Patent Description:
The present invention relates to methods for screening alpha-amylases having high wash performance at low temperature.

Alpha-amylases (alpha-<NUM>,<NUM>-glucan-<NUM>-glucanohydrolases, E. <NUM>) constitute a group of enzymes, which catalyses hydrolysis of starch and other linear and branched <NUM>,<NUM>-gluosidic oligo- and polysaccharides.

There is a long history of industrial use of alpha-amylases in several known applications such as detergent, baking, brewing, starch liquefaction and saccharification e.g. in preparation of high fructose syrups or as part of ethanol production from starch. These and other applications of alpha-amylases are known and utilize alpha-amylases derived from microorganisms, in particular bacterial alpha-amylases.

Among the first bacterial alpha-amylases to be used were an alpha-amylase from B. licheniformis, also known as Termamyl which have been extensively characterized and the crystal structure has been determined for this enzyme. Alkaline amylases, such as SP707, form a particular group of alpha-amylases that have found use in detergents. Many of these known bacterial amylases have been modified in order to improve their functionality in a particular application.

Methods of increasing the thermostability of alpha-amylases have been well studied. Suzuki et al. (<NUM>) disclose chimeric alpha-amylases, in which specified regions of a B. amyloliquefaciens alpha-amylase have been substituted for the corresponding regions of a B. licheniformis alpha-amylase. The chimeric alpha-amylases were constructed with the purpose of identifying regions responsible for thermostability. Such regions were found to include amino acid residues <NUM>-<NUM> and amino acid residues <NUM>-<NUM> of the B. amyloliquefaciens alpha-amylase. Igarashi et al. <NUM> show that the thermostability of AmyS-type amylases can be increased by the deletion of two amino acid residues, R179-G180, (AmyS numbering) from a loop (F <NUM> to A184). However, Shiau et al. (<NUM>) showed that an AmyS enzyme with deletion in the same loop has a lower specific activity for corn starch hydrolysis at high-temperature than the parent enzyme, negating one of the principal advantages of AmyS amylases.

<CIT> discloses an alpha-amylase of Bacillus sp. The enzyme has activity at lower temperatures, e.g., <NUM>-<NUM>.

<CIT> discloses a deletion variant <NUM>+ <NUM> with a W284 modification, wherein the mutations exhibit altered specific activity, especially at temperatures from <NUM>-<NUM>, for use in cleaning compositions. The enzyme of is used for hydrolysis of granular starch into a soluble starch hydrolysate at a temperature below the initial gelatinization temperature of the granular starch.

<CIT> discloses low-temperature amylases that can be applied for washing directly under room temperature.

Conference presentation abstract (Smolenice Castle, Slovakia (September <NUM>) concerning the importance of starch affinity for industrial applications.

Conference presentation slides (Smolenice Castle, Slovakia (September <NUM>) concerning the importance of starch affinity for industrial applications.

For environmental reasons it has been increasingly important to lower the temperature in washing, dishwashing and/or cleaning processes. However, most enzymes including amylases have a temperature optimum which is above the temperature usually used in low temperature washing. Alpha-amylase is a key enzyme for use in detergent compositions and its use has become increasingly important for removal of starchy stains during laundry washing or dishwashing. Therefore, it is important to find alpha-amylase variants, which retain their wash performance, stain removal effect and/or activity when the temperature is lowered. However, despite the efficiency of current detergents enzyme compositions, there are many stains that are difficult to completely remove. These problems are compounded by the increased use of low (e.g., cold water) wash temperatures and shorter washing cycles. Thus, it is desirable to have amylolytic enzymes that can function under low temperature and at the same time preserve or increase other desirable properties such as specific activity (amylolytic activity), stability and/or wash performance.

Thus, it is an object of the present invention to provide improved methods for screening alpha-amylases and variants which have high wash performance at low temperatures.

In a first aspect the present invention relates to a method for screening alpha-amylases having high wash performance at low temperatures of <NUM>-<NUM>, comprising the steps of:.

wherein the alpha-amylases have less than <NUM>% of the binding to starch than the alpha-amylase having SEQ ID NO: <NUM>.

In a further aspect the invention relates to a method for selecting variants of a parent alpha-amylase, wherein the alpha-amylases have at least <NUM>% sequence identity to SEQ ID NO: <NUM>; and comprising the steps of:.

The invention is based on the observation that there exists an inverse correlation between the binding of an amylase to raw starch and wash performance at low temepratures. For example using data for a number of experimental detergent amylase samples a clear correlation between binding to rice starch and wash performance in AMSA testing. Thus, it has surprisingly been found that alpha-amylases having a low binding to a starch have a high performance in detergents at low temperatures.

Alpha-amylase activity: The term "alpha-amylase activity" means the activity of alpha-<NUM>,<NUM>-glucan-<NUM>-glucanohydrolases, E. <NUM>, which constitute a group of enzymes, which catalyze hydrolysis of starch and other linear and branched <NUM>,<NUM>-glucosidic oligo- and polysaccharides.

Substrate binding: The term "Substrate binding"or "binding to a solid substrate" or grammatically equivalent terms are understood as the property of an alpha-amylase to bind to a solid substrate including a substrate immobilized to a solid material. The term is a relative term and it is in general expressed as a relative binding in comparison with a reference alpha-amylase. For variants, substrate binding is in general measured relativety to the parent alpha-amylse that has served as starting point for the variants. Substrate binding may be measured by incubating a solution of the alpha-amylase with the solid substrate, removing the substrate and determining the fraction of the initial amount of alpha-amylase that remain attached to the solid substrate, Preferred methods for determining the substrate binding to a solid substrate is disclosed below in the Materials and Methods section.

Variant: The term "variant" means a polypeptide having alpha-amylase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding <NUM>-<NUM> amino acids adjacent to an amino acid occupying a position.

Mutant: The term "mutant" means a polynucleotide encoding a variant.

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

Parent or Parent alpha-amylase: The term "parent" or "parent alpha-amylase" means an alpha-amylase to which an alteration is made to produce the enzyme variants. The parent may be a naturally occurring (wild-type) polypeptide or a variant thereof. In relation to variants the parent polypeptide is the polypeptide having exactly the same sequence as the variant except for the residues specifically altered in the variant.

Isolated variant: The term "isolated variant" means a variant that is modified by the hand of man. In one aspect, the variant is at least <NUM>% pure, e.g., at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, and at least <NUM>% pure, as determined by SDS-PAGE.

Substantially pure variant: The term "substantially pure variant" means a preparation that contains at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, and at most <NUM>% by weight of other polypeptide material with which it is natively or recombinantly associated. Preferably, the variant is at least <NUM>% pure, e.g., at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>%, at least <NUM>% pure, and <NUM>% pure by weight of the total polypeptide material present in the preparation. The variants are preferably in a substantially pure form. This can be accomplished, for example, by preparing the variant by well known recombinant methods or by classical purification methods.

Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc..

Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having alpha-amylase activity.

Sequence Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (<NPL>) as implemented in the Needle program of the EMBOSS package (<NPL>), preferably version <NUM>. <NUM> or later. The optional parameters used are gap open penalty of <NUM>, gap extension penalty of <NUM>, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:<MAT>.

For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, <NUM>, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , <NUM>, supra), preferably version <NUM>. <NUM> or later. The optional parameters used are gap open penalty of <NUM>, gap extension penalty of <NUM>, and the EDNAFULL (EMBOSS version of NCBI NUC4. <NUM>) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: <MAT>.

Fragment: The term "fragment" means a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has alpha-amylase activity.

Subsequence: The term "subsequence" means a polynucleotide having one or more (several) nucleotides deleted from the <NUM>'- and/or <NUM>'-end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having alpha-amylase activity.

Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term "isolated polynucleotide" means a polynucleotide that is modified by the hand of man. In one aspect, the isolated polynucleotide is at least <NUM>% pure, e.g., at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, and at least <NUM>% pure, as determined by agarose electrophoresis. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

Substantially pure polynucleotide: The term "substantially pure polynucleotide" means a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered polypeptide production systems. Thus, a substantially pure polynucleotide contains at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, at most <NUM>%, and at most <NUM>% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring <NUM>'- and <NUM>'- untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least <NUM>% pure, e.g., at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, and at least <NUM>% pure by weight. The polynucleotides are preferably in a substantially pure form.

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

cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which 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 expression of a coding sequence.

Control sequences: The term "control sequences" means all components necessary for the expression of a polynucleotide encoding a variant. Each control sequence may 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, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.

Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

Expression: The term "expression" includes any step involved in the production of the variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to additional nucleotides that provide for its expression.

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

Starch removing process: The expression "starch removing process" relates to any kind of process whereby starch is removed (or converted) such as in washing processes where starch is removed from textile e.g. textile cleaning such as laundry. A starch removing process could also be hard surface cleaning such as dish wash or it could be cleaning processes in general such as industrial or institutional cleaning. The expression also comprises other starch removing processes or starch conversion, ethanol production, starch liquefaction, textile desizing, paper and pulp production, beer making and detergents in general.

Improved property: The term "improved property" means a characteristic associated with a variant that is improved compared to the parent. Such improved properties include, but are not limited to, thermal activity, thermostability, pH activity, pH stability, substrate/cofactor specificity, improved surface properties, product specificity, increased stability or solubility in the presence of pretreated biomass, improved stability under storage conditions, and chemical stability.

Wash performance: In the present context the term "wash performance" is used as an enzyme's ability to remove starch or starch-containing stains present on the object to be cleaned during e.g. laundry or hard surface cleaning, such as dish wash. The wash performance may be quantified by calculating the so-called intensity value (Int) defined in the description of AMSA or in the beaker wash performance test in the Methods section below.

Improved wash performance: The term "improved wash performance" is defined herein as a variant enzyme displaying an alteration of the wash performance of an amylase variant relative to the wash performance of the parent amylase or relative to an alpha-amylase having the identical amino acid sequence of said variant but not having the deletion at one or more of the specified positions or relative to the activity of an alpha-amylase having the amino acid sequence shown in SEQ ID NO <NUM>, e.g. by increased stain removal. The term "wash performance" includes cleaning in general e.g. hard surface cleaning as in dish wash, but also wash performance on textiles such as laundry, and also industrial and institutional cleaning.

Low temperature: "Low temperature" is a temperature of <NUM>-<NUM>, preferably <NUM>-<NUM>, more preferably <NUM>-<NUM>, more preferably <NUM>-<NUM>, most preferably <NUM>-<NUM>, and in particular <NUM>-<NUM>. In a preferred embodiment, "Low temperature" is a temperature of <NUM>-<NUM>, preferably <NUM>-<NUM>, more preferably <NUM>-<NUM>, most preferably <NUM>-<NUM>, and in particular <NUM>-<NUM>.

In a first aspect the present invention relates to a method for screening alpha-amylases having high wash performance at low temperature of <NUM>-<NUM>, wherein the alpha-amylases have at least <NUM>% sequence identity to SEQ ID NO: <NUM>; and comprising the steps of:.

The method is suitable for screening new alpha-amylases in order to find enzymes that have good wash performance at low temperature. The method may also with advantage be automized and adapted to a high throughput screening procedure.

In one embodiment the method is suitable for finding new alpha-amylases. One convenient way is to test a number of candidate alpha-amylases and include one known detergent alpha-amylase as a standard and then selecting new candidate alpha-amylases having a lower binding to the substrate than the standard detergent alpha-amylase. The selected alpha-amylases will be the candidates having high wash performance and can be further tested for other properties important for detergent enzymes, such as stability, specific activity etc..

Several detergent amylases are known from the literature including wild type amylases such as the alpha-amylases having SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>: variants of any of these such as the variants disclosed in <CIT> and <CIT>, and any of these known detergent amylases may suitably be included in the method as a standard detergent alpha-amylase.

The alpha-amylases in this embodiment are selected to have lower binding to the starch substrate than the selected standard detergent amylase shown in SEQ ID NO: <NUM> having less than <NUM>% of the binding; preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, more preferred less than <NUM>% of the binding, more preferred less than <NUM>% of the binding and most preferred less than <NUM>% of the binding.

Alternatively using SP722, the alpha-amylase having SEQ ID NO: <NUM>, as the standard detergent alpha-amylase the method may be used for selecting alpha-amylases having improved wash performance at low temperature by selecting for alpha-amylases having lower binding to the substrate than SP722 preferably less than <NUM>% of the binding; preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, more preferred less than <NUM>% of the binding and most preferred less than <NUM>% of the binding.

Alternatively using SP707, the alpha-amylase having SEQ ID NO: <NUM>, as the standard detergent alpha-amylase the method may be used for selecting alpha-amylases having improved wash performance at low temperature by selecting for alpha-amylases having lower binding to the substrate than SP707, preferably less than <NUM>% of the binding; preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the bindingpreferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, more preferred less than <NUM>% of the binding and most preferred less than <NUM>% of the binding.

Alternatively using AA560, the alpha-amylase having SEQ ID NO: <NUM>, as the standard detergent alpha-amylase the method may be used for selecting alpha-amylases having improved wash performance at low temperature by selecting for alpha-amylases having lower binding to the substrate than AA560 preferably less than <NUM>% of the binding; preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, more preferred less than <NUM>% of the binding and most preferred less than <NUM>% of the binding.

Alternatively using SP690, the alpha-amylase having SEQ ID NO: <NUM>, as the standard detergent alpha-amylase the method may be used for selecting alpha-amylases having improved wash performance at low temperature by selecting for alpha-amylases having lower binding to the substrate than SP690 preferably less than <NUM>% of the binding; preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, more preferred less than <NUM>% of the binding and most preferred less than <NUM>% of the binding.

Alternatively using KSM-AP1378, the alpha-amylase having SEQ ID NO: <NUM>, as the standard detergent alpha-amylase the method may be used for selecting alpha-amylases having improved wash performance at low temperature by selecting for alpha-amylases having lower binding to the substrate than KSM-AP1378, preferably less than <NUM>% of the binding; preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, more preferred less than <NUM>% of the binding and most preferred less than <NUM>% of the binding.

Alternatively using SP7-<NUM>, the alpha-amylase having SEQ ID NO: <NUM>, as the standard detergent alpha-amylase the method may be used for selecting alpha-amylases having improved wash performance at low temperature by selecting for alpha-amylases having lower binding to the substrate than SP7-<NUM>, preferably less than <NUM>% of the binding; preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, more preferred less than <NUM>% of the binding and most preferred less than <NUM>% of the binding.

In a further aspect the invention relates to a method for selecting variants of a parent alpha-amylase, wherein the alpha-amylases have at least <NUM>% sequence identity to SEQ ID NO: <NUM>; comprising the steps of.

In this preferred embodiment the parent alpha-amylase may be a detergent alpha-amylase i.e. an alpha-amylase having activity under conditions usually applied in cleaning and laundry, such as activity in the presence of surfactants, chelators and other components routinely used in detergents, and activity at an alkaline pH such as in the range of <NUM>-<NUM>, such as <NUM>-<NUM>.

The variants may be prepared by substituting, deleting or inserting one or more amino acid residue in one or more amino acids located at the surface of the parent alpha-amylase.

The skilled person will know how to identify residues to be modified by identifying residues located at the surface of the parent molecule. In this connection residues located on the surface of the molecule is understood as residues where at least a part of the residue are in direct contact with the surrounding medium in a natural form of the molecule. It will be appreciated that the skilled person can extract this kind of information from <NUM>-D structures of the parent molecule in case that a <NUM>-D structure of the parent molecule is available or in case that a <NUM>-D structure of the parent is not available by aligning the parent alpha-amylase with an alpha-amylase for which the <NUM>-D structure is available and identifying residues located on the surface of the molecule as residues of the parent alpha-amylase that in the alignment corresponds to residued located on the surface in the alpha-amylase. As more than one <NUM>-D structure of alpha-amylases are known in the art the identification of residues located on the surface of a given parent alpha-amylase should be based on alignment of the parent alpha-amylase with the 3D structure for the alpha-amylase having the highest sequence identity to the parent alpha-amylase.

The variants are generated by substituting, deleting or inserting one or more amino acid residue in one or more amino acids located at the surface of the parent alpha-amylase of one or more amino acids residued in positions located on the surface of the parent alpha-amylase, such as substituting, deleting or inserting at least one residues located on the surface, such as at least two residues, e.g.at least three residues, e.g. at least four residues, e. g at least five residues, e.g. at least six residues, e.g. at least seven residues, e.g. at least eight residues, e.g. at least nine residues, e.g. at least ten residues, such as up to fiftheen residue e.g. up to twenty residues or up to thirty residues located on the surface of the parent alpha-amylase.

The variants may additionally contain further substitutions, deletions or insertions in one or more amino acid residue not located on the surface of the molecule. Many such substitutions, deletions or insertions have been disclosed in the prior art and may also be used. As examples of such preferred additional substitution, insertion or deletion can be mentioned the deletion of residues D183 and G184 in the parent alpha-amylase, as disclosed in <CIT>.

Preferably variants are generated having at least <NUM>% sequence identity to SEQ ID NO: <NUM>, preferably at least <NUM>% sequence identity, more preferred at least <NUM> % sequence identity, more preferred at least <NUM>% sequence identity and most preferred at least <NUM>% sequence identity.

The variants are selected to have less binding to the substrate than the parent alpha-amylase such are having less than <NUM>% of the binding of SEQ ID NO: <NUM>, such as less than <NUM>% of the binding; preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, preferably less than <NUM>% of the binding, more preferred less than <NUM>% of the binding and most preferred less than <NUM>% of the binding.

The variants which may be screened according to the method of the invention include variants wherein one or more residues located on the surface of the parent alpha-amylases and being part of a substrate binding site have been substituted or deleted. Such substrate bindig sites present on the surface of parent alpha-amylases have been disclosed in the literature for a few parent alpha-amylases and the skilled person will appreciate that such substrate binding sites may be identified in other parent alpha-amylases by aligning a given parent alpha-amylase with another alpha-amylase sequence for which such substrate binging sites have been identified.

As examples of residues being part of a substrate binding site can be mentioned:.

The variants which may be screened according to the method of the invention also include variants including one or more modifications that introduce one or more bulky amino acid residues in a position that is close to a residue being part of a substrate binding site, such as the residues mentioned in a)-f) above. In this connection a bulky amino acid residue can be selected among amino acids having a large side chain, such as Tyr, Trp, Phe, His and Ile, where Tyr and Trp are preferred, Close to a residue being part of a substrate binding site is intended to mean that the modified residue is located less than <NUM>Å from the residue being part of a substrate binding site, preferably less than 6Å and most preferred less than 3Å from such residues. It is within the skilled persons capabilities to identify such residues based on available structure information.

Other preferred variants which may be screened according to the method of the invention include variants having at least <NUM>% sequence identity to SEQ ID NO: <NUM>, preferably at least <NUM>% sequence identity, more preferred at least <NUM>% sequence identity, more preferred at least <NUM>% sequence identity, more preferred at least <NUM>% sequence identity, more preferred at least <NUM>% sequence identity, more preferred at least <NUM>% identity, more preferred at least <NUM>% sequence identity, but less than <NUM>% sequence identity and comprising the following substitutions:.

For the purpose of the present invention binding of alpha-amylases to a solid or immobilized substrate can in principle be determined using any such method for determining binding to a substrate known in the art. In general such method involves contacting an alpha-amylase to a solid or immobilized substrate and determining the fraction of alpha-amylase bound to the substrates.

The solid or immobilized substrate may be any substrate for alpha-amylases that is substratially insoluble under the conditions of the test. The solid substrate may be a starch such as wheat starch, maize starch or rice starch, where rice starch is preferred. Alternatively may the determination be made using immobilized substrate which includes any soluble or insoluble substrates for alpha-amylases immobilized on a solid matrix. According to the invention binding of an alpha-amylase to its substrate is determined as the fraction of bound amylase measured at <NUM> in the presence of starch at pH <NUM>. Binding of an alpha-amylase to its substrate can be determined by incubating an aqueous solution of the alpha-amylase with the starch and determining the fraction of bound amylase using the method disclosed below.

For purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM> or SEQ ID NO: <NUM> 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 disclosed in SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM> or SEQ ID NO: <NUM> and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: <NUM> is determined using the Needleman-Wunsch algorithm (<NPL>) as implemented in the Needle program of the EMBOSS package (<NPL>), preferably version <NUM>. <NUM> or later.

Identification of the corresponding amino acid residue in another alpha-amylase can be confirmed by an alignment of multiple polypeptide sequences using "ClustalW" (<NPL>).

When the other enzyme has diverged from the mature polypeptide of SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, or SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM> or SEQ ID NO: <NUM> such that traditional sequence-based comparison fails to detect their relationship (<NPL>), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (<NPL>). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide has one or more representatives in the protein structure databases. Programs such as GenTHREADER (<NPL>; <NPL>) utilize 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 <NPL>, 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 such models can be assessed for accuracy using a variety of tools developed for that purpose.

For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example the SCOP superfamilies of proteins 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 (<NPL>) or combinatorial extension (<NPL>), and implementations of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., <NPL>).

In describing the alpha-amylase variants, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single letter or three letter amino acid abbreviation is employed.

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 <NUM> is designated as "Thr226Ala" or "T226A". Multiple mutations are separated by addition marks ("+"), e.g., "Gly205Arg + Ser411Phe" or "G205R + S411F", representing substitutions at positions <NUM> and <NUM> of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

For an amino acid deletion, the following nomenclature is used: Original amino acid, position*. Accordingly, the deletion of glycine at position <NUM> is designated as "Gly195*" or "G195*". Multiple deletions are separated by addition marks ("+"), e.g., "Gly195* + Ser411*" or "G195* + S411*".

Insertions. For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position <NUM> is designated "Gly195GlyLys" or "G195GK". An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #<NUM>, inserted amino acid #<NUM>; etc.]. For example, the insertion of lysine and alanine after glycine at position <NUM> is indicated as "Gly195GlyLysAla" or "G195GKA".

In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:.

Multiple alterations. Variants comprising multiple alterations are separated by addition marks ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" representing a substitution of tyrosine and glutamic acid for arginine and glycine at positions <NUM> and <NUM>, respectively.

Different substitutions. Where different substitutions can be introduced at a position, the different substitutions are separated by a comma, e.g., "Arg170Tyr,Glu" represents a substitution of arginine with tyrosine or glutamic acid at position <NUM>. Thus, "Tyr167Gly,Ala + Arg170Gly,Ala" designates the following variants:.

The parent alpha-amylase may be a polypeptide with at least <NUM>% sequence identity with the mature polypeptide of SEQ ID NO: <NUM>, SP707 disclosed in <NPL>.

In another aspect, the parent has a sequence identity to the mature polypeptide of SEQ ID NO: <NUM> of at least <NUM>%, e.g., at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or <NUM>%, which have alpha-amylase activity. In one aspect, the amino acid sequence of the parent differs by no more than ten amino acids, e.g., by five amino acids, by four amino acids, by three amino acids, by two amino acids, and by one amino acid from the mature polypeptide of SEQ ID NO: <NUM>.

The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: <NUM>. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ ID NO: <NUM>.

In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO: <NUM>.

The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a cell in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

The parent may be a bacterial alpha-amylase. For example, the parent may be a gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces alpha-amylase, or a gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma alpha-amylase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis alpha-amylase.

In another aspect, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus alpha-amylase.

In another aspect, the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans alpha-amylase.

The parent may be a fungal alpha-amylase. For example, the parent may be a yeast alpha-amylase such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia alpha-amylase. For example, the parent may be a filamentous fungal alpha-amylase such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria alpha-amylase.

In another aspect, the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis alpha-amylase.

In another aspect, the parent is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride alpha-amylase.

In another aspect, the parent is a Bacillus sp. alpha-amylase, e.g., the alpha-amylase of SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>.

The aforementioned species encompass both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc,) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. The polynucleotide encoding a parent may then be derived by similarly screening a genomic or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected with a probe(s), the polynucleotide may be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al. , <NUM>, supra).

The parent may be a hybrid polypeptide in which a portion of one polypeptide is fused at the N-terminus or the C-terminus of a portion of another polypeptide.

The parent also may be a fused polypeptide or cleavable fusion polypeptide in which one polypeptide is fused at the N-terminus or the C-terminus of another polypeptide. A fused polypeptide is produced by fusing a polynucleotide encoding one polypeptide to a polynucleotide encoding another polypeptide. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator. Fusion proteins may also be constructed using intein technology in which fusions are created post-translationally (<NPL>; <NPL>).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in <NPL>; <NPL>; <NPL>; <NPL>; and <NPL>; <NPL>; <NPL>; <NPL>; and <NPL>.

Detergent compositions may comprise an enzyme in combination with one or more additional cleaning composition components.

The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below. The choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product. Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.

Polypeptides found according to the screening method of the invention may be added to a detergent composition in an amount corresponding to <NUM>-<NUM> of protein, such as <NUM>-<NUM> of protein, preferably <NUM>-<NUM> of protein, more preferably <NUM>-<NUM> of protein, even more preferably <NUM>-<NUM> of protein, most preferably <NUM>-<NUM> of protein, and even most preferably <NUM>-<NUM> of protein per liter of wash liquor.

The enzyme(s) of the detergent composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as <NUM>-formylphenyl boronic acid, and the composition may be formulated as described in, for example, <CIT> and <CIT>.

A polypeptide may also be incorporated in the detergent formulations disclosed in <CIT>.

The detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant(s) is typically present at a level of from about <NUM>% to <NUM>% by weight, such as about <NUM>% to about <NUM>%, or about <NUM>% to about <NUM>%, or about <NUM>% to about <NUM>%. The surfactant(s) is chosen based on the desired cleaning application, and includes any conventional surfactant(s) known in the art. Any surfactant known in the art for use in laundry detergents may be utilized.

When included therein the detergent will usually contain from about <NUM>% to about <NUM>% by weight, such as from about <NUM>% to about <NUM>%, including from about <NUM>% to about <NUM>%, or from about <NUM>% to about <NUM>% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-<NUM>,<NUM>-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.

When included therein the detergent will usually contain from about <NUM> % to about <NUM>% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alklydimethylehanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, and combinations thereof, Alkyl quaternary ammonium compounds, Alkoxylated quaternary ammonium (AQA),.

When included therein the detergent will usually contain from about <NUM>% to about <NUM>% by weight of a non-ionic surfactant, for example from about <NUM>% to about <NUM>%, in particular from about <NUM>% to about <NUM>%, from about <NUM>% to about <NUM>%, such as from about <NUM>% to about <NUM>%, or from about <NUM>% to about <NUM>%. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

When included therein the detergent will usually contain from about 0_% to about <NUM>% by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide and N-(tallow-alkyl)-N,N-bis(<NUM>-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

When included therein the detergent will usually contain from about 0_% to about <NUM>% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, and sulfobetaine, and combinations thereof.

A hydrotrope is a compound that solubilises hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and a hydrophobic character (so-called amphiphilic properties as known from surfactants); however the molecular structure of hydrotropes generally do not favor spontaneous self-aggregation, see e.g. review by <NPL>. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming miceller, lamellar or other well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care, food, to technical applications. Use of hydrotropes in detergent compositions allow for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain <NUM>-<NUM>% by weight, such as about <NUM> to about <NUM>%, or about <NUM>% to about <NUM>%, of a hydrotrope. Any hydrotrope known in the art for use in laundry detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonates (STS), sodium xylene sulfonates (SXS), sodium cumene sulfonates (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.

The detergent composition may contain about <NUM>-<NUM>% by weight, such as about <NUM>% to about <NUM>% of a detergent builder or co-builder, or a mixture thereof. In a dish wash deteregent, the level of builder is typically <NUM>-<NUM>%, particularly <NUM>-<NUM>%. The builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry or dish washing detergents or detergents used for industrial or institutional (?) cleaning may be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-<NUM> from Hoechst), ethanolamines such as <NUM>-aminoethan-<NUM>-ol (MEA), iminodiethanol (DEA) and <NUM>,<NUM>',<NUM>"-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), and combinations thereof.

The detergent composition may also contain <NUM>-<NUM>% by weight, such as about <NUM>% to about <NUM>%, of a detergent co-builder, or a mixture thereof. The detergent composition may include include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA). Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid. Additional specific examples include <NUM>,<NUM>',<NUM>"-nitrilotriacetic acid (NTA), etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), <NUM>-hydroxyethane-<NUM>,<NUM>-diylbis(phosphonic acid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonic acid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonic acid) (DTPMPA), N-(<NUM>-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid- N,N-diacetic acid (ASDA), aspartic acid-N- monopropionic acid (ASMP) , iminodisuccinic acid (IDA), N-(<NUM>-sulfomethyl) aspartic acid (SMAS), N- (<NUM>-sulfoethyl) aspartic acid (SEAS), N- (<NUM>- sulfomethyl) glutamic acid (SMGL), N- (<NUM>- sulfoethyl) glutamic acid (SEGL), N- methyliminodiacetic acid (MIDA), α- alanine-N,N-diacetic acid (α -ALDA) , serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA) , anthranilic acid- N ,N - diacetic acid (ANDA), sulfanilic acid-N, N-diacetic acid (SLDA) , taurine-N, N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine (DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., <CIT>, <CIT> Incrustation inhibitors such as phosphonates.

The detergent may contain <NUM>-<NUM>% by weight, such as about <NUM> % to about <NUM>%, of a bleaching system. Any bleaching system known in the art for use in laundry + dish wash + l&l detergents may be utilized. Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborates, preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone (R), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra-hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator. By Bleach activator is meant herin a compound which reacts with peroxygen bleach like hydrogen peroxide to form a Peracid. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herin include those belonging to the class of esters amides, imides or anhydrides, Suitable examples are tetracetyl athylene diamine (TAED), sodium <NUM>,<NUM>,<NUM> trimethyl hexanoyloxybenzene sulphonat, diperoxy dodecanoic acid, <NUM>-(dodecanoyloxy)benzenesulfonate (LOBS), <NUM>-(decanoyloxy)benzenesulfonate, <NUM>-(decanoyloxy)benzoate (DOBS), <NUM>-(<NUM>,<NUM>,<NUM>-trimethylhexanoyloxy)benzenesulfonate (ISONOBS), tetraacetylethylenediamine (TAED) and <NUM>-(nonanoyloxy)benzenesulfonate (NOBS), and/or those disclosed in <CIT>. A particular family of bleach activators of interest was disclosed in <CIT> and particulary preferred in that family is acetyl triethyl citrate (ATC). ATC or a short chain triglyceride like Triacin has the advantage that it is environmental friendly as it eventually degrades into citric acid and alcohol. Furthermore acethyl triethyl citrate and triacetin has a good hydrolytical stability in the product upon storage and it is an efficient bleach activator. Finally ATC provides a good building capacity to the laundry additive. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The bleaching system may also comprise peracids such as <NUM>-(phthaloylamino)percapronic acid (PAP). The bleaching system may also include a bleach catalyst. In some embodiments the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formulae:
<CHM>
<CHM>
(iii) and mixtures thereof; wherein each R<NUM> is independently a branched alkyl group containing from <NUM> to <NUM> carbons or linear alkyl group containing from <NUM> to <NUM> carbons, preferably each R<NUM> is independently a branched alkyl group containing from <NUM> to <NUM> carbons or linear alkyl group containing from <NUM> to <NUM> carbons, more preferably each R<NUM> is independently selected from the group consisting of <NUM>-propylheptyl, <NUM>-butyloctyl, <NUM>-pentylnonyl, <NUM>-hexyldecyl, n- dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso- tridecyl and iso-pentadecyl. Other exemplary bleaching systems are described, e.g., in <CIT>, <CIT>, <CIT>, <CIT>. Suitable photobleaches may for example be sulfonated zinc phthalocyanine.

The detergent may contain <NUM>-<NUM>% by weight, such as <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>% or <NUM>-<NUM>% of a polymer. Any polymer known in the art for use in laundry, dish wash and l&l detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide antiredeposition, fiber protection, soil release, dye transfer inhibition and/or grease cleaning properties. Exemplary antiredeposition polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), and polycarboxylates such as PAA, PAA/PMA, and lauryl methacrylate/acrylic acid copolymers. Exemplary fiber protection polymers include hydrophobically modified CMC (HM-CMC) and silicones. Exemplary soil release polymers include copolymers of terephthalic acid and oligomeric glycols. Exemplary dye transfer inhibition polymers include PVP, poly(vinylimidazole) (PVI) and poly(vinylpyridin-N-oxide) (PVPO or PVPNO). Other exemplary polymers are disclosed in, e.g., <CIT>.

The detergent compositions may also include fabric hueing agents such as dyes or pigments which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions thus altering the tint of said fabric through absorption/reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C. ) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in <CIT>, <CIT>, <CIT> and <CIT>. The detergent composition preferably comprises from about <NUM> wt% to about <NUM> wt%, from about <NUM> wt% to about <NUM> wt%, or even from about <NUM> wt% to about <NUM> wt% fabric hueing agent. The composition may comprise from <NUM> wt% to <NUM> wt% fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g., <CIT>, <CIT>.

The detergent additive as well as the detergent composition may comprise one or more [additional] enzymes such as a protease, lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

In general the properties of the selected enzyme(s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in <CIT>, <CIT>, <CIT>, <CIT> and <CIT>.

Especially suitable cellulases are the alkaline or neutral cellulases having color care benefits. Examples of such cellulases are cellulases described in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>. Other examples are cellulase variants such as those described in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> and <CIT>.

Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc. ), and KAC-<NUM>(B)™ (Kao Corporation).

Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin <NUM>, subtilisin <NUM> and subtilisin <NUM> (described in <CIT>). Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in <CIT> and <CIT>.

Examples of useful proteases are the variants described in <CIT>, <CIT>, <CIT>, and <CIT>, especially the variants with substitutions in one or more of the following positions: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>.

Preferred commercially available protease enzymes include Alcalase™, Savinase™, Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.

Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) as described in <CIT> and <CIT> or from H. insolens as described in <CIT>, a Pseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes (<CIT>), P. cepacia (<CIT>), P. stutzeri (<CIT>), P. fluorescens, Pseudomonas sp. strain SD <NUM> (<CIT> and <CIT>), P. wisconsinensis (<CIT>), a Bacillus lipase, e.g., from B. subtilis (<NPL>), B. stearothermophilus (<CIT>) or B. pumilus (<CIT>).

Other examples are lipase variants such as those described in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> and <CIT>.

Preferred commercially available lipase enzymes include Lipolase™, Lipolase Ultra™, and Lipex™ (Novozymes A/S).

Amylases: Suitable amylases (α and/or β) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, α-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in <CIT>.

Examples of useful amylases are the variants described in <CIT>, <CIT>, <CIT>, and <CIT>, especially the variants with substitutions in one or more of the following positions: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>.

Commercially available amylases are Stainzyme™, Stainzyme™ Plus, Natalase™, Duramyl™, Termamyl™, Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ , Powerase™ and Purastar™ (from Genencor International Inc.

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in <CIT>, <CIT>, and <CIT>.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive, i.e., a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in <CIT> and <CIT> and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of <NUM> to <NUM>; ethoxylated nonylphenols having from <NUM> to <NUM> ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from <NUM> to <NUM> carbon atoms and in which there are <NUM> to <NUM> ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in <CIT>. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in <CIT>.

Any detergent components known in the art for use in laundry, dish wash or l&l detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry, dish wash or l&l detergents may be utilized. The choice of such ingredients is well within the skill of the artisan. Dispersants - The detergent compositions can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in <NPL>. Dye Transfer inhibiting Agents - The detergent compositions may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about <NUM> % to about <NUM>%, from about <NUM>% to about <NUM>% or even from about <NUM>% to about <NUM>% by weight of the composition. Fluorescent whitening agent - The detergent compositions will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about <NUM>,<NUM>% to about <NUM>,<NUM>%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of: <NUM>,<NUM>'-bis-(<NUM>-diethanolamino-<NUM>-anilino-s-triazin-<NUM>-ylamino) stilbene-<NUM>,<NUM>'-disulphonate; <NUM>,<NUM>'-bis-(<NUM>,<NUM>-dianilino-s-triazin-<NUM>-ylamino) stilbene-<NUM>'-disulphonate; <NUM>,<NUM>'-bis-(<NUM>-anilino-<NUM>(N-methyl-N-<NUM>-hydroxy-ethylamino)-s-triazin-<NUM>-ylamino) stilbene-<NUM>,<NUM>'-disulphonate, <NUM>,<NUM>'-bis-(<NUM>-phenyl-<NUM>,<NUM>,<NUM>-triazol-<NUM>-yl)stilbene-<NUM>,<NUM>'-disulphonate; <NUM>,<NUM>'-bis-(<NUM>-anilino-<NUM>(<NUM>-methyl-<NUM>-hydroxy-ethylamino)-s-triazin-<NUM>-ylamino) stilbene-<NUM>,<NUM>'-disulphonate and <NUM>-(stilbyl-<NUM>"-naptho-<NUM>. ,<NUM>':<NUM>,<NUM>)-<NUM>,<NUM>,<NUM>-trizole-<NUM>"-sulphonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of <NUM>,<NUM>'-bis-(<NUM>-morpholino-<NUM> anilino-s-triazin-<NUM>-ylamino) stilbene disulphonate. Tinopal CBS is the disodium salt of <NUM>,<NUM>'-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use in the invention include the <NUM>-<NUM>-diaryl pyrazolines and the <NUM>-alkylaminocoumarins. Suitable fluorescent brightener levels include lower levels of from about <NUM>, from <NUM>, from about <NUM> or even from about <NUM> wt % to upper levels of <NUM> or even <NUM> wt%. Soil release polymers - The detergent compositions may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example <NPL>. Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in <CIT>. Furthermore random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in <CIT>, <CIT> and <CIT>. Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives such as those described in <CIT> or <CIT>. Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof. Anti-redeposition agents - The detergent compositions may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents. Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, structurants for liquid detergents and/or structure elasticizing agents.

In order to assess the wash performance of the alpha-amylase variants in a detergent base composition, washing experiments may be performed. The enzymes are tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the textile swatch to be washed against all the slot openings. During the washing time, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution in contact with the textile and apply mechanical stress in a regular, periodic oscillating manner. For further description see <CIT>, especially the paragraph "Special method embodiments" at page <NUM>-<NUM>.

A test solution comprising water (<NUM>°dH), <NUM>/L detergent, e.g. model detergent A as described below, or <NUM> HCO<NUM>-, and the enzyme, e.g. at concentration of <NUM>,<NUM><NUM>, <NUM>, <NUM>, <NUM> and/or <NUM> enzyme protein/L, is prepared. Fabrics stained with starch (e.g. CS-<NUM> from Center For Testmaterials BV, P. Box <NUM>, <NUM> KT, Vlaardingen, The Netherlands) is added and washed for <NUM> minutes at <NUM>, or alternatively <NUM> minutes at <NUM> °, or alternatively <NUM> minutes at <NUM> or alternatively <NUM> minutes at <NUM> or <NUM> as specified in the examples. After thorough rinse under running tap water and drying in the dark, the light intensity or reflectance values of the stained fabrics are subsequently measured as a measure for wash performance. The test with <NUM> enzyme protein/L is used as a blank to obtain a delta remission value (ΔREM). Alternatively, the wash performance is compared to that of the parent alpha-amylase, with the performance result of the parent alpha-amylase is assigned the value of <NUM> and the results of the variants are compared to this value. Preferably mechanical action is applied during the wash step, e.g. in the form of shaking, rotating or stirring the wash solution with the fabrics.

The AMSA wash performance experiments were conducted under the experimental conditions specified below:.

Water hardness was adjusted to <NUM>°dH by addition of CaCl<NUM>, MgCl<NUM>, and NaHCOs (Ca<NUM>+:Mg<NUM>+:HCO<NUM>- = <NUM>:<NUM>:<NUM>) to the test system. After washing the textiles were flushed in tap water and dried.

Approximate wash pH in <NUM> °dH water (Ca: Mg: HCO<NUM> =<NUM>:<NUM>:<NUM>).

Water hardness was adjusted to <NUM> °dH by addition of CaCl<NUM>, MgCl<NUM>, and NaHCO<NUM> (Ca: Mg: HCO<NUM> =<NUM>:<NUM>:<NUM>) to the test system. After washing the textiles were flushed in tap water and dried.

The wash performance is measured as the brightness of the colour of the textile washed. Brightness can also be expressed as the intensity of the light reflected from the sample when illuminated with white light. When the sample is stained the intensity of the reflected light is lower, than that of a clean sample. Therefore the intensity of the reflected light can be used to measure wash performance.

Colour measurements are made with a professional flatbed scanner (Kodak iQsmart, Kodak), which is used to capture an image of the washed textile.

To extract a value for the light intensity from the scanned images, <NUM>-bit pixel values from the image are converted into values for red, green and blue (RGB). The intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector: <MAT>.

Textile sample CS-<NUM> (rice starch on cotton) is obtained from Center For Testmaterials BV, P. Box <NUM>, <NUM> KT Vlaardingen, the Netherlands.

This assay is a small scale model of a top loaded washing machine and used to evaluate the washing performance of amylases.

The beaker wash performance test, using <NUM> beakers and a paddle stirrer providing oscillating rotational motion, <NUM>° in each direction, with a frequency of <NUM> per minute, comprises the following steps: providing <NUM> wash solution (<NUM>, <NUM> °dH, , pH <NUM>) containing <NUM> NaHCOs and <NUM>/L enzyme; adding two swatches of CS-<NUM> (5x5 cm) and two swatches of EMPA <NUM> (5x5 cm) to the wash solution to start the wash; setting the agitation speed to <NUM> rpm; stopping the agitation after <NUM> minutes, rinsing the swatches under cold running tap water; drying the rinsed swatches in the dark over night; and evaluating the wash performance by measuring the remission of incident light at <NUM> using Color Eye as described below.

Water bath (<NUM>) with circulation; glass beakers (<NUM>); one rotating arm per beaker with capacity of <NUM> of washing solution; test swatches: CS-<NUM> (rice starch on cotton) from Center for Testmaterials BV, Vlaardingen, The Netherlands and EMPA <NUM> (rice starch on cotton/polyester) from EMPA Testmaterials AG, St. Gallen, Switzerland, the swatches are cut into 5x5 cm.

Wash solution: <NUM> NaHCOs buffer, pH <NUM>, water hardness: <NUM> °dH, Calcium: Magnesium ratio <NUM>:<NUM>. Amylase stock solution: <NUM> enzyme protein per mL. - A solution of <NUM> % (w/v) Triton X-<NUM> and <NUM> CaCl<NUM> in ultrapure water (MilliQ water) is used for dilution of amylase (amylase dilution buffer).

Wash performance is expressed as a delta remission value (ΔRem). Light reflectance evaluations of the swatches were done using a Macbeth Color Eye <NUM> reflectance spectrophotometer with very small oval aperture, i.e. <NUM><NUM> (-<NUM> x <NUM>). The measurements were made without UV in the incident light and remission at <NUM> was extracted. The swatch to be measured was placed on top of another swatch of the same type before being measured to reduce reflection from the piston pushing the swatch up against the measuring opening. Delta remission values for individual swatches were calculated by subtracting the remission value of the swatch washed without added amylase (control) from the remission value of the swatch washed with amylase.

Mini-wash robot is a small scale model of washing machine and used to evaluate the washing performance of amylases. To <NUM> beakers are added <NUM> wash solution, which is heated to <NUM> or <NUM>. Then enzyme (Concentrations: <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM> enzyme protein/L) is added. Textile (CS-<NUM>; rice starch on cotton) on rack is submerged into the wash solution containing a certain enzyme concentration and washed for <NUM> minutes. After wash the textile on rack is dried in drying cupboards without heat. The remission of the textile is measured at <NUM> by use of ZEISS MCS <NUM> VIS Spectrophotometer. Delta remission values for individual textile were calculated by subtracting the remission value of the textile washed without added amylase (control) from the remission value of the textile washed with amylase.

The alpha-amylase activity may be determined by a method employing the G7-pNP substrate. G7-pNP which is an abbreviation for <NUM>,<NUM>-ethylidene(G<NUM>)-p-nitrophenyl(G<NUM>)-α,D-maltoheptaoside, a blocked oligosaccharide which can be cleaved by an endo-amylase, such as an alpha-amylase. Following the cleavage, the alpha-Glucosidase included in the kit digest the hydrolysed substrate further to liberate a free p-nitrophenol (pNP) molecule which has a yellow color and thus can be measured by visible spectophometry at λ = <NUM> (<NUM>-<NUM>). Kits containing G7-pNP substrate and alpha-Glucosidase is manufactured by Roche/Hitachi (cat.

The G7-pNP substrate from this kit contains <NUM> <NUM>,<NUM>-ethylidene- G7-pNP and <NUM> HEPES (<NUM>-[<NUM>-(<NUM>-hydroxyethyl)-<NUM>-piperazinyl]-ethanesulfonic acid), pH <NUM>).

The alpha-Glucosidase reagent contains <NUM> HEPES, <NUM> NaCl, <NUM> MgCl<NUM>, <NUM> CaCl<NUM>, ≥ <NUM> kU/L alpha-glucosidase).

The substrate working solution is made by mixing <NUM> of the alpha-Glucosidase reagent with <NUM> of the G7-pNP substrate. This substrate working solution is made immediately before use.

Dilution buffer: <NUM> MOPS, <NUM>% (w/v) Triton X100 (polyethylene glycol p-(<NUM>,<NUM>,<NUM>,<NUM>-tetramethylbutyl)-phenyl ether (C<NUM>H<NUM>O(C<NUM>H<NUM>O)n (n = <NUM>-<NUM>))), <NUM> CaCl<NUM>, pH <NUM>.

The amylase sample to be analyzed was diluted in dilution buffer to ensure the pH in the diluted sample is <NUM>. The assay was performed by transferring <NUM>µl diluted enzyme samples to <NUM> well microtiter plate and adding 80µl substrate working solution. The solution was mixed and preincubated <NUM> minute at room temperature and absorption is measured every <NUM> sec. over <NUM> minutes at OD <NUM>.

The slope (absorbance per minute) of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions. The amylase sample should be diluted to a level where the slope is below <NUM> absorbance units per minute.

Amylase variants are incubated in the presence or absence of insoluble raw rice starch at a selected pH value, in the range of pH <NUM> to <NUM> depending on the intended pH value for the application of the alpha-amylases to be selected,; e.g. for detergent applications the pH is suitably selected in the alkaline area such as pH <NUM> or pH10. <NUM>; at a selected time, in general between <NUM> minutes and one hour, preferably in the range of <NUM> to <NUM> such as <NUM> or <NUM> minutes; and at a sselected temperature, in general in the range of <NUM> to <NUM>, preferably at <NUM>. After centrifugation, amylase activity is determined in the supernatants. Difference in activity in the samples incubated in the presence and absence of rice starch is a measure of binding of amylase to insoluble starch.

Rice starch (Sigma Inc, Cat No. S7260), HEPES, Calcium chloride, Triton X-<NUM>, Glycine, EnzChek Ultra Amylase Assay Kit (Life Technologies, Cat No. E33651), <NUM> microwell plates for incubation and dilution (Nunc, Cat No. <NUM>) and <NUM> well half-area black plates for fluorescence measurements (Corning, Cat No. <NUM>).

Assay buffer contained <NUM> HEPES (pH <NUM>), <NUM> CaCl<NUM> and <NUM>% Triton X-<NUM>. Enzyme protein solutions were diluted to <NUM>/ml with the assay buffer. High pH binding buffer contained <NUM> Glycine-NaOH (pH <NUM>) and <NUM>% Triton X-<NUM>. Rice starch solution (<NUM>%) was prepared in the assay buffer for the variants of SEQ ID NO: <NUM> and in high pH binding buffer for the variants OF SEQ ID NO: <NUM>. EnzCheck Ultra Amylase substrate solution was prepared according to the manufacturer's instructions and diluted to <NUM>µg/ml in the assay buffer.

Using the method of measuring the binding to starch and the AMSA wash performance test described in the methods section, a number of amylases and variants thereof were analyzed for binding to amylose and wash performance. The binding analysis was done at a pH of <NUM>, binding period of <NUM> minutes and at a temperature of <NUM>. The binding was tested using a <NUM> % (w/v) amylose (Sigma A0512), and the wash performance test was carried out as described for the AMSA wash performance test using Model detergent A and an enzyme dosage of <NUM> enzyme protein/L wash solution and a washing temperature of <NUM>. Wash time was <NUM> minutes. The results are presented in table <NUM> below.

From these results it is clearly seen that there is an inverse correlation between the fraction bound to the starch and the wash performance.

Same principle as described in Example <NUM>, only a suspension of <NUM> % (w/v) rice starch (Sigma S7260) was used instead of amylose. The binding analysis was done at a pH of <NUM>, binding period of <NUM> minutes and at a temperature of <NUM>.

Same principle as described in Example <NUM>, only a suspension of <NUM> % (w/v) amylopectin (from Fluka) from corn was used instead of amylose.

Variants of B. licheniformis alpha-amylase having SEQ ID NO: <NUM>, were made. The variants tested had lower binding to starch and better wash performance in comparison with the parent alpha-amylase having SEQ ID NO: <NUM>. The binding analysis was done at a pH of <NUM> with <NUM>% (w/v) insoluble rice starch, binding period of <NUM> minutes and at a temperature of <NUM>. The wash performance of the variants were tested by use of mini-wash using the conditions described under "Wash performance alpha-amylases using Mini-wash robot" and showed that the variants have improved wash performance using <NUM>/L at <NUM> compared with the parent alpha-amylase from Bacillus licheniformis. Washing time was <NUM> minutes.

Additional variants of the alpha-amylase having SEQ ID NO: <NUM> was generated and the variants tested for substrate binding and AMSA wash performance tested using <NUM> enzyme protein/L at <NUM> for <NUM> minutes in Model detergent X. The binding analysis was done at a pH of <NUM> with <NUM>% (w/v) insoluble rice starch, binding period of <NUM> minutes and at a temperature of <NUM>. The wash performance is indicated relative to the wash performance of the parent alpha-amylase having SEQ ID NO: <NUM> with the modifications H183*+G184*.

Further variants of the alpha-amylase having SEQ ID NO: <NUM> was generated and the variants tested for substrate binding and wash performance at low temperature, and variants selected having lower substrate binding than the parent alpha-amylase having SEQ ID NO: <NUM> with the modifications H183*+G184*. AMSA wash tests were performed using <NUM> enzyme protein/L at <NUM> for <NUM> minutes in Model detergent X. The wash performance is indicated relative to the wash performance of the parent alpha-amylase having SEQ ID NO: <NUM> with the modifications H183*+G184*.

Further variants of the alpha-amylase having SEQ ID NO: <NUM> was generated and the variants tested for substrate binding and wash performance at low temperature, and variants selected having lower substrate binding than the parent alpha-amylase having SEQ ID NO: <NUM> with the modifications H183*+G184*. AMSA wash tests were performed using <NUM> enzyme protein/L at <NUM> for <NUM> minutes in Model detergent X. The binding analysis was done at a pH of <NUM>, binding period of <NUM> minutes and at a temperature of <NUM>. The wash performance is indicated relative to the wash performance of the parent alpha-amylase having SEQ ID NO: <NUM> with the modifications H183*+G184*.

Claim 1:
A method for screening alpha-amylases having high wash performance at low temperature of <NUM>-<NUM>, wherein the alpha-amylases have at least <NUM>% sequence identity to SEQ ID NO: <NUM>; and comprising the steps of:
a) determining the binding of the alpha-amylases to solid or immobilized starch;
b) selecting alpha-amylases having a low binding to starch;
wherein the alpha-amylases have less than <NUM>% of the binding to starch than the alpha-amylase having SEQ ID NO: <NUM>.