X can be any number. The term "granular starch" refers to raw starch, that is, uncooked starch, for example, that has not been subjected to gelatinization.

The term "liquefaction" refers to the step in the conversion of starch in which gelatinized starch is hydrolyzed to give soluble low molecular weight dextrins. As used herein, the term "saccharification" refers to the enzymatic conversion of starch to glucose. The term "degree of polymerization" (DP) refers to the number (n) of anhydrous glucopyranose units in a given saccharide. Examples of DPI are the monosaccharides glucose and fructose. Examples of DP2 are the disaccharides maltose and sucrose.

As used herein, the term "dry solids content" (ds) refers to the total solids of a suspension on a percent dry weight basis. The term "suspension" refers to an aqueous mixture containing insoluble solids.

The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process in the ethanol-producing microorganism and at least one enzyme, such as PS4 or a variant thereof, is present during the same process step. SSF refers to the simultaneous hydrolysis of granular starch substrates to saccharides and the fermentation of the saccharides in alcohol, for example, in the same reactor vessel.

As used herein, "ethanologenic microorganism" refers to a microorganism with the ability to convert a sugar or oligosaccharide to ethanol.

1.2. Abbreviations

The following abbreviations apply unless otherwise stated:

ADA azodicarbonamide

cDNA complementary DNA

CGTase cyclodextrin glucanotransferase

DEAE diethylaminoethanol

dH2O deionized water

Deoxyribonucleic acid DNA

ds-DNA double-stranded DNA

EC enzyme commission for enzyme classification

FGSC Fungal Genetics Stock Center

G121F glycine residue (G) at position 121 of SEQ ID NO: 2 is replaced with a phenylalanine residue (F), where amino acids are designated by single letter abbreviations commonly known in the art.

HPLC high performance liquid chromatography

messenger ribonucleic acid mRNA

PCR polymerase chain reaction

PDB protein database

PEG polyethylene glycol

ppm parts per million

G4 P. saccharophila- forming PS4 amylase

RT-PCR polymerase chain reaction-reverse transcriptase

G4 P. saccharophila- forming SAS amylase

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

1X SSC 0.15 M NaCl, 0.015 M Sodium Citrate, pH 7.0

SSF simultaneous saccharification and fermentation

t '/ 2 half-life

Tm melting temperature (° C) at which 50% of the protein present melts

ATm ° C increase in Tm

w / v weight / volume

w / w weight / weight

2. Polypeptide variants of SEQ ID NO: 1

In one aspect, a polypeptide is provided that has a substitution at one or more positions that produces an altered property, which can be any combination of altered exospecificity, endospecificity, or thermostability, or an alteration in handling properties, relative to SEQ ID. NO: 1. Such polypeptide variants are also referred to herein for convenience as "polypeptide variant", "polypeptide variant" or "variant". In one aspect, polypeptides as defined herein have an improved anti-aging effect compared to amylase of SEQ ID NO: 1. In another aspect, polypeptides as defined herein have improved thermostability compared with the amylase of SEQ ID NO: 1. In highly preferred embodiments, they have the added advantage of higher thermostability, or higher exoamylase activity or higher pH stability, or any combination.

In one aspect, the variants defined herein exhibit enzymatic activity. In one aspect, the polypeptide variants comprise amylase activity. In a further aspect, the polypeptide variants comprise exoamylase activity. In a further aspect, the polypeptide variants exhibit non-maltogenic exoamylase activity. In one aspect, the variants defined herein can be derived from Pseudomonas saccharophila • -amylase (PS4). In one aspect, the variants defined herein are a maltotetraose-forming maltotetrahydrolase, also referred to as EC 3.2.1.60; G4-forming amylase; G4-amylase or glucan 1,4-alpha-maltotetrahydrolase.

The compositions, including food additives, food products, bakery products, improver compositions, food products, including animal feed, etc. comprising such polypeptide variants defined herein, such as those having non-maltogenic exoamylase activity, as well as methods of making and using such polypeptides and compositions, are provided herein.

As noted above, the polypeptide variants may comprise one or more improved handling properties, preferably improved baking properties. Therefore, the polypeptide variants are such that the food products thus treated have one or more of (preferably all of) lower firmness, higher elasticity, higher cohesion, lower friability, or higher pliability. Such improved handling or baking properties exhibited by variant polypeptides are described in more detail below.

Furthermore, there is provided a treatment of food products, in particular doughs and bakery products with such polypeptides, and such that such food products have the desired qualities set forth above.

In addition, other uses of the variants described herein and compositions comprising these variants are provided, such as in the preparation of detergents, such as sweeteners, syrups, etc. Compositions include the polypeptide along with at least one other component. In particular, feed or animal feed additives comprising the polypeptides are provided.

An isolated and / or purified polypeptide comprising a polypeptide variant defined herein is provided. In one embodiment, the polypeptide variant is a mature form of the polypeptide (SEQ ID NO: 1), where the 21 amino acid leader sequence is cleaved, such that the N-terminal end of the polypeptide begins at the acid residue. aspartic (D). In one aspect, the variants include a C-terminal starch binding domain. A representative amino acid sequence of a starch-binding domain comprises or consists of an amino acid sequence of SEQ ID NO: 36. Other variants include variants where between one and about 25 amino acid residues have been added or deleted with respect to SEQ ID NO: 1. In one aspect, the variant has the amino acid sequence of SEQ ID NO: 1, where any number between one and about 25 amino acids has been substituted. In a further aspect, the variant has the amino acid sequence of s Eq ID NO: 1, where any number between three and twelve amino acids has been substituted. In a further aspect, the variant has the amino acid sequence of SEQ ID NO: 1, where any number between five and nine amino acids has been substituted.

In one aspect, at least two have been substituted, in another aspect at least three, and in still another aspect at least five amino acids of SEQ ID NO: 1.

In a further aspect, the length of the polypeptide variant is 390 to 540 amino acids. In a further aspect, the length of the polypeptide variant is 410 to 440 amino acids. In a further aspect, the length of the polypeptide variant is 420 to 435 amino acids. In a further aspect, the length of the polypeptide variant is 429 to 430 amino acids.

Described herein is a polypeptide having amylase activity comprising an amino acid sequence having:

a) at least 65% sequence identity with the amino acid sequence of SEQ. ID NO: 1 and wherein the polypeptide comprises one or more amino acid substitutions at the following positions: 88 or 205, and / or

b) at least 78% sequence identity with the amino acid sequence of SEQ ID NO: 1, and where the polypeptide comprises one or more amino acid substitutions at the following positions: 16, 48, 97, 105, 235, 240 , 248, 266, 311, 347, 350, 362, 364, 369, 393, 395, 396, 400, 401, 403, 412 or 409, and / or

c) at least 78% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises one or more of the following amino acid substitutions: 42K / A / V / N / I / H / F, 34Q, 100Q / K / N / R, 272D, 392 K / D / E / Y / N / Q / R / T / G or 399C / H, and / or

d) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises one or more amino acid substitutions at the following positions: 44, 96, 204, 354 or 377, and / or

e) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises the following amino acid substitution: 392S

wherein an amino acid substitution refers to the corresponding amino acid of SEQ ID NO: 1 and the positions are with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In a further aspect, the polypeptide defined herein comprises one or more amino acid substitutions at the following positions: 88, 205, 235, 240, 248, 266, 311, 377, or 409 and / or one or more of the following substitutions amino acids: 42K / A / V / N / I / H / F, 34Q, 100Q / K / N / R, 272D or 392K / D / E / Y / N / Q / R / S / T / G. In a further aspect, the polypeptide defined herein further comprises one or more amino acid substitutions at the following positions: 88, 205, 235, 240, 311, or 409 and / or one or more of the following amino acid substitutions: 42K / N / I / H / F, 272D, or 392 K / D / E / y / n / Q / R / S / T / G. In a further aspect, the polypeptide defined herein comprises amino acid substitutions at least four, five, or all of the following positions: 88, 205, 235, 240, 311, or 409 and / or has at least one, or two of the following amino acid substitutions: 42K / N / I / H / F, 272D or 392 K / D / E / Y / N / Q / R / S / T / G. In a further aspect, the polypeptide defined herein further comprises one or more of the following amino acids 33Y, 34N, 70D, 121F, 134R, 141P, 146G, 157L, 161A, 178F, 179T, 223E / S / K / A, 229P, 307K, 309P and 334P. In a further aspect, the polypeptide defined herein further has the following amino acids 33Y, 34N, 70D, 121F, 134R, 141P, 146G, 157L, 161A, 178F, 179T, 229P, 307K, 309P, and 334P. In a further aspect, the polypeptide defined herein has at least one of the amino acid substitutions in a) of claim 1 and further comprises one or more amino acid substitutions at the following positions: 44, 96, 204, 354 or 377 In a further aspect, the polypeptide defined herein has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO. : 1. In a further aspect, the polypeptide defined herein has amino acid 34Q. In a further aspect, the polypeptide defined herein has the amino acid 42K / F / H / I / N / A / V. In a further aspect, the polypeptide defined herein has the amino acid 42K / F / H / I / N. In a further aspect, the polypeptide defined herein has amino acid 42K. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 88. In a further aspect, the polypeptide defined herein has the amino acid 88L / Y / K. In a further aspect, the polypeptide defined herein has amino acid 88L. In a further aspect, the polypeptide defined herein has amino acid 88Y. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 205. In a further aspect, the polypeptide defined herein has the amino acid 205L / K / M / N / Q / R / V / Y . In a further aspect, the polypeptide defined herein has the amino acid 205L / K / M / N / Q / R / V. In a further aspect, the polypeptide defined herein has amino acid 205L. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 235. In a further aspect, the polypeptide defined herein has the amino acid 235H / K / R / Q / S. In a further aspect, the polypeptide defined herein has the amino acid 235H / K / R / Q. In a further aspect, the polypeptide defined herein has the amino acid 235H / K / R. In a further aspect, the polypeptide defined herein has amino acid 235R. In a further aspect, the polypeptide defined herein has amino acid 235H. In a further aspect, the polypeptide defined herein has amino acid 235K. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 240. In a further aspect, the polypeptide defined herein has the amino acid 240E / H / M / D / S. In a further aspect, the polypeptide defined herein has the amino acid 240E / H / M / D. In a further aspect, the polypeptide defined herein has amino acid 240E. In a further aspect, the polypeptide defined herein has amino acid 272D. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 311. In a further aspect, the polypeptide defined herein has amino acid 311P. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 409. In a further aspect, the polypeptide defined herein has amino acid 409H / Q / T / E. In a further aspect, the polypeptide defined herein has amino acid 409E. In a further aspect, the polypeptide defined herein has the amino acid 100Q / K / N / R. In a further aspect, the polypeptide defined herein has amino acid 100Q. In a further aspect, the polypeptide defined herein has the amino acid 392D / E / K / Y / N / Q / R / S / T / G. In a further aspect, the polypeptide defined herein has the amino acid 392D / E / K / Y / N / Q / R / S / T. In a further aspect, the polypeptide defined herein has the amino acid 392D / E / K / Y. In a further aspect, the polypeptide defined herein has amino acid 392D. In a further aspect, the polypeptide defined herein has amino acid 392E.

In a further aspect, the polypeptide defined herein has amino acid 392K. In a further aspect, the polypeptide defined herein has amino acid 392Y. In a further aspect, the polypeptide defined herein has both of the following amino acids 235R and 311P. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 16. In a further aspect, the polypeptide defined herein has amino acid 16 / A / E / K. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 48. In a further aspect, the polypeptide defined herein has amino acid 48 / C / L. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 97. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 105. In a further aspect, the defined polypeptide herein it has the amino acid 105 / N / R. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 248. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 266. In a further aspect, the defined polypeptide herein it comprises an amino acid substitution at position 347. In a further aspect, the polypeptide defined herein has amino acid 347 / C / D / H / K. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 350. In a further aspect, the polypeptide defined herein has the amino acid 350E / H / N. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 354. In a further aspect, the polypeptide defined herein has amino acid 354D / E. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 362. In a further aspect, the polypeptide defined herein has amino acid 362E / H / P. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 364. In a further aspect, the polypeptide defined herein has amino acid 364E / K / NQ. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 369. In a further aspect, the polypeptide defined herein has amino acid 369I / N. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 377. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 393. In a further aspect, the defined polypeptide herein it has the amino acid 393D / E / K. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 395. In a further aspect, the polypeptide defined herein has amino acid 395C / E / K. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 396. In a further aspect, the polypeptide defined herein has amino acid 396D / E. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 399. In a further aspect, the polypeptide defined herein has amino acid 399C / H. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 400. In a further aspect, the polypeptide defined herein has amino acid 400S / W. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 401. In a further aspect, the polypeptide defined herein has amino acid 401D / K. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 403. In a further aspect, the polypeptide defined herein has amino acid 403E / T / V. In a further aspect, the polypeptide defined herein comprises an amino acid substitution at position 412. In a further aspect, the polypeptide defined herein has amino acid 412D / N. In a further aspect, the polypeptide defined herein further comprises one or more of the following amino acids 121F, 134R, 141P, 229P, or 307K. In a further aspect, the polypeptide defined herein has one or more of the following amino acids 42K, 88L, 205L, 235R, 240E, 272D, 311P, 392D, or 409E. In a further aspect, the polypeptide defined herein comprises the following amino acids 42K, 88L, 235R, 311P, 392D and 223S, 223K or 223A. In a further aspect, the polypeptide defined herein further comprises one or more selected from the group consisting of 272D, 409E, 205L, and 240E. In a further aspect, the polypeptide defined herein has at least four, five, six, seven, or eight of the following amino acids 42K, 88L, 205L, 235R, 240E, 272D, 311P, 392D, or 409E. In a further aspect, the polypeptide defined herein has amino acid 223A / K / S. In a further aspect, the polypeptide defined herein has the following amino acids 42K, 88L, 223S, 235R, 311P, and 392D. In a further aspect, the polypeptide defined herein has the following amino acids 42K, 88L, 223K, 235R, 272D, 311P, and 392D. In a further aspect, the polypeptide defined herein has the following amino acids 42K, 88L, 223S, 235R, 311P, 392D, and 409E. In a further aspect, the polypeptide defined herein has the following amino acids 42K, 88L, 205L, 223S, 235R, 311P, 392D, and 409e. In a further aspect, the polypeptide defined herein has the following amino acids 42K, 88L, 205L, 223K, 240E, 235R, 311P, 392D, and 409E. In a further aspect, the polypeptide defined herein has the following amino acids 42K, 88L, 205L, 223A, T235R, 311P, 392D, and 409E. In a further aspect, the polypeptide defined herein has one or more amino acids at the N-terminus. In one aspect, the polypeptide defined herein has the amino acid M at the N-terminus. In a further aspect, the polypeptide defined herein comprises one or more amino acid substitutions at the following positions: 88, 205, 235, or 409 and / or one or more of the following amino acid substitutions: 42K, 34Q, 100Q, 223A / S, 240E, 311P, 392D, or 409E with reference to the position numbering of the sequence shown as SEQ ID NO: 1. In a further aspect, the polypeptide defined herein comprises one or more of the following substitutions of amino acids: 42K, 34Q, 88K / L / Y, 100Q, 205L, 223A / S / K, 235 ^k / H / Q / R, 240E / D, 248H, 266T, 311P, 377D / E / P, 392K / D / Y or 409E. In a further aspect, the polypeptide defined herein comprises one or more of the following amino acid substitutions: 34Q, 42K, 88L, 100Q, 205L, 223A / S / K, 235K / R, 240E, 311P, 392D, or 409E. In a further aspect, the polypeptide defined herein comprises the following amino acid substitutions: 235R and 311P. In a further aspect, the polypeptide defined herein comprises the 42K amino acid substitution. In a further aspect, the polypeptide defined herein comprises the following amino acid substitutions: 42K and 223S. In a further aspect, the polypeptide defined herein comprises the following amino acid substitutions: 42K, 223S, and 392D. In a further aspect, the polypeptide defined herein comprises the following amino acid substitutions: 42K, 88L, 223A, 235R, 311P, and 392D. In a further aspect, the polypeptide defined herein comprises the following amino acid substitutions: 42K, 88L, 205L, 223S, 235R, 240E, 311P, 392D, and 409E. In a further aspect, the polypeptide defined herein comprises the following amino acid substitutions: 42K, 88L, 205L, 223K, 235R, 240E, 311P, 392D, and 409E. In a further aspect, the polypeptide defined herein comprises the following amino acid substitutions: 42K, 88L, 205L, 223S, 235R, 311P, and 392D. In a further aspect, the polypeptide defined herein comprises the following amino acid substitutions: 34Q, 42K, 88L, 205L, 223K, 235R, 240E, 311P, 392D, and 409E. In a further aspect, the polypeptide defined herein comprises the following amino acid substitutions: 42K, 88L, 100Q, 205L, 223K, 235R, 240E, 311 P, 392D, and 409E.

Representative embodiments of the polypeptides defined herein comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. Other representative embodiments of the polypeptides defined herein comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. Other representative embodiments of the polypeptides defined herein have an amino acid sequence selected from the group consisting of SEQ ID NO : 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ I D NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO : 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. Other representative embodiments of the polypeptides defined herein have an amino acid sequence selected from the group consisting of SeQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and optionally one or more amino acids at the N-terminus. In one aspect, the polypeptides defined herein comprise the sequence HGGDEIILQFHWN (SEQ ID NO: 37) at positions corresponding to positions 13-26 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence DGF-X1-AIW-X2-P-X3-PWRD-X4-SSW (SEQ ID NO: 38), where X1 is S or T, X2 is M or L , X3 is V or P and X4 is any natural amino acid residue, preferably an L-amino acid, at positions corresponding to positions 49-66 with reference to the position numbering of the sequence shown as SEQ ID NO: 1 .

In one aspect, the polypeptides defined herein comprise the sequence GGEGYFW (SEQ ID NO: 39) at positions corresponding to positions 79-85 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence VPNH (SEQ ID NO: 40) at positions corresponding to positions 114-117 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence CDDGD (SEQ ID NO: 41) at positions corresponding to positions 150-154 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence AGGFRFDFVRG (SEQ ID NO: 42) at positions corresponding to positions 187-197 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence FALK (SEQ ID NO: 43) at positions corresponding to positions 256-259 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence WREVAVTFVDNHD (SEQ ID NO: 44) at positions corresponding to positions 282-294 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence GYSPG (SEQ ID NO: 45) at positions corresponding to positions 296-300 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence GQH (SEQ ID NO: 46) at positions corresponding to positions 304-306 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence AYAYI (SEQ ID NO: 47) at positions corresponding to positions 318-322 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence SPGTP (SEQ ID NO: 48) at positions corresponding to positions 325-329 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence VYW (SEQ ID NO: 49) at positions corresponding to positions 331-333 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the sequence HMYDWG (SEQ ID NO: 50) at positions corresponding to positions 335-340 with reference to the position numbering of the sequence shown as SEQ ID NO: 1.

In another aspect, the polypeptide variant defined herein has the sequence of SEQ ID NO: 1, where any number between one and about 25 amino acids has been substituted. Representative examples of polypeptide variants having unique or diverse amino acid substitutions are shown in the following TABLE A:

The polypeptide variants defined herein may have altered thermostability, altered endo-amylase activity, altered exo-amylase activity, and / or altered exo-a activity ratio. endo amylase compared to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10. In one aspect, the polypeptides defined herein have a better anti-aging effect compared to amylase of SEQ ID NO: 1 In another aspect, the polypeptides defined herein have better thermostability compared to amylase of SEQ ID NO: 1.

The polypeptide variants defined herein can have up to 25, 23, 21, 19, 17, 15, 13, 11, 9, 8, 7, 6, 5 amino acid deletions, additions, insertions, or substitutions compared to the sequence amino acids of SEQ ID NO: 1.

The present description also refers to each and every G4-amylase backbone having SEQ ID NO: 2, 4, 6 or 8 that comprises the substitutions further defined herein.

Nucleic acids encoding the above polypeptide variants are also provided. In one embodiment, a nucleic acid encoding a variant of the polypeptide defined herein is a nucleotide sequence encoding proteins comprising an amino acid sequence of residues 1-429 of SEQ ID NO: 1 set forth in SEQ ID NO: 52. For example, the nucleotide sequence SEQ ID NO: 3 encodes the backbone of G4 amylase with SEQ ID NO: 2. As is well understood by those of skill in the art, the genetic code is degenerate, which it means that multiple codons in some cases can code for the same acidic amino acid. Nucleic acids can include genomic DNA, mRNA, and cDNA encoding a 2.1 polypeptide variant. Characterization of the polypeptide variant

Enzyme variants can be characterized by their primary nucleic acid and polypeptide sequences, by three-dimensional structural modeling, and / or by their specific activity. Additional characteristics of polypeptide variants as defined herein include, for example, stability, pH range, oxidation stability, and thermostability. Enzyme expression and activity levels can be assessed using standard assays known to those of skill in the field. In another aspect, the variants demonstrate improved performance characteristics relative to the polypeptide with SEQ I ^d NO: 1, such as better stability at high temperatures, eg, 65-85 ° C. In one aspect, the polypeptide variants defined herein are advantageous for use in liquefaction or other processes that require elevated temperatures, such as baking. For example, a thermostable polypeptide variant defined herein can degrade starch at temperatures of from about 55 ° C to about 85 ° C or higher.

An expression characteristic means an altered level of expression of the variant, when the variant occurs in a particular host cell. The term generally refers to the amount of active variant that is recoverable from a fermentation broth using standard techniques in the art, during a given period of time. The term can also refer to the amount or rate of variant produced within the host cell or secreted by the host cell. The term can also refer to the rate of translation of the mRNA encoding the variant of the enzyme.

A nucleic acid complementary to a nucleic acid encoding any of the polypeptide variants defined herein is provided herein. Additionally, a nucleic acid capable of hybridizing to complement is provided. In another embodiment, the sequence for use in the methods and compositions described herein is a synthetic sequence. Includes, but is not limited to, sequences obtained with optimal codon usage for expression in host organisms, such as yeast.

3. Production of the polypeptide variants defined herein

The polypeptide variants provided herein can be produced synthetically or by recombinant expression in a host cell, according to procedures well known in the art. The expressed polypeptide variant defined herein is optionally isolated prior to use. In another embodiment, a polypeptide variant defined herein is purified after expression. Methods of genetic modification and recombinant production of polypeptide variants are described, for example, in U.S. Pat. Nos. 7,371,552, 7,166,453; 6,890,572; and 6,667,065; and U.S. Published Applications Nos 2007/0141693; 2007/0072270; 2007/0020731; 2007/0020727; 2006/0073583; 2006/0019347; 2006/0018997; 2006/0008890; 2006/0008888; and 2005/0137111. Relevant teachings of these disclosures include polynucleotide sequences encoding the polypeptide, primers, vectors, selection methods, host cells, purification and reconstitution of expressed polypeptide variants, and the characterization of polypeptide variants defined herein, including Useful buffers, pH ranges, Ca2 + concentrations, substrate concentrations, and enzyme concentrations for enzyme assays.

In another embodiment, suitable host cells include a Gram positive bacterium selected from the group consisting of Bacillus subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amiloliquefaciens, B. coagulans, B. circulans, B. lautus, B. thuringiensis, Streptomyces lividans, or S. murinus; or a Gram negative bacterium, where said Gram negative bacterium is an Escherichia coli or Pseudomonas species. In one aspect, the host cell is a B. subtilus or B. licheniformis. In one embodiment, the host cell is B. subtilis, and the expressed protein is genetically engineered to comprise a signal sequence from B.

subtilis, which are discussed in more detail below. In one aspect, the host cell expresses the polynucleotide set forth in the claims.

In some embodiments, a host cell is genetically engineered to express a variant of the polypeptide defined herein with an amino acid sequence that is at least about 78%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the polypeptide of SEQ ID NO: 1. In some embodiments, the polynucleotide that encodes a variant of the polypeptide defined herein will have a nucleic acid sequence that encodes the protein of SEQ ID NO: 1 or a nucleic acid sequence that is at least about 78% .80 %, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a nucleic acid encoding the protein of SEQ ID NO: 1. In one embodiment, the sequence of nucleic acids have at least about 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid of SEQ ID NO: 52.

3.1. Cartoon vector

In one aspect, the invention relates to a vector comprising a polynucleotide. In one aspect, a bacterial cell comprises the vector. In some embodiments, a DNA construct comprising a nucleic acid encoding a variant is transferred to a host cell in an expression vector comprising regulatory sequences operably linked to a coding sequence. The vector can be any vector that can be integrated into a fungal host cell genome and replicates when introduced into the host cell. The University of Missouri FGSC Catalog of Strains lists suitable vectors. Additional examples of suitable expression vectors and / or integration vectors are provided in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001); Bennett et al., MORE GENE MAN ⁱ P ^u LATIONS IN FUNGI, Academic Press, San Diego (1991), pp. 396-428 and US Patent No. 5,874,276. Examples of vectors include pFB6, pBR322, PUC18, pUC100 and pENTR / D, pDON ™ 201, pDONR ™ 221, pENTR ™, pGEM®3Z and pGEM®4Z. Examples for use in bacterial cells include pBR322 and pUC19, which allow replication in E. coli, and pE194, for example, which allow replication in Bacillus.

In some embodiments, a nucleic acid encoding a variant is operably linked to a suitable promoter, which enables transcription in the host cell. The promoter can be derived from genes encoding proteins homologous or heterologous to the host cell. Suitable non-limiting examples of promoters include cbh1, cbh2, egl1, and egl2 promoters. In one embodiment, the promoter is one that is native to the host cell. For example, when P. saccharophila is the host, the promoter is a native P, saccharophila P promoter. An "inducible promoter" is a promoter that is active under environmental or developmental regulation. In another embodiment, the promoter is one that is heterologous to the host cell.

In some embodiments, the coding sequence is operably linked to a DNA sequence that codes for a signal sequence. A representative signal peptide is SEQ ID NO: 11, which is the native signal sequence of the PS4 precursor. In other embodiments, the DNA encoding the signal sequence is replaced with a nucleotide sequence encoding a signal sequence from a species other than P. saccharophila. In this embodiment, the polynucleotide encoding the signal sequence is immediately upstream and in frame of the polynucleotide encoding the polypeptide. The signal sequence can be selected from the same species as the host cell. In a non-limiting example, the signal sequence is a cyclodextrin glucanotransferase signal sequence. (CGTase; EC 2.4.1.19) from Bacillus sp, and the polypeptide variants described herein are expressed in a B. subtilis host cell. A methionine residue can be added to the N-terminal end of the signal sequence.

In some embodiments, a signal sequence and a promoter sequence comprising a DNA construct or vector for introduction into a fungal host cell are derived from the same source. In some embodiments, the expression vector also includes a termination sequence. In one embodiment, the terminator sequence and the promoter sequence are derived from the same source. In another embodiment, the termination sequence is homologous to the host cell.

In some embodiments, an expression vector includes a selectable marker. Examples of suitable selectable markers include those that confer resistance to antimicrobial agents, eg, hygromycin or phleomycin. Nutritional selective markers are also suitable and include amdS, argB, and pyr4. In one embodiment, the selective marker is the amdS gene, which encodes the enzyme acetamidase that allows transformed cells to grow in acetamide as a nitrogen source. The use of an A. nidulans amdS gene as a selective marker is described in Kelley et al, EMBO J. 4 :. 475-479 (1985) and Penttila et al, Gene 61: 155-164 (1987).

A suitable expression vector comprising a DNA construct with a polynucleotide encoding a variant can be any vector that is capable of autonomously replicating in a given host organism or integrating into the host's DNA. In some embodiments, the expression vector is a plasmid. In some embodiments, two types of expression vectors are contemplated to obtain gene expression. The first expression vector comprises DNA sequences in which the promoter, the coding region and the terminator originate from the gene to express. In some embodiments, gene truncation is achieved by deleting unwanted DNA sequences, for example, DNA encoding the C-terminal starch-binding domain, to allow the domain to be expressed under the control of its own transcriptional and translational regulatory sequences. The second type of expression vector is preassembled and contains sequences necessary for high-level transcription and a selectable marker. In some embodiments, the coding region for a gene or part of it is inserted into this general purpose expression vector so that it is under the transcriptional control of the promoter and terminator sequences of the expression construct. In some embodiments, the genes or part of them are inserted downstream of the strong cbh1 promoter.

3.2. Transformation, expression and culture of host cells

Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, eg, lipoinfection-mediated and DEAE-Dextrin-mediated transfection; incubation with calcium phosphate DNA precipitate; high-speed bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, for example, Ausubel et al. (1987), supra, chapter 9; Sambrook et al. (2001), supra; and Campbell et al., Curr. Genet. 16: 53-56 (1989). Expression of the heterologous protein in Trichoderma is described, for example, in U.S. Pat. No. 6,022,725; U.S. Patent No. 6,268,328; Harkki et al., Enzyme Microb. Technol. 13: 227-233 (1991); Harkki et al., Biotechnol. 7: 596-603 (1989); EP 244,234; and EP 215,594. In one embodiment, genetically stable transformants are constructed with vector systems whereby nucleic acid encoding a variant is stably integrated into a host cell chromosome. The transformants are then purified by known techniques.

In a non-limiting example, stable transformants, which include an amdS marker, are distinguished from unstable transformants by their faster growth rate and the formation of circular colonies with a regular rather than irregular contour on solid culture medium containing acetamide. . In addition, in some cases, an additional stability test is carried out by culturing the transformants on a solid non-selective medium, for example, a medium lacking acetamide, harvesting the spores from this culture medium, and determination of the percentage of these spores that will later germinate and grow in selective medium containing acetamide. Other methods known in the art can be used to select transformants.

3.3. Identification of the activity

To assess the expression of a variant in a host cell, assays can measure expressed protein, corresponding mRNA, or • -amylase activity. For example, appropriate assays include Northern and Southern blotting, RT-PCR (reverse transcriptase polymerase chain reaction), and in situ hybridization, using an appropriately labeled hybridization probe. Suitable assays also include measurement activity in a sample. Suitable assays for variant exo-activity include, but are not limited to, the Betamyl® assay (Megazyme, Ireland). Suitable assays for the endo-activity of the variant include, but are not limited to, the Phadebas blue assay (Pharmacia and Upjohn Diagnostics AB). The assays also include HPLC analysis of liquefacts prepared in the presence of the variant. HPLC can be used to measure amylase activity by separating the DP-3 and DP-4 saccharides from other components of the assay.

Improved handling properties

The polypeptide variants described herein preferably, preferably improved baking properties. Therefore, the polypeptide variants have improved properties when compared to SEQ ID NO: 1, such as one or more of improved thermostability, improved pH stability, or improved exospecificity. The polypeptide variants described herein preferably also have better handling properties, such that a food product treated with a polypeptide variant has one or all of lower firmness, higher elasticity, higher cohesion, lower friability, or higher pliability. compared to a food product that has been treated with SEQ ID NO: 1.

Without wishing to be bound by any particular theory, mutations at particular positions are considered to have individual and cumulative effects on the properties of a polypeptide comprising such mutations.

Thermostability and pH stability

Preferably, the polypeptide variant is thermostable; preferably it has higher thermostability than SEQ ID NO: 1.

In wheat and other cereals the external amylopectin side chains are in the range of 12-19 DP. Therefore, enzymatic hydrolysis of amylopectin side chains, for example, by described polypeptide variants having non-maltogenic exoamylase activity, can markedly reduce their crystallization tendencies.

The starch in wheat and other cereals that are used for baking purposes is present in the form of starch granules that are generally resistant to enzymatic attack by amylases. Therefore starch modification is primarily limited to damaged starch and progresses very slowly during dough making and initial baking until gelatinization begins at about 60 ° C. As a consequence, only amylases with a high degree of thermostability are capable of modifying starch efficiently during baking. And, in general, the efficiency of amylases increases with increasing thermostability. This is so because the more heat stable the enzyme is, the longer it can be active during baking and therefore will provide more anti-aging effect.

Accordingly, the use of variant polypeptides as described herein when added to starch at any stage of its processing into a food product, for example, before, during, or after baking into bread can retard or prevent or slow retrogradation. . Such use is described in more detail below.

As used herein, the term "thermostable" refers to the ability of the enzyme to retain activity after exposure to elevated temperatures. Preferably, the polypeptide variant is capable of degrading starch at temperatures of from about 55 ° C to about 80 ° C or more. Conveniently, the enzyme retains its activity after exposure to temperatures up to about 95 ° C.

The thermostability of an enzyme such as a non-maltogenic exoamylase is measured by its half-life. Consequently, the polypeptide variants described herein have extended half-lives relative to the parent enzyme preferably by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. 100%, 200% or more, preferably at elevated temperatures of 55 ° C to about 95 ° C or more, preferably about 80 ° C.

As used herein, the half-life (t1 / 2) is the time (in minutes) during which half of the enzyme activity is inactivated under defined heat conditions. In preferred embodiments, the half-life is tested at 80 degrees C, preferably; the sample is heated for 1-10 minutes at 80 ° C or higher. The half-life value is then calculated by measuring residual amylase activity, by any of the methods described herein. Preferably, a half-life test is carried out as described in more detail in the Examples.

Preferably, the polypeptide variants described herein are active during baking and hydrolyze starch during and after gelatinization of the starch granules beginning at temperatures of about 55 degrees C. The more heat stable the non-maltogenic exoamylase is, the more time can be active and therefore will provide more anti-aging effect. However, during baking at temperatures above about 85 degrees C, inactivation of the enzyme can occur. If this happens, the non-maltogenic exoamylase can be gradually inactivated so that there is substantially no activity after the baking process in the final bread. Therefore, preferably, the non-mallogenic exoamylases suitable for use as described have an optimum temperature above 50 degrees C and below 98 degrees C.

The thermostability of the polypeptide variants described herein can be improved through the use of genetic engineering of proteins to be more thermostable and therefore more suitable for the uses described herein; therefore the use of polypeptide variants modified to be more thermostable by genetic manipulation of proteins is encompassed.

Preferably, the polypeptide variant is stable at pH; more preferably, it has greater pH stability than SEQ ID NO: 1. As used herein the term "pH stable" refers to the ability of the enzyme to retain activity over a wide pH range. Preferably, the polypeptide variant is capable of degrading starch at a pH of from about 5 to about 10.5. In one embodiment, the degree of pH stability can be tested by measuring the half-life of the enzyme under specific pH conditions. In another embodiment, the degree of pH stability can be tested by measuring the activity or specific activity of the enzyme under specific pH conditions. Specific pH conditions can be any pH from pH 5 to pH 10.5.

Consequently, the polypeptide variant may have a longer half-life or higher activity (according to the assay) when compared to SEQ ID NO: 1 under identical conditions. Polypeptide variants can have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or longer half-life compared to SEQ ID NO. : 1 under identical pH conditions. Alternatively or additionally they may have higher activity when compared to SEQ ID NO: 1 under identical pH conditions.

Exospecificity

It is known that some non-maltogenic exoamylases may have some degree of endoamylase activity. In some cases, it may be necessary to reduce or eliminate this type of activity as endoamylase activity can possibly adversely affect the quality of the final bread product by producing a sticky or rubbery crumb due to the accumulation of branched dextrins.

Exospecificity can be usefully measured by determining the ratio of total amylase activity to total endo-amylase activity. This relationship is referred to herein as an "Index of Exospecificity". In preferred embodiments, an enzyme is considered an exoamylase if it has an exospecificity index of 20 or more. That is, its total amylase activity (which includes exo-amylase activity) is 20 times or greater than its endoamylase activity. In highly preferred embodiments, the exospecificity index of the exoamylases is 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more. In highly preferred embodiments, the exospecificity index is 150 or more, 200 or more, 300 or more, 400 or more, 500 or more, or 600 or more.

Total amylase activity and endoamylase activity can be measured by any means known in the art. For example, total amylase activity can be measured by testing the total number of reducing ends released from a starch substrate. Alternatively, the use of a Betamyl assay is described in more detail in the Examples, and for convenience, the amylase activity analyzed in the Examples is described in terms of "Betamyl units" in the Tables.

Endoamylase activity can be tested using a Phadebas kit (Pharmacia and Upjohn). This uses a cross-linked starch marked with blue (marked with an azo dye); only internal cuts of the starch molecule release the mark, while external cuts do not. Dye release can be measured spectrophotometrically. Accordingly, the Phadebas kit measures endoamylase activity, and for convenience, the results of such an assay are referred to herein as "Phadebas units".

In a highly preferred embodiment, consequently the exospecificity index is expressed in terms of Betamil units / Phadebas units, also referred to as "B / Phad".

Exospecificity can also be analyzed according to the methods described in the prior art, for example, in our International Patent Publication number WO99 / 50399. This measures exospecificity by means of a ratio of endoamylase activity to exoamylase activity. Therefore, in a preferred aspect, the polypeptide variant described herein will have less than 0.5 endoamylase units (EAU) per unit of exoamylase activity. Preferably, the non-maltogenic exoamylases that are suitable for use in accordance with the present invention have less than 0.05 EAU per unit of exoamylase activity and more preferably less than 0.01 EAU per unit of exoamylase activity.

The polypeptide variant described herein will preferably have exospecificity, for example, as measured by the exospecificity indices, described above, in a manner compatible with exoamylases. Furthermore, it preferably has either greater or increased exospecificity when compared to SEQ ID NO: 1. Consequently, for example, the polypeptide variant may have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or greater index of exospecificity when compared with SEQ ID NO: 1, preferably under identical conditions. They can be 1.5x or more, 2x or more, 5x or more, 10x or more, 50x or more, 100x or more, when compared to SEQ ID NO: 1, preferably under identical conditions.

3.4. Methods to purify variants

In general, a variant produced in cell culture is secreted into the medium and can be purified or isolated, for example, by removing unwanted components from the cell culture medium. In some cases, a variant can be recovered from a cell lysate. In such cases, the enzyme is purified from the cells in which it was produced using routine techniques employed by those of skill in the art. Examples include, but are not limited to, affinity chromatography, ion exchange chromatographic methods, including high performance ion exchange, hydrophobic interaction chromatography, two phase partition, ethanol precipitation, reverse phase HPLC, silica chromatography or a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

4. Compositions and uses of variants

A polypeptide variant produced and purified by the methods described above is useful for a variety of industrial applications. In one embodiment, the polypeptide variant defined herein is useful in a starch conversion process, particularly in a starch liquefaction process, for example, corn starch, wheat starch, or barley starch. The desired end product can be any product that can be produced by enzymatic conversion of the starch substrate. For example, the desired product can be a syrup rich in saccharides useful for fermentation, in particular maltotriose, glucose and / or maltose. The final product can then be used directly in a fermentation process to produce fuel or drinkable alcohol (i.e. drinkable alcohol). The person skilled in the art is aware of the various fermentation conditions that can be used in the production of ethanol or other final fermentation products. A microbial organism capable of fermenting maltotriose and / or less complex sugars, such as S. cerevisiae or a genetically modified variant of it is particularly useful. Suitable genetically altered variants of S. cerevisiae particularly useful for maltotriose fermentation include variants that express AGT1 permease (Stambuck et al., Lett. Appl. Microbiol. 43: 370-76 (2006)), MTT1 and MTT1alt (Dietvorst et al. ., Yeast 22: 775-88 (2005)), or MAL32 (Dietvorst et al., Yeast 24: 27-38 (2007)). The present polypeptide variants are also useful in food preparation compositions and methods, when enzymes that express amylase activity at high temperatures are desired.

The desirability of using a particular polypeptide variant will depend on the general properties exhibited by the polypeptide variant with respect to the requirements of a particular application. As a general matter, polypeptide variants useful for a starch conversion process have substantial endo-amylase activity compared to wild-type PS4 or SEQ ID NO: 1, and / or have lower exo to endo-amylase activity in comparison with wild-type PS4 or SEQ ID NO: 1. Such polypeptide variants can be particularly useful in a liquefaction process, when used alone or in combination with other amylase variants, wherein internal cleavage of complex branching saccharides reduces the viscosity of the substrate. Some polypeptide variants useful for liquefaction, however, are expected to have endo-amylase activity comparable to or even less than wild-type PS4. Useful polypeptide variants include those having more or less the exo-amylase activity of wild-type PS4 polypeptide or SEQ ID NO: 1, according to the application. Compositions can include one or a combination of amylase variants, each of which can display a different set of properties.

4.1. Preparation of starch substrates

Those skilled in the art are well aware of the available methods that can be used to prepare starch substrates for use in the processes described herein. For example, a useful starch substrate can be obtained from tubers, roots, stems, legumes, cereals, or whole grain. More specifically, granular starch comes from plants that produce high amounts of starch. For example, granular starch can be obtained from corn, cobs, wheat, barley, rye, millet, sago, cassava, tapioca, sorghum, rice, peas, beans, bananas, or potatoes. Corn contains approximately 60 to 68% starch; barley contains approximately 55-65% starch; millet contains approximately 75 to 80% starch; wheat contains approximately 60 to 65% starch; and polished rice contains 70-72% starch. Specifically contemplated starch substrates are corn starch, wheat starch, and barley starch. The starch of a grain can be ground or whole and includes corn solids, such as grains, bran and / or cobs. The starch can be raw highly refined starch or raw material from starch refinery processes. Various starches are also commercially available. For example, cornstarch is available from Cerestar, Sigma, and Katayama Chemical Industry Co. (Japan); Wheat starch is available from Sigma; Sweet potato starch is available from Wako Pure Chemical Industry Co. (Japan); and potato starch is available from Nakaari Chemical Pharmaceutical Co. (Japan).

Maltodextrins are useful as starch substrates in embodiments of the present invention. Maltodextrins comprise hydrolysis products of starch having about 20 or less dextrose (glucose) units. Typical commercial maltodextrins contain mixtures of polysaccharides that include from about three to about nineteen linked dextrose units. Maltodextrins are defined by the FDA as products that have a dextrose equivalence (DE) of less than 20. They are generally recognized as safe food ingredients (GRAS) for human consumption. Dextrose equivalence (DE) is a measure of reducing energy compared to a dextrose (glucose) standard of 100. The higher the DE, the greater the degree of depolymerization of the starch, resulting in a smaller average polymer size. (polysaccharide), and the greater the sweetness. A particularly useful maltodextrin is MALTRIN® M040 made from corn starch, available from Grain Processing Corp. (Muscatine, Iowa): DE 4.0-7.0; bulk density 0.51 g / cc; water content measured at 6.38% by weight.

The starch substrate can be a raw, ground whole grain starch, containing non-starch fractions, eg, germ residues and fibers. The grinding may comprise wet grinding or dry grinding. In wet milling, the whole grain is immersed in water or dilute acid to separate the grain into its component parts, fibers, eg starch, protein, germ, oil, grain fibers. Wet milling efficiently separates germ and flour (i.e. starch and protein granules) and is especially suitable for syrup production. In dry milling, whole grains are ground into a fine powder and processed without breaking the grain into its component parts. The grain dry ground in this way will comprise significant amounts of non-starch carbohydrate compounds, in addition to starch. Most of the ethanol comes from dry milling. Alternatively, the starch to be processed can be a highly refined starch grade, eg, at least about 90%, at least 95%, at least 97%, or at least 99.5% pure.

4.2. Saccharification, gelatinization and liquefaction of starch

As used herein, the term "liquefaction" or "liquefying" means a process by which starch is converted into less viscous, shorter chain dextrins. This process involves gelatinization of the starch simultaneously with or followed by the addition of a variant polypeptide described herein. A thermostable polypeptide variant described herein is preferably used for this application. Additional liquefaction inducing enzymes can optionally be added.

In some embodiments, the starch or maltodextrin substrate prepared as described above is suspended with water. The starch or maltodextrin suspension may contain starch as dry solids weight percent of about 10-55%, about 20-45%, about 30-45%, about 30-40%, or optionally about 30 -35%. • -amylases, eg, bacterial alphaamylases including Bacillus alpha-amylases, are usually supplied, for example, at about 1500 units per kg of starch dry matter. To optimize stability and activity of • -amylase, the pH of the suspension can be adjusted to the optimum pH for the polypeptide variant used. Other • -amylases can be added and may require different optimal conditions. Bacterial • -amylase remaining in the suspension after liquefaction can be inactivated by lowering the pH in a subsequent reaction step or by removing calcium from the suspension.

The starch suspension plus the polypeptide variant can be pumped continuously through a jet cooker, which is steam heated from about 85 ° C to 105 ° C. Gelatinization occurs very rapidly under these conditions, and enzyme activity, combined with significant shear forces, begins hydrolysis of the starch substrate. The residence time in the jet cooker is very short. The partially gelatinized starch can be passed into a series of holding tubes maintained at about 85-105 ° C and held for about 5 min. to complete the gelatinization process. These tanks may contain baffles to discourage back mixing. As used herein, the term "secondary liquefaction" refers to the stage of liquefaction subsequent to primary liquefaction, when the suspension is allowed to cool to room temperature. This cooling step can be from about 30 minutes to about 180 minutes, for example, about 90 minutes to 120 minutes.

A variant of the polypeptide defined herein can be added to the liquefied starch obtained by the above procedure or to a maltodextrin suspension at about 0.01 to about 1.0 kg / MTDS. 1 kg / MTDS = 0.1% by weight of dissolved solids. In one embodiment, a polypeptide defined herein can be added to a liquefied starch or maltodextrin suspension at a treatment level ranging from about 0.001% by weight to about 0.01% by weight based on the dissolved solids. In a typical embodiment, a polypeptide defined herein can be added to a liquefied starch or maltodextrin suspension at a treatment level ranging from about 0.0025% by weight to about 0.01% by weight on the basis of dissolved solids. In one embodiment, the polypeptide defined herein is immobilized, and the liquefied starch or maltodextrin substrate is passed over the immobilized polypeptide defined herein and converted into the product in a continuous reaction. In this embodiment, the polypeptide defined herein can be immobilized with additional enzymes, such as a pullulanase.

The production of maltotetraose may further comprise contacting the liquefied starch or other source of maltodextrins with an isoamylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, or any combination of these.

Also provided is a method of preparing a saccharide syrup (eg, maltotetraose), which comprises adding a variant of the polypeptide defined herein or a composition comprising the variant in a starch liquefied and saccharifying the starch liquefied to form the saccharide syrup. The polypeptide variant defined herein can be added to the starch liquefied in a range from about 0.001% by weight to about 0.1% by weight based on dissolved solids. In one embodiment, the variant is added to the starch liquefier in a range from about 0.0025% by weight to about 0.01% by weight based on dissolved solids. Units of concentration are also expressed herein as kg of polypeptide variant per metric ton of dry solids (MTDS), where 1 kg / MTDS = 0.1% by weight of dissolved solids. The liquefied starch solution can be a slurry of liquefied starch at about 20-35% w / w dry solids. The starch can be obtained from corn, cobs, wheat, barley, rye, millet, sago, cassava, tapioca, sorghum, rice, peas, beans, bananas or potatoes. The starch liquefied can be saccharified at about 55 ° C to about 65 ° C such as about 60 ° C to about 65 ° C. The starch liquefied can be saccharified at about pH 5.0 to about pH 7.0. A pullulanase, isoamylase, pullulanase, protease, cellulase, hemicellulase, lipase, cutinase, or any combination of these, can be added with the polypeptide variant to the starch liquefied. In one embodiment, the saccharide syrup can be fermented to produce ethanol. The saccharide syrup produced by the method may comprise at least about 40%, about 45%, about 50%, about 55%, or about 60% by weight of maltotetraose based on total saccharide content.

In another aspect a method of preparing a saccharide syrup is provided, including the addition of a variant of the polypeptide defined herein and an alpha-amylase to the granular starch and hydrolysis of the granular starch to form the saccharide syrup. In one embodiment, the granular starch liquefact is produced by an alpha-amylase. In one embodiment, the granular starch liquefact is an acid-produced liquefied. In one embodiment the polypeptide variant defined herein is added to granular starch in a range from about 0.001% by weight to about 0.1% by weight based on dissolved solids. In another embodiment the polypeptide variant defined herein is added to granular starch in a range from about 0.0025% by weight to about 0.01% by weight based on dissolved solids. The Granular starch can be obtained from corns, cobs, wheat, barley, rye, millet, sago, cassava, tapioca, sorghum, rice, peas, beans, bananas or potatoes.

In a particular embodiment the granular starch is saccharified at 55 ° C to 65 ° C such as 60 ° C to 65 ° C. In another embodiment the granular starch is saccharified at pH 5.0 to pH 7.0. It is envisioned that the method may also include fermentation of the saccharide syrup to produce ethanol.

In one embodiment the method includes a step of adding an enzyme having debranching activity in granular starch. The enzyme having debranching activity can include but is not limited to an isoamylase, a pullulanase, an isopululanase, a neopululanase, or any combination thereof. It is also envisioned that the method may optionally include a further step of adding a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a pectate lyase or any of their combinations to the granular starch.

In one embodiment the saccharide syrup includes at least 35% by weight of maltotetraose based on the total saccharide content. Alternatively, the saccharide syrup includes at least 45% by weight of maltotetraose based on the total saccharide content. In another embodiment the saccharide syrup includes at least 50% by weight of maltotetraose based on the total saccharide content. In a further embodiment the saccharide syrup includes from 45% by weight to 60% by weight of maltotetraose based on the total saccharide content.

It is envisioned that the polypeptide variant defined herein by the method may be immobilized.

In another aspect a method of obtaining IMO is provided, including the addition of a) a variant of the polypeptide defined herein, b) an alpha-amylase, and c) a transglucosidase for starch in the form of a starch liquefact or granular starch and saccharify the starch to form IMO. It is envisioned that the IMO can be formed to an IMO number of at least 30, at least 40 and / or at least 45. In one embodiment the starch is obtained from corn, cob, wheat, barley, rye, millet, sago , cassava, tapioca, sorghum, rice, peas, beans, bananas or potatoes.

In another aspect a method of obtaining IMO is provided, which includes adding a) a variant of the polypeptide, b) an alpha-amylase, and c) a transglucosidase to the starch in the form of a starch liquefied or granular starch and saccharifying the starch to form IMO. Any number of transgucosidase (TG) enzymes can be used, for example, TRANSGLUCOSIDASE L-500® (Danisco US Inc., Genencor Division).

It is envisioned that the IMO can be formed in an IMO number of at least 30, at least 40 and / or at least 45. In one embodiment the starch is obtained from corn, cob, wheat, barley, rye, millet, sago, cassava, tapioca, sorghum, rice, peas, beans, bananas or potatoes.

4.3. Fermentation processes

Yeast typically Saccharomyces spp. it is added to the dough and fermentation continues for 24-96 hours, such as typically 35 to 60 hours. The temperature is between about 26-34 ° C, typically about 32 ° C, and the pH is about pH 3-6, usually around a pH of about 4-5.

In one embodiment, a batch fermentation process is used in a closed system, where the composition of the medium is fixed at the beginning of fermentation and is not altered during fermentation. At the beginning of the fermentation, the medium is inoculated with the desired microbial organism. In this method, fermentation is allowed to occur without the addition of any components to the system. Typically, a batch fermentation qualifies as a "batch" with respect to the addition of the carbon source, and attempts are often made to control factors such as pH and oxygen concentration. The metabolite and biomass compositions of the batch system are constantly changing until the time fermentation is stopped. Within batch cultures, cells progress through a static lag phase to a high growth log phase and finally to a stationary phase, where the growth rate slows or stops. If left untreated, cells in the stationary phase eventually die. In general, the logarithmic phase cells are responsible for the mass of production of the product.

A suitable variation on the standard batch system is the "batch fed" fermentation system. In this variation of a typical batch system, substrate is added in increments as fermentation progresses. Batch fed systems are useful when catabolite repression is likely to inhibit cell metabolism and when it is desirable to have limited amounts of substrate in the medium. Measurement of actual substrate concentration in batch fed systems is difficult and therefore estimated on the based on changes in measurable factors, such as pH, dissolved oxygen, and the partial pressure of waste gases, such as CO2. Batch-fed and batch-fed fermentations are well known in the art. Continuous fermentation is an open system in the that a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed during before him prosecution. Continuous fermentation generally maintains cultures at a constant high density, where cells are primarily logarithmic phase growth. Continuous fermentation allows the modulation of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, a limiting nutrient, such as the carbon source or nitrogen source, is held at a fixed rate and all other parameters are allowed to moderate. In other systems, a number of factors that affect growth can be continuously altered while the cell concentration, measured by mean turbidity, remains constant. Continuous systems strive to maintain steady state growing conditions. Therefore, the loss of cells due to the medium being extracted must be balanced with the rate of cell growth in fermentation. Nutrient and growth factor modulation methods for continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

After fermentation, the mixture is distilled to extract ethanol. The ethanol obtained according to the process of the description can be used as, for example, combustible ethanol; Beverage ethanol, that is, neutral drinkable alcohol or industrial ethanol. The leftover from fermentation is grain, which is normally used for animal feed, either in liquid or dry form. Additional details on how to carry out liquefaction, saccharification, fermentation, distillation, and recovery of ethanol are well known to experts. According to the process of the invention, saccharification and fermentation can be carried out simultaneously or separately.

5. Compositions and methods for the preparation of bakery and food

In one aspect, the compositions, including additives, food products, bakery products, improver compositions, food products, including animal food, etc. comprising such altered variants as described herein, such as those having non-maltogenic exoamylase activity, as well as methods of obtaining and using such polypeptides and compositions are provided.

As noted above, the polypeptide variants described above may comprise one or more improved handling properties, preferably improved baking properties. Thus, the polypeptide variants described above can provide food products so treated having one or more of (preferably all of) lower firmness, higher elasticity, higher cohesion, lower friability, or higher pliability. Such improved handling or baking properties exhibited by variant polypeptides are described in more detail below. In one aspect, a variant is provided, in which the half-life (t1 / 2), preferably at 60 degrees C, increases by 15% or more, preferably 50% or more, most preferably 100% or more, compared to the amino acid sequence of SEQ ID NO: 1.

In one aspect, a variant is provided, as described herein, wherein a product treated with the variant has one or more, preferably all of the following properties: (a) inferior firmness; (b) superior elasticity; (c) superior cohesion; (d) lower friability; and (e) increased folding capacity compared to a food product that has been treated with the amino acid sequence of SEQ Id NO: 1.

In one aspect, a variant is provided, as described herein, in which the elasticity. Cohesion or pliability of the food product independently increases by 15% or more, preferably 50% or more, most preferably 100% or more, compared to a food product that has been treated with the amino acid sequence of the SEQ ID NO: 1.

In one aspect, a variant is provided, as described herein, in which each is elastic. Cohesion and foldability of a food product treated with the variant is increased compared to a food product that has been treated with the amino acid sequence of SEQ ID NO: 1.

In one aspect, a variant is provided, as described herein, wherein the firmness or friability of the food product is independently decreased by 15% or more, preferably 50% or more, most preferably 100% or Furthermore, with respect to a food product, it has been treated with the amino acid sequence of SEQ ID NO: 1.

In one aspect, a variant is provided, as described herein, in which each firmness and friability of a food product treated with the polypeptide is decreased compared to a food product that has been treated with the amino acid sequence of the SEQ ID NO: 1,

In one aspect, there is provided a variant, as described herein comprising a fragment of at least 20 residues of a polypeptide as set forth in the claims, wherein the polypeptide has non-maltogenic exoamylase activity.

In one aspect, a variant, as described herein, has non-maltogenic exoamylase activity. Such activity can be determined by the methods described in US 6,667,065.

In one aspect, the treatment of food products, particularly doughs and bakery products with such polypeptides, and such that the food products exhibit the desired qualities set forth above. In one aspect, a process for treating a starch is provided which comprises contacting the starch with a variant, as described herein, and allowing the polypeptide to generate one or more linear products from the starch. In one aspect, the use of a variant, as described herein, for preparing a food product or animal feed is provided. In one aspect, there is provided a process for preparing a food or animal food product which comprises mixing a variant, as described herein with a food or animal food ingredient. In one aspect, the use or process is provided, wherein the food product comprises a dough or dough product, preferably a processed dough product. In one aspect, the use or process is provided, in which the food product is a bakery product. In one aspect, a process for making a bakery product is provided comprising: (a) providing a starch medium; (b) adding a variant to the starch medium, as described herein; and (c) applying heat to the starch medium during or after step (b) to produce a bakery product. In one aspect, there is provided a food product, animal food product, dough product or a bakery product obtained by a process defined in the claims.

In one aspect, the use of a variant, as described herein, in a dough product is provided to retard or reduce aging, preferably deleterious retrogradation, of the dough product.

In one aspect, there is provided the use of a variant, as described herein, in a dough product to improve some or more of the firmness, elasticity, cohesion, friability, or pliability of the dough product.

In one aspect, a combination of a variant, as described herein, together with one or more of the following:

(a) Maltogenic alpha-amylase also called glucan 1,4 - ^ - maltohydrolase (EC 3.2.1.133) is provided from Bacillus stearothermophilus, or a variant, homologous or mutants thereof having maltogenic alpha-amylase activity; (b) a baker's xylanase (EC 3.2.1.8) from for example Bacillus sp., Aspergillus sp., Thermomyces sp. or Trichoderma sp .;

(c) α-amylase (EC 3.2.1.1) from Bacillus amiloliquefaciens or Aspergillus sp. or a variant, homologue or mutants thereof having alpha-amylase activity; and

(d) a lipase such as glycolipase from Fusarium heterosporum.

The variants described herein preferably comprise one or more improved handling properties compared to a parent polypeptide or a wild-type polypeptide, such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably ^s E ^q ID NO: 1. Improved handling properties in preferred embodiments may comprise improved baking properties.

Accordingly, the variant described herein in one aspect is such that a food product treated with the polypeptide variant has an improved handling or baking-preferred property compared to a food product that has been treated with a parent polypeptide or a wild type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably ^s E ^q ID NO: 1. The handling or baking property can be selected from the group consisting of: firmness, elasticity, cohesion, friability and folding capacity.

These handling properties can be analyzed by any means known in the art. For example, firmness, elasticity and cohesion can be determined by analyzing bread slices by analyzing the texture profile using for example a texture analyzer, as for example described in 11 Firmness

The variants described herein are in one aspect such that a food product treated with the polypeptide variant has inferior firmness compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of the SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1.

Firmness in preferred embodiments is inversely correlated with the softness of the food product; consequently, a higher softness may reflect a lower firmness, and vice versa.

Firmness can be measured using the "Firmness Assessment Protocol" set out in Example 11.

In one aspect, the variants described herein are such that a food product treated with the polypeptide variant has at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 100%, 200% or more lower firmness compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1. A food product treated with The polypeptide variants described herein may have 1 , 1x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more of lower firmness compared to a food product that has been treated with a parent polypeptide or a wild type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1.

Elasticity

In one aspect, the variants described herein are such that a food product treated with the polypeptide variant has superior elasticity compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1.

Elasticity can be measured using the "Elasticity Assessment Protocol" set out in 11.

Consequently in one aspect, the variants described herein are such that a food product treated with the polypeptide variant has at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more superior elasticity compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1. A food product treated with the polypeptide variant can have a 1.1x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more superior elasticity compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ iD No: 1.

Cohesion

In one aspect, the variants described herein are such that a food product treated with the polypeptide variant has superior cohesion compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of the SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1.

Cohesion can be measured using the "Cohesion Assessment Protocol" set out in 11.

Accordingly, in one aspect, the variants disclosed herein are such that a food product treated with the variant polypeptide disclosed herein has 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 100%, 200% or more of higher cohesion compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1. A food product treated with the polypeptide variant can have 1.1x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x , 8x, 9x, 10x or more of higher cohesion compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1.

Friability

In one aspect, the variants described herein are such that a food product treated with the polypeptide variant has inferior friability compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of the SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1.

Friability can be measured by the "Friability Evaluation Protocol" set forth in Example 13.

Consequently in one aspect, the variants described herein are such that a food product treated with the polypeptide variant has 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more lower friability compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1. A food product treated with the polypeptide variant can have a 1.1x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more lower friability compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1.

Folding capacity

In one aspect, the variants described herein are such that a food product treated with the polypeptide variant has greater folding capacity compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide. of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1.

Pliability is preferably measured by the "Pliability Evaluation Protocol" set forth in Example 14.

Consequently, the variants described herein are such that a food product treated with the polypeptide variant has 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more of increased folding capacity compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4 , 6, 8 or 10, preferably SEQ ID NO: 1. A food product treated with the polypeptide variant can have a 1.1x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x , 10x or more greater folding capacity compared to a food product that has been treated with a parent polypeptide or a wild-type polypeptide such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SEQ ID NO: 1.

In one aspect, the use of the polypeptide variants described herein in combination with a xylanase to improve foldability is provided.

Furthermore, a method of preparing a food product is provided, the method comprising: (a) obtaining a non-maltogenic exoamylase; (b) introducing a mutation at any or more of the non-maltogenic exoamylase positions as set forth herein; (c) mixing the resulting polypeptide with a food ingredient.

The polypeptide variants can be used to improve the bulk of bakery products such as bread. Without wishing to be bound by any particular theory, this is considered to result from the reduction in the viscosity of the dough during heating (such as baking) as a result of amylase shortening the amylose molecules. This allows the carbon dioxide generated by the fermentation to increase the size of the loaf with less hindrance.

Consequently, food products comprising or treating with the polypeptide variants are expanded in volume when compared to products that have not been treated in this way, or treated with the parent polypeptides such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably SeQ ID NO: 1. In other words, food products have a larger volume of air per volume of food product. Alternatively, or in addition, food products treated with polypeptide variants as described herein have a lower density, or weight (or mass) per volume ratio. In particularly preferred embodiments, polypeptide variants are used to improve bread volume. Enhancing or expanding volume is beneficial because it reduces the gummyness or starch content of foods. Light foods are preferred by consumers, and the customer experience is enhanced. In preferred embodiments, the use of the polypeptide variants increases the volume by 10%, 20%, 30%, 40%, 50% or more.

The polypeptide and nucleic acid variants described herein can be used as -or in the preparation of a food. In particular, they can be added to a food, that is, as a food additive. The term "food" is considered to include both prepared food as well as an ingredient for a food, such as flour. In a preferred aspect, the food is for human consumption. The food can be in the form of a solution or as a solid - according to the use and / or the mode of application and / or the mode of administration. The polypeptide and nucleic acid variants can be used as a food ingredient. As used herein, the term "food ingredient" includes a formulation, which is or can be added to functional or edible foods and includes formulations that can be used at low levels in a wide variety of products that require, for example , acidification or emulsion. The food ingredient can be in the form of a solution or as a solid - according to the use and / or the mode of application and / or the mode of administration. The polypeptide and nucleic acid variants described herein can be - or can be added to - food supplements. The polypeptide and nucleic acid variants described herein may be - or may be added to - functional foods. As used herein, the term "functional food" means a food that is capable of providing not only a nutritional effect and / or a taste satisfaction, but is also capable of providing an additional beneficial effect to the consumer. Although there is no legal definition of a functional food, most stakeholders in this area agree that they are marketed foods that have specific health effects.

The polypeptide variants can also be used in the manufacture of a food product or edible. Typical groceries include dairy products, meat products, poultry products, fish products, and dough products. The dough product can be any processed dough product, including fried, oil-fried, roasted, baked, steamed, and boiled doughs, such as steamed bread and rice cakes. In highly preferred embodiments, the food product is a bakery product.

Preferably, the foodstuff is a bakery product. Typical (baked) bakery products include bread - such as breads, rolls, buns, donuts, pizza bases, etc. pastries, pretzels, tortillas, cakes, sweet cookies, biscuits, pretzels, etc.

Food products preferably benefit from one or more of the handling or baking properties of the polypeptide variants described herein. The property of handling or baking Improved can be selected from the group consisting of: improved firmness, improved elasticity, improved cohesion, improved friability, and improved pliability.

Furthermore, in one aspect, there is provided a method of modifying a food additive comprising a non-maltogenic exoamylase, the method comprising introducing a mutation at one or more of the positions of the non-maltogenic exoamylase set forth herein. The same method can be used to modify a food ingredient, or a food supplement, a food product, or an edible.

In one aspect, the use of polypeptide variants that are capable of retarding the slump of starch media, such as starch gels, is provided. Polypeptide variants are especially capable of retarding the deleterious retrogradation of starch.

Most starch granules are composed of a mixture of two polymers: an essentially linear amylose content and a highly branched amylopectin. Amylopectin is a very large, branched molecule consisting of chains of α-D-glucopyranosyl units linked by (1-4) bonds, wherein said chains are linked by α-D- (1-6) bonds to form branches. Amylopectin is present in all natural starches, constituting approximately 75% of most common starches. Amylose is essentially a straight chain of linked α-D-glucopyranosyl (1-4) units having few α-D- (1-6) branches. Most starches contain about 25% amylose.

Starch granules heated in the presence of water undergo an order-disorder phase transition called gelatinization, where the liquid is absorbed by the swelling granules. Gelatinization temperatures vary for different starches. After cooling the freshly baked bread, the amylose fraction, within hours, retrogrades to develop a network. This process is beneficial as it creates a desirable crumb structure with a low degree of firmness and better slicing properties. The more gradual crystallization of amylopectin occurs within the gelatinized starch granules during the days after baking. In this process, amylopectin is considered to strengthen the amylose network in which the starch granules are embedded. This reinforcement leads to an increase in the firmness of the breadcrumb. This reinforcement is one of the main causes of bread aging.

The quality of baked goods is known to gradually deteriorate during storage. As a consequence of starch recrystallization (also called retrogradation), the water-holding capacity of the crumb is changed with important implications for organoleptic and dietary properties. The crumb loses softness and elasticity and becomes firm and brittle. Increased crumb firmness is often used as a measure of the aging process of bread.

The rate of detrimental retrogradation of amylopectin depends on the length of the amylopectin side chains. Consequently, enzymatic hydrolysis of amylopectin side chains, for example, by the polypeptide variants described herein that have non-maltogenic exoamylase activity, can markedly reduce their crystallization tendencies.

Accordingly, in one aspect, the use of polypeptide variants described herein when added to starch at any stage of its transformation into a food product, for example, before, during or after baking in bread can delay or prevent or slow down retrograde. Such use is described in more detail below.

In one aspect, a method is provided for enhancing the ability of a non-maltogenic exoamylase to prevent aging, preferably deleterious retrogradation, of a dough product, the method comprising introducing a mutation at any or more of the positions of non-maltogenic exoamylase discussed in this document.

For the evaluation of the anti-aging inhibitory effect of the polypeptide variants, such a variant having non-maltogenic exoamylase activity described herein, the firmness of the crumb can be measured 1, 3 and 7 days after baking by means of an Analyzer. Instron 4301 Universal Food Texture Machine or similar equipment known in the art.

Another method traditionally used in the art and used to evaluate the effect on starch retrogradation of a polypeptide variant such as a variant having exoamylase activity is based on DSC (Differential Scanning Calorimetry). Herein, the enthalpy of fusion of retrograde amylopectin is measured in breadcrumbs or crumbs of a dough from the model baked dough system with or without enzymes (control). The applied DSC equipment can be a DSC 820 Mettler-Toledo run with a temperature gradient of 10 ° C per minute, from 20 to 95 ° C. For the preparation of the samples, 10 to 20 mg of crumb are weighed and transferred to Mettler-Toledo aluminum plates that are hermetically sealed.

Model system doughs can contain standard wheat flour and optimal amounts of water or buffer with or without the polypeptide variant. Mix 10 or 50 g in a Brabender farinograph for 6 or 7 min, respectively. The mass samples are placed in glass test tubes (15 * 0.8 cm) with a cap. These test tubes are subjected to a baking process in a water bath starting at 30 min. from incubation at 33 ° C followed by heating 33 to 95 ° C with a gradient of 1.1 ° C per minute. and, finally, 5 min. incubation at 95 ° C. The tubes are then stored in a thermostat at 20 ° C prior to DSC analysis.

In one embodiment, the variants described herein have a reduced enthalpy of fusion, compared to the control. In a further embodiment, the variants have 10% or more reduced enthalpy of fusion. In still another aspect, they have 20% or more, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced enthalpy of fusion when compared to the control.

In one aspect, the use of polypeptide variants in the preparation of food products, in particular starch products, is provided. The method comprises forming the starch product by adding a non-maltogenic exoamylase enzyme such as a polypeptide variant to a starch medium. If the starch medium is a dough, then the dough is prepared by mixing flour, water, the non-maltogenic exoamylase which is a polypeptide variant described herein, and optionally other possible ingredients and additives. The term "starch" is to be understood in the sense of starch per se or a component thereof, especially amylopectin. The term "starch medium" means any suitable medium comprising starch. The term "starch product" means any product that contains or is based on or is derived from starch. Preferably, the starch product contains or is based on or is derived from starch obtained from wheat flour. The term "flour" as used herein is a synonym for finely ground wheat or other grain flour. Preferably, however, the term means flour obtained from wheat per se and not from another grain. Thus, and unless otherwise stated, references to "wheat flour" as used herein preferably mean references to wheat flour per se, as well as to wheat flour when present in a medium. , just like a dough.

A preferred flour is wheat flour or rye flour or mixtures of wheat and rye flour. However, dough comprising flour derived from other types of cereals such as for example rice, corn, barley and durra is also contemplated. Preferably the starch product is a bakery product. Most preferably the starch product is a bread product. Even more preferably, the starch product is a baked farinaceous bakery product. The term "baked farinaceous bakery product" refers to any baked product based on a dough obtainable by mixing flour, water, and a leavening agent under dough-forming conditions. Obviously you can add other components to the dough mix.

Consequently, if the starch product is a baked farinaceous bakery product, the process then comprises mixing - in any suitable order - flour, water, and a leavening agent under dough-forming conditions and further adding a polypeptide variant. described herein, optionally in the form of a premix. The leavening agent can be a chemical leavening agent such as sodium bicarbonate or any strain of Saccharomyces cerevisiae (Baker's yeast).

The polypeptide variant described herein can be added together with any dough ingredient including the ingredient mix water or dough or with any additive or additive mix. The dough can be prepared by any conventional dough-making method common in the bakery industry or any other dough-based product making industry.

The baking of farinaceous bakery products such as for example white bread, bread made from sifted rye flour and wheat flour, rolls and the like is normally accomplished by baking the bread dough at oven temperatures in the range of 180 to 250 ° C for approximately 15 to 60 minutes. During the baking process a strong temperature gradient (200 to 120 ° C) is prevalent in the outer dough layers where the characteristic crust of the baked product develops. However, due to the consumption of heat due to the generation of steam, the temperature in the crumb is only about 100 ° C at the end of the baking process.

Also provided is a process for making a bread product comprising: (a) providing a starch medium; (b) adding to the starch medium a variant polypeptide described herein; and (c) applying heat to the starch medium during or after step (b) to produce a bread product. A process for obtaining a bread product is provided which comprises adding a described polypeptide variant to a starch medium.

In one aspect, the polypeptide variant can be added as a liquid preparation or as a dry powdery composition comprising the enzyme as the only active component or in admixture with one or more dough ingredients or dough additive.

In a further aspect, improver compositions are provided, including bread improver compositions and dough improver compositions. These comprise a polypeptide variant, optionally together with an additional ingredient, or an additional enzyme, or both.

In one aspect, a dough improver composition is provided, wherein the improver composition comprises a variant set forth in any one of the claims, and at least one dough ingredient or additional dough additive.

In one aspect, there is provided a composition comprising a flour and a variant set forth in any one of the claims.

In a further aspect, the use of bread and dough improver compositions in baking is provided. In a further aspect, a baked or dough product or dough obtained from the bread improver composition or dough improver composition is provided. In another aspect, there is provided a product or dough baked or obtained through the use of a bread improver composition or a dough improver composition.

A dough can be prepared by mixing flour, water, a dough improver composition comprising a polypeptide variant (as described above) and optionally other ingredients and additives.

The dough improver composition can be added along with any dough ingredient including flour, water, or other optional ingredients or additives. The dough improver composition can be added before the flour or water or other optional ingredients and additives. The dough improver composition can be added after the flour or water, or other optional ingredients and additives. The dough can be prepared by any conventional dough preparation method common in the bakery industry or any other flour dough-based product manufacturing industry.

The dough improver composition can be added as a liquid preparation or in the form of a dry powder composition comprising the composition as the only active component or in admixture with one or more other ingredients or dough additives.

The amount of the polypeptide variant that is added is usually in an amount that results in the presence in the finished dough of 50 to 100,000 units per kg of flour, preferably 100 to 50,000 units per kg of flour. Preferably, the amount is in the range of 200 to 20,000 units per kg of flour. Alternatively, the polypeptide variant is added in an amount that results in the presence in the finished dough of 0.02-50 ppm of flour-based enzyme (0.02-50 mg enzyme per kg of flour), preferably 0.2 - 10 ppm.

In the present context, 1 unit of non-maltogenic exoamylase is defined as the amount of enzyme that releases hydrolysis products equivalent to 1 pmol of reducing sugar per min when incubated at 50 degrees C in a test tube with 4 ml of 10 mg waxy corn starch ml / in 50 mM MES, 2 mM calcium chloride, pH 6.0 as described hereinafter.

The dough as described herein generally comprises wheat semolina or wheat flour and / or other types of semolina, flour or starch such as corn flour, cornstarch, corn flour, rice flour, rye flour. , rye semolina, oatmeal, oatmeal, soybean meal, sorghum semolina, sorghum meal, potato semolina, potato flour or potato starch. The dough can be fresh, frozen, or partially baked.

The dough can be a proofed dough or a dough to be proofed. The dough can be leavened in various ways, such as by adding chemical leavening agents, for example sodium bicarbonate or by adding a leavening agent (leavening dough), but it is preferred to proof the dough by adding a suitable yeast culture , such as a culture of Saccharomyces cerevisiae (baker's yeast), for example, a commercially available strain of S. cerevisiae.

The dough may comprise fats, such as granulated fat or shortening. The dough may further comprise an additional emulsifier such as mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxyethylene stearates, or lysolecithin.

A premix comprising flour is also provided in conjunction with the blend described herein. The premix may contain dough improver additives and / or bread improver additives, for example any of the additives, including enzymes, mentioned herein.

In order to further improve the properties of the baked product and impart distinctive qualities to the baked product, additional dough ingredients and / or dough additives can be incorporated into the dough. Typically, such additionally added components may include dough ingredients such as salt, grains, fats and oils, sugar or sweetener, dietary fibers, protein sources such as powdered milk, soy, gluten or eggs, and dough additives such as emulsifiers, other enzymes, hydrocolloids, flavoring agents, oxidizing agents, minerals, and vitamins.

Emulsifiers are useful as dough enhancers and crumb softeners. As dough builders, emulsifiers can provide tolerance with respect to standing time and tolerance to shock during fermentation. Furthermore, dough enhancers will improve the tolerance of a given dough to variations in fermentation time. Most dough boosters also improve puffiness which means increasing the volume of leavened to baked goods. Lastly, the dough boosters will emulsify all the fats present in the recipe mix.

Suitable emulsifiers include lecithin, polyoxyethylene stearate, mono- and diglycerides of edible fatty acids, acetic acid esters of monoglycerides and diglycerides of edible fatty acids, lactic acid esters of mono- and diglycerides of edible fatty acids, esters of citric acid. of edible fatty acid mono- and diglycerides, diacetyl tartaric acid esters of edible fatty acid monoglycerides and diglycerides, sucrose esters of edible fatty acids, sodium stearoyl-2-lactylate, and calcium stearoyl-2-lactylate.

The additional dough additive or ingredient may be added along with any dough ingredient including flour, water or other optional ingredients or additives, or the dough improver composition. The additive or additional dough ingredient may be added before the flour, water, other ingredients and optional additives or the dough improver composition. The additive or additional dough ingredient may be added after the flour, water, other ingredients and optional additives or the dough improver composition.

The additive or additional dough ingredient may conveniently be a liquid preparation. However, the additive or additional dough ingredient may conveniently be in the form of a dry composition.

In one aspect the additive or additional dough ingredient is at least 1% of the weight of the flour component of the dough. In a further aspect, the additive or additional bulk ingredient is at least 2%, preferably at least 3%, preferably at least 4%, preferably at least 5%, preferably at least 6%. If the additive is a fat, then normally the fat may be present in an amount of 1 to 5%, usually 1 to 3%, more usually about 2%.

For commercial and domestic use of flour for baking and food production, it is important to maintain an adequate level of • -amylase activity in the flour. An activity level that is too high can produce a product that is sticky and / or mushy and therefore not marketable. Flour with insufficient • -amylase activity may not contain enough sugar for proper yeast function, resulting in dry, friable bread or baked goods. Accordingly, a variant, as described herein, by itself or in combination with another • -amylase or other variant, can be added to the flour to increase the level of endogenous • -amylase activity in the flour. The variant of this embodiment can have an optimum temperature in the presence of starch, for example in the ranges of about 30-90 ° C, 40-80 ° C, 40-50 ° C, 45-65 ° C, or 50 -60 ° C. The optimum pH in a 1% solution of soluble starch can be for example between pH 4.5 to 6.0.

Cereals, such as barley, oats, wheat, as well as plant components, such as corn, hops, and rice, are also used for brewing, both in industrial and home brewing. The components used in the brewing industry can be unmalted or they can be malted, that is, partially sprouted, which produces an increase in the levels of enzymes, including • -amylase. For successful brewing, adequate levels of enzyme • -amylase activity are necessary to ensure proper levels of sugars for fermentation. A variant, by itself or in combination with another • -amylase, can consequently be added to the components used to brew beer.

As used herein, the term "flour" means ground or comminuted cereal grain. The term "flour" can also mean sago products or tubers that have been ground or crushed. In some embodiments, the flour may also contain components in addition to ground or ground grain or plant matter. An example of an additional component, although not intended to be limiting, is a leavening agent. Cereal grains include wheat, oats, rye, and barley. Tuber products include tapioca flour, cassava flour, and flan powder. The term "flour" also includes ground corn flour, corn grits, rice flour, whole wheat flour, self-rising flour, tapioca flour, cassava flour, ground rice, enriched flour, and flan powder.

As used herein, the term "supplies" means grains and plant components that are crushed or broken. For example, barley used in beer production is a grain that has been coarsely ground or ground to a consistency suitable for producing a paste for fermentation. As used herein, the term "supplies" includes any of the aforementioned types of plants and grains in ground or coarsely ground forms. The methods described herein can be used to determine levels of amylase activity in both flours and supplies.

One or more additional enzymes can be used in combination with the polypeptide variants described herein. Such combinations can for example be added to food, dough preparation, food product or starch composition.

Baking amylases can come from a fungus, bacteria, or plant. It can be an alpha-amylase (EC 3.2.1.1). The lapha-amylase can be a fungal alpha-amylase from Aspergillus or Trichoderma, for example from Aspergillus oryzae. Or it can be a Bacillus alpha-amylase, for example B. amiloliquefaciens or B. licheniformis. Either it can be a plant betaamylase (EC 3.2.1.2), for example beta-amylase from barley malt or soybean or it can be an exoamylase used for anti-aging, for example non-maltogenic exo-amylase (EC 3.2.1.60) grown from Pseudomonas saccharophila maltotetrahydrolase (SEQ ID NO: 10) or maltogenic amylase (EC 3.2.1.133) grown from Bacillus stearothermophilus which is further described below.

In one aspect, the additional amylase is a maltogenic alpha-amylase. A "maltogenic alpha-amylase" (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is capable of hydrolyzing amylose and amylopectin to maltose in the alpha configuration. A maltogenic alpha-amylase from Bacillus (EP 120 693) is commercially available under the trade name Novamyl (Novozymes, Denmark) and is widely used in the bakery industry as an anti-aging agent due to its ability to reduce starch retrogradation. Novamyl is described in detail in International Patent Publication WO 91/04669. The maltogenic alpha-amylase actions Novamyl shares several characteristics with cyclodextrin glucanotransferases (CGTases), including sequence homology (Henrissat B, Bairoch A; Biochem. J., 316, 695-696 (1996)) and the formation of transglycosylation products (Christophersen, C., et al., 1997, starch, vol. 50, No. 1, 39-45). The Novamyl may in particular comprise Novamyl 1500 MG. Other documents describing Novamyl and its uses include Christophersen, C., Pedersen, S., and Christensen, T., (1993) Method for production of maltose an a limit dextrin, the limit dextrin, and use of the limit dextrin. Denmark, I WO 95/10627. It is further described in U.S. Patent No. No. 4,598,048 and U.S. Patent No. No. 4,604,355. Preferred examples of anti-aging amylases are exo-amylases, for example, non-maltogenic exo-amylase or G4-amylase (EC 3.2.1.60) developed from Pseudomonas saccharophila maltotetraohydrolase having SEQ ID NO: 1 or maltogenic amylase ( EC 3.2.1.133) developed from Bacillus stearothermophilus having SEQ ID NO: 51.

An anti-aging amylase reduces aging after baking and leads to a reduction in the increase in firmness and a reduction in the decrease in elasticity from 1 to 7 days after baking relative to an enzyme-free control. An anti-aging amylase can be analyzed by performing a baking test and using texture analysis to determine the development of firmness and elasticity over time. Texture analysis is described in Example 11.

Additional enzymes can be selected from, for example, any combination of the following: (a) Novamyl, or a variant, homologous, or mutants thereof having maltogenic alpha-amylase activity; (b) a xylanase such as GRINDAMYL ™ POWERBake 900 (Danisco A / S); (c) a bacterial α-amylase such as Max-Life U4 (Danisco A / S); and (d) a lipase such as GRINDAMIL ™ POWERBake 4050 (Danisco A / S).

In one embodiment a polypeptide variant described herein is used in combination with at least one enzyme selected from the list consisting of oxidoreductases, hydrolases, lipases, esterases, glycosidases, amylases, pullulanases, xylanases, cellulases, hemicellulases, enzymes starch degradants, proteases and lipoxygenases. In one embodiment, the composition comprises at least one polypeptide variant described herein and a Bacillus maltogenic amylase, disclosed in WO91 / 04669. One embodiment comprises a polypeptide variant described herein and flour.

Other enzymes that can be added to the dough include oxidoreductases, hydrolases, such as lipases and esterases, as well as glycosidases such as α-amylase, pullulanase, and xylanase. Oxidoreductases, such as for example glucose oxidase and hexose oxidase, can be used for dough strengthening and volume control of baked goods and xylanases and other dough hemicellulases can be added to improve dough handling properties, firmness of the crumb and the volume of the bread. Lipases are useful as dough builders and crumb softeners, and α-amylases and other amylolytic enzymes can be incorporated into dough to control bread volume and further reduce crumb firmness.

Other enzymes that can be used can be selected from the group consisting of a cellulase, a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase.

A variant, as defined herein, can also be added alone or in combination with other amylases, including other amylase variants to prevent or delay aging, ie, crumb firmness of baked goods. The amount of anti-aging amylase will normally be in the range of 0.01-10 mg of enzyme protein per kg of flour, eg 0.5 mg / kg ds. Additional anti-aging amylases can be used in combination with a variant polypeptide described herein include an endo-amylase, eg, a bacterial Bacillus endo-amylase. The additional amylase may be a maltogenic • -amylase (EC 3.2.1.133), eg from Bacillus. Novamil® is an example of maltogenic • -amylase from B. stearothermophilus strain NCIB 11837 and is described in Christophersen et al., Starch 50: 39-45 (1997). Other examples of anti-aging endo-amylases include Bacillus-derived bacterial amylases, such as B. licheniformis es or B. amiloliquefaciens. The anti-aging amylase can be an exo-amylase, such as • -amylase, for example, from plant sources, such as soybeans, or from microbial sources, such as Bacillus.

The baking composition comprising a polypeptide variant described herein may further comprise a phospholipase. Phospholipase can have A1 or A2 activity to remove fatty acid from phospholipids, forming a lysophospholipid. It may or may not have lipase activity, that is, activity on triglyceride substrates. Phospholipase typically has an optimal temperature in the range of 30-90 ° C, for example, 30-70 ° C. The added phospholipases can be of animal origin, eg, from pancreas, eg, bovine or porcine pancreas, snake venom or bee venom. Alternatively, the phospholipase can be of microbial origin, eg, from filamentous fungi, yeast, or bacteria, such as the genus or species. Examples of phospholipase sources include Aspergillus, A. niger; Dictyostelium, D. discoideum; Mucor, M. javanicus, M. mucedo, M. subtilissimus; Neurospora, N. crassa; Rhizomucor, R. pusillus; Rhizopus, R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, S. libertiana; Trichophyton, T. rubrum; Whetzelinia, W. sclerotiorum; Bacillus, B. megaterium, B. subtilis; Citrobacter, C. freundii; Enterobacter, E. aerogenes, E. cloacae; Edwardsiella, E. takes; Etwinia, E. herbicola; Escherichia, E. coli; Klebsiella, K. pneumoniae; Proteus, P. vulgaris; Providencia, P. stuartii; Salmonella, S. typhimurium; Serratia, S. liquefasciens, S. marcescens; Shigella, S. flexneri; Streptomyces, S. violeceoruber; Yersinia, Y. enterocolitica; Fusarium, and F. oxysporum (eg, strain DSM 2672).

The phospholipase is added in an amount that improves the softness of the bread during the initial period after baking, in particular the first 24 hours. The amount of phospholipase will typically be in the range of about 0.01 to 10 mg of enzyme protein per kg of flour, eg 0.1 to 5 mg / kg. Phospholipase activity will generally be in the range of about 20 to 1000 Lipase Unit (LU) / kg of flour, where a lipase unit is defined as the amount of enzyme required to release 1 pmol of butyric acid per minute at 30 ° C, pH 7.0, with gum arabic as emulsifier and tributyrin as substrate.

The dough compositions generally comprise wheat semolina or wheat flour and / or other types of semolina, flour or starch such as corn flour, cornstarch, corn flour, rice flour, rye flour, rye semolina, oatmeal, oatmeal, soybean meal, sorghum semolina, sorghum meal, potato semolina, potato flour, or potato starch. The dough can be fresh, frozen, or partially baked. The dough can be a proofed dough or a dough to be proofed. The dough can be leavened in various ways, such as by adding chemical leavening agents, for example sodium bicarbonate or by adding a leavening agent, i.e. leavening dough. The dough can also be leavened by the addition of a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), for example, a commercially available strain of S. cerevisiae.

The dough may also comprise other conventional dough ingredients, for example, proteins, such as powdered milk, gluten, and soy; eggs (for example, whole eggs, egg yolks, or egg whites); an oxidant, such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA), or ammonium persulfate; an amino acid such as L-cysteine; a sugar; or a salt, such as sodium chloride, calcium acetate, sodium sulfate, or calcium sulfate. The fat mass may further comprise, for example, triglycerides, such as granulated fat or shortening. The dough may further comprise an emulsifier such as mono- or diglycerides, diacetyl tartaric acid esters of mono or diglycerides, sugar esters of fatty acids, polyglycerol fatty acid esters, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides , polyoxyethylene stearates or lysolecithin. In particular, the dough can be made without the addition of emulsifiers.

Optionally, an additional enzyme can be used in conjunction with anti-aging amylase and phospholipase. The additional enzyme can be a second amylase, such as an amyloglucosidase, a • -amylase, a cyclodextrin glucanotransferase, or the additional enzyme can be a peptidase, in particular an exopeptidase, a transglutaminase, a lipase, a cellulase, a hemicellulase, in particular a pentosanase, such as xylanase, a protease, a protein disulfide isomerase, for example, a protein disulfide isomerase, as described in WO 95/00636, for example, a glycosyltransferase, a branching enzyme (branching enzyme 1, 4 - ^ - glucan), a 4 - ^ - glucanotransferase (dextrin glycosyltransferase) or an oxidoreductase, for example a peroxidase, a laccase, a glucose oxidase, a pyranose oxidase, a lipoxygenase, an L-amino acid oxidase or a carbohydrate oxidase . The additional enzyme can be of any origin, including mammals and plants, and in particular of microbial origin (bacterial, yeast or fungal) and can be obtained by techniques conventionally used in the art.

The xylanase is usually of microbial origin, for example derived from a bacterium or fungus, such as an Aspergillus strain, in particular A. aculeatus, A. niger (cf. WO 91/19782), A. awamori (for example , WO 91/18977), or A. tubigensis (eg WO 92/01793); from a Trichoderma strain, eg T. reesei, or from a Humicola strain, eg H. insolens (eg WO 92/17573). Pentopan® and Novozym 384® are commercially available xylanase preparations produced from Trichoderma reesei. The amyloglucosidase can be an amyloglucosidase from A. niger (such as AMG®). Other useful amylase products include Grindamil® A 1000 or A 5000 (available from Danisco, Denmark) and Amylase® H or Amylase P (available from Gist-Brocades, The Netherlands). The glucose oxidase can be a fungal glucose oxidase, in particular a glucose oxidase from Aspergillus niger (such as Gluzyme®). An example of a protease is Neutrase®. An example of lipase can be derived from the strains of Thermomyces (Humicola), Rhizomucor, Candida, Aspergillus, Rhizopus, or Pseudomonas, in particular from Thermomyces lanuginosus (Humicola lanuginosa), Rhizomucor miehei, Candida antarctica, Asperizopus delemarger, or Rhizopus delemarger. arrhizus, or Pseudomonas cepacia. In specific embodiments, the lipase can be Lipase A or Lipase B derived from Candida Antarctica as described in WO 88/02775, for example, or the lipase can be derived from Rhizomucor miehei as described in EP 238,023, for example, or Humicola lanuginosa, described in EP 305,216, for example, or Pseudomonas cepacia as described in the EP 214,761 and WO 89/01032, for example.

The process can be used for any type of baked product prepared from dough, whether it is of a soft or crisp character, whether it is white, light or dark. Examples are bread, particularly white, wholemeal or rye bread, usually in the form of loaves or rolls, such as, but not limited to, French baguette bread, pita bread, omelettes, cakes, pancakes, biscuits, cookies, shortcrust dough, crusty bread, steamed bread, pizza and the like.

In another embodiment, a polypeptide variant described herein can be used in a premix, comprising flour together with an anti-aging amylase, a phospholipase, and a phospholipid. The premix may contain other dough improver and / or bread improver additives, for example any of the additives, including the enzymes, mentioned above. In one aspect, the polypeptide variant described herein is a component of an enzyme preparation comprising an anti-aging amylase and a phospholipase, for use as a baking additive.

The enzyme preparation is optionally in the form of an agglomerated granulate or powder. The preparation can have a narrow particle size distribution of more than 95% (by weight) of the particles in the range of 25 to 500 pm. Agglomerated granules and powders can be prepared by conventional methods, for example, by spraying the polypeptide described herein onto a carrier in a fluid bed granulator. The carrier may consist of particle nuclei having a suitable particle size. The carrier can be soluble or insoluble, for example, a salt (such as NaCl or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits , or soy.

Another aspect contemplates the envelope of particles comprising a polypeptide variant described herein, ie, • -amylase particles. To prepare the enveloped amylase particles, the enzyme is contacted with a food grade lipid in sufficient quantity to suspend all of the • -amylase particles. Food grade lipids, as used herein, can be any natural organic compound, which is insoluble in water but is soluble in nonpolar organic solvents such as hydrocarbons or diethyl ether. Suitable food grade lipids include, but are not limited to, triglycerides, either in the form of fats or oils that are saturated or unsaturated. Examples of fatty acids and combinations of these that make up saturated triglycerides include, but are not limited to, butyric acid (derived from milk fat), palmitic (derived from animal and vegetable fat), and / or stearic (derived from animal fat). and vegetable). Examples of fatty acids and combinations of these that make up unsaturated triglycerides include, but are not limited to, palmitoleic (derived from animal and vegetable fat), oleic (derived from animal and vegetable fat), linoleic (derived from vegetable oils), and / or linolenic (derived from linseed oil). Other suitable food grade lipids include, but are not limited to, monoglycerides and diglycerides derived from the aforementioned triglycerides, phospholipids and glycolipids.

Food grade lipid, particularly in liquid form, is contacted with a powdered form of the • -amylase particles such that the lipid material covers at least a portion of the surface of at least a majority , for example, 100% of the • -amylase particles. In this way, each particle of • -amylase is individually wrapped in a lipid. For example, all or substantially all of the • -amylase particles are provided with a continuous thin lipid envelope film. This can be achieved by first pouring a quantity of lipids into a container, and then suspending the fine particles, continuing so that the lipid completely moistens the surface of each • -amylase particle. After a short period of agitation, the enveloped • -amylase particles, which carry a substantial amount of the lipids on their surfaces, are recovered. The thickness of the coating thus applied to the • -amylase particles can be controlled by selecting the type of lipid used and repeating the operation in order to build a thicker film, when desired.

Storage, handling and incorporation of the loaded delivery vehicle can be accomplished by means of a packaged mixture. The packaged mixture may comprise the enveloped • -amylase. However, the packaging mix may further contain additional ingredients as required by the manufacturer or the baker. After the wrapped • -amylase has been incorporated into the dough, the baker continues through the normal process of producing said product.

The advantages of wrapping the • -amylase particles are two times. First, the food grade lipid protects the enzyme from thermal denaturation during the baking process for enzymes that are heat labile. Consequently, while the • -amylase is stabilized and protected during the fermentation and baking stages, it is released from the protective layer in the final baked product, where it hydrolyzes the glycosidic bonds in polyglycans. The loaded delivery vehicle also provides a sustained release of the active enzyme in the baked good. That is, after the baking process, the active • -amylase is continuously released from the protective layer at a counteracting rate, and therefore reduces the rate of aging mechanisms.

In general, the amount of lipids applied to the • -amylase particles can vary from a few percent of the total weight of the • -amylase to many times the weight, according to the nature of the lipid, the way in which applies to the • -amylase particles, the composition of the dough mixture to be treated, and the severity of the dough mixing operation involved.

The loaded delivery vehicle, ie, the lipid envelope enzyme, is added to the ingredients used to prepare a baked good in an amount effective to extend the shelf life of the baked good. The baker calculates the amount of wrapped • -amylase, prepared as described above, that will be required to achieve the desired anti-aging effect. The amount of • -amylase with envelope required is calculated based on the concentration of the enzyme with envelope in the ratio of • -amylase to the specified flour. A wide range of concentrations has been found to be effective, although, as discussed, the observable improvements in anti-aging do not correspond linearly with the concentration of • -amylase, but above certain trough levels, large increases in the concentration of • -amylase produce little further enhancement. The concentration of • -amylase actually used in a particular bakery production could be much higher than the minimum necessary to provide the baker with some security against inadvertent measurement errors by the baker. The lower limit of enzyme concentration is determined by the minimum anti-aging effect that the baker wishes to achieve.

A method of preparing a baked good may comprise: (a) preparing lipid-coated • -amylase particles, wherein substantially all of the • -amylase particles are coated; (b) mixing a dough containing flour; (c) adding the lipid-coated • -amylase particles before mixing is complete and finishing mixing before removing the lipid layer from the • -amylase; (d) fermenting the dough; and (e) baking the dough to provide the baked product, wherein the • -amylase is inactive during the mixing, leavening and baking steps and is active in the baked product.

The wrapped • -amylase can be added to the dough during the mix cycle, for example near the end of the mix cycle. The wrapped • -amylase is added at a point in the mixing step that allows sufficient distribution of the wrapped • -amylase throughout the dough; however, the mixing step is completed before the protective layer is peeled off the • -amylase particle. Depending on the type and volume of the dough, and the action and speed of the mixer, it may take at any time from one to six minutes or more to mix the wrapped • -amylase into the dough, but two to four minutes is the average. Therefore, several variables can accurately determine the procedure. First, the amount of wrapped • -amylase must have a total volume sufficient to allow the wrapped • -amylase to spread throughout the dough mixture. If the wrapped • -amylase preparation is highly concentrated, it may be necessary to add oil to the premix before adding the • -amylase with wrap to the dough. Recipes and production processes may require specific modifications; however, generally good results can be achieved when 25% of the specified oil in a bread dough formula is kept out of the dough and used as a carrier for a concentrated wrapper • -amylase when added close to the end of the mix cycle. In bread or other baked goods, especially those with a low fat content, for example French-type breads, a mixture of • -amylase with wrap of about 1% by weight of dry flour is sufficient to mix the • - amylase properly wrapped with the dough. The range of suitable percentages is wide and depends on the formula, finished product and the requirements of the individual baker's production methodology. Second, the sheathed • -amylase suspension should be added to the mix long enough for complete mixing into the dough, but not for such a time that excessive mechanical action will strip the protective lipid layer off the particles. of • -amylase with envelope.

6. Compositions for textile desizing and use

Compositions and methods of treating fabrics (eg, to de-sizing a fabric) using a polypeptide variant described herein are also contemplated. In one aspect, a method of de-gluing tissues is provided, which comprises contacting the polypeptide variant described herein with a tissue for a time sufficient to de-do the tissue.

Fabric treatment methods are well known in the art (see, for example, U.S. Patent No.

6,077,316). For example, in one aspect, the feel and appearance of a fabric is improved by a method comprising contacting the fabric with the polypeptide variant described herein in a solution. In one aspect, the fabric is treated with the solution under pressure.

In one aspect, a polypeptide variant described herein is applied during or after weaving of a fabric, or during the desizing step, or one or more additional steps of tissue processing. During the weaving of the textile material, the threads are exposed to considerable mechanical stress. Before weaving on power looms, warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and to prevent breakage. A polypeptide variant described herein can be applied during or after weaving to remove these sizing starch or starch derivatives. After weaving, a polypeptide variant described herein can be used to remove sizing ACPA prior to further processing the fabric to ensure a homogeneous and waterproof result.

A polypeptide variant described herein can be used alone or with other chemical de-sizing reagents and / or de-sizing enzymes to de-size fabrics, including cotton-containing fabrics, as detergent additives, for example, in aqueous compositions. A polypeptide variant described herein can also be used in compositions and methods for producing a stonewashed appearance on indigo dyed denim fabrics and garments. For the manufacture of clothing, the fabric can be cut and sewn into clothing or garments, which are then subjected to finishing. In particular, for the manufacture of jeans, different enzyme finishing methods have been developed. The finishing of denim garments normally begins with an enzymatic desizing stage, during which the garments are subjected to the action of amylolytic enzymes to provide softness to the fabric and make the cotton more accessible to subsequent enzymatic finishing stages. A variant of the polypeptide described herein can be used in methods of finishing denim garments (eg, a "biological pre-wash process"), enzymatic desizing and providing softness to fabrics, and / or finishing process. .

Examples

Example 1. Cloning of PS4 and G4-amylases

Pseudomonas sacharophila was grown overnight in LB medium and chromosomal DNA was isolated by standard methods (Sambrook J, 1989). A 2190 bp fragment containing the PS4 open reading frame (Zhou et al., 1989) was amplified from P. sacharophila chromosomal DNA by PCR using primers P1 and P2 (see Table 3). The resulting fragment was used as a template in a nested PCR with primers P3 and P4, amplifying the PS4 open reading frame without its signal sequence and introducing an NcoI site at the 5 'end of the gene and a BamHI site at the end. 3'. Along with the NcoI site a codon for an N-terminal methionine was introduced, allowing intracellular expression of PS4. The 1605 bp fragment was cloned into pCRBLUNT TOPO (Invitrogen) and the integrity of the construct was analyzed by sequencing. The Bacillus E.coli carrier vector pDP66K (Penninga et al., 1996) was modified to allow the expression of PS4 under the control of the P32 promoter and the ctgasse signal sequence. The resulting plasmid, pCSmta, is transformed into B. subtilis.

A second expression construct was prepared in which the starch binding domain of PS4 was removed. In a PCR with primers P3 and P6 (Table 3) in pCSmta, a truncated version of the mta gene was generated. The full-length mta gene in pCSmta was exchanged with the truncated version that resulted in the plasmid pCSmta-SBD. G4 amylases from Pseudomonas sp. AM1 (2006), Pseudomonas sp. 7193, Pseudomonas mendocina (strain ymp) and Hahella chejuensis (strain KCTC 2396) were cloned in a Bacillus expression vector with the signal sequence by gene synthesis (GeneScript; NJ, USA) of the sequences that encode mature proteins with a M added at the N terminus as shown in SEQ ID NO 3, 5, 7 and 9.

Example 2. Site-directed mutagenesis

Site-directed mutagenesis was used to produce variant polypeptide as disclosed herein. Mutations were introduced into a nucleic acid encoding pMS 382 having SEQ ID: 1, using the Quick Change ™ method (Stratagene, California), according to the instructions supplied with the kit with some modifications. Briefly, a single colony was taken and inoculated into 3 ml of LB (22 g / l Lennox L Base Broth, Sigma) supplemented with 50 g / ml kanamycin (Sigma) in a 10 ml Falcon tube. After incubation overnight at 37 ° C at 200 rpm, the culture was centrifuged at 5000 rpm for 5 min. The medium was removed and a double-stranded DNA template was prepared using QIAGEN columns (QIAGEN). The primers were designed according to the manufacturer's protocol. For example, TABLE 1 indicates the primers that were used to generate new variants based on the nucleotide sequence of pMS382 as shown in SEQ ID NO: 52.

A PCR was then performed to synthesize the mutant strand. The PCR reaction mix contained the following:

2.5 • l 10 X QuickChange Multi-Reaction Buffer

0.75 • l QuickSolution

X • l primers (10 pmol for 28-35 nt primers; 7 pmol for 24-27 nt primers; or 5 pmol for 20-23 nt primers)

1 • I dNTP mix

X • l ds-DNAe template (200 ng)

1 • l QuickChange multienzyme mix (2.5 AU l) (PfuTurbo DNA polymerase)

X • l dH2O (to a final volume of 25 • l)

The PCR reaction was performed on an Eppendorf thermal cycler for 35 cycles of denaturation (962C for 1 min), primer annealing (62.8 ° C for 1 min), and elongation (65 ° C for 15 min), and then it was kept at 4 ° C. For each amplification reaction, 2 l of restriction enzyme Dpnl (10 AU l) were added, and the mixture was incubated at 37 ° C for ~ 3 h.

The Dpnl-treated DNA was then used to transform XL10-Gold® ultracompetent cells (Stratagene). The XL10-Gold® cells were thawed on ice. For each mutagenesis reaction, 45 µl cells were added to a pre-cooled Falcon tube. Subsequently, 2 µl of beta-mercaptoethanol mixture was added to each tube. The mixture was incubated on ice for 10 min with spinning every 2 min. Then 1.5 µl of Dpnl-treated DNA was added to each aliquot of cells, and the mixture was incubated on ice for 30 min. The sample was subjected to a thermal pulse for 30 sec at 42 ° C, and was placed on ice for another 2 min. 0.5 ml of prewarmed NZY + broth was added to each sample and incubation was carried out at 37 ° C for 1 h with shaking at 225-250 rpm. 200 µl of each transformation reaction was plated on LB plates (33.6 g / l Lennox L Agar, Sigma) supplemented with 1% starch and 50 µg / ml kanamycin. The plates were incubated overnight at 37 ° C. Individual colonies harboring the desired mutations were identified by DNA sequencing and subjected to plasmid preps to harvest plasmids with the desired mutations.

Example 3. Transformation into Bacillus subtilis.

Bacillus subtilis (strain DB104A; Smith et al., Gene 70, 351-361 (1988)) was transformed with the mutated plasmid DNA according to the following protocol.

A. Media for protoplast preparation and transformation

2 x SMM per liter: 342 g sucrose (1 M); 4.72 g of sodium maleate (0.04 M); 8.12 g of MgC12-6H20

(0.04M); pH 6.5 with concentrated NaOH. Distribute in 50 ml portions and autoclave for 10 min.

4 x YT (1/2 NaCl) 2 g of yeast extract 3.2 g of Tryptone 0.5 g of NaCl per 100 ml.

SMMP mix equal volumes of 2 x SMM and 4 x YT.

PEG 10 g of polyethylene glycol 6000 (BDH) or 8000 (Sigma) in 25 ml 1 x SMM (Autoclave for 10 min.).

B. Media for plating / regeneration

agar 4% Difco minimal agar. Autoclave for 15 min.

Sodium succinate 270 g / 1 (1 M), pH 7.3 with HCl. Autoclave for 15 min.

phosphate buffer 3.5 g of K2HPO4 1.5 g of KH2PO4 per 100 ml. Autoclave for 15 min

MgCl2 20.3 g of MgC12-6H2O per 100 ml (1 M).

casaminoacids solution at 5% (p / v). Autoclave for 15 min.

yeast extract 10 g per 100 ml, autoclave for 15 min.

glucose solution at 20% (w / v). Autoclave for 10 min.

DM3 regeneration medium: mix at 60 ° C (water bath; 500 ml bottle):

250 ml of sodium succinate

50 ml of casamino acids

25 ml yeast extract

50 ml phosphate buffer

15 ml glucose

10 ml MgCl2

100 ml molten agar

Add appropriate antibiotics: chloramphenicol and tetracycline, 5 • g / ml; erythromycin, I • g / ml. Selection on kanamycin is problematic in DM3 medium: concentrations of 250 g / ml may be required.

C. Preparation of protoplasts

Use detergent-free glass or plastic containers.

Inoculate 10 ml of 2 x YT medium into a 100 ml single colony flask. Grow an overnight culture at 25-30 ° C on a shaker (200 rev / min).

Dilute the overnight culture 20 times in 100 ml of 2 x fresh YT medium (250 ml flask) and grow to OD600 = 0.4-0.5 (approx. 2 hrs) at 37 ° C on a shaker (200 -250 rev / min).

Harvest the cells by centrifugation (9000 g, 20 min, 4 ° C).

Remove the supernatant with a pipette and resuspend the cells in 5 ml of SMMP 5 mg lysozyme, sterile filtered.

Incubate at 37 ° C on a shaker in a water bath (100 rpm).

After 30 min and then at 15 min intervals, examine 25 • I samples under the microscope. Continue the incubation until 99% of the cells are formed as protoplasts (globular appearance). Harvest the protoplasts by centrifugation (4000 g, 20 min, RT) and pipet off the supernatant. Gently resuspend the pellet in 1-2 ml of SMMP.

The protoplasts are now ready to use. Portions (eg 0.15 ml) can be frozen at -80 ° C for future use (no addition of glycerol is required). Although this may result in some reduction in the capacity for transformation, 106 transformants can be obtained per ug of DNA with frozen protoplasts). D. Transformation

Transfer 450 µl of PEG to a microtube.

Mix 1-10 µl of DNA (0.2 • g) with 150 µl of protoplasts and add the mixture to the microtube with PEG. Mix immediately, but gently.

Leave for 2 min at room temperature, then add 1.5 ml of SMMP and mix.

Harvest the protoplasts by microcentrifuge (10 min, 13,000 rpm (10,000-12,000 g)) and remove the supernatant. Remove the remaining drops with a tissue.

Add 300 µl of SMMP (do not vortex) and incubate for 60-90 min at 37 ° C in a shaker with a water bath (100 rpm) to allow the expression of antibiotic resistance markers. (The protoplasts are sufficiently resuspended through the shaking action of the water bath). Make appropriate dilutions in 1 x SSM and plate 0.1 ml on DM3 plates.

Example 4. Fermentation of variants in shake flasks.

The shake flask substrate is prepared by dissolving the following in water:

Yeast extract 2% (w / v)

Soy flour 2% (p / v)

NaCl 0.5% (p / v)

Dipotassium phosphate 0.5% (p / v)

Antifoam agent 0.05% (w / v)

The substrate was adjusted to pH 6.8 with 4N sulfuric acid or sodium hydroxide before autoclaving. 100 ml of substrate was placed in a 500 ml flask with a baffle and autoclaved for 30 minutes. Subsequently, 6 ml of sterile dextrose syrup was added. Dextrose syrup is prepared by mixing a 50% w / v volume of dextrose with one volume of water followed by autoclaving for 20 minutes.

Shake flasks are inoculated with the variants and incubated for 24 hours at 35 ° C and 180 rpm in an incubator. After incubation, the cells are separated from the broth by centrifugation (10,000 g in 10 minutes) and finally, the cells are removed from the supernatant by microfiltration at 0.2 pm. The cell-free supernatant is used for assays and application assays.

Table 1. Primers used to generate variants based on the nucleotide sequence of pMS382 as shown in SEQ ID NO: 52.

Example 5. Amylase assays

Betamyl assay

One Betamyl unit is defined as the activity that degrades 0.0351 mmol per 1 min of maltopentaose coupled with PNP such that 0.0351 mmol of PNP per 1 min can be released by excess alpha-glucosidase in the assay mixture. The assay mix contains 50 ul of 50 nM Na citrate, 5 mM CaCl2, pH 6.5 with 25 ul of enzyme sample and 25 ul of Betamyl substrate (Glc5-PNP and alpha-glucosidase) from Megazyme, Ireland ( 1 vial dissolved in 10 ml of water). The assay mix was incubated for 30 min at 40 ° C and then stopped by adding 150 ul of 4% Tris. Absorbance at 420 nm was measured using an ELISA reader and Betamyl activity was calculated based on Activity = A420 * d in Betamyl units / ml of enzyme sample tested. For the dosage in the baking tests 1 BMK = 1000 Betamyl units were used.

Endo-amylase assay

The endo-amylase assay is identical to the Phadebas assay performed according to the manufacturer (Pharmacia & Upjohn Diagnostics AB).

Exo-specificity

The relationship between amylase exo-activity and Phadebas activity was used to assess exo-specificity. Example 6. Thermal stability determinations of the process based on residual activity

Enzyme samples in appropriate dilutions were incubated in 50 mM Na acetate buffer, pH 5.0 with 0.5 M NaCl at room temperature (control) or for 4 minutes at 80 ° C (heated sample), and immediately after that the control sample and the heated sample were analyzed using the Betamyl assay. The residual activity was calculated as the Betamyl activity of the heated sample divided by the Betamyl activity of the control sample.

Example 7. Stabilizing effects of mutations

Using the procedure described in Example 6 the stabilizing effects of the Q42K, R88L, S205L, E223S, Q311P, S409E and T235K mutations are shown in Table 2 as increased residual activity after heating. Similarly, the stabilizing effect of A392D is shown in Table 3, of S205L, S223A and S205L combined with S409 in Table 4, of N34Q and G100Q in Table 5 and of Q240E in Table 6. In tables 2- 6, the variants have the same sequence as mentioned in the first line except for the subsequent mutation (s) performed as described in the second column.

Table 2. Stabilizing effect of Q42K, R88L, S205L, E223S, Q311P, S409E and T235K combined with Q311P

Table 3. Stabilizing effect of A392D

Table 4. Stabilizing effect of S205L, S223A and S205L combined with S409E

Table 5. Stabilizing effect of N34Q and G100Q

Table 6. Stabilizing effect of Q240E

Example 10. Recipe for baking tests

Baking tests were carried out with a standard white bread dough and sponge recipe for US toast bread. The sponge dough is prepared from 1500 g of "Gold Medal" flour from General Mills, USA, 890 g of water, 40 g of soybean oil, 7.5 g of GRINDSTED ™ SSL P55 Veg, 10 g of DiMODAN ™ PH300 emulsifier, 26 g of dry yeast and ascorbic acid (20 ppm final concentration). The sponge is mixed for 1 min at low speed and subsequently 3 min at speed 2 in a Hobart spiral mixer. The sponge is subsequently fermented for 3 hours at 25 ° C, 85% RH.

Then 500 g of "Gold Medal" flour, 14 g of dry yeast, 5 g of calcium propionate, 240 g of high fructose corn syrup (42%), 5 g of calcium propionate, 250 g of water and ascorbic acid (30 ppm final concentration) and 40 g of salt. The resulting mass is mixed for 0.5 min at low speed and then for 10.5 min at high speed in a Hobart spiral mixer.

The dough is allowed to rest for 5 min at room temperature, and then 794 g dough pieces are weighed, molded in a cross-grain former, and transferred to containers. After 60 min of fermentation at 43 ° C at 80% RH, the doughs are baked for 21 min at 200 ° C in a Reed tray oven.

Example 11. Protocol for the evaluation of firmness, resistance and cohesiveness

Analysis of the bread texture profile

Firmness, strength and cohesiveness are determined by analyzing bread slices by Texture Profile Analysis using a Texture Analyzer from Stable Micro Systems, UK. The calculation of firmness, strength, and cohesiveness is done in accordance with the preset standard supplied by Stable Micro System, UK. The probe used is a 50mm round aluminum probe.

Firmness is determined at 40% compression during the first compression. The figure is the force required to compress the slice to 40% of the total thickness. The lower the value, the softer the bread. Firmness is expressed, for example, in grams.

Example 12. Evaluation of anti-rancidity effects of variant G4 amylase polypeptides

The bread was baked as described in Example 10 with pMS1776, pMS1934, pMS2020, pMS2022 and pMS2062 in dosages ranging from 7.5 to 20 BMK / kg corresponding to 7,500 to 20,000 Betamyl units / kg.

The firmness of the bread is tested according to the protocol presented in Example 11 at various times after baking. As controls, baked bread was also baked with standard dosages of 600 ppm Novamyl 1500 and / or 416.66 ppm of a composition comprising SEQ ID NO: 1.

Figs. 1 and 2 show the firmness results. In relation to the controls with standard dosages of 600 ppm of Novamyl 1500 and / or 416.66 ppm of a composition comprising SEQ ID NO: 1, a much stronger reduction in the firmness rate is obtained from day 3 to day 10 after baking with pMS1776, pMS1934, pMS2020, pMS2022 and pMS2062. These breads had also greatly improved in strength and cohesiveness relative to controls.

This indicates that variant polypeptides, as described herein, greatly improved anti-rancidity effects compared to Novamyl 1500 and a composition comprising SEQ ID NO: 1.

Example 13. Protocol for the evaluation of friability (Resistance to friability)

Two slices of bread are placed on a piece of paper. Each slice is divided into 4 squares by vertical and subsequently horizontal breaks of the slice.

The break is done by crushing the crumb with your fingers. First the slice is separated from the center of the upper surface of the bread to the center of the lower surface of the bread. Then each half of the original slice is pulled away from the crust side to the inside of the slice. The small pieces of crumb, which are separated from the 4 squares, are removed by shaking each piece at least 3 times moving the hand up and down.

The weight of the separated small crumb pieces is determined as a measure of friability. This test can be called "Friability Assessment Protocol".

Example 14. Protocol for the evaluation of the folding capacity

The bread for toast is cut using an automatic bread cutter with a preset slice thickness of 15 mm. The slice is folded by hand from the top of the slice to the bottom, such that the direction of the fold is from side to side.

The folding ability is visually evaluated using the following scoring system:

This test can be referred to as the "Pliability Assessment Protocol".

Example 15. Preparation of DP4 syrup

Materials:

SPEZYME FRED® granular corn starch liquefied (34% ds, 9.1DE) treated with bacterial alpha-amylase (Danisco US Inc., Genencor Division).

Pullulanase (OPTIMAX L-1000, Danisco US Inc., Genencor Division)

Experimental:

The pH of the starch smoothie was adjusted to 5.2 with NaOH and then each 100 g of smoothie was incubated at 60 ° C during the DP4 saccharification dosing enzymes at 0.03 BMK / gds. When pullulanase was used, it was dosed at 0.5 ASPU / gds. The reaction was carried out up to 40 hours, stopped for periodic sampling to check the maximum DP4. When the maximum DP4 was observed, the reaction was stopped by heating in boiling water.

Results:

As shown in Table 1, the results indicated that PMS2062 produced DP4 as a major sugar product. As also noted, the addition of pullulanase (PU) (PMS2062 PU) resulted in a faster reduction of higher sugars (see, for example, DP11 +), while the level of DP4 was minimally affected.

Sequences

SEQ ID NO: 1. Mature protein sequence of pMS382

DQAGKSPAGVRYHGGDEIILQGFHWMVVREAPYNWYNILRQQASTIAADGFSAIWMPVPWRDFSSWTDGDKSGGGEGYFWHDF NKNGRYGSDAQLRQAAGALGGAGVKVLYDWPNHMNRFYPDKEINLPAGQRFWRNDCFDFGNGPNDCDDGDRFLGGEADLNTG HPQIYGMFRDEFTNLR3GYGAGGFRFDFVRGYAPERVDSWMSDSADSSFCVGELWKEF3EYPFWDWRNTASWQQIIKDWSDRA kcpvfdfalkerm qhgsvadwkhglngnp dprwrevavtfvdnhdt gyspgqnggqhkwplqd glirqayayiltspgtp wy WPHMYDWGYGDFIRQLIQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLWALNSDLANPGQVASGSFSEAVNASNGQVRV WRSGSGDGGGHDGG

SEQ ID NO: 2. Mature amylase G4 protein from Pseudomonas sp. AM1 (2006) with M added at the N end (Q1EJP2 protein):

1 MD1AGKSPGG VRYHGGDEII LQGFHWNVIR ESPNNWYNTL RDMAPTIAAD

51 GFSAIWMPVP WRDFSSWSDG AMSGGGEGYF WHDFNKNGRY GSDTQLKQAA

101 GALNNAQVKV LYDW PMHMN RGYPDKQIML PAGQGFWRMD CADPGMYPND

151 CDDGDRFMGG DADLNTANPQ VYGMFRDEFA NLRSNYGAGG FRFDFVRGYA

201 GERVDSWMGA AHDNAFCVGE LWKAPAEYPS WDWRNTASWQ QVIKDWSDRA

251 KCFVFDFALK ERMQNGSIAD WKNGLNGNPD PRWREVAVTF VDNHDAGYSP

301 GQNGGQHHWA LQDGLIRQAY AYILTSPGTP WYWS HMYDW GYGDFIRQLI

351 QVRRTAGVRA DSAISFHSGY SGLVATVSGS QQTL WALNS DLAMPGQVAS

401 GSFSEAVNAS NGQVRVWRSG SGDGGGNDGG EGGLVNVNFR CDNGVTQMGD

451 SVYAVGNVSQ LGNWSPASAV RLTDTSSYPT WKGSIALPDG QNVEKKCLIR

501 HEADATLVRQ WQSGGNNQVQ AAAGASTSGS F *

SEQ ID NO: 3. Nucleotide DNA sequence encoding amylase G4 from Pseudomonas sp. AM1 (2006) (Q1EJP2) with M added at the N-terminus (Q1 EJP2 DNA):

atggatctggcaggcaaatcaccgggcggcgttagat atcatggcggcgatgaaattattctgcaaggctttcattggaatgt tattagagaatcaccgaataattggtataatacactgagagatatggcaccgacaattgcagcagatggcttttcagcaattt ggatgccggttccgtggagagatttttcatcatggtcagatggcgcaaattcaggcggcggcgaaggctatttttggcatgat tttaataaaaatggcagatatggctcagatacacaactgaaacaagcagcaggcgcactgaataatgcacaagttaaagttct gtatgatgttgttccgaatcatatgaatagaggctatccggataaacaaattaatctgccggcaggccaaggcttttggagaa atgattgcgcagatccgggcaattatccgaatgattgcgatgatggcgatagatttatgggcggcgatgctgatctgaataca gcaaatccgcaagtttatggcatgtttagagatgaatttgcaaatctgagatcaaattatggcgcaggcggctttagatttga ttttgttagaggctatgcaggcgaaagagttgattcatggatgggcgcagcacatgataatgcattttgcgttggcgaactgt ggaaagcaccggcagaatatccgtcatgggattggagaaatacagcatcatggcaacaagttattaaagattggtcagataga gcaaaatgcccggtttttgattttgcactgaaagaaagaatgcaaaatggctcaattgcagattggaaaaatggcctgaatgg caatccggatccgagatggagagaagttgcagttacatttgttgataatcatgatgcaggctattcaccgggccaaaatggcg gccaacatcattgggcactgcaagatggcctgattagacaagcatatgcatatattctgacatcaccgggcacac cggttgtt tattggtcacatatgtatgattggggctatggcgattttattagacaactgattcaagttagaagaacagcaggcgttagagc agattcagcaatttcatttcattcaggctattcaggcctggttgcaacagtttcaggctcacaacaaacactggttgttgcac tgaatagcgatctggcaaatccgggccaagttgcatcaggctcattttcagaagcagttaatgcatcaaatggccaagttaga gtttggagatcaggctcaggcgatggcggcggcaatgatggcggcgaaggcggectggttaatgttaattttagatgcgataa tggcgttacacaaatgggcgattcagtttatgcagttggcaatgtttcacaactgggcaattggtcaccggcatcagcagtta gactgacagatacatcatcatatecgacatggaaaggctcaattgcactgccggatggccaaaatgttgaatggaaatgcctg attagaaatgaagcagatgcaacactggttagacaatggcaatcaggcggcaataatcaagttcaagcagcagcaggcgcatc aacatcaggctcattttaa

SEQ ID NO: 4. Mature amylase G4 protein from Pseudomonas sp. 7193 with M added at the N-terminus (A5CVD5 protein):

1 MDIiAGKSPGG VRYHGGDEII LQGFHWNVIR ESPNNWYHTL RDMAPTIAAD

51 GFSAIWMPVP WRDFSSWSDG ANSGGGEGYF WHDFIJKNGRY GSDTQLKQAA

101 GALNNAQVKV LYDAVPHHMN RGYPDKQINL PAGQGFWRND CADPGNYPND

151 CDDGDRFMGG DADLNTANPQ VYGMFRDEFA NLRSNYGAGG FRFDFVRGYA

201 GERVDSWMGD GACQRLLRGR ALEGTGRIPE LGLAQYGQLA AVIKDWSDRA

251 KVPGCSNFAL KGAHAERLPS PTGRTASTAT PMPRHREVAV TFVDNHDTGY

301 SPGQNGGQHH WALRDDLVRQ AYAYILASPG TPVVYWSHMY DWGHGPLIRQ

351 LIQIRRAAGV RADSAIEFHS GYSGLVATVR GTAQTLVMAL GSNLSSPAEV

401 SSGSFSQALN QDSGQLRIWT TGSIGGDEGD GGGDGTMVSV NFRCDNGITQ

451 PGDSVYAVG5 LAQLG3WSPA MAVRLTDVSN YPTWKGAISL PAGQAVEHKC

501 IVRSEADPTQ VRQWQAGDIIII RVTAGAGATT IGRL *

SEQ ID NO: 5. Nucleotide DNA sequence encoding Pseudomonas sp. G4 amylase. 7193 (A5CVD5) with M added at the N terminus (A5CVD5 DNA):

atggatctggcaggcaaatcaccgggcggcgttagatat catggcggcgatgaaattattctgcaaggctttcattggaatgt tattagagaatcaccgaataattggtataatacactgagagatatggcaccgacaattgcagcagatggcttttcagcaattt ggatgccggttccgtggagagatttttcatcatggtcagatggcgcaaattcaggcggcggcgaaggctatttttggcatgat tttaataaaaatggcagatatggctcagatacacaactgaaacaagcagcaggcgcactgaataatgcacaagttaaagttct gtatgatgcagttccgaatcatatgaatagaggctatccggataaacaaattaatctgccggcaggccaaggcttttggagaa atgattgcgcagatccgggcaattatccgaatgattgcgatgatggcgatagatttatgggcggcgatgctgatctgaataca gcaaatccgcaagtttatggcatgtttagagatgaatttgcaaatctgagatcaaattatggcgcaggcggctttagatttga ttttgttagaggctatgcaggcgaaagagttgattcatggatgggcgatggcgcatgccaaagactgctgagaggcagagcac tggaaggcacaggcagaattccggaactgggcctggcacaatatggccaactggcagcagttattaaagattggtcagataga gcaaaagttccgggctgctcaaattttgcactgaaaggcgcacatgcagaaagactgccgtcaccgacaggcagaacagcatc aacagcaacaccgatgccgagatggagagaagttgcagttacatttgttgataatcatgatacaggctattcaccgggccaaa atggcggccaacatcattgggcactgagagatgatctggttagacaagcatatgcatatattctggcatcaccgg gcacaccg gttgtttattggtcacatatgtatgattggggtcatggaccgctgattagacaactgattcaaattagaagagcagcaggcgt tagagcagattcagcaattgaatttcattcaggctattcaggcctggttgcaacagttagaggcacagcacaaacactggtta tggcactgggctcaaatctgtcatcaccggcagaagtttcatcaggctcattttcacaagcactgaatcaagattcaggccaa ctgagaatttggacaacaggctcaacaggcggcgatgaaggcgatggcggcggcgatggcacaatggtttcagttaattttag atgcgataatggcattacacaaccgggcgattcagtttatgcagttggctcactggcacaactgggctcatggtcaccggcaa atgcagttagactgacagatgtttcaaattatccgacatggaaaggcgcaatttcactgccggcaggccaagcagttgaatgg aaatgcattgttagatcagaagcagatccgacacaagttagacaatggcaagcaggcgataataatagagttacagcaggcgc aggcgcaacaacaattggcagactgtaa

SEQ ID NO: 6. Mature amylase G4 protein from Pseudomonas mendocina (strain ymp) with M added at the N-terminus (protein A4XX23):

1 MDAPGKTASG VRYHGGDEII LQGFHWNTVR TSSNWYATLA 5MAPTLAADG

51 FSAIWMPVPW RDFSSW3DPG NGTSGGGEGY FWHDFNKHGR YGSDSLLRQA

101 A5ALNAAGVK PIYDVVPNHM NRGYPDKEIN LPAGQGLWRH DCNDPGNYAN

151 DCDDGDRFMG GDADLNTGHP QHYAMFRDEF ARLRSQYGAG GFRFDFVRGY

201 AGERVA3WMS DAHDHGFCLG ELWKAPGEYP SWDWRNGASW QQILKDWSDR

251 AKCTVFDFAL KERMQNGGIA DWRHGLNGNP DARWREVAVT FVDNHDTGYS

301 PGPHGGQHHW PLPDARLKQA YAYIL5SPGT PWYWPHMYD WGHGDFIRQL

351 IQIRRAAGVK AASAIQFHTG FSGLVATISG SQQQLLIALD SNLSSPGQVA

401 SGDFTQALNT DMGAIRIWRS GQGGGDGQGN LVSVNFRCDH GVTQWGDSVY

451 ALGNVTQLGN W3PAGAVRLT DTSAYPTWKG SIALPAGQQV QWKCIVR3ES

501 HP TQVKTWQP GGNHSVTVAS GASIAGSF *

SEQ ID NO: 7. Nucleotide DNA sequence encoding Pseudomonas mendocina amylase G4 (ymp strain) (A4XX23) with M added at the N-terminus: (A4XX23 DNA):

atggatgcaccgggcaaaacagcatcaggcgttagatatcatggcggcgatgaaattattctgcaaggctttcattggaatac agttagaacatcatcaaattggtatgcaacactggcatcaatggcaccgacactggcagcagatggcttttcagcaatttgga tgccggttccgtggagagatttttcatcatggtcagatccgggcaatggcacatcaggcggcggcgaaggctatttttggcat gattttaataaaaatggcagatatggctcagattcactgctgagacaagcagcatcagcactgaatgcagcaggcgttaaacc gatttatgatgttgttccgaatcatatgaatagaggctatccggataaagaaattaatctgccggcaggccaaggcctgtgga gacatgattgcaatgatccgggcaattatgcaaatgattgcgatgatggcgatagatttatgggcggcgatgctgatctgaat acaggccatccgcaaaattatgcaatgtttagagatgaatttgcaagactgagatcacaatatggcgcaggcggctttagatt tgattttgttagaggctatgcaggcgaaagagttgcatcatggatgtcagatgcacatgataatggcttttgcctgggcgaac tgtggaaagcaccgggcgaatatccgtcatgggattggagaaatggcgcatcatggcaacaaattctgaaagattggtcagat agagcaaaatgcacagtttttgattttgcactgaaagaaagaatgcaaaatggcggcattgcagattggagacatggcctgaa tggcaatccggatgcaagatggagagaagttgcagttacatttgttgataatcatgatacaggctattcaccgggcccgcatg gcggccaacatcattggccgctgccggatgcaagactgaaacaagcatatgcatatattctgtcatcaccgggcac accggtt gtttattggccgcatatgtatgattggggtcatggagattttattagacaactgattcaaattagaagagcagcaggcgttaa agcagcatcagcaattcaatttcatacaggcttttcaggcctggttgcaacaatttcaggctcacaacaacaactgctgattg cactggattcaaatctgtcatcaccgggccaagttgcatcaggcgattttacacaagcactgaatacagataatggcgcaatt agaatttggagatcaggccaaggcggcggcgatggccaaggcaatctggtttcagttaattttagatgcgataatggcgttac acaatggggcgattcagtttatgcactgggcaatgttacacaactgggcaattggtcaccggcaggcgcagttagactgacag atacatcagcatatccgacatggaaaggctcaattgcactgccggcaggccaacaagttcaatggaaatgcattgttagatca gaatcaaatccgacacaagttaaaacatggcaaccgggcggcaataattcagttacagttgcatcaggcgcatcaacagcagg ctcattttaa

SEQ ID NO: 8. Hahella chejuensis G4 amylase mature protein (strain KCTC 2396) with M added at the N-terminus (Q2SEA8 protein):

1 MESSGKSGAG VRFHGGDEII LCGFHWNWR TAERNWYNIL QSKAQQISED

51 GFTAIWMFVP WP.DM35IÍQAS 3DTRFGGEGY FWADMDKNSR YGDDGQLKQA

101 ASALKNKGVK VIYDIVPHHH DRGHSHD5LN LPSGQGYYRS DC3SCDDGDP

151 FMDGGSDFST AHPDVYDLFK NELVNLKTNY SAGGFRFDFV RGYAPERI5A

201 WMSASLDSGY CVGELWKGPS EYPSWDWRHS ASWQEILKDF TDASDCSVFD

251 FALKERMQNG SISDWRYGLN GHPSAQWREV AVTFVDWHDT GYSPGPLGGQ

301 HHWALPDWKR KMAYAYILSS PGTPWYWPH MYDWGMRDFI RHLIQLRKSA

351 GVKAYSGVQF HDGFSGLVGT TSGSHGKLLF AIDSNF33FN QVAGGAWNLA

401 VNEDMGRIRI WRQ *

SEQ ID NO: 9. Nucleotide DNA sequence encoding Hahella chejuensis amylase G4 (strain KCTC 2396) with M added at the N-terminus (DNA from Q2 SEA8):

atggaatcatcaggcaaatcaggcgcaggcgttagatttcatggcggcgatgaaattattctgcaaggctttcattggaacgt tgttagaacagcagaaagaaactggtacaacatcctgcaatcaaaagcacaacaaatttcagaagatggctttacagcaattt ggatgccggttccgtggagagataattcatcatggcaagcatcatcagatacaagatttggcggcgaaggctatttttgggca gatatggataaaaatteaagatatggcgatgatggccaactgaaacaagcagcatcagcactgaaaaataaaggcgttaaagt tatttatgatattgttccgaatcatcatgatagaggccattcaaatgattcactgaatctgccgtcaggccaaggctattata gatcagattgctcatcatgcgatgatggcgatccgtttatggatggcggctcagatttttcaacagcacatccggatgtttac gatctgtttaaaaacgaactggttaacctgaaaacaaactactcagcaggcggctttagatttgattttgttagaggctatgc □ ccggaaagaatttcagcatggatgtcagcatcactggattcaggctattgcgttggcgaactgtggaaaggcccgtcagaat atccgtcatgggattggagacattcagcatcatggcaagaaattctgaaagattttacagatgcatcagattgctcagttttt gattttgcactgaaagaaagaatgcaaaatggctcaatttcagattggagatatggcctgaatggcaatccgtcagcacaatg gagagaagttgcagttacatttgttgataatcatgatacaggctattcaccgggcccgctgggcggccaacatcattgggcac tgecggattggaaaagaaaaatggcatatgcatatattctgtcatcaecgggcacaccggttgtttattggccgca tatgtat gattggggcatgagagattttattagaaatctgattcaactgagaaaatcagcaggcgttaaagcatattcaggcgttcaatt tcatgatggcttttcaggcctggttggcacaacatcaggctcaaatggcaaactgctgtttgcaattgattcaaatttgatattgcatcaggatcaggatcaggatcaggatcatt SEQ ID NO: 10.> gi | 77787 | pir || S05667 mature protein 1,4-alpha-maltotetraohydrolase glucan (EC 3.2.1.60) without the signal sequence - Pseudomonas saccharophila

DQAGKSPAGVRYHGGDEIILQGFHWNVVREAPNDWYNILRQQASTIAADGFSAIWMPVPWRDFSSWTDGGKSGGGEGYFWHDF NKHGRYGSDAQLRQAAGALGGAGVKVLYDWPNHMNRGYPDKEINLPAGQGFWRHDCADPGNYPNDCDDGDRFIGGESDLNTG HPQIYGMFRDELANLRSGYGAGGFRFDFVRGYARERVDSWMSDSADSSFCVGELWKGPSEYPSWDWRNTASWQQIIKDWSDRA KCPVFDFALKERMQNGSVADWKHGLNGNPDPRWREVAVTFVDNHDTGYSPGQNGGQHHWALQDGLIRQAYAYILTSPGTPWY WSHMYDWGYGDFIRQLIQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLWALHSDLANPGQVASGSFSEAVNASKGQVRV WRSGSGDGGGNDGGEGGLVHVNFRCDNGVTQMGDSVYAVGNVSQLGNWSPASAVRLTDTSSYPTWKGSIALPDGQNVEWKCLI RMEADATLVRQWQSGGNNQVQAAAGAS T5GSF

SEQ ID NO: 11. Glycan signal peptide 1,4-alpha-maltotetraohydrolase (EC 3.2.1.60) - Pseudomonas saccharophila

MSHILRAAVLAAVLLPFPALA

SEQ ID NO: 12. Mature protein sequence of pMS1776

1 DQAGKSPAGV RYHGGDEIIL QGFHWt IW R E APYHWYNILR QKASTIAADG

51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFtJKtJGLYG SDAQLRQAAG

lo: ALGGAGVKVL YDWPMHI1HR FYPDKEIMLP AGQRFWRNDC PDPGMGPNDC

: DDCDRFLCC-E ADLNTGHPQI YGHFRDEFTíI LRSGYGAGGF RFDFVRGYAF

201 ERVDSWMSD3 ADSSFCVGEL WK5P5EYFFW DWRNRASWQQ IIKDWSDRAK

252 CPVFDFALKE RMQNGSVADW KHGLHGHFDF RWREVAVTFV DHHDTGYSPG

202 QHGGQHKHFL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ

251 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALHSD LDNPGQVASG

'101 SFSEAVNA5H GQVRVWRSGS GDGGGtIDGG *

SEQ ID NO: 13. Mature protein sequence of pMS1934

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG

51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKMGLYG SDAQLRQAAG

101 ALGGAGVKVL YDWPMHMMR FYPDKEIMLP AGQRFWRNDC PDPGNGPNDC

151 DDGDRFLGGE ADLMTGHPQI YGHFRDEFTN LRSGYGAGGF RFDFVRGYAP

201 ERVDSWMSDS ADSSFCVGEL WKKPSEYPPW DWRNRASWQQ IIKDWSDRAK

251 CPVFDFALKE RMQNGSVADW KDGLNGNPDP RWREVAVTFV DHHDTGYSPG

301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ

351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALHSD LDNPGQVASG

401 SFSEAVNASIT GQVRVWRSGS GDGGGIIDGG *

SEQ ID NO: 14 Mature protein sequence of pMS2020

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG

51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKMGLYG SDAQLRQAAG

101 ALGGAGVKVL YDWPMHMMR FYPDKEIMLP AGQRFWRNDC PDPGMGPNDC

151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP

201 ERVDSWMSDS ADSSFCVGEL WKSP3EYPPW DWRNRASWQQ IIKDWSDRAK

251 CPVFDFALKE RMQNGSVADW KHGLHGHPDP RWREVAVTFV DHHDTGYSPG

301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ

351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALHSD LDNPGQVASG

401 SFSEAVHAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 15 Mature protein sequence of pMS2022

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG

51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKMGLYG SDAQLRQAAG

101 ALGGAGVKVL YDVVPNHMNR FYPDKEIMLP AGQRFWRNDC PDPGMGPNDC

151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP

201 ERVDLWMSDS ADSSFCVGEL WKSPSEYPPW DWRNRASWQQ IIKDWSDRAK

251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DHHDTGYSPG

301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ

351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG

401 SFSEAVNAEII GQVRVWRSGS GDGGGIIDGG *

S E Q ID NO: 16 S e cu e n c e of p M ad u ra s p ro tein a s 2062

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP February 01 ERVDSWMSDS ADSSFCVGEL WKKPSEYPPW DWRNRASWQE IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ March 51 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 17 Mature protein sequence of pMS2171

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP February 01 ERVDLWMSDS ADS SFCVGEL WKAPSEYPPW DWRNRASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG 401 SFSEAVNAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 18 Mature protein sequence of pMS465:

l DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QQASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKSPSEYPPW DWRNTASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 19 Mature protein sequence of pMS1042:

l DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QQASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKEPSEYPPW DWRNTASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 20 Mature protein sequence of pMS1104:

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKEPSEYPPW DWRNTASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

S E Q ID N O: 21 P M S 1153 M ad u re p ro tein a s S e cu e nce:

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QQASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKEPSEYPPW DWRNTASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 22 Mature protein sequence of pMS1286:

i DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QQASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKEPSEYPPW DWRNRASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 23 Mature protein sequence of pMS1284:

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QQASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKEPSEYPPW DWRNRASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 24 Mature protein sequence of pMS1290:

i DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QQASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKEPSEYPPW DWRNRASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 25 Mature protein sequence of pMS1484:

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QQASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP

201 ERVDLWMSDS ADSSFCVGEL WKEPSEYPPW DWRNTASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

S E Q ID N O: 26 P M S 1579 M ad u ra s p ro tein a s S e cu e n cia:

i DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QQASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKEPSEYPPW DWRNTASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 27 Mature protein sequence of pMS1105:

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKSPSEYPPW DWRNTASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 28 Mature protein sequence of pMS1723:

i DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKSPSEYPPW DWRNTASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 29 Mature protein sequence of pMS2104:

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDSWMSDS ADSSFCVGEL WKAPSEYPPW DWRNRASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 30 Mature protein sequence of pMS2138:

i DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDLWMSDS ADSSFCVGEL WKSPSEYPPW DWRNRASWQQ IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

S E Q ID N O: 31 S e cu e n c e of pM S 2124 m ad u re p ro tein a s:

l DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDLWMSDS ADSSFCVGEL WKKPSEYPPW DWRNRASWQE IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG 401 SFSEAVNAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 32 Mature protein sequence of pMS2177:

i DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYQWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDLWMSDS ADSSFCVGEL WKKPSEYPPW DWRNRASWQE IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG 401 SFSEAVNAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 33 Mature protein sequence of pMS2178:

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAQ 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDLWMSDS ADSSFCVGEL WKKPSEYPPW DWRNRASWQE IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG 401 SFSEAVNAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 34 Mature protein sequence of pMS2118:

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDLWMSDS ADSSFCVGEL WKSPSEYPPW DWRNRASWQE IIKDWSDRAK 251 CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG 401 SFSEAVNAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 35. Linker Sequence

GSGDGGGNDGG

SEQ ID NO: 36: PS4 SBD Sequence:

1 EGGLVNVNFR CDNGVTQMGD SVYAVGNVSQ LGNWSPASAV RLTDTSSYPT 51 WKGSIALPDG QNVEWKCLIR NEADATLVRQ WQSGGNNQVQ AAAGASTSGS 101 F *

SEQ ID NO: 37

HGGDEIILQFHWN

SEQ ID NO: 38

DGF-X1-AIW-X2-P-X3-PWRD-X4-SSW

SEQ ID NO: 39

GGEGYFW

SEQ ID NO: 40

VPNH SEQ ID NO: 41

CDDGD SEQ ID NO: 42

AGGFRFDFVRG SEQ ID NO: 43

FALK SEQ ID NO: 44

WREVAVTFVDNHD SEQ ID NO: 45

GYSPG SEQ ID NO: 46

GQH SEQ ID NO: 47

AYAYI SEQ ID NO: 48

SPGTP SEQ ID NO: 49

VYW SEQ ID NO: 50

HMYDWG SEQ ID NO: 51: Novamyl:

SSSASVKGDVIYQIIIDRFYDGDTTNNNPAKSYGLYDPTKSKWKMYWGGDLEGVRQKLPYLKQLGVTTIWLSPVLDNLDTLAG TDNTGYHGYWTRDFKQIEEHFGNWTTFDTLVNDAHQNGIKVIVDFVPNHSTPFKANDSTFAEGGALYNNGTYMGNYFDDATKG YFHHNGDISNWDDPYEAQWKNFTDPAGFSLADLSQENGTIAQYLTDAAVQLVAHGADGLPIDAVKHFNSGFSKSLADKLYQKK DIFLVGEWYGDDPGTANHLEKVRYANNSGVNVLDFDLNTVIRNVFGTFTQTMYDLNNMVNQTGNEYKYKENLITFIDNHDMSR FLSVNSNKANLHQALAFILTSRGTPSIYYGTEQYMAGGNDPYNRGMMPAFDTTTTAFKEVSTLAGLRRNNAAIQYGTTTQRWI NNDVYIYERKFFNDVVLVAINRNTQSSYSISGLQTALPNGSYADYLSGLLGGNGISVSNGSVASFTLAPGAVSVWQYSTSASA PQIGSVAPNMGIPGNWTIDGKGFGTTQGTVTFGGVTATVKSWTSNRIEVYVPNMAAGLTDVKVTAGGVSSNLYSYNILSGTQ TSVVFTVKSAPPTNLGDKIYLTGNIPELGNWSTDTSGAVNNAQGPLLAPNYPDWFYVFSVPAGKTIQFKFFIKRADGTIQWEN GSNHVATTPTGATGNITVTWQN

Claims (15)

1. A polypeptide having amylase activity comprising an amino acid sequence that has at least 78% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises an amino acid substitution at position 311, with reference to the sequence position numbering shown as SEQ ID NO: 1, wherein the polypeptide has improved thermostability compared to amylase of SEQ ID NO: 1. 2. The polypeptide according to claim 1, wherein the polypeptide comprises one or more amino acid substitutions at the following positions: 235, 88, 205, 240, 248, 266, 377 or 409 and / or one or more of the following amino acid substitutions: 42K / A / V / N / I / H / F, 34Q, 100Q / K / N / R, 272D or 392K / D / E / Y / N / Q / R / S / T / G . 3. The polypeptide according to claim 1 or 2, wherein the polypeptide further comprises amino acid 223S / K / A. The polypeptide according to any of claims 1-3, having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with respect to amino acid sequence of SEQ ID NO: 1. 5. The polypeptide according to any of claims 1-4 comprising SEQ ID NO: 19. 6. The polypeptide according to any of claims 1-3 having a linker fused at the C-terminus. 7. The polypeptide according to any of claims 1-6 having amylase exoactivity, preferably non-maltogenic amylase exoactivity. 8. A nucleic acid capable of encoding a polypeptide according to any of claims 1-7. 9. An expression vector vector comprising a nucleic acid according to claim 8, or capable of expressing a polypeptide according to any of claims 1-7. 10. A plasmid comprising a nucleic acid according to claim 8. 11. A host cell comprising an expression vector according to claim 9 or plasmid according to claim 10. 12. A host cell transformed with an expression vector according to claim 9 or a plasmid according to claim 10. 13. Use of a polypeptide according to any of claims 1-7 as a food or feed additive. 14. A method of preparing a saccharide syrup, which comprises adding a polypeptide according to any of claims 1-7 to a granular starch smoothie to form the saccharide syrup. 15. A food product comprising a polypeptide according to any of claims 1-7.