ES2728939T3 - Alpha-amylase variants with improved stability - Google Patents

Abstract

Isolated alpha-amylase variant, comprising a substitution in three or more positions corresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179 , 180, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416 and 427, in particular, in the positions that correspond to positions 129, 177 and 179, in the that the variant has at least 90% and less than 100% sequence identity to the mature polypeptide of SEQ ID No: 6, and the variant has alpha-amylase activity, and where the variant comprises a set of substitutions, using SEQ ID no: 1 for numbering, consisting of: E129V + K177L + R179E, and in which the alterations of I181 * + G182 * + N193F present in SEQ ID no: 6 and the variants have a improved thermostability compared to alpha-amylase described as SEQ ID no: 6.

Description

DESCRIPTION

Alpha-amylase variants with improved stability

Reference to a sequence listing

[0001] This application contains a list of sequences in computer readable format.

Background of the invention

Field of the Invention

[0002] The present invention relates to alpha-amylase variants that have an improved property, eg, improved stability, polynucleotides encoding the variants, methods for producing the variants and methods for using the variants. Description of the related technique

[0003] Alpha-amylases (alpha-1,4-glucan-4-glucanhydrolases, E.C. 3.2.1.1) constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glucosidic oligosaccharides and polysaccharides.

[0004] Alpha-amylases are used commercially for a variety of purposes, such as in the initial stages of starch processing ( eg, liquefaction); in wet milling processes and in the production of alcohol from carbohydrate sources. They are also used as cleaning agents or complements in detergent matrices, in the textile industry for starch decoupling, in baking applications, in the beverage industry, in oil fields in drilling processes, in recycling processes, p .ej., to allocate paper, and in animal feed.

[0005] WO2002 / 010355 describes Termamyl-type alpha-amylases that show altered stability at high temperatures and low calcium concentrations.

[0006] Hatada et al., (Enzyme and Microbial Technol. 39 (2006) 1333-1340) describe a stable oxidative alpha-amylase from a deep-sea Bacillus isolate.

[0007] JP 62-104580 describes a heat-resistant alpha-amylase derived from Bacillus stearothermophilus, however, the positions corresponding to positions 129, 177 and 179 of the present invention have not been modified.

Summary of the Invention

[0008] The present invention provides alpha-amylase variants with improved properties, eg, improved thermostability compared to its original enzyme.

[0009] The present invention relates to an isolated alpha-amylase variant, which comprises a substitution in three or more positions corresponding to any of positions 59, 89, 91.96, 108, 112, 129, 157, 165 , 166, 168, 171, 177, 179, 180, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416 and 427, in particular, in the positions corresponding to positions 129, 177 and 179, in which the variant has at least 90% and less than 100% sequence identity with the mature polypeptide of SEQ ID #: 6, and the variant has alpha- activity amylase, and where the variant comprises a set of substitutions, using SEQ ID No.: 1 for numbering, consisting of: E129V K177L R179E, and in which the alterations of I181 * G182 * N193F present in the SEQ ID #: 6 and the variants have improved thermostability compared to the alpha-amylase described as SEQ ID #: 6.

[0010] The present invention also relates to isolated polynucleotides encoding a variant of alpha-amylase, nucleic acid constructs, vectors and host cells comprising the polynucleotides, and methods for producing a variant of a progenitor alpha-amylase.

[0011] The present invention also relates to the use of variants in the liquefaction of starch. In addition, the present invention relates to a process for producing a fermentation product, which comprises

a) liquefying a starch-containing material with a variant of any of claims 1-3 to produce a liquefied mixture;

b) saccharify the liquefied mixture to produce fermentable sugars; Y

c) ferment the fermentable sugars in the presence of a fermenting organism.

Brief description of the figures

[0012] Figure 1 shows an alignment of the catalytic domains of the alpha-amylases with the amino acid sequence of SEQ ID Nos: 1, 2, 3, 4, 5 and 7. SEQ ID #: 1 is an alpha-amylase from Bacillus stearothermophilus; SEQ ID NO: 2 is an amylase from Bacillus flavothermus, AMY1048 described in WO 2005/001064; SEQ ID #: 3 is Bacillus alpha-amylase TS-22; SEQ ID #: 4 is Bacillus alpha-amylase TS-23 described in J. Appl. Microbiology, 1997, 82: 325-334. (SWALL: q59222); SEQ ID #: 5 is an alpha-amylase described in WO 2004/091544; and SEQ ID #: 7 is another alpha-amylase from Bacillus stearothermophilus.

Detailed description of the invention

[0013] The present disclosure relates to isolated variants of a parent alpha-amylase, which comprise a substitution in three or more positions corresponding to positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166 , 168, 171, 177, 179, 180, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281,284, 416 and 427, where the variant has at least 65 % and less than 100% sequence identity with at least one of the mature polypeptides of SEQ ID #: 1; and the variant has alpha-amylase activity.

Definitions

[0014] Allelic variant: The term "allelic variant" means any two or more alternative forms of a gene that occupy the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Genetic mutations can be silent (without changes in the encoded polypeptide) or they can encode polypeptides that have altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0015] Alpha-amylases (alpha-1,4-glucan-4-glucanhydrolases, E.C. 3.2.1.1) are a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glucosidic oligosaccharides and polysaccharides.

[0016] Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of its polypeptide product. The limits of the coding sequence are generally determined by an open reading frame, which normally begins with the ATG start codon or alternative start codons such as GTG and TTG, and ends with a termination codon such as TAA, TAG and TGA. The coding sequence can be a DNA, cDNA, synthetic or recombinant polynucleotide.

[0017] Control sequences: The term "control sequences" means all the components necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence can be native or foreign to the polynucleotide encoding the variant, or native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a peptide sequence and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Control sequences can be provided with linkers in order to introduce specific restriction sites that facilitate the binding of control sequences with the polynucleotide coding region encoding a polypeptide.

[0018] Expression: The term "expression" includes any step involved in the production of the polypeptide that includes, among others, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0019] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide its expression.

[0020] Host cell: The term "host cell" means any type of cell that is susceptible to transformation, transfection, transduction and the like with a nucleic acid construct or an expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any descendant of a stem cell that is not identical to the stem cell due to mutations that occur during replication.

[0021] Enhanced property: The term "enhanced property" means a characteristic associated with an improved variant compared to the original alpha-amylase. Such improved properties include thermostability.

[0022] Isolated variant: The terms "isolated" and "purified" mean a polypeptide or polynucleotide that is removed from at least one component with which it is naturally associated. For example, a variant can be at least 1% pure, for example, at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure and at least 90% pure, as determined by SDS-PAGE and a polynucleotide may have at least 1% pure, for example, at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure and at least 90% pure, as determined by agarose electrophoresis.

[0023] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modification, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide consists of amino acids 1 to 483, 1 to 486 or 1 to 493 of SEQ ID #: 1. It is known in the art that a host cell can produce a mixture of two or more polypeptides. different mature ( ie, with a different C-terminal and / or N-terminal amino acid) expressed by the same polynucleotide.

[0024] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a nucleotide sequence encoding a mature polypeptide that has alpha-amylase activity.

[0025] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single or double stranded, that is isolated from a gene that occurs naturally or is modified to contain Nucleic acid segments in a way that would not otherwise exist in nature or that is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for the expression of a coding sequence.

[0026] Operationally linked: The term "operably linked" means a configuration in which a control sequence is placed in an appropriate position with respect to the coding sequence of the polynucleotide sequence, such that the control sequence directs the expression of the coding sequence of a polypeptide.

[0027] Progenitor: The term "progenitor" alpha-amylase means an alpha-amylase in which an alteration is made to produce a variant of the present invention. The parent can be a polypeptide of natural origin (wild type) or a variant thereof, prepared by suitable means. The parent can also be an allelic variant.

[0028] Polypeptide fragment: The term "polypeptide fragment" means a polypeptide having one or more (several) amino acids removed from the amino and / or carboxyl ends of a mature polypeptide; in which the fragment has alpha-amylase activity. In one aspect, a fragment contains at least 483 amino acid residues, e.g. eg, at least 486 and at least 493 amino acid residues, of the mature polypeptide of SEQ ID #: 1.

[0029] Sequence identity: The relationship between two amino acid sequences or between two nucleotide sequences is described by the "sequence identity" parameter.

[0030] For the purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. Optional parameters used are the gap opening penalty of 10, the gap extension penalty of 0.5 and the replacement matrix EBLOSUM62 (EMBOSS version of BLOSUM62). The result of Needle labeled "longer identity" (obtained through the -nobrief option) is used as the percentage of identity and is calculated as follows:

(Identical waste x 100) / (Alignment length - Total number of gaps in the alignment)

[0031] For the purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the package's Needle program. EMBOSS (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. Optional parameters used are the gap opening penalty of 10, gap extension penalty of 0.5 and the replacement matrix EDNAFULL (NCBI EMBOSS NUC4.4). The result of Needle labeled "longer identity" (obtained through the -nobrief option) is used as the percentage of identity and is calculated as follows:

(Identical deoxyribonucleotides x 100) / (Alignment length - Total number of alignment gaps)

[0032] Subsequence: The term "subsequence" means a polynucleotide sequence that has one or more (several) nucleotides removed from the 5 'and / or 3' ends of a coding sequence of a mature polypeptide; where the sub sequence encodes a polypeptide fragment that has alpha-amylase activity.

[0033] Variant: The term "variant" means a polypeptide that has alpha-amylase activity that comprises an alteration, that is, a substitution, insertion and / or deletion of one or more (several) amino acid residues in one or more (Various positions. A substitution means a replacement of an amino acid that occupies a position with a different amino acid; a deletion means the removal of an amino acid that occupies a position; and an insertion means adding 1-5 amino acids right after an amino acid that occupies a position.

[0034] Wild type: The term "wild type" means an alpha-amylase expressed by a naturally occurring microorganism, such as a bacterium, yeast or filamentous fungus found in nature.

Conventions for the designation of variants

[0035] For the purposes of the present invention, the mature polypeptide described in SEQ ID #: 1 is used to determine the corresponding amino acid residue in another alpha-amylase. The amino acid sequence of another alpha-amylase is aligned with the mature polypeptide described in SEQ ID #: 1 and, depending on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide described in SEQ ID #: 1 can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS : European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later.

[0036] The identification of the corresponding amino acid residue in another alpha-amylase can be confirmed by an alignment of multiple polypeptide sequences using "ClustalW" (Larkin et al., 2007, Bioinformatics 23: 2947-2948).

[0037] When the other enzyme has diverged from the mature polypeptide of SEQ ID #: 1, so that the traditional sequence-based comparison does not detect its relationship (Lindahl & Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based search can be achieved using search programs that use probabilistic representations of polypeptide families (profiles) to search the databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is able to detect remote counterparts (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily of the polypeptide has one or more (several) representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) use information from a variety of sources (PSI-BLAST, secondary structure prediction , structural alignment profiles and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown structure with the Superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the polypeptide, and the accuracy of such models can be evaluated using a variety of tools developed for that purpose.

[0038] For proteins of known structure, various tools and resources are available to recover and generate structural alignments. For example, SCOP protein superfamilies have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms, such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Eng. 11 : 739-747), and implementations of these algorithms can also be used to query databases of structures with a structure of interest in order to discover possible structural counterparts (for example, Holm and Park, 2000, Bioinformatics 16: 566 -567). These structural alignments can be used to predict the corresponding amino acid residues structurally and functionally in proteins within the same structural superfamily. This information, together with information derived from homology models and profile searches, can be used to predict which residues must be mutated when moving mutations of interest from a protein to a nearby or remote homolog.

[0039] In describing the alpha-amylase variants of the present invention, the nomenclature described below is adapted to facilitate reference. In all cases, the single letter or three letter abbreviation for amino acids accepted by the IUPAC is used.

[0040] Substitutions. For an amino acid substitution, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine with alanine at position 226 is designated as "Thr226Ala" or "T226A". Multiple mutations are separated by sum signs ("+"), p. eg, "Gly205Arg Ser411Phe" or "G205R S411F", which represent mutations at positions 205 and 411 replacing glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

[0041] Deletions. For an amino acid deletion, the following nomenclature is used: original amino acid, position, *. Therefore, the glycine deletion at position 195 is designated as "Gly195 *" or "G195 *". Multiple deletions are separated by sum signs ("+"), p. eg, "Gly195 * Ser411 *" or "G195 * S411 *".

[0042] Insertions. For an amino acid insertion, the following nomenclature is used: original amino acid, position, original amino acid, new inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is designated "Gly195GlyLys" or "G195GK". Multiple insertions of amino acids are designated [original amino acid, position, original amino acid, new amino acid inserted # 1, new amino acid inserted # 2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as "Gly195GlyLysAla" or "G195GKA".

[0043] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position number of the amino acid residue that precedes the inserted amino acid (s). In the previous example, the sequence would be:

[0044] Multiple alterations. Variants comprising multiple alterations are separated by sum signs ("+"), p. eg, "Arg170Tyr Gly195Glu" or "R170Y G195E" representing a substitution of tyrosine and glutamic acid with arginine and glycine at positions 170 and 195, respectively.

[0045] Different alterations. When different alterations can be introduced in one position, the different alterations are separated by a comma, e.g. eg, "Arg170Tyr, Glu" represents a substitution of arginine with tyrosine or glutamic acid at position 170. Therefore, "Tyr167Gly, Ala Arg170Gly, Ala" designates the following variants:

Tyr167Gly Arg170Gly, Tyr167Gly Arg170Ala, Tyr167Ala Arg170Gly and Tyr167Ala Arg170Ala.

Preparation of variants

[0046] The present disclosure also relates to methods for obtaining a variant with alpha-amylase activity, comprising: (a) introducing into a parent alpha-amylase a substitution in three or more (several) positions corresponding to positions 59 , 89, 91.96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274,276, 281,284 , 416 and 427, where the variant has alpha-amylase activity; and (b) recover the variant.

[0047] Variants can be prepared according to any mutagenesis method known in the art, such as directed mutagenesis, synthetic gene construction, semisynthetic gene construction, random mutagenesis, shuffling, etc.

[0048] Targeted mutagenesis is a technique in which one or more (several) mutations are created at a defined site in a polynucleotide molecule that encodes the progenitor alpha-amylase. The technique can be performed in vitro or in vivo.

[0049] The construction of the synthetic gene involves in vitro synthesis of a polynucleotide molecule designed to encode a molecule of polypeptides of interest. Gene synthesis can be performed using several techniques, such as microchip-based multiplex technology described by Tian et al., 2004, Nature 432: 1050-1054, and similar technologies in which oligonucleotides are synthesized and assembled into photoprogrammable microfluidic chips. .

[0050] Targeted mutagenesis can be achieved in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. In vitro directed mutagenesis can also be performed by cassette mutagenesis that involves cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the progenitor alpha-amylase and subsequent binding of an oligonucleotide containing the mutation in the polynucleotide Generally, the restriction enzyme that performs the digestion in the plasmid and the oligonucleotide is the same, allowing the cohesive ends of the plasmid and the insert to join together. See, for example, Scherer and Davis, 1979, Proc. Natl Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Research 18: 7349-4966.

[0051] Targeted mutagenesis can be achieved in vivo by methods known in the art. See, for example, publication of US patent application. UU. No. 2004/0171154; Storici et al., 2001, Nature Biotechnology 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

[0052] Any method of directed mutagenesis can be used in the present invention. There are many commercial kits available that can be used to prepare variants of a progenitor alpha-amylase.

[0053] Substitutions, deletions and / or insertions of single or multiple amino acids can be made and tested using known methods of mutagenesis, recombination and / or random mixing, followed by a relevant selection procedure, such as those described by Reidhaar-Olson and Sauer , 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display ( e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0054] Mutagenesis / shuffling methods can be combined with high-performance automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells. Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and sequenced quickly using standard methods in the art. These methods allow to quickly determine the importance of individual amino acid residues in a polypeptide of interest.

[0055] The construction of semi-synthetic genes is achieved by combining aspects of the construction of synthetic genes and / or directed mutagenesis and / or random and / or shuffled mutagenesis. The semi-synthetic construction is typified by a process that uses synthesized polynucleotide fragments, in combination with PCR techniques. Defined regions of de novo genes can thus be synthesized while other regions can be amplified using site-specific mutagenic primers, and even while other regions can undergo error-prone PCR or non-error-prone PCR amplification. The polynucleotide fragments can then be shuffled.

Variants

[0056] The present disclosure refers to variants comprising a substitution in three or more (several) positions corresponding to positions 59, 89, 91.96, 108, 112, 129, 157, 165, 166, 168, 171 , 177, 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416 and 427, where the variant has alpha-amylase activity.

[0057] In one aspect of the disclosure, the variant has a sequence identity of at least 65%, for example, at least 70%, at least 75%, 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%, but less than 100%, with the amino acid sequence of the parent alphaamylase.

[0058] In another aspect of the disclosure, the variant has a sequence identity of at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%, but less than 100% with the mature polypeptide of SEQ ID No .: 1.

[0059] In another aspect of the disclosure, the variant has a sequence identity of at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%, but less than 100% with the mature polypeptide of SEQ ID No .: 2.

[0060] In another aspect of the disclosure, the variant has a sequence identity of at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%, but less than 100% with the mature polypeptide of SEQ ID No .: 3.

[0061] In another aspect of the disclosure, the variant has a sequence identity of at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%, but less than 100% with the mature polypeptide of SEQ ID No .: 4.

[0062] In another aspect of the disclosure, the variant has a sequence identity of at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%, but less than 100% with the mature polypeptide of SEQ ID No .: 5.

[0063] In another aspect the variant has a sequence identity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%, but less than 100% with maturity Polypeptide of SEQ ID #: 6.

[0064] In another aspect of the disclosure, the variant has a sequence identity of at least 65%, for example, at minus 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%, but less than 100% with the mature polypeptide of SEQ ID #: 7.

[0065] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

59 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Tyr, in particular with Ala, Gln,

Glu, Gly, Ile, Leu, Prot or Thr.

[0066] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

89 with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Arg, His or

Lys.

[0067] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

91 with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ile, Leu or

Val.

[0068] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

96 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ile, Leu or

Val.

[0069] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

108 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ala.

[0070] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

112 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ala, Asp or

Glu.

[0071] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

129 with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ala, Thr or

Val.

[0072] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

157 with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with His, Lys,

Phe or Tyr.

[0073] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

165 with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Asn.

[0074] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

166 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr or Val, in particular with Phe.

[0075] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

168 with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ala.

[0076] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

171 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Glu.

[0077] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

177 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Arg, Leu or

Met

[0078] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 179 with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Gln, Glu,

Ile, Leu, Lys or Val.

[0079] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

180 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Glu or Val.

[0080] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

181 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Glu, Gly or Val.

[0081] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

184 with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ser.

[0082] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 208 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Phe or

Tyr.

[0083] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to the position

220 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in particular with Pro.

[0084] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 224 with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Leu.

[0085] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 242 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met , Phe, Pro, Thr, Trp, Tyr or Val, in particular with Ala, Asp, Glu, Gln or Met.

[0086] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 254 with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ala, Ser or Thr.

[0087] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 269 with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, lie, Leu, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Gln or Glu.

[0088] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 270 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Leu, Thr or Val.

[0089] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 274 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Arg, Gln, Glu, Lys or Phe.

[0090] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 276 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met , Phe, Pro, Ser, Thr, Trp or Val, in particular with Phe.

[0091] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 281 with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Asn or Ser.

[0092] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 284 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with His, Thr or Val.

[0093] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 416 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ala, Asn, Asp, Ser, Thr or Val.

[0094] In one aspect of the disclosure, the variant comprises a substitution in a position corresponding to position 427 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe , Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ile, Met or Val.

[0095] In one aspect of the disclosure, the variant comprises a substitution in three positions corresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177 , 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281,284, 416 and 427. In particular, the variant comprises a replacement at the positions corresponding to positions 129 , 177 and 179 or positions 220, 242, and 254.

[0096] In another aspect of the disclosure, the variant comprises a substitution in four positions corresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177 , 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281,284, 416 and 427. In particular, the variant comprises a replacement at the positions corresponding to positions 129 , 177, 179 and 208; positions 129, 177, 179 and 242; positions 129, 177, 179 and 284; positions 208, 220, 224 and 254; positions 220, 224, 242 and 254; or positions 220, 224, 254 and 284.

[0097] In another aspect of the disclosure, the variant comprises a substitution in five positions corresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177 , 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281,284, 416 and 427. In particular, the variant comprises a replacement at positions corresponding to positions 129 , 177, 179,208 and 242; positions 129, 177, 179, 208 and 284; positions 208, 220, 224, 242 and 254; or positions 208, 220, 224, 254 and 284.

[0098] In another aspect of the disclosure, the variant comprises a substitution in six positions corresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177 , 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281,284, 416 and 427. In particular, the variant comprises a replacement at the positions corresponding to positions 129 , 177, 179,208, 242 and 284 or positions 129, 177, 179, 220, 224 and 254.

[0099] In another aspect of the disclosure, the variant comprises a substitution in seven positions corresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177 , 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416 and 427.

[0100] In another aspect of the disclosure, the variant comprises a substitution in eight positions corresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177 , 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416 and 427.

[0101] In another aspect of the disclosure, the variant comprises a substitution in nine positions corresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416 and 427.

[0102] In another aspect of the disclosure, the variant comprises a substitution in ten positions corresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177 , 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416 and 427.

[0103] The variants preferably have from 3 to 20, for example, from 3 to 10 and from 6 to 10, alterations such as, for example, 3, 4, 5, 6, 7, 8, 9 or 10 alterations. In one aspect of the disclosure, the alterations are substitutions.

[0104] In one aspect of the disclosure, the variant alpha-amylases comprise or consist of a set of substitutions selected from the group consisting of:

V59A G108A;

S242Q M284V;

V59A M284V;

G108A M284V;

V59A G108A M284V;

V59A G108A S242Q M284V;

E129V K177L R179E;

K220P N224L Q254S;

E129V K177L R179E M284V;

V59A E129V K177L R179E H208Y M284V;

V59A H208Y K220P N224L Q254S M284V;

E129V K177L R179E K220P N224L Q254S;

V59A Q89R E129V K177L R179E H208Y K220P N224L Q254S;

V59A E129V K177L R179E H208Y K220P N224L S242Q Q254S;

V59A Q89R E129V K177L R179E H208Y K220P N224L Q254S M284V;

V59A E129V K177L R179E H208Y K220P N224L S242Q Q254S M284V;

V59A Q89R G108A E129V K177L R179E H208Y K220P N224L Q254S M284V; Y

V59A G108A E129V K177L R179E H208Y K220P N224L S242Q Q254S M284V.

[0105] The variants may further comprise a deletion in one or more positions, for example, two, three or four, positions corresponding to positions 179, 180, 181 and 182. For example, the variants may comprise a deletion in positions which correspond to positions 181 and 182.

[0106] The variants may also further comprise an alteration, preferably a substitution, in a position corresponding to position 193. For example, the alteration in a position corresponding to position 193 may be a substitution with Phe.

[0107] The variants may also further comprise a deletion of the amino acid at the position corresponding to positions 376 and / or 377.

[0108] In another aspect, the variants further comprise an additional alteration, preferably a substitution, in a position corresponding to position 200, such as a substitution in a position corresponding to position 200 with Leu, Ile or Thr.

[0109] Variants of the present invention preferably consist of amino acids 483 to 515, 483 to 493 or 483 to 486.

Polynucleotides

[0110] The present invention also relates to isolated polynucleotides encoding any of the variants of the present invention.

Nucleic Acid Constructs

[0111] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more (several) control sequences that direct expression of the coding sequence in a host cell suitable under conditions compatible with the control sequences. .

[0112] An isolated polynucleotide encoding a variant can be manipulated in a variety of ways to deal with the expression of the variant. Manipulation of the polynucleotide before insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0113] The control sequence may be a promoter sequence, which is recognized by a host cell for polynucleotide expression. The promoter sequence contains transcriptional control sequences that mediate variant expression. The promoter can be any nucleic acid sequence that shows transcriptional activity in the host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides, either homologous or heterologous to the host cell .

[0114] Examples of promoters suitable for directing transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are promoters obtained from the alpha-amylase (amyQ) gene of Bacillus amyloliquefaciens, the gene of alpha-amylase (amyL) from Bacillus licheniformis , the penicillinase (penP) gene from Bacillus licheniformis, the maltogenic amylase (amyM) gene from Bacillus stearothermophilus, the levansacarase (sacB) gene from Bacillus subtilis, and xB genes of Bacillus subtilis, the E. coli lac operon , the Streptomyces coelicolor agarase (dagA) gene and the beta-lactamase prokaryotic gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA. US 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. US 80: 21 25). Other promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

[0115] Examples of promoters suitable for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the Aspergillus nidulans acetamidase genes , neutral alpha-amylase of Aspergillus niger, alpha-amylase stable against acid Aspergillus niger glucoamylase (glaA) of Aspergillus niger or Aspergillus awamori, TAKA amylase of Aspergillus oryzae, protease alkaline Aspergillus oryzae triose phosphate isomerase Aspergillus oryzae, trypsin - like protease from Fusarium oxysporum (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase , beta-glucosidase beta-glucosidase Trichoderma reesei, cellobiohydrolase I from Trichoderma reesei, cellobiohydrolase II from Trichoderma reesei, endoglucanase I from Trichoderma reesei, endo Trichoderma reesei glucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei as tr2, as well as Trichoderma re-tripeder as well as Tr2 (a modified promoter that includes a gene encoding a neutral alpha-amylase in Aspergilli in which the untranslated leader has been replaced by an untranslated leader of a gene encoding triosa phosphate isomerase in Aspergilli; Non-limiting examples include modified promoters that include the gene encoding neutral alpha-amylase in Aspergillus niger in which the untranslated leader has been replaced by an untranslated leader of the gene encoding the triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated and hybrid promoters thereof.

[0116] In a yeast host, useful promoters are obtained from the enolase (ENO-1) genes of Saccharomyces cerevisiae, galactokinase (GAL1) of Saccharomyces cerevisiae, alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP) from Saccharomyces cerevisiae, triosa phosphate isomerase (TPI) from Saccharomyces cerevisiae, metallothionein (CUP1) from Saccharomyces cerevisiae and 3- phosphoglycerate kinase from Saccharomyces cerevisiae. Other promoters useful for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488 ..

[0117] The control sequence may also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operatively linked to the 3 ′ terminal of the polynucleotide encoding the variant. Any terminator that is functional in the host cell can be used in the present invention.

[0118] Preferred terminators for host cells filamentous fungal are obtained from the genes of anthranilate synthase Aspergillus nidulans, Aspergillus niger glucoamylase, alpha-glucosidase from Aspergillus niger, TAKA amylase of Aspergillus oryzae and trypsin - like protease Fusarium oxysporum .

[0119] Preferred terminators for yeast host cells are obtained from the enochase genes of Saccharomyces cerevisiae, cytochrome C (CYC1) Saccharomyces cerevisiae and glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae. Other terminators useful for yeast host cells are described by Romanos et al., 1992, supra.

[0120] The control sequence may also be a suitable leader sequence, an untranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operatively linked to the 5 ′ terminal of the polynucleotide encoding the variant. Any leader sequence that is functional in the host cell can be used in the present invention.

[0121] Preferred leaders for filamentous fungal host cells are obtained from the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase genes.

[0122] Suitable leaders for yeast host cells are obtained from the enolase (ENO - 1), Saccharomyces cerevisiae 3 - phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor of Saccharomyces cerevisiae , and alcohol dehydrogenase / glyceraldehyde-3 -phosphate dehydrogenase (ADH2 / GAP) from Saccharomyces cerevisiae.

[0123] The control sequence may also be a polyadenylation sequence, a sequence operatively linked to the 3 'terminal of the polypeptide coding sequence and, when transcribed, the host cell recognizes it as a signal to add polyadenosine residues to the mRNA. transcribed Any polyadenylation sequence that is functional in the host cell can be used in the present invention.

[0124] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the Aspergillus nidulans anthranylate synthase genes , Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase , Aspergillus oryinae type trypsinase and trypsin amylase Fusarium oxysporum.

[0125] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0126] The control sequence may also be a signal peptide coding region that encodes an amino acid sequence linked to the amino-terminal of a variant and directs the encoded polypeptide to the cell's secretory pathway. The 5 'end of the polynucleotide coding sequence may inherently contain a signal peptide coding region naturally linked in a translation reading frame with the segment of the coding region encoding the secreted variant. Alternatively, the 5 'end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The coding region of the foreign signal peptide may be necessary when the coding sequence does not contain a signal peptide coding region naturally. Alternatively, the coding region of the foreign signal peptide can simply replace the coding region of the natural signal peptide to increase secretion of the variant. However, any signal peptide coding region that directs the expressed polypeptide towards the secretory pathway of a host cell can be used in the present invention.

[0127] The effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the Bacillus maltogenic amylase genes NCIB 11837, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase , alpha-amylase of Bacillus stearothermophilus, neutral proteases ( nprT, nprS, nprM) of Bacillus stearothermophilus and prsA of Bacillus subtilis. Other signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0128] The signal signal peptide coding sequences effective for the filamentous fungal host cells are the signal peptide coding sequences obtained from the neutral Aspergillus niger amylase genes, Aspergillus niger glucoamylase , Aspergillus oryzae TAKA amylase , Humicola cellulase insolens, endoglucanase V from Humicola insolens, lipase from Humicola lanuginosa and aspartic proteinase from Rhizomucor miehei.

[0129] Useful signal peptides for yeast host cells are obtained from the alpha factor genes of Saccharomyces cerevisiae and invertase of Saccharomyces cerevisiae. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0130] The control sequence may also be a propeptide coding region encoding an amino acid sequence positioned at the amino terminal of a variant. The resulting polypeptide is known as proenzyme or propolypeptide (or zymogen in some cases). A propolypeptide is generally inactive and can be converted into a mature active polypeptide by catalytic or autocatalytic cleavage of the propolypeptide propeptide. The coding region of the propeptide can be obtained from the genes for Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase and Saccharomyces cerevisiae alpha factor .

[0131] When both the signal peptide and propeptide regions are present in the amino-terminal of a polypeptide, the propeptide region is placed next to the amino-terminal of a polypeptide and the signal peptide region is placed next to the amino terminal of the propeptide region.

[0132] It may also be desirable to add regulatory sequences that allow regulation of variant expression in relation to host cell growth. Examples of regulatory systems are those that cause gene expression to be activated or deactivated in response to a physical or chemical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac and trp operator systems . In yeasts, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter , the Aspergillus oryzae TAKA alpha-amylase promoter and the Aspergillus oryzae glucoamylase promoter can be used as regulatory sequences. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the variant would be operatively linked to the regulatory sequence.

Expression vectors

[0133] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter and transcriptional and translational stop signals. The various nucleotide and control sequences described above may be linked to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow insertion or replacement of the polynucleotide encoding the variant at said sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. When creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked to the appropriate control sequences for expression.

[0134] The recombinant expression vector can be any vector ( eg, a plasmid or a virus) that can conveniently undergo recombinant DNA procedures and can produce polynucleotide expression. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. Vectors can be linear or closed circular plasmids.

[0135] The vector may be an autonomous replication vector, that is, a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, e.g. eg, a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector may contain any means to guarantee self-replication. Alternatively, the vector may be such that, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome (s) in which it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon can be used.

[0136] The vectors of the present invention preferably contain one or more (several) selectable markers that allow easy selection of transformed, transfected, transduced or similar cells. A selectable marker is a gene whose product provides biocidal or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0137] Examples of selectable bacterial markers are the Bacillus subtilis or Bacillus licheniformis dal genes , or markers that confer resistance to antibiotics, for example, resistance to ampicillin, chloramphenicol, kanamycin or tetracycline. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5) 'decarboxylase phosphate), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. The amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the Streptomyces hygroscopicus bar gene are preferred for use in an Aspergillus cell .

[0138] The vectors of the present invention preferably contain an element or elements that allow the integration of the vector into the genome of the host cell or autonomous replication of the vector in the cell independent of the genome.

[0139] For integration into the genome of the host cell, the vector may be based on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences to direct integration by homologous recombination into the genome of the host cell at one or more precise locations on the chromosome (s). To increase the probability of integration in a precise place, the integration elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs and more preferably 800 to 10,000 pairs of bases, which have a high degree of sequence identity with the corresponding target sequence to increase the probability of homologous recombination. The integration elements may be any sequence that is homologous to the target sequence in the genome of the host cell. In addition, the integration elements may be non-coding or coding nucleotide sequences. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0140] For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicator that mediates autonomous replication that works in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a nucleotide sequence that allows a plasmid or vector to replicate in vivo.

[0141] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177 and pACYC184 that allow replication in E. coli ,, and pUB110, pE194, pTA1060 and pAMR1 that allow replication in Bacillus.

[0142] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3 and the combination of ARS4 and CEN6.

[0143] Examples of useful origins of replication in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67 .; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175. ; WO 00/24883). The isolation of the AMA1 gene and the construction of plasmids or vectors comprising the gene can be achieved according to the methods described in WO 00/24883.

[0144] More than one copy of a polynucleotide of the present invention can be inserted into the host cell to increase the production of an alpha-amylase variant. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including a selectable marker gene amplifiable with the polynucleotide where cells containing amplified copies of the selectable marker gene, and therefore additional copies of the polynucleotide, can be selected by culturing the cells in the presence of the appropriate selectable agent.

[0145] The methods used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, eg, Sambrook et al. 1989, supra ) to obtain substantially pure alpha-amylase variants.

Host cells

[0146] The present invention also relates to recombinant host cells, which comprise a polynucleotide encoding a variant, which are favorably used in the recombinant production of the variant. A vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above. The choice of a host cell will depend largely on the gene encoding the polypeptide and its source.

[0147] The host cell can be any cell useful in the recombinant production of a variant, eg, a prokaryotic or a eukaryotic.

[0148] The prokaryotic host cell can be any gram-positive or gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, among others, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0149] The bacterial host cell can be any Bacillus cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus leutus, Bacillus leutus, Bacillus leutus, Bacillus leutus, Bacillus leutus, Bacillus leutus, Bacillus leutus , Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis.

[0150] The bacterial host cell can also be any Streptococcus cell , including, among others, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis and Streptococcus equi subsp. Zooepidemicus

[0151] The bacterial host cell can also be any Streptomyces cell including, among others, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus and Streptomyces lividans cells .

[0152] The introduction of DNA into a Bacillus cell can, for example, be accomplished by transformation of protoplasts (see, eg, Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), using competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell can, for example, be carried out by transformation of protoplasts (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell can be carried out, for example, by transformation of protoplasts and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Prague) 49: 399-405), by conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585) or by transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell can, for example, be carried out by electroporation (see, eg, Choi et al., 2006, J. Microbiol. Methods 64: 391-397), or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell can, for example, be carried out by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by transformation of protoplasts (see, e.g., Catt and Jollick, 1991, Microbes 68: 189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation (See, eg, Clewell, 1981, Microbiol. Rev. 45: 409-436.) However, any method known in the art can be used to introduce DNA into a host cell.

[0153] The host cell can also be a eukaryotic, such as a mammalian, insect, plant or fungal cell.

[0154] In one aspect, the host cell is a fungal cell. "Fungi" as used herein includes the rows Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, as well as Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8a edition, 1995, CAB International, University Press, Cambridge, United Kingdom).

[0155] In another aspect, the fungal host cell is a yeast cell. "Yeast", as used herein, includes ascosporogenic yeast (Endomycetales), basidiosporogenic yeast and yeast belonging to fungi imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast will be defined as described in Biology and Activities of Yeast (Skinner, FA, Passmore, SM, and Davenport, RR, eds, Soc App Bacteriol Symposium Series No. 9, 1980).

[0156] In another aspect, the yeast host cell is a cell of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia.

[0157] In another aspect, the yeast host cell is a cell of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oiacharomyces lipcharomyces or Yarcharomyces lipcharomyces oiacharomyces.

[0158] In another aspect, the fungal host cell is a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the Eumycota and Oomycota subdivision (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is necessarily aerobic. By contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by germination of a single-celled talus and carbon catabolism can be fermentative.

[0159] In another aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaportheph, Mucor, Necorraix, Necorraix, Necorraix, Mycorrix, Necorraix, Mycorus Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or T richoderma

[0160] In another aspect, the filamentous fungal host cell is a cell of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkanderais ceriporiosis ceriporiosis Ceripus cerisoriois ceriporiosis Ceripus cerpsoriosis Ceripus pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridiousumusus, Fusarium graumusariumusus, Fusarium graumusariumususus, Fusarium graumiumusususus, Fusarium graumiumususus, Fusarium graumiumususus, Fusarium graumusususus, Fusarium graumusususus , Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicolaporamoraphylophoramoraphylamoraphylopusumium, Mythioramoraphylopusumus, Pheromotopus, Pneumotransmitter, Pneumonia chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride

[0161] Fungal cells can be transformed by a process that involves the formation of protoplasts, the transformation of protoplasts and the regeneration of the cell wall in a manner known per se. Suitable procedures for the transformation of Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proc. Natl Acad. Sci. USA 81: 1470-1474. Suitable methods for the transformation of the Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast can be transformed using the procedures described by Becker and Guarente, In Abelson, JN and Simon, MI, eds, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pages 182-187, Academic Press, Inc., NY; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl Acad. Sci. USA 75: 1920

Production methods

[0162] The present invention also relates to methods of producing a variant, comprising: (a) culturing a host cell of the present invention under conditions suitable for expression of the variant; and (b) recover the variant of the culture medium.

[0163] Host cells are cultured in a nutrient medium suitable for variant production using methods known in the art. For example, the cell can be cultured by shaking flask culture and small or large scale fermentations (including continuous fermentations, by batch, by fed or solid-state batch) in laboratory or industrial fermenters performed in a suitable medium and under conditions suitable that allow the polypeptide to be expressed and / or isolated. The culture takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using methods known in the art. Suitable media are available from commercial suppliers or can be prepared in accordance with the published compositions ( e.g., in the catalogs of the American Type Culture Collection). If the polypeptide is secreted in the nutrient medium, it can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0164] In an alternative aspect, the variant is not recovered, but rather a host cell of the present invention expressing a variant is used as the source of the variant.

[0165] The variant can be detected using methods known in the art that are specific to the variants. These detection methods may include the use of specific antibodies, the formation of an enzyme product or the disappearance of an enzyme substrate. For example, an enzymatic assay can be used to determine the activity of the polypeptide as described herein in the examples.

[0166] The variant can be recovered by methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by conventional procedures that include, but are not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

[0167] A variant of the present invention can be purified by a variety of procedures known in the art including, but not limited to, chromatography ( eg, ion exchange, affinity, hydrophobia, chromato-focusing and size exclusion), electrophoretic procedures ( e.g. , preparative isoelectric focusing), differential solubility ( e.g., ammonium sulfate precipitate), SDS-PAGE or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden , editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

Plants

[0168] The present disclosure also refers to plants, e.g. eg, a transgenic plant, a plant part or a plant cell, comprising an isolated polynucleotide encoding a variant of the present invention to express and produce the variant in recoverable amounts. The variant can be recovered from the plant or part of the plant. Alternatively, the plant or part of the plant containing the recombinant variant can be used as such to improve the quality of a food or feed, eg, to improve nutritional value, palatability and rheological properties, or to destroy a antinutritive factor

[0169] The transgenic plant can be dicotyledonous or monocotyledonous. Examples of monocotyledonous plants are grasses, such as poa pratense (blue grass, poa), forage grass such as Festuca, Lolium, temperate grass such as Agrostis and cereals, p. eg, wheat, oats, rye, barley, rice, sorghum and corn.

[0170] Examples of dicotyledonous plants are tobacco, legumes, such as lupines, potatoes, sugar beets, peas, beans and soybeans; and cruciferous plants (family Brassicaceae), such as cauliflower, rapeseed and the closely related model organism of Arabidopsis thaliana.

[0171] Examples of plant parts are stem, callus, leaves, root, fruits, seeds and tubers, as well as the individual tissues comprising these parts, e.g. eg, epidermis, mesophyll, parenchyma, vascular tissues and meristems. Specific compartments of plant cells, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm, are also considered a part of the plant. In addition, any plant cell, regardless of tissue origin, is considered a part of the plant. Similarly, parts of the plant, such as specific isolated cells and tissues to facilitate the use of the invention, are also considered parts of the plant, e.g. eg, embryos, endosperms, aleurone and teguments.

[0172] Descendants of such plants, parts of plants and plant cells are also included within the scope of the present invention.

[0173] The transgenic plant or plant cell that expresses a variant of the present invention can be constructed according to methods known in the art. In summary, the plant cell or plant is constructed by incorporating one or more (several) expression constructs encoding a polypeptide in the host genome of the plant or the chloroplast genome and propagating the resulting modified plant or cell to a plant or cell. transgenic vegetable

[0174] The expression construct is conveniently a nucleic acid construct comprising a polynucleotide encoding a variant of the present invention operably linked to appropriate regulatory sequences required for the expression of the nucleotide sequence in the plant or part of the plant chosen. . In addition, the expression construct may comprise a selectable marker useful for identifying the host cells in which the expression construct has been integrated, and the DNA sequences necessary for the introduction of the construct into the plant in question (the latter depends on the method of introduction of DNA to be used).

[0175] The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, based on when, where and how it is desired to express the variant. For example, the expression of the gene that encodes a variant can be constitutive or inducible, or it can be developmental, phase or tissue specific, and the genetic product can be directed to specific tissues or plant parts such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiol. 86: 506.

[0176] For constitutive expression, promoters 35S-CaMV, ubiquitin 1 from corn and actin 1 from rice can be used (Franck et al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters can be, for example, a promoter of storage sink tissues such as seeds, potato tubers and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or tissue metabolic sink such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a specific seed promoter such as the glutelin, prolamine, globulin or albumin promoters of rice (Wu et al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter of legume B4 and the unknown seed protein gene of Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152: 708 -711), a promoter of a seed oily body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941), the napA storage protein promoter of Brassica napus, or any other specific promoter of seed known in the art, e.g. eg, as described in WO 91/14772. In addition, the promoter can be a leaf-specific promoter such as the rice or tomato rbcs promoter (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the promoter of the adenine methyltransferase virus gene. chlorella (Mitra and Higgins, 1994, Plant Mol. Biol.

26: 85-93), the rice aldP promoter (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or a wound-inducible promoter such as the potato pin2 promoter ( Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Similarly, the promoter can be induced by abiotic treatments such as temperature, dryness or changes in salinity or induced by exogenously applied substances that activate the promoter, e.g. eg, ethanol, estrogens, plant hormones such as ethylene, abscisic acid and gibberellic acid, and heavy metals.

[0177] A promoter enhancer element can also be used to achieve higher expression of a polypeptide in the plant. For example, the promoter enhancer element may be an intron that is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention. For example, Xu et a /., 1993, supra, describe the use of the first intron of the rice actin 1 gene to enhance expression.

[0178] The selectable marker gene and any other part of the expression construct can be chosen from those available in the art.

[0179] The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium- mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio / Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

[0180] Currently, gene transfer mediated by Agrobacterium tumefacienses the method chosen to generate transgenic dicotyledons (for a review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can also be used to transform monocotyledonous, although other transformation methods are often used for these plants. At present, the method chosen to generate transgenic monocotyledons is the bombardment of particles (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant J. 2: 275- 281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992, Bio / Technology 10: 667-674). An alternative method for monocot transformation is based on the transformation of protoplasts as described by Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428. Additional transformation methods for use in accordance with the present disclosure include those described in US Pat. UU. Nos. 6,395,966 and 7,151,204.

[0181] After transformation, the transformants that have incorporated the expression construct are selected and regenerated in whole plants according to methods well known in the art. Often, the transformation procedure is designed for the selective removal of selection genes during regeneration or in subsequent generations using, for example, cotransformation with two separate T-DNA constructs or site-specific cleavage of the selection gene by a specific recombinase.

[0182] In addition to the direct transformation of a particular plant genotype with a construct prepared in accordance with the present invention, transgenic plants can be made by crossing a plant having a construct of the present invention with a second plant lacking the construct. For example, a construct that encodes a variant or a portion thereof can be introduced into a particular plant variety by crossing, without the need to directly transform a plant of that given variety. Therefore, the present invention encompasses not only a plant directly regenerated from cells that have been transformed according to the present invention, but also the progeny of said plants. As used herein, descendant may refer to the offspring of any generation of an original plant prepared in accordance with the present invention. Said offspring may include a DNA construct prepared in accordance with the present invention, or a portion of a DNA construct prepared in accordance with the present invention. Crossing results in a transgene of the present invention that is introduced into a plant line by cross-pollination of a starting line with a donor plant line that includes a transgene of the present invention. Non-limiting examples of such steps are explained in more detail in US Pat. UU. No. 7,151,204.

[0183] Plants can be generated by a backcross conversion process. For example, plants include plants called genotypes, lines, innate or hybrids converted by backcrossing.

[0184] Genetic markers can be used to aid in the introgression of one or more transgenes of the invention from one genetic background to another. Marker-assisted selection offers advantages over conventional reproduction, since it can be used to avoid errors caused by phenotypic variations. In addition, genetic markers can provide data on the relative degree of elite germplasm in the individual offspring of a particular crossing. For example, when a plant with a desired trait that on the other hand has an agronomically undesirable genetic background crosses an elite origin, genetic markers can be used to select the progeny that not only possess the trait of interest, but also have a relatively large proportion of desired germplasm. Proportion of desired germplasm. In this way, the number of generations required for the introgression of one or more traits in a particular genetic background is minimized.

[0185] The present invention also relates to methods for producing a variant of the present invention, comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding a variant of the present invention under favorable conditions for the production of the variant; and (b) recover the variant.

Compositions

[0186] The present invention also relates to compositions comprising an alpha-amylase variant and at least one additional enzyme. The additional enzyme (s) can be selected from the group consisting of beta-amylase, cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase), glucoamylase, hemicellulase ( e.g., xylanase), isoamylase , isomerase, lipase, phytase, protease, pululanase and / or other enzymes useful in a commercial process together with an alpha-amylase. The additional enzyme can also be a second alpha-amylase. Such enzymes are known in the art in starch processing, sugar conversion, fermentation of alcohol and other useful end products, commercial detergents and aids for cleaning, stain removal, treatment or decoupling of tissues, and the like.

Methods of use of alpha-amylase variants: industrial applications

[0187] Variants of the present invention possess valuable properties that allow a variety of industrial applications. In particular, the variants can be used in detergents, in particular laundry detergent compositions and dishwashing detergent compositions, hard surface cleaning compositions, and for decoupling textile products, fabrics or garments, pulp and paper production, production of beer, ethanol production and starch conversion processes.

[0188] Alpha-amylase variants can be used for starch processes, in particular starch conversion, especially starch liquefaction (see, e.g., U.S. Patent No. 3,912,590, EP 252730 and EP 063909, WO 99/19467 and WO 96/28567). Compositions for starch conversion purposes are also contemplated, which in addition to the variant of the invention also comprise AMG, pululanase and other alpha-amylases.

[0189] In addition, the variants are particularly useful in the production of sweeteners and ethanol (see, e.g., Patent No. 5,231,017), as ethanol for fuels, for consumption and for industrial use, from starch or whole grains.

[0190] Variants can also be used for the decoupling of textile products, fabrics and garments (see, e.g., WO 95/21247, U.S. Patent No. 4,643,736 and EP 119920), processing or beer distillation, and in the production of pulp and paper or related processes.

Starch processing

[0191] Native starch consists of microscopic granules, which are insoluble in water at room temperature. When an aqueous suspension of starch is heated, the granules swell and eventually burst, dispersing the starch molecules in the solution. During this "gelatinization" process there is a dramatic increase in viscosity. Since the level of Solids is 30-40 % in a typical industrial process, the starch must be diluted or "liquefied" so that it can be properly processed. This viscosity reduction is mainly achieved by enzymatic degradation in current commercial practice.

[0192] Conventional starch conversion processes, such as liquefaction and saccharification processes, are described, e.g. e.g., in US Pat. UU. No. 3,912,590, EP 252730 and EP 063909.

[0193] In one embodiment, the conversion process that degrades starch to low molecular weight carbohydrate components, such as sugars or fat substitutes, includes a branching step.

[0194] In the case of converting starch into sugar, the starch is depolymerized. Said depolymerization process consists of, e.g. eg, a pretreatment stage and two or three consecutive process stages, that is, a liquefaction process, a saccharification process and, depending on the desired final product, an optional isomerization process.

[0195] When the desired final sugar product is, p. eg, high fructose syrup, dextrose syrup can be converted into fructose. After the saccharification process, the pH is increased to a value in the range of 6-8, p. eg, pH 7.5, and calcium is removed by ion exchange. Dextrose syrup is then converted into high fructose syrup using, e.g. eg, an immobilized glucose isomerase.

Generation of fermentation products.

[0196] In general, the production of alcohol (ethanol) from whole grains can be separated into four main steps: grinding, liquefaction, saccharification and fermentation.

[0197] The grain is ground to open the structure and allow the processing to continue. Two processes used are wet or dry milling. In dry grinding, all the grain is ground and used in the remaining part of the process. Wet milling provides a very good separation of germ and flour (starch and protein granules) and, with some exceptions, is applied in places where there is a parallel production of syrups.

[0198] In the liquefaction process, the starch granules are solubilized to maltodextrins by hydrolysis, mainly of a GP greater than 4. Hydrolysis can be carried out by acidic or enzymatically treatment by means of an alpha-amylase. Acid hydrolysis is used in a limited way. The raw material may be ground whole grain or a lateral flow of starch processing.

[0199] During a typical enzymatic liquefaction, long chain starch is degraded into shorter branched and linear units (maltodextrins) by an alpha-amylase. Enzymatic liquefaction is generally carried out as a three-stage hot suspension process. The suspension is heated to a temperature of 60-95 ° C ( e.g., 77-86 ° C, 80-85 ° C, or 83-85 ° C) and the enzyme (s) is added ( n). The liquefaction process is carried out at 85 ° C for 1-2 hours. The pH is generally between 5.5 and 6.2. In order to ensure optimal enzyme stability under these conditions, 1 mM calcium is added (to provide approximately 40 ppm of free calcium ions). After such treatment, the liquefied starch will have a "dextrose equivalent" (SD) of 10-15.

[0200] The suspension is subsequently sprayed at a temperature of 95-140 ° C, p. For example, 105-125 ° C, cooled to 60-95 ° C and more enzymes were added to obtain the final hydrolysis. The liquefaction process is carried out at pH 4.5-6.5, typically at a pH between 5 and 6. The ground and liquefied grain is also known as a mixture.

[0201] The material containing liquefied starch is saccharified in the presence of saccharifying enzymes such as glucoamylases. The saccharification process can last from 12 to 120 hours ( e.g., from 12 to 90 hours, from 12 to 60 hours and from 12 to 48 hours).

[0202] However, it is common to perform a pre-carification stage for about 30 minutes to 2 hours ( eg, 30 to 90 minutes) at a temperature of 30 to 65 ° C, typically around 60 ° C, followed of complete saccharification during fermentation, which is called simultaneous saccharification and fermentation (SSF). The pH is usually between 4.2-4.8, p. eg, pH 4.5. In a simultaneous process of saccharification and fermentation (SSF), there is no retention stage for saccharification, but rather yeast and enzymes are added together.

[0203] In a typical saccharification process, the maltodextrins produced during liquefaction are converted to dextrose by the addition of a glucoamylase and a de-branching enzyme, such as an isoamylase (U.S. Patent No. 4,335,208) or a pululanase . The temperature drops to 60 ° C, before the addition of glucoamylase and the de-branching enzyme. The saccharification process is carried out for 24-72 hours.

[0204] Before the addition of saccharifying enzymes, the pH is reduced to less than 4.5, while maintaining a high temperature (above 95 ° C), to inactivate the alpha-amylase in liquefaction. This process reduces the formation of a short oligosaccharide called a "panosa precursor," which cannot be adequately hydrolyzed by the de-branching enzyme. Normally, about 0.2-0.5% of the saccharification product is the branched bony trisaccharide (Glc pa1-6Glc pa1-4Glc), which cannot be degraded by a pululanase. If the active amylase of liquefaction remains present during saccharification ( that is , there is no denaturation), the amount of bog can be up to 1-2%, which is highly undesirable since it significantly decreases the yield of the saccharification.

[0205] Fermentable sugars ( eg, dextrins, monosaccharides, particularly glucose) are produced from enzymatic saccharification. These fermentable sugars can be purified and / or become useful sugar products. In addition, sugars can be used as fermentation raw material in a microbial fermentation process to generate end products, such as alcohol ( e.g., ethanol and butanol), organic acids ( e.g., succinic acid and lactic acid), sugar alcohols ( e.g., glycerol), ascorbic acid intermediates ( e.g., gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate and 2-keto-L-gulonic acid), amino acids ( e.g., lysine), proteins ( e.g. ., antibodies and fragments thereof).

[0206] In one embodiment, the fermentable sugars obtained during the stages of the liquefaction process are used to produce alcohol and particularly ethanol. In the production of ethanol, an SSF process is commonly used in which saccharifying enzymes and fermenting organisms ( e.g., yeast) are added at the same time and then carried out at a temperature of 30-40 ° C.

[0207] The organism used in the fermentation will depend on the desired final product. Typically, if ethanol is the desired end product, the yeast will be used as a fermentation organism. In some preferred embodiments, the ethanol producing microorganism is a yeast and specifically Saccharomyces such as S. cerevisiae strains (U.S. Patent No. 4,316,956). A variety of S. cerevisiae are commercially available and include, among others, FALI (Fleischmann's Yeast), SUpErSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) and Angel alcohol yeast (Angel Yeast Company, China). The amount of starting yeast used in the methods is an amount effective to produce a commercially significant amount of ethanol in an appropriate amount of time, ( e.g., to produce at least 10% ethanol from a substrate that have between 25-40% of DS in less than 72 hours). Yeast cells are generally supplied in amounts of about 104 to about 1012, and preferably about 107 to about 1010 of viable yeast count per ml of fermentation broth. After adding the yeast to the mixture, it is typically subjected to fermentation for approximately 24-96 hours, e.g. eg, 35-60 hours. The temperature is between about 26-34 ° C, typically at about 32 ° C, and the pH is pH 3-6, p. eg, approximately pH 4-5.

[0208] Fermentation may include, in addition to a fermenting microorganism ( eg, yeast), additional nutrients and enzymes, including phytases. The use of yeast in fermentation is well known in the art.

[0209] In other embodiments, the use of appropriate fermentation microorganisms, as is known in the art, may result in a final fermentation product that includes, e.g. eg, glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lactic acid, amino acids and derivatives of the same. More specifically when lactic acid is the desired end product, a Lactobacillus sp. ( L. Casei); when glycerol or 1,3-propanediol are the desired end products E. coli can be used; and when the desired end products are 2-keto-D-gluconate, 2,5-diketo-D-gluconate and 2-keto-L-gulonic acid, Pantoea citrea can be used as a fermenting microorganism. The list listed above is composed only of examples and one skilled in the art will know a multitude of fermenting microorganisms that can be used to obtain a desired final product.

Processes to generate fermentation products from non-gelatinized material containing starch.

[0210] The disclosure refers to processes for generating fermentation products from starch-containing material without gelatinization ( ie, uncooked) of starch-containing material. The fermentation product, for example, ethanol, can be produced without liquefying the aqueous suspension containing water and the material containing starch. In one embodiment, a process of the invention includes saccharifying the starch-containing material ( e.g., ground), e.g. eg, granular starch, below the initial gelatinization temperature, preferably in the presence of alpha-amylase and / or enzymes generating carbohydrate sources to produce sugars that can be fermented to give rise to the fermentation product by a suitable fermenting organism . In this embodiment, the desired fermentation product, e.g. For example, ethanol is produced from cereal grains, such as corn, preferably ground, not gelatinized ( that is , uncooked). Accordingly, in the first aspect, the invention relates to processes for generating fermentation products from starch-containing material that comprise simultaneously saccharifying and fermenting the starch-containing material using a carbohydrate-generating enzyme and a fermentation organism. at a temperature below the initial gelatinization temperature of said starch-containing material. In one embodiment a protease is also present. The protease can be any acidic fungal protease or metalloprotease. The fermentation product, p. eg, ethanol, optionally can be recovered after fermentation, e.g. eg, by distillation. Normally, amylase (s), such as glucoamylase (s) and / or other enzymes generating carbohydrate sources, and / or alpha-amylase (s), is present during fermentation Examples of glucoamylases and other enzymes that generate carbohydrate sources include glucoamylases that hydrolyze raw starch. Examples of alpha-amylases include acidic alpha-amylases, such as acidic fungal alphaamylases. Examples of fermenting organisms include yeast e.g. eg, a strain of Saccharomyces cerevisiae. The term "initial gelatinization temperature" means the lowest temperature at which starch gelatinization begins. In general, water-heated starch begins to gelatin between approximately 50 ° C and 75 ° C; The exact temperature of the gelatinization depends on the specific starch and can easily be determined by one skilled in the art. Therefore, the initial gelatinization temperature may vary according to the plant species, the particular variety of the plant species and the growth conditions. In the context of this invention, the initial gelatinization temperature of a given starch-containing material can be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein and Lii, 1992 , Starch / Starke 44 (12): 461-466. Before starting the process, a suspension of starch-containing material, such as granular starch, having 10-55% w / w dry solids (DS), preferably 25-45% w / w solids, can be prepared dried, more preferably 30-40% w / w dry solids of starch-containing material. The suspension may include water and / or process water, vinegar (distillation residue), sewage water, condensate or evaporator distillate, water resulting from distillation or process water from other fermentation product plants. Because the process of the invention is carried out below the initial gelatinization temperature, and therefore, a significant increase in viscosity does not occur, high levels of distillation residues can be used if desired. In one embodiment, the aqueous suspension contains from about 1 to about 70% vol. , preferably 15-60% vol. , especially about 30 to 50% vol. of water and / or process water, vinasse (distillation residue), sewage water, condensate or evaporator distillate, water resulting from distillation or process water from other plants fermentation products, or combinations thereof or the like. The starch-containing material can be prepared by reducing the particle size, preferably by dry or wet grinding, to 0.05 to 3.0 mm, preferably 0.1 0.5 mm. After having undergone a process of the invention, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or preferably at least 99% of the dried solids in the starch-containing material are converted into a soluble starch hydrolyzate. A process in this aspect of the invention is carried out at a temperature below the initial gelatinization temperature, which means that the temperature is typically in the range between 30-75 ° C, preferably between 45-60 ° C . In a preferred embodiment, the process is carried out at a temperature of 25 ° C to 40 ° C, such as 28 ° C to 35 ° C, such as 30 ° C to 34 ° C, preferably around 32 ° C. In one embodiment, the process is carried out so that the sugar level, for example, the glucose level, is maintained at a low level, such as below 6% w / w, such as below about 3% w / w, such as below about 2% w / w, such as below about 1% w / w, such as below about 0.5% w / w or below 0.25% p / p, such as below about 0.1% p / p. Such low sugar levels can be achieved simply by using tight amounts of enzymes and fermenting organisms. One skilled in the art can easily determine which dose / amounts of enzyme and fermenting organism to use. The amounts of enzyme and fermenting organism can also be selected to maintain low concentrations of maltose in the fermentation broth. For example, the level of maltose can be maintained below about 0.5% w / w, such as below about 0.2% w / w. The process of the invention can be carried out at a pH of about 3 and 7, preferably a pH of 3.5 to 6, or more preferably a pH of 4 to 5. In one embodiment, fermentation takes place over a period of 6 at 120 hours, in particular from 24 to 96 hours.

Processes to generate fermentation products from gelatinized material containing starch.

[0211] In this aspect, the invention relates to processes for generating fermentation products, especially ethanol, from a starch-containing material, a process that includes a liquefaction stage and fermentation and saccharification stages sequentially or simultaneously. Accordingly, the invention relates to processes for generating fermentation products from a starch-containing material comprising the steps of:

(a) liquefaction of the starch-containing material in the presence of an alpha-amylase variant, or

(b) saccharification of the liquified material obtained in step (a) using an enzyme generating carbohydrate sources;

(c) fermentation using a fermenting organism.

[0212] In one aspect, a pululanase, such as a family of GH57 pululanase, is also used in the liquefaction stage. In one embodiment, a protease, such as an acidic fungal protease or a metalloprotease, is added before, during and / or after liquefaction. In one embodiment, the metalloprotease is derived from a strain of Thermoascus, for example, a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670. In one embodiment, the carbohydrate-generating enzyme is a glucoamylase derived from a strain. of Aspergillus, for example, Aspergillus niger or Aspergillus awamori, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia especially Athelia rolfsii; a strain of Trametes, for example, Trametes cingulata; a strain of the genus Pachykytospora, for example, a strain of Pachykytospora papyracea; or a strain of the genus Leucopaxillus, for example, Leucopaxillus giganteus; or a strain of the genus Peniophora, for example, a strain of the species Peniophora rufomarginata; or a mixture thereof. The saccharification stage (b) and the fermentation stage (c) can be carried out sequentially or simultaneously. A pululanase and / or a metalloprotease can be added during saccharification and / or fermentation when the process is carried out as a sequential saccharification and fermentation process and before or during fermentation when steps (b) and (c) are carried out. simultaneously (SSF process). Pululanase and / or metalloprotease can also be added favorably before liquefaction (pre-liquefaction treatment), that is, before step (a) or during it, and / or after liquefaction (post-liquefaction treatment), that is, after step (a). Pululanase is added more favorably before or during liquefaction, that is, before or during step (a). The fermentation product, especially ethanol, can optionally be recovered after fermentation, e.g. eg, by distillation. The fermenting organism is preferably yeast, preferably a strain of Saccharomyces cerevisiae. In a particular embodiment, the process of the invention further comprises, before step (a), the steps of:

x) reduce the particle size of the starch-containing material, preferably by grinding ( eg, using a hammer mill);

and) form a suspension comprising water and the starch-containing material.

[0213] In one embodiment, the particle size is smaller than a mesh 7, p. eg, a 6 mesh. A 7 mesh is generally used in conventional prior art processes. The aqueous suspension may contain 10-55, e.g. eg, 25-45 and 30-40,% w / w dry solids (DS) of the starch-containing material. The suspension is heated above the gelatinization temperature and a variant of alpha-amylase can be added to start liquefaction (dilution). In one embodiment, the suspension can be sprayed to further gelatinize the suspension before being subjected to alpha-amylase in step (a). In one embodiment, the liquefaction can be carried out as a three-stage hot suspension process. The suspension is heated to between 60-95 ° C, preferably between 70-90 ° C, preferably between 80-85 ° C at pH 4-6, preferably 4.5-5.5, and the alpha-amylase variant is added, optionally together with a pululanase and / or protease, preferably metalloprotease, to initiate liquefaction (dilution). In one embodiment, the suspension can be sprayed at a temperature between 95-140 ° C, preferably 100-135 ° C, such as 105-125 ° C, for about 1-15 minutes, preferably for about 3-10 minutes , especially about 5 minutes. The suspension is cooled to 60-95 ° C and more alpha-amylase variant is added and, optionally, variant of pululanase and / or protease, preferably metalloprotease, is added to end hydrolysis (secondary liquefaction). The liquefaction process is generally carried out at pH 4.0-6, in particular at a pH of 4.5 to 5.5. The saccharification step (b) can be carried out using conditions well known in the art. For example, a complete saccharification process can last from about 24 to about 72 hours, however, it is common to do a precarification typically 40 to 90 minutes at a temperature between 30 and 65 ° C, typically around 60 ° C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). The saccharification is typically performed at temperatures of 20-75 ° C, preferably 40-70 ° C, typically around 60 ° C, and at a pH between 4 and 5, usually around pH 4.5. The most widely used process to generate a fermentation product, especially ethanol, is a simultaneous process of saccharification and fermentation (SSF), in which there is no retention stage for saccharification, which means that a fermenting organism, such as Yeast, and enzyme (s), can be added together. The SSF can typically be carried out at a temperature of 25 ° C to 40 ° C, such as 28 ° C to 35 ° C, such as 30 ° C to 34 ° C, preferably approximately 32 ° C. In one embodiment, the fermentation lasts from 6 to 120 hours, in particular 24 to 96 hours.

Brewing

[0214] Alpha-amylase variants can also be used in a brewing and similar fermentation process; Alpha-amylases will typically be added during the maceration process. The process is substantially similar to the milling, liquefaction, saccharification and fermentation processes described above.

Processing a starch suspension with vinasse

[0215] The ground material containing starch is combined with water and recycled diluted vinegar, resulting in an aqueous suspension. The suspension may comprise between 15 and 55% w / w DS ( e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to 40%, 20 to 35% and from 30 to 36% DS). In some embodiments, the recycled diluted vinegar (distillation residue) is in the range of about 10 to 70% v / v ( e.g., 10 to 60%, 10 to 50%, 10 to 40% , from 10 to 30%, from 10 to 20%, from 20 to 60%, from 20 to 50%, from 20 to 40% and also from 20 to 30%).

[0216] Once the ground material containing starch is combined with water and distillation residue, the pH is not adjusted in the suspension. In addition, the pH is not adjusted after adding a phytase and, optionally, an alpha-amylase to the suspension. In one embodiment, the pH of the suspension will be in the range of about pH 4.5 to about less than pH 6.0 ( e.g., pH 4.5 to 5.8, pH 4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5 and pH 5.2 to 5.9). The pH of the suspension may be between approximately pH 4.5 and 5.2, depending on the amount of diluted vinegar added to the suspension and the type of material comprising the diluted vinegar. For example, the pH of the diluted vinegar can be between pH 3.8 and pH 4.5.

[0217] During ethanol production, acids can be added to lower the pH in the beer well, to reduce the risk of microbial contamination before distillation.

[0218] In some embodiments, a phytase is added to the suspension. In other embodiments, in addition to phytase, an alpha-amylase is added to the suspension. In some embodiments, a phytase and an alpha-amylase are sequentially added to the suspension. In other embodiments, a phytase and an alpha-amylase are added simultaneously. In some embodiments, the suspension comprising a phytase and, optionally, an alpha-amylase, is incubated (pretreated) for a period of about 5 minutes to about 8 hours ( e.g., 5 minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours and 15 minutes to 4 hours). In other embodiments, the suspension is incubated at a temperature in the range of about 40 to 115 ° C ( e.g., 45 to 80 ° C, 50 to 70 ° C, 50 to 75 ° C, 60 at 110 ° C, 60 to 95 ° C, 70 to 110 ° C, 70 to 85 ° C and 77 to 86 ° C).

[0219] In other embodiments, the suspension is incubated at a temperature of about 0 to about 30 ° C ( eg, 0 to 25 ° C, 0 to 20 ° C, 0 to 15 ° C, of 0 to 10 ° C and 0 to 5 ° C) below the starch gelatinization temperature of the starch-containing material. In some embodiments, the temperature is below about 68 ° C, below about 65 ° C, below about 62 ° C, below about 60 ° C and below about 55 ° C. In some embodiments, the temperature is greater than approximately 45 ° C, greater than approximately 50 ° C, greater than approximately 55 ° C and greater than approximately 60 ° C. In some embodiments, incubation of the suspension comprising a phytase and an alpha-amylase at a temperature below the gelatinization temperature of starch is called primary liquefaction (1 °).

[0220] In one embodiment, the ground material containing starch is corn or millet. The suspension comprises from 25 to 40% of DS, the pH is in the range of 4.8 to 5.2, and the suspension is incubated with a phytase and, optionally, an alpha-amylase for a time of 5 minutes to 2 hours, in a temperature range from 60 to 75 ° C.

[0221] Currently, it is believed that commercially available microbial alpha-amylases used in the liquefaction process are generally not stable enough to produce a substrate of liquefied starch from a dry milling process using whole grain ground temperature above about 80 ° C at a pH level that is less than pH 5.6. The stability of many commercially available alpha-amylases is reduced to a pH of less than about 4.0.

[0222] At an additional liquefaction stage, the starch-containing or pretreated material is exposed to an increase in temperature, such as from about 0 to about 45 ° C above the starch gelatinization temperature of the starch-containing material ( e.g., 70 ° C to 120 ° C, 70 ° C to 110 ° C and 70 ° C to 90 ° C) for a period of time from about 2 minutes to about 6 hours (for example, 2 minutes to 4 hours, 90 minutes, 140 minutes and 90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1 hour and 2 hours. The temperature can be increased by a conventional high temperature jet cooking system for a short period of time, for example, from 1 to 15 minutes. The starch can then be further hydrolyzed at a temperature that varies between approximately 75 ° C and 95 ° C ( e.g., from 80 ° C to 90 ° C and from 80 ° C to 85 ° C) over a period of approximately 15 to 150 minutes ( e.g., 30 to 120 minutes). In a preferred embodiment, the pH is not adjusted during these process steps and the pH of the liquefied mixture is in the range of approximately pH 4.0 to pH 5.8 ( eg, pH 4.5 to 5.8, pH 4.8 to 5.4 and pH 5.0 to 5.2). In some embodiments, a second dose of thermostable alpha-amylase is added to the secondary liquefaction stage, but in other embodiments there is no additional dose of alpha-amylase.

[0223] The incubation and liquefaction steps may be followed by saccharification and fermentation steps well known in the art.

Distillation

[0224] Optionally, after fermentation, an alcohol ( eg, ethanol) can be extracted by, for example, distillation and, optionally, following one or more process steps.

[0225] In some embodiments, the ethanol production generated by the methods provided herein is at least 8%, at least 10%, at least 12%, at least 14%, at least 15% , at least 16%, at least 17% and at least 18% (v / v) and at least 23% v / v. The ethanol obtained in accordance with the process provided herein can be used, for example, as fuel ethanol, drinking ethanol, that is, drinking neutral alcohols, or industrial ethanol.

By-products

[0226] What is left of the fermentation is the grain, which is normally used in animal feed in liquid or dry form. In further embodiments, the final product may include fermentation co-products such as dry distillery grains (DDG) and dry distillery grains with soluble (DDGS), which can be used, for example, as animal feed.

[0227] Other details on how to carry out liquefaction, saccharification, fermentation, distillation and recovery of ethanol are well known to the experts.

[0228] According to the process provided herein, saccharification and fermentation can be carried out simultaneously or separately.

Pulp and paper production

[0229] Alpha-amylase variants can also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced paper and cardboard, especially when new pulp production occurs at a pH greater than 7 and when the amylases facilitate the disintegration of the waste material by degradation of the reinforcing starch. Alpha-amylase variants are especially useful in a process for producing a papermaking pulp from printed paper coated with starch. The process can be performed as described in WO 95/14807 and comprises the following steps:

a) disintegrate the paper to produce a pulp,

b) dealing with an enzyme that degrades starch before, during or after step a), and

c) separate the ink particles from the pulp after steps a) and b).

[0230] Alpha-amylase variants may also be useful for modifying starch when enzymatically modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alpha-amylase variants it is possible to modify the starch in the presence of the filler, which allows a simpler integrated process.

Decoupling of fabrics, fabrics and garments.

[0231] Alpha-amylase variants can also be very useful in decoupling fabrics, fabrics or garments. In the textile processing industry, alpha-amylases are traditionally used as auxiliaries in the decoupling process to facilitate the removal of the glue containing starch, which has served as a protective coating on the weft threads when knitting. Complete removal of the glue coating after weaving is important to ensure optimal results in subsequent processes, in which the fabric is washed, discolored and dyed. The enzymatic decomposition of starch is preferred because it does not imply any detrimental effect on the fiber material. To reduce the cost of processing and increase the grinding performance, the decoupling process is sometimes combined with the washing and bleaching stages. In such cases, non-enzymatic auxiliaries, such as alkaline or oxidizing agents, are typically used to break down starch, because traditional alpha-amylases are not very compatible with high pH levels and bleaching agents. The non-enzymatic decomposition of the starch glue causes some damage to the fiber due to the quite aggressive chemicals used. Consequently, it would be desirable to use the alpha-amylase variants, since they have improved performance in alkaline solutions. Alpha-amylase variants can be used alone or in combination with a cellulase when the cellulose-containing fabric or fabric is unclogged.

[0232] The decoupling and bleaching processes are well known in the art. These processes are described on p. eg, WO 95/21247, US Pat. UU. No. 4,643,736, EP 119920.

Cleaning processes and detergent compositions.

[0233] Alpha-amylase variants can be added as a component of a detergent composition for various cleaning or washing processes, including laundry and dishwashing. For example, the variants can be used in the detergent compositions described in WO 96/23874 and WO 97/07202.

[0234] Alpha-amylase variants can be incorporated into detergents with conventionally used concentrations. For example, a variant of the invention can be incorporated in an amount corresponding to 0.00001-10 mg (calculated as pure active enzyme protein) of alpha-amylase per liter of washing liquid / dishwashing using conventional detergent dosage levels.

[0235] The detergent composition may be formulated, for example, as a detergent composition for hand or machine laundry, which includes an additive composition for laundry suitable for pretreatment of stained fabrics and a fabric softening composition, or it may be formulated as a detergent composition for use in general cleaning operations of hard surfaces in the home, or formulated for dishwashing operations by hand or machine.

[0236] The detergent composition may further comprise another enzyme (s), such as lipase, peroxidase, protease, another amylolytic enzyme, e.g. eg, another alpha-amylase, glucoamylase, maltogenic amylase, CGTase, cellulase, mannanase (such as Mannaway ™ from Novozymes, Denmark), pectinase, pectin lyase, cutinase and / or laccase.

[0237] In general, the properties of the chosen enzyme (s) must be compatible with the selected detergent ( ie, optimum pH, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the Enzyme (s) must be present in effective amounts.

[0238] The detergent enzymes or enzymes may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all these enzymes. A detergent additive, e.g. For example, a separate additive or a combined additive can be formulated, for example, as a granulate, a liquid, a suspension, etc. Preferred detergent additive formulations are granules, in particular non-powder granules, liquids, in particular stabilized liquids or suspensions.

[0239] Non-powdered granules can be produced, e.g. eg, as described in US Pat. UU. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly (oxyethylene) products (polyethylene glycol, PEG) with average molar weights of 1000 to 20,000; ethoxylated nonylphenols having 16 to 50 units of ethylene oxide; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and in which there are 15 to 80 units of ethylene oxide; fatty alcohols; fatty acids; and mono-, di- and triglycerides of fatty acids. Examples of coating materials that form films suitable for application by fluidized bed techniques are given GB 1483591. Liquid enzyme preparations can, for example, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to The established methods. Protected enzymes can be prepared according to the method described in EP 238216.

[0240] The detergent composition may be in any convenient form, e.g. eg, a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent can be aqueous, typically with a content of up to about 70% water and 0 to about 30% organic solvent, or non-aqueous.

[0241] The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and / or anionic and / or cationic and / or zwitterionic. Surfactants are typically present at a level of about 0.1% to 60% by weight.

[0242] When included, the detergent will generally contain from about 1% to about 40% of an anionic surfactant, such as linear alkylbenzenesulfonate, alpha-olefin sulfonate, alkyl sulfate (fatty alcohol sulfate), ethoxy sulfate alcohol, secondary alkanesulfonate, alpha sulfonated fatty acid methyl ester, alkyl or alkenyl succinic acid or soap.

[0243] When included, the detergent will generally contain from about 0.2% to about 40% of a non-ionic surfactant, such as alcohol ethoxylate, nonylphenol ethoxylate, alkyl polyglucoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, monoethanolamide of fatty acid, polyhydroxy fatty acid alkylamide or N-acyl derivatives N-alkyl glucosamine ("glucamides").

[0244] The detergent may contain from 0 to about 65% of a detergent enhancing or complexing agent, such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl or alkenyl succinic acid, soluble silicates or layered silicates ( e.g., Hoechst SKS-6).

[0245] The detergent may comprise one or more polymers. Examples are carboxymethyl cellulose, poly (vinyl pyrrolidone), poly (ethylene glycol), poly (vinyl alcohol), N-oxide of (poly) vinyl pyridine, poly (vinylimidazole), polycarboxylates such as polyacrylates, copolymers of maleic and acrylic acid and lauryl methacrylate / Acrylic acid copolymers.

[0246] The detergent may contain a bleaching system, which may comprise a source of H2O2 such as perborate or percarbonate that can be combined with a peracid-forming bleach activator, such as tetraacetylethylenediamine or nonanoyl oxybenzene sulphonate. Alternatively, the bleaching system may comprise peroxyacids, for example, of the amide, imide or sulfone type.

[0247] The enzyme (s) of the detergent composition can be stabilized using conventional stabilizing agents, e.g. eg, a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid or a boric acid derivative, e.g. eg, an aromatic borate ester or a phenylboronic acid derivative such as 4-formylphenylboronic acid, and the composition can be formulated as described in, e.g. eg, WO 92/19708 and WO 92/19709.

[0248] The detergent may also contain other conventional detergent ingredients such as, e.g. eg, fabric conditioners that include clays, foam booster, foam suppressants, anti-corrosive agents, soil suspension agents, agents against dirt redeposition, colorants, bactericides, optical brighteners, hydrotropes, tarnish inhibitors or perfumes.

[0249] The detergent compositions may comprise any enzyme in an amount corresponding to 0.01-100 mg of enzymatic protein per liter of washing liquid, preferably 0.055 mg of enzymatic protein per liter of washing liquid, in particular 0.1-1 mg of protein. enzymatic per liter of washing liquid.

[0250] One or more of the enzyme variants described herein may also be incorporated into the detergent formulations described in WO 97/07202.

[0251] This description includes more details in the following examples.

Examples

Assay to determine residual alpha-amylase activity

[0252] The residual alpha-amylase activity is determined by a method that uses the EnzChek® substrate. The substrate in the EnzChek® Ultra amylase assay kit (E33651, Invitrogen, La Jolla, CA, USA) is a derivative of corn starch, DQ ™ starch, which is corn starch labeled with BODIPY® FL dye to a degree that fluorescence is deactivated.

[0253] A vial containing approx. 1 mg of lyophilized substrate is dissolved in 100 microliters of 50 mM sodium acetate (pH 4.0). The vial is vortexed for 20 seconds and left at room temperature, in the dark, with occasional mixing until dissolved. Then 900 microliters of 100 mM acetate, 0.01% (w / v) TRITON® X100, CaCl20.12 mM, pH 5.5 are added, vortexed and stored at room temperature, in the dark until ready to use. The substrate functional solution is prepared by diluting 10 times in residual activity buffer (100 mM acetate, 0.01% (w / v) TRITON® X100, CaCl20.12 mM, pH 5.5) which gives a substrate concentration of 100 micrograms / ml. Immediately after incubation, the enzyme is diluted to a concentration of 15 ng enzyme protein / ml in 100 mM acetate, 0.01% (w / v) TRITON® X100, CaCl20.12 mM, pH 5.5.

[0254] For the assay, 25 microliters of the functional solution of the substrate are mixed for 10 seconds with 25 microliters of the enzyme diluted in a 384-well black microtiter plate. The fluorescence intensity is measured (excitation: 485 nm, emission: 555 nm) once per minute for 15 minutes in each well at 25 ° C and Vmax. It is calculated as the slope of the fluorescence intensity plot versus time. The graph should be linear and the residual activity test has been adjusted so that the reference diluted enzyme solution is within the linear range of the activity test.

Glucoamylase Activity (AGU)

[0255] A glucoamylase unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromol of maltose per minute under standard conditions of 37 ° C, pH 4.3, substrate: 23.2 mM maltose, buffer: 0.1 M acetate, time reaction 5 minutes.

[0256] A self-analysis system can be used. Mutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present becomes beta-D-glucose. Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming NADH, which is determined using a photometer at 340 nm as a measure of the original glucose concentration.

Example 1

Stability of alpha-amylase variants

[0257] The stability of a reference alpha-amylase ( Bacillus stearothermophilus alpha-amylase with mutations I181 * G182 * N193F truncated to 491 amino acids (SEQ ID #: 6)) and its alpha-amylase variants was determined incubating the reference alpha-amylase and variants at pH 4.5 and 5.5 and temperatures of 75 ° C and 85 ° C with CaCl20.12 mM and then determining the residual activity using the EnzChek® substrate (amylase assay kit EnzChek® Ultra, E33651, Molecular Probes).

[0258] The purified enzyme samples were diluted to working concentrations of 0.5 and 1 or 5 and 10 ppm (micrograms / ml) in enzyme dilution buffer (10 mM acetate, 0.01% Triton X100, CaCl20.12 mM, pH 5.0). Twenty microliters of enzyme sample were transferred to a 48-well MTP PCR and 180 microliters of stability buffer (150 mM acetate, 150 mM MES, 0.01% Triton X100, CaCl20.12 mM, pH 4.5 or 5.5) to each well and mixed. The assay was performed using two enzyme concentrations in duplicates. Before incubation at 75 ° C or 85 ° C, 20 microliters were removed and stored on ice as control samples. Incubation was performed in a PCR machine at 75 ° C and 85 ° C.

[0259] After incubation, the samples were diluted to 15 ng / ml in a buffer of residual activity (100 mM acetate, 0.01% Triton X100, 0.12 mM CaCl2, pH 5.5) and 25 microliters of diluted enzyme were transferred to a black 384 well microtiter plate. Residual activity was determined using the EnzChek substrate by adding 25 microliters of substrate solution (100 micrograms / ml) to each well. Fluorescence was determined every minute for 15 minutes using an excitation filter at 485-P nm and an emission filter at 555 nm (the fluorescence reader is Polarstar, BMG). The residual activity was normalized to the control samples for each configuration.

[0260] Assuming that the logarithmic half-life (T / (min)) has been calculated using the equation: T / (min) = T (min) * LN (0.5) / Ln (% RA / 100), where T is the incubation time of the test in minutes, and% RA is the% of residual activity determined in the test.

[0261] Using this assay configuration, the half-life for the reference alpha-amylase and its variant was determined, as shown in Table 1.

Table 1

[0262] The results demonstrate that the alpha-amylase variants have a significantly higher half-life and stability than the reference alpha-amylase.

Example 2

Ethanol production using alpha-amylase variants

[0263] Four small-scale purees of the reference alpha-amylase and two alpha-amylase variants described in example 1 were prepared as follows: approximately 54 g of ground corn, approximately 51 g of tap water and approximately 45 g of distillation residue were mixed in a 250 ml plastic bottle to a total weight of the suspension of 150 g. The pH of the corn suspension was adjusted to 4.5. Enzymes were added to the suspensions with 2 micrograms of amylase per gram of dry solids. For liquefaction, the alpha-amylases were added to the bottles and the bottles were thoroughly mixed and placed in a preheated water bath at 85 ° C. The samples were kept in the water bath for 2 hours at pH 4.5 while stirring every 10 minutes for the first 30 minutes and then every 30 minutes for the rest of the 2-hour liquefaction. The samples were then cooled in a bath of

Claims (13)

1. Isolated alpha-amylase variant, which includes a substitution in three or more positions corresponding to any of positions 59, 89, 91.96, 108, 112, 129, 157, 165, 166, 168, 171, 177 , 179, 180, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416 and 427, in particular, in the positions corresponding to positions 129, 177 and 179, in which the variant has at least 90% and less than 100% sequence identity with the mature polypeptide of SEQ ID #: 6, and the variant has alpha-amylase activity, and where the variant comprises a set of substitutions, using SEQ ID #: 1 for numbering, consisting of: E129V K177L R179E, and in which the alterations of I181 * G182 * N193F present in SEQ ID #: 6 and the variants have improved thermostability compared to the alpha-amylase described as SEQ ID #: 6. 2. A variant according to claim 1, wherein the progenitor alpha-amylase has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and 100% sequence identity with the mature polypeptide of SEQ ID. No .: 6. 3. Variant according to any of claims 1-2, wherein the variant consists of 483 to 515, 483 to 493 or 483 to 486 amino acids. 4. Composition comprising a variant according to any of claims 1-3 and one or more enzymes selected from the group consisting of beta-amylase, cellulase, beta-glucosidase, cellobiohydrolase and endoglucanase, glucoamylase, hemicellulase, xylanase, isoamylase, isomerase, lipase, phytase, protease and pululanase. 5. Use of a variant according to any of claims 1-3 to liquefy a starch-containing material. 6. Method for producing liquefied starch, which comprises liquefying a starch-containing material with a variant according to any of claims 1-3. 7. Process of generating a fermentation product, which includes to. liquefying a starch-containing material with a variant of any one of claims 1 to 3 to produce a liquefied mixture; b. saccharify the liquefied mixture to produce fermentable sugars; Y C. ferment the fermentable sugars in the presence of a fermenting organism. 8. Process for generating a fermentation product, comprising contacting a starch substrate with a variant according to any one of claims 1-3, a glucoamylase and a fermenting organism. 9. Isolated polynucleotide encoding the variant according to any of claims 1-3. 10. Nucleic acid construct comprising the polynucleotide according to claim 9. 11. Expression vector comprising the nucleic acid construct according to claim 10. 12. Host cell comprising the nucleic acid construct according to claim 10. 13. Method for producing an alpha-amylase variant, comprising: to. culturing the host cell according to claim 12 under conditions suitable for expression of the variant; and b. recover the variant of the culture medium.