DE10309803B4 - α-amylase variants with improved alkalinity - Google Patents

Abstract

hydrolytically active α-amylase variant with at least two amino acids exchanged across from the starting molecule, characterized in that in Position 13 according to the count of α-amylase from B. amyloliquefaciens (SEQ ID NO: 4) a proline residue (P) and in position 414 is a serine residue (S).

Description

The The present invention relates to α-amylase variants with an improved activity in alkaline medium and various technical applications for these α-amylase variants, in particular Detergents and cleaners with these α-amylase variants.

α-amylases (E.C. 3.2.1.1) hydrolyze internal α-1,4-glycosidic bonds of strength and starchy ones Polymers to form dextrins and β-1,6 branched oligosaccharides. by virtue of this strength-splitting one activity become α-amylases for numerous technical applications, including as an active component in detergents and cleaners. Because washing and cleaning agents predominantly have alkaline pH values, this particular α-amylases needed which are active in the alkaline medium. Most technically important α-amylases are from Microorganisms, i. Fungi or bacteria, especially those of the Genera Aspergillus and Bacillus are produced and secreted. outgoing Of these microbiological sources, they can mostly (gen) technical adapted accordingly Process on an industrial scale be won.

For the determination of suitable α-amylases for the respective areas, there is still an intensive search for new α-amylases from natural sources. Many such enzymes have already been described in the prior art; their number continues to increase. For use at alkaline pH values, in particular the alkaline α-amylases which are naturally formed by species of the genus Bacillus are of importance. Of particular technical importance are the α-amylases from Bacillus licheniformis, B. amyloliquefaciens and B. stearothermophilus. They are referred to in the present application as BLA, BAA or BStA. The wild-type B. licheniformis enzyme is available from Novozymes under the name Termamyl ^® and from Genencor under the name Purastar® ^® ST. The wild-type α-amylase from B. amyloliquefaciens is marketed by Novozymes under the name BAN ^® and the wild-type α-amylase from B. stearothermophilus under the names BSG ^®, likewise from Novozymes.

Another suitable for use in alkaline media α-amylase (referred to in the present application as LAMY) is the company Kao in the applications WO 94/26881 A1 ( EP 670367 A1 ) and WO 97/00324 A1. It also comes from an alkaline microorganism known as Bacillus sp. KSM-AP1378 (FERM BP-3048). Further, the α-amylase S707, originally from Bacillus sp. # 707 and with the publication "Nucleotide sequence of the maltohexaose-producing amylase gene from an alkalophilic Bacillus sp. # 707 and structural similarity to liquefying type α-amylases "by Tsukamoto et al., Biochem., Biophys. Res., Comm. (1988), 151 (1), 25-31.

Further examples for especially useful in detergents and cleaners amylolytic Enzymes are the α-amylase from Bacillus sp. A 7-7 (DSM 12368, referred to herein as A 7-7) in WO 02/10356 A2, and the enzyme from B. agaradherens (DSM 9948), at the same time as α-amylase and are considered as cyclodextrin glucanotransferase (CGTase) can and has been published in WO 02/44350 A2. Further α-amylases are those which correspond to the sequence space defined in the application WO 03/002711 A2 belong.

Out The application WO 00/60060 A2 Novozymes go the two α-amylases AA560 (from Bacillus sp. DSM 12649) and AA349 (from Bacillus sp DSM 12648), which both have an alkaline pH optimum. Two more termamyl-like α-amylases with the designations SP690 and SP722 as well as variants of these enzymes are disclosed, for example, with the application WO 99/23211 A1. You vote for the count the amino acid positions coincide with AA560 and AA349.

A another Bacillus amylase is described by Lin et al. in the publication "Production and property of a raw-starch-degrading Amylase from the thermophilic and alkaliphilic Bacillus sp. TS-23 "(1998), in Biotechnol. Appl. Biochem. Vol. 28 (1), pages 61 to 68. It is here as TS-23 designated. The associated DNA and amino acid sequences are, for example, in the database Swiss-Prot (Geneva Bioinformatics (GeneBio) S.A., Geneva, Switzerland; http://www.genebio.com/sprot.html) under number Q59222 and in GenBank (National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, MD, USA) deposited under the number U22045.

A comparison of the sequences of these enzymes requires an alignment in which the mutually corresponding amino acids come to lie one above the other. The amino acid positions concerning the present invention, which will be discussed later in detail, can be compared with these most important enzymes via the following, from the alignment of the 1 derived table. The Nu specified in the first line amyloliquefaciens (BAA) is the α-amylase according to SEQ ID NO. 4th

The amino acids are given in the international common one-letter code; Abbreviations of the enzymes as in the text; kA stands for: not specified. Table 1: Assignment of the amino acid substitutions of the present application in the count according to SEQ ID NO. 4 to the homologous positions of the most important α-amylases described in the prior art.

The naturally occurring α-amylase molecules in a variety of ways, especially molecular biology modifications been developed to meet specific applications to optimize. Such optimizations relate, for example the substrate specificities, the stability of the enzyme under different conditions or the enzymatic activity even.

As a molecular biologically important type of modification, the gene fusion or production of fusion proteins (hybrids) can be considered. Thus, for example, in the application WO 99/57250 A1 discloses that detergent enzymes can be increased in their contributions to the washing performance by coupling to cellulose-binding domains (CBD). In the patent application WO 99/43793 A1 sequence similarities between Novamyl ^® (see below) and known cyclodextrin glucanotransferases (CGTases) utilized to produce hybrids of both enzymes. The use of hybrids of α-amylases from B. amyloliquefaciens and B. licheniformis proceeds from the application DE 10138753 out. Hybrids of the B. stearothermophilus and B. licheniformis α-amylases are disclosed, for example, in the application EP 208491 A2 disclosed.

One but still important principle, such advancements is to perform point mutations, i.e. Deletions, insertions and especially substitutions of individual Amino acids. For example, the random mutagenesis method of the application WO 99/20768 A1 leads to α-amylase variants, which are particularly stable in the presence of detergent ingredients are. For practically all technically important wild-type enzymes (see above) are now numerous variants improved by point mutations have been described, these variations are not usually on limited the enzymes involved but are for related molecules similar Achieve performance improvements.

For example, the application WO 94/02597 A1 teaches to replace methionine residues by substitution with other amino acids, thereby lowering the sensitivity to oxidation and improving activity at higher temperatures. This is illustrated by variants of the B. licheniformis α-amylase. Further improvements to this Termamyl ^® molecule go out, for example from WO 95/26397 A1. Such optimization products of the α-amylase from B. licheniformis are Duramyl ^® under the trade name of the Fa. Novozymes available (SOFW-Journal, 123, (1997), pp 723-731). Another optimization product is Termamyl ^® ultra with lower calcium binding.

The α-amylases the application WO 97/43424 A1 show point mutations of the calcium-binding amino acids a changed one Calcium ion binding behavior and thus altered enzymatic properties. This finding is based on the 3D structure of the α-amylase B. licheniformis, but was also on other α-amylases applicable. The application WO 96/23874 A1 is based on findings from the 3D structure, namely different molecules, including also hybrid amylases, and beats numerous positions in which α-amylases, in particular the "classical" molecules BLA, BStA and BAA changed can be to change their enzymatic properties.

For example, from the application WO 96/23873 A1 variants of α-amylases from various Bacilli, including B. stearothermophilus, Bacillus sp. # 707 and the already improved molecules according to WO 95/26397 A1, which have higher oxidation stability, higher thermal stability and / or lower calcium dependence. The amylases of the application WO 99/02702 A1 with additional disulfide bridges are more stable at higher temperatures than the starting molecule. The enzymes of application WO 99/23211 A1 are more stable at high pH values and / or in the presence of calcium ions and / or more active at higher temperatures. Derivative variants, in particular of the α-amylase from B. stearothermophilus, for example, emerge from the application WO 02/02726 A1 for use in detergents and cleaners. The corresponding commercial product from Novozymes named Novamyl ^®.

Variants of the enzyme LAMY (from Bacillus sp KSM-AP1378) discloses the application EP 985731 A1 ,

Out the application WO 00/60060 A2 from the company. Novozymes, the α-amylases AA560 and AA349 are also available through point mutation Variants of these enzymes.

As the wild-type enzymes can also hybrid amylases additionally be further developed by point mutagenesis. This is disclosed, for example the application to be regarded as a further development of WO 96/23874 A1 WO 97/41213 A1. In the applications WO 00/60059 A2 and WO 00/60060 A2 variants are disclosed, which changed in terms of a Fission pattern at the substrate strength have been developed and therefore especially for starch processing suitable. In these writings are numerous point mutations both of native α-amylases, as well as hybrid amylases.

Compared to the here presented documents from the prior art are in following are the positions concerning this application and substitutions are reviewed.

Out the application WO 97/41213 A1 show variants that in the count after BLA exchanges D325N, R375E in combination with another Substitution and L3V, in combination with several others have defined exchanges.

The Application WO 00/60059 A2 discloses a variety of possibilities, α-amylases to change by point mutations. Including an exchange in position S168 (according to BLA) is proposed below, specifically the variant S168Y.

From the registration EP 985731 A1 show α-amylase variants that can be obtained by mutation in position A202 according to LAMY; in particular by exchange with T, I, L, A, V or S. This is in the count according to SEQ ID NO. 4 affected the relevant position A230.

The application WO 96/23874 A1 discloses numerous variants which influence the enzymatic activity, which have been suggested by comparing the 3D structures of various α-amylases. However, this does not include an exchange in a position concerning the present application. The corresponding patent US 5989169 also describes a method for detecting, by comparison of the 3D structures of different α-amylases, those positions concerning the calcium dependence or the stability of the enzymes in question. Only in this special context are there, besides numerous other positions, the amino acid positions A199 and Q443 of BLA called. A preliminary description of the fact that these positions could possibly relate to the alkali activity is not to be seen therein.

The two applications WO 91/00343 A2 and EP 409299 A2 relate to α-amylase variants with reduced stability. In this context, inter alia, exchanges in positions W163 and S166 according to the BAA count are proposed.

The application WO 95/10603 A1, in turn, discloses a variety of substitutions which, inter alia, are intended to influence the contributions of the respective enzymes to the washing performance, indicated in the BLA count. These include exchanges for which only areas in general are indicated; However, from the areas 142-182, 172-178, 260-269 and 369-383 mentioned there, only position 375 is concretely described. For this purpose, the replacement R375Y is disclosed. In particular, in the last-mentioned range, the position 383 is indicated as a limit, without this position, however, being given any significance, so that only the amino acids located between the edge positions seem to be meant. At the very least, it is not disclosed by any example that it is this position which has an influence on alkalinity activity. As a further relevant position, this document shows position 3; we recommend replacing the L3V.

The application WO 02/092797 A2 appears particularly complex. Here, in the "Examples" section, the amino acid exchanges now compiled in Table 2 are "revealed" in the BLA count. Table 2: In WO 02/092797 A2 "disclosed" amino acid substitutions.

Out Thus, this application does not substitute amino acid positions 174 and 383, corresponding to 203 and 414 in the count according to SEQ ID NO. 4 forth. On the other hand means in WO 02/092797 A2 only, these variants have been produced; however, information on biochemical properties is lacking. For no However, these positions are concretely influenced by usability described alkaline conditions.

In the above-mentioned application WO 99/23211 A1 are also variants enzymes SP690 and SP722, which contain the positions W167, S170, D271 and K385 relate, where, according to claim, respectively against all remaining amino acids is to be substituted; which means in this generality that here in principle every amino acid possible is. Concretely, only the replacement K385R, i.e., K385R, is disclosed. the substitution of one basic amino acid for another basic one amino acid at this position, in combination with other point mutations.

In summary, it should be noted that in the prior art, no exchanges in the positions 13 and 203 according to the counting of SEQ ID NO. 4 are prescribed. In position 203, as for example from Table 1 and from the alignment of 1 shows that in most α-amylases an isoleucine residue (I) is highly conserved. Position 13 is within the propeptide. For position 414, so far only the replacement of one basic amino acid with another basic amino acid has been disclosed, which correlates with the amino acid lysine (K) being present in most other α-amylases at this site. In contrast, a substitution for another amino acid, in particular against the amino acid serine, has not yet been described. Each of the other positions considered in the present invention has already been varied in the prior art, albeit rarely to the new amino acids specifically suggested herein, and by no means in these positional combinations.

From this introductory presentation it is also clear that there is an uninterrupted need to find or specifically develop new α-amylases in nature, especially with regard to diverse possible uses. In particular, for use in detergents and cleaners there is a need for further development of the various cleaning purposes, the changing habits and demands of consumers, such as lower cleaning temperatures or special dosage forms or from the development of other ingredients, the enzymes in their performance affect, such as bleaching compounds. Most importantly, most commercially available detergents and cleaners have a strong alkaline pH, so that improving the activity of the enzymes at high pHs poses a permanent challenge to the skin tion of detergents and cleaning agents.

It Thus, the task was to find new α-amylases or α-amylase variants to develop, which show better conditions, in the frame of washing and cleaning formulations to develop the highest possible activity and thus to improve their washing or cleaning performance. Which also includes in particular an improved activity under alkaline conditions.

A Sub-task was in view of the industrial applicability this development in it, the for such α-amylases coding nucleic acids to disposal to ask about the enzymes involved molecular biology advancements and technical manufacturing processes.

The solution The object is achieved according to the invention by providing α-amylase variants with at least two amino acid exchanges across from the starting molecule, characterized in that in position 13 according to the count of α-amylase from B. amyloliquefaciens (SEQ ID NO: 4) a proline residue (P) and in Position 414, a serine residue (S) is present. Another aspect to solution The object is achieved according to the invention by providing α-amylase variants with at least two amino acids exchanged across from the starting molecule, characterized in that an exchange in position 203 and a replacement at position 474 according to the count of B. amyloliquefaciens α-amylase (SEQ ID NO 4). Both aspects are through appropriate Variants illustrated in the examples for the present Application are described and correspondingly preferred embodiments of the present application (see below).

Α-Amylases according to the invention and as explained in the introduction are enzymes of the classification EC 3.2.1.1 which hydrolyze internal α-1,4-glycosidic bonds of starch and starch-like polymers to form dextrins and β-1,6-branched oligosaccharides. The invention is applicable in principle to all known and still to be found α-amylases, insofar as they can be homologated with the B. licheniformis α-amylase. Such a homologation is in 1 represents the technically most important α-amylases and allows or makes it easier to find the corresponding amino acid positions of the invention in the other amylases. Preferably, as explained below, the invention is applied to those α-amylases which are already known to be used in technical fields, preferably in those in which higher enzymatic activities, in particular at alkaline pH values, are desired. Examples of art-established α-amylases are given in the introduction.

Under Variants are proteins according to the invention to understand that are different from starting proteins in terms of amino acid sequence differ. The equivalent at the nucleic acid level is the mutant. For example, BAA L13P by a variant of BAA, all amino acids except for in position 13 has in common with the BAA. This corresponds to the DNA level Exchange of the codon CTT (37-39 according to SEQ ID NO. 3) against CCT (according to SEQ ID NO. 5).

By amino acid substitutions are meant substitutions of one amino acid against another amino acid. According to the invention, such substitutions are indicated by designation of the positions in which the exchange takes place, if appropriate combined with the relevant amino acids in the internationally used single-letter code. "Substitution in position 13" means, for example, that a variant in the position which has position 13 in the sequence of a reference protein has a different amino acid. Exchange does not mean that this amino acid has been changed objectively, but merely designates a sequence difference For example, "L13P" means that the reference enzyme at position 13 has the amino acid leucine, while the variant under consideration has the amino acid proline at the homologizable position. "13P" means that any, that is usually a naturally given, amino acid is replaced with a proline at a position corresponding to position 13. An example of such a homologous exchange would be L-18P in BLA, ie B. licheniformis α-amylase formed because of 1 allows the position 13 of BAA to be homologated with BLA position -18.

In principle, the amino acid substitutions proposed by the present application are not limited to being the only substitutions in which the variant in question differs from the wild-type molecule. It is known in the art that the beneficial properties of single point mutations can complement each other. A with respect to certain properties such as calcium-binding or stability to surfactants optimized α-amylase according to the invention additionally be further developed through the substitutions presented here. Thus, embodiments of the present invention include all variants which, in addition to other exchanges with the wild-type molecule, also comprise the exchanges according to the invention. This is especially true for molecules obtained via fusion mutation (hybridization) (see below).

According to the invention, the non-processed α-amylase from B. amyloliquefaciens, whose nucleotide sequence is shown in SEQ ID NO. 1 and its amino acid sequence in SEQ ID NO. is specified. This amino acid sequence also goes from the first few lines in the alignments of 1 and 2 (BAA). The variants according to the invention are described by way of this comparison. It may be necessary to take into account that in the in vivo synthesis of this amylase by B. amyloliquefaciens, the first 31 amino acids are split off from the remaining mature protein as so-called propetide. For some enzymes, such as AA349 and AA560, only the mature parts are given in the patent literature (see above). Here, homologization can only be carried out via the respective positions of the mature region and is corresponding by sequence comparison 1 possible.

Inventive α-amylase variants are characterized in that Position 13 according to the count of α-amylase from B. amyloliquefaciens (SEQ ID NO: 4) a proline residue (P) and in Position 414, a serine residue (S) is present.

As illustrated in the prior art, no exchanges in the position 13 according to the counting of SEQ ID NO. 4 described above. To 1 it is within the propeptide for all considered α-amylases. In the context of the present invention, it is particularly surprising that an amino acid exchange in one position of the propeptide can exert a positive influence on the subsequent activity of the relevant protein. Without wishing to be bound by this theory, one might suspect that this is due to folding effects of the resulting polypeptide chain. Proline is known to have strong influence on secondary and tertiary structure in proteins.

In item 414, most other α-amylases have a basic amino acid (see Tables 1 and 4) 1 ). Accordingly, the state of the art in this position so far suggests only the replacement of one basic amino acid with another basic amino acid. It is thus completely surprising that the exchange for an amino acid with a hydroxyl group influences the activity, especially in the alkaline medium.

In The examples of the present application are three variants of the α-amylase B. licheniformis (BAA A42, BAA A29 and BAA B1) described in detail. You have across from this comparative molecule (the Wild-type enzyme), measured at pH 6.5 over significantly higher specific activities (see Example 5). additionally indicates the variant BAA A42 the wild-type molecule one shifted by a whole pH unit in the alkaline range pH optimum on. Also features she over a significantly increased relative activity at pH 10 (see Example 6).

These three variants have two similarities, namely the two amino acid substitutions L13P and N414S. Thus, according to the invention, it can be assumed that these both exchanges the activity of α-amylases, in particular their alkalinity to increase.

Preferred α-amylase variants this aspect of the invention are characterized in that additionally at least an amino acid exchange in one of the positions 194, 197 or 356 according to the count of B. amyloliquefaciens α-amylase (SEQ ID NO. 4) is present, preferably in two, more preferably in old three of these positions.

The exchanges at positions 194 and 197 are in a range that is highly conserved in many α-amylases; this goes for example 1 (205 and 208). It is therefore surprising that at these positions it is even possible to successfully vary, and yet functional molecules are obtained. In particular, an increase in performance was not expected. Also the position 356 (368 according to 1 ) is highly conserved: there is an aspartic acid residue in most other α-amylases and a glutamic acid residue only in the BAA.

The The success of these exchanges is demonstrated by the BAA variant A42, whose DNA and amino acid sequences below SEQ ID NO. 5 and 6 respectively. From Example 5 goes the performance increase and from example 6 the most high performance in alkaline medium.

Preferred α-amylase variants with these substitutions are characterized in that in position 194 an arginine (R) and / or in position 197 a proline (P) and / or in position 356 an aspartic acid residue (D) is present.

Especially it is preferably a variant with the replacement 13P / 194R / 197P / 356D / 414S.

This is surprising of those mentioned positive characteristics of the variant BAA A42 supported.

In another preferred embodiment is an α-amylase variant with the exchanges 13P and 414S characterized in that additionally at least an amino acid exchange in one of the positions 32, 230, 297 or 406 according to the count of the α-amylase B. amyloliquefaciens (SEQ ID NO: 4) is present, preferably in two, more preferably in three, most preferably in all four of these positions.

Position 32 is the first amino acid of the mature protein in BAA, and represents the third amino acid of the mature protein in most other α-amylases. It may play some role in the folding of the protein. Position 230 (242 according to FIG 1 ) is naturally occupied by an alanine residue in most other α-amylases and is in a highly conserved region. So it is surprising that in such a position a successful exchange can take place. The latter also applies to position 297 (309) 1 ), which in the BAA is naturally occupied by an N, ie an amino acid having an acid amide side chain. In position 406 (418 according to 1 ) is naturally a K at the BAA; the most important α-amylases of the prior art have there the amino acid residues R, Y or H, so rather show a nonuniform image; This is especially because Y has a hydroxyl group.

In a preferred embodiment are α-amylase variants characterized in that in position 32 an alanine (A) and / or at position 230 a valine (V) and / or at position 297 an aspartic acid (D) and / or at position 406 an arginine residue is present.

Especially for position It was not to be expected that this would be a room fuller amino acid successfully introduced as alanine can be. It is surprising also that the better suitability of the variants of the invention for alkaline Conditions apparently only slightly with the change to more alkaline overall or stronger acidic amino acids accompanied. Because the change in position 297 means for the BAA the transition from a neutral to an acidic amino acid and in position 406 from a basic to a stronger basic amino acid.

Especially preferred are such α-amylase variants, which are characterized in that they are variants with replacement 13P / 32A / 230V / 297D / 406R / 414S.

One such molecule is provided with the variant BAA A29 and in the examples examined for its biochemical properties. she has particularly high activity values compared to the wild-type enzyme, i. the molecule without these six exchanges on.

As As already stated above, another aspect of the solution exists Task in the provision of α-amylase variants with at least two amino acid exchanges across from the starting molecule, characterized in that an exchange in position 203 and a replacement at position 474 according to the count of B. amyloliquefaciens α-amylase (SEQ ID NO 4).

in this connection these can be exchanges that take place on a wild-type molecule or, in particular, those already existing and possibly in a certain direction improved variant additionally made have been.

The position 203 is how 1 shows, in a relatively conserved region of the α-amylases; in particular, isoleucine is present at this point in all other important α-amylases. For item 474, the situation looks more indifferent, as here are in the comparative enzymes of the prior art various amino acids, especially K, H, N or Q. However, there is no indication that these positions for the alkali stability and in particular the alkalinity activity could be of importance.

In a preferred embodiment, such α-amylase variants are characterized in that that in position 203 a leucine (L) and / or in position 474 a glutamine residue (Q) is present.

Because about this Both substitutions, the variant of the invention differs BAA B1 from BAA A29. This preserves they, as Example 6 shows, with a not measurably changed pH optimum a higher relative activity at pH 10.

In a preferred embodiment these exchanges in positions 203 and 474 will not be at one Wild-type molecule but on a molecule made, which already the exchanges according to the invention shown above in positions 13 and 414 and additionally in positions 32, 230, 297 and / or 406. Because for these are the illustrated advantageous properties already shown above.

claimed Thus, such α-amylase variants, the additional on the exchanges mentioned in headings 13 and 414, in combination characterized by those in positions 32, 230, 297 and / or 406 are that in addition at least an amino acid exchange in one of the positions 203 or 474 according to the count of B. amyloliquefaciens α-amylase (SEQ ID NO. 4), preferably in both positions.

Corresponding from the above, those α-amylase variants are also preferred characterized in that at position 203 a leucine (L) and / or at position 474 is a glutamine residue (Q).

A particularly preferred embodiment This and the first aspect of the invention is an α-amylase variant, which is characterized in that it is a variant Replacement 13P / 32A / 203L / 230V / 297D / 406R / 414S / 474Q.

One Representative of such a molecule is that obtained according to Examples 1 to 3 and in the examples 4 to 6 characterized variant BAA B1, which is also the name B. amyloliquefaciens α-amylase L13P / V32A / I203L / A230V / N297S / K406R / N414S / K474Q wearing. She stands opposite the wild-type molecule BAA in particular by an increased activity from (Example 5).

As Illustrated in the prior art are numerous α-amylases for the Use for various technical purposes, especially as active Components in detergents and cleaning agents established. These include fungal and especially bacterial enzymes. Especially common are those of Gram-positive bacteria, especially the genus Bacillus adapted to an alkaline environment. Because they reject cheap in advance Properties for these fields of application. According to the invention they can by additional Point mutations will be further developed. In the scope of the present application falls thus generally any α-amylase variant, characterized by the amino acid variations identified above is, preferably each, for already described an appropriate use, for example also the molecules AA560, AA349, SP690 and SP722.

Prefers this is any α-amylase variant, which is characterized in that the starting molecule of an α-amylase derives originally from a Bacillus species, preferably from B. amyloliquefaciens, B. licheniformis, B. agaradherens, B. stearothermophilus, B. sp. KSM-AP 1378, B. sp. # 707 or B. sp. A 7-7 (DSM 12368), especially preferably from B. amyloliquefaciens, B. agaradherens (DSM 9948) or Sp. A 7-7 (DSM 12368), especially from B. amyloliquefaciens.

These Enzymes have been treated in detail in the introduction. Especially preferred among these are, on the one hand, those which differ from the α-amylases from B. agaradherens (DSM 9948) and from B. sp. A 7-7 (DSM 12368) derived. The first-named molecule, simultaneously called α-amylase and as cyclodextrin glucanotransferase (CGTase) is described in the application WO 02/44350 A2 described. The α-amylase from Bacillus sp. A 7-7 (DSM 12368), referred to herein as A 7-7 is, it is apparent from the application WO 02/10356 A2. For both So far, no performance improvements have been described about point mutations so that the Variations according to the invention therefor particularly promising starting points should be. To the other variants of the B. amyloliquefaciens enzyme (BAA) preferred because these are prepared according to the examples set forth herein have been and have the said advantageous properties.

As mentioned, the present invention is not limited to natural or largely unmodified enzymes but can be applied in particular to hybrid amylases. Because the generation of α-amylases having positive properties via fusion mutagenesis has now been described in detail in the prior art.

Accordingly is any α-amylase variant of the invention claimed in addition characterized in that it at the parent molecule a hybrid α-amylase is preferably based on at least one of the α-amylases from B. amyloliquefaciens, B. licheniformis, B. agaradherens, B. stearothermophilus, B. sp. KSM-AP 1378, B. sp. # 707 or B. sp. A 7-7 (DSM 12368), more preferably based on at least two of the α-amylases from B. amyloliquefaciens, B. licheniformis, B. agaradherens, B. stearothermophilus, B. sp. KSM-AP 1378, B. sp. # 707 or B. sp. A 7-7 (DSM 12368).

In particular from the application DE 10138753 Hybrids of α-amylases from B. amyloliquefaciens and B. licheniformis, which show positive properties especially when used in detergents and cleaners.

Especially Thus, each α-amylase variant according to the invention is preferred is characterized in that it in the hybrid α-amylase is one of whose subsequences of the α-amylases from B. amyloliquefaciens and derived from B. licheniformis.

Especially preferred is an α-amylase variant, if it is characterized in that it is a variant the α-amylase of B. amyloliquefaciens with one of the amino acid exchange combinations L13P / W194R / S197P / E356D / N414S, L13PN32A / A230V / N297D / K406R / N414S or L13PN32A / I203L / A230V / N297D / K406R / N414S / K474Q preferably with the amino acid exchange combination L13P / W194R / S197P / E356D / N414S.

Because these molecules are under the designations BAA 42, BAA 29 and BAA B1 in the examples of the described in the present application. Among them, the enzyme has the amino acid exchange combination L13P / W194R / S197P / E356D / N414S (BAA A42) has a particularly high alkalinity activity under Proof.

Preferably are these α-amylase variants characterized in that it is the α-amylase variant from B. amyloliquefaciens A42 (SEQ ID NO: 6), from B. amyloliquefaciens A29 (SEQ ID NO: 8) or from B. amyloliquefaciens B1 (SEQ ID NO. 10), preferably the α-amylase variant from B. amyloliquefaciens A42 (SEQ ID NO. 6).

In In fact, as shown in Example 1, the molecules BAA A42, A29 and B1 are not from trunks the species B. amyloliquefaciens itself. Originally came only the sequence for the wild-type molecule from B. amyloliquefaciens, as for example under the number DSM 7 from the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1 b, 38124 Braunschweig (http://www.dsmz.de) can be obtained. This is under SEQ ID NO.3 respectively 4 indicated. The relevant mutant B. amyloliquefaciens strains may be, for example be generated by the fact that the illustrated pGBAA plasmids preferably derived therefrom plasmids with larger flanking Regions are transferred to the BAA gene in the wild-type cells. Analogously, the Genes from SEQ ID NO. 5, 7 and 9 also synthesized and in corresponding Vectors are introduced. By homologous recombination of the mutant Gene in the associated The site in the chromosome becomes the claimed mutant of B. amyloliquefaciens which permanently expresses the mutated gene. This procedure is particularly advantageous if the protein in question also to be produced by a strain of this species.

a own subject of the invention represent the nucleic acids, the for one of the previously described α-amylase variants encode.

this includes are both RNA and DNA molecules and DNA analogs to understand both the coding and the codogenic strand, in each reading frame. For example, it would be possible with the invention exploit connected doctrine to have an interfering corresponding RNA to make a regulation of corresponding α-amylases or the To increase lifespan of genetic information by putting in an in vivo slower degradable DNA analogue is transferred. The nucleic acids open, like at the below Invention objects can be seen, the present invention to some extent the molecular biological dimension. They are according to the above accordingly preferred.

Preferably, these are to be understood as meaning DNA molecules. Because of these, it is possible to produce the α-amylase variants according to the invention by molecular biological methods known per se and / or optionally further modified.

starting points for this nucleic acid can be the DNA sequence information given in the Sequence Listing, to over chemical re-synthesis to obtain the genes in question. In contrast, can but it may also be advantageous to already available in vivo nucleic acids resorting, such as those for encode the known in the art α-amylases. Another Alternatively, according to Example 1, a total DNA preparation from certain ones contemplated To make microorganisms and over PCR to isolate the endogenous α-amylase genes. As a primer can in principle, those from SEQ ID NO. 3 also 5'- and 3'-sequence sections to be read be used.

A is preferred nucleic acid according to the invention thus characterized in that they differ from a nucleic acid according to SEQ ID NO. 3 derives.

among them are not just those that are explained as explained above this sequence have been obtained, but also all mutants, which have been generated in positions other than just those who be described with the present application.

A preferred embodiment applies to those actually studied in the present application Examples back.

Especially Thus, each nucleic acid according to the invention is preferred, which is characterized is, that you different from a nucleic acid according to SEQ ID NO. 5, SEQ ID NO. 7 or SEQ ID NO. 9 derives, preferably one these sequences themselves.

A particularly advantageous form to handle nucleic acids provide Vectors because they have areas that make it possible to to characterize the particular gene of interest (about flanking areas for Seqzenzierprimer), and by transformation if necessary to activate again or to express it when the derived Protein examined and produced in larger quantities shall be. In vectors, it may be particularly eukaryotic Host cells such as yeast are artificial Act chromosomes; in bacteria, vectors are usually plasmids.

a own subject of the invention for the realization of the present Invention thus represent vectors that a range with a Nucleic acid described above contain.

In a preferred embodiment such a vector is characterized by being a cloning vector is.

Because in this form, the DNA in question can be stored as stated above be or over for example introduction further mutations further changed which, for example, further developed inventive α-amylase variants can be generated.

In another preferred embodiment such a vector is characterized by being an expression vector is.

Because This can be an α-amylase variant according to the invention in a convenient molecular biological way even in larger quantities be generated.

In further advantageous embodiments the said vectors have origins of replication for different microorganisms on, causing them as so-called shuttle vectors in different Host cells can be introduced and maintained stable there. It is also possible To produce vectors that can be used both as cloning and as Expression vector can be considered.

Corresponding the above and the methodology established in the prior art the molecular-biological dimension also extends to organisms, especially on microorganisms.

a own subject of the invention thus represent cells that by are characterized in that they contain one of the above-identified nucleic acids, preferably on a vector indicated above.

These include, for example, those which transforms with a corresponding cloning vector have been derived or derived from such. This is used in particular for the characterization and / or storage of the gene in question. In contrast, it is particularly important for the realization of the present invention to actually obtain the enzyme in question; because of its improved properties, there is a special commercial interest. For example, this formation can be permanent and at high production rates when the transgene of interest is under the control of a constitutive and strong promoter. Alternatively, it is also possible to place the gene for the α-amylase variant under the control of an inducible and advantageously no less potent promoter. As a result, the production can be selectively switched on from the outside, for example, if there are favorable conditions for this. This is the case with fermentative production, for example, when a certain cell density has been reached.

Prefers is thus each cell referred to above, which is characterized is, that you one of the above-designated α-amylase variants forms or can be stimulated to their formation, preferably using a nucleic acid described above, more preferably below Use of an expression vector referred to above.

A additionally advantageous embodiment refers to the fact that the most industrially important α-amylases originally as enzymes have been found by the microorganisms in question, in particular Bacteria are secreted into the surrounding medium. Because in vivo acts most of them are digestive enzymes. Accordingly, it is advantageous when the industrially produced enzymes of the invention also in the surrounding Be secreted medium. Because from this they can without cell disruption and thus be worked up comparatively easily. This can be, for example be achieved in that the relevant genes are supplemented by appropriate sequences - if they are not already available are the for a Leader peptide code, which causes the cell concerned auszuschleusen them. An alternative with Gram-negative bacteria is, for example in it, the outer membrane to Partially open release of proteins, such as this in the application WO 01/81597 A1.

A preferred cell of the invention is therefore characterized in that it is a bacterium in particular one which secretes the formed α-amylase variant into the surrounding medium.

Especially the characterization of genes and enzymes is carried out on a laboratory scale in usually over Inter-cloning and / or expression in Gram-negative bacteria. This is also confirmed by the examples of the present application. There are but also established production methods based on gram-negative ones Build bacteria (see above).

The hitherto described cells of the invention are preferred thus characterized in that it is gram-negative Bacteria, preferably of the genera Escherichia coli or Klebsiella, especially derivatives of E. coli K12, E. coli B or K. planticola, and especially derivatives of strains E. coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or K. planticola (Rf).

in the The state of the art is especially for the production of enzymes from gram-positive organisms, for these also Gram-positive expression hosts select. But also in terms of characterization on a laboratory scale it is not uncommon to work with Gram-positive bacteria. Is particularly advantageous it, if a particular enzyme is also produced by the host, from which it originally comes because of this the respective Reguklationssignale i.A. optimal be implemented.

The thus previously described cells of the invention are not less preferably characterized in that it is grampositive Bacteria, in particular the genera Staphylococcus, Corynebacterium or Bacillus, in particular the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B. alcalophilus, B. licheniformis, B. amyloliquefaciens, B. stearothermophilus, B. agaradherens, B. globigii or B. lentus.

this Subject of the invention are, after all, still all methods for the production of an α-amylase variant according to the invention. Because this is the object of economic interest indeed to disposal posed. Such a production can for example via a chemical synthesis of the protein in question.

In contrast, are in accordance with the known and molecular biological techniques presented above however, those methods which rely on the nucleic acids set forth above particularly preferably using a vector shown above and most preferably using one described above Cell.

In in connection with the present invention means, which are characterized in that they have an α-amylase variant described above contain.

Herewith is meant any agent in which the subject α-amylase in any form, that is, in the desired brought hydrolytic contact to its substrate starch or starch-like polysaccharide or for this is prepared. The desired Contact usually takes place in an aqueous environment, conveniently is buffered to an appropriate pH and optionally further favoring Contains factors. These include For example, other enzymes that further the immediate reaction products implement, for example, with regard to starch liquefaction for the food or Animal feed production or for an ethanol production. Furthermore, this includes low molecular weight Compounds, for example, in resulting oligosaccharides as cyclodextrins are included, or low molecular weight compounds, which further solubilize the cleavage products or favor the overall process Have effect, such as surfactants in the context of a detergent formulation. It may also be agents in which the desired amylolytic activity only with a big one Time Delay is to be induced, as in temporary adhesive bonding, after which the α-amylase variant the adhesive in question early added but only after a long time by increasing the water content actually becomes active. This also applies, for example, to detergents and cleaners to, which only after a phase of storage at the moment of dilution in the wash liquor opposite to become active in the substrate.

Especially Relevant to the invention are such agents, which characterized are that it is washing or cleaning agents.

The properties of the invention and important ingredients for Such washing and Detergents are detailed below. At this Job takes place first an overview of the most important and therefore inventively particularly relevant forms such detergents and cleaners.

Preferably is a invention relevant washing or cleaning agent thereby characterized in that it 0.000001 weight percent to 5 weight percent, and increasingly preferred 0.00001 to 4 wt .-%, 0.0001 to 3 wt .-%, 0.001 to 2 wt .-% or 0.01 to 1 wt .-% of the α-amylase variant contains.

Preferably is a invention relevant washing or cleaning agent thereby characterized in that it additionally contains other enzymes, especially hydrolytic enzymes or oxidoreductases, especially preferably further amylases, proteases, lipases, cutinases, hemicellulases, Cellulases, β-glucanases, oxidases, Peroxidases, laccases.

Preferably is a invention relevant washing or cleaning agent thereby characterized in that the α-amylase variant stabilized by one of the other ingredients of the agent and / or in their contribution to the washing or cleaning performance of the agent is increased.

Preferably is a invention relevant washing or cleaning agent thereby characterized in that it is generally solid, preferably after a compaction step for at least one of the components contained, more preferably, that it is in total is compacted.

Preferably is a invention relevant washing or cleaning agent thereby characterized in that it totally liquid, gel or pasty is, preferably encapsulated for at least one of the contained Components, particularly preferably encapsulated at least one the enzymes contained, most preferably encapsulated the α-amylase variant.

An important application for amylases is as active components in detergents or cleaners for cleaning textiles or solid surfaces such as crockery, floors or implements. In these applications, the amylolytic activity serves to hydrolytically dissolve or dissolve carbohydrate-containing impurities, and especially starch-based ones, from the subsurface. They can be used alone, in suitable media or in detergents or cleaners for application. The conditions to be chosen for this, such as temperature, pH, ionic strength Ke, redox ratios or mechanical influences should be optimized for the particular cleaning problem, so in terms of soiling and on the substrate. For example, normal temperatures for detergents and cleaners range from 10 ° C for manual agents above 40 ° C and 60 ° C up to 95 ° for mechanical or technical applications. Since the temperature is usually infinitely adjustable in modern washing machines and dishwashers, all intermediate stages of the temperature are included. Preferably, the ingredients of the respective agents are coordinated. The other conditions can also be interpreted very specific to the particular cleaning purpose of the other, below executed components of the funds.

preferred Invention-relevant washing and cleaning agents are characterized from that under any the conditions definable in this way the washing or cleaning performance this agent by adding an α-amylase variant according to the invention over the Recipe without this variant is improved. Synergies are preferred in terms of cleaning performance.

A α-amylase variant according to the invention can be used both in funds for bulk consumers or technical users as well as in consumer products application All of the established and / or suitable dosage forms in the prior art also embodiments of the present invention. With the invention relevant Washing or cleaning agents are therefore all conceivable types of detergents meaning both concentrates and undiluted agents; to the Use on a commercial scale, in the washing machine or hand wash, or cleaning. These include, for example, detergents for textiles, Carpets, or natural fibers, for the term detergent according to the present invention is used. This includes for example, dishwashing detergent for dishwashers or manual dishwashing detergent or cleaner for hard surfaces like metal, glass, porcelain, ceramics, tiles, stone, painted Surfaces, Plastics, wood or leather; For such is the term detergent according to the present invention used. Any type of washing or cleaning agent is relevant to the invention, if they are an amylase according to the invention enriched.

suitable Forms of invention-relevant means include all according to the state the technique established and / or all appropriate dosage forms. To counting For example, solid, powdery, liquid, gel or pasty Means, optionally also of several phases, compressed or not compressed; furthermore belong for example: extrudates, granules, tablets or pouches, both in bulk containers as well as portionwise packed.

α-amylases, are in relevant to the invention, for example, with individual or more of the following ingredients combined: nonionic, anionic and / or cationic surfactants, bleaches, bleach activators, Bleach catalysts, builders and / or cobuilders, solvents, Thickeners, sequestrants, electrolytes, optical brighteners, Grayness inhibitors, corrosion inhibitors, especially silver protectants, soil release agents, Color transfer (or transfer) inhibitors, Foam inhibitors, abrasives, dyes, fragrances, antimicrobial Active ingredients, UV protectants, Enzymes such as proteases, (optionally other) amylases, Lipases, cellulases, hemicellulases or oxidases, stabilizers, in particular enzyme stabilizers, and other components made up known in the art.

The nonionic surfactants used are preferably alkoxylated, advantageously ethoxylated, in particular primary, alcohols having preferably 8 to 18 carbon atoms and on average 1 to 12 moles of ethylene oxide (EO) per mole of alcohol, in which the alcohol radical can be linear or preferably methyl-branched in the 2-position , or may contain linear and methyl-branched radicals in the mixture, as they are usually present in Oxoalkoholresten. In particular, however, alcohol ethoxylates with linear radicals of alcohols of native origin having 12 to 18 carbon atoms, for example of coconut, palm, tallow or oleyl alcohol, and on average 2 to 8 EO per mole of alcohol are preferred. The preferred ethoxylated alcohols include, for example, C _12-14 alcohols with 3 EO or 4 EO, C _9-11 alcohols with 7 EO, C _13-15 alcohols with 3 EO, 5 EO, 7 EO or 8 EO, C _12-18 alcohols with 3 EO, 5 EO or 7 EO and mixtures of these, such as mixtures of C _12-14 -alcohol with 3 EO and C _12-18 -alcohol with 5 EO. The degrees of ethoxylation given represent statistical means which, for a particular product, may be an integer or a fractional number. Preferred alcohol ethoxylates have a narrow homolog distribution (narrow range ethoxylates, NRE). In addition to these nonionic surfactants, fatty alcohols with more than 12 EO can also be used. Examples include tallow fatty alcohol with 14 EO, 25 EO, 30 EO or 40 EO.

Another class of preferred nonionic surfactants used either as the sole nonionic surfactant or in combination with other nonionic surfactants are alkoxylated te, preferably ethoxylated or ethoxylated and propoxylated fatty acid alkyl esters, preferably having 1 to 4 carbon atoms in the alkyl chain, in particular fatty acid methyl esters.

Another class of nonionic surfactants that can be used to advantage are the alkyl polyglycosides (APG). Usable Alkypolyglycoside meet the general formula RO (G) _z , in which R is a linear or branched, especially in the 2-position methyl branched, saturated or unsaturated, aliphatic radical having 8 to 22, preferably 12 to 18 carbon atoms and G is the Is a symbol which represents a glycose unit having 5 or 6 C atoms, preferably glucose. The degree of glycosylation z is between 1.0 and 4.0, preferably between 1.0 and 2.0 and in particular between 1.1 and 1.4. Preference is given to using linear alkyl polyglucosides, that is to say alkyl polyglycosides in which the polyglycosyl radical is a glucose radical and the alkyl radical is an n-alkyl radical.

Also nonionic surfactants of the amine oxide type, for example N-cocoalkyl-N, N-dimethylamine oxide and N-tallow alkyl-N, N-dihydroxyethylamine oxide, and the fatty acid alkanolamide can be suitable. The proportion of these nonionic surfactants is preferably no over that of the ethoxylated fatty alcohols, especially in not more than half from that.

in der RCO für einen aliphatischen Acylrest mit 6 bis 22 Kohlenstoffatomen, R¹ für Wasserstoff, einen Alkyl- oder Hydroxyalkylrest mit 1 bis 4 Kohlenstoffatomen und [Z] für einen linearen oder verzweigten Polyhydroxyalkylrest mit 3 bis 10 Kohlenstoffatomen und 3 bis 10 Hydroxylgruppen steht. Bei den Polyhydroxyfettsäureamiden handelt es sich um bekannte Stoffe, die üblicherweise durch reduktive Aminierung eines reduzierenden Zuckers mit Ammoniak, einem Alkylamin oder einem Alkanolamin und nachfolgende Acylierung mit einer Fettsäure, einem Fettsäurealkylester oder einem Fettsäurechlorid erhalten werden können.Further suitable surfactants are polyhydroxy fatty acid amides of the formula (II)

wherein RCO is an aliphatic acyl group having 6 to 22 carbon atoms, R ^{1 is} hydrogen, an alkyl or hydroxyalkyl group having 1 to 4 carbon atoms and [Z] is a linear or branched polyhydroxyalkyl group having 3 to 10 carbon atoms and 3 to 10 hydroxyl groups. The polyhydroxy fatty acid amides are known substances which can usually be obtained by reductive amination of a reducing sugar with ammonia, an alkylamine or an alkanolamine and subsequent acylation with a fatty acid, a fatty acid alkyl ester or a fatty acid chloride.

in der R für einen linearen oder verzweigten Alkyl- oder Alkenylrest mit 7 bis 12 Kohlenstoffatomen, R¹ für einen linearen, verzweigten oder cyclischen Alkylrest oder einen Arylrest mit 2 bis 8 Kohlenstoffatomen und R² für einen linearen, verzweigten oder cyclischen Alkylrest oder einen Arylrest oder einen Oxy-Alkylrest mit 1 bis 8 Kohlenstoffatomen steht, wobei C_1-4-Alkyl- oder Phenylreste bevorzugt sind und [Z] für einen linearen Polyhydroxyalkylrest steht, dessen Alkylkette mit mindestens zwei Hydroxylgruppen substituiert ist, oder alkoxylierte, vorzugsweise ethoxylierte oder propoxylierte Derivate dieses Restes.The group of polyhydroxy fatty acid amides also includes compounds of the formula (III)

in the R is a linear or branched alkyl or alkenyl radical having 7 to 12 carbon atoms, R ^{1 is} a linear, branched or cyclic alkyl radical or an aryl radical having 2 to 8 carbon atoms and R ^{2 is} a linear, branched or cyclic alkyl radical or an aryl radical or an oxyalkyl radical having 1 to 8 carbon atoms, with C _1-4 alkyl or phenyl radicals being preferred and [Z] being a linear polyhydroxyalkyl radical whose alkyl chain is substituted by at least two hydroxyl groups, or alkoxylated, preferably ethoxylated or propoxylated Derivatives of this residue.

[Z] is preferably prepared by reductive amination of a reducing Sugar, for example glucose, fructose, maltose, lactose, Galactose, mannose or xylose. The N-alkoxy or N-aryloxy-substituted Connections can for example, by reaction with fatty acid methyl esters in the presence an alkoxide can be converted as a catalyst into the desired polyhydroxy fatty acid amides.

As anionic surfactants, for example, those of the sulfonate type and sulfates are used. The surfactants of the sulfonate type are preferably C _9-13 -alkylbenzenesulfonates, olefinsulfonates, ie mixtures of alkene and hydroxyalkanesulfonates and disulfonates, as are obtained, for example, from C _12-18 -monoolefins having terminal or internal double bonds by sulfonation with gaseous sulfur trioxide and subsequent alkaline or acid hydrolysis of the sulfonation products into consideration. Also suitable are alkanesulfonates which are obtained from C _12-18 alkanes, for example by sulfochlorination or sulfoxidation with subsequent hydrolysis or neutralization. Likewise suitable are the esters of α-sulfo fatty acids (ester sulfonates), for example the α-sulfonated methyl esters of hydrogenated coconut, palm kernel or tallow fatty acids.

Further suitable anionic surfactants are sulfated fatty acid glycerol esters. Fatty acid glycerol esters are to be understood as meaning the mono-, di- and triesters and mixtures thereof, as used in the preparation by Veres obtained from a monoglycerol with 1 to 3 moles of fatty acid or in the transesterification of triglycerides with 0.3 to 2 moles of glycerol. Preferred sulfated fatty acid glycerol esters are the sulfonation products of saturated fatty acids having 6 to 22 carbon atoms, for example caproic acid, caprylic acid, capric acid, myristic acid, lauric acid, palmitic acid, stearic acid or behenic acid.

Alk (en) ylsulfates are the alkali metal salts and in particular the sodium salts of the sulfuric monoesters of C ₁₂ -C ₁₈ fatty alcohols, for example coconut fatty alcohol, tallow fatty alcohol, lauryl, myristyl, cetyl or stearyl alcohol or the C ₁₀ -C ₂₀ oxo alcohols and those half-esters of secondary alcohols of these chain lengths are preferred. Also preferred are alk (en) ylsulfates of said chain length, which contain a synthetic, produced on a petrochemical basis straight-chain alkyl radical, which have an analogous degradation behavior as the adequate compounds based on oleochemical raw materials. Of washing technology interest, the C ₁₂ -C ₁₆ alkyl sulfates and C ₁₂ -C ₁₅ alkyl sulfates and C ₁₄ -C ₁₅ alkyl sulfates are preferred. 2,3-alkyl sulfates are also suitable anionic surfactants.

The sulfuric acid monoesters of straight-chain or branched C _7-21 -alcohols ethoxylated with from 1 to 6 mol of ethylene oxide, such as 2-methyl-branched C _9-11- alcohols having on average 3.5 mol of ethylene oxide (EO) or C _12-18 . Fatty alcohols with 1 to 4 EO are suitable. Due to their high foaming behavior, they are only used in detergents in relatively small amounts, for example in amounts of up to 5% by weight, usually from 1 to 5% by weight.

Further suitable anionic surfactants are also the salts of alkylsulfosuccinic acid, which are also referred to as sulfosuccinates or as sulfosuccinic acid esters and the monoesters and / or diesters of sulfosuccinic acid with alcohols, preferably fatty alcohols and in particular ethoxylated fatty alcohols. Preferred sulfosuccinates contain C _8-18 fatty alcohol residues or mixtures of these. Particularly preferred sulfosuccinates contain a fatty alcohol residue derived from ethoxylated fatty alcohols, which in themselves constitute nonionic surfactants (see description below). Sulfosuccinates, whose fatty alcohol residues are derived from ethoxylated fatty alcohols with a narrow homolog distribution, are again particularly preferred. Likewise, it is also possible to use alk (en) ylsuccinic acid having preferably 8 to 18 carbon atoms in the alk (en) yl chain or salts thereof.

When other anionic surfactants are especially soaps into consideration. Suitable are saturated fatty acid soaps, like the salts of lauric acid, myristic, palmitic acid, stearic acid, hydrogenated erucic acid and behenic acid and in particular of natural fatty acids, for example, coconut, palm kernel or tallow fatty acids, derived soap mixtures.

The including anionic surfactants the soaps can in the form of their sodium, potassium or Ammonium salts and as soluble Salts of organic bases, such as mono-, di- or triethanolamine, are present. The anionic surfactants are preferably in the form of their sodium or potassium salts, especially in the form of the sodium salts.

The Surfactants can in the invention relevant cleaning or washing agents in total in an amount of preferably 5% by weight to 50% by weight, in particular from 8 wt .-% to 30 wt .-%, based on the finished composition be.

Inventive agents may contain bleaching agents. Among the compounds serving as bleaches in water H ₂ O ₂ , sodium percarbonate, sodium perborate tetrahydrate and sodium perborate monohydrate are of particular importance. Other useful bleaching agents are, for example, peroxopyrophosphates, citrate perhydrates and H ₂ O ₂ -producing peracidic salts or peracids, such as persulfates or persulfuric acid. Also useful is the urea peroxohydrate percarbamide, which can be described by the formula H ₂ N-CO-NH ₂ .H ₂ O ₂ . In particular, when using the means for cleaning hard surfaces, for example in automatic dishwashing, they may, if desired, also contain bleaching agents from the group of organic bleaches, although their use is also possible in principle for laundry detergents. Typical organic bleaches are the diacyl peroxides, such as dibenzoyl peroxide. Other typical organic bleaches are the peroxyacids, examples of which include the alkyl peroxyacids and the aryl peroxyacids. Preferred representatives are the peroxybenzoic acid and its ring-substituted derivatives, such as alkylperoxybenzoic acids, but also peroxy-α-naphthoic acid and magnesium monoperphthalate, the aliphatic or substituted aliphatic peroxyacids, such as peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid (Phthalimidoperoxyhexansäure, PAP), o-carboxybenzamidoperoxycaproic acid, N-nonenylamidoperadipic acid and N-nonenylamidopersuccinates; and aliphatic and araliphatic peroxydicarboxylic acids such as 1,12-diperoxycarboxylic acid Re, 1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, the diperoxyphthalic acids, 2-decyldiperoxybutane-1,4-diacid, N, N-terephthaloyl-di (6-aminopercaproic acid) can be used.

Of the Content of the bleaching agent may be 1 to 40% by weight and especially 10 to 20 wt .-%, wherein advantageously perborate monohydrate or percarbonate is used. A synergistic use of amylase with percarbonate or of amylase with percarboxylic acid with applications WO 99/63036 or WO 99/63037.

In order to achieve an improved bleaching effect when washing at temperatures of 60 ° C and below, and especially during the laundry pretreatment, the agents may also contain bleach activators. As bleach activators, it is possible to use compounds which, under perhydrolysis conditions, give aliphatic peroxycarboxylic acids having preferably 1 to 10 C atoms, in particular 2 to 4 C atoms, and / or optionally substituted perbenzoic acid. Suitable substances are those which carry O- and / or N-acyl groups of the stated C atom number and / or optionally substituted benzoyl groups. Preference is given to polyacylated alkylenediamines, in particular tetraacetylethylenediamine (TAED), acylated triazine derivatives, in particular 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT), acylated glycolurils, in particular 1,3,4,6 Tetraacetylglycoluril (TAGU), N-acylimides, in particular N-nonanoylsuccinimide (NOSI), acylated phenolsulfonates, in particular n-nonanoyl or isononanoyloxybenzenesulfonate (n- or iso-NOBS), acylated hydroxycarboxylic acids, such as triethyl-O-acetylcitrate (TEOC), Carboxylic acid anhydrides, in particular phthalic anhydride, isatoic anhydride and / or succinic anhydride, carboxylic acid amides such as N-methyldiacetamide, glycolide, acylated polyhydric alcohols, in particular triacetin, ethylene glycol diacetate, isopropenyl acetate, 2,5-diacetoxy-2,5-dihydrofuran and those from the German patent applications DE 196 16 693 and DE 19616 767 known enol esters and acetylated sorbitol and mannitol or their in the European patent application EP 0 525 239 mixtures described (SORMAN), acylated sugar derivatives, in particular pentaacetylglucose (PAG), pentaacetylfructose, tetraacetylxylose and octaacetyllactose as well as acetylated, optionally N-alkylated glucamine or gluconolactone, triazole or triazole derivatives and / or particulate caprolactams and / or caprolactam derivatives, preferably N-acylated lactams, for example, N-benzoyl caprolactam and N-acetyl caprolactam, which are known from international patent applications WO 94/27970, WO 94/28102, WO 94/28103, WO 95/00626, WO 95/14759 and WO 95/17498. The from the German patent application DE 196 16 769 known hydrophilic substituted acyl acetals and in the German patent application DE 196 16 770 and the international patent application WO 95/14075 acyllactams are also preferably used. Also from the German patent application DE 4443 177 known combinations of conventional bleach activators can be used. Likewise, nitrile derivatives such as cyanopyridines, nitrile quats, for example N-alkylammonium acetonitriles, and / or cyanamide derivatives can be used. Preferred bleach activators are sodium 4- (octanoyloxy) benzenesulfonate, n-nonanoyl or isononanoyloxybenzenesulfonate (n- or iso-NOBS), undecenoyloxybenzenesulfonate (UDOBS), sodium dodecanoyloxybenzenesulfonate (DOBS), decanoyloxybenzoic acid (DOBA, OBC 10) and / or dodecanoyloxybenzenesulfonate ( OBS 12), as well as N-methylmorpholinum acetonitrile (MMA). Such bleach activators can be used in the customary amount range of from 0.01 to 20% by weight, preferably in amounts of from 0.1 to 15% by weight, in particular from 1% to 10% by weight, based on the total composition, be included.

In addition to or in place of the conventional bleach activators, so-called bleach catalysts may also be included. These substances are bleach-enhancing transition metal salts or transition metal complexes such as Mn, Fe, Co, Ru or Mo-salene complexes or carbonyl complexes. Mn, Fe, Co, Ru, Mo, Ti, V and Cu complexes with N-containing tripod ligands and Co, Fe, Cu and Ru amine complexes are also suitable as bleach catalysts, wherein those compounds are preferably used, which in the DE 197 09 284 A1 are described. According to WO 99/63038 also acetonitrile derivatives and according to WO 99/63041 bleach-activating transition metal complex compounds in combination with amylases can develop a bleach-activating effect.

relevant invention Means usually contain one or more builders, in particular Zeolites, silicates, carbonates, organic cobuilders and - where no ecological reasons speak against their commitment - too the phosphates. The latter are especially in detergents for mechanical Wash the dishes preferred builders to be used.

Mention should be made here crystalline, layered sodium silicates of the general formula NaMSi _x O _{2x + 1} · yH ₂ O, where M is sodium or hydrogen, x is a number from 1.6 to 4, preferably 1.9 to 4.0 and y Number is from 0 to 20 and preferred values for x are 2, 3 or 4. Such crystalline layered silicates are described, for example, in the European patent application EP 0 164 514 described. preferred crystalline layered silicates of the formula given are those in which M is sodium and x is 2 or 3. In particular, both β- and δ-sodium disilicates Na ₂ Si ₂ O ₅ .yH ₂ O are preferred. Such compounds are commercially available, for example, under the name ^SKS® (from Clariant). So it is mainly SKS-6 ^® is a δ-sodium having the formula Na ₂ Si ₂ O ₅ · yH ₂ O, SKS-7 ^® primarily the β-sodium disilicate. Reaction with acids (for example citric acid or carbonic acid) gives kanemite NaHSi ₂ O ₅ .yH ₂ O, commercially available under the names SKS- ^9® or SKS- ^10® (from Clariant) from the δ-sodium disilicate. It may also be advantageous to use chemical modifications of these phyllosilicates. For example, the alkalinity of the layered silicates can be suitably influenced. Phyllosilicates doped with phosphate or with carbonate have altered crystal morphologies in comparison with the δ-sodium disilicate, dissolve more rapidly and show an increased calcium binding capacity in comparison with δ-sodium disilicate. Thus, phyllosilicates of the general empirical formula xNa ₂ O.ySiO ₂ .zP ₂ O ₅ , in which the ratio x to y of a number 0.35 to 0.6, the ratio x to z of a number of 1.75 to 1200 and the Ratio y to z correspond to a number from 4 to 2800, in the patent application DE 19601 063 described. The solubility of the layered silicates can also be increased by using particularly finely divided layered silicates. Also compounds from the crystalline layer silicates with other ingredients can be used. In particular, compounds with cellulose derivatives which have advantages in the disintegrating effect and are used in particular in detergent tablets, and compounds with polycarboxylates, for example citric acid, or polymeric polycarboxylates, for example copolymers of acrylic acid, may be mentioned.

It is also possible to use amorphous sodium silicates with a Na ₂ O: SiO ₂ modulus of from 1: 2 to 1: 3.3, preferably from 1: 2 to 1: 2.8 and in particular from 1: 2 to 1: 2.6, which Delayed and have secondary washing properties. The dissolution delay compared with conventional amorphous sodium silicates may have been caused in various ways, for example by surface treatment, compounding, compaction / densification or by overdrying. In the context of this invention, the term "amorphous" is also understood to mean "X-ray amorphous". This means that the silicates do not yield sharp X-ray reflections typical of crystalline substances in X-ray diffraction experiments, but at most one or more maxima of the scattered X-rays having a width of several degrees of diffraction angle. However, it may well even lead to particularly good builder properties if the silicate particles provide washed-out or even sharp diffraction maxima in electron diffraction experiments. This is to be interpreted as meaning that the products have microcrystalline regions of size 10 to a few hundred nm, values of up to max. 50 nm and in particular up to max. 20 nm are preferred. Particularly preferred are compacted / compacted amorphous silicates, compounded amorphous silicates and overdried X-ray amorphous silicates.

An optionally usable, finely crystalline, synthetic and bound water-containing zeolite is preferably zeolite A and / or P. As zeolite P zeolite MAP ^® (commercial product from Crosfield) is particularly preferred. Also suitable, however, are zeolite X and mixtures of A, X and / or P. Commercially available and preferably usable in the context of the present invention is, for example, a cocrystal of zeolite X and zeolite A (about 80% by weight of zeolite X) ), which is sold by the company CONDEA Augusta SpA under the brand name VEGOBOND AX ^® and by the formula nNa ₂ O · (1 - n) K ₂ O · Al ₂ O ₃ · (2 - 2.5) SiO ₂ · (3.5-5.5) H ₂ O can be described. Suitable zeolites have an average particle size of less than 10 μm (volume distribution, measuring method: Coulter Counter) and preferably contain 18 to 22% by weight, in particular 20 to 22% by weight, of bound water.

Of course it is also a use of the well-known phosphates as builders possible, if such use is not avoided for environmental reasons should be. Among the variety of commercially available Phosphates have the alkali metal phosphates, with particular preference of Pentasodium or Pentakalium triphosphate (sodium or potassium tripolyphosphate) in the washing and cleaning industry the greatest importance.

Alkali metal phosphates is the summary term for the alkali metal (especially sodium and potassium) salts of various phosphoric acids, in which one can distinguish metaphosphoric acids (HPO ₃ ) _n and orthophosphoric H ₃ PO _{4 in} addition to higher molecular weight representatives. The phosphates combine several advantages: they act as alkali carriers, prevent lime deposits on machine parts or lime incrustations in fabrics and also contribute to the cleaning performance.

Sodium dihydrogen phosphate, NaH ₂ PO ₄ , exists as a dihydrate (density 1.91 gcm ^-3 , melting point 60 °) and as a monohydrate (density 2.04 gcm ^-3 ). Both salts are white powders which are very soluble in water and which lose their water of crystallization when heated and at 200 ° C into the weak acid diphosphate (disodium hydrogen diphosphate, Na ₂ H ₂ P ₂ O ₇ ), at higher temperature in sodium trimetaphosphate (Na ₃ P ₃ O ₉₎ and Maddrell's salt (see below), pass. NaH ₂ PO _{4 is} acidic; It arises when phosphoric acid is adjusted to a pH of 4.5 with sodium hydroxide solution and the mash is sprayed. Potassium dihydrogen phosphate (potassium phosphate primary or monobasic potassium phosphate, KDP), KH ₂ PO ₄ , is a white salt of 2.33 gcm ^-3 density, has a melting point of 253 ° [decomposition to form potassium polyphosphate (KPO ₃ ) _x ] and is light soluble in water.

Disodium hydrogen phosphate (secondary sodium phosphate), Na ₂ HPO ₄ , is a colorless, very slightly water-soluble crystalline salt. It exists anhydrous and with 2 moles (density 2.066 gcm ^-3 , loss of water at 95 °), 7 moles (density 1.68 gcm ^-3 , melting point 48 ° with loss of 5 H ₂ O) and 12 moles water ( Density 1.52 gcm ^-3 , melting point 35 ° with loss of 5 H ₂ O) becomes anhydrous at 100 ° C and, upon increased heating, passes into the diphosphate Na ₄ P ₂ O ₇ . Disodium hydrogen phosphate is prepared by neutralization of phosphoric acid with soda solution using phenolphthalein as an indicator. Dipotassium hydrogen phosphate (secondary or dibasic potassium phosphate), K ₂ HPO ₄ , is an amorphous, white salt that is readily soluble in water.

Trisodium phosphate, tertiary sodium phosphate, Na ₃ PO ₄ , are colorless crystals which have a density of 1.62 gcm ^-3 as dodecahydrate and a melting point of 73-76 ° C (decomposition), as decahydrate (corresponding to 19-20% P ₂ O ₅ ) have a melting point of 100 ° C and in anhydrous form (corresponding to 39-40% P ₂ O ₅ ) have a density of 2.536 gcm ^-3 . Trisodium phosphate is readily soluble in water under alkaline reaction and is prepared by evaporating a solution of exactly 1 mole of disodium phosphate and 1 mole of NaOH. Tripotassium phosphate (tertiary or tribasic potassium phosphate), K ₃ PO ₄ , is a white, deliquescent, granular powder of density 2.56 gcm ^-3 , has a melting point of 1340 ° and is readily soluble in water with an alkaline reaction. It is produced, for example, by heating Thomasschlacke with coal and potassium sulfate. Despite the higher price, the more soluble, therefore highly effective, potassium phosphates are often preferred over the corresponding sodium compounds in the detergent industry.

Tetrasodium diphosphate (sodium pyrophosphate), Na ₄ P ₂ O ₇ , exists in anhydrous form (density 2.534 gcm ^-3 , melting point 988 °, also indicated 880 °) and as decahydrate (density 1.815-1.836 gcm ^-3 , melting point 94 ° with loss of water) , Both substances are colorless crystals which are soluble in water with an alkaline reaction. Na ₄ P ₂ O ₇ is formed on heating of disodium phosphate to> 200 ° C or by reacting phosphoric acid with soda in a stoichiometric ratio and dewatering the solution by spraying. The decahydrate complexes heavy metal salts and hardness agents and therefore reduces the hardness of the water. Potassium diphosphate (potassium pyrophosphate), K ₄ P ₂ O ₇ , exists in the form of the trihydrate and is a colorless, hygroscopic powder with a density of 2.33 gcm ^-3 , which is soluble in water, the pH being 1% Solution at 25 ° 10.4.

Condensation of NaH ₂ PO ₄ or of KH ₂ PO ₄ gives rise to higher molecular weight sodium and potassium phosphates, in which one can distinguish cyclic representatives, the sodium or potassium metaphosphates and chain-type, the sodium or potassium polyphosphates. In particular, for the latter are a variety of names in use: melting or annealing phosphates, Graham's salt, Kurrolsches and Maddrell's salt. All higher sodium and potassium phosphates are collectively referred to as condensed phosphates.

The industrially important pentasodium triphosphate, Na ₅ P ₃ O ₁₀ (sodium tripolyphosphate), is a water-free or with 6 H ₂ O crystallizing a non-hygroscopic white water-soluble salt of the general formula NaO- [P (O) (ONa) -O] _n -Na with n = 3. In 100 g of water dissolve at room temperature about 17 g, at 60 ° about 20 g, at 100 ° around 32 g of the salt water-free salt; after two hours of heating the solution to 100 ° caused by hydrolysis about 8% orthophosphate and 15% diphosphate. In the preparation of pentasodium triphosphate, phosphoric acid is reacted with soda solution or sodium hydroxide solution in a stoichiometric ratio and the solution is dehydrated by spraying. Similar to Graham's salt and sodium diphosphate, pentasodium triphosphate dissolves many insoluble metal compounds (including lime soaps, etc.). Pentakaliumtriphosphat, K ₅ P ₃ O ₁₀ (potassium tripolyphosphate), for example, in the form of a 50 wt .-% solution (> 23% P ₂ O ₅ , 25% K ₂ O) in the trade. The potassium polyphosphates are widely used in the washing and cleaning industry. There are also sodium potassium tripolyphosphates which can also be used in the context of the present invention. These arise, for example, when hydrolyzed sodium trimetaphosphate with KOH: (NaPO ₃ ) ₃ + 2KOH → Na ₃ K ₂ P ₃ O ₁₀ + H ₂ O

These are exact according to the invention such as sodium tripolyphosphate, potassium tripolyphosphate or mixtures can be used from these two; also mixtures of sodium tripolyphosphate and sodium potassium tripolyphosphate or mixtures of potassium tripolyphosphate and sodium potassium tripolyphosphate or mixtures of sodium tripolyphosphate and potassium tripolyphosphate and sodium potassium tripolyphosphate used according to the invention.

When organic cobuilders can in the invention washing and cleaning agents in particular Polycarboxylates or polycarboxylic acids, polymeric polycarboxylates, polyaspartic acid, polyacetals, optionally oxidized dextrins, other organic cobuilders (see below) as well as Phosphonates are used. These classes of substances are hereafter described.

useful organic builders For example, they can be used in the form of their sodium salts polycarboxylic where among polycarboxylic acids such carboxylic acids be understood that carry more than one acid function. For example these are citric acid, adipic acid, Succinic acid, Glutaric acid, malic acid, tartaric acid, maleic acid, fumaric acid, sugar acids, aminocarboxylic acids, nitrilotriacetic acid (NTA), if such an application for ecological reasons not is to be avoided, as well as mixtures of these. Preferred salts are the salts of polycarboxylic acids like citric acid, adipic acid, Succinic acid, glutaric, Tartaric acid, sugar acids and mixtures of these.

Also the acids in itself can be used. They typically have besides their builder effect also the property of an acidifying component and thus serve to set a lower and milder one pH value of washing or Detergents, if not by the mixture of other components resulting pH value desired is. In particular, systemic and environmentally friendly acids such as citric acid, Acetic acid, Tartaric acid, malic acid, lactic acid, glycolic acid, succinic acid, glutaric acid, adipic acid, gluconic acid and to name any mixtures of these. But also mineral acids, in particular sulfuric acid or bases, in particular ammonium or alkali metal hydroxides can as Serve pH regulators. Such regulators are relevant to the invention Means in amounts of preferably not more than 20 wt .-%, in particular from 1.2% to 17% by weight.

When Builders are further suitable polymeric polycarboxylates, these are for example, the alkali metal salts of polyacrylic acid or polymethacrylic acid, for example, those having a molecular weight of 500 to 70,000 g / mol.

For the purposes of this document, the molecular weights stated for polymeric polycarboxylates are weight-average molar masses M _{w of} the particular acid form, which were determined in principle by means of gel permeation chromatography (GPC), a UV detector being used. The measurement was carried out against an external polyacrylic acid standard, which provides realistic molecular weight values due to its structural relationship with the polymers investigated. These data differ significantly from the molecular weight data, in which polystyrene sulfonic acids are used as standard. The molar masses measured against polystyrenesulfonic acids are generally significantly higher than the molecular weights specified in this document.

suitable Polymers are in particular polyacrylates, which preferably have a molecular mass of 2,000 to 20,000 g / mol. Because of their superior solubility can from this group again the short-chain polyacrylates, the molecular weights from 2,000 to 10,000 g / mol, and more preferably from 3,000 to 5,000 g / mol, be preferred.

Suitable are furthermore copolymeric polycarboxylates, especially those of acrylic acid with methacrylic acid and the acrylic acid or methacrylic acid with maleic acid. Particularly suitable are copolymers of acrylic acid with maleic which contain 50 to 90% by weight of acrylic acid and 50 to 10% by weight of maleic acid. Your molecular weight, based on free acids, is generally 2,000 to 70,000 g / mol, preferably 20,000 to 50 000 g / mol and in particular 30 000 to 40 000 g / mol. The (co) polymers Polycarboxylates can either as a powder or as an aqueous one solution be used. The content of the agents in (co) polymeric polycarboxylates may be from 0.5 to 20 wt .-%, in particular 1 to 10 wt .-%, amount.

to Improvement of water solubility can the polymers also include allylsulfonic acids, such as allyloxybenzenesulfonic acid and methallylsulfonic acid, as Contain monomer.

Especially Biodegradable polymers of more than two are also preferred various monomer units, for example, those as monomers Salts of acrylic acid and maleic acid and vinyl alcohol or vinyl alcohol derivatives or salts as monomers the acrylic acid and 2-alkylallyl sulfonic acid and sugar derivatives.

Further preferred copolymers are those which are preferably used as monomers Acrolein and acrylic acid / acrylic acid salts or acrolein and vinyl acetate.

As well are as further preferred builders polymeric aminodicarboxylic acids whose Salts or their precursors to call. Particularly preferred are polyaspartic acids or their salts and derivatives.

Further Suitable builders are polyacetals, which by reaction of dialdehydes with polyol carboxylic acids containing 5 to 7 carbon atoms and at least 3 hydroxyl groups can be obtained. preferred Polyacetals are made from dialdehydes such as glyoxal, glutaraldehyde, terephthalaldehyde and mixtures thereof and from polyol carboxylic acids such as gluconic acid and / or glucoheptonic receive.

Further suitable organic builders are dextrins, for example Oligomers or polymers of carbohydrates by partial hydrolysis of starches can be obtained. The Hydrolysis can be carried out according to usual, for example acidic or enzyme-catalyzed processes. Preferably These are hydrolysis products with average molecular weights in the range from 400 to 500,000 g / mol. It is a polysaccharide with a Dextrose equivalent (DE) in the range from 0.5 to 40, in particular from 2 to 30 preferred where DE is a common one Measure of the reducing Effect of a polysaccharide compared to dextrose is which a DE of 100 owns. Useful are both maltodextrins with a DE between 3 and 20 and dry glucose syrup with a DE between 20 and 37 as well as so-called yellow dextrins and white dextrins with higher Molar masses in the range of 2,000 to 30,000 g / mol.

The oxidized derivatives of such dextrins are their reaction products with oxidizing agents which are capable of oxidizing at least one alcohol function of the saccharide ring to the carboxylic acid function. Particularly preferred organic builders for agents according to the invention are oxidized starches or their derivatives from the applications EP 472,042 , WO 97/25399, and EP 755 944 ,

Also Oxydisuccinates and other derivatives of disuccinates, preferably Ethylenediamine disuccinate are other suitable cobuilders. there becomes ethylenediamine-N, N'-disuccinate (EDDS) preferably used in the form of its sodium or magnesium salts. Also preferred in this context are glycerol disuccinates and glycerol trisuccinates. Suitable amounts are zeolithhaltigen and / or silicate-containing formulations between 3 and 15 wt .-%.

Further useful organic cobuilders are, for example, acetylated hydroxy or their salts, which may also be in lactone form may be present and which at least 4 carbon atoms and at least one hydroxy group and a maximum of two acid groups contain.

A another substance class with cobuilder properties are the phosphonates These are in particular hydroxyalkane or Aminoalkanephosphonates. Among the hydroxyalkane phosphonates is the 1-hydroxyethane-1,1-diphosphonate (HEDP) of particular importance as a co-builder. It is preferably used as the sodium salt, wherein the disodium salt neutral and the tetrasodium salt is alkaline (pH 9). As aminoalkanephosphonates preferably ethylenediamine tetramethylenephosphonate (EDTMP), Diethylentriaminpentamethylenphosphonat (DTPMP) and their higher homologues in question. They are preferably in the form of neutral reacting Sodium salts, for example as the hexasodium salt of EDTMP or as hepta- and octa-sodium salt of DTPMP used. As a builder is used from the class of phosphonates preferably HEDP. The aminoalkanephosphonates also have a pronounced heavy metal binding capacity. Accordingly It may, especially if the agents also contain bleach, preferred be aminoalkanephosphonates, in particular DTPMP use, or mixtures to use from the said phosphonates.

Furthermore can all compounds that are capable of complexes with alkaline earth ions train as co-builders.

Builder substances may optionally be present in the inventive agents in amounts of up to 90 Wt .-% be included. They are preferably contained in amounts of up to 75% by weight. Inventive detergents have builder contents of, in particular, from 5% by weight to 50% by weight. In inventively relevant agents for the cleaning of hard surfaces, in particular for the automated cleaning of dishes, the content of builder substances is in particular 5 wt .-% to 88 wt .-%, wherein preferably no water-insoluble builder materials are used in such agents. In a preferred embodiment of invention-relevant means for the particular machine cleaning of dishes are 20 wt .-% to 40 wt .-% of water-soluble organic builder, in particular alkali, 5 wt .-% to 15 wt .-% alkali carbonate and 20 wt .-% bis 40 wt .-% Alkalidisilikat included.

Solvent, in the liquid until gel-shaped Compositions of detergents and cleaners are used can, are for example from the group monohydric or polyhydric alcohols, alkanolamines or glycol ethers, provided that they are within the specified concentration range are miscible with water. Preferably, the solvents selected from ethanol, n- or i-propanol, butanols, ethylene glycol methyl ether, Ethylene glycol ethyl ether, ethylene glycol propyl ether, ethylene glycol mono-n-butyl ether, Diethylene glycol methyl ether, diethylene glycol ethyl ether, propylene glycol methyl, ethyl or propyl ether, dipropylene glycol monomethyl, or ethyl ether, Di-isopropylene glycol monomethyl, or ethyl ether, methoxy, ethoxy or butoxytriglycol, 1-butoxyethoxy-2-propanol, 3-methyl-3-methoxybutanol, Propylene glycol t-butyl ether as well as mixtures of these solvents.

solvent can in the invention relevant liquid until gel-shaped Detergents and cleaning agents in amounts of between 0.1 and 20% by weight, but preferably below 15 wt .-% and in particular below 10 % By weight.

to Adjustment of viscosity can invention-relevant compositions one or more thickeners, or thickening systems are added. These high molecular weight Substances that are also called swelling agents usually suck the liquids They swell and then swell to finally pass into viscous true or colloidal solutions.

suitable Thickeners are inorganic or polymeric organic compounds. The inorganic thickeners include, for example, polysilicic acids, clay minerals such as montmorillonites, zeolites, silicas and bentonites. The organic ones Thickeners come from the groups of natural polymers, modified natural Polymers and fully synthetic polymers. Such from nature originating polymers are, for example, agar-agar, carrageenan, tragacanth, Gum arabic, alginates, pectins, polyoses, guar flour, locust bean gum, Strength, Dextrins, gelatin and casein. Modified natural substances, which as Thickeners used mainly come from the group of modified starches and celluloses. Examples are carboxymethylcellulose and other cellulose ethers, hydroxyethyl and propylcellulose as well Called Kernmehlether. Fully synthetic thickeners are polymers such as polyacrylic and Polymethacrylic compounds, vinyl polymers, polycarboxylic acids, polyethers, Polyimines, polyamides and polyurethanes.

The Thickeners can in an amount up to 5% by weight, preferably from 0.05 to 2% by weight, and particularly preferably from 0.1 to 1.5 wt .-%, based on the finished composition, be included.

The Inventive washing or cleaning agent may optionally as another usual Ingredients sequestering agents, electrolytes and other auxiliaries contain.

relevant invention Textile detergents can as optical brightener derivatives of Diaminostilbendisulfonsäure or containing their alkali metal salts. For example, salts are suitable 4,4'-bis (2-anilino-4-morpholino-1,3,5-triazinyl-6-amino) stilbene-2,2'-disulphonic acid or similarly constructed compounds that replace the morpholino group a diethanolamino group, a methylamino group, an anilino group or carry a 2-methoxyethylamino group. Furthermore, brighteners the type of substituted Diphenylstyryle be present, for example the alkali salts of 4,4'-bis (2-sulfostyryl) -diphenyl, 4,4'-bis (4-chloro-3-sulfostyryl) or 4- (4-chlorostyryl) -4 '- (2-sulfostyryl) -diphenyls. Mixtures of the aforementioned optical brightener can be used become.

Graying inhibitors have the task of keeping suspended from the textile fiber dirt suspended in the fleet. Water-soluble colloids of mostly organic nature are suitable for this purpose, for example starch, glue, gelatin, salts of ether carboxylic acids or ether sulfonic acids of starch or of cellulose or salts of acidic sulfuric acid esters of cellulose or starch. Also, water-soluble polyamides containing acidic groups are suitable for this purpose. Furthermore, other than the above-mentioned starch derivatives can be used, for example aldehyde starches. Preference is given to cellulose ethers, such as carboxy methylcellulose (Na salt), methylcellulose, hydroxyalkylcellulose and mixed ethers, such as methylhydroxyethylcellulose, methylhydroxypropylcellulose, methylcarboxymethylcellulose and mixtures thereof, for example in amounts of from 0.1 to 5% by weight, based on the agents.

To effect silver corrosion protection, silver corrosion inhibitors can be used in dishwashing detergents that are relevant to the invention. Such are known in the art, for example benzotriazoles, ferric chloride or CoSO ₄ . For example, from the European patent EP 0 736 084 B1 Are known for common use with enzymes particularly suitable silver corrosion inhibitors manganese, titanium, zirconium, hafnium, vanadium, cobalt or cerium salts and / or complexes in which said metals in one of the oxidation states II, III , IV, V or VI. Examples of such compounds are MnSO ₄ , V ₂ O ₅ , V ₂ O ₄ , VO ₂ , TiOSO ₄ , K ₂ TiF ₆ , K ₂ ZrF ₆ , Co (NO ₃ ) ₂ , Co (NO ₃ ) ₃ , and the like mixtures.

"Soil-release" agents or "soil repellents" are mostly polymers, when used in a laundry detergent laundry detergent dirt-repellent Give properties and / or the soil removal capacity of the other detergent ingredients support. A comparable effect can also be found in their use in cleaning agents for hard surfaces to be watched.

Particularly effective and long-known soil release agents are copolyesters with dicarboxylic acid, alkylene glycol and polyalkylene glycol units. Examples of these are copolymers or copolymers of polyethylene terephthalate and polyoxyethylene glycol (DT 16 17 141, or DT 22 00 911). In German Offenlegungsschrift DT 22 53 063 acidic agents are mentioned which contain, inter alia, a copolymer of a dibasic carboxylic acid and an alkylene or cycloalkylene polyglycol. Polymers of ethylene terephthalate and polyethylene oxide terephthalate and their use in detergents are described in the German publications DE 28 57 292 and DE 33 24 258 and the European Patent EP 0 253 567 described. The European patent EP 066 944 relates to agents containing a copolyester of ethylene glycol, polyethylene glycol, aromatic dicarboxylic acid and sulfonated aromatic dicarboxylic acid in certain molar ratios. From the European patent EP 0 185 427 For example, methyl or ethyl group end-capped polyesters having ethylene and / or propylene terephthalate and polyethylene oxide terephthalate units and detergents containing such soil release polymer are known. The European patent EP 0 241 984 relates to a polyester which in addition to oxyethylene groups and terephthalic acid units also contains substituted ethylene units and glycerol units. From the European patent EP 0 241 985 are known polyester containing in addition to oxyethylene groups and terephthalic acid units 1,2-propylene, 1,2-butylene and / or 3-methoxy-1,2-propylene and glycerol units and endgruppenverschlossen with C ₁ - to C ₄ alkyl groups are. From the European patent application EP 0 272 033 are at least partially by C _1-4 alkyl or acyl radicals end-capped polyester with poly-propylene terephthalate and polyoxyethylene terephthalate units known. The European patent EP 0 274 907 describes sulfoethyl end-capped terephthalate-containing soil release polyesters. According to the European patent application EP 0 357 280 are prepared by sulfonation of unsaturated end groups soil release polyester with terephthalate, alkylene glycol and poly-C _2-4 glycol units. International Patent Application WO 95/32232 relates to acidic, aromatic soil release polymers. International Patent Application WO 97/31085 discloses non-polymeric soil repellent active ingredients for multi-functional cotton materials: a first entity, which may be cationic, for example, is capable of adsorption to the cotton surface by electrostatic interaction, and a second Unit that is hydrophobic is responsible for the retention of the drug at the water / cotton interface.

To the for the use in invention-relevant laundry detergents in question upcoming color transfer inhibitors belong in particular polyvinylpyrrolidones, polyvinylimidazoles, polymers N-oxides such as poly (vinylpyridine-N-oxide) and copolymers of vinylpyrrolidone with vinylimidazole.

When used in automated cleaning processes, it may be advantageous to add foam inhibitors to the compositions. As foam inhibitors are, for example, soaps of natural or synthetic origin, which have a high proportion of C ₁₈ -C ₂₄ fatty acids. Suitable non-surfactant foam inhibitors are, for example, organopolysiloxanes and mixtures thereof with microfine, optionally silanized silica and paraffins, waxes, microcrystalline waxes and mixtures thereof with silanated silica or bistearylethylenediamide. It is also advantageous to use mixtures of various foam inhibitors, for example those of silicones, paraffins or waxes. The foam inhibitors, in particular silicone- and / or paraffin-containing foam inhibitors, are preferably bound to a granular, water-soluble or dispersible carrier substance. In particular, mixtures of paraffins and bistearylethylenediamides are preferred.

One Invention-relevant cleaning agent for hard surfaces can about that In addition, abrasive ingredients, in particular from the group including quartz flours, wood flours, flours, chalks and glass microspheres and mixtures thereof. Abrasives are in the invention relevant Cleaning agents preferably not more than 20 wt .-%, in particular from 5% to 15% by weight.

color and fragrances are added to detergents and cleaners to the aesthetic Improve the impression of the products and the consumer in addition to the Washing and cleaning performance a visually and sensory "typical and unmistakable" product available put. As perfume oils respectively Fragrances can individual fragrance compounds, for example the synthetic ones Products of the ester, ether, aldehyde, ketone, alcohol and type Hydrocarbons are used. Fragrance compounds from Type of esters are, for example, benzyl acetate, phenoxyethyl isobutyrate, p-tert-butylcyclohexyl acetate, linalyl acetate, dimethylbenzyl-carbinyl acetate, Phenylethyl acetate, linalyl benzoate, benzyl formate, ethyl methyl phenyl glycinate, Allyl cyclohexyl propionate, styrallyl propionate and benzyl salicylate. Count to the Ethern for example, benzyl ethyl ether, to the aldehydes for example the linear alkanals with 8-18 C atoms, citral, citronellal, citronellyloxyacetaldehyde, Cyclamenaldehyde, Hydroxycitronellal, Lilial and Bourgeonal, to the Ketones, for example, the ionone, α-isomethylionone and methyl cedryl ketone, to the alcohols anethole, citronellol, eugenol, Geraniol, linalool, phenylethyl alcohol and terpineol, to the hydrocarbons belong mainly the terpenes like limonene and pinene. However, mixtures are preferred different fragrances used, which together create an appealing Create a fragrance. Such perfume oils can also natural Contain fragrance mixtures, as they are accessible from plant sources, for example, pine, citrus, jasmine, patchouly, rose or ylang-ylang oil. Also Muscat, sage, chamomile, clove, lemon balm, mint, cinnamon, lime, juniper, vetiver, olibanum, galbanum and labdanum oils are also suitable Orange blossom oil, neroliol, Orange peel oil and sandalwood oil. Usually is the content of detergents and cleaners to dyes below 0.01 wt% while Perfumes can make up to 2 wt .-% of the total formulation.

The Fragrances can be incorporated directly into the detergents and cleaning agents, but it can also be advantageous to apply the fragrances to carriers, the liability of the perfume reinforce on the items to be cleaned and by a slower release of fragrance for long-lasting fragrance, in particular of treated textiles. As such carrier materials, for example Cyclodextrins proven, the cyclodextrin-perfume complexes additionally can still be coated with other excipients. A further preferred carrier for fragrances is the described zeolite X, which instead of or in mixture can also absorb fragrances with surfactants. Therefore, preference is given Detergents and cleaners containing the described zeolite X and Perfumes, preferably at least partially attached to the zeolite are absorbed.

preferred Dyes whose selection does not present any difficulty to the skilled person have a high storage stability and insensitivity to the rest Ingredients of the agents and against light and no pronounced substantivity to textile fibers, so as not to stain them.

to fight of microorganisms can Detergents or cleaning agents contain antimicrobial agents. Here one differentiates depending on the antimicrobial spectrum and Mechanism of action between bacteriostats and bactericides, fungistatics and fungicides, etc. Important substances from these groups are, for example Benzalkonium chlorides, alkylaryl sulfonates, halophenols and phenol mercuriacetate. The Terms antimicrobial action and antimicrobial agent have the usual meaning within the scope of the teaching according to the invention, for example, by K. H. Wallhäußer in "Praxis der Sterilisation, Disinfection - preservation : Identification of germs - occupational hygiene "(5th ed. - Stuttgart; New York Thieme, 1995), all of which are described therein Substances with antimicrobial action can be used. suitable antimicrobial agents are preferably selected from the groups of alcohols, amines, aldehydes, antimicrobial acids respectively their salts, carboxylic esters, acid amides, Phenols, phenol derivatives, diphenyls, diphenylalkanes, urea derivatives, Oxygen, nitrogen acetals and formals, benzamidines, isothiazolines, Phthalimide derivatives, pyridine derivatives, antimicrobial surface active agents Compounds, guanidines, antimicrobial amphoteric compounds, Quinolines, 1,2-dibromo-2,4-dicyanobutane, iodo-2-propyl-butyl-carbamate, iodine, iodophors, Peroxo compounds, halogen compounds and any mixtures the preceding one.

The antimicrobial agent may be selected from ethanol, n-propanol, i-propanol, 1,3-butanediol, phenoxyethanol, 1,2-propylene glycol, glycerol, undecylenic acid, benzoic acid, salicylic acid, dihydracetic acid, o-phenylphenol, N-methylmorpholine-acetonitrile ( MMA), 2-benzyl-4-chlorophenol, 2,2'-methylenebis (6-bromo-4-chlorophenol), 4,4'-dichloro-2'-hydroxydiphenyl ether (Dichlosan), 2,4,4 'trichloro-2'-hydroxydi phenylether (trichlosan), chlorhexidine, N- (4-chlorophenyl) -N- (3,4-dichlorophenyl) -urea, N, N '- (1,10-decanediyldi-1-pyridinyl-4-ylidene) - bis (1-octanamine) dihydrochloride, N, N'-bis (4-chlorophenyl) -3,12-diimino-2,4,11,13-tetraaza-tetradecandiimidamide, glucoprotamines, antimicrobial quaternary surface active compounds, guanidines incl . the bi- and polyguanidines such as 1,6-bis (2-ethylhexyl-biguanido hexane) dihydrochloride, 1,6-di- (N _1, N ₁ '-phenyldiguanido-N _5, N _5') hexane-tetrahydrochloride, 1,6-di- (N ₁ , N ₁ '-phenyl-N ₁ , N ₁ -methyldiguanido-N ₅ , N ₅ ') -hexane dihydrochloride, 1,6-di- (N ₁ , N ₁ '- o -chlorophenyldiguanido-N ₅ , N ₅ ') -hexane dihydrochloride, 1,6-di- (N ₁ , N ₁ '-2,6-dichlorophenyldiguanido-N ₅ , N ₅ ') hexane dihydrochloride, 1,6-di- [N ₁ , N ₁ '-beta (p-methoxyphenyl) diguanido-N ₅ , N ₅ '] -hexane dihydrochloride, 1,6-di- (N ₁ , N ₁ ' -alpha-methyl-.beta.-phenyldiguanido-N ₅ , N ₅ ') -hexane dihydrochloride, 1,6-di- (N ₁ , N ₁ ' -p-nitrophenyldiguanido-N ₅ , N ₅ ') hexane dihydrochloride, omega: omega-di- (N ₁ , N ₁ '-phenyldiguanido-N ₅ , N ₅ ') -di-n-propyl ether dihydrochloride, omega: omega'-di- (N ₁ , N ₁ '- p-chlorophenyldiguanido-N ₅ , N ₅ ') di-n-propyl ether tetrahydrochloride, 1,6-di- (N ₁ , N ₁ ' -2,4-dichlorophenyldiguanido-N ₅ , N ₅ ') hexane-tetrahydrochloride , 1,6-di- (N ₁ , N ₁ '-p-methylphenyldiguanido-N ₅ , N ₅ ') hexane dihydrochloride, 1,6-di- (N ₁ , N ₁ '-2,4,5- trichlorophenyldiguanido-N ₅ , N ₅ ') hexane-tetrahydrochloride, 1,6-di- [N ₁ , N ₁ ' -alpha (p-chlorophenyl) -ethyldiguanido-N ₅ , N ₅ '] hexane dihydrochloride, omega: omega Di- (N ₁ , N ₁ '-p-chlorophenyldiguanido-N ₅ , N ₅ ') m-xylenedihydrochloride, 1,12-di- (N ₁ , N ₁ '-p-chlorophenyldiguanido-N ₅ , N ₅ ') dodecane dihydrochloride, 1,10-di- (N _1, N _1' -phenyldiguanido-N _5, N ₅ ') -decane tetrahydrochloride, 1,12-di- (N _1, N _1' -phenyldiguanido -N ₅ , N ₅ ') dodecane tetrahydrochloride, 1,6-di- (N ₁ , N ₁ ' -o-chlorophenyldiguanido-N ₅ , N ₅ ') hexane dihydrochloride, 1,6-di (N ₁ , N ₁ '- o -chlorophenyldiguanido-N ₅ , N ₅ ') hexane-tetrahydroch loride, ethylene bis (1-tolyl biguanide), ethylene bis (p-tolyl biguanide), ethylene bis (3,5-dimethylphenyl biguanide), ethylene bis (p-tert-amylphenyl biguanide), ethylene bis (nonylphenylbiguanide), ethylene bis (phenylbiguanide), ethylene bis (N-butylphenylbiguanide), ethylene bis (2,5-diethoxyphenylbiguanide), ethylene bis (2,4-dimethylphenyl biguanide), ethylene bis (o-diphenylbiguanide), ethylene bis (mixed amyl naphthyl biguanide), N-butyl ethylene bis (phenylbiguanide), trimethylene bis (o-tolyl biguanide), N-butyl trimethyl bis (phenyl biguanide) and the like Salts such as acetates, gluconates, hydrochlorides, hydrobromides, citrates, bisulfites, fluorides, polymaleates, N-cocosalkyl sarcosinates, phosphites, hypophosphites, perfluorooctanoates, silicates, sorbates, salicylates, maleates, tartrates, fumarates, ethylenediaminetetraacetates, iminodiacetates, cinnamates, thiocyanates, arginates, Pyromellitate, Tetracarboxybutyrate, benzoates, glutarates, monofluorophosphates, perfluoropropionates and any mixtures thereof. Also suitable are halogenated xylene and cresol derivatives, such as p-chlorometacresol or p-chloro-meta-xylene, and natural antimicrobial agents of plant origin (for example, from spices or herbs), animal and microbial origin. Preferably, antimicrobial surface-active quaternary compounds, a natural antimicrobial agent of plant origin and / or a natural antimicrobial agent of animal origin, most preferably at least one natural antimicrobial agent of plant origin from the group comprising caffeine, theobromine and theophylline and essential oils such as eugenol, thymol and geraniol, and / or at least one natural antimicrobial agent of animal origin from the group, comprising enzymes such as protein from milk, lysozyme and lactoperoxidase, and / or at least one antimicrobial surface-active quaternary compound with an ammonium, sulfonium, phosphonium, iodonium - or Arsoniumgruppe, peroxo compounds and chlorine compounds are used. Also substances of microbial origin, so-called bacteriocins, can be used.

The quaternary ammonium compounds (QAV) suitable as antimicrobial agents have the general formula (R ¹ ) (R ² ) (R ³ ) (R ⁴ ) N ⁺ X ^- , in which R ¹ to R ^{4 are the} same or different C ₁ -C ₂₂ -alkyl radicals, C ₇ -C ₂₈ -aralkyl radicals, or heterocyclic radicals, or in the case of an aromatic compound such as pyridine-even three groups together form with the nitrogen atom forming the heterocycle, for example a pyridinium or imidazolinium compound, and X ^- Halide ions, sulfate ions, hydroxide ions or similar anions. For optimum antimicrobial activity, preferably at least one of the radicals has a chain length of 8 to 18, in particular 12 to 16, carbon atoms.

QAC are tertiary by implementation Amines with alkylating agents, such as methyl chloride, benzyl chloride, Dimethyl sulfate, dodecyl bromide, but also ethylene oxide produced. The alkylation of tertiary amines with a long alkyl radical and two methyl groups succeeds especially easy, also the quaternization of tertiary amines with two long ones Residues and a methyl group can be with the help of methyl chloride mild conditions become. Amines, over have three long alkyl radicals or hydroxy-substituted alkyl radicals little reactive and are preferably quaternized with dimethyl sulfate.

Suitable QAVs are, for example, benzalkonium chloride (N-alkyl-N, N-dimethylbenzylammonium chloride, CAS No. 8001-54-5), benzalkone B (m, p-dichlorobenzyl-dimethyl-C 12 -alkylammonium chloride, CAS No. 58390-78 6), benzoxonium chloride (benzyldodecyl-bis (2-hydroxyethyl) ammonium chloride), cetrimonium bromide (N-hexadecyl-N, N-trimethylammonium bromide, CAS No. 57-09-0), benzetonium chloride (N, N). Dimethyl N- [2- [2- [p- (1,1,3,3-tetramethylbutyl) phenoxy] ethoxy] ethyl] benzyl ammonium chloride, CAS no. 12.1-54-0), dialkyldimethylammonium chlorides such as di-n-decyldimethylammonium chloride (CAS No. 7173-51-5-5), didecyldi-methylammonium bromide (CAS No. 2390-68-3), dioctyldimethylammonium chloride , 1-cetylpyridinium chloride (CAS No. 123-03-5) and thiazoline iodide (CAS No. 15764-48-1) and mixtures thereof. Particularly preferred QACs are the benzalkonium chlorides having C ₈ -C ₁₈ -alkyl radicals, in particular C ₁₂ -C ₁₄ -alkylbenzyl-dimethyl-ammonium chloride.

Benzalkonium halides and / or substituted benzalkonium halides are for example commercially available as Barquat ^® ex Lonza, Marquat® ^® ex Mason, Variquat ^® ex Witco / Sherex and Hyamine ^® ex Lonza and as Bardac ^® ex Lonza. Other commercially obtainable antimicrobial agents are N- (3-chloroallyl) hexaminium chloride such as Dowicide and Dowicil ^{® ®} ex Dow, benzethonium chloride such as Hyamine ^® 1622 ex Rohm & Haas, methylbenzethonium as Hyamine ^® 10X ex Rohm & Haas, cetylpyridinium chloride such as Cepacol ex Menell Labs ,

The antimicrobial agents are added in amounts of 0.0001% by weight 1 wt .-%, preferably from 0.001 wt .-% to 0.8 wt .-%, particularly preferably from 0.005 wt.% to 0.3 wt.% and especially from 0.01 to 0.2 % By weight used.

The Means can UV absorbents (UV absorbers) contained on the treated Apply textiles and the light resistance of the fibers and / or the light resistance improve other recipe ingredients. Under UV absorber are organic substances (sunscreen) to be understood in the Are able to absorb ultraviolet rays and the recorded Energy in the form of longer-wave Radiation, for example heat to give up again.

Compounds having these desired properties include, for example, the non-radiative deactivating compounds and derivatives of benzophenone having substituents in the 2- and / or 4-position. Also suitable are substituted benzotriazoles, in the 3-position phenyl-substituted acrylates (cinnamic acid derivatives, optionally with cyano groups in the 2-position), salicylates, organic Ni complexes and natural substances such as umbelliferone and the body's own urocanic acid. Of particular importance are biphenyl and especially Stilbenderivate as described for example in the EP 0728749 A are described and commercially available as Tinosorb FD ^® or Tinosorb FR ^® are available ex Ciba. As UV-B absorbers may be mentioned: 3-Benzylidencampher or 3-Benzylidennorcampher and its derivatives, for example 3- (4-methylbenzylidene) camphor, as in EP 0693471 B1 described; 4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4- (dimethylamino) benzoate, 2-octyl 4- (dimethylamino) benzoate and 4- (dimethylamino) benzoic acid ester; Esters of cinnamic acid, preferably 4-methoxycinnamic acid 2-ethylhexyl ester, 4-methoxycinnamic acid propyl ester, 4-methoxycinnamic acid isoamyl ester, 2-cyano-3,3-phenylcinnamic acid 2-ethylhexyl ester (octocrylene); Esters of salicylic acid, preferably 2-ethylhexyl salicylate, 4-isopropylbenzyl salicylate, homomenthyl salicylate; Derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4'-methylbenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone; Esters of benzalmalonic acid, preferably di-2-ethylhexyl 4-methoxybenzmalonate; Triazine derivatives such as 2,4,6-trianilino (p-carbo-2'-ethyl-1'-hexyloxy) -1,3,5-triazine and octyl triazone, as described in U.S. Pat EP 0818450 A1 Dioctyl Butamido Triazone or described (Uvasorb HEB ^®); Propane-1,3-diones such as 1- (4-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1,3-dione; Ketotricyclo (5.2.1.0) decane derivatives as described in U.S.P. EP 0694521 B1 described. Also suitable are 2-phenylbenzimidazole-5-sulfonic acid and its alkali metal, alkaline earth metal, ammonium, alkylammonium, alkanolammonium and glucammonium salts; Sulfonic acid derivatives of benzophenones, preferably 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and its salts; Sulfonic acid derivatives of 3-Benzylidencamphers, such as 4- (2-oxo-3-bomylidenemethyl) benzenesulfonic acid and 2-methyl-5- (2-oxo-3-bomylidene) sulfonic acid and salts thereof.

As a typical UV-A filter in particular derivatives of benzoylmethane are suitable, such as 1- (4'-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1,3-dione, 4-tert-butyl 4'-methoxydibenzoylmethane (Parsol 1789), 1-phenyl-3- (4'-isopropylphenyl) -propane-1,3-dione, and enamine compounds as described in U.S.P. DE 19712033 A1 (BASF).

Of course, the UV-A and UV-B filters can also be used in mixtures. In addition to the soluble substances mentioned, insoluble photoprotective pigments, namely finely dispersed, preferably nano-metal oxides or salts, are also suitable for this purpose. Examples of suitable metal oxides are in particular zinc oxide and titanium dioxide and, in addition, oxides of iron, zirconium, silicon, manganese, aluminum and cerium and mixtures thereof. As salts silicates (talc), barium sulfate or zinc stearate can be used. The oxides and salts are already in the form of pigments for skin care and skin-protecting emulsions and decorative cosmetics. The particles should have an average diameter of less than 100 nm, preferably between 5 and 50 nm and in particular between 15 and 30 nm. They may have a spherical shape, but it is also possible to use those particles which have an ellipsoidal or otherwise deviating shape from the spherical shape. The pigments can also be surface-treated, ie hydrophilized or hydrophobized. Typical examples are coated titanium dioxides, such as titanium dioxide T 805 (Degussa) or Eusolex ^® T2000 (Merck;. As hydrophobic coating agents are here preferably silicones and more preferably trialkoxyoctylsilanes or simethicones Preferably micronized zinc oxide is used Further suitable UV photoprotective filters. the review by P. Finkel in SÖFW Journal, Volume 122 (1996), p 543.

The UV absorbents are commonly used in amounts of 0.01% by weight to 5% by weight, preferably of 0.03% by weight to 1 wt .-%, used.

In order to increase the washing or cleaning performance, agents which are relevant to the invention may contain further enzymes in addition to the α-amylase variants according to the invention, it being possible in principle to use all enzymes established for this purpose in the prior art. These include in particular proteases, other amylases, lipases, hemicellulases, cellulases or oxidoreductases, and preferably mixtures thereof. These enzymes are basically of natural origin; Starting from the natural molecules, improved variants are available for use in detergents and cleaners, which are preferably used accordingly. Inventive agents preferably contain enzymes in total amounts of 1 × 10 ^-6 to 5 weight percent based on active protein. The protein concentration can be determined by known methods, for example the BCA method (bicinchoninic acid, 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method (AG Gornall, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766).

Among the proteases, those of the subtilisin type are preferable. Examples thereof are the subtilisins BPN 'and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, subtilisin DY and the enzymes thermitase, proteinase K and the subtilases, but not the subtilisins in the narrower sense Proteases TW3 and TW7. Subtilisin Carlsberg in a developed form under the trade names Alcalase ^® from Novozymes A / S, Bagsvaerd, Denmark. The subtilisins 147 and 309 are sold under the trade names Esperase ^®, or Savinase ^® from Novozymes. Listed under the name BLAP ^® variants of the protease from Bacillus lentus DSM 5483 (WO 91/02792 A1) are derived, in particular, in WO 92/21760 A1, WO 95/23221 A1, WO 02/088340 A2 and PCT / EP02 / 11725 (not yet published). Other useful proteases from various Bacillus sp. and B. gibsonii are from the unpublished patent applications DE 10162727 . DE 10163883 . DE 10163884 and DE 10162728 out.

Other usable proteases are, for example, under the trade names Durazym ^®, relase ^®, Everlase® ^®, Nafizym, Natalase ^®, Kannase® ^® and Ovozymes ^® from Novozymes, under the trade names Purafect ^®, Purafect ^® OxP and Properase.RTM ^® by the company Genencor, that under the trade name Protosol® ^® from Advanced Biochemicals Ltd., Thane, India, under the trade name Wuxi ^® from Wuxi Snyder Bioproducts Ltd., China, under the trade names Proleather® ^® and protease P ^® by the company Amano Pharmaceuticals Ltd., Nagoya, Japan, and the enzyme available under the name Proteinase K-16 from Kao Corp., Tokyo, Japan.

Examples of suitable amylases are the α-amylases from Bacillus licheniformis, from B. amyloliquefaciens or from B. stearothermophilus and their further developments for use in detergents and cleaners. The enzyme from B. licheniformis is available from Novozymes under the name Termamyl ^® and from Genencor under the name Purastar® ^® ST. Development products of this α-amylase are available from Novozymes under the trade names Duramyl ^® and Termamyl ^® ultra, from Genencor under the name Purastar® ^® OxAm and from Daiwa Seiko Inc., Tokyo, Japan, as Keistase ^®. The α-amylase from B. amyloliquefaciens is marketed by Novozymes under the name BAN ^®, and variants derived from the α-amylase from B. stearothermophilus under the names BSG ^® and Novamyl ^®, likewise from Novozymes.

Furthermore, the α-amylase from Bacillus sp. Disclosed in the application WO 02/10356 A2 for this purpose. A 7-7 (DSM 12368) and the cyclodextrin glucanotransferase (CGTase) from B. agaradherens (DSM 9948) described in the application WO 02/44350 A2. Furthermore, the amylolytic enzymes which belong to the sequence space of α-amylases can be used, which are described in the application WO 03/002711 A2 is defined, and those in the as yet unpublished application DE 10163748 A1 to be discribed. Likewise, fusion products of said molecules can be used, for example those from the application DE 10138753 A1 ,

In addition, the enhancements available under the trade names Fungamyl.RTM ^® by Novozymes of α-amylase from Aspergillus niger and A. oryzae, which are. Another commercial product is, for example, the amylase ^LT® .

Agents of the invention may contain lipases or cutinases, in particular because of their triglyceride-cleaving activities, but also to generate in situ peracids from suitable precursors. These include, for example, the lipases originally obtainable from Humicola lanuginosa (Thermomyces lanuginosus) or further developed, in particular those with the amino acid exchange D96L. They are sold, for example, by Novozymes under the trade names Lipolase ^®, Lipolase Ultra ^®, LipoPrime® ^®, Lipozyme® ^® and Lipex ^®. Furthermore, for example, the cutinases can be used, which were originally isolated from Fusarium solani pisi and Humicola insolens. Likewise useable lipases are available from Amano under the designations Lipase CE ^®, Lipase P ^®, Lipase B ^®, or lipase CES ^®, Lipase AKG ^®, Bacillis sp. ^Lipase® , Lipase ^AP® , Lipase ^MAP® and Lipase ^{AML® are} available. From the company Genencor, for example, the lipases, or cutinases can be used, the initial enzymes were originally isolated from Pseudomonas mendocina and Fusarium solanii. Other important commercial products are the originally marketed by Gist-Brocades preparations M1 Lipase ^® and Lipomax® ^® and the enzymes marketed by Meito Sangyo KK, Japan under the names Lipase MY-30 ^®, Lipase OF ^® and lipase PL ^® to mention also the product Lumafast® ^® from Genencor.

relevant invention Means can especially if they are for the treatment of textiles are thought to contain cellulases, depending on the purpose as pure enzymes, as enzyme preparations or in the form of Mixtures in which the individual components advantageously with regard to their different performance aspects. To count these performance aspects in particular contributions to the primary washing performance, for secondary washing performance of the agent (anti-redeposition effect or graying inhibition) and softening (fabric effect), to exerting a "stone washed" effect.

A useful fungal, endoglucanase (EG) -rich cellulase preparation, or its further developments are offered by Novozymes under the trade name Celluzyme ^®. The products Endolase® ^® and Carezyme ^®, likewise available from Novozymes, are based on the 50 kD EG and 43 kD EG from H. insolens DSM 1800. Further commercial products of this company are Cellusoft® ^® and Renozyme ^®. The latter is based on the application WO 96/29397 A1. Performance-enhanced cellulase variants are disclosed, for example, in the application WO 98/12307 A1. Likewise, the cellulases disclosed in the application WO 97/14804 A1 can be used; For example, it revealed 20 kD EG Melanocarpus, available from AB Enzymes, Finland, under the trade names Ecostone® ^® and Biotouch ^®. Further commercial products from AB Enzymes are Econase® ^® and ECOPULP ^®. Other suitable cellulases from Bacillus sp. CBS 670.93 and CBS 669.93 are disclosed in WO 96/34092 A2, wherein those derived from Bacillus sp. CBS 670.93 from the company Genencor under the trade name Puradax ^{® is} available. Further commercial products of the company Genencor are "Genencor detergent cellulase L" and IndiAge ^® Neutra.

Compositions of relevance to the invention may contain, in particular for the removal of certain problem soiling, further enzymes which are combined under the term hemicellulases. These include, for example, mannanases, xanthan lyases, pectin lyases (= pectinases), pectin esterases, pectate lyases, xyloglucanases (= xylanases), pullulanases and β-glucanases. Suitable mannanases are available, for example under the name Gamanase ^® and Pektinex AR ^® from Novozymes, under the name Rohapec ^® B1 from AB Enzymes and under the name Pyrolase® ^® from Diversa Corp., San Diego, CA, United States. A suitable β-glucanase from a B. alcalophilus is disclosed, for example, in the application WO 99/06573 A1. The obtained from B. subtilis β-glucanase is available under the name Cereflo ^® from Novozymes. To increase the bleaching effect, detergents and cleaning agents which are relevant to the invention may be oxidoreductases, for example oxidases, oxygenases, catalases, peroxidases, such as halo-, chloro-, bromo-, lignin, glucose or manganese peroxidases, dioxygenases or laccases (phenol oxidases, polyphenol oxidases). contain. Suitable commercial products Denilite® ^® 1 and 2 from Novozymes should be mentioned. Advantageously, it is additionally preferred to add organic, particularly preferably aromatic, compounds which interact with the enzymes, in order to enhance the activity of the relevant oxidoreductases (enhancers) or, in the case of greatly differing redox potentials, between the oxidizing enzymes and soiling to ensure the flow of electrons (mediators).

The Enzymes used in agents relevant to the invention are either derived originally from microorganisms, such as the genera Bacillus, Streptomyces, Humicola, or Pseudomonas, and / or are known per se biotechnological processes produced by suitable microorganisms, for example by transgenic expression hosts of the genera Bacillus or filamentous Fungi.

The Purification of the enzymes concerned is conveniently done by itself established methods, for example about precipitation, sedimentation, concentration, Filtration of the liquid Phases, microfiltration, ultrafiltration, exposure to chemicals, Deodorization or suitable combinations of these steps.

relevant invention Means can the enzymes are added in any form known in the art become. These include for example, by granulation, extrusion or lyophilization obtained solid preparations or, especially in liquid or gelatinous Means, solutions the enzymes, preferably as possible concentrated, low in water and / or added with stabilizers.

alternative can the enzymes for both the solid as well for the liquid Dosage form can be encapsulated, for example by spray drying or extrusion of the enzyme solution together with a, preferably natural polymer or in the form of capsules, for example those in which the enzymes are as in enclosed in a solidified gel or in core-shell type, in which an enzyme-containing core with a water, air and / or Chemical-impermeable Protective layer coated is. In superimposed layers additional active ingredients, For example, stabilizers, emulsifiers, pigments, bleach or Dyes are applied. Such capsules are after known methods, for example by shaking or Roll granulation or applied in fluid-bed processes. Advantageously Such granules, for example by applying polymeric film-forming agents, low in dust and storage-stable due to the coating.

Farther Is it possible, to assemble two or more enzymes together, so that a single Granules several enzyme activities having.

One protein contained in an invention-relevant agent and / or Enzyme can be especially during storage against damage such as inactivation, denaturation or decay about by physical influences, Oxidation or proteolytic cleavage are protected. In microbial Obtaining the proteins and / or enzymes is inhibiting the Proteolysis particularly preferred, especially if the means Contain proteases. Preferred agents according to the invention contain this Purpose stabilizers.

A Group of stabilizers are reversible protease inhibitors. Become frequent therefor Benzamidine hydrochloride, borax, boric acids, boronic acids or their salts or esters used, including especially derivatives with aromatic groups, such as ortho, meta or para-substituted phenylboronic, in particular 4-formylphenylboronic acid, or the salts or esters of the compounds mentioned. Also peptide aldehydes, the is called Oligopeptide with reduced C-terminus, especially those from From 2 to 50 monomers are used for this purpose. To the peptidic reversible protease inhibitors include, among others, ovomucoid and Leupeptin. Also specific, reversible peptide inhibitors for the protease Subtilisin as well as fusion proteins from proteases and specific Peptide inhibitors are for this suitable.

Other enzyme stabilizers are amino alcohols such as mono-, di-, triethanol- and -propanolamine and mixtures thereof, aliphatic carboxylic acids up to C ₁₂ , such as succinic acid, other dicarboxylic acids or salts of said acids. End-capped fatty acid amide alkoxylates are also suitable for this purpose. Certain organic acids used as builders are capable, as disclosed in WO 97/18287, of additionally stabilizing a contained enzyme.

low aliphatic alcohols, but especially polyols, such as Glycerine, ethylene glycol, propylene glycol or sorbitol are other often used enzyme stabilizers. Di-glycerol phosphate also protects against Denaturation due to physical influences. Likewise, calcium and / or magnesium salts, such as calcium acetate or calcium formate.

Polyamide oligomers or polymeric compounds such as lignin, water-soluble vinyl copolymers or cellulose ethers, acrylic polymers and / or polyamides stabilize the enzyme preparation, inter alia, against physical influences or pH fluctuations. Polyamine N-oxide containing polymers act simultaneously as enzyme stabilizers and as color transfer inhibitors. Other polymeric stabilizers are linear C ₈ -C ₁₈ polyoxyalkylenes. Also, alkylpolyglycosides can stabilize the enzymatic components of the agent according to the invention and, preferably, are capable of additionally increasing their performance. Crosslinked N-containing compounds preferably fulfill a dual function as soil release agents and as enzyme stabilizers. Hydrophobic, nonionic polymer stabilizes in particular an optionally contained cellulase.

reducing agent and increase antioxidants the stability of the enzymes oxidative decay; therefor For example, sulfur-containing reducing agents are familiar. Other Examples are sodium sulfite and reducing sugars.

Especially preferably combinatons of stabilizers are used, for example made of polyols, boric acid and / or borax, the combination of boric acid or borate, reducing Salts and succinic acid or other dicarboxylic acids or the combination of boric acid or borate with polyols or polyamino compounds and with reducing Salt. The effect of peptide-aldehyde stabilizers is favorably by combining with boric acid and / or boric acid derivatives and polyols increased and even further by the extra Effect of divalent cations, such as calcium ions.

relevant invention Means are characterized in a preferred embodiment characterized that they, For example, the active ingredients contained temporally or spatially from each other release, consist of more than one phase. there These can be phases in different phases, but especially in phases in the same physical states act.

Inventive agents, which are composed of more than one solid component, can be prepared in a simple manner by mixing together various solid components, in particular powders, granules or extrudates with different ingredients and / or different release behavior in total loose form. The preparation of solidifying agents which are relevant to the invention from one or more phases can be carried out in a known manner, for example by spray drying or granulation, the enzymes and any further thermally sensitive ingredients such as, for example, bleaching agents optionally being added separately later. For the preparation of inventive compositions having an increased bulk density, in particular in the range of 650 g / l to 950 g / l, is one of the European patent EP 0 486 592 known method comprising an extrusion step is preferred. Another preferred preparation by means of a granulation process is in the European patent specification EP 0 642 576 described.

proteins can for fixed Agent for example in dried, granulated, encapsulated or more encapsulated and in addition dried form can be used. They can be separated, that is as their own Phase, or with other components together in the same phase, be added with or without compaction. Should be microencapsulated Enzymes are processed in solid form, so the water can with From the prior art known from the Working up resulting aqueous solutions removed be, like spray-drying, Centrifuging or by solubilization. The way this way obtained particles usually have a particle size between 50 and 200 μm.

The encapsulated form lends itself to protecting the enzymes from other ingredients, such as bleaches, or to allow for controlled release. Depending on the size of these capsules, a distinction is made according to milli, micro and nano capsules, with microcapsules for enzymes being particularly preferred. Such capsules, for example, with the patent applications WO 97/24177 and DE 199 18 267 disclosed. Another possible encapsulation method is that suitable for use in detergents or cleaning enzymes, starting from a mixture of the enzyme solution with a solution or suspension of starch or a starch derivative, are encapsulated in starch, or the starch derivative. Such an encapsulation process is with the German application DE 199 56 382 described.

There may also be at least two solid phases connected to each other. Thus, there is a possibility to provide a solid means relevant to the invention, in the pressing or compacting into tablets. Such tablets may be monophase or polyphase. Thus, this dosage form also offers the possibility to present a fixed invention-relevant agent with two solid phases. For the preparation of agents relevant to the invention in tablet form, which may be monophasic or multiphase, monochromatic or multicolor and / or consist of a plurality of layers, preferably all components - optionally one layer - are mixed together in a mixer and the mixture by means Conventional tablet presses, such as eccentric or rotary presses, pressed with compressive forces in the range of about 50 to 100 kN / cm ² , preferably at 60 to 70 kN / cm ² . Particularly in the case of multilayer tablets, it may be advantageous if at least one layer is pre-compressed. This is preferably carried out at pressing forces between 5 and 20 kN / cm ² , in particular at 10 to 15 kN / cm ² . Preferably, a tablet produced in this way has a weight of from 10 g to 50 g, in particular from 15 g to 40 g. The spatial form of the tablets is arbitrary and can be round, oval or angular, with intermediate forms are also possible.

Especially it is advantageous if in multiphase means at least one the phase contains an amylase-sensitive material, in particular starch or is at least partially surrounded or coated by this. On this way, this phase is mechanically stabilized and / or against influences protected from the outside and simultaneously over an active in the wash liquor amylase attacked, so that the release of the Ingredients is facilitated.

Also preferred means relevant to the invention are characterized in that that she totally liquid, gel or pasty available. The proteins contained, preferably a protein according to the invention, become such agents preferably starting from a state of the Technology performed Protein recovery and preparation in concentrated aqueous or non-aqueous solution, for example in liquid form, as a solution, Suspension or emulsion, but also in gel form or encapsulated or added as a dried powder. Such detergents or cleaners in the form of solutions in usual solvents are usually made by simply mixing the ingredients, in substance or as a solution in an automatic mixer can be given.

A Form of suitable agents are those liquid, gel or pasty Means to which a protein essential to the invention and / or a the other contained proteins and / or one of the other contained Ingredients encapsulated, preferably in the form of microcapsules has been added. Especially suitable are those with Capsules of amylase-sensitive material. Such a common use of amylase-sensitive materials and the invention essential amylolytic enzyme in a detergent or cleaning agent can Synergy effects show, for example, that the starch-splitting enzyme splits the microcapsules supported and thus the release process of encapsulated ingredients so that their release is not during the Storage and / or not at the beginning of the cleaning process, but only takes place at a certain time. On this mechanism can complex washing and cleaning agent systems with a wide variety of Ingredients and various types of capsules are based, especially preferred embodiments of the present invention.

One Comparable effect is given when the ingredients of the washing or cleaning agent to at least two different Distribute phases; For example, two or more fixed, with each other connected phases of a tablet-shaped washing or cleaning agent, or different granules within the same powdered agent. Two- or multi-phase cleaners are suitable for use both in machine dishwashers as well as in detergents prior art. The activity of a amylolytic enzyme in an earlier activated phase is a prerequisite for the Activation of a later Phase, when surrounded by an amylase-sensitive envelope or coating or the amylase-sensitive material is an integral part the solid phase, in its partial or complete hydrolysis disintegrated the phase in question. The use of the invention essential Enzyme for This purpose thus constitutes a preferred embodiment of the present invention Invention.

The Ingredients of detergents and cleaners are able to Suitably to assist one another in their performance. The synergistic use of amylase and dye transfer inhibitors Increase the cleaning performance, for example, with the application WO 99/63035. It is also known that polymers that are simultaneously can be used as co-builders, for example alkyl-poly-glycosides, the activity and the stability stabilize and increase the amount of enzymes contained, so from the application WO 98/45396. Thus, it is preferable if a α-amylase variant according to the invention through one of the rest, listed above Modified ingredients, in particular stabilized and / or in his Contribution to the washing or cleaning performance of the agent is increased. Corresponding formulations for agents according to the invention thus represent particularly preferred embodiments of the present invention Invention.

In the context of appropriate means, α-amylase variants according to the invention can serve for the activation of one's own or other phases if they are used alone or together with at least one other detergent-active or cleaning-active ingredient in one of more than one phase standing washing or cleaning agents are provided. Similarly, they may also serve to release ingredients from capsules when provided in encapsulated form in a detergent or cleaning agent.

A Another object of the present invention provide methods for cleaning textiles or hard surfaces, characterized in that in at least one of the method steps an above-described α-amylase variant according to the invention becomes active.

Because in this form, the invention is realized in that the inventively improved enzymatic properties, in particular the alkalinity activity an improvement in principle exploited every cleaning process become. Each cleaning process is enriched with the activity in question, when added in at least one process step. such Methods are used, for example, with machines such as common household dishwashers or household washing machines realized.

Further suitable are such processes, which are characterized that the α-amylase variant via a above-described agent is used.

Especially suitable is any process which is characterized in that the α-amylase in the respective Process step in an amount of 0.01 mg to 400 mg per corresponding Process step is used, preferably from 0.02 mg to 300 mg, more preferably from 0.03 mg to 100 mg.

At best, This results in concentration values of 0.0005 to 20 mg per 1, preferably 0.005 to 10 mg per liter, more preferably 0.005 to 8 mg of the amylolytic protein per liter wash liquor. The protein concentration can be done using known methods, for example the BCA method (Bicinchoninic acid, 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method (A.G. Gornall, C.S. Bardawill and M.M. David, J. Biol. Chem. 177 (1948), pp. 751-766).

Corresponding the previous versions The present invention is also characterized by the use of α-amylase variants according to the invention realized. Because even here come the favorable properties of the relevant Enzyme for action. These apply in particular for cleaning purposes.

a own invention subject thus represents the use of one of above-described α-amylase variant according to the invention for cleaning textiles or hard surfaces.

there It can be used as the only effective component. Preferably this is done together with at least one other cleaning active or the cleaning effect supporting agent.

Prefers is thus such a use characterized is that the α-amylase variant via a above-described agent is used.

conveniently, such a use is characterized in that, per application, preferably per application in a dishwasher or a washing machine 0.01 mg to 400 mg of the α-amylase variant are used, preferably 0.02 mg to 300 mg, more preferably 0.03 mg to 100 mg.

Because This results in the wash liquor conveniently the above Concentration values. This dosage may vary depending on the cleaning problem be made by the manufacturer of the product or by the end user.

A another embodiment represents the use of an α-amylase variant according to the invention for the treatment of Raw materials or intermediates in textile production, especially for desizing cotton.

Raw materials and intermediate products of textile production, for example those based on cotton, are provided with starch during their production and further processing in order to enable better processing. This process, applied to yarns, to intermediate products, as well as to textiles, is called sizing. To remove the size, ie the starch-containing protective layer (desizing, desizing), amylolytic proteins according to the invention are suitable. This in particular especially when alkaline conditions prevail or when other ingredients such as surfactants are present.

Examples

All molecular biological work steps follow standard methods, such as for example, in the handbook of Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual ", Cold Spring Harbor Laboratory Press, New York, 1989, or equivalent relevant Plants are specified. Enzymes and kits were tested according to the Information from the respective manufacturer used.

example 1

Representation of the B. amyloliquefaciens amylase mutants by error-prone mutagenesis

Starting from a total DNA embossment of B. amyloliquefaciens, as obtained under the number DSM 7 from the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1 b, 38124 Braunschweig (http://www.dsmz.de) can, PCR was about 1700 bp fragment containing the 1545 bp gene for the α-amylase contained. This fragment, after restriction with BamHI and SpeI, was ligated into the expression vector pCYTEX P1 which had previously been cut with the same enzymes to give the in 3 shown vector pCYTBAA was obtained. Subsequently, standard site-directed mutagenesis introduced an NdeI restriction site upstream of the start codon of the BAA gene. Now, the BAA fragment obtained by restriction with NdeI and PstI was ligated into the NdeI / PstI site of the vector pG-PFE, whereby the in 4 vector pGBAA-WT was obtained. In principle, any other cloning vector for the insertion of the α-amylase gene could have been used analogously. By sequencing, it was finally verified (1) that the orientation of the insert as in 4 (ie, the gene still had the completely correct wild-type sequence as shown in SEQ ID NO. 3 is shown.

A preparation of this vector pGBAA-WT has now been prepared as described by H. Zhao et al. (1999) "Methods for Optimizing Industrial Enzyme by Directed Evolution" in Industrial Microbiology and Biotechnology, eds., Demain, AL et al., Washington DC, ASM Press, pp. 597-604, is subjected to error-prone PCR In particular, the following substances were combined for a 100 μl batch: 10 μl 10 × mutagenic buffer (70 mmol / l MgCl ₂ , 500 mmol / l KCl, 100 mmol / l Tris-HCl buffer (pH 8), 0.1% by weight gelatin), 10 μl dNTP mix (2 mmol / l dGTP and dATP, 10 mmol / l dCTP and dTTP each), 50 pmol of each primer (SEQ ID NO: 1 and 2), 5-15 ng of template DNA, an appropriate amount of ddH ₂ O and 3 μl of MnCl ₂ (5 mmol / l), followed by 5 units (E) of commercially available Taq. Polymerase (from QIAGEN, Hilden, Germany) were added and the batch was subjected to the following temperature program: 60 s denaturation at 95 ° C., 25 cycles each (a) 30 s at 95 ° C, (b) 30 s at 50 ° C and (c) 45 s at 72 ° C and finally 120 s at 72 ° C.

The PCR product was purified after completion of the reaction and cloned into suitable cloning and / or expression vectors. In this case, the QIAquick ^® PCR Purification Kit (QIAGEN, Hilden Fa.) Was added to purify selected and the PCR product was, after restriction with the enzymes NdeI and PstI into the Nde I - Pst I - restriction site of the vector pGASTON (E. Henke, PhD thesis, Institute of Technical Biochemistry, University of Stuttgart).

to Expression of the obtained BAA variants were initially competent Cells of E. coli XL1-blue (available from Stratagene, LaJolla, USA) with the vector pJL3 (MoBi-Tech, Göttingen, Germany) transformed to over bacteriocin release protein (BRP), the enzyme yield later released into the supernatant to increase. Subsequently they were transformed with the pGASTON plasmids in question and isolated on microtiter plates.

Example 2

Screening of B. amyloliquefaciens α-amylase mutants for improved alkalinity of the derived enzyme

Preparation of the enzyme solution

The the amylase expressing cells were disrupted by the whole microtiter plates twice in liquid nitrogen (-196 ° C) frozen and for 45 min at 37 ° C were thawed. The cell pellet was purified by centrifugation (1000 g, 15 min, 25 ° C) the microtiter plates from the supernatant separated and this enzyme-containing supernatant used directly for the activity test.

Perform the activity test

For an assay is run at pH 7 (standard) in a 50 ml Falcon reaction vessel to 30 ml of double distilled water (ddH ₂ O) 4 Phadebas ^® tablets (crosslinked starch with covalently-bound dye; available from Pharmacia & Upjohn. GmbH, Diagnostics, Freiburg: Order No. 10-5380-32) and suspended. For performance at pH 10 4 Phadebas ^® tablets as described above are suspended in a glass filter (pore size 1 or 2) from the liquid phase separated and washed twice with 10 ml _ddH2O. The retentate is resuspended in 30 ml of a 0.1 mol / l glycine / NaOH buffer (pH 10) with 0.25 mmol / l CaCl ₂ .

150 .mu.l of the resulting suspensions for the respective pH values were pipetted into each well of a good slurrying MAR5 N50 ^® -Filtermikrotiterplatte (Fa. Millipore, Schwalbach). These were mixed with 50-75 ul enzyme-containing solution, mixed and incubated for 15 min at 37 ° C. The liquid phases were transferred by centrifugation from the filter microtiter plate (15 min, 1000 g, 25 ° C) in an underlying second microtiter plate and the absorption of the thus obtained liquid phases at a wavelength of 620 nm measured.

On thus, three mutants of the α-amylase gene were identified: the tribes A42, A29 and B1.

Example 3

Cultivation of the trunks, extraction of the associated Gene and its sequencing.

The Strains identified in this way were prepared according to routine methods taken under antibiotic selection in culture, the relevant pGASTON mutant plasmids isolated and over Sequencing determines the deviations from the starting DNA.

The DNA and amino acid sequences of the α-amylases BAA A42, BAA A29 and BAA B1 obtained thereby are shown in the sequence listing under SEQ ID NO. 5 and 6, SEQ ID NO. 7 and 8, or under SEQ ID NO. 9 and 10 indicated. The associated amino acid sequences are also aligned with the 2 compared to the wild-type sequence.

These Variants differ from the wild-type B. amyloliquefaciens α-amylase through exchanges in individual positions of 13, 32, 194, 197, 203, 230, 297, 356, 406, 414 and 474 in the count according to SEQ ID NO. 4. Concrete the following exchanges were identified: L13P, V32A, W194R, S197P, I203L, A230V, N297D, E356D, K406R, N414S and K474Q.

• – B. amyloliquefaciens-α-Amylase L13P/W194R/S197P/E356D/N414S, als
• – B. amyloliquefaciens-α-Amylase L13P/V32A/A230V/N297S/K406R/N414S, beziehungsweise als
• – B. amyloliquefaciens-α-Amylase L13P/V32A/I203L/A230V/N297S/K406R/N414S/K474Q bezeichnet werden.

In detail, BAA A42 has the exchanges L13P, W194R, S197P, E356D and N414S, BAA A29 through the exchanges L13P, V32A, A230V, N297S, K406R and N414S, and BAA B1 through the exchanges L13P, V32A, I203L, A230V, N297S , K406R, N414S and K474Q. Therefore, instead of as α-amylases BAA A42, A29 and B1 also as

• B. amyloliquefaciens α-amylase L13P / W194R / S197P / E356D / N414S, as
• B. amyloliquefaciens α-amylase L13P / V32A / A230V / N297S / K406R / N414S, respectively
• B. amyloliquefaciens α-amylase L13P / V32A / I203L / A230V / N297S / K406R / N414S / K474Q.

It is noteworthy that the exchanges in positions 13 and 414 (L13P and N414S) in all three α-amylase variants occur. A42 has three additional exchanges in positions 194, 197 and 356 (W194R, S197P and E356D). A29 has four additional exchanges with those in positions 13 and 414, namely at positions 32, 230, 297 and 406 (V32A, A230V, N297S and K406R). Compared to A29, B1 also has two other amino acid substitutions, namely positions 203 and 474, specifically the variations I203L and K474Q.

Example 4

Cleaning and characterization the respective B. amyloliquefaciens amylase variant

Purification of α-amylase

The these three α-amylases producing strains were taken by standard methods in liquid culture and the transgenically formed α-amylase isolated. Purification of α-amylase was done according to a protocol by Candussio, A., et al. (1990): "Biochemical and genetic analysis of a maltopentaose-producing amylase from alkaliphilic Grampositive bacterium ", Eur. J. Biochem., Vol. 191, pages 177-185.

Protein content and activity determination

The the purification accompanying protein content determinations were made with the Micro-BCA Protein Assay Kit from Pierce Biotechnology, Rockford, IL, USA (Order No. 23235) and BSA as standard according to the manufacturer's instructions. The amylase activities were determined as indicated in Example 2. this was average activity yields from 5 to 10%.

Example 5

activity determination

1 Unit (E) of amylase activity is defined as the amount of enzyme which produces 1 μmol glucosidic acid per 1 minute Bonds at 37 ° C hydrolyzed.

Phadebas ^® test

A Phadebas ^® tablet is suspended in 10 ml ddH ₂ O. Of these, 500 .mu.l aliquots per measurement are mixed with 50 .mu.l of appropriately diluted sample (activity concentration <1000 U / l). After incubation for 15 min at 37 ° C., the pH of the buffer substances contained in the tablet being 6.5, the reaction is stopped by the addition of 150 μl of 0.5 mol / l NaOH. To determine the blank value, 500 .mu.l of Phadebas suspension are incubated for 15 min at 37.degree. C. and, after the immediate addition of 150 .mu.l of 0.5 M NaOH, 50 .mu.l of sample are added. After centrifugation for 2 min at 14,000 rpm (Centrifuge 5417C from Eppendort, Hamburg), 500 μl of the supernatant are diluted in a cuvette 1: 2 with ddH ₂ O and the absorbance at a wavelength of 620 nm is measured against the corresponding blank value obtained. The activity can be read from a calibration curve previously prepared with an amylase preparation of known activity.

In this way, the specific activities indicated in Table 1 were determined, which are a characteristic size for each enzyme. Table 3: Specific activities of the invention B. amyloliquefaciens amylase variants among Bedingugen the Phadebas ^® tests

Example 6

pH activity profiles

To record a pH activity profile, 8 Phadebas ^® tablets are suspended in 40 ml ddH ₂ O. The solid substrate is separated by filtration through a glass frit (pore size 0 or 1) and taken up in 40 ml ddH ₂ O. 5 ml each of the well-slurried substrate suspension are mixed with 5 ml of the 0.1 mol / l buffer with the appropriate pH. Here, for measurements in the pH range of 4 to 6 acetate buffer, for the range 7 to 8 Tris / HCl buffer, for pH 9 and pH 10 glycine / NaOH buffer and for measurements at pH 11 carbonate buffer used. For each pH A triple determination is performed similarly to the protocol for the Phadebas ^® -Schnelltest. In addition, the blank values at pH 6 and pH 10 are also determined in triplicate.

The values obtained also represent enzyme-specific properties. TABLE 4 pH Optima and relative activity at pH 10 of variants A42, A29 and B1 compared to wild-type BAA

Here shows that in particular the variant BAA 42 opposite the starting molecule, but also opposite the other variants have a significantly improved alkalinity. This can only be done on one or both exchanges in the positions 203 and / or 474, specifically I203L and K474Q be due.

description the figures

1 : Alignment of the α-amylase from Bacillus amyloliquefaciens with the most important other α-amylases of the prior art.

In this mean:

BAA

α-amylase from B. amyloliquefaciens

BLA

α-amylase from B. licheniformis

LAMY

α-amylase from Bacillus sp. KSM-AP1378

S707

α-amylase from Bacillus sp. # 707

BStA

α-amylase from B. stearothermophilus

TS-23

α-amylase from Bacillus sp. TS-23

Of the The beginning of mature proteins is uniform in the count of this alignment at positions 41 and 43 respectively.

2 : Alignment of wild-type α-amylase from B. amyloliquefaciens (BAA) with the α-amylases particularly preferred according to the invention.

In this mean:

BAA

α-amylase from B. amyloliquefaciens, Wild-type enzyme

BAA_42

α-amylase from B. amyloliquefaciens, Variant A42

BAA_A29

α-amylase from B. amyloliquefaciens, Variant A29

BAA_B1

α-amylase from B. amyloliquefaciens, Variant B1

3 : The cloning vector pCYTBAA

As in Example 1, the wild-type gene of α-amylase was shown from B. amyloliquefaciens (SEQ ID NO: 3) prior to conversion to an expression vector intercloned to an additional Introduce restriction site.

4 : The expression vector pGBAA-WT

As shown in Example 1, the wild-type gene of the B. amyloliquefaciens α-amylase (SEQ ID NO: 3) was transferred to this expression vector after intermediate cloning and subjected to error-prone PCR, whereby the BAA variants according to the invention were obtained. SEQUENCE LISTING

Claims (30)

Hydrolytically active α-amylase variant having at least two amino acid exchanges with respect to the starting molecule, characterized in that a proline residue (P) is inserted into position 13 according to the counting of the α-amylase from B. amyloliquefaciens (SEQ ID NO: 4) and in position 414 Serine residue (S) is present. α-amylase variant according to claim 1, characterized in that in addition at least one amino acid exchange in one of the positions 194, 197 or 356 according to the count of B. amyloliquefaciens α-amylase (SEQ ID NO: 4) is present, preferably in two, particularly preferred in all three of these positions. α-amylase variant according to claim 2, characterized in that in position 194 an arginine (R) and / or at position 197 a proline (P) and / or at position 356 an aspartic acid residue (D) is present. α-amylase variant according to claim 1, characterized in that in addition at least one amino acid exchange in one of the positions 32, 230, 297 or 406 according to the count of the α-amylase B. amyloliquefaciens (SEQ ID NO: 4) is present, preferably in two, more preferably in three, most preferably in all four of these positions. α-amylase variant according to claim 4, characterized in that in position 32 an alanine (A) and / or in position 230 a valine (V) and / or in position 297 an aspartic acid (D) and / or at position 406 an arginine residue is present. Hydrolytically active α-amylase variant with at least two amino acid exchanges across from the starting molecule, characterized in that a Exchange at position 203 and exchange at position 474 according to the α-amylase count from B. amyloliquefaciens (SEQ ID NO: 4). α-amylase variant according to claim 6, characterized in that in position 203 a leucine (L) and / or in position 474 a glutamine residue (Q) is present. α-amylase variant according to one of the claims 4 or 5, characterized in that additionally at least one amino acid exchange in one of the positions 203 or 474 according to the count of B. amyloliquefaciens α-amylase (SEQ ID NO 4), preferably in both positions. α-amylase variant according to claim 8, characterized in that in position 203 a leucine (L) and / or in position 474 a glutamine residue (Q) is present. α-amylase variant according to one of the claims 1 to 9, characterized in that the starting molecule of an α-amylase derives originally from a Bacillus species, preferably from B. amyloliquefaciens, B. licheniformis, B. agaradherens, B. stearothermophilus, B. sp. KSM-AP 1378, B. sp. # 707 or B. sp. A 7-7 (DSM 12368), especially preferably from B. amyloliquefaciens, B. agaradherens (DSM 9948) or Sp. A 7-7 (DSM 12368), especially from B. amyloliquefaciens. α-amylase variant according to one of the claims 1 to 9, characterized in that the starting molecule is a Hybrid α-amylase is preferably based on at least one of the α-amylases from B. amyloliquefaciens, B. licheniformis, B. agaradherens, B. stearothermophilus, B. sp. KSM-AP 1378, B. sp. # 707 or B. sp. A 7-7 (DSM 12368), more preferably based on at least two the α-amylases from B. amyloliquefaciens, B. licheniformis, B. agaradherens, B. stearothermophilus, B. sp. KSM-AP 1378, B. sp. # 707 or B. sp. A 7-7 (DSM 12368). α-amylase variant according to claim 11, characterized in that it is in the hybrid α-amylase is one of whose subsequences of the α-amylases from B. amyloliquefaciens and derived from B. licheniformis. α-amylase variant according to one of the claims 1 to 10, characterized in that it is a variant the α-amylase of B. amyloliquefaciens with one of the amino acid exchange combinations L13P / W194R / S197P / E356D / N414S, L13P / V32A / A230V / N297D / K406R / N414S or L13P / V32A / I203L / A230V / N297D / K406R / N414S / K474Q from B. amyloliquefaciens A42 (SEQ ID NO: 6), from B. amyloliquefaciens A29 (SEQ ID NO: 8) or off amyloliquefaciens B1 (SEQ ID NO: 10), preferably with the amino acid exchange combination L13P / W194R / S197P / E356D / N414S from B. amyloliquefaciens A42 (SEQ ID NO. 6). Nucleic acid, the for one of the α-amylase variants according to one of the claims 1 to 13 coded. nucleic acid according to claim 14, characterized in that it is derived from a nucleic acid according to SEQ ID NO. 3 derives. nucleic acid according to claim 14, characterized in that it is derived from a nucleic acid according to SEQ ID NO. 5, SEQ ID NO. 7 or SEQ ID NO. 9 derives, preferably one these sequences themselves. Vector that resembles an area with a nucleic acid one of the claims 14 to 16 contains. Vector according to Claim 17, characterized that he is a cloning vector. Vector according to Claim 17, characterized that he is an expression vector. Cell, characterized in that it contains one of the nucleic acids designated in claims 14 to 16, preferably on a vector according to any one of claims 17 to 19, wherein the cell has no is human embryonic stem cell. Cell according to claim 20, characterized in that that she one of the claims 1 to 13 designated α-amylase variants forms or can be stimulated to their formation, preferably using a nucleic acid according to one of the claims 14 to 16, particularly preferably using an expression vector according to claim 19. Cell according to claim 21, characterized in that that it this is a bacterium, especially one that the formed α-amylase variant secreted into the surrounding medium. Cell according to one of Claims 20 to 22, characterized that it this is a Gram-negative bacterium, preferably the genera Escherichia coli or Klebsiella, in particular derivatives E. coli K12, E. coli B or K. planticola, and more particularly to derivatives of the strains E. coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or K. planticola (Rf). Cell according to one of Claims 20 to 22, characterized that it These are gram-positive bacteria, especially the genera Staphylococcus, Corynebacterium or Bacillus, in particular the Species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B. alcalophilus, B. licheniformis, B. amyloliquefaciens, Stearothermophilus, B. agaradherens, B. globigii or B. lentus. Process for the preparation of an α-amylase variant according to one the claims 1 to 13, preferably using a nucleic acid according to one of the claims 14 to 16, more preferably using a vector according to one of the claims 17 to 19, most preferably using a cell according to one of the claims 20 to 24. Use of an α-amylase variant according to one of the claims 1 to 13 for cleaning textiles or hard surfaces. Use according to claim 26, characterized that the α-amylase variant has a washing or cleaning agent is used. Use according to claim 26 or 27, characterized that pro Application, preferably per application in a dishwasher or a washing machine 0.01 mg to 400 mg of the α-amylase variant used are preferably 0.02 mg to 300 mg, more preferably 0.03 mg to 100 mg. Use of an α-amylase variant according to one of the claims 1 to 13 for the treatment of raw materials or intermediates in textile production, especially for desizing cotton.