ES2704159T3 - Amylase polypeptides - Google Patents

Abstract

A polypeptide having amylase activity comprising an amino acid sequence having at least 65% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises an amino acid substitution at the following position: 88, with reference to the numbering of the position of the sequence shown as SEQ ID NO: 1, wherein the polypeptide has improved thermostability compared to the 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 food products for animals.

BACKGROUND OF THE INVENTION

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

The crystallization of amylopectin 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 the 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 leathery crust.

Enzymatic hydrolysis (eg, by amylases) of amylopectin side chains can reduce crystallization and increase 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 composed 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 units units of a-D-glucopyranosyl units linked by bonds (1-4), in which the chains are joined by a-D- (1-6) bonds to form branches. Amylose is a linear chain of linked α-D-glucopyranosyl units (14) that have few a-D- (1-6) branches.

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

The activity of endoamylase activity may adversely affect the quality of the final bread product by the production of a sticky or gummy crumb due to the accumulation of branched dextrins. The exoamylase activity is preferred, because it carries out the desired modification of starch which leads to delayed aging, with less of the negative effects associated with the endo-amylase activity. The reduction of endoamylase activity can lead to greater exospecificity, which can reduce branched dextrins and produce a better quality bread.

The conversion of vegetable starches, especially corn starch, to ethanol is a rapidly expanding industry. The 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 corn starch, to maltotetraose and lower sugars, such as glucose or maltose, is a rapidly expanding industry. The current process consists of two stages catalyzed by sequential enzymes that result in the production of glucose or maltose. The yeast can be used to ferment glucose to ethanol.

The first stage is catalyzed by the enzyme is the liquefaction of the starch. Typically, a suspension of starch is gelatinized by rapid heating to 85 ° C or more. • -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 decompose the starch, the viscosity decreases. Because liquefaction is typically carried out at elevated temperatures, thermostable • amylases, such as a Bacillus sp. Amylase, are preferred for this step.

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

The 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 flavor or aroma. The G4 syrup has a high moisture retention in foods and exhibits less damaging Maillard reaction products due to lower glucose and maltose content. The G4 syrup also has a higher viscosity than sucrose, which improves the texture of the food. 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, the G4 syrup also affects the osmotic pressure less than sucrose. Together, these qualities make the G4 syrup ideal as an ingredient in food and medical products. G4 syrup is useful in other industries, too. For example, the G4 syrup imparts gloss and can be used advantageously 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 that 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 improved by Mg2 + supplementation and overexpression of AGT1 permease (Stambuck et al, Lett Appl Microbiol 43: 370-76 (2006)), overexpression of MTT1 and MTT1alt for 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 completely, if the liquefaction step produced sufficient levels of mono-, di-, or tri-saccharides and S. cerevisiae or its variants genetically modified for the fermentation step were used.

Pseudomonas saccharophila expresses a maltotetraohydrolase-forming maltotetraose (EC 3.2.1.60, amylase-forming G4; 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-terminal end. shows both endo and exo-a-amylase activity The endoa-amylase activity is useful for decreasing the viscosity of the gelatinized starch, and the exo-α-amylase activity is useful for decomposing the maltodextrins into smaller saccharides. 2007/148224 describes PS4 variant polypeptides having non-maltogenic exo-amylase activity.

Synthesis of the invention

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, 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 a better thermostability compared to the amylase of SEQ ID NO: 1.

A polypeptide variant is provided which is set forth in the claims. In particular, the present invention provides a polypeptide having amylase activity comprising an amino acid sequence having at least 65% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises an amino acid substitution at the following position: 88, with reference to the numbering of the position of the sequence shown as SEQ ID NO: 1; wherein the polypeptide has improved thermostability compared to the amylase of SEQ ID NO: 1. In a further aspect, the use of such polypeptide variant is provided, which is included in and as food additives, food products, products of bakery products, improving compositions, food products, including animal feed, etc. as set forth in the claims. In a further aspect, nucleic acids encoding and relating to polypeptide variants are provided, as set out 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 and constitute a part of this specification and illustrate various embodiments.

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

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

Detailed description of the invention

According to 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 dictates 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,

Polypeptides 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 in the following positions: 88 or 205, and / or at least 78% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises one or more amino acid substitutions in 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 numbering of the position of the sequence shown as SEQ ID NO: 1.

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

Also described is the use of polypeptides having a degree of sequence identity or sequence homology with the amino acid sequence defined herein or with a polypeptide having the specific properties defined herein. In particular, peptides having a degree of sequence identity are described with SEQ ID NO: 1, which is defined below or homologues thereof. Herein, the term "homologous" means an entity sequence identity having the present amino acid sequence or the present nucleotide sequences, wherein the amino acid sequence present 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 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 sequence present. Normally, the counterparts will understand the same active sites etc. than the present amino acid sequence. Although homology can also be considered in terms of similarity (ie, amino acid residues having similar chemical properties / functions), in the present context it is preferred to express homology in terms of sequence identity.

Sequence identity comparisons can be performed with the naked eye, or more usually, with the help of readily available sequence comparison programs. These commercially available software use complex comparison algorithms to align two or more sequences that better reflect the 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 the alignment of identical or similar amino acids and the penalty of the insertion of the gaps, gap extensions and alignments of the non-similar amino acids. The scoring system of the comparison algorithms includes:

i) the assignment of a penalty score each time a gap is inserted (penalty score of the gap),

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

iii) the assignment of high scores after the alignment of identical amino acids, and

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

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

The scores given for the alignment of the 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 is substituted with 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 is substituted with another polar amino acid is higher compared to being substituted with a hydrophobic amino acid. Therefore, the scoring matrix will assign the highest identical amino acid score, lowest non-identical amino acid score, but similar and even lowest score for non-identical non-identical 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)).

The appropriate computer programs to carry out such 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 the sequence alignment is BLAST (basic local alignment search tool), which is available on the National Center for Biotechnology Information website which can currently be found at http://www.ncbi.nlm.nih.gov/ and which was described in first place in Altschul et al (1990) J. Mol Biol 215; 403-410.

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

In one embodiment, it is preferred to use the ClustalW software to perform sequence alignments. Preferably, the alignment with ClustalW is carried out with the following parameters for the alignment in pairs:

ClustalW2 is, for example, available on the Internet by the European Institute of Bioinformatics on the website www.ebi.ac.uk EMBL-EBIen 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 the Align X program of Vector NTI (Invitrogen) to perform sequence alignments. In one embodiment, Exp10 has been used with the predetermined configuration: Gap breach penalty: 10

Extension penalty of the gap: 0.05

Penalty range of gap separation: 8

Scoring matrix: blosum62mt2

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

The sequences, in particular those of the variants, homologs 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. The deliberate amino acid substitutions can be made on the basis of polarity similarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues provided that 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 be produced ie substitution of equals, such as basic by basic , acid by acid, polar by polar, etc. The non-conservative substitution can also occur, i.e., from one kind of residue to another, or alternatively involving the inclusion of non-natural 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.

The 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).

The replacements can also be made by non-natural 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 *, p-phenylalanine *, L -alyl-glycine *, 13-alanine *, La-amino butyric acid *, LY-amino butyric acid *, La-amino isobutyric acid *, L-α-amino caproic acid #, 7-amino heptanoic acid *, 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 (1,2,3,4-tetrahydroisoquinoline-3-acid) carboxyl) *, L-diaminopropionic acid # and L-Phe (4-benzy) *. 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 .

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

Disclosed herein are variants of polypeptides, which are Pseudomonas saccharophila amylase (PS4), variants 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 the amino acid sequence with this.

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 skilled 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 activity of the endo-action amylase 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. "Amylase activity of endo action", "endo-activity", "activity "specific endo" and "endo-specificity" are synonymous, when the terms refer to the polypeptide variants as defined in the claims The same is valid for the corresponding terms for exo activity "Maltotetrahydrolase-forming maltotetraose; 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 the maltotetrahydrolase of Pseudomonas saccharophila.

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 in one or more locations in the amino acid or nucleotide sequence, respectively. The phrases "polypeptide variant", "polypeptide", "variant" and "enzyme variant" means 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 having a certain percentage, eg, 65%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of sequence identity with SEQ ID NO: 1. As used herein, "original enzymes", "original sequence", "original polypeptide" means enzymes and polypeptides from which the polypeptide variants are based, eg, SEQ ID NO: 1. An "original 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 may differ from the signal sequence of the 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 covered by the present description, the following nomenclature will be adopted to facilitate the reference. When the substitution includes a number and a letter, for example, 141P, then it refers to {position according to the numbering system / substituted amino acid}. Accordingly, for example, substitution of an amino acid to proline at position 141 is designated 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 / substituted amino acid system}. Accordingly, for example, the substitution of alanine with proline at position 141 is designated as A141P.

When two or more substitutions are possible in a particular position, it will be referred to as contiguous letters, which may optionally be separated by bar marks "/", for example, G303ED or G303E / D.

The positions and amino acid 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 identity percentage and to determine thereafter which amino acid is aligned to correspond to an amino acid of a specific position of SEQ ID NO: 1. Such alignment and use of a sequence as a First reference is simply a matter of routine for a person skilled in the art.

The "nucleic acid variants" can include sequences that are complementary to the 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 nucleotide sequences presented in this document, in particular, to SEQ ID NO: 52 (DNA pMS 382) More particularly, the term variant encompasses sequences that are complementary to the sequences that are capable of hybridizing under very stringent conditions, for example, 65 ° C and 0.1 x SSC, to the nucleotide sequences presented herein, in particular, to SEQ ID NO: 52 ( ^a D ⁿ pMS 382.) The melting point (Tm) of a nucleic acid variant can be approximately 1, 2, or 3 ° C lower than the Tm of nucleic acid wild type co. Nucleic acid variants include a polynucleotide having a certain percentage, for example, 80%, 85%, 90%, 95%, or 99%, of sequence identity with the nucleic acid encoding S ^e Q 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 sequence naturally associated and found in nature, for example, genomic sequences.

"Purified" means that the material is in a relatively pure state, for example, 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 (t-ic), where half the enzymatic activity is lost by the half-life. The value of the half-life is calculated under certain conditions by measuring the activity of the residual amylase. 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, homologs, fragments and derivatives thereof. The nucleotide sequence may be of genomic, synthetic or recombinant origin and may be double-stranded or single-stranded, whether it represents the sense or anti-sense chain. 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 a polypeptide having a certain percentage, for example, 80%, 85%, 90%, 95%, or 99%, of sequence identity with another sequence. The percent identity means that, when aligned, that percentage of bases or amino acid residues are the same as when comparing the two sequences. The amino acid sequences are not identical, where an amino acid is substituted, deleted or added in comparison with the sequence present. The percentage of sequence identity is typically measured with respect to the mature sequence of the present protein, i.e., for example after the removal of a signal sequence. Typically, homologs 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 chain is linked to a complementary strand through base pairing, as well as the amplification process that is carried out in the reaction technologies. in 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 chain comprising both ribonucleotides and deoxyribonucleotides. The nucleic acid variant can be optimized by codons 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 use of codons for host organisms, such as a yeast host cell or other expression host of choice.

As used herein, the "transformed cell" includes cells, including bacterial and fungal cells, that have been transformed by the use of recombinant DNA techniques. The transformation typically occurs by inserting one or more nucleotide sequences in a cell. The inserted nucleotide sequence can be a heterologous nucleotide sequence, i.e., it is a sequence that is not natural for the cell to be transformed, such as a fusion protein.

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

As used herein, the term "starch" refers to any compound material of the complex polysaccharide carbohydrates of plants, such as corn, amylose compound and amylopectin with the formula (C6H1üO5) x, where X can be any number. The term "granular starch" refers to raw starch, ie, uncooked starch, for example, that has not been subjected to gelatinization.

The term "liquefaction" refers to the step in the conversion of the starch in which the starch is gelatinized to hydrolyze to give soluble dextrins of low molecular weight. 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 DP1 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 dry weight percent 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, are present during the same process step. SSF refers to the simultaneous hydrolysis of substrates from granular starch to saccharides and the fermentation of 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 indicated:

ADA azodicarbonamide

cDNA complementary DNA

CGTase cyclodextrin glucanotransferase

DEAE diethylaminoethanol

dH2O deionized water

DNA deoxyribonucleic acid

ds-DNA double-stranded DNA

EC commission of enzymes for the classification of enzymes

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 the amino acids are designated with the single letter abbreviations commonly known in the art

HPLC high performance liquid chromatography

mRNA ribonucleic acid messenger

PCR polymerase chain reaction

PDB protein database

PEG polyethylene glycol

ppm parts per million

PS4 amylase forming G4 P. saccharophila

RT-PCR polymerase chain reaction - reverse transcriptase

SAS amylase forming G4 P. saccharophila

SDS-PAGE polyacrylamide gel electrophoresis with sodium dodecyl sulfate

1X SSC 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0

SSF saccharification and simultaneous fermentation

ty2 half life

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

ATm ° C increase in theTm

w / v weight / volume

w / w weight / weight

2. Variants of polypeptides of SEQ ID NO: 1

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

In one aspect, the variants defined herein exhibit enzymatic activity. In one aspect, 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 may be derived from the phenylephrine-amylase of Pseudomonas saccharophila (PS4). In one aspect, the variants defined herein are a maltotetrahydrolase that promotes maltotetraose, also called EC 3.2.1.60; G4-forming amylase; G4-amylase or glucan 1,4-alpha-maltotetrahydrolase.

The compositions, which include food additives, food products, bakery products, enhancing compositions, food products, including animal feed, etc. comprising such variants of polypeptides 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 so treated have one or more of (preferably all of) a lower firmness, a higher elasticity, a higher cohesion, a lower friability or a greater folding capacity. Such improved handling or baking properties exhibited by the variant polypeptides are described in more detail below.

In addition, a treatment of food products, in particular doughs and bakery products with such polypeptides, is provided, and such food products have the desired qualities discussed above.

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

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 leader sequence of 21 amino acids is cleaved, such that the N-terminus of the polypeptide begins at the acid residue aspartic (D). In one aspect, the variants include a C-terminal starch binding domain. An amino acid sequence representative of a starch binding domain comprises or consists of an amino acid sequence of SEQ ID NO: 36. Other variants include those 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 SEQ 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 yet 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 variant of the polypeptide is 429 to 430 amino acids.

In one aspect, the present invention relates to a polypeptide having amylase activity comprising an amino acid sequence having at least 65% sequence identity with the amino acid sequence of SEQ. ID NO: 1 and wherein the polypeptide comprises an amino acid substitution at the following position: 88, with reference to the numbering of the position of the sequence shown as SEQ ID NO: 1; wherein the polypeptide has an improved thermostability purchased with the amylase of SEQ ID NO: 1.

In a further aspect, the polypeptide defined herein comprises one or more amino acid substitutions in the following positions: 88, 205, 235, 240, 248, 266, 311, 377 or 409 and / or one or more of the following substitutions of 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 in 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 in 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 with 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 the 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 the amino acid 88L. In a further aspect, the polypeptide defined herein has the 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 amino acid 205UK / M / N / Q / R / V / Y. In a further aspect, the polypeptide defined herein has the amino acid 205L / K / M / N / G / R / V. In a further aspect, the polypeptide defined herein has amino acid 205L. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids 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 the amino acid 235H. In a further aspect, the polypeptide defined herein has the amino acid 235K. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 240. In a further aspect, the polypeptide defined herein has 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 a substitution of amino acids at position 311. In a further aspect, the polypeptide defined herein has the amino acid 311P. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 409. In a further aspect, the polypeptide defined herein has the amino acid 409H / Q / T / E. In a further aspect, the polypeptide defined herein has the 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 1000. 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 the amino acid 392D. In a further aspect, the polypeptide defined herein has the amino acid 392E.

In a further aspect, the polypeptide defined herein has the amino acid 392K. In a further aspect, the polypeptide defined herein has the 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 a substitution of amino acids at position 16. In a further aspect, the polypeptide defined herein has the amino acid 16 / A / E / K. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 48. In a further aspect, the polypeptide defined herein has the amino acid 48 / C / L. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 97. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 105. In a further aspect, the polypeptide defined in the present it has the amino acid 105 / N / R. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 248. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 266. In a further aspect, the polypeptide defined herein it comprises an amino acid substitution at position 347. In a further aspect, the polypeptide defined herein has the amino acid 347 / C / D / H / K. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids 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 a substitution of amino acids 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 a substitution of amino acids at position 362. In a further aspect, the polypeptide defined herein has the amino acid 362E / H / P. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 364. In a further aspect, the polypeptide defined herein has the amino acid 364E / K / NQ. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 369. In a further aspect, the polypeptide defined herein has the amino acid 369I / N. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 377. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 393. In a further aspect, the polypeptide defined herein has the amino acid 393D / E / K. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 395. In a further aspect, the polypeptide defined herein has the amino acid 395C / E / K. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 396. In a further aspect, the polypeptide defined herein has the amino acid 396D / E. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 399. In a further aspect, the polypeptide defined herein has the amino acid 399C / H. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 400. In a further aspect, the polypeptide defined herein has the amino acid 400S / W. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 401. In a further aspect, the polypeptide defined herein has the amino acid 401D / K. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 403. In a further aspect, the polypeptide defined herein has the amino acid 403E / T / V. In a further aspect, the polypeptide defined herein comprises a substitution of amino acids at position 412. In a further aspect, the polypeptide defined herein has the 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, 229 ^p, 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 409 ^e. 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, T235 ^r, 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 in 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 numbering of the position 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, 235K / H / Q / R, 240E / D, 248H, 266T, 311P, 377D / E / P, 392K / D / And or 409E. In a further aspect, the polypeptide defined herein comprises one or more of the following amino acid substitutions: 34Q, 42K, 88 ^l, 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 replacement of 42K amino acids. 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, 311P, 392D and 409E.

Representative embodiments of the polypeptides defined herein comprise a sequence of amino acids 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 a sequence of amino acids 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 a sequence of amino acids selected from the group consisting of the ^s E ^q 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 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 to 26 with reference to the position numbering of the sequence shown as E ^{s q} ID NO: one.

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 the 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 the positions corresponding to positions 79-85 with reference to the numbering of the position of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the VPNH sequence (SEQ ID NO: 40) at the positions corresponding to positions 114-117 with reference to the numbering of the position 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 the positions corresponding to positions 150-154 with reference to the numbering of the position 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 the positions corresponding to positions 187-197 with reference to the numbering of the position 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 the positions corresponding to positions 256-259 with reference to the numbering of the position 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 the positions corresponding to positions 282-294 with reference to the numbering of the position 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 the positions corresponding to positions 296-300 with reference to the numbering of the position 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 the positions corresponding to positions 304-306 with reference to the numbering of the position 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 the positions corresponding to positions 318-322 with reference to the numbering of the position of the sequence shown as SEQ ID NO: 1.

In one aspect, the polypeptides defined herein comprise the SPGTP sequence (SEQ ID NO: 48) at the positions corresponding to positions 325-329 with reference to the numbering of the position 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 the positions corresponding to positions 331-333 with reference to the numbering of the position 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 the positions corresponding to positions 335-340 with reference to the numbering of the position of the sequence shown as SEQ ID NO: 1.

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

The polypeptide variants defined herein may have an altered thermostability, an altered endo-amylase activity, an altered exo-amylase activity, and / or an altered exo-to-endoamylase activity ratio as 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 the amylase of SEQ ID NO: 1. In another aspect, the polypeptides defined in present have a better thermostability compared to the 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 of amino acids of SEQ ID NO: 1.

The present disclosure also relates to each and every skeleton of G4-amylase having SEQ ID NO: 2, 4, 6 or 8 comprising the substitutions defined herein further.

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 the protein 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 skeleton of the G4 amylase with SEQ ID NO: 2. As is well understood by those skilled 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 variant of polypeptide 2.1. Characterization of the polypeptide variant

The variants of the enzyme can be characterized by their primary nucleic acid and polypeptide sequences, by three-dimensional structural modeling, and / or by their specific activity. Additional features of the polypeptide variants as defined herein include for example, stability, pH range, stability to oxidation, and thermostability. The levels of expression and activity of the enzyme can be assessed using standard assays known to those skilled in the art. In another aspect, the variants demonstrate improved performance characteristics relative to the polypeptide with SEQ ID 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 may degrade the starch at temperatures from about 55 ° C to about 85 ° C or more.

An expression characteristic means an altered level of expression of the variant, when the variant is produced 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, for a given period of time. The expression may also refer to the amount or rate of variant produced within the host cell or secreted by the host cell. The expression can also refer to the translation rate of the mRNA encoding the enzyme variant.

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 with the complement is provided. In another embodiment, the sequence for use in the methods and compositions described herein is a synthetic sequence. It includes, but is not limited to, the sequences obtained with the optimal use of codons for expression in host organisms, such as yeast.

3. Production of the polypeptide variants defined herein

The polypeptide variants provided herein may 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 before use. In another embodiment, variant of the polypeptide 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. Nros 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 descriptions, including the polynucleotide sequences encoding the polypeptide, primers, vectors, selection methods, host cells, purification and reconstitution of expressed polypeptide variants and characterization of polypeptide variants defined herein, which they include useful buffers, pH ranges, Ca2 + concentrations, substrate concentrations and enzyme concentrations for enzymatic assays, are incorporated herein. 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, wherein 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 B. subtilis signal sequence, which are discussed in more detail below. In one aspect, the host cell expresses the polynucleotide set out 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 having 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 encoding a variant of the polypeptide defined herein will have a nucleic acid sequence encoding the protein of SEQ ID NO: 1 or a nucleic acid sequence having 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 has 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. 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 genome of the fungal host cell and replicates when it is introduced into the host cell. The FGSC catalog of strains from the University of Missouri 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 MANIPULATIONS IN FUNGI, Academic Press, San Diego (1991), p. 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 allows replication in Bacillus.

In some embodiments, a nucleic acid encoding a variant is operably linked to a suitable promoter, which allows 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 promoters cbh1, cbh2, egl1, and egl2. 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 promoter of P, saccharophila P native. 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 encoding 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 that encodes 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 signal sequence of cyclodextrin glucanotransferase. (CGTase, EC 2.4.1.19) of Bacillus sp, and the polypeptide variants described herein are expressed in a host cell of B. subtilis. 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 to be introduced into a fungal host cell are derived from the same source. In some embodiments, the expression vector also includes a terminator 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, for example, hygromycin or phleomycin. Selective nutritional 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 which allows the transformed cells to grow in acetamide as a nitrogen source. The use of an amdS gene of A. nidulans 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 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 for expression. In some embodiments, the truncation of genes is obtained by deleting sequences ^to D ⁿ unwanted, for example, DNA encoding the binding domain starch-C-terminal, to allow the domain to be expressed under the control of their own regulatory transcription and translation sequences. The second type of expression vector is pre-assembled and contains sequences necessary for high-level transcription and a selectable marker. In some embodiments, the coding region for a gene or part thereof is inserted into this commonly used 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 these are inserted downstream of the strong cbh1 promoter.

3.2. Transformation, expression and culture of host cells

The introduction of a DNA or vector construct into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, for example, lipoinfection mediated transfection mediated by DEAE-Dextrin; incubation with DNA precipitate with calcium phosphate; high speed bombardment with microprojectiles coated with DNA; and fusion of protoplasts. The techniques of general transformation 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 the nucleic acid encoding a variant is stably integrated into a chromosome of the host cell. The transformants are then purified by known techniques.

In a non-limiting example, stable transformants, including 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 in solid culture medium containing acetamide. In addition, in some cases, further stability testing is carried out by culturing the transformants in a solid non-selective medium, for example, a medium lacking acetamide, harvesting the spores from this culture medium and the 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 evaluate the expression of a variant in a host cell, the assays can measure the expressed protein, corresponding mRNA, or the α-amylase activity. For example, appropriate assays include Northern and Southern blots, 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 the exo-activity of the variant include, but are not limited to, the Betamyl® assay (Megazyme, Ireland). Suitable assays of 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 the HPLC analysis of liquefies prepared in the presence of the variant. HPLC can be used to measure the activity of amylase by separating the DP-3 and DP-4 saccharides from other test components.

Improved handling properties

The polypeptide variants described herein preferably, preferably improved baking properties. Therefore, 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 exo-specificity. The polypeptide variants described herein preferably also have better handling properties, so that a food product treated with a polypeptide variant has one or all of lower firmness, higher elasticity, greater cohesion, lower friability or greater folding capacity. in comparison with a food product that has been treated with SEQ ID NO: 1.

Without wishing to be bound to any particular theory, it is considered that mutations in particular positions have individual and cumulative effects on the properties of a polypeptide comprising said mutations.

Thermostability and stability at pH

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

In wheat and other cereals the external side chains of amylopectin are in the range of 12-19 DP. Therefore, enzymatic hydrolysis of the amylopectin side chains, for example, by variants of polypeptides described that have 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 the modification of the starch is mainly limited to the damaged starch and progresses very slowly during the preparation of the dough and initial baking until the gelatinization starts at about 60 ° C. As a consequence of this only the amylases with a high degree of thermostability are able to modify the starch efficiently during baking. And, in general, the efficiency of amylases increases with the increase in thermostability. This is so because the more thermostable 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 the starch at any stage of its processing into a food product, eg, before, during or after baking in bread can retard or prevent or slow down the retrogradation . Such use is described in more detail below.

As used herein, the term "thermostable" refers to the ability of the enzyme to maintain activity after exposure to elevated temperatures. Preferably, the polypeptide variant is capable of degrading starch at temperatures 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. Accordingly, the polypeptide variants described herein have extended half-lives with respect to the original enzyme preferably at 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 at about 80 ° C.

As used herein, the half-life (t1 / 2) is the time (in minutes) during which half the activity of the enzyme 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 value of the half-life is then calculated by measuring the activity of the residual amylase, by any of the methods described herein. Preferably, a half-life assay is carried out as described in more detail in the Examples.

Preferably, the polypeptide variants described herein are active during baking and hydrolyze the starch during and after the gelatinization of the starch granules starting at temperatures of about 55 degrees C. The more thermostable 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, non-maleogenic 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 by the use of genetic manipulation of proteins to be more thermostable and therefore more suitable for the uses described herein; therefore, the use of modified polypeptide variants to be more thermostable by genetic engineering of proteins is encompassed.

Preferably, the polypeptide variant is stable at pH; more preferably, it has a higher 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 range of pH. Preferably, the polypeptide variant is capable of degrading the starch at a pH of 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 stability to the pH can be tested by measuring the activity or the specific activity of the enzyme under specific pH conditions. The specific pH conditions can be any pH from pH 5 to pH 10.5.

Accordingly, the polypeptide variant can have a longer half-life or a higher activity (according to the assay) when compared to SEQ ID NO: 1 under identical conditions. The 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 since the endoamylase activity may possibly adversely affect the quality of the final bread product by producing a sticky or gummy 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 in this document as an "Exospecificity Index". 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 exo-specificity 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.

The activity of total amylase and endoamylase activity can be measured by any means known in the art. For example, the 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.

The endoamylase activity can be analyzed by 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 brand, while the external cuts do not. The release of dye can be measured by spectrophotometry. Consequently, the Phadebas kit measures the endoamylase activity, and for convenience, the results of such an assay are referred to herein as "Phadebas units".

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

The 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 relationship between endoamylase activity and exoamylase activity. Therefore, in a preferred aspect, the polypeptide variant described herein will have less than 0.5 units of endoamylase (EAU) per unit of exoamylase activity. Preferably, the non-maltogenic exoamylases which are suitable for use according to 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 the exoamylases. In addition, it preferably has either increased or increased exospecificity when compared to SEQ ID NO: 1. Accordingly, for example, the polypeptide variant can have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or higher exospecificity index when it is compared to SEQ ID NO: 1, preferably under identical conditions. They may have 1.5x or greater, 2x or higher, 5x or higher, 10x or higher, 50x or higher, 100x or higher, 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 skilled in the art. Examples include, but are not limited to, affinity chromatography, ion exchange chromatographic methods, including high resolution 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 process of liquefying a starch, for example, corn starch, wheat starch or barley starch. The desired final product can be any product that can be produced by the enzymatic conversion of the starch substrate. For example, the desired product may 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 alcohol for fuel or potable (ie, potable 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 thereof is particularly useful. Suitable genetically altered variants of S. cerevisiae particularly useful for maltotriose fermentation include variants expressing 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 compositions and methods of food preparation, when desired, enzymes that express amylase activity at high temperatures.

The desirability of using a particular polypeptide variant will depend on the general properties shown 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 as compared to wild-type PS4 or SEQ ID NO: 1, and / or have an exo-to-endo-amylase activity in Comparison with wild type PS4 or SEQ ID NO: 1. Such polypeptide variants may be particularly useful in a liquefaction process, when used alone or in combination with other amylase variants, wherein the internal cleavage of complex branching saccharides reduces the viscosity of the substrate. It is expected that some polypeptide variants useful for liquefaction, however, have a comparable endo-amylase activity or even lower than wild-type PS4. Useful polypeptide variants include those that have more or less the exo-amylase activity of the wild type PS4 polypeptide or SEQ ID NO: 1, according to the application. The compositions may include one or a combination of amylase variants, each of which may show a different set of properties.

4.1. Preparation of starch substrates

Those skilled in the art are well aware of the methods available 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 grains. More specifically, granular starch comes from plants that produce high amounts of starch. For example, granular starch can be obtained from maize, corn, wheat, barley, rye, millet, sago, cassava, tapioca, sorghum, rice, peas, beans, bananas or potatoes. Corn contains approximately 60 to 68% starch; the barley contains approximately 55-65% starch; millet contains approximately 75 to 80% starch; wheat contains approximately 60 to 65% starch; and the polished rice contains 70-72% starch. The starch substrates specifically contemplated 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 ears. The starch can be raw highly refined starch or raw material from starch refinery processes. Various starches are also available commercially. For example, corn starch 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 the embodiments of the present invention. Maltodextrins comprise starch hydrolysis products having approximately 20 or less dextrose (glucose) units. Typical commercial maltodextrins contain mixtures of polysaccharides that include from about three to about nineteen bound dextrose units. Maltodextrins are defined by the FDA as products that have a dextrose equivalence (DE) of less than 20. In general, they are 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 greater the DE, the greater the degree of depolymerization of the starch, resulting in a lower average polymer size. (polysaccharide), and greater sweetness. A particularly useful maltodextrin is MALTRIN® M040 obtained from corn starch, available from Grain Processing Corp. (Muscatine, Iowa): DE4.0-7.0; bulk density of 0.51 g / cc; water content measured at 6.38% by weight.

The starch substrate can be a ground whole-grain raw starch, which contains non-starch fractions, eg, germ residues and fibers. The milling may comprise wet milling or dry milling. In wet grinding, the whole grain is dipped in water or diluted acid to separate the grain into its component parts, fibers, for example, starch, proteins, germ, oil, grain fibers. Wet milling efficiently separates the germ and flour (ie, starch and protein granules) and is especially suitable for the production of syrups. In dry milling, whole grains are ground into a fine powder and processed without fractionating the grain into its component parts. The dry milled grain will thus comprise significant amounts of non-starchy carbohydrate compounds, in addition to starch. Most ethanol comes from dry grinding. Alternatively, the starch for processing can be a highly refined starch quality, for example, 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 "liquefy" means a process whereby the starch is converted to shorter, less viscous chain dextrins. This process involves the gelatinization of the starch simultaneously with or followed by the addition of a polypeptide variant described herein. A thermostable polypeptide variant described herein is preferably used for this application. Additional liquefaction inducing enzymes may 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 one percent by weight dry solids of about 10-55%, about 20-45%, about 30-45%, about 30-40%, or, optionally, about 30. -35%. The α-amylases, for example, bacterial alpha-amylases that include Bacillus alpha-amylases, are usually supplied, for example, in approximately 1500 units per kg of dry matter of starch. 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 may be added and may require different optimal conditions. The bacterial • -amylase remaining in the suspension after liquefaction can be deactivated by reducing 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 heated by steam of about 85 ° C to 105 ° C. Gelatinization occurs very rapidly under these conditions, and enzymatic activity, combined with significant shearing forces, begins hydrolysis of the starch substrate. The residence time in the jet cooker is very short. The partially gelatinized starch can be passed in a series of holding tubes held at about 85 to 105 ° C and held for about 5 min. to complete the gelatinization process. These tanks may contain deflectors to discourage backmixing. As used herein, the term "secondary liquefaction" refers to the liquefaction step 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 may be added to the liquefied starch obtained by the above method 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 may be added to a suspension of liquefied starch or maltodextrin at a treatment level in the range of about 0.001 wt% to about 0.01 wt% based on the dissolved solids. In a typical embodiment, a polypeptide defined herein may be added to a suspension of liquefied starch or maltodextrin at a treatment level in a range of about 0.0025 wt% to about 0.01 wt% on the base 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 the product is converted into a continuous reaction. In this embodiment, the polypeptide defined herein can be immobilized with additional enzymes, such as 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 thereof.

Also provided is a method of obtaining 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 variant of the polypeptide defined herein may be added to the starch liquefied in a range of about 0.001 wt% to about 0.1 wt% based on the dissolved solids. In one embodiment, the variant is added to the starch liquefied in a range of about 0.0025 wt% to about 0.01 wt% based on the dissolved solids. The 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 liquified starch solution can be a suspension of liquefied starch at about 20-35% w / w dry solids. Starch can be obtained from maize, corn, 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 thereof, can be added with the variant of the polypeptide 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 on the basis of the total saccharide content.

In another aspect there is provided a method of preparing a saccharide syrup, which includes the addition of a variant of the polypeptide defined herein and an alpha-amylase to the granular starch and the hydrolysis of the granular starch to form the saccharide syrup. In one embodiment, the granular starch liquefied is produced by an alpha-amylase. In one embodiment, the liquefied granular starch is an acid produced liquefied product. In one embodiment the variant of the polypeptide defined herein is added to the granular starch in a range from about 0.001 wt% to about 0.1 wt% based on the dissolved solids. In another embodiment the variant of the polypeptide defined herein is added to the granular starch in a range of about 0.0025 wt% to about 0.01 wt% based on the dissolved solids. The granular starch can be obtained starch from corn, corn, 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 envisaged 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 the granular starch. The enzyme having debranching activity may include but is not limited to an isoamylase, a pullulanase, an isopululanase, a neopululanase or any combination thereof. It is also envisaged that the method may optionally include an additional step of adding a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a pectate lyase or any combination thereof to the granular starch.

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

It is envisaged that the variant of the polypeptide defined in the present method may be immobilized.

In another aspect a method for 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 the starch in the form of a starch liquefied or granular starch and saccharify the starch to form IMO. It is envisaged that the IMO may be formed into an IMO number of at least 30, at least 40 and / or at least 45. In one embodiment the starch is obtained from maize, ears, wheat, barley, rye, millet, sago , cassava, tapioca, sorghum, rice, peas, beans, bananas or potatoes.

In another aspect a method for obtaining IMO is provided, including the addition of a) a variant of the polypeptide, b) an alpha-amylase, and c) a transglucosidase to the starch in the form of a liquefied starch 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 envisaged 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 maize, ears, wheat, barley, rye, millet, sago, cassava, tapioca, sorghum, rice, peas, beans, bananas or potatoes.

4.3. Fermentation processes

The yeast typically of Saccharomyces spp. it is added to the dough and the fermentation continues for 24-96 hours, such as typically 35 to 60 hours. The temperature is between about 26-34 ° C, typically at 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 set at the beginning of the 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 component 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 change constantly until the fermentation stops. Within the batch cultures, the cells progress through a phase of static latency to a logarithmic phase of high growth and finally to a stationary phase, where the rate of growth decreases or stops. If left untreated, cells in the stationary phase eventually die. In general, cells in logarithmic phase are responsible for the mass of production of the product.

One suitable variation in the standard batch system is the "batch-fed" fermentation system.In this variation of a typical batch system, the substrate is added in increments as fermentation progresses. useful when the repression of catabolites probably inhibits the metabolism of the cells and when it is convenient to have limited amounts of substrate in the medium.The measurement of the actual substrate concentration in batch-fed systems is difficult and therefore is estimated on the basis of changes in measurable factors, such as pH, dissolved oxygen and partial pressure of waste gases, such as CO2 Batch and batch-fed fermentations are well known in the art.Continuous fermentation is an open system in the that a continuously defined fermentation medium is added to a bioreactor and an equal amount of conditioned medium is removed simultaneously during processing. Continuous fermentation generally maintains the cultures at a high constant density, where the cells are mainly growing in logarithmic phase. 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 carbon source or nitrogen source, is maintained at a fixed rate and all other parameters are allowed to moderate, in other systems, a number of factors affecting the growth can be altered continuously while the cellular concentration, measured by the average turbidity, remains constant. Continuous systems strive to maintain steady-state growth conditions. Therefore, cell loss due to the medium that is extracted must be balanced with the rate of cell growth in the fermentation. Methods of modulating nutrients and growth factors 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 the ethanol. The ethanol obtained according to the process of the description can be used as, for example, fuel ethanol; ethanol for beverage, that is, potable neutral alcohol or industrial ethanol. The surplus of the fermentation is the grain, which is normally used for animal feed, either in liquid or dry form. Additional details on how to carry out the liquefaction, saccharification, fermentation, distillation, and recovery of ethanol are well known to the 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, which include additives, food products, bakery products, enhancing compositions, food products, including animal feed, 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 providing the compositions.

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

In one aspect, there is provided a variant, as described herein, in which a product treated with the variant has one or more, preferably all of the following properties: (a) lower firmness; (b) superior elasticity; (c) superior cohesion; (d) lower friability; and (e) greater 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, as described herein, is provided in which the elasticity. Cohesion or folding capacity of the food product increases independently 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, as described herein, is provided in which each of elasticity. Cohesion and folding capacity 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, there is provided a variant, as described herein, in which the firmness or friability of the food product decreases independently by 15% or more, preferably 50% or more, most preferably 100% or more, with respect to a food product has been treated with the amino acid sequence of SEQ ID NO: 1.

In one aspect, there is provided a variant, 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, which is incorporated herein by reference.

In one aspect, the treatment of food products, in particular doughs and bakery products with such polypeptides, and so that the food products exhibit the desired qualities set forth above. In one aspect, there is provided a process for treating a starch comprising 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, is provided for preparing a food product or animal feed. In one aspect, there is provided a process for preparing a food product or animal feed comprising mixing a variant, as described herein, with an ingredient for food or animal feed. In one aspect, the use or a process is provided, wherein the food product comprises a dough or dough product, preferably a dough product processed. In one aspect, the use or a process is provided, in which the food product is a bakery product. In one aspect, a process is provided for obtaining a bakery product comprising: (a) providing a starch medium; (b) adding to the starch medium a variant, 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 bakery product obtained by a process defined in the claims.

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

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

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

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

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

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

The variants described herein preferably comprise one or more improved manipulation properties compared to an original polypeptide or a wild-type polypeptide, such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 to 10, preferably ^s E ^q ID NO: 1. the 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 property or preferably a baking property compared to a food product that has been treated with an original polypeptide or a polypeptide such as a wild - type 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 may be selected from 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 analysis of bread slices by analysis of the texture profile using for example a texture analyzer, as for example described in FIG.

The variants described herein are in one aspect so that a food product treated with the polypeptide variant has lower firmness as compared to a food product that has been treated with an original 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.

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

Firmness can be measured using the "Firmness Assessment Protocol" described 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%, 3%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 200% or more lower firmness compared to a food product that has been treated with an original 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. a food product treated with 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 an original 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 N ^o : 1.

Elasticity

In one aspect, the variants described herein are such that a food product treated with the polypeptide variant has a superior elasticity compared to a food product that has been treated with an original 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 N ^o : 1.

The elasticity can be measured by means of the "Elasticity evaluation protocol" described in 11.

Accordingly 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 of superior elasticity compared to a food product that has been treated with an original 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 of superior elasticity compared to a food product that has been treated with an original 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 ⁱ DN ^or : 1.

Cohesion

In one aspect, the variants described herein are such that a food product treated with the polypeptide variant has superior cohesion as compared to a food product that has been treated with an original 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 described herein are such that a food product treated with the variant of the polypeptide described herein has 10%, 20%, 30%, 40%, 50%, 60%, 70% 80%, 90%, 100%, 200% or more of superior cohesion compared to a food product that has been treated with an original 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 i D 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 superior cohesion compared to a food product that has been treated with an original 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 as compared to a food product that has been treated with an original 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 N ^o : 1.

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

Accordingly 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 of inferior friability compared to a food product that has been treated with an original 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 of inferior friability compared to a food product that has been treated with an original 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 N ^o : 1.

Folding capacity

In one aspect, the variants described herein are such that a food product treated with the polypeptide variant has a greater folding ability compared to a food product that has been treated with an original 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.

The folding capacity is preferably measured by the "Folding Capacity Evaluation Protocol" set forth in Example 14.

Accordingly, 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 greater folding capacity compared to a food product that has been treated with an original 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 of greater folding capacity compared to a food product that has been treated with an original 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 N ^o : 1.

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

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

Variants of polypeptides can be used to improve the volume of bakery products such as bread. Without wishing to be bound by any particular theory, it is considered that this comes from the reduction of viscosity of the dough during heating (such as baking) as a result of the amylase that shortens the amylose molecules. This allows the carbon dioxide generated by the fermentation to increase the size of the bread with fewer impediments.

Accordingly, the food products comprising or treating the polypeptide variants expand in volume when compared to products that have not been treated in this way, or treated with the original polypeptides such as a polypeptide of SEQ ID NO: 1 or SEQ ID NOs: 2, 4, 6, 8 or 10, preferably S ^e Q ⁱ D NO: 1. In other words, the food products have a larger air volume by volume of the 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, the polypeptide variants are used to improve bread volume. The improvement or expansion of volume is beneficial because it reduces the gumminess or the starch content of the food. Light foods are preferred by consumers, and the customer experience is increased. 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 may be used as -o 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 the prepared food as well as an ingredient for a food, such as a flour. In a preferred aspect, the food is for human consumption. The food may 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. Variants of polypeptides and nucleic acids 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 may 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 may be - or may 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.

Variants of polypeptides can also be used in the manufacture of a food product or a foodstuff. Typical foods include dairy products, meat products, poultry products, fish products and dough products. The dough product can be any processed dough product, which includes fried doughs, fried with oil, roasted, baked, steamed and boiled, 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 bakery products (baked) include bread - such as breads, rolls, buns, donuts, pizza bases, etc. pastries, pretzels, tortillas, cakes, sweet cookies, biscuits, salty cookies, etc.

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

In addition, in one aspect, there is provided a method for modifying a food additive comprising a non-maltogenic exoamylase, the method comprising introducing a mutation into one or more of the non-maltogenic exoamylase positions 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, there is provided the use of polypeptide variants that are capable of retarding the slump of starch media, such as starch gels. The polypeptide variants are especially capable of retarding the damaging retrogradation of the 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 aD-glucopyranosyl units linked by linkages (1-4), wherein said chains are linked by 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 linear chain of linked aD-glucopyranosyl (1-4) units that have few branches aD- (1-6). Most starches contain approximately 25% amylose.

The 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. The gelatinization temperatures vary for different starches. After cooling the freshly baked bread the amylose fraction, within hours, retrograded to develop a network. This process is beneficial because it creates a desirable crumb structure with a low degree of firmness and better slicing properties. The more gradual crystallization of amylopectin takes place within the gelatinized starch granules during the days after baking. In this process, amylopectin is considered to reinforce the amylose network in which the starch granules are embedded. This reinforcement leads to an increase in the firmness of the bread crumb. This reinforcement is one of the main causes of bread aging.

It is known that the quality of baked goods deteriorates gradually during storage. As a consequence of starch recrystallization (also called retrogradation), the water retention capacity of the crumb is changed with important implications on the 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 bread aging process.

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

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

In one aspect, there is provided a method for improving the ability of a non-maltogenic exoamylase to prevent aging, preferably the damaging retrogradation, of a dough product, the method comprising the introduction of a mutation in any or several of the positions of non-maltogenic exoamylase exposed in this document.

For the evaluation of the antiaging inhibitory effect of the polypeptide variants, such 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 of food texture Universal Instron 4301 or similar equipment known in the art.

Another method traditionally used in the art and used to evaluate the effect on retrogradation of the starch of a polypeptide variant such as a variant having exoamylase activity is based on DSC (differential scanning calorimetry). At present, the enthalpy of fusion of the retrograde amylopectin in bread crumb or crumb of a mass of the model system of baked dough with or without enzymes (control) is measured. The DSC equipment applied can be a run of DSC 820 Mettler-Toledo 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.

The masses of the model system may contain standard wheat flour and optimum amounts of water or buffer with or without the polypeptide variant. They are mixed in a Brabender 10 or 50 g farinograph for 6 or 7 min, respectively. The samples of the masses are placed in glass test tubes (15 * 0.8 cm) with a lid. These test tubes are subjected to a baking process in a water bath starting with 30 min. incubation at 33 ° C followed by heating 33 at 95 ° C with a gradient of 1.1 ° C per minute. and, finally, 5 min. incubation at 95 ° C. Subsequently, the tubes are stored in a thermostat at 20 ° C before the DSC analysis.

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

In one aspect, the use of polypeptide variants is provided in the preparation of food products, in particular, starch products. The method comprises forming the starch product by the addition of 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, non-maltogenic exoamylase which is a variant of polypeptide described herein and optionally other possible ingredients and additives. The term "starch" should 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 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 of finely ground wheat flour or other grain. 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 means references to wheat flour per se, as well as to wheat flour when present in a medium , such as a mass.

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. More 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 fermenting agent under dough forming conditions. Obviously other components can be added to the dough mixture.

Accordingly, 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 mass forming conditions and further addition of a polypeptide variant described herein, optionally in the form of a premix. The leavening agent may be a chemical leavening agent such as sodium bicarbonate or any strain of Saccharomyces cerevisiae (Baker's yeast).

The polypeptide variant described herein may be added together with any dough ingredient including the water or dough mixture of ingredients or with any additive or mixture of additives. The dough can be prepared by any conventional dough preparation method common in the bakery industry or in any other flour dough manufacturing industry.

Baking of farinaceous bakery products such as for example white bread, bread made of rye flour and sifted wheat flour, bagels and the like is normally achieved by baking 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 (200-120 ° C) is prevalent in the outer layers of dough where the characteristic crust of the baked product develops. However, because of the heat consumption due to steam generation, the temperature in the crumb is only about 100 ° C at the end of the baking process.

Also provided is a process for obtaining a bread product comprising: (a) providing a starch medium; (b) adding the starch medium a polypeptide variant 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 comprising adding a variant of described polypeptide to a starch medium is provided.

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

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

In one aspect, there is provided an improving composition for a dough, wherein the improving composition comprises a variant disclosed in any of the claims, and at least one ingredient of the dough or additive of additional dough.

In one aspect, a composition comprising a flour and an exposed variant is provided in any of the claims.

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

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

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

The dough-improving composition may be added as a liquid preparation or in the form of a dry powder composition comprising the composition as the sole 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 the presence in the finished dough occurs from 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 enzyme based on the flour (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 μm or reducing sugar per min when incubated at 50 degrees C in a test tube with 4 ml of 10 mg of 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, corn starch, corn flour, rice flour, rye flour. , rye groats, oatmeal, oatmeal, soy flour, sorghum semolina, sorghum flour, potato semolina, potato flour or potato starch. The dough can be fresh, frozen, or partially baked.

The dough can be a leavened dough or a dough that must be leavening. The dough can be leached in several ways, such as by the addition of chemical leavening agents, for example, sodium bicarbonate or by the addition of a leavening (fermentation dough), but it is preferred to leave 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 fat or granulated butter. The dough may further comprise an additional emulsifier such as mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, esters of lactic acid of monoglycerides, esters of acetic acid of monoglycerides, stearates of polyoxyethylene, or lysolecithin.

A premix comprising flour is also provided together with the combination described herein. The premix may contain dough-improving additives and / or bread improving 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, ingredients of the dough and / or additional dough additives may 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 milk powder, gluten soy or eggs and dough additives such as emulsifiers, other enzymes, hydrocolloids, flavoring agents, oxidizing agents, minerals and vitamins.

Emulsifiers are useful as dough reinforcers and crumb softeners. As dough reinforcers, the emulsifiers can provide tolerance with respect to the resting time and shock tolerance during fermentation. In addition, dough reinforcers will improve the tolerance of a given mass to variations in fermentation time. Most dough reinforcers also improve foaming which means increasing the volume of fermented baked products. Finally, the dough reinforcers will emulsify all the fats present in the recipe mixture.

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

The additive or ingredient of the additional dough can be added together with any dough ingredients that include flour, water or other optional ingredients or additives, or the dough-improving composition. The additive or additional dough ingredient can be added before the flour, water, other ingredients and optional additives or the dough-improving composition. The additional dough additive or ingredient can be added after the flour, water, other ingredients and optional additives or the dough improving composition.

The additional dough additive or 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 additional dough additive or ingredient is at least 1% of the weight of the flour component of the dough. In a further aspect, the additional dough additive or 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 use and domestic use of flour to bake and produce food, it is important to maintain an adequate level of • -amylase activity in the flour. A level of activity that is too high can produce a product that is sticky and / or pasty and therefore not marketable. Flour with insufficient • amylase activity may not contain enough sugar for proper yeast function, resulting in friable, dry bread or baked goods. Accordingly, a variant, as described herein, by itself or in combination with another α-amylase or other variant, can be added to the flour 10 increasing the activity level of endogenous α-amylase 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 solution of 1% 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 can be malted, that is, partially germinated, which produces an increase in enzyme levels, which include • -amylase. For successful brewing, adequate levels of enzyme-amylase activity are required to ensure appropriate levels of sugars for fermentation. A variant, by itself or in combination with another • -amylase, can therefore be added to the components used to brew beer.

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

As used herein, the term "supplies" means grains and components of the plant that are crushed or broken. For example, the barley used in the production of beer is a grain that has been ground or coarsely ground to obtain an appropriate consistency to produce a paste for fermentation. As used herein, the term "supplies" includes any of the above-mentioned types of plants and grains in crushed or ground forms into large pieces. The methods described herein can be used to determine the 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 for example can be added to the food, preparation of the dough, 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 lafa 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. Or it can be a plant beta-amylase (EC 3.2.1.2), for example barley malt beta-amylase or soy bean or it can be an exoamylase used for anti-aging, for example non-maltogenic exo-amylase (EC 3.2.1.60) developed from maltotetrahydrolase from Pseudomonas saccharophila (SEQ ID NO: 10) or maltogenic amylase (EC 3.2.1.133) developed from Bacillus stearothermophilus which is described further below.

In one aspect, the additional amylase is a maltogenic alpha-amylase. A "maltogenic alpha-amylase" (glucan 1,4-alpha-maltohydrolase, EC 3.2.1.133) is capable of hydrolyzing amylose and amylopectin to maltose in the alpha configuration. A Bacillus maltogenic alpha-amylase (EP 120 693) is commercially available under the tradename Novamyl (Novozymes, Denmark) and is widely used in the bakery industry as an anti-aging agent due to its ability to reduce retrogradation of starch. 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), which include 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 can comprise in particular 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 US Patent No. 4,598,048 and US Patent 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 maltotetrahydrolase. saccharophila 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 in relation to a control without enzyme. 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. The 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, homologue, 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 degrading starch, proteases and lipoxygenases. In one embodiment, the composition comprises at least one variant of polypeptide described herein and a maltogenic amylase of Bacillus, described in WO91 / 04669. One embodiment comprises a variant polypeptide 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 the strengthening of the dough and volume control of the baked goods and xylanases and other hemicellulases dough can be added to improve the handling properties of the dough, firmness of the crumb and the volume of the bread. Lipases are useful as dough softeners and crumb softeners and a-amylases and other amylolytic enzymes can be incorporated into the dough to control bread volume and further reduce firmness of the crumb.

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

A variant, as defined herein, may also be added alone or in combination with other amylases, which include other amylase variants to prevent or retard aging, i.e., firmness of the crumb of the baked products. The amount of anti-aging amylase will normally be in the range of 0.01-10 mg of enzyme protein per kg of flour, for example, 0.5 mg / kg ds. Additional anti-aging amylases can be used in combination with a polypeptide variant described herein include an endo-amylase, for example, a Bacillus bacterial endo-amylase. The additional amylase may be a maltogenic • -amylase (EC 3.2.1.133), for example, from Bacillus. Novamil® is an example of maltogenic • -amylase of 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 bacterial -amylases derived from Bacillus, such as B. licheniformis or B. amiloliquefaciens. The anti-aging amylase can be an exo-amylase, such as • -amylase, for example, from plant sources, such as soybean or from microbial sources, such as Bacillus.

The baking composition comprising a polypeptide variant described herein may further comprise a phospholipase. The phospholipase may have A1 or A2 activity to remove the fatty acid from the phospholipids, which forms a lysophospholipid. It may or may not have lipase activity, that is, activity on triglyceride substrates. Phospholipase typically has an optimum temperature in the range of 30-90 ° C, for example, 30-70 ° C. The added phospholipases can be of animal origin, for example, from pancreas, for example, bovine or porcine pancreas, snake venom or bee venom. Alternatively, the phospholipase can be of microbial origin, for example, 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. tarda; 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 (for example, strain DSM 2672).

The phospholipase is added in an amount that improves the softness of the bread during the initial period after baking, particularly 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. The activity of the phospholipase will generally be in the range of approximately 20 to 1000 Lipase Unit (LU) / kg of flour, where one unit of lipase 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, corn starch, corn flour, rice flour, rye flour, rye semolina, oatmeal, oatmeal, soy flour, sorghum semolina, sorghum flour, potato semolina, potato flour or potato starch. The dough can be fresh, frozen, or partially baked. The dough can be a leavened dough or a dough that must be leavening. The dough can be leached in several ways, such as by the addition of chemical leavening agents, for example, sodium bicarbonate or by the addition of a leavening agent, i.e., fermentation dough. The dough can also be leaved 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 ingredients of the dough, e.g., proteins, such as milk powder, 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 fat or granulated butter. 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 esters of fatty acids, esters of lactic acid of monoglycerides, esters of acetic acid 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 together with the anti-aging amylase and the 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 disulfide isomerase protein, for example, a disulfide isomerase protein, as described in WO 95/00636, for example, a glycosyltransferase, a branching enzyme (branching enzyme 1, 4-γ-glucan), a 4-γ-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 normally of microbial origin, for example, derived from a bacterium or fungus, such as an Aspergillus strain, in particular from A. aculeatus, A. niger (see WO 91/19782), A. awamori (for example , WO 91/18977), or A. tubigensis (for example, WO 92/01793); of a strain of Trichoderma, for example, T. reesei, or of a strain of Humicola, for example, H. insolens (for example, 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 Thermomyces lanuginosus (Humicola lanuginosa), Rhizomucor miehei, Candida antarctica, Aspergillus niger, Rhizopus delemar or Rhizopus. arrhizus, or Pseudomonas cepacia. In the form of 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 EP 214,761 and WO 89/01032, for example.

The process can be used for any type of baked product prepared from dough, either of a soft or crispy character, whether white, light or dark. Examples are bread, in particular white, wholemeal or rye bread, usually in the form of breads or rolls, such as, but not limited to, French baguette bread, pita bread, tortillas, cakes, pancakes, biscuits, cookies, broken 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-improving additives and / or bread improvers, for example, any of the additives, which include 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 | jm. Granules and agglomerated powders can be prepared by conventional methods, for example, by spraying the polypeptide described herein on a carrier in a fluid bed granulator. The carrier can consist of particle cores 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 wrapping of particles comprising a variant polypeptide described herein, ie, particles of • -amylase. To prepare the wrapped amylase particles, the enzyme is contacted with a food-grade lipid in sufficient quantity to suspend all of the α-amylase particles. Food grade lipids, as used herein, can be any natural organic compound, which is insoluble in water but is soluble in non-polar 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 which comprise the 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 which comprise the 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.

The food grade lipid, in particular in liquid form, is contacted with a powder form of the α-amylase particles in such a way that the lipid material covers at least a portion of the surface of at least a majority , for example, 100% of the particles of • -amylase. In this way, each particle of • -amylase is individually wrapped in a lipid. For example, all or substantially all of the α-amylase particles are provided with a thin and continuous 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 wet the surface of each particle of • -amylase. After a short period of agitation, the wrapped γ-amylase particles, which carry a substantial amount of the lipids on their surfaces, are recovered. The thickness of the coating thus applied to the α-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.

The storage, handling and incorporation of the loaded administration vehicle can be achieved by means of a packaged mixture. The packaged mixture may comprise the packaged • amylase. However, the packaging mix may also contain additional ingredients as required by the manufacturer or the baker. After the packaged amylase has been incorporated into the dough, the baker continues through the normal production process of said product.

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

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

The loaded delivery vehicle, i.e., lipid-coated enzyme, is added to the ingredients used to prepare a baked product in an amount effective to extend the shelf life of the baked product. The baker calculates the amount of • -amylase with wrap, prepared as described above, which will be required to achieve the desired anti-aging effect. The amount of shell-bound • -amylase required is calculated on the basis of the concentration of the coated enzyme in the ratio of • -amylase to the specified flour. It has been found that a wide range of concentrations is effective, although, as has been discussed, the observable improvements in anti-aging do not correspond linearly with the concentration of • -amylase, but above certain minimum levels, large increases in the concentration of • -amylase produce little further improvement. 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 unintentional measuring errors by the baker. The lower limit of concentration of the enzyme is determined by the minimum anti-aging effect that the baker wishes to achieve.

A method for preparing a baked product may comprise: (a) preparing lipid-coated amylase particles, wherein substantially all of the α-amylase particles are coated; (b) mixing a dough containing flour; (c) adding the lipid-coated α-amylase particles before the mixture is complete and finishing the mixture before removing the lipid layer of the α-amylase; (d) ferment the dough; and (e) bake the dough to provide the baked product, in which the • -amylase is inactive during the mixing, fermentation and baking stages and is active in the baked product.

The coated -amylase can be added to the dough during the mixing cycle, for example, near the end of the mixing cycle. The • -amylase with wrapping is added at a point in the mixing stage that allows for the sufficient distribution of the • -amylase with wrapping throughout the mass; however, the mixing step is terminated before the protective layer is detached from the α-amylase particle. According to the type and volume of the dough, and the action and speed of the mixer, it may be required at any time from one to six minutes or more to mix the • -amylase with casing in the dough, but two to four minutes is the average. Therefore, several variables can accurately determine the procedure. First, the amount of coated • -amylase should have a sufficient total volume to allow the coated • -amylase to spread throughout the mass mixture. If the packaging preparation of • -amylase is highly concentrated, it may be necessary to add oil to the premix before adding the packaged • -amylase to the dough. The recipes and production processes may require specific modifications; however, good results generally can be achieved when 25% of the oil specified in a bread dough formula is kept out of the dough and used as a carrier for a concentrated amylase with concentrate wrap when added of the end of the mixing cycle. In bread or other baked goods, especially those with a low fat content, for example, French-type breads, a mixture of • -amylase with about 1% by weight of dry flour wrap is sufficient to mix the • - amylase with proper packing with the dough. The range of suitable percentages is broad and depends on the formula, finished product and the requirements of the individual baker's production methodology. Second, the suspension of the packaged • -amylase should be added to the mixture with sufficient time for the complete mixture in the mass, but not for a time such that excessive mechanical action releases the protective lipid layer of the particles of • -amylase with wrapping.

6. Compositions for textile desizing and use

Also contemplated are compositions and methods of treating fabrics (e.g., to peel a tissue) using a polypeptide variant described herein. In one aspect, a method of decoiling tissue is provided, comprising contacting the polypeptide variant described herein with a tissue for a sufficient time to de-tissue 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 tissue weaving, or during the desizing stage, or one or more additional tissue processing steps. During weaving of the textile material, the yarns are exposed to considerable mechanical stress. Before weaving on mechanical looms, the 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 may be applied during or after the tissue to remove these starch or starch derivatives. After weaving, a polypeptide variant described herein can be used to remove ACPA from sizing prior to further processing the fabric to ensure a homogeneous and waterproof result.

The polypeptide variant described herein can be used alone or with other chemical desizing reagents and / or desizing enzymes to de-weave fabrics, including cotton-containing fabrics, as detergent additives, for example, in aqueous compositions. A polypeptide variant described herein may also be used in compositions and methods to produce a stone-washed appearance on the fabric and denim-dyed garments. For the manufacture of clothing, the fabric can be cut and sewed clothing or clothing, which then undergo the finish. In particular, for the manufacture of jeans, different methods of enzymatic finishing have been developed. The finishing of denim garments usually begins with an enzymatic desizing step, during which the garments are subjected to the action of amylolitic enzymes to provide softness to the fabric and make the cotton more accessible to the subsequent enzymatic finishing stages. A variant of the polypeptide described herein can be used in denim garment finishing methods (e.g., a "biological pre-wash process"), enzymatic desizing and provision of 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 open reading frame PS4 (Zhou et al., 1989) was amplified from chromosomal DNA of P. sacharophila by PCR using primers P1 and P2 (see Table 3). The resulting fragment was used as a template in a PCR nested with primers P3 and P4, amplifying the open reading frame of PS4 without its signal sequence and introducing an Ncol site at the 5 'end of the gene and a BamHI site at the 3 'end. Together with the NcoI site, a codon was introduced for an N-terminal methionine, allowing the intracellular expression of PS4. The 1605 bp fragment was cloned into pCRBLUNTTOPO (Invitrogen) and the integrity of the construct was analyzed by sequencing. The Bacillus E.coli transport vector pDP66K (Penninga et al., 1996) was modified to allow the expression of PS4 under the control of the P32 promoter and the ctgasa signal sequence. The resulting plasmid, pCSmta is transformed into B. subtilis.

A second expression construct was prepared in which the starch ligation 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 into a Bacillus expression vector with the signal sequence by gene synthesis (GeneScript; NJ, USA) of the sequences encoding the 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 in 3 ml of LB (22 g / l of 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 (QIAGEN) columns. 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.

Next, a PCR was performed to synthesize the mutant strand. The PCR reaction mixture contained the following:

2.5 • l 10 X QuickChange multi-reaction buffer

0.75 • l QuickSolution

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

1 • I mix dNTP

X • l ds-dnae template (200 ng)

1 • l QuickChange multienzyme mixture (2.5 U / ^ l) (PfuTurbo DNA polymerase)

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

The PCR reaction was performed in an Eppendorf thermal cycler for 35 cycles of denaturation (96 ° C for 1 min), annealing of primers (62.8 ° C for 1 min), and elongation (65 ° C for 15 min), and then it was maintained at 4 ° C. For each amplification reaction, 2 • l of restriction enzyme DpnI (10 U / l) was added, and the mixture was incubated at 37 ° C for ~ 3 h.

DNA treated with Dpnl was then used to transform ultracompetent XL10-Gold® cells (Stratagene). The XL10-Gold® cells were thawed on ice. For each mutagenesis reaction, 45 cells were added to a pre-cooled Falcon tube. Subsequently, 2 • l of beta-mercaptoethanol mixture was added to each tube. The mixture was incubated on ice for 10 min with spinning every 2 min. Then 1.50 • l 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 of 30 sec at 42 ° C, and placed on ice for another 2 min. 0.5 ml of pre-warmed NZY + broth was added to each sample and incubation was carried out at 37 ° C for 1 h with agitation at 225-250 rpm. 200 • l of each transformation reaction was plated on plates with LB (33.6 g / l Lennox L Agar, Sigma) supplemented with 1% starch and 50 • g / 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 in Bacillus subtilis.

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

A. Means 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 (0.04 g)

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

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

NaCl)

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. Means 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

MgCl 2 20.3 g of MgC12'6H2O per 100 ml (1 M).

Casamino acids 5% solution (w / 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.

Regeneration medium DM3: mix at 60 ° C (water bath, 500 ml bottle):

250 ml of sodium succinate

50 ml of casamino acids

25 ml of yeast extract

50 ml of phosphate buffer

15 ml glucose

10 ml of MgCl2

100 ml melted agar

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

C. Preparation of protoplasts

Use plastic or glass containers free of detergent.

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

Dilute overnight culture 20 times in 100 ml of 2 x fresh YT medium (250 ml flask) and culture to OD600 = 0.4-0.5 (approx 2 hs) 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, filtered in sterile form.

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 by the microscope. Continue incubation until 99% of the cells are formed as protoplasts (globular aspect). Harvest the protoplasts by centrifugation (4000 g, 20 min, RT) and remove the supernatant with a pipette. Re-suspend the pellet gently 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 (glycerol addition is not required). Although this may result in a certain reduction in transformation capacity, 106 transformants can be obtained per ug of DNA with frozen protoplasts). D. Transformation

Transfer 450 • l of PEG to a microtube.

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

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

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

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

Example 4. Fermentation of variants in shake flasks.

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

Yeast extract 2% (w / v)

Soy flour 2% (w / v)

NaCl 0.5% (w / v)

Dipotassium phosphate 0.5% (w / v)

Antifoaming agent 0.05% (w / v)

The substrate was adjusted to pH 6.8 with 4 N sulfuric acid or sodium hydroxide before being autoclaved. 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. The dextrose syrup is prepared by mixing a volume of 50% w / v dextrose with a volume of water followed by autoclaving for 20 minutes.

The 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 ^ m. The cell-free supernatant is used for assays and application tests.

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 test

A Betamyl unit is defined as the activity that degrades 0.0351 mmoles per 1 min of maltopentaose coupled with PNP in such a way that 0.0351 mmoles of PNP for 1 min can be released by excess of alpha-glucosidase in the test mixture. The assay mixture contains 50 ul of 50 nM Na citrate, 5 mM CaCl 2, pH 6.5 with 25 ul of enzyme sample and 25 ul of substrate Betamyl (Glc5-PNP and alpha-glucosidase) from Megazyme, Ireland ( 1 vial dissolved in 10 ml of water). The test mixture was incubated for 30 min at 40 ° C and then stopped by adding 150 ul of 4% Tris. The absorbance at 420 nm was measured using an ELISA reader and the activity of Betamyl 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 activity and Phadebas activity was used to evaluate exo-specificity. Example 6. Determinations of thermal stability 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 the control sample and the heated sample were analyzed using the test Betamyl. 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 mutations Q42K, R88L, S205L, E223S, Q311P, S409E and T235K are shown in Table 2 as an increase in 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 the one mentioned in the first line except for the one or more subsequent mutations 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 trials

The baking trials were carried out with a sponge recipe (preferment) and standard white bread dough for US toast bread. The sponge mass 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 emulsifier DIMODAN ™ PH300, 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 are added to the sponge. 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 left to rest for 5 min at room temperature, and then pieces of dough of 794 g are weighed, molded in a cross grain moulder and transferred to containers. After 60 min of fermentation at 43 ° C to 80% RH the doughs are baked for 21 min at 200 ° C in a Reed tray oven.

Example 11. Protocol for the assessment of firmness, strength and cohesiveness

Analysis of bread texture profile

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

The 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. The firmness is expressed, for example, in grams.

Example 12. Evaluation of anti-rancid effects of G4 amylase variant 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 results of the firmness. With respect 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 of day 3 per day is obtained 10 after baking with pMS1776, pMS1934, pMS2020, pMS2022 and pMS2062. These breads had also improved a lot in terms of resistance and cohesiveness in relation to the controls.

This indicates that the variant polypeptides, as described herein, greatly improved the anti-freezing 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 made by crumbling the crumb with your fingers. First the slice is separated from the center of the upper surface of the bread at the center of the bottom surface of the bread. Then, each half of the original slice is separated from the side of the rind inside the slice. The small crumb pieces, which are separated from the 4 squares, are removed by shaking each piece at least 3 times by moving the hand up and down.

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

Example 14. Protocol for the evaluation of folding capacity

Toast bread 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, so that the direction of the fold is from side to side.

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

This test can be called "Folding capacity evaluation protocol".

Example 15. Preparation of DP4 syrup

Materials:

SPEZYME FRED® liquefied granular corn starch (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 liquor was adjusted to 5.2 with NaOH and then every 100 g of liquefied was incubated at 60 ° C during saccharification with DP4 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 a DP4 as a main sugar product. As also indicated, the addition of pullulanase (PU) (PMS2062 PU) resulted in a more rapid reduction of the higher sugars (see, for example, DP11 +), while the level of DP4 was minimally affected.

Sequences

SEQ ID NO: 1. Sequence of mature proteins of pMS382

DQAGKSPAGVRYHGGDEIILQGFHWNVVREAPYNWY4IILRQQASTIAADGFSAIWMPVPWRDFSSWTDGDKSGGGEGYFWHDF NKHGRYGSDAQLRQAAGALGGAGVKVLYDWPNHMNRFYPDKEINLPAGQRFWRNDCPDFGNGPNDCDDGDRFLGGEADLNTG HPQIYGMFRDEFTNLRSGYGAGGFRFDFVRGYAPERVDSWMSDSADSSFCVGELWKEPSEYPPWDWRNTASWQQIIKDWSDRA KCFVFDFALKERMQNGSVADWKHGLNGNPDPRWREVAVTFVDNHDTGYSPGQHGGQHKWPLQDGLIRQAYAYILTSPGTPWY WPHMYDWGYGDFIRQLIQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLWALNSDLANPGQVASGSFSEAVHASNGQVRV WRSGSGDGGGHDGG

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

1 MDLAGKSPGG VRYHGGDEII LQGFHWNVIR ESPNNWYNTL RDMAPTIAAD

51 GFSAIWMPVP WRDFSSW5DG ANSGGGEGYF WHDFNKNGRY G5DTQLKQAA

101 GALNNAQVKV LYDWF14HMII RGYPDKQIML PAGQGFWRMD CADPGNYPND

151 CDDGDRFMGG DADLNTANPQ VYGMFRDEFA NLRSNYGAGG FRFDFVRGYA

201 GERVDSWMGA AHDMAFCVGE LWKAPAEYFS WDWRNTASWQ QV1KDWSDRA

251 KCFVFDFALK ERMQNGSIAD WKNGLWGNFD PRWREVAVTF VDNHDAGYSP

301 GQNGGQHHWA LQDGLIRQAY AYILTSPGTP WYWSHMYDW GYGDFIRQLI

351 QVRRTAGVRA DSAISFHSGY SGLVATVSGS QQTLWALNS DLAMPGQVAS

401 GSFSEAVNAS WGQVRVWRSG SGDGGGNDGG EGGLVNVNFR CDNGVTQMGD

451 SVYAVGNVSQ LGNWSPASAV RLTDTSSYPT WKGSIALPDG QWVEWKCLIR

501 NEADATLVRQ WQSGGNMQVQ AAAGASTSGS F *

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

atggatctggcaggc & aatcacegggcggcgttagatatcatggcggcgatgaaattattctgca & ggctttcattggaatgt tattagagaatcaccgaataattggtataatacactgagagatatggcaccgacaattgcagcagatggcttttcagcaattt gga.tgccggttccgtggagagatttttcatcatggtcagatggcgcaaattcaggcggcggcgaaggctatttttggcatgat tttaataaaaatggcagatatggctcagatacacaactgaaacaagcagcaggcgcactgaataatgcacaagttaaagttct atgattgcgcagatccgggcaattatccgaatgattgcgatgatggcgatagatttatgggcggcgatgctgatctgaataca gtatgatgttgttccgaatcatatgaatagaggctatccggataaacaaattaatctgccggcaggccaaggcttttggagaa gcaaa tc cgcaag TTTA tg tg gca TTTA gaga tg aa TTTG caaa tc tg aga tc aaa tta tg gcgcaggcggc TTTA ga TTTG to ttttgttagaggctatgcaggcgaaagagttgattcatggatgggcgcagcacatgataatgcattttgcgttggcgaactgt gg aaa gcaccggcagaa ta TCCG tca ttgga tggga gaaa tacagca tca tggcaacaag tta tta AAGA TTGG tcaga taga g ca aaa tg cccgg tttttg a ttttg cac tg aaagaaagaa tg caaaa tg gc tc aa ttg caga ttg gaaaaa tg gcc tg aa tg g caatccggatccgagatggagagaagttgcagttacatttgttgataatcatgatgcaggctattcaccgggccaaaatggcg gccaaca tc to ttg gcc tg caaga ggcac tg tg tg to gacaagca tta ta ca ta ta tc aca ttc tg tt accgggcacaccgg ttg tattggtcacatatgtatgattggggctatggcgattttattagacaactgattcaagttagaagaacagcaggcgttagagc agattcagcaatttcatttcattcaggctattcaggcctggttgcaacagtttcaggctcacaacaaacactggttgttgcac tga.atagcga.tctggcaaatccgggccaagttgcatcaggctcattttcagaagcagttaatgcatcaaatggccaagttaga tggcgttacacaaatgggcgattcagtttatgcagttggcaatgtttcacaactgggcaattggtcaccggcatcagcagtta gtttggagatcaggctcaggcgatggcggcggcaatgatggcggcgaaggcggcctggttaatgttaattttagatgcgataa gactgacagatacatcatcatatccgacatggaaaggctcaattgcactgccggatggccaaaatgttgaatggaaatgcctg to ttagaaa tgcaacactgg tgaagcaga ttagacaa TGGCAA tcaggcggcaa taa tc ttcaagcagcagcaggcgca tcaag aacatcaggctcattttaa

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

1 MDLAGK5PGG VRYHGGDEII LQGFHWNVIR ESPNNWYHTL RDMAPTIAAD

51 GFSAIWMPVP WRDFSSWSDG ANSGGGEGYF WHDFNKHGRY GSDTQLKQAA

101 GALNNAQVKV LYDAVPNHMN RGYPDKQINL PAGQGFWRMD CADPGNYPND

151 CDDGDRFMGG DADLNTANPQ VYGMFRDEFA NLRSNYGAGG FRFDFVRGYA

201 GERVDSWMGD GACQRLLRGR ALEGTGRIPE LGLAQYGQLA AVIKDWSDRA

251 KVPGCSNFAL KGAHAERLPS PTGRTASTAT PMPRWREVAV TFVDNHDTGY

301 SPGQNGGQHH WALRDDLVRQ AYAYILASPG TPVVYHSHMY DHGHGPLIRQ

351 LIQIRRAAGV RADSAIEFHS GYSGLVATVR GTAQTLVMAL GSWLSSPAEV

401 SSGSFSQALN QDSGQLRIWT TGSIGGDEGD GGGDGTMVSV NFRCD14GITQ

451 PGDSVYAVGS LAQLGSWSPA MAVRLTDVSN YPTWKGAISL PAGQAVEWKC

501 IVRSEADPTQ VRQWQAGDNI4 RVTAGAGATT IGRL *

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

atggatctggcaggcaaateaccgggcggcgttagatatcatggcggcgatgaaattattctgcaaggctttcattggaatgt tattagagaatcaccgaataattggtataatacactgagagatatggcaccgacaattgcagcagatggcttttcagcaattt ggatgccggttccgtggagagatttttcatcatggtcagatggcgcaaattcaggcggcggcgaaggctatttttggcatgat tttaataaaaatggcagatatggctcagatacacaactgaaacaagcagcaggcgcactgaataatgcacaagttaaagttct gtatgatgcagttccgaatcatatgaatagaggctatccggataaacaaattaatctgccggcaggccaaggcttttggagaa atgattgcgcagatccgggcaattatccgaatgattgcgatgatggcgatagatttatgggcggcgatgctgatctgaataca gcaaatccgcaagtttatggcatgtttagagatgaatttgcaaatctgagatcaaattatggcgcaggcggctttagatttga ttttgttagaggctatgcaggcgaaagagttgattcatggatgggcgatggcgcatgccaaagactgctgagaggcagagcac tggaaggcacaggcagaattccggaactgggcctggcacaatatggccaactggcagcagttattaaagattggtcagataga gcaaaagttccgggctgctcaaattttgcactgaaaggcgcacatgcagaaagactgccgtcaccgacaggcagaacagcatc aacagcaacaccgatgccgagatggagagaagttgcagttacatttgttgataatcatgatacaggctattcaccgggccaaa atggcggccaacatcattgggcactgagagatgatctggttagacaagcatatgcatatattctggcatcaccggg cacaccg gttgtttattggtcacatatgtatgattggggtcatggaccgetgattagacaactgattcaaattagaagagcagcaggcgt tagagcagattcagcaattgaatttcattcaggctattcaggcctggttgcaacagttagaggcacagcacaaacactggtta tggcactgggctcaaatctgtcatcaecggcagaagtttcatcaggctcattttcacaagcactgaatcaagattcaggccaa ctgagaatttggacaacaggctcaacaggcggcgatgaaggcgatggcggcggcgatggcacaatggtttcagttaattttag atgcgataatggcattacacaaccgggcgattcagtttatgcagttggctcactggcacaactgggctcatggtcaccggcaa atgcagttagactgacagatgtttcaaattatccgacatggaaaggcgcaatttcactgccggcaggccaagcagttgaatgg aaatgcattgttagatcagaagcagatccgacacaagttagacaatggcaagcaggcgataataatagagttacagcaggcgc aggcgcaacaacaattggcagactgtaa

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

1 MDAPGKTASG VRYHGGDEII LQGFHWNTVR TSSNWYATLA SMAPTLAADG

51 FSAIWMPVPW RDFSSWSDPG HGTSGGGEGY FWHDFNKNGR YGSDSLLRQA

101 ASALNAAGVK PIYDVVPNHM NRGYPDKEIN LPAGQGLWRH DCNDPGNYAN

151 DCDDGDRFMG GDADLNTGHP QNYAMFRDEF ARLRSQYGAG GFRFDFVRGY

201 AGERVASWMS DAHDMGFCLG ELWKAPGEYP SWDWRNGASW QQILKDWSDR

251 AKCTVFDFAL KERMQHGGIA DWRHGLMGNP DARWREVAVT FVDNHDTGYS

301 PGPHGGQHHW PLPDARLKQA YAYILSSPGT PWYWPHMYD WGHGDFIRQL

351 IQIRRAAGVK AASAIQFHTG FSGLVATISG SQQQLLIALD SNLSSPGQVA

401 SGDFIQALWT DLIGAIRIWRS GQGGGDGQGM LVSVNFRCDH GVTQWGDSVY

451 ALGNVTQLGN WSPAGAVRLT DTSAYPTWKG SIALPAGQQV QWKCIVRSES

501 MPTQVKTWQP GGMHSVTVAS GASTAGSF *

SEQ ID NO: 7. Nucleotide DNA sequence coding for G4 amylase from Pseudomonas mendocin (strain ymp) (A4XX23) with M added at the N-terminus: (at 4x X23 DNA):

atggatgcaccgggcaaaacagcatcaggcgttagatateatggcggcgatgaaattattctgcaaggctttcattggaatac agttagaacatcatcaaattggtatgcaacactggcatcaatggcaccgacactggcagcagatggcttttcagcaatttgga tgccggttccgtggagagatttttcatcatggtcagatccgggcaatggcacatcaggcggcggcgaaggctatttttggcat gattttaataaaaatggcagatatggctcagattcactgctgagacaagcagcatcagcactgaatgcagcaggcgttaaacc gatttatgatgttgttccgaatcatatgaatagaggctatccggataaagaaattaatctgccggcaggccaaggcctgtgga gacatgattgcaatgatccgggcaattatgcaaatgattgcgatgatggcgatagatttatgggcggcgatgctgatctgaat acaggccatccgcaaaattatgcaatgtttagagatgaatttgcaagactgagatcacaatatggcgcaggcggctttagatt tgattttgttagaggctatgcaggcgaaagagttgcatcatggatgtcagatgcacatgataatggcttttgcctgggcgaac tgtggaaagcaccgggcgaatatccgtcatgggattggagaaatggcgcatcatggcaacaaattctgaaagattggtcagat agagcaaaatgcacagtttttgattttgcactgaaagaaagaatgcaaaatggcggcattgcagattggagacatggcctgaa tggcaatccggatgcaagatggagagaagttgcagttacatttgttgataatcatgatacaggctattcaccgggccegcatg gcggccaacatcattggccgctgccggatgeaagactgaaacaagcatatgcatatattctgtcatcaccgggcac accggtt gtttattggccgcatatgtatgattggggtcatggagattttattagacaactgattcaaattagaagagcagcaggcgttaa agcagcatcagcaattcaatttcatacaggcttttcaggcctggttgcaacaatttcaggctcacaacaacaactgctgattg cactggattcaaatctgtcatcacegggccaagttgcatcaggcgattttacacaagcactgaatacagataatggcgcaatt agaatttggagatcaggccaaggcggcggcgat ggeeaaggcaatctggtttc agttaattttagatgcgataatggcgttac acaatggggcgattcagtttatgcactgggcaatgttacacaactgggcaattggtcaccggcaggcgcagttagactgacag atacatcagcatatccgacatggaaaggctcaattgcactgccggcaggccaacaagttcaatggaaatgcattgttagatca gaatcaaatccgacacaagttaaaacatggcaaccgggcggcaataattcagttacagttgcatcaggcgcatcaacagcagg ctcattttaa

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

1 MESSGKSGAG VRFHGGDEII LQGFHWNWR TAERNWYNIL QSKAQQISED

51 GFTAIWMPVP WRDNSSWQAS SDTRFGGEGY FWADMDKNSR YGDDGQLKQA

101 ASALKNKGVK VIYDIVPHHH DRGHSHDSLN LPSGQGYYRS DCSSCDDGDP

151 FMDGGSDF3T AHPDVYDLFK MELVNLKTNY SAGGFRFDFV RGYAPERISA

201 WMSASLDSGY CVGELWKGPS EYPSWDWRHS ASWQEILKDF TDASDCSVFD

251 FALKERMQNG SISDWRYGLM GHPSAQWREV AVTFVDNHDT GYSPGPLGGQ

301 HHWALPDWKR KMAYAYILSS PGTPWYWPH MYDWGMRDFI RNLIQLRKSA

351 GVKAYSGVQF HDGFSGLVGT TSGSNGKLLF AIDSNFSSPW QVAGGAWHLA

401 VNEDNGRIRI WRQ *

SEQ ID NO: 9. Nucleotide DNA sequence encoding the G4 amylase of Hahella chejuensis (strain KCTC 2396) with added M at the N-terminus (DNA of Q2SEA8):

atggaatcatcaggcaaatcaggcgcaggcgttagatttcatggcggcgatgaaattattctgcaaggctttcattggaacgt tgttagaacagcagaaagaaactggtacaacatcetgeaatcaaaagcacaacaaatttcagaagatggetttacageaattt ggatgccggttccgtggagagataattcatcatggcaagcatcatcagatacaagatttggcggcgaaggctatttttgggca gatatggataaaaattcaagatatggcgatgatggccaactgaaacaagcagcatcagcactgaaaaataaaggcgttaaagt tatttatgatattgttccgaatcatcatgatagaggccattcaaatgattcactgaatctgecgtcaggccaaggctattata gatcagattgctcatcatgcgatgatggcgatccgtttatggatggcggctcagatttttcaacagcacatccggatgtttac gatctgtttaaaaacgaactggttaacctgaaaacaaactactcagcaggcggctttagatttgattttgttagaggctatgc accggaaagaatttcagcatggatgtcagcatcactggattcaggctattgcgttggcgaactgtggaaaggcccgtcagaat atccgteatgggattggagacattcagcateatggcaagaaattctgaaagattttacagatgcatcagattgctcagttttt gattttgcactgaaagaaagaatgcaaaatggctcaatttcagattggagatatggcctgaatggcaatccgtcagcacaatg gagagaagttgcagttacatttgttgataatcatgatacaggctattcaccgggcccgctgggcggccaacatcattgggcac tgccggattggaaaagaaaaatggcatatgcatatattctgtcatcaecgggcacaccggttgtttattggccgca tatgtat gattggggcatgagagattttattaga.aatctgattcaactgagaaaatca.gcaggcgttaaagcatattcaggcgttcaatt tcatgatggcttttcaggcctggttggcacaacatcaggctcaaatggcaaactgetgtttgcaattgattcaaatttttcat caccgaatcaagttgcaggcggcgcatggaatctggcagttaatgaagataatggcagaattagaatttggagacaataa SEQ ID NO: 10.> gi | 77787 | pir || S05667 mature protein glucan 1,4-alpha-maltotetraohydrolase (EC 3.2.1.60) without the signal sequence - Pseudomonas saccharophila

DQAGKSPAGVRYHGGDEIILQGFHWNVVREAPNDWYNILRQQASTIAADGFSAIWMPVPWRDFSSWTDGGKSGGGEGYFWHDF NKNGRYGSDAQLRQAAGALGGAGVKVLYDWPNHMNRGYPDKEINLPAGQGFWRNDCADPGNYPNDCDDGDRFIGGESDLNTG HPQIYGMFRDELANLRSGYGAGGFRFDFVRGYAPERVDSWMSDSADSSFCVGELWKGPSEYPSWDWRNTASWQQIIKDWSDRA KCPVFDFALKERMQNGSVADWKHGLNGNPDPRWREVAVTFVDNHDTGYSPGQNGGQHHWALQDGLIRQAYAYILTSPGTPWY WSHMYDWGYGDFIRQLIQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLWALNSDLANPGQVASGSFSEAVNASNGQVRV WRSGSGDGGGNDGGEGGLVMVNFRCDNGVTQMGDSVYAVGHVSQLGNWSPASAVRLTDTSSYPTWKGSIALPDGQNVEWKCLI RMEADATLVRQWQSGGNNQVQAAAGAS TSG3F

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

MSHILRAAVLAAVLLPFPALA

SEQ ID NO: 12. Sequence of mature proteins of pMS1776

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG

51 F5AIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFMKNGLYG SDAQLRQAAG

101 ALGGAGVKVL YDWPNHHMR FYPDKEINLP AGQRFWRMDC PDPGMGPMDC

151 DDGDRFLGGE ADLHTGHPQT YGMFRDEFTH LRSGYGAGGF RFDFVRGYAP

201 ERVDSWMSDS ADSEFCVGEL WKSPSEYPPW DWRNRASWQQ IIKDWSDRAK

251 CPVFDFALKE RMQHGSVADW KHGLHGMFDF RWREVAVTFV DHHDTGYSPG

301 ■ QITGGQHKHFL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ

351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG

401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 13. Sequence of mature proteins of pMS1934

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG

51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFMKNGLYG SDAQLRQAAG

101 ALGGAGVKVL YDVVPNHMNR FYPDKEIMLP AGQRFWRNDC PDPGMGPMDC

151 DDGDRFLGGE ADLMTGHPQI YGMFRDEFTH LRSGYGAGGF RFDFVRGYAP

201 ERVDSWMSDS ADSSFCVGEL WKKPSEYPPW DWRNRASWQQ IIKDWSDRAK

251 CPVFDFALKE RMQNGSVADW KDGLMGMPDP RWREVAVTFV DNHDTGYSPG

301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ

351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG

401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 14 Sequence of mature proteins of pMS2020

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG

51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFMKNGLYG SDAQLRQAAG

101 ALGGAGVKVL YDWPHHMNR FYPDKEINLP AGQRFWRNDC PDPGMGPMDC

151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTH LRSGYGAGGF RFDFVRGYAP

201 ERVDSWMSDS ADSSFCVGEL WKSPSEYPPW DWRNRASWQQ IIKDWSDRAK

251 CPVFDFALKE RMQHGSVADW KHGLNGHPDF RWREVAVTFV DNHDTGYSPG

301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ

351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG

401 SFSEAVHAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 15 Sequence of mature proteins of pMS2022

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APYNWYNILR QKASTIAADG

51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFHKHGLYG SDAQLRQAAG

101 ALGGAGVKVL YDWPNHMNR FYPDKEINLP AGQRFWRNDC PDPGMGPMDC

151 DDGDRFLGGE ADLMTGHPQI YGMFRDEFTH LRSGYGAGGF RFDFVRGYAP

201 ERVDLWMSDS ADSSFCVGEL WKSPSEYPPW DWRNRASWQQ IIKDWSDRAK

251 CPVFDFALKE RMQTIGSVADW KHGLNGNFDF RWREVAVTFV DHHDTGY3PG

301 QNGGQHKWPL PDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ

351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LDNPGQVASG

401 SFSEAVHAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 16 Sequence of mature proteins of pMS2062

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 WKKPSEYPPW DWRNRASWQE 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: 17 Sequence of mature proteins of pMS2171

1 DQAGKSPAGV RYHGGDEI IL QGFHWNWRE APYNWYNILR QKASTIAADG 51 FSAIWMPVPW RDFSSWTDGD KSGGGEGYFW HDFNKNGLYG SDAQLRQAAG 101 ALGGAGVKVL YDVVPNHMNR FYPDKEINLP AGQRFWRNDC PDPGNGPNDC 151 DDGDRFLGGE ADLNTGHPQI YGMFRDEFTN LRSGYGAGGF RFDFVRGYAP 201 ERVDLWMSDS ADSSFCVGEL 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 Sequence of mature proteins of pMS465:

1 DQAGKSPAGV RYHGGDEI IL 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 Sequence of mature proteins of pMS1042:

1 DQAGKSPAGV RYHGGDEI IL 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 Sequence of mature proteins of pMS1104:

1 DQAGKSPAGV RYHGGDEI IL 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 *

SEQ ID NO: 21 Sequence of mature proteins of pMS1153:

l 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 Sequence of mature proteins of pMS1286:

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 QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 23 Sequence of mature proteins 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 Sequence of mature proteins 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 Sequence of mature proteins 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 *

SEQ ID NO: 26 Sequence of mature proteins of pMS1579:

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 QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ 351 VRRTAGVRAD SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG 401 SFSEAVNAEN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 27 Sequence of mature proteins of pMS1105:

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 LANPGQVASG 401 SFSEAVNASN GQVRVWRSGS GDGGGNDGG *

SEQ ID NO: 28 Sequence of mature proteins 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 Sequence of mature proteins 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 Sequence of mature proteins of pMS2138:

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 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 *

SEQ ID NO: 31 Sequence of mature proteins of pMS2124:

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 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 Sequence of mature proteins 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 Sequence of mature proteins 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 Sequence of mature proteins 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. Linking Sequence

GSGDGGGNDGG SEQ ID NO: 36: Sequence of PS4 SBD:

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 YFHHNGDISNWDDRYEAQWKNFTDPAGFSLADLSQENGTIAQYLTDAAVQLVAHGADGLRIDAVKHFNSGFSKSLADKLYQKK DIFLVGEWYGDDPGTANHLEKVRYANNSGVNVLDFDLNTVIRNVFGTFTQTMYDLNNMVNQTGNEYKYKENLITFIDNHDMSR FLSVNSNKANLHQALAFILTSRGTPSIYYGTEQYMAGGNDPYNRGMMPAFDTTTTAFKEVSTLAGLRRNNAAIQYGTTTQRWI NNDVYIYERKFFNDVVLVAINRNTQSSYSISGLQTALPNGSYADYLSGLLGGNGISVSNGSVASFTLAPGAVSVWQYSTSASA PQIGSVAPNMGIPGNWTIDGKGFGTTQGTVTFGGVTATVKSWTSNRIEVYVPNMAAGLTDVKVTAGGVSSNLYSYNILSGTQ TSVVFTVKSAPPTNLGDKIYLTGNIPELGNWSTDTSGAVNNAQGPLLAPNYPDWFYVFSVPAGKTIQFKFFIKRADGTIQWEN GSNHVATTPTGATGNITVTWQN

Claims (14)

A polypeptide having amylase activity comprising an amino acid sequence having at least 65% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide comprises an amino acid substitution in the following position: 88, with reference to the numbering of the position of the sequence shown as SEQ ID NO: 1, wherein the polypeptide has improved thermostability compared to the amylase of SEQ ID NO: 1. The polypeptide according to claim 1, wherein the polypeptide comprises one or more amino acid substitutions in the following positions: 88, 205, 235, 240, 311 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 223A / K / S. 4. 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 the amino acid sequence of SEQ ID NO: 1. 5. The polypeptide according to claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21. 6. The polypeptide according to any of claims 1-5 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. A vector expression 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, preferably transformed with, an expression vector according to claim 9 or plasmid according to claim 10. 12. Use of a polypeptide according to any of claims 1-7 as a food or feed additive. 13. A method of preparing a saccharide syrup, comprising adding a polypeptide according to any of claims 1-7 to a granular starch blend to form the saccharide syrup. 14. A food product comprising a polypeptide according to any of claims 1-7.